HYALURONAN Volume 1 - Chemical, Biochemical and Biological Aspects
Editors: JOHN F. KENNEDY BSe, PhD, OSe, EurChem CChem FRSc, CBiol FIBiol, FCIWEM, FCMI, FIFST Director of Birmingham Carbohydrate and Protein Technology Group, School of Chemical Sciences,The University ofBinningham, BirminghamB15 2IT, England, UK, Director of Chembiotech Ltd, University of Birmingham Research Park, Birmingham B15 2SQ, England, UK, Director of Inovamed Ltd, Chembiotech Laboratories, University of Birmingham Research Park, Vincent Drive, Birmingham B15 2SQ, England, UK, and Professor of Applied Chemistry, The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd, LUI 2AW, Wales, UK
GLYN O. PHILLIPS BSe, PhD, OSe, HODOSe, HODLIB, CChem FRSC Chairman of Research Transfer Ltd, Newtech Innovation Centre, Professorial Fellow, The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd, LUI 2AW, Wales, UK, and Professor of Chemistry, The University of Salford, England, UK
PETER A. WILLIAMS BSe, PhD, CChem FRSC Director of the Centre for Water Soluble Polymers, The North East Wales Institute of Higher Education, Plas Coch, Mold LUI 2AW, Wales, UK, Director of the Centre for Advanced and Renewable Materials at Institute and University of Wales, Bangor, The North East Wales Institute of Higher Education, P1as Coch, Mold LUI 2AW, Wales, UK Professor of Polymer and Colloid Chemistry, The North East Wales Institute of Higher Education, Plas Coch, Mold . LL11 2A W, Wales, UK
Road, Wrexham, Clwyd,
the North East Wales Road, Wrexham, Clwyd,
Road, Wrexham, Clwyd,
Guest Editor: VINCE C. HASCALL
PhD
Co-Direetor of the Orthopaedic Surgery Musculoskeletal Research Center, Department of Biomedical Engineering ND-20, Lerner Research Institute. Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA Adjunct Professor Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA Adjunct Professor Department of Biochemistry, Rush Presbyterian S1. Lukes Medical Center, Chicago, Illinois, 60612 USA
WOODHEAD PUBLISHING LIMITED
Published by Woodhead Publishing Ltd, Abington Hall, Abington, Cambridge CB I 6AH, England www.woodhead-publishing.com First published 2002 © 2002, Woodhead Publishing Ltd The authors have asserted their moral rights Conditions of sale This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from the publisher. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN I 85573 5709 (2 volume set) Printed in Great Britain by MFK Group Ltd
CONTENTS Preface GO Phillips
xvii
Introductory remarks E A Balazs
xix
PART 1: OVERVIEW OF THE HISTORY AND DEVELOPMENT OF HYALURONAN l.
Hyaluronan before 2000 T C Laurent
2.
Karl Meyer - Discoverer of hyaluronan Biomatrix Inc
3.
17
Alexander G ("Sandy") Ogston (1911-1996) T C Laurent
4.
3
25
Albert Dorfman N R Schwarz and L Roden
29
PART 2: CHARACTERISATION AND SOLUTION PROPERTIES OF HYALURONAN S.
Predictive and experimental behaviour of hyaluronan in solution and solid state K Haxaire, E Buhler, M Milas, S Perez and M Rinaudo.
6.
37
Aqueous SEC, light scattering and viscometry of ultra-high molar mass Hyaluronan R Mendichi and A G Schieroni
7.
47
Molecular characterisation of hyaluronan and hylan using GPC MALLS and asymmetrical Flow FFF-MALLS SAl-Assaf, P A Williams and G 0 Phillips
8.
55
Force measurements on surfaces bearing covalently linked hyaluronan M Morra and C Cassinelli
67
iv
Contents
9.
The intrinsic viscosity of hyaluronan M K Cowman and S Matsuoka
10.
75
Viscosity of polymer solutions revisited S Matsuoka and M K Cowman
79
11. Conformational and rheological properties of hyaluronan K Nishinari, Y Mo, R Takahashi, K Kubota and A Okamoto 12.
89
The heat dependence of hyaluronan conformation G Armand, K Fan and E A Balazs .
99
13. Temperature effect on the dynamic rheological characteristics of hyaluronan, hylan A and Synvisc J M Hoefling, M K Cowman, S Matsuoka and E Balazs
14.
Tapping mode atomic force microscopy of hyaluronan and hylan A M K Cowman, M Li, A Oyal and S Kanai
15.
103 109
Biological properties of hyaluronan are controlled and sequestered by tertiary structures J E Scott and F Heatley
16.
117
Analysis of the concentrated solution properties of hyaluronan by confocal-FRAP show no evidence of chain-chain association T Hardingham, B CHeng and P Gribbon.
17.
Hyaluronic acid self-association in the presence and absence of salts T M McIntire and 0 A Brant.
18.
123 137
Comparison of the reactivity of different reactive oxidative species (ROS) towards hyaluronan B J Parsons, SAl-Assaf, S Navaratnam and G 0 Phillips
19.
141
Polysaccharide fragmentation induced by hydroxyl radicals and hypochlorite M 0 Rees, C L Hawkins and M J Davies
20.
151
Getting to grips with HA-protein interactions C.O Blundell, J 0 Kahmann, A Perczel, 0 J Mahoney, M R Cordell, P Teriete, I 0 Campbell and A J Day
161
Contents
v
PART 3: RHEOLOGICAL BEHAVIOUR OF HYALURONAN 21
Effect of metal ions on the rheological flow profiles of hyaluronate solutions C J Knill, J F Kennedy, Y Latif and D C Elwood
22.
. 175
Rheological behaviour of hyaluronan, healon and hylan in aqueous solutions M Milas, M Rinaudo, I Roure, SAl-Assaf, G 0 Phillips and P A Williams
23.
181
Rheological creep experiments utilizing mixtures of 1 % hylan A solution and 0.5 % hylan B gel slurry J .M Hoefling, S Matsuoka and E A Balazs
24.
Rheology of hyaluronan products
o Wik, B Agerup and H B Wik 25
. 195
201
Structural change in hydrogelation of hyaluronan induced by annealing the solution in sol state M Takahashi, T Iseki, H Hattori, T Hatakeyama and H Hatakeyama
26
. 205
The roles of extensional and shear flows of synovial fluid and replacement systems in joint protection C Backus, S P Carrington, L R Fisher, J A Odell and D A Rodigues
. 209
PART 4: BIOSYNTHESIS AND BIOLOGICAL DEGRADATION OF HYALURONAN 27
The production of hyaluronic acid from Streptococci D C Ellwood
28.
. 221
Hyaluronan synthases: mechanistic studies and biotechnological applications P L DeAngelis
29.
. 227
Hyaluronan synthase expression in human endometrium during the menstrual cycle M Tellbach, LA Salamonsen, G Brownlee, T Brown and M-P Van Damme
237
vi
Contents
30.
In vivo investigation of hyaluronan synthase function during vertebrate embryogenesis J Y Lee and A P Spicer
31.
. 245
Influence of substrate and enzyme concentrations on hyaluronan hydrolysis kinetics catalysed by hyaluronidase T Asteriou, B Deschrevel, F Gouley, and J C Vincent
32.
· 249
Human hyaluronidase polymorphism and evidence for conserved hyaluronidase potential N-glycosylation sites in mammalians and non mammalian species B Fiszer-Szafarz, A Litynska and L.Zou
· 253
PART 5: NOVEL MODIFIED FORMS OF HYALURONAN
33.
Hyaluronan linear and crosslinked derivatives as potential/actual biomaterials V Crescenzi, A Francescangeli, D Renier and D Bellini
34.
· 261
Novel biomaterials based on cross-linked hyaluronan structural investigations L Michielin, C Bevilacqua, S Paoletti, A Gamini R Toffanin and F Micali
35.
A novel crosslinking process for hyaluronan X Zhao, J Fraser and C Alexander .
36.
293
Hyaluronan DNA matrix for gene transfer W Chen, D Checkla and P Dehayza
39.
285
Derivatized hyaluronan for gels and nanochemically patterned surfaces R Barbucci, D Pasqui and G Leone.
38.
· 277
A biocompatible gel of hyaluronan A Okamoto and T Miyoshi
37.
· 269
· 305
Thermal properties of hyaluronic acid-based polyurethane derivatives associated with water H Hatakeyama, Y Asano, T Hatakeyama and J F Kennedy
40.
· 313
Phase transition of sodium hyaluronate, hylan and polyurethanes derived from hyaluronic acid in the presence of water T Hatakeyama and H Hatakeyama
· 323
Contents
VB
PART 6: CELL SURFACES AND HYALURONAN RECEPTORS 41.
CD 44: The link between hyaluronan and the cytoskeleton C.B Knudson, G A Nofal, G Chow and R S Peterson
42.
. 331
Hyaluronan binding by cell surface CD44 J Lesley, N English, V C Hascall, M Tarnrni and R Hyman
43.
. 341
Inhibition of tumor growth in vivo and anchorage-independent growth in vitro by perturbing hyaluronan-cell interactions B P Toole, R M Peterson and S Ghatak
44.
.
. 349
Novel Endothelial Hyaluronan Receptors D G Jackson, R Prevo, J Ni and S Banerji
45.
. 355
An insight into cellular signalling mediated by hyaluronan binding protein (HABPl) T B Deb, M Majumdar, A Bharadwa], B K Jha and K Datta
46.
RHAMM (CD168) co-associates with and regulates ERK kinase R Harrison, F S Wang and E A Turley
47.
. 365
. 373
Poly I:C induces mononuclear leukocyte-adhesive hyaluronan structures on colon smooth muscle cells: Icd and versican facilitate adhesion C A de la Motte, V C Hascall, J A Drazba and S A Strong .
48.
The generation of hyaluronan-dependent pericellular matrix in vitro J R E Fraser
49.
. 381
. 389
Purification and characterization of the hyaluronan receptor for endocytosis (HARE) PH Weigel, C McGulary, B Zhou and J A Weigel
50.
.
. 401
Identification of hyaluronan as crystal binding molecule at the surface of migrating and proliferating MDCK cells C.F Verkoelen, B G vd Boom, M S J Schepers and J C Romijn
•
. 411
Vlll
Contents
PART 7: THE ACTION OF HYALURONAN IN CELLS
51.
Anti-cancer activity of hyaluronan Me Filion, S Menard, B Filion, J Roy, S Reader and N C Phillips
52.
. 419
Pro-inflammatory activity of contaminating DNA in hyaluronan preparations M C Filion and N C Phillips .
53.
. 429
Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72 H Xu, T Ito, A Tawada, H Maeda, H Yarnanokuchi, K Isahara, K Yoshida, Y Uchiyama and A Asari
54.
. 435
The impact of hyaluronan on the in vitro invasive properties of human breast cancer cell lines with CD44 expression A Herrera-Gayol and S Jothy
55
. 443
Identification of a novel intracellular hyaluronan-bfndlng protein, IHABP4 L Hung, N Grarnmatikakis, M Yoneda, S D Banerjee and B P Toole
56.
. 447
The presence and processing of intercellular hyaluronan in proliferating cells S P Evanko and T N Wight
57.
451
Low molecular weight oligosaccharides of hyaluronan potently activate dendritic cells C C Termeer, P Prehm and J C Simon
58.
. 457
Signal transduction pathways in hyaluronan induced proliferation of endothelial cells M Slevin, S Kumar and J Gaffney..
59.
. 469
Control of hyaluronan (RA) generation in renal proximal tubular epithelial cells G Stuart, S Jones, M Jones and A 0 Phillips
60.
. 473
Mechanical injury of human peritoneal mesothelial cells (HPMC) is accompanied by an increase in hyaluronan synthesis S Yung , G J Thomas and M Davies
. 481
Contents
61.
IX
Apoptosis and hyaluronan-enriched extracellular matrix degradation in cumulus cell-oocyte complex: implication in fertility M D Giacomo, A Camaioni and A Salustri
62.
489
Proteoglycan enhances the formation of the SHAP-Hyaluronan complex and its effect in hyaluronan-rich matrix M Zhao, M Yoneda, L Zhuo, L Huang, H Watanabe, Y Yamada, S Nagasawa, H Nishimura and K Kimata .
63.
. 497
Enhanced thromboxane synthesis through the induction of cycleoxygenase-2 by hyaluronan in renal cells L KSun
. 501
PART 8: KERATINOCYTES AND HYALURONAN
64
Hyaluronan metabolism and distribution in stratified differentiated cultures of epidermal keratinocytes S Pasonen-Seppanen, R Tammi, M Tammi, M Hogg, V C Hascall, and D K MacCallum
65.
. 51 I
Intracellular Hyaluronan in epidermal keratinocytes R Tammi, K Rilla, J P Pieairnaki, M Hogg, 0 K MacCallum, V C Hascall and M Tammi
66.
517
Evaluation of the influence of hyaluronan and hyaluronan fragments on human keratinocytes during UV irradiation D Gerlach, C Huschka and W Wohlrab .
67.
525
Effect of hyaluronan on matrix metalloprotease expression in fibroblasts and keratocytes N Isnard, J. M Legeais, G Renard and L Robert
68.
531
Aging and regulation of hyaluronan biosynthesis comparative studies on human skin fibroblasts and corneal keratocytes L Robert, I Fodil, N Isnard, F Dupuy, A M Robert and G Renard.
69.
537
Effects of KGF and TGF-b on Hyaluronan synthesis and distribution in extra-, peri-, and intra-cellular compartments of epidermal keratinocytes S Karvinen, M Tammi and R Tammi
. 545
x
70.
Contents
Hyaluronan stimulates keratinocyte migratiou and activates the transcription factor AP·l in keratinocytes through the JNK pathway K Torronen, M Yabal, K Rilla, K Kaamiranta, R Tammi, M J Lammi and M Tammi
71.
. 551
Hyaluronan synthase 2 (HAS2) regulates migration of epidermal keratinocytes K Rilla, M Lammi, R Sironen, V C Hascall, R Midura, M Tammi and R Tammi. .
72.
. 557
EGF regulates HAS2 expression controls epidermal thickness and stimulates keratinocyte migration M Tammi, J P Pienirnaki, K Rilla, C Fullop. M J Lammi, R Sironen, R Midura, V C Hascall, M Luukkonen, K Torronen, T Lehto and R Tammi . 561
THE CELLUCON TRUST Incorporating
Cellucon Conferences International Educational Scientific Meetings on Wood and Cellulosics and Other Carbohydrate Polymers
Cellucon Conferences as an organisation was initiated in 1982, and Cellucon '84, which was the original conference, set out to establish the strength of British expertise in the international field of cellulose and its derivatives. This laid the foundation for subsequent conferences on carbohydrate etc. polymer topics in Wales (1986), Japan (1988), Wales (1989), Czechoslovakia (1990), USA (1991), Wales (1992), Sweden (1993), Wales (1994), Finland (1998), Japan (1999), and Wales 2000. These conferences have had truly international audiences drawn from the major industries involved in the production and use of cellulose pulp and fibre derivatives of cellulose, plus representatives of academic institutions and government research centres. This diverse audience has allowed the cross-fertilisation of many ideas, which has done much to give the field of cellulose in its diverse forms the higher profile that it rightly deserves. More recently other carbohydrate polymers have been the centre of focus, particularly hya1uronan, with the conference in 2000 - Hyaluronan 2000 - being the first major international conference on this majorly important carbohydrate polymer. Cellucon Conferences are organised by The Cellucon Trust, an official UK. charitable Trust with world-wide objectives in education in wood and cellulosics. The Cellucon Trust is continuing to extend the knowledge of all aspects of cellulose, lignin, hyaluronan and other national polymers world-wide. At least one book has been published from each Cellucon Conference as the proceedings thereof This volume arises from the 2000 conference held in Wrexham, Wales and the conference planned to be held in the USA in 2003 again on hyaluronan, will generate further useful books in this area. THE CELLUCON TRUST TRUSTEES AND DIRECTORS Prof G.O. Phillips (Chairman) Prof. IF. Kennedy (Deputy Chairman and Treasurer) Prof P.A. Williams (Secretary General)
Research Transfer Ltd, UK. The North East Wales Institute, UK, and The University of Birmingham, UK. The North East Wales Institute, UK.
The Cellucon Trust is a registered charity, UK. Registration No: 328582 and a company limited by guarantee, UK. Registration No: 2483804 with its registered offices at Chembiotech Laboratories, The University of Birmingham Research Park, Vincent Drive, Birmingham, B15 2SQ, UK
The 12th International Cellucon Conference
YA L URONA N
H 2000 An International Meeting Celebrating the
80'" Birthday of Endre A Balazs
ACKNOWLEDGEMENTS This book is one of the two volumes arising from the International Conference - HYALURONAN 2000 - which was held at The North East Wales Institute for Higher Education, Wrexham, Wales, UK. This meeting owes its success to the invaluable work of its Executive Committee, Scientific Committee and International Advisory Board, and its Generous Supporters and Exhibitors.
SUPPORTERS AND EXHIBITORS Acordia, USA Anika Therapeutics Inc, USA Bingham Dana LLP, USA Biomatrix Inc, USA Fermentech Medical Limited, UK Fidia SpA, Italy Fidia Advanced Biopolymers SrI, Italy Genzyme Corporation, USA I-Med Pharma Inc, USA Lifecore Biomedical, USA Orquest Inc, USA Q-Med, Sweden Seikagaku Corporation, Japan Vitrolife UK Ltd Wyeth-Ayerst Laboratories,USA
EXECUTIVE COMMITTEE G. O. Phillips (Chairman) J. F. Kennedy (Deputy Chairman & Treasurer) P .A Williams (Secretary General) S. AI-Assaf M. Davies T. Hardingham H. Hughes (Administration Secretariat) C. J. Knill (Scientific Secretariat)
Research Transfer Ltd, Wales Univ of Birmingham Res Park, UK The North East Wales Institute, Wales The North East Wales Institute, Wales College of Medicine, Wales University of Manchester, UK The North East Wales Institute, Wales Univ of Birmingham Res Park, UK
SCIENTIFIC COMMITTEE AND INTERNATIONAL ADVISORY BOARD V.c. Hascall (Chainnan) C. Abetangelo P.A Band TJ. Brown B. Caters on M.K. Cowman AJ. Day J.L. Delinger M. Ferguson K. Harding H. Hatakeyama N.E. Larsen T.C. Laurent K. Moore K. Nishinari B.J.Parsons A Okamoto M. Rinaudo S. Takiami B.P.Poole C. Weiss
Cleveland Clinic Foundation, USA University of Padova, Italy Biomatrix Inc, USA Monash University, Australia University of Wales College of Medicine, UK Polytechnic University, USA Oxford University, UK Biomatrix Inc, USA University of Manchester, UK University of Wales College of Medicine, UK Fukui University of Technology, Japan Biomatrix Inc, USA University of Uppsala, Sweden. University of Wales College of Medicine, UK Osaka University, Japan The North East Wales Institute, Wales Denki Kagoku Kogyu Japan University of Grenoble, France Gunma University, Japan Tufts University, USA Mount Sinai Medical Centre, USA
THE CELLUCON CONFERENCES 1984 Cellucon '84 UK.
CELLULOSE AND ITS DERIVATIVES Chemistry, Biochemistry and Applications
1986 Cellucon '86 UK.
WOOD AND CELLULOSICS Industrial Technology, Biotechnology, Structure and Properties
1988 Cellucon '88 Japan
CELLULOSICS AND WOOD Fundamentals and Applications
1989 Cellucon '89 UK.
CELLULOSE: SOURCES AND EXPLOITATION Industrial Utilisation, Biotechnology and Physico-Chemical Properties
1990 Ce11ucon '90 Czechoslovakia
CELLULOSE New Trends in the Complex Utilisation of Lignocellulosics (phytomass)
1991 Cellucon '91 USA
CELLULOSE A Joint Meeting of: ACS Cellulose, Paper and Textile Division, The Cel1ucon Trust, and 111h Syracuse Cellulose Conference
1992 Cellucon '93 UK.
SELECTIVE PURIFICATION AND SEPERATION PROCESSES
1993 Ce11ucon '93 Sweden
CELLULOSE AND CELLULOSE DERIVATIVES Physico-Chemical Aspects and Industrial Applications
1994 Cellucon '94 UK.
CHEMISTRY AND PROCESSING OF WOOD AND PLANT FIBROUS MATERIALS The Chemistry and Processing of Wood and Plant Fibrous Materials
1998 Ce11ucon '98 Finland
PULP AND PAPER MAKING Fibre and Surface Properties and other Aspects of Cellulose Technology
1999 Ce11ucon '99 Japan
RECENT ADVANCES IN ENVIRONMENTALLY COMPATffiLE POLYMERS
2000 Hyaluronan 2000 UK.
HYALURONAN 2000 An International Meeting Celebrating the 801h Birthday of Endre A Balazs
2003 Hyaluronan 2003 USA
HYALURONAN 2003
The proceedings of each conference were formerly published by Ellis Horwood, Simon and Schuster International Group, Prentice Hall, Campus 400, Maylands Avenue, Hemel Hempstead, Herts, HP2 7EZ, UK. and from 1993 are published by Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CBl 6AH, UK.
PREFACE
Together, these two volumes are the most comprehensive account of the chemistry, biology and medical aspects ofhyaluronan now available. They are based on the international congress "HYALURONAN 2000" held at The North East Wales Institute. Wrexham, Wales, which attracted 350 specialists from 23 countries, and who delivered 221 presentations. The timing was deliberate to enable the achievements of one century to be evaluated and the opportunities ofthe new to be identified. A principal objective was to celebrate the so" birthday ofDr Endre A Balazs and his unique contribution to the subject. There is not a worker in the hyaluronan field who is not familiar with his name. For the past 50 years he has pioneered both basic hyaluronan research and its clinical use in a spectrum of medical applications. In this meeting we honoured his gigantic contribution to the field and in his continuing to innovate even after his so" birthday. Friends, scientific and medical colleagues as well as commercial competitors, when they had notice of this Meeting, immediately signified that they wished to be present to mark his contribution to the subject. Chemists, cell biologists, clinicians and a range of medico-scientific researchers are now building on the foundations, which he has laid. It is fitting, therefore, that in both of these volumes there is an invited Introduction and Final Evaluation from Dr Balazs. The papers presented by his research group in these volumes testify also to the breadth ofhis current contribution. All aspects ofthe science ofhyaluronan are included. The initial contributions on the chemistry, rheology, chemical modification and characterisation provide a foundation for existing and potential new applications. These depend on the basic structure, its physical and solution properties, such as viscoelasticity, intermolecular interactions and conformation, as well as the specific participation of functional chemical groupings. The recognition that hyaluronan can induce cell-signalling functions has led to an explosion in associated cell biology investigations. This information is of vital importance because it points to mechanisms for wound healing and tissue regulating functions. There are already products entering the market based on these observations and the clinical contributions cover these aspects also. The tissue supplementation and application in osteoarthritis is now an established clinical application, and rightly is given due prominence in these volumes, along with the associated control of and interaction with pain receptors. After years of basic research into a material which seemed only to have shock absorbing and protective functions within the matrix, and was little more than a scientific novelty, the subject has now exploded. Those who doubt the link between basic research and practical application should scrutinise the now ubiquitous role of hyaluronan, as exemplified in the following subjects which were covered at the Meeting:
xviii
Preface
•
Hyaluronan receptors and signalling
•
Inhibition oftumor progression by perturbing hyaluronan-tumor cell interactions
•
Mechanoprotective actions of elastoviscous hylans on articular pain receptors
•
Hyaluronan as a drug vehicle in breast cancer
•
Anti-cancer activity ofhyaluronan
•
Hyaluronan in corneal wound healing
•
The role ofhyaluronan in inflammation and repair oflung injury
•
Functions ofhyaluronan in wound repair
•
HA based dermal and epidermal grafts for diabetic foot ulcers
•
Hyaluronan and hylans in the treatment of osteoarthritis
•
Hyaluronan and rheumatoid arthritis
•
Hyaluronan based medical formulations for control of post surgical adhesion development.
•
Clinical application of serum hyaluronan for liver diseases and its significance
There can be little doubt, therefore, that these volumes will constitute a landmark in hyaluronan science and application. My thanks go to my co-Editors, the Chairs and members of the Scientific, Executive, Industrial and International committees for their hard work and vision. Haydn Hughes, the Organising Secretary, bore the main burden and deserves all our appreciation for his quiet efficiency.
Glyn O. Phillips Chair, Executive Committee
Introductory Remarks Endre A. Balazs Matrix Biology Institute. 65 Railroad Avenue. Ridgefield, New Jersey 07657 USA
It is wonderful to see so many friendly and familiar faces in this audience -friends and colleagues who have been with us, forming this community of scientists, for the past half century. We grew up together scientifically and learned this science as we went along, with very little help from the outside. Many of us grew old together, sharing friendship, fellowship and success. I greet you all with the warmth of many pleasant memories. It is also encouraging to see so many new faces in the audience, young scientists whom we will know better during the next week. I greet them with the warmth of great expectations, because they represent the future, the fulfillment of many of our dreams. I must sadly remind you that since the last meeting in Stockholm, we lost a great friend and a great scientist, Sven Gardel!. He was not only a brilliant and very influential researcher, but also a great teacher and educator. Many of us learned from his teaching and counsel during half a century. Our thanks and appreciation to the organizers of this meeting for the work they did. We know that organizing a meeting with more than 300 participants and more than 200 presentations of lectures and posters is not an easy task. The diversity and richness of the program clearly reflects their successful work. I am here to introduce Torvard Laurent's presentation. To put this in perspective, I have to take you back to a sunny September day fifty-two years ago in 1948 in the Karolinska Institute of Stockholm. On this day Torvard entered my laboratory, my life and, most importantly, the life and adventures of hyaluronan and the intercellular matrix. I had already been hooked on this subject for ten years, because, like Torvard, I was also introduced to the intricacies of the "intercellular substance" at the age of 18 as a first-year medical student. Little did we know on that September day in 1948 that a lifelong friendship and scientific fellowship was born. We only knew that at that time very few people in the world were interested in our subject. What was happening in the world of hyaluronan research in 1948? Haddian and Pirie at the Worcester Foundation in Massachusetts had just published a paper on the preparation of hyaluronan. Some years later this would trigger Roger leanloz' definitive work at the same institution on the primary structure of hyaluronan. Three years earlier, Gunnar Blix (with Snellman) published the first determination of intrinsic viscosity of hyaluronan prepared from bovine synovial fluid. They concluded that the behavior of hyaluronan solution was Newtonian. This conclusion was the result of the low molecular weight and low intrinsic viscosity of their preparation.
xx
Introductory remarks
Karl Meyer's pioneering research on hyaluronan was interrupted by the war effort. His work was concentrated on penicillin and Iysosyme during that time. In the late 1940s he had just started to pick up his work on hyaluronidase. But his work in the late 1930s triggered the interest of several prominent chemists to determine the primary structure of hyaluronan by methylation and periodate oxidation methods: M.A.G. Kay, in Stacey's laboratory in Birmingham, UK; 1. Felling in Kurt Mayer's laboratory in Geneva; and Roger Jeanloz in the Worcester Foundation in Massachusetts started their work in 1948-49. Lars Sundblad at G. Blix' laboratory in Uppsala and J.E. Stanier in A. G. Ogston's laboratory in Oxford would soon start their doctoral research work on synovialfluid hyaluronan. The hot topics of the day were Duran-Renal's spreading factor that was already identified as hyaluronidase. In 1948 Albert Dorfman published the first study on the kinetics of enzymatic hydrolysis of hyaluronan. Between 1935 and 1948 it was widely reported that most human and animal tumors contained hyaluronidase, and yet others also contained hyaluronan. The role of hyaluronidase in tumors was implicated, but it was noted that some invasive tumors had high hyaluronidase content while others had none. I also contributed to the confusion at that time by publishing that the necrotic part of invasive tumors contained higher levels of hyaluronidase activity than the living parts and that plant cells also have hyaluronan-degrading activity during mitosis. Bengt Sylven, a histopathologist at Karolinska Institute, working with histochemical methods, was convinced that sulfated glycosaminoglycans of the intercellular matrix and mast cells were part of the body's early defense system against growth and spread of tumors. This was the scientific environment in which Torvard started his work with me in 1948. I am very proud that during the years we worked together closely in Stockholm and Boston, I succeeded in infecting him with the spiritual virus of hyaluronan and intercellular matrix curiosity, which yielded a wealth of new knowledge though his own research activity and through that of two generations of his students. As a chain reaction, the number of scientists interested in the field grew logarithmically, and under his tutelage the research on hyaluronan and other macromolecular components of the intercellular matrix and its function in health and diseases flourished. We all must be grateful for his loyalty to this molecule and its role in the life of multi-cellular organisms. It is not an exaggeration to say that without his highly significant contribution to our field of science, the coming week's events could not have occurred.
PARTl OVERVIEW OF THE HISTORY AND DEVELOPMENT OF HYALURONAN
HYALURONAN BEFORE 2000 Torvard C Laurentt llnstiuae ofMedical Bioc1u!mistry and Microbiology, University of Uppsala.
BMC. Box582. SE·752 23 Uppsala; Sweden.
ABSTRACT In a review of the history of hyaluronan the author has tried to call attention to the key discoveries which have lead to paradigm shifts in the research on this unique polysaccharide. The selection is no doubt subjective.
KEYWORDS Hyaluronan, hyaluronate, hyaluronic acid, Endre Balazs
INTRODUCTION It is a great honour to be asked to give a key note lecture at the symposium "Hyaluronan 2000". However, realizing that I have nothing new to contribute to the field I have chosen to speak about "Hyaluronan before 2000". At the present pace of science the active researcher seldom has time to go back in literature to look for key discoveries and therefore I will try to make a summary.The selection is based on my own experience and may not agree with the view of other observers. However, first I would like to tell you how I entered the field. At this conference we are honouring Endre A. Balazs, who became 80 years in January. Endre - or Bandi as we always have said - was my teacher.
HOW IT STARTED In 1948 I entered Medical School in Stockholm and studied Anatomy and Histology for a year. In September 1949, at the age of 18, I got the opportunity to be unpaid instructor at the Department of Experimental Histology parallel to my medical studies. An Hungarian scientist, Bandi Balazs, immediately engaged me in research. He had left Hungary in 1947, before the communists took over, and had come to Stockholm to work on the biological role of extracellular polysaccharides and especially hyaluronan. My first assignment was to prepare hyaluronan from umbilical cords to be used in fibroblast cultures. Cell culturing was then performed very differently from what you are
4
Overview of the history and development ofhyaluronan
used to now. Before antibiotics everything had to be done under strictly sterile conditions as in an operating theater. Cells were grown in hanging fibrin clots. Fibro-blasts were obtained from pieces of embryonic chicken hearts, which were placed in the fibrin clots, and the cellular growth rates were determined by planimetry of enlargements of the cultures, which estimated how far the cells had migrated. Hyaluronan was extracted from umbilical cords and precipitated by alcohol. It was freed from proteins by shaking the extracts with chloroform and isoamyl alcohol (Sewag technique). We were looking for techniques to sterilize the highly viscous hyaluronan solutions. They could not be ultraftltered. In the end we used autoclaving. This work led to three early observations, which should tum out to be of rather basic character. Firstly, when we extracted hyaluronan from the umbilical cords at different ionic conditions we got material of very different viscosities. The highest viscosity was obtained when we extracted with distilled water. We then realized that the viscosity of hyaluronan changed with pH and ionic strength. Today this is common knowledge but at that time it had only been observed for synthetic polyelectrolytes by Raymond Fuoss. We wrote a note in Journal of Polymer Chemistry with the title "The viscosity function of hyaluronic acid as a polyelectrolyte"(l). This started my interest in the physical chemical properties of hyaluronan. Secondly, when we tried to sterilize hyaluronan by UV-irradiation, it lost all its viscosity. It was later shown that irradiation with electrons also degraded hyaluronan and other polysaccharides (2). We now know that we observed one of the first examples of free radical degradation of hyaluronan. The third observation concerned the biological effects of hyaluronan and some sulphated polysaccharides, heparin, heparan sulphate (which at that time was called heparin monosulphuric acid) and synthetically sulphated hyaluronan (3). We compared effects on cell growth, anticoagulant activity, antithrombine activity and antihyaluronidase activity. The main purpose was to clarify if heparin actually was a sulphated hyaluronan, as had been stated by Asboe-Hansen, but we concluded that it was not so. However, hyaluronan promoted cell growth, in contrast to the sulphated polysaccharides, and this was probably one of the first observations that hyaluronan interacts with cellular functions - today we know that this occurs via cellular receptors. Interestingly, this was probably also one of the first studies of the biological activity of heparan sulphate. The work was performed in a short time between September 1949 and December 1950, i.e, during slightly more than a year. Then Bandi moved to Boston, the head of our department died and I moved to the Chemistry Department where I continued with Physical Chemistry. Also, more of my time was taken up by clinical training. In retrospect the year with Bandi more than 50 years ago dramatically changed my life. I met a charismatic person who induced enthusiasm in the work and who looked upon things in an unorthodox way far away from what you could read in the text books. Bandi and I were going to work together for another four years during two periods in Boston between 1953 and 1961.
Hyaluronan before 2000
5
DISCOVERY OF HYALURONAN AND HYALURONIDASE Karl Meyer discovered hyaluronan in 1934 when he worked in the eye clinic of Columbia University (4). He isolated the compound from the vitreous body of bovine eyes under acid conditions and gave it the name hyaluronic acid after hyalos - a greek word for glassy - and uronic acid, a constituent of the polymer. It should be noted that other polysaccharides (chondroitin sulphate and heparin) had previously been isolated. Furthermore, already in 1918, Levene and Lopez-Suarez (5) isolated a polysaccharide from vitreous body and umbilical cord which contained glucosamine, glucuronic acid and some sulphate. They named the polysaccharide mucoitin sulphuric acid but with our present knowledge it must have been hyaluronan with some sulphate impurity. During the next ten years hyaluronan was isolated from various tissues by Karl Meyer and others. For example, it was found in joint fluid, skin, umbilical cord and rooster comb. Most notably it was discovered in the capsules of streptococci by Kendall et al. (6) in 1937. Since then it has been found in practically every tissue in vertebrates. Independently and preceeding the discovery of hyaluronan a factor was described by Duran-Reynals in testis (7). It was later named spreading factor. Similar activities were found in bee venom, extracts of leeches etc. When the spreading factors were injected in skin together with India-ink they caused a rapid spread of the black stain. The factors were identified as enzymes, which degrade hyaluronan, and were named hyaluronidases (8). Even in mammalian blood a hyaluronidase was present but it only acted at acid pH.
PREPARATION OF HYALURONAN
The original preparation procedures for hyaluronan followed what was conventional for polysaccharides, i.e, proteins were removed by the Sewag-technique or by proteolytic digestion. The polymer was then fractionally precipitated by alcohol. A great step forward in separating differently charged polysaccharides was taken by John Scott when he developed fractionated precipitation with a cationic detergent (CPC, cetylpyridinium chloride) by varying the salt concentration (9). Hyaluronan could be efficiently separated from sulphated polysaccharides. The method could also be used for molecular weight fractionation. In principle similar results can be obtained by ion exchange chromatography. THE STRUCTURE AND CONFORMATION OF HYALURONAN The chemical structure of the polysaccharide was essentially solved by Karl Meyer and associates in the 1950's (see e.g. 10). We now know that hyaluronan is a long polymer built from disaccharides consisting of N-acetyl-D-glucosamine and D-glucuronic acid linked by Bl-4 and Bl-31inkages. Karl Meyer did not follow the conventional approach to analyze the intact polysaccharide. Instead he obtained by hyaluronidase digestion
6
Overview of the history and development ofhyaluronan
specific disaccharides or oligosaccharides which he characterized and from this information he could deduce the structure of the intact polymer. A conformational analysis of hyaluronan 'fibers' was first attempted by X-ray crystallographers. There was a heated debate between different groups at a meeting in Turku in 1972, regarding the helix structure of hyaluronan. Apparently different types of helices can be formed by hyaluronan dependent on counter ion composition and water content Various structures were published during the 70' s and 80' s. A break-through was made by John Scott when he on the basis of lack of reactivity to periodate oxidation suggested a structure in solution containing intra-chain hydrogen bonds (11). He later verified this hypothesis by NMR analysis (12) and the conformation could be accommodated in a two-fold helical structure described by Atkins et al. already in 1972 (13). PHYSICAL CHEMICAL CHARACTERIZATION
Fifty years ago we did neither know the chemical structure of hyaluronan nor its macromolecular properties, i.e. molecular weight, homogeneity, molecular shape, hydration, charge and interaction with other molecules. This became the interest in the following 10 years of A.G. Ogston and his collaborators in Oxford, of Balazs and coworkers in Boston, of myself in Stockholm and of a few other laboratories. Our main difficulty was to prepare hyaluronan free of proteins and other components before physical measurements. There was always a risk of degrading the polymer during the purification process. Ogston used the technique of ultrafiltration assuming that free proteins went through the filter and that proteins bound to hyaluronan were retained in the top solution. He studied a 'complex' which contained 30% protein (14). Other investigators used various physical, chemical and enzymatic means which removed proteins down to a few percent However, the general results of the physical chemical analyses gave a consistent picture of the hyaluronan molecule. The molecular weight was usually several millions, although many samples were polydisperse. Light-scattering showed the molecule to behave as a randomly coiled, relatively stiff, chain molecule with a radius of gyration of the order of 200 nm (15). The chain stiffness is due to the intrachain hydrogen bonds mentioned above. The random coil structure was later confirmed by the molecular weight/viscosity relationship. Ogston and Stanier (14) using sedimentation, diffusion and shear rate dependence of viscosity and birefringence drew the conclusion that the molecule behaved as a large hydrated sphere which was compatible with a random coil configuration. ANALYTICAL TECHNIQUES
The only way of analysing hyaluronan quantitatively was from the beginning to isolate the polysaccharide in pure form and measure its content of uronic acid and/or N-acetyl
Hyaluronan before 2000
7
glucosamine. The methods of choice were the Dische carbazole technique for uronic acid (16) and the Elson-Morgan reaction for hexosamine (17). The importance of the carbazole technique for routine analysis cannot be overestimated. The analyses of hyaluronan required milligram quantities. The next step in the development came with the introduction of specific enzymes. The streptomyces hyaluronidase is specific for hyaluronan (18) and produces unsaturated hexa- and tetrasaccharides. This can be utilized to analyze the hyaluronan content in the presence of other polysaccharides and impurities and the 'unsaturated' state of the uronic acid can be used to lower the detection limit of the product. The enzymatic technique increased the sensitivity to microgram quantities of hyaluronan. The final step came with the use of affinity proteins recognizing hyaluronan. Tengblad (19) used hyaluronan binding proteins from cartilage and Delpech (20) subsequently used hyaluronectin from brain. These proteins could be used in assays similar to immunoassays and now nanogram quantities of hyaluronan could be measured directly in tissue fluids. Tengblad's technique formed a basis for much of the work performed in Uppsala since then.
VISUALIZATION OF HYALURONAN The detection of hyaluronan in tissue sections is closely related to analysis of the polymer in tissue fluids. From the beginning unspecific staining with basic dyes were used. John Scott increased the specificity by the same principle he had used in fractionation of anionic polysaccharides with detergents. He stained with Alcian Blue at different ionic concentrations (21) and could thereby differentiate between polysaccharides. Lately he has gone over to use Cupromeronic Blue. However, hyaluronan can also with advantage be localized by specific affinity proteins in tissue sections. The first such reports came in 1985 (22,23). The technique has been used with great success and has given us a detailed information of hyaluronan distribution in various organs. Hyaluronan can also be visualized by electron microscopy. The first picture was published by Jerome Gross (24) but did not show any details. The paper by Fessler and Fessler (25) can be regarded as the first interpretable study. It showed hyaluronan as a long extended single chain. Another ingenious way of visualizing pericellular hyaluronan was described by Robert Fraser (26). He added a suspension of particles to fibroblast cultures. The particles were excluded from a thick layer surrounding the fibroblasts. This pericellular coat turned out to be hyaluronidase sensitive.
ENTANGLEMENT AND RHEOLOGY From the dimensions of the largest hyaluronan molecules one can estimate that they
8
Overview of the history and development ofhyaluronan
should fill the solution completely at concentrations of the order of 1 gil. At high concentrations the molecules will entangle and the solution consists of a continuous network of chains. The entanglement point is clearly visible as a point, where the specific viscosity increases dramatically when the concentration is further increased. Another property which changes dramatically with concentration is the shear dependence of the viscosity. The latter was first described by Ogston and Stanier (13). Also the elastic behaviour of the solution increases with increasing concentration and molecular weight Flow elasticity of pure hyaluronan was first shown by Jensen and Koefoed (27) and a thorough analysis of the viscoelastic behaviour was made by Gibbs et al. (28). Is this dramatic behaviour only a function of mechanical entanglement or could it also be due to chemical interactions between the chains? Already in early papers by Ogston it was discussed if some kind of interactions via proteins could occur. Clear evidence that there occurred a chain-chain interaction was obtained by Welsh et al (29) when they showed that the elasticity could be counteracted by addition of short hyaluronan chains (60 disaccharides) to the solution. Apparently these competed with the interactions between longer chains. In the more recent work by John Scott it has become clear that the conformation of hyaluronan, which displays hydrophobic patches along the chain, is well suited for forming helices with neighbouring molecules which are stabilized by hydrophobic forces (30). It is therefore most probable that chain-chain interaction to a large extent contributes to the rheological properties of hyaluronan.
PHYSIOLOGY OF HYALURONAN NETWORKS The discovery that hyaluronan chains entangle at concentrations, which may occur in many tissues, raised the hypothesis that hyaluronan actually exercised its physiological activity via the properties of a continuous three-dimensional chain network. Various properties of the networks were discussed: Viscosity. The very high visco-elasticity of concentrated high-molecular weight hyaluronan solutions, as well as the shear dependence, were connected with lubrication of joints and other tissues. Hyaluronan seems always to be present in spaces separating mobile tissues, e.g. in joints and between muscles. Osmotic pressure. The osmotic pressure of hyaluronan solutions is strongly concentration dependent and at higher concentrations the colloid osmotic pressure is larger than that of an albumin solution (31). This property was assumed to be of importance for the water homeostasis in the tissues. Flow resistance . A tight chain network exerts a very high resistance towards water flow. That hyaluronan really forms flow barriers in tissues was first shown by Day (32). Excluded volume. A three-dimensional network removes space for other macromolecules. The available volume can be measured by equilibrium dialysis between a hyaluronan solution and a buffer solution and it turns out that the effect corresponds to what one can calculate that it should be (33) according to a theoretical relationship
Hyaluronan before 2000
9
deduced by Ogston (34). The exclusion effect has been discussed in connection with protein partition between the vascular space and extracellular tissue space but it has also been discussed in connection with deposition of physiological or pathological material in connective tissues. Polymer exclusion decreases the solubility of proteins (35). Diffusionbarrier . The movement of macromolecules through a hyaluronan solution can be measured by sedimentation or diffusion analyses. The larger a particle is the more it becomes retarded in its movements (36). This effect has been connected with formation of diffusion barriers in the tissues. For example, the pericellular layer of hyaluronan could protect cells from large macromolecules or other cells.
HYALURONAN·BINDING PROTEINS (HYALADHERINS)
Proteoglycans. Until 1972 it was believed that hyaluronan was an inert compound in tissues that did not specifically interact with other macromolecules. In this year Hardingham and Muir (37) showed that hyaluronan can aggregate cartilage proteogtycans. Thorough studies by Hascall and Heinegard (38) documented that there is a specific binding between hyaluronan, the N-terminal globular part of the proteoglycan and a link protein. This is a very firm association and many proteoglycans bind to the same hyaluronan chain forming large aggregates in cartilage and other tissues. Hyaluronan receptors. Also in 1972, Pessac and Defendi (39) and Wasteson et al. (40) demonstrated that certain cells in suspension aggregate when hyaluronan is added. This was the first report that hyluronan interacts specifically with cell surfaces. Subsequently, Underhill and Toole (41) described in 1979 that hyaluronan actually binds to cells and the responsible 'receptor' was purified in 1985 (42). In 1989 two groups reported that the lymphocyte homing receptor, CD44, was a hyaluronan binding protein with homology to the link protein in cartilage (43,44). It was soon shown that the receptor of Underhill and Toole was identical to CD44' Another hyaluronan binding protein was isolated from the supematant of cultures of 3T3 cells by Turley et al. in 1982 (45) and named RHAMM (receptor for hyaluronan mediating motility). Following these initial discoveries a number of other hyaladherins have been defmed. CELL BIOLOGICAL ROLE OF HYALURONAN Until the discovery of hyaladherins, hyaluronan was thought to influence cell behaviour entirely through physical interactions. Evidence that hyaluronan might playa role in biological processes was purely circumstantial and to a large extent built upon the presence or absence of hyaluronan during biological processes. Much of the speculations were based on unspecific histological investigations. A very successful research line started in Boston in the beginning of the 1970' s. Bryan Toole and Jerome Gross. (46) showed that during regeneration of the newt limb hyaluronan was first synthesized and subsequently removed by hyaluronidase, when
10
Overview of the history and development of hyaluronan
instead chondroitin sulphate was formed, A similar pattern was seen in the developing chick cornea (47). Toole has pointed out that accumulation of hyaluronan coincided with periods of cellular migration in the tissues. As mentioned above Toole also pioneered studies on membrane-bound hyaladherins and with the introduction of hyaluronan 'receptors' we have a much firmer basis for the concept that hyaluronan plays a role in regulating cellular activity, e.g, motility (48). We have seen an explosion of publications dealing with the role of hyaluronan in cellular migration, mitosis, inflammation, cancer, angiogenesis, fertilization etc. in the last decade.
BIOSYNTHESIS OF HYALURONAN Research on the biosynthesis of hyaluronan has gone through three phases. The dominating person in the first phase was Albert Dorfman. He and his collaborators described during the early 1950's the origin of the monosaccharides to be incorporated into the hyaluronan chain in streptococci. However, it was Glaser and Brown in 1955 who for the first time demonstrated hyaluronan synthesis in a cell free system (49). They used a particulate enzyme from Rous chicken sarcoma and incorporated 14
•
30
Adhesion formation and hyaluronan
such as NF-kappa B fect.
16,
whereas high molecular weight (6000xl0 3) HA has no such ef-
3500
3000
2500
2000
1500
1000
500
Wave number [cm'"] Fig.3. Infrared spectra of both non-crosslinked (curve "0 h") and crosslinked (curves "1-21 h") thin HA films. The crosslinked samples show a new absorption at a wave number of 1700 cm· 1 (arrows), assigned to the carbonyl group most likely of ester bond. The non-crosslinked control (0 h) shows no additional peak. The intensity of the absorption increases with the crosslinking time.
3,5
o
3,0
~
s
2,5
«
2,0
~
FigA. Quantification of ester formation. The figure shows the intensity ratio of the absorbance at 2925 em" to that at 1700 ern" in correspondence to the cross linking time.
o
1,5
10
crosslinking time [hi
Also, angiogenesis is stimulated only by low molecular weight HA fragments, whilst high molecular weight HA suppresses angiogenesis 17. Generally, HA surfaces prevent mammalian cell adhesion 18, a phenomenon utilised in abdominal surgery to reduce postoperative adhesions 19. On the other hand, many tumour cells 20.21 as well as mast cells 22 adhere more easily on HA substrata. Malignant tumours express elevated levels of HA, and many tumour cells express elevated levels of the main HA binding protein, CD44 23. Since rat calvaria osteoblasts used in our study are known to express CD44 14, the impaired attachment of the cells indicates that CD44 or other related HA receptor proteins may not recognise modified HA, dependent on its degree of erosslinking. In summary, we have shown that chemical alteration of HA changes its cell adhesion properties. This might have important consequences for tissue engineering procedures,
Cell adhesion on crosslinked surfaces
31
especially when HA is used in multicomponcnt biomaterials, e.g. in mineralised collagen-hyaluronan membranes 24. The biocompatibility can be optimised particularly by the time of crosslinking of the HA component, until requirements of both favourable cell adhesion and water-insolubility are matched.
ACKNOWLEDGEMENTS We are indebted to the technical assistance of Mr Rene Born for infrared measurements. This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG).
REFERENCES 1. T. C. Laurent, U. B. Laurent & J. R. Fraser. The structure and function ofhyaluronan: An overview.lmmunol Cell Biol, 1996,74(2), AI-7. 2. J. Y. Lee & A. P. Spicer. Hyaluronan: a multifunctional, megaDalton, stealth molecule. Curl' Opin Cell Biol, 2000, 12(5),581-586. 3. B. P. Toole. Hyaluronan and its binding proteins, the hyaladhcrins. Curl' Opin Cell BioI, 1990,2(5), 839-844. 4. P. W. Kincade, Z. Zheng, S. Katoh & 1. Hanson. The importance of cellular enviromnent to function of the CD44 matrix receptor. Curl' Opin Cell Biol, 1997, 9(5), 635-642. 5. S. P. Evanko & T. N. Wight. Intracellular Localization of Hyaluronan in Proliferating Cells. J Histochem Cytochem, 1999,47(10), 1331-1342. 6. 1. Huang, N. Grammatikakis, M. Yoneda, S. D. Banerjee & B. P. Toole. Molecular characterization of a novel intracellular hyaluronan-binding protein. J Biol Chern, 2000, . 7. M. Wiig, S. O. Abrahamsson & G. Lundborg. Effects ofhyaluronan on cell proliferation and collagen synthesis: a study of rabbit flexor tendons in vitro. J Hand Surg [Am}, 1996,21(4),599-604. 8. E. A. Balazs. Sodium hyaluronate and viscosurgery. In: Miller D, Stegmann R, eds. Healon (sodium hyaluronate). A guide to its use in ophthalmic surgery. New York: Wiley, 1983; 5-28. 9. T. J. Liesegang. Viscoelastic substances in ophthalmology. Surv Ophthalmoi, 1990, 34(4),268-293. 10. P. Bulpitt & D. Aeschlimann. New strategy for chemical modification of hyaluronic acid: preparation of functionalized derivatives and their usc in the formation of novel biocompatible hydrogels. J Biomed Mater Res, 1999,47(2), 152-169. 11. N. E. Larsen, C. T. Pollak, K. Reiner, E. Leshchiner & E. A. Balazs. Hylan gel biomaterial: dermal and immunologic compatibility. J Biomed Mater Res, 1993, 27(9), 1129-1134. 12. Y. S. Choi, S. R. Hong, Y. M. Lee, K. W. Song, M. H. Park & Y. S. Nam. Studies on gelatin-containing artificial skin: II. Preparation and characterization of crosslinked gelatin-hyaluronate sponge. J Biomed Mater Res, 1999,48(5),631-639. 13. K. Tomihata & Y. Ikada. Crosslinking of hyaluronic acid with water-soluble carbodiimide. J Biomed Mater Res, 1997,37(2),243-251. 14. E. Schulze, M. Witt, M. Kasper, C. W. Lowik & R. H. Funk. Immunohistochemical investigations on the differentiation marker protein Ell in rat calvaria, calvaria cell
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Adhesion formation and hyaluronan
culture and the osteoblastic cell line ROS 17/2.8. Histochem Cell Bioi, 1999, 111(1),61-69. 15. M. E. Adams, A. 1. Lussier & J. G. Peyron. A risk-benefit assessment of injections of hyaluronan and its derivatives in the treatment of osteoarthritis of the knee [In Process Citation]. Drug Saj, 2000, 23(2), 115-130. 16. B. Oertli, B. Beck-Schimmer, X. Fan & R. P. Wuthrich. Mechanisms of hyaluronan-induced up-regulation ofICAM-l and YCAM-l expression by murine kidney tubular epithelial cells: hyaluronan triggers cell adhesion molecule expression through a mechanism involving activation of nuclear factor-kappa B and activating protein-I. J Immunol, 1998, 161(7),3431-3437. 17. M. Slevin, J. Krupinski, S. Kumar & J. Gaffney. Angiogenic oligosaccharides of hyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplasmic signal transduction pathway resulting in proliferation. Lab Invest, 1998, 78(8), 987-1003. 18. M. Morra & C. Cassineli. Non-fouling properties of polysaccharide-coated surfaces. J Biomater Sci Polym Ed, 1999,10(10), 1107-1124. 19. T. Sawada, K. Hasegawa, K. Tsukada & S. Kawakami. Adhesion preventive effect of hyaluronic acid after intraperitoneal surgery in mice. Hum Reprod, 1999, 14(6), 1470-1472. 20. D. Peck & C. M. Isacke. CD44 phosphorylation regulates melanoma cell and fibroblast migration on, but not attachment to, a hyaluronan substratum. Curr Bioi, 1996, 6(7), 884-890. 21. K. Takahashi, I. Stamenkovic, M. Cutler, A. Dasgupta & K. K. Tanabe. Keratan sulfate modification of CD44 modulates adhesion to hyaluronate. J Bioi Chem, 1996,271(16),9490-9496. 22. M. Fukui, K. Whittlesey, D. D. Metcalfe & J. Dastych. Human mast cells express the hyaluronic-acid-binding isoform of CD44 and adhere to hyaluronic acid. Clin Immunol, 2000, 94(3), 173-178. 23. Z. Rudzki & S. Jothy. CD44 and the adhesion of neoplastic cells. Mol Pathol, 1997, 50(2), 57-71. 24. M. Gelinsky, B. Knepper-Nicolai, K. Flade, W. Pompe, U. Hempel, C. Roehlecke & M. Witt. Mineralized collagen-hyaluronate membranes. Materials Week, Miinchen, Germany, Sept. 25-28, 2000.
CELL ATTACHMENT AND GROWTH ON SOLID HYALURONAN (RYLAN B GEL) Endre A. Balazs 2, lIana K. EUezer-Pye', Rita A. Dennebaum', Nancy E. Larsen! & Julie L. Whetstone2 I
Biomatrix, Inc.• 65 Railroad Avenue, Ridgefield. New Jersey 07657. USA
'Matrix Biology Institute, 65 Railroad Avenue. Ridgefield. New Jersey 07657. USA
ABSTRACT Rylan B is a water-insoluble hyaluronan produced by bis-ethyl sulfone covalent crosslinks. Rylan B gels containing 0.5% hyaluronan polymers are heat stable, but degradable by various hyaluronidase. They are more resistant to degradation by free radicals than high molecular weight (average MW > 4 million) hyaluronan of hylan A (avg. MW 6 million). Cells after trypsin treatment were seeded on the surface of hylan B gels imbibed with tissue culture media supplemented with fetal bovine serum. Cells from eight established cell lines originating from fibroblasts, epithelial or endothelial cells, chondrocytes, tumor cells and stem cells were used. All but the endothelial-origin cells attach to the gel, but only the L929 fibroblasts and stem cells multiplied. Fibronectins (plasma or cellular) added to the media-imbibed gel promoted the spread of the cells of some of these cell lines, while sulfated glycosaminoglycans inhibited the spread and growth of some of these cells. Some poly-lysines, on the other hand, promoted their growth. First explant chicken embryonic cells were also cultured on hylan B gels. Embryonic fibroblasts from the heart migrated and multiplied on the gel surface when homologous embryo extract was added to the culture medium. The results form these in vitro cell culture studies suggest that hylan B gel matrices may be modified by the addition of various types of cell attachment molecules as a means to promote cell attachment and growth.
INTRODUCTION Different cell types were cultured on a variety of materials to evaluate their potential as cell support systems. Hyaluronan (hyaluronic acid, HA) is a biocompatible, natural polysaccharide that may be modified to produce new materials. Hylan B gel' (produced by Biomatrix, Inc., Ridgefield, NJ) is a water-insoluble, crosslinked hyaluronan derivative that is heat stable and more resistant to degradation by free radicals than high molecular weight hyaluronan. A variety of cell lines were cultured on hylan B alone and on hylan B treated with various cell attachment molecules. First explant chicken embryonic cells were also cultured on hylan B gels. Using inverted-phase microscopy, the attachment, spread, and growth of cells on hylan B was monitored. The purpose of this study was to evaluate the potential of hylan B as a support to promote cell growth.
34
Adhesion formation and hyaluronan
METHODS CeU Lines Eight anchorage-dependent cell lines (L929 fibroblasts, BLO-II fibroblast-like cells, CPAE endothelial cells, CRFK and BSC-I epithelial cells, Monkey chondrocytes, LA-4 adenoma cells, and NE stem cells) were used in these experiments. Following confluency and subsequent trypsinization, cells were seeded on the surface of hylan B gel slabs. All slabs were equilibrated with tissue culture medium supplemented with 10% fetal bovine serum (medium for BLO-II cells was supplemented with 20% fetal bovine serum). 24 hours prior to the addition of cells, the hylan B gel slabs were treated in one of three ways: unaltered (10% or 20% EMEM only), 0.007 mg cellular fibronectin, or 0.007 mg plasma fibronectin. These substances were added to the surface of the gels and incubated for 24 hours at 37°C. One cell line was also seeded upon a gel slab coated with 0.007 mg poly-L-lysine. Cells were maintained at 37°C and monitored for attachment, spread, and growth using inverted-phase microscopy.
Chicken Embryonic CeUs Chicken embryos were sacrificed at 10 days. Heart muscle was obtained and cultured on hylan B gel slabs supplemented with homologous embryo extract. Using inverted-phase microscopy, cell behavior was monitored.
RESULTS Cell Lines Of the eight cell lines employed, all but the endothelial cells attached to untreated and plasma fibronectin-treated hylan B gel. Plasma fibronectin enhanced attachment of both BSC-l epithelial cells and L929 fibroblasts. Added cellular fibronectin did not affect the attachment of the cell lines tested. Spreading of cells on untreated gels was similar to fibronectin-treated samples, with L929 fibroblasts displaying an increased spreading with plasma fibronectin, and BLO-II spreading more readily on cellular fibronectin gel. BLO-ll cell spreading on cellular fibronectin gel was inhibited by the addition of various sulfated glycosaminoglycans (heparan sulfate, keratan sulfate). Slabs with poly-L-lysine facilitated the spreading of CRFK epithelial cells (Fig. 1). Two cell lines, L929 fibroblasts and NE stem cells, were found to proliferate on the gel slabs as determined by cell counts using a hemacytometer (Fig. 2). BLO-II cells appear to proliferate on cellular fibronectin gels as indicated by changes in cell morphology and increased gel surface coverage (Fig. 3).
Chicken Embryonic Cells Embryonic fibroblasts from the hearts of chicken embryos migrate and multiply on the gel surface, forming a reticulated network overriding the heart tissue (Fig. 4).
Cell attachment and growth
35
Figure 1. CRFK Epithelial Cells
48 hours
72 hours
With poly-L-lysine, 48 hours
With poly-L-lysine, 72 hours
36
Adhesion formation and hyaluronan
Figure 2. NE Stem Cells
48 hours
5 days
The effect of fibronectins on cell spreading and growth
Cell Spreading
CeUson hyianB gel
Cell Growth
cellular plasma fibronectin fibronectin
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- : no spread, no growth +: spread, growth NT: not tested
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Cell attachment and growth
Figure 3. BLO-ll Fibroblast-like Cells with Cellular Fibronectin
24 hours
6 days
Figure 4. Chicken Embryonic Fibroblasts (From lO-day-old Embryo Heart Tissue)
18 days
32 days
37
38
Adhesion formation and hyaluronan
CONCLUSIONS The results from these studies indicate that hylan B gel provides a suitable scaffold for the growth of fibroblasts (L929), stem cell (NE), and embryonic cells (chicken). Other cell types such as the epithelial cells (CRFK) and fibroblast-like cells (BLO-II) grow on hylan B gel slab only when additional components are present (i.e. cellular fibronectin, poiy-L-Iysine). The results suggest that hylan B gel matrices may be optimized as cellular scaffolds by the addition of substances such as cellular fibronectin or poly-L-Iysine to enhance cell attachment. REFERENCES 1. E.A. Balazs & E.A. Leshchiner, Hyaluronan, its crosslinked derivative-hylan- and their medical applications, In: Cellulosics Utilization: Research and Rewards in Cellulosics (Proceedings of Nisshinbo International Conference on Cellulosics Utilization in the Near Future), H. Inagaki & G.O. Phillips (eds.), Elsevier Applied Science, New York, 1989, pp 233-241.
MOLECULAR CHARACTERISATION OF HYALURONAN AND DYLAN USING GPC-MALLS AND ASYMMETRICAL Flow FFFMALLS S. AI-Assaf*, P.A. Williams and G.O. Phillips The North Hast Wales Institute, Centre for Water Soluble Polymers, Wrexham LLl I 24 W, UK. E-mail s.alassaf@ne.~i.ac.uk
ABSTRACT Gel permeation chromatography (OPC) and asymmetrical flow-FFF coupled to a multiangle laser light scattering detector have been used to determine the molecular weight and molecular weight distributions of hyaluronan, Healon and Hylan. It was shown that OPC-MALLS and FFF-MALLS gave comparable results for hyaluronan samples in the molecular weight range of9.0 x 104 to 2 X 106 • For Healon and Hylan it 6 was not possible to obtain molecular weight distribution by OPC but values of 5 x 10 7 and 1 x 10 were obtained using flow-FFF-MALLS and these values compare well to those obtained by static light scattering and low shear viscometry. It was concluded that FFF-MALLS is an effective method to determine the entire molecular weight range ofhyaluronan and Hylan. KEYWORDS Hyaluronan, characterisation, molecular weight distribution, OPC, FFF INTRODUCTION Due to the widening base of cosmetic, pharmaceutical and medical applications of hyaluronan and its derivatives I, macromolecular characterisation has become of great analytical importance. Balazs and co-workers 2 have developed a new family of hyaluronan derivatives named Hylan. Hylan can be produced, depending on the reaction conditions, over a range of molecular weight from 2 - 24 million. Hylan A (water soluble) is produced by cross-linking hyaluronan in situ in cock's comb with formaldehyde. Recently we reported static light scattering measurements on a range of Hylan samples with different molecular weights 3. The results were compared to capillary and low shear viscometry data and showed that successive filtration of Hylan low and high molecular weight did not influence the intrinsic viscosity value determined by capillary viscometry and were similar to that of the unfiltered sample. The intrinsic viscosity values, determined using low shear viscomet'J' for Hylan with Mw ca. 10 x 106 and 1.8 x 106 were found to be 8188 and 2070 em g-I. The comparative values, using capillary viscometry were 5146 and 2017 cm3 g-I demonstrating the effect of shear in the capillary viscometer. Filtration through a 1 um filter resulted in the average Mw of 10 x 106 for a range of hylan samples. The filtration showed pronounced effects on the results obtained by static light scattering where the Mw of Hylan (10 x 106 ) with RMS-radius of276nm was reduced when filtered through a 0.45 um filter to ca. 3 x 106 with RMS-radius of 167 nm. Repeated filtration through a
56
Characterisation and solution properties of hyaluronan
0.45~m filter did not reduce the Mw any further. On the other hand filtration through a O.2J.lm filter caused a further reduction but was not always possible for high molecular weight Hylan. There is a need for an absolute size characterisation method which can cover the wide molecular weight range of hyaluronan samples and its derivatives, with good resolution. This paper reports and compares results on the determination of molecular weight and molecular weight distribution of hyaluronan, Healon and Rylan using GPC-MALLS and asymmetrical Flow FFF-MALLS.
MATERIALS Hyaluronan HAl (Lot No. F17S0762) and HA2 (lot HA(P) were obtained from Dr Akio Okamoto, Denki Kagasku Kogyo K.K., Japan. HAl was autoclaved, for various times, and then freeze-dried to produce RAJ, HAS and RA6. HA4 (originally labeled Auto Back 2) was donated by Biomatrix Inc, (NJ, USA). The sample was dialysed against water for 5 days then autoclaved at 128C for 30 minutes and then freeze-dried. Rylan samples and Healon GV were kindly donated by Biomatrix Inc, (NJ, USA). All samples were dissolved in, a 0.22 lim filtered aqueous solution of, O.ISM NaCI and left to tumble at 4°C until complete dissolution.
EXPERIMENTAL GPC-MALLS Background Gel permeation chromatography (GPC) is a widely used separation technique for the determination of molecular weight and molecular weight distribution of polymers. It involves a column packed with a porous material with certain pore size and when a polydisperse polymer (consists of different size molecules) is passed through the column separation occurs based on hydrodynamic size. Molecules that are large are excluded from some of the pores, whereas small molecules can penetrate most of the pores. The large molecules, therefore, move quickly through the column. One of the major disadvantages of GPC is that it has limited application for high molecular weight polymer samples 4,5 since the molecules may be completely excluded from all the pores of the column packing material. In addition, high molecular weight polymers may degrade due to the high shear forces generated in the column. For some polymers, adsorption onto the packing material also presents a problem. Multi angle laser light scattering (MALLS) is one of the few absolute methods available for the determination of molecular weight and size over broad range. It utilises the principle that the intensity of light scattered elastically by a molecule (Raleigh scattering) is directly proportional to the product of the weight average molecular weight and concentration ofthe polymer 6 (equation below):
The term given between two brackets represents P( () which is a general form of a scattering function. K is an optical constant given by (K=21f 2 no (dn/dc)2 / A 4 NA), C is
Molecular characterisation
57
the concentration, RfJ is the excess Rayleigh ratio which is the measured quantity, fJ is the scattering angle, M w is the weight average molecular weight, A2 is the second virial coefficient, n, is the refractive index of the solvent, dn/dc the refractive index increment of the polymer in solution.Z is the wavelength of light, NA is Avogadro's number. When size exclusion chromatography is coupled to an on-line absolute molecular weight determining device (such as MALLS) and a concentration sensitive detector (refractive index or photometric) it is possible to measure the scattering intensity and sample concentration for each slice (fraction) in the fractionated peak. Thus information about the weight average molecular weight (Mw), number average molecular weight (M,,), molecular we~ht distribution, polydispersity index (MwlMn ) and radius of gyration can be obtained . GPC-MALLS System The system utilised a Waters (Division of Millipore, USA) Solvent Delivery System Model 6000A or P-500 dual piston syringe pump (Pharmacia Biotech). Two sets of columns were used. The first set employed a stainless steel column- Hemabio linear (lOl1m)packed with hydrophilic modified Hema gel (hydroxyethyl methacrylate copolymer) obtained from Polymer Standard Services, Germany. The second set employed two columns packed with composite crosslinked agarose called Superose" 6HR 10/30 (with bead diameter of 11-15 um) and Superose" 12HR 10/30 (with a bead diameter of 8-12 um) connected in series. According to the manufacturer (Pharmacia Biotech, Sweden) the separation range is from 1000 to 5 X 106, based on globular proteins. Injection into the GPC column was made with a manual Rheodyne Model 7125 syringe loading sample injector equipped with either a 100, 250 or 1000111 sample loop, a concentration dependent detector Wyatt OptilabDSP interferometric refractomter operated at 632.8 nm equipped with a Ia-mm PIOO cell (Wyatt Technology Corporation, USA). DAWN DSP laser light scattering photometer was equipped with a 632.8 nm He-Ne laser (Wyatt Technology Corporation., USA) with 15 detectors calibrated with filtered toluene and normalised with pullulan (23.8K) obtained from Polymer Standards Services. A value of 0.162 was used for the refractive index increment (dn/dc) 7. Data accumulation for the detectors used Wyatt Technology ASTRA 4.5 software. All measurements were performed at room temperature. Flow-field flow fractionation Background Field flow fractionation (FFF) is a family of fractionation techniques capable of separating particles and macromolecules according to their size. Asymmetrical flowfield flow fractionation (flow-FFF) is a variant of FFF that was first developed by Wahlund & Giddings 8. It separates molecules or particles according to the difference in diffusion coefficients and does not suffer from problems such as adsorption, molecular weight limit and shear degradation encountered by other techniques such as GPC. It has been shown recently that flow-FFF is an effective technique for the determination of the molecular weight distribution of polymers such as glutenin 9, amphiphilic graft copolymers 10 , carrageenan and xanthan 11, hydroxypropylmethyl
58
Characterisation and solution properties of hyaluronan
cellulose 12 and globular proteins, polystyrene latex beads and anionic polystyrene sulfonates 13. In this technique, the fractionation is achieved in a thin flat trapezoid shaped channel (see Figure 1). The channel consists of an upper (solid) wall made from polymethyl methacrylate and a lower porous wall, called the accumulation wall (Figure 1). An ultrafiltration membrane, permeable to the carrier liquid but not to the sample molecules, is placed on top of a porous frit. The membrane usually has a cut off of 104 and this determines the lower limit of molecular weight that can be fractionated. The top and bottom walls are clamped together. When an aqueous liquid is pumped through the channel it creates a secondary flow vector perpendicular to the primary axial flow vector (channel flow). The channel flow creates a parabolic flow velocity distribution across the thickness of the channel and transports the sample component down the channel to the outlet end where they are detected (Figure 1). The secondary flow, termed crossflow, drives any sample molecule or particle down to the membrane surface. This crossflow is counteracted by molecular diffusion caused by Brownian motion so that an exponential concentration distribution layer of polymer molecules is established. The thickness of this layer, therefore, depends on the magnitude of the diffusion coefficients. The larger polymer molecules tend to reside closer to the wall whereas the smaller ones diffuse towards the channel centre. Since the channel flow has a parabolic velocity profile the smaller species which are furthest from the accumulation wall move faster along the channel and elute first followed by the slowly diffusing component (high molecular weight). Therefore the elution profile in flow-FFF is opposite to that achieved by GPC. The basic theory of the asymmetrical flow-FFF and other retention modes are given in detailS, 14. Channel flow
Channel flow inlet
outlet,
':at..
'~~ - - - _ ~ I;;:~:""tec-~-'~-~--(Vout)
Focusing
poinl (z')
. ._ _•
~
c32f.l
Solid Wall
Cbaone
Width (W)
Figure 1. (top) Asymmetrical flow field flow fractionation channel and (bottom) separation principle in flow-FFF. For all FFF experiments a pre-experiment time was used in order to obtain a baseline for all detectors and was followed by activating the FFF software to start the fractionation procedure. A complete sample fractionation includes three necessary stages, namely injection/focusing/relaxation, elution and rinsing. The first stage is injection- focusing-relaxation mode (see Figure 2A). In this mode the liquid carrier, delivered by the main pump, enters the channel via the carrier outlet and exits the
Molecular characterisation
59
channel via the crossflow outlets (no flow at the carrier inlet). Following this the sample is injected into the channel, by a separate (injection) pump connected to the carrier reservoir, and enters at the injection point at a rate of 0.1ml/min. The sample is then concentrated at the focusing point (injection point) and left to relax for few seconds while the injection pump is off. The injection time was determined by the volume of the connecting tubes from the injection pump and the volume of the sample loop. At the end of stage 1 the elution (stage 2) starts by means of the control valve controlled by the software. In this phase, the liquid carrier enters the channel via the inlet and can exit through the channel outlet and crossflow outlet (see Figure 2B). For low molecular weight hyaluronan a crossflow of 3mVmin was used and lower cross flow (from 1.0 to 2.0 mVmin) were used for high molecular weight (> 3 million). Once the baseline for all detectors is back at the pre-determined baseline this is an indication that all the expected sample components are eluted, the flow direction is switched to the rinsing position (stage 3). In this stage, the carrier liquid enters the channel via the outlet and exit via the inlet to waste. This procedure is maintained for at least 10min at a flow rate of Iml/min. By switching to elution position the system is ready for the next run. All measurements were performed at room temperature unless otherwise stated. A
Carder
in
1
C,ossf:ow
ou11et
8
-_., ,.. ...
.
. : .... F r it: ., ~ ......... -
p.....-
.....,...-- MerTlbrane
I
Cro~sflow
outfet
Figure 2. Schematic representation ofthe flow in the asymmetrical FFF. (A) during injection/focusing/relaxation mode, (B) elution mode". FFF-MALLS System The flow-FFF system was supplied by ConSensus (Germany). The Channel has a trapezoidal geometry where the length of the channel is 28.6cm, the trapezoid breadths were 2.12 and 0.47cm respectively. The cut off area at the inlet end was 2.25cm2 and the total area of membrane enclosed by the spacer was 36.09cm2. The channel thickness was 190mm and the resulting channel volume 0.68ml. A Nadir UF-IOCIO ultrafiltration membrane of regenerated cellulose (Hoechst, Germany) was placed on the accummulation wall. Inlet and outlet holes are drilled in the upper wall to coincide with the tips of the cut out channel. Sample injection is carried out through an
60
Characterisation and solution properties ofhyaluronan
injection port that is 2.05 em downstream from the liquid carrier inlet port. The whole set (upper and lower wall) was clamped together with bolts tightened by approximately 4 Nm. The channel and crossflow were controlled using CSC V2.0 software supplied by ConSensus, Germany. The flow in the channel was generated by a Constant Metric 3200 pump and the injection of the sample was made with a Knauer pump (Microstar KlOO) connected to a rheodyne injector with an injection loop of 50, 100 or 250 ,.J.!. The solvent was filtered through using a 0.22 urn cellulose nitrate filter and was degassed before entering the channel using a ERC 3215a degasser. An in-line filter (0.22 um cellulose nitrate) was fitted between the pump and the flow-FFFchannel. The outlet end of the channel was connected to a DAWN-DSP (multiangle laser light scattering detector) and Wyatt OptilabDSP interferometric refractomter as described above. RESULTS AND DISCUSSION
Figure 3 shows the molecular weight distribution of different hyaluronan samples in the range of 1.8 x 104 - 2 X 106 obtained by GPC-MALLS. ~
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _----j .
't!,~:"'Ul9
......
,
Norm
~Lug
-~
~:~UlJ
_.
_u.-YF-o..:l
-e-
-,-
~~';:/'IJ
_YUK
~:~~11 Norm-LOll 1.orVer
Molecular weight (gfmol)
Figure 3. Differential molecular weight distribution of hyaluronan obtained by GPC-MALLS which employed superose 6HR + 12HR columns connected in series, flow rate 0.5mVmin. 6
A (HAl (Mw 2.0 x 106 , injected mass lOOJ1l of9 x 10-4 g/OO];B (HA2 (Mw 1.5 x 10 , injected 4 mass lOOJ1l of9 x 10-4 g/OO]; C [HA3 (Mw 8.0 x 106, injected mass 100J1lof9 x 10- g/OO];D (HA4 4 (Mw 4.4 x 106, injected mass 100J1lof 9 x 10-4g/OO]; E [HAS (Mw 9.0 X 10 , injected mass 250J1l 3 of 2 x 10-3 g/OO];F [HA6 (Mw 1.8 x 104, injected mass 250111 of 2 x 10- g/OO].
The results shown above agree well when compared to other techniques such as capillary viscometry and static light scattering. However, the GPC-MALLS technique is capable of covering only a limited range of hyaluronan molecular weight (up to 3 million). Above 3 million due to the size exclusion the high molecular weight is underestimated. Furthermore, the efficiency of a GPC column is markedly influenced by the carrier flow rate, column condition, sample concentration and pH. This clearly shown in Figure 4 for hyaluronan HA4 (Mw 4.4 x lOs). All runs shown in Figure 4 gave the same weight average molecular of 4.4 x io' ± 2%. However, the distribution is quite different and the polydispersity index increases with decreasing the flow rate which is an indication of a better separation.
61
Molecular characterisation 12.0
~
A
~-+-
1uloHler 6
..
. ~ - - - __.,._~-
-~
Nann'" I_og
~:,~~~;;':,:ag
C
Nonn = Log
1::Jt crder
440·QPC1
r:~:'~'r~e~ug
D O~O ._-~- ----.-~"""""......-.:;;;. 1.Oxlof' MolecuJar weight (gImol) Fi~ure
4. Molecular weight distribution obtained for hyaluronan HA4 (M, 4.4 x 10 ) by GPC-MALLS under different conditions.
A (column (Hemabiolinear, flow rate I mlImin, Cone. 2 x 10"" glml, Iml injected volume, at pH2; B, (Column-Hemabiolinear, flow rate I mlImin, Cone. 2 x 10-4J'ml, Iml injected volume, at pH6); C (Column-Hemabiolinear, flow rate 0.5 ml/min, Cone. 2 x 10 g/ml, Iml injected volume, at pH6; D (Superose 6HR and superose 12HR eonnected in series), flow rate 0.5 mlImin, Cone. 9 x 10"" g/ml, injected volume 1001-11.
In order to examine the applicability of flow-FFF to the separation of hyaluronan we used a hyaluronan sample of low molecular weight that can be easily separated by GPC. Figure SA shows the molecular weight distribution obtained by GPC and compares with the distribution of repeated runs (B and C) obtained by flow-FFF. Weight average molecular weight values of 9.08 and 9.41 x 104 were obtained and compare well with that of9.0 x 104 obtained by GPC (see Table 1).
I
J
I
3.0
2.0
1.0 -
1.0.10"
Molecular weight (gImol)
Figure 5. Differential molecular weight distribution for hyaluronan HA5. A, obtained by GPC-MALLS, conditions as in Figure 3E. B, obtained by FFF-MALLS, Cone. 1.77 x 10-3 glm!, 2501J,1 injected mass. 3m! erossflow during focusing. Elution mode- lmllmin ehannel flow and O.3ml/min erossflow; C as in B (repeated run).
Table 1. Weight average molecular weight and radius of gyration for hyaluronan sample HA5 obtained using GPC-MALLS and flow FFF-MALLS. Sample
Method
A
GPC-MALLS FFF-MALLS FFF-MALLS
B
C
Flow rate (ml/min) 0.5 1.0 1.0
Mwt 9.01 ±0.15 x 104 9.08 ± 0.15 x 104 9.41 ± 0.25 x 104
KgI Dm 30.0 ± 5.7 28.1 ± 6.8 26.1± 8.8
Figure 5 5 5
62
Characterisation and solution properties ofhyaluronan
The separation by GPC was less effective in comparison to that of flow-FFF. Furthermore, quite good reproducibility was obtained for the flow-FFF runs which 4 showed fractions from - 1 x 10 to -8 X 105.. In order to examine the quality of separation a plot of molecular weight as a function of the elution volume is usually used. Figure 6 clearly shows that good separation has been achieved. -------"'--'=---==,:::..J
1.0JC10~
1.0x10"
-,
1.0x10"
"'''
is related to the persistence length q from the equation: (9)
The length of a disaccharide unit is assumed to be 1 nm. The molecular mass per unit length, ML, ofhyaluronan molecule is 40111 = 401 nm", Milas, Rinaudo and Borseli" have used 410.
M
3.75xl04 3.5xlO' l.oxl O" 3.0x1Ob 6.0xlOb 1.2xlO'
Table I Molecular size ofhyaluronan in salt solution ,12 qnm Lnm c* cgs L/q L/diameter [TI] cgs nm 4.5 3.26 94 29 21 132 1.8x1O,2 7.97 872 790 3.16x1O,3 110 6.9 126 14.6 429 3,990 2,664 9.38x1O-4 273 9.3 18.8 4,407 5.67xl0-4 10.6 706 7,481 397 24.8 14,963 7,672 3.26xlO-4 603 12.1 1237 32.8 29,925 13,358 1.87xlO-4 913 13.9 2153
Table I represent the persistence length and other parameters calculated for hyaluronan of various molecular lengths. The first row with molecular weight of 3.75 x 104 is at the border of the two regimes, each with its own set ofK and a. This molecular weight is, therefore, at the lower limit of the high molecular weight range in which a is 0.8. For this molecular weight, the length L is only 3.2 times the diameter. As a complete circle would require 7t times the diameter, this molecule can just make a complete circle. This is the minimum molecular weight necessary to form a loop. The radial distribution of local concentration in the cloud ought to be very different above and below this critical molecular weight. Above it, it is a Gaussian cloud, and the MarkHouwink-Sakurada formula is followed by a of 0.5 if in the theta solvent, or a higher value if in a good solvent. Below it, the density at a distance r from center is inversely proportional to 4xr, instead of 4m for the longer chains. The average internal concentration obtained is inversely proportional to the effective length of a molecular rod, rather than to ll2. So [TI] should be proportional to M. The short and wriggling molecules are actually better described as flexible rods. [TI] should be proportional to M2v , or M I for theta solvent, and M L2 for a good solvent. Recall the experimental value for the exponent was 1.16. 4 The persistence length q for hyaluronan of M=3.75xl0 has been calculated to be 4.5 nm, or 5 time as large as the length of disaccharide unit that is 1 nm. This ratio,
84
Characterisation and solution properties ofhyaluronan
however, is not limited to the stiff chain polymers. A similar set of quantities has been calculated for polyisoprene. The critical molecular weight and the absolute q are both an order of magnitude smaller than for hyaluronan, but the ratio of q to the bond length is about 4 for the critical molecular weight where L / diameter is 7t. The Semi-Dilute Solution By applying the Stokes-Einstein equation to the dilute solution of the "mass points" in a cloud of a dynamic polymer chain, the specific viscosity was shown to be equal to 2.5 c/c*. To arrive at this result, the cloud was allowed to pass the fluid around individual "mass points" with a normal laminar flow. No hydrodynamic interactions were assumed among the "mass points" other than, indirectly, from their Gaussian distribution in space. The Newtonian flow was restricted to be slow enough so the conformational probability was not altered by the flow field. The only serious assumption made has been that the concentration is low enough that no clouds were allowed to touch each other. However, a true Gaussian cloud would "feel" the presence of others at infinitesimal concentrations. Clouds of real polymer chains, too, can be considered to reach a great distance in excess of the length of a chain, as a conformation of the molecule far removed from the center of the cloud is a statistical possibility. If only the value of average internal density were of interest, this is perhaps a moot point, because the most significant part of the Gaussian distribution is limited to within some distance from the center of the cloud. However, whether to account or neglect intermolecular interaction at very low concentrations is a very serious question. The non-draining hydrodynamic sphere model, too, can account for the interaction to start at 0 concentration, by allowing the partial overlap as a possibility over the time. With the hydrodynamic volume model, various kinds of interactions between the spheres were speculated in the past, sometimes resulting in some polynomials without placing a physically significant meaning to each term, calling the polynomial a generalized hydrodynamic theory. 19 Initially at concentration of nearly 0, the internal density is at c*. The cloud is at the unperturbed state with the highest possible conformational entropy. When the concentration is increased to c, the internal density increases to a higher level than c* because, even though the concentration c is still below the average internal density, the outer region of the cloud, where the local density is lower than c*, will increase. Conformational rearrangement that follows will raise the average density higher than c*. If, for the sake of argument, the internal density were to remain unchanged at unperturbed c*, then Tlsp will always remain as c[Tl] at all concentration levels. Experimentally, this is found not to be the case. We now attempt to estimate that part of the viscosity increase, dTlsp, which is solely due to the increased internal density in the cloud. There are three kinds of concentrations to be dealt with: c, c; , and c*. c starts at 0, and so does the difference between c; and c*, that we define dc; = Cj - c*. All changes in these concentration values are a unique function of the respective relative volume. Thus, dc; = c. Now, the viscosity is proportional to the average concentration, even on local levels. dTlsp =_1=_ dc c __ c[ '1']] C* C* or,
(10)
Viscosity of polymer solutions
~11sp =C[11]·~=0.4c2[11]2
c* and for the total 11sp, one obtains:
85
(11)
which is the Huggins equation. The theoretical value of 0.4 for the Huggins constant, kH, has its origin in the value of2/5 from the Stokes-Einstein formula. This value should hold even for non-spherical particles, as long as they are randomly oriented. Actually, the values of k H ranging between 0.3 and 0.5 are found in the extensive compilation for many golymers by Stickler and Sutherlin. 20 The values for hyaluronan, obtained by Shimada, 2 are typically from 0.35 to 0.40 in the high molecular weight 21 range. For polyisoprene, the value of 0.42 was found by Patel and Takahashi throughout the molecular weight range of their investigation. From our experience, we find it difficult to determine the values of kH and [11] by assuming a linear equation from real data that actually curve upward with concentration. However, we were able to obtain kH nearly always of 0.4 if the eq.13 below was directly applied to raw data. The same Huggins formula can be derived from the model of hydrodynamic volume. Specifically, by considering the overlap of two hydrodynamic volumes to be the volume fraction of a volume fraction, the square term in the Huggins equation results. However, the actual value of the hydrodynamic volume fraction can be two orders greater than unity, which would be difficult to visualize physically. Also, in the overlapped region the hydrodynamic volume must be assumed to expand with increased concentration. The reason why the solid sphere model works in spite of these unreal operations is that it is the average density of the cloud that is being utilized for the calculation. Eq. 12 was derived, assuming the interaction to occur between two neighboring molecules. For one .molecule to interact with two neighbors at the same time, an additional increase in Cj must be included. In this case, the choice of two neighbors is combinatorial, so it must be divided by 2! to avoid redundancy. For this, ~211sp= 2/2. c[llH0.4c[1l]J For a greater number of interacting neighbor molecules, there will be a greater number of terms. Specifically, for one molecule interacting with up to three neighbors, the specific viscosity llsp is given by: (13) with value of 0.4 for k H. If the neighbor interaction were assumed to extend to all existing molecules, the polynomial is extended to infinite terms,
which is precisely the exponential function, (15)
86
Characterisation and solution properties ofhyaluronan
This is the Martin equation. 22 Previously, wei have subscribed to the Martin equation, based on the data that did not extend to the large enough value of c[n]. A critical examination for the choice between this equation vs an equation based on a limited number of neighbor interactions, such as Eq. 13, requires a set of very accurate Newtonian viscosity data obtained for high molecular weight polymers at relatively high concentration. Data obtained by Berriaud, Milas, and Rinaud0 27 extend to unusually high values of c[n], with added assurance that the Newtonian viscosity is being measured. These authors fitted their data with an empirical expression: (16)
7.0 6.0 I --eq.15 5.0 -eq.13 4.0 o Berriaud et al 3.0 --------- ---T I 2.0 1.0 0.0 -1.0 0.0 -1.0
i
+----------..~""'-"--------t--------.-----1
--ji --- -------1.0
2.0
log c[eta]
Fig. 1. Comparison ofeq. 13, eq. IS, and data [eq. 16, Ref. 27] fornsp vs c[n].
Eqs. 13, 15, and 16 are compared in Fig. I, indicating that Eq.l3, but not Eq, 15, is in better agreement with the data. In addition to Berriaud et al's data on hyaluronan, data on polyisoprene in hydrocarbon solvent, obtained by Patel and Takabashi;" data on polystyrene, polyisoprene, polybutadiene, by Raspaud et al,24 data on semi-rigid polyhexyl isocyanates by Ohshima et al,25 and on straight and rigid polyphenylenes by Kwei et al26 have been examined. Fit with eq. 13 is excellent with these data. In all these data, we fitted eq.13 directly to raw data, without going through the procedure of obtaining kH and [n] by drawing a straight line for Huggins and Kraemer equations for the low concentration range. We have shown that Eq. 13 is a universal curve for many types of polymers. It is based on the viscosity behavior of dynamic polymer chains modeled by clouds of mass points. These clouds allow for fluid to flow through. The flow around the points within the cloud was assumed to obey the Stokes Einstein law. No hydrodynamic interactions
Viscosity of polymer solutions
87
between these mass points were incorporated. The average density of the cloud is affected by the concentration. Overlapping of clouds increases the internal density of the cloud, thereby constraining the original unperturbed conformation, Although we have not discussed, the elastic modulus can be calculated based on the change in Cj such that G will depend on c2 for v=O.5, or a higher power if v>0.5. Eq. 13 can be applied to cc*, as clouds are touching their neighbors at all levels of concentration. The concept of molecular entanglement was not needed and not used in the development of the theory. The applicability of our model to rigid polyphenylene molecules endorses this contention.
Rigid, Sem i-rigid and Flexible Polym ers
7....--------------------, 6
-1-----
5-+---------------------~¥-
4
-+-------------------,.,.....IK-'~--~
3-+--------------2 - + - - - - - - - - - - - - - - -....1 ) 6 ' " - - - - - - - - - - - - 1 1 O-+------- ~'¥"-------------__i -1 -1
-0.5
o
0.5 log c[eta]
1
1.5
2
I I I ____-I
Fig. 2 Comparison of eq. 13 with experiment for many polymers.
ACKNOWLEDGEMENT Helpful discussions with Professors E. A. Balazs and T. K. Kwei are gratefully acknowledged. This work was partially funded by Biomatrix, Inc.
REFERENCES (1) Matsuoka, S. Relaxation Phenomena in Polymers, Hanser, Munich, New York, 1992. pp 170. (2) Eyring, H. Phys. Rev. 1932 39, 746. (3) Flory, P. J. Principles ofPolymer Chemistry, Cornell University Press, Ithaca, 1953 pp 601-621. (4) Milas, M.; Rinaudo, M.; BorseIi, J. J. Brazil AAS. 1993,45(11),46-48. (5) Arnott, S.; Mitra, A. K.; Raghnathan, S.; J. Mol. Bio!. 1983 169, 861-872. (6) Kirkwood, J. G.; Riseman, J. J. Chem. Phys. 1948,16,565. (7) Mark, H.; Tobolsky, A. V. Physical Chemistry of High Polymer Systems. Interscience, 1950, p.344
88
Characterisation and solution properties ofhyaluronan
(8) Turner, R.E., Lin, P., Cowman, M.K.; Arch. Biochem. Biophys. 1988,265,484-495. (9) Balazs, E.A (1965) in The Amino Sugars: The Chemistry and Biology of Compounds Containing Amino Sugars (Balazs, E.A & Jeanloz, R.W., eds) Vol. 2A, ppAO1460, Academic Press, New York. (10) Laurent, T. C.; Ryan, M.; Piertruszkiewicz, A. Biochim. Biophys. Acta. 1960,42, 476 (11) Cleland, R. L., and Wang, J. L., Biopolymers. 1970, 9, 799 (12) Shimada, E.; Matsumura, G.J. Biochem. 1975,78, 513-517 (13) Bothner, H.; Waaler, T.; Wik, O. Int. J. Bioi. Macromol. 1988, 10,287-291. (14) Yanaki, T.; Yamaguchi, M. Chem. Pharm. Bull. 1994,42 (8), 1651-1654. (15) Cleland, R. L. Biopolymers. 1970,9,811-824; ibid 1971,10,1925-1948. (16) Peterlin, A J. Polymer Sci. 1950,5,473. (17) Kuhn, H.; Kuhn, W. J. Polymer Sci. 1950,5, 519. (18) Fujita, H. Polymer Solutions, Elsevier 1990 pl40 (19) Frisch, H. L.; Simha, R. Rheology - Theory and Practice, Eirich, F. R., ed. 1956, Academic Press, New York, Chapter 14, pp525 - 614 (20) Stickler, M.; Sutherlin, N, in "Polymer Handbook", ed. Immergut, E. H.; Brandrup, J., John Wiley & Sons, New York, 1989. (21) Patel, S. S.; Takahashi, K.; Macromolecules 1992,25,4382-4391. (22) Martin Eq see for example, Tyrrell, Matthew, Rheology of Polymeric Liquids, C. W Macosko, Rheology Principles, Measurements, and Applications Wiley-VCH 1994 NYpp481 (23) Fouissac, E.; Milas, M.; Rinaudo, M. Macromolecules. 1993,26,6945-6951. (24) Raspaud, E, Lairez, D., Adam, M; Macromol. 199528, 978 (25)Ohshima, A Yamagata, A; Sato, T.; Teramoto, A; Macromolecules 1999, 32, 8645 (26) Kwei, T. K.; Nakazawa, M; Matsuoka, S; Cowman, M. K.; Okamoto, Y. Macromolecules 2000 33235 (27) Berriaud, N; Milas, M; Rinaudo, M; Int. J. Bioi. Macromol. 1994,16,137-142 (28) Davies, A.; Gonnally, J.; Wyn-Jones, E.; Wedlock, D. J.; Phillips, G. 0.; Int. J. Bioi. Macromol. 1982 4 436438..
CONFORMATIONAL AND RHEOLOGICAL PROPERTIES OF HYALURONAN Katsuyoshi Nishinari·), Yunqian Mo" Ryo Takahashr', Kenji Kubota 2 & Akio Okamoto3 I Department
ofFood and Nutrition, Faculty ofHuman Life Science,
Osaka City University, Sumiyoshi, Osaka 558-8585, Japan 2 Department
ofBiological and Chemical Engineering, Faculty ofEngineering, Gunma University, Kiryu, Gunma 376-8515, Japan 3Research
Center, Denki Kagaku Kogyo, Co.Ltd.,
Asahi-machi, Machidashi, Tokyo 194-8560, Japan
ABSTRACT
Effects of' urea, sodium chloride (NaCl), guanidine hydrochloride (GuHCI) or sucrose on the viscoelasticity of sodium hyaluronate (NaHA) solutions were studied. Urea did not change both storage and loss moduli so much, NaCl and GuHCI decreased both moduli, while sucrose increased both moduli. The critical overlap concentration C* was determined as an inflection point in the plot of zero shear specific viscosity vs concentration for NaHA solutions with and without urea, NaCI, GuHCI or sucrose. It is suggested that sodium ions or guanidinium ions shield the electrostatic repulsion of NaHA molecules, hence reduce the coil dimension, and C" shifted to higher concentrations. However, sucrose enhances the entanglement coupling between NaHA molecules and promotes the creation of hydrogen bonds, and then C* for NaHA solutions with sucrose shifts to lower concentrations. Both C* and magnitude of zero shear specific viscosity did not depend so much on the concentration of added urea either in the presence or in the absence of O.2M NaCI. This is in agreement with experimental results by light scattering. The radius of gyration Rg and hydrodynamic radius Rh were determined as a function of urea and sucrose concentration in the presence of O.2M NaCl by light scattering. Although the addition of sucrose reduces the coil dimensions of NaHA and stiffens NaHA molecules in dilute solutions, it increases both storage and loss moduli of concentrated NaHA solutions because of the enhancement of the formation of the temporary network via newly created hydrogen bonds. Both moduli of hyaluronan solution with urea were higher than those without urea in the lower angular frequency region, and tend to become nearly the same in the higher angular frequency region. The crossover angular frequency of both moduli decreases only a little with the addition of urea. It was found that the disruption of the hydrogen bonds due to urea, even if it occurs, does not affect significantly the rheological behavior of hyaluronan solutions. Intermolecular hydrogen bonds which lead to the formation of network in many gelling polysaccharides do not seem to exist in hyaluronan solutions. KEYWORDS
urea, guanidine hydrochloride, hydrogenbonds, rheology, light scattering
90
Characterisation and solution properties ofhyaluronan
INTRODUCTION
Hyaluronan (HA) is a major macromolecular component of the intercellular matrix of most connective tissues such as synovial fluid, cartilage, eye vitreous humor. Because of its unique viscoelastic properties, it is used as a medicine for arthritis and a surgical aid in ophthalmic surgery'", Hyaluronan is a linear polysaccharide consisting of disaccharide repeating sequence. The two saccharide residues are D-glucuronic acid and N-acetyl-D-glucosamine, which are linked P(l-3) and P(l-4) to each other4-6. The rheological characteristics of hyaluronan solutions have been widely studied, especially the relationships of its lubricating and shock-absorbing functionalities in synovial fluid to its viscoelasticity", The flow properties of normal synovial fluid extracted from human and cattle joints have been extensively studied8, JO, and are shown to be shear-thinning at high shear rates. By comparison of dynamic viscosity and static viscosity of synovial fluid at infinite time scales, Myers et al found that the viscosity at zero frequency and zero shear-rate agreed with each other and synovial fluids act only as viscous liquids in human joints under very slow motion 7 • It is reported that in osteoarthritis joints, the viscoelasticity of synovial fluid decreases and low molecular weight HA appears. It is important, therefore, to study the effect of the addition of the low molecular weight HA on high molecular weight HA solutions, Morris et al found that the addition of segmented chains of HA prepared by enzymatic degradation reduces drastically storage and loss moduliII, whilst Fujii et al. did not observe the similar phenomenon using segmented chains prepared by physical methods such as ultrasonic degradation or pyrolysis'f. It was suggested that surviving enzyme continued to degrade long molecular chains ofHA in the previous experiment", Several authors have reported that hyaluronan molecular coil expands at low ionic strength and high pH causing the increase in intrinsic viscosityI4.16. However, Preston et al. were unable to verify the expansion of the hyaluronan molecule with decreasing ionic strength by light-scattering measurements'". Cleland reported that the intrinsic viscosity and the radius of gyration of a hyaluronan preparation were 1400ml/g, 453ml/g and 75.8nm, 62.5nm in O.OlM and 2.0M NaCI solution, respectively 16. Morris et al. showed that increasing ionic strength of hyaluronan solutions is analogous to lowering pHII. A reduction in viscosity results from the suppression of electrostatic repulsion between the dissociated carboxyl groups, Scott proposed that the carboxyl group in hyaluronan forms a hydrogen bond with one of the hydroxyl groups in the uronic residue l 8 ; and it was confirmed!'. This concept "super hydrogen-bond" may be used to explain the extended coil dimensions at physiological pH and ionic strength. In the present work, the influence of urea on viscoelasticity of HA solution was examined to understand the role of hydrogen bonds because urea is known to be a hydrogen bond breaker. Then, the effects of urea on the rheological properties of hyaluronan solutions were compared with those of guanidine hydrochloride, which is also known to be a hydrogen bond breaker, and is also an electrolyte like sodium chloride. EXPERIMENTAL
Preparation of sample solutions Three samples of powdered HA, extracted from the culture medium of Streptococcus equi and purified were kindly supplied by Denki Kagaku Kogyo Co., Ltd. and Hoya Co.,Ltd.. Molecular weights of those samples determined from intrinsic viscosity using
Conformational and rheological properties
91
the Mark-Houwink parameters'" are 1.6x10 6 and 2.02x10 6 and 4.83 x10 5, respectively. NaC1, GuHC1, and urea of the reagent grade were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and ICN Biomedicals, Inc. (Ohio,USA) respectively and were used without further purification. HA solutions of various concentrations were prepared by dissolving in NaCl, GuHCI, and/or urea by weighing and were stirred for one day at room temperature. Special care toward the contamination of bacteria was taken. Light scattering measurements Light scattering measurements were carried out using a homemade spectrometer" for the NaHA solution in the presence of urea in 0.2M NaCI solution. Weight average molecular weight for the light scattering measurements is 4.83 x105 • Sample solutions were dialyzed against the respective urea and 0.2M NaCI solutions thoroughly before the measurements. Light source was Ar ion laser operated at 488.0 nm. Temperature of the sample solution was regulated within ±O.Ol°C. Correlation functions of the scattered light intensity were obtained by ALV-5000JE multiple-tau digital correlator. The radii of gyration were determined by the conventional angular dependence of scattered intensity. The hydrodynamic radii of HA were determined from the average decay rate obtained by using the third cumulant expansion method and the Stokes-Einstein equation. Corrections for the refractive index and the solvent viscosity were made by use of an Abbe's refractometer at 589 nm and an Ubbelohde capillary viscometer. It was not possible to carry out light scattering measurements without NaCI with sufficient accuracy because of the too low scattered intensity as is normally the case for an aqueous polyelectrolyte solution without salt. Intrinsic viscosity measurement The intrinsic viscosity [11] was determined using an Ubbelohde capillary viscometer at 25°C. The flow time for water was about ~141s at 25°C. A unit-thermalbath (Yamato Science, Inc. Tokyo, Japan) was used to regulate the temperature at 25±0.02°C. Dynamic and steady viscoelastic measurements Dynamic and steady viscoelastic measurements were performed by a RFSn (Rheometries Fluids Spectrometer; Rheometries, Inc., NJ, USA). The diameter and angle of the cone were 2.5cm and O.lrad, respectively. The strain was 5% and angular frequencies ranged from 0.01 to 100 radls in the dynamic measurements. The shear rates ranged from 0.01 to 1000 S·l in the steady state measurements. Temperature was fixed at 20°C. RESULTS AND DISCUSSION Light scattering The coil dimensions of NaHA solution with urea in 0.2M NaCl solutions were studied by light scattering. Radius of gyration (Rg), hydrodynamic radius (Rh) and the ratio (RglRh) in the presence of urea are shown together with those in the presence of sucrose in Fig. I.
92
Characterisation and solution properties ofhyaluronan 2.5 ~
• • •
2.0
l!:",
0
II:: 1.5
Oct
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• 0
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0
• • •
• • •
ir,"'" 0.5 -co
II::
•
•
s
•
••
0
0
0.5
1.0
• •
sucrose RgfRh
0
• e (mol)
sucrose RgfRg,o sucrose RhfRh,O
0
0.0 0.0
•
~ 3.0 3.1
urea RgfRg,o
0
urea RhfRh,O
0
urea RlRh
Figure 1. Radius of gyration and hydrodynamic radius of NaHA as a function of concentration of added urea and sucrose in the presence of O.2M NaCl.
The results suggest that urea does not affect significantly the coil dimensions of HA molecules within experimental error, although a slight decrease in Rg and Rg/Rt, was observed. These results show a clear contrast with our recent results for NaHA with sucrose in O.2M NaCI solutions, where both Rg and Rn decrease and Rg/RJ. increases appreciably with the increase of sucrose concentration". The magnitude of Rg and Rn and the slight decrease of Rg/Rt, by the addition of urea indicate that HA molecules are in the form of an extended coil affected by the excluded-volume effect and the solubility of HA becomes worse with the addition of urea causing a reduction in the excluded-volume effect. No intrinsic interaction between urea and HA molecules other than the excluded-volume effect might work at least in the experimental concentration region, and less solubility simply results in an increase in intermolecular interaction. Therefore,.the concentration dependence of 11spO would be stronger with the addition of urea in the dilute region, and the behavior in the entangled region is not affected so much by the presence of urea (especially without NaCl). It has been known that HA molecules have both the hydrophilic portions consisting of equatorial OH groups of saccharides and the hydrophobic portions consisting of axial CH groupS22. Hydrogen bonds are formed intramolecularly between the continuing monosaccharides and work to stiffen HA molecular chains. Such a stiffuess was evaluated quantitatively by use of wormlike chain model recentlY3,24. It was suggested that the stiffness of HA chains in solution originates from intramolecular hydrogen bonding by NMR measurements'". It was also suggested that the majority of stiff chains survive even after the change in ionic strength, temperature or by the addition of urea; that is, the addition of urea can not disrupt effectively the intramolecular hydrogen bonds between HA molecules, but no experimental data in the presence of urea was shown2s. Less solubility and larger intermolecular interaction should be due to the destruction of intramolecular hydrogen bonds, and chain dimensions must decrease much more than the experimental observations. The presence ofNaCI might lower this effect of urea. Intrinsic viscosity For dilute solutions (C C*) regions are 1.6 and 4.0, respectively, and C*[l1] and Tlspo at C = C" is evaluated ae- 3.2 and 24
96
Characterisation and solution properties ofhyaluronan
105 10 4 10 3
• • •
IMurea 3Murea 6Murea
., 10 2 Q,
I='
10 1
slope=1.6
C*[ f)J=3.2
10° 10- 1 10- 1
10°
C[ f)J
10 2
10 1
Figure 6. Zero shear specific viscosity for NaHA solutions with urea in the presence of 0.2MNaCI as a function of the coil overlap parameter C[TJ] (Temperature, 20°C; Molecular weight, 1.6 x 106) .
no urea 0"
................
O.2MNaCIG"
+ IMurea 0"
................
+lMUrea G"
········0·····..·
+3MureaO"
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+6Murea 0"
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10 -2 -f-'.............-........""t-........""r-..........................""" 10- 3 10-2 10- 1 100 10 1 10 210-3 10- 2
10- 1 100
10 1
10 2
w (rad/s) w (rad/s) Figure 7. (a) The angular frequency dependence of G' and G" for lwt% NaHA solutions with and without urea of various concentrations (b) The angular frequency dependence of G' and G" for Iwt% NaHA solutions with and without urea of various concentrations in the presence of 0.2MNaCI (Temperature, 20°C; Molecular weight, 2.02 x 106) . respectively. These values are in agreement with our previous results". Any intrinsic characteristics of the addition of urea were not observed. Fig.7(a) and (b) show the angular frequency dependence of storage shear modulus G' and loss shear modulus G" for 1wt% HA solutions in the presence of urea without NaCI and with 0.2M NaCl, respectively. At low frequency, both G' and Gil increased, and the crossover frequency of G' and G" shifted to lower angular frequencies with increasing concentration of urea. The similar shift was observed by the addition of
Conformational and rheological properties
97
sucrose", however, it was more pronounced than the shift by urea; the shift by urea was less than half by sucrose. Although the decrease of crossover frequency with adding urea might correspond to the strengthening the transient network structure formed by the chain entanglements, this is not so pronounced as in the case of sucrose. The increase in G'(Fig.7) and T1sp (Fig.2) of 1wt"10 HA by the addition of urea might be attributed to the breaking of intermolecular hydrogen bonds between the groups separated by many intervening segments along the chain, which have been called long range hydrogen bonds, used to explain the similar increase in the intrinsic viscosity of poly(vinyl alcohol) solutions in the presence of urea 29. It was, however, not possible to detect the change in the radius of gyration without adding NaCI by light scattering, and this should be explored in the future. CONCLUSION The effect of urea on the viscoelastic properties of HA solution was examined. The addition of urea decreases the expansion of HA molecular coils and promotes the intermolecular interaction in dilute solution presumably by a reduced excluded-volume effect due to poorer solvent conditions. The presence of NaCI could lower this effect of urea On the contrary, in concentrated and entangled solutions, urea has almost no effect on the rheological behavior at all. The addition of urea does not affect the alleged hydrogen bonds in the HA molecules significantly. This finding is in a sharp contrast with the results of the addition of sucrose in HA solution. REFERENCES 1. E. A. Balazs, In: Disorders of the Knee, A. Helfet, (ed.), 1974, T. B. Lippincott Company, Philadelphia, pp.63-75. 2. D. J. Tate, P. D. Oliver, M. V. Miceli, R. Stem, S. Shuster & D. A. Newsome, 'Age - dependent change in the hyaluronic acid content of the human chorioretinal complex', Arch. Ophthalmol., 1993, HI, 963-967. 3. N. Yerushalmi, A. Arad & R. Margalit, 'Molecular and cellular studies of hyaluronic acid - modified liposomes as bioadhesive carriers for topical drug delivery in wound healing', Arch. Biochem. Biophys, 1994, 313, 267-273. 4. R. L. Cleland, 'Ionic polysaccharides. IV. free - rotation dimensions for disaccharide polymers. Comparison with experiment for hyaluronic acid', Biopolymers, 1970,9,811-824. 5. W. T. Winter, P. J. Smith & S. Arnott, 'Hyaluronic acid: structure of a fully extended 3-fold helical sodium salt and comparison with the less extended 4-fold helical forms', J. Mol. Bioi., 1975, 99,219-235. 6. J. K. Sheehan, K. H. Gardner & T. E. D. Atkins, 'Hyaluronic acid: a double- helical structure in the presence of potassium at low pH and found also with the cations ammonium, rubidium and caesium',J. Mol. Bioi., 1977, H7, H3-135. 7. R. R. Myers, S. Negami & R. K. White, 'Dynamic mechanical properties of synovial fluid', Biorheology, 1966, 3, 197-209. 8. V. Tirtaetmadja, D. V. Boger & J. R. E. Fraser, 'The dynamic and steady shear properties of synovial fluid and of the components making up synovial fluid', Rheol. Acta, 1984,23,311-321. 9. J. E. Gomez & G. B. Thurston, 'Comparisons of the oscillatory shear viscoelasticity and composition of pathological synovial fluids' Biorheology, 1993,30,409-427. 10. J. Schurz, 'Rheology of polymer solutions of the network type', Prog. Polym. Sci.,
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Characterisation and solution properties ofhyaluronan
1991, 16, 1-53. 11. E. R. Morris, D. A. Rees & E. J. Welsh, 'Conformation and dynamic interactions in hyaluronate solutions', J. Mol. Bioi., 1980, 138,383-400. 12. K. Fujii, M. Kawata, Y. Kobayashi, A. Okamoto & K. Nishinari, 'Effects of the addition of hyaluronate segments with different chain lengths on the viscoelasticity of hyaluronic acid solutions', Biopolymers, 1996, 38, 583-591. 13. E. R. Morris, Private communication 14. E. A Balazs & T. C. Laurent, 'Viscosity function of hyaluronic acid as a polyelectrolyte', J. Polym. Sci. Lett. Ed, 1951, 6, 665-668. 15. T. C. Laurent, 'Studies on hyaluronic acid in the vitreous body', J. Biol. Chem., 1955,216,263-270. 16. R. L. Cleland, 'Ionic polysaccharides. II. Comparison of polyelectrolyte behavior of hyaluronate with that of carboxymethyl cellulose', Biopolymers, 1968, 6, 1519-1529. 17. B. N. Preston, M. Davies & A G. Ogston, 'The composition and physicochemical properties of hyaluronic acids prepared from ox synovial fluid and from a case of mesothelioma', Biochem. J., 1965,96,449-471. 18. J. E. Scott, 'Periodate oxidation, pKa and conformation of hexuronic acids in polyuronides and mucopolysaccharides', Biochim. Biophys. Acta, 1968, 170, 471-473. 19. T. C. Laurent, M. Ryan & P. Kizwica, 'Fractionation of hyaluronic acid: the polydispersity of hyaluronic acid from the bovine vitreous body', Biochim. Biophys. Acta, 1960,42,476-485. 20. D. Ito and K. Kubota, 'Solution properties and thermal behavior of poly (N-n-propylacrylamide) in water', Macromolecules, 1997,30, 7828-7835. 21. Y. Mo, T. Takaya, K. Nishinari, K. Kubota & A. Okamoto, 'Effects of sodium chloride, guanidine hydrochloride, and sucrose on the viscoelastic properties of sodium hyaluronate solutions', Biopolymers, 1999, 50, 23-34. 22. B. Weissmann & K. Meyer, 'The structure of hyalobiuronic acid and of hyaluronic acid from umbilical cord',J. Am. Chem. Soc., 1954, 76,1753-1757. 23. K. Hayashi, F. Tsutsumi, T. Nakajima, T. Norisuye & A. Teramoto, 'Chain-stiffaess and excluded-volume effects in solutions of sodium hyaluronate at high ionic strength', Macromolecules, 1995,28, 3824-3830. 24. R. Takahashi, K. Kubota, M. Kawata & A Okamoto, 'Effect of molecular weight distribution on the solution properties of sodium hyaluronate in 0.2M NaCI solution', Biopolymers, 1999,50,87-98. 25. A Darke, E. G. Finer, R. Moorhouse & D. A Rees, 'Studies of hyaluronate solutions by nuclear magnetic relaxation measurements. Detection of covalently-defined, stiff segments within the flexible chains', J. Mol. Biol., 1975, 99, 477-486. 26. Y. Kobayashi, AOkamoto, & K. Nishinari, 'Viscoelasticity of hyaluronic acid with different molecular weights', Biorheology, 1994, 31, 235-244. 27. W. W. Graessley, 'The entanglement concept in polymer rheology', Adv. Polymer Sci, 1974, 16, 125-179. 28. S. B. Ross-Murphy, V. J. Morris & E. R. Morris, 'Molecular viscoelasticity of xanthan polysaccharide', Faraday Symp. Chem. Soc., 1983, 18, 115-129. 29. H. Maeda, T. Kawai & S. Sekii, 'Intra-and intermolecular hydrogen bonds in polyvinyl alcohol solutions', J. Polymer Sci., 1959, 35,288-292.
CARTILAGE REPAIR WITH BONE MARROW IN A HYALURONAN-BASED SCAFFOLD Luis A. Solchaga', Victor M. Goldberg2 and Arnold I. Caplan' ISkeletal Research Center, Department ofBiology, Case Western Reserve University, 2080 Adelbert Road, Cleveland, OH 44106, USA. 2Department
ofOrthopaedics, Case Western Reserve University School ofMedicine and University Hospitals ofCleveland. 11100 Euclid Avenue, Cleveland, OH 44106, USA.
ABSTRACT The repair of osteochondral defects can be enhanced by providing structural support for the reparative activity. We hypothesize that the impregnation of specialized materials with autologous bone marrow will provide progenitor cells and cytokines to accelerate the healing response and, therefore, improve the quality of the repair. New Zealand White rabbits received bilateral, osteochondral defects on the femoral condyle. One leg received fibronectin-coated ACP""', a hyaluronan-based polymer, and the other flbronectin-coated, bone marrow-loaded ACp™. Rabbits were sacrificed 3, 4, 12 and 24 weeks after surgery. The reparative tissue was assessed by histology. Both treatment groups presented similar appearance at 4, 12 and 24 weeks after surgery. Four weeks after surgery, the defects presented new bone filling the defect with a layer of hyaline cartilage on top that integrated well with the adjacent cartilage. At the 12 and 24-week time points the defects presented bone filling the defect area beyond the level of the tidemark and a layer of hyaline cartilage about half as thick as the adjacent normal cartilage. At the 3-week time point , the defects that received bone marrowloaded implants presented more bone and the surface layer contained more cartilage. The early events of the regeneration process are accelerated by the inclusion of bone marrow; however, because the access to the marrow space is open in osteochondral defects, the medium- and long-term results obtained appeared to be similar. KEYWORDS Hyaluronan, tissue engineering, cartilage, bone marrow, repair, regeneration. INTRODUCTION The natural repair process of osteochondral defects can be enhanced through the use of biocompatible, biodegradable materials to serve as scaffolding for the regeneration process I. These materials provide structural support for the reparative activity of mesenchymal progenitor cells recruited into the area, presumably from the underlying bone marrow. The failure or success of the reparative process can be determined by the acquisition of mechanical stability in the damaged area through synthesis of extracellular matrix by the cells recruited into the defect. We hypothesize that impregnation of the implant material with bone marrow at the time of surgery will provide progenitor cells and bioactive factors required for the regeneration and accelerate the healing response.
64
Application ofhyaluronan in tissue engineering
METHODS - Preparation ofthe implants: The ACpTM sponge was cut into 2-mm diameter cylinders and then pre-coated by immersion into solution of fibronectin. After a I-h incubation at 4°C, the implants were removed from the fibronectin solution and dried overnight'. During the surgical procedure, autologous bone marrow, obtained as described below, was combined with the ACpTM sponges in a 5-mL tube. Negative pressure was applied to the tube to allow complete infiltration of the delivery vehicles with the cell suspension. The composites were incubated at room temperature for 20 min prior to implantation. Control implants were hydrated in sterile saline solution prior to implantation. - Surgical procedure: A total of 33 4-month-old New Zealand White rabbits were used in this study. All procedures followed an Institutional Animal Care and Use Committeeapproved protocol. Under anesthesia, the knee was exposed trough a skin incision; the capsule was incised and the medial femoral condyle exposed after luxation of the patella. A full-thickness defect (3-mm diameter x 3-mm deep) through articular cartilage and into the subchondral bone was prepared on the center of the medial femoral condyle. The implants were then placed into the defect and, after reducing the patella, the capsule, muscle and skin were closed with 4-0 dexon suture. Each rabbit received ACpTM sponge in one knee and BM-loaded ACpTM sponge in the contralateral knee. - Procurement ofbone marrow: In the same surgical act, the proximal medial surface of the rabbit tibia was exposed through a small incision. Subcutaneous tissue and periosteum were incised to expose the bony surface. The tibia was perforated with a 16gauge needle, and bone marrow (4 - 5 mL) was then aspirated from the tibial shaft through a pre-heparinized plastic tubing affixed to a 1O-mL syringe containing heparin. - Histologic processing: Rabbits were killed at 3, 4, 12and 24weeks after surgery. The knee joint was approached as described above and the distal femoral condyles dissected. The specimens were fixed in formalin, demineralized with RDO, and embedded in paraffin. Sections of 6 urn were cut and stained with Toluidine Blue. Representative sections were scored independently and blindly by 4 investigators with a 29-point scale. - Statistical analysis: The histologic scores were compared with a Wilcoxon signed rank test. P values minor than 0.05 were considered significant.
RESULTS At the 3-week time point (Fig. lA, E), the ACpTM-treated defects presented bone formation in the bottom half of the defect with a superficial layer of non-mineralized tissue composed of loose fibrous tissue, hypertrophic and hyaline-like cartilage. The 8M-loaded ACpTM-treated defects exhibited bone formation in the bottom 3/4 of the defect with a superficial layer composed of hypertrophic and hyaline-like cartilage. Four weeks after surgery (Fig. IB,F), the repair tissue in defects treated with ACpTM did not fill the defect area up to the level of the surrounding cartilage. Most of the defects presented new bone filling the defect with a layer of hyaline cartilage that integrated well with the adjacent cartilage. In some cases, hypertrophic cartilage was present between the bone and the hyaline cartilage. The defects treated with BM-Ioaded ACpTM presented a very similar appearance to those of the ACp''M-treated group.
Cartilage repair with bone marrow
Figure 1.
65
Light microscopy of the defects after implantation. A, B, C, D: ACP"" sponge. E, F, G, H: Bone marrow-loaded ACP"" sponge. A, E: 3 weeks after implantation. B, F: 4 weeks after implantation. C, G: 12 weeks after implantation. D, H: 24 weeks after implantation. Table I. Histologic scores by category (median (range». 4 weeks
Catezorv % hyaline cartilaae surface reaularftv degenerative changes structural
ACI)~
~CP~+8M
12 weeks ACP~
4.7 3.3 2.7 -11 (Fidia Advanced Biopolymers srl, Abano Terme, Italy). Two different forms ofHYAFP®-11were used for cell-cultivation and grafting: 1. Three-dimensional non-woven fleece, made of 20J.1m-thick fibres with a specific weight of n 100 g/mz , (Hyalograft 3D '), used for cultivation of autologous fibroblasts (Fig. 1); 2. Transparent, 20J.1m-thick membranes with laser-drilled microperforations (40J.1m in diameter), (Laserskin"), used for cultivation ofautologous keratinocytes (Fig. 2). Mentioned materials were obtained from Fidia Advanced Biopolymers srl, Abano Terme, Italy.
Fig. 1 Fig.l
Fig.2
Fig.2 Scanning electron microscope view of fibroblasts attached to Hyalograft 3D™_ fibres, an ideal environment for adhesion, proliferation and subsequent production of dermal extracellular matrix Electron microscopic view ofkeratinocytes grown on Laserskinf-membrane
Cell culturing procedures Autologous fibroblasts
Human keratinocytes and fibroblasts were isolated from 2x2 cm skin biopsies taken with an electric dermatome. During transportation to the laboratory, the biopsy was kept in a nutritional medium (Dulbecco s Modified Eagles Medium, DMEM containing 5% fetal calf serum (FCS) and antibiotics). On arrival, the skin was rinsed in phosphate-buffered saline (PBS), the deep dermal layer was removed and the tissue was cut into small fragments, which were transferred into petri dishes containing 20 ml dispase (5 mg/ml). The de-epidermalized fragments of dermis were rinsed with PBS and further minced with a scalpel. Human fibroblasts were isolated by overnight digestion with a solution of 80 U/ml of type 1 collagenase (Worthington Biochemical Corp. NJ) in DMEM 10% FCS, at 37°C 5% COz. Cells were propagated in DMEM 10% FCS, the culture medium was renewed twice a week. Cells were trypsinized at 80% confluency and splitted 1:3 for subsequent passaging.
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Application ofhyaluronan in tissue engineering TN
Fibroblasts were seeded onto the Hyalograft 3D matrix at a density of 8,0-10,0 x 104 cells/em" (size unit: 8 x 8 em) in DMEM 10% FCS, culture medium was changed twice a week. Starting day 8 from seeding, the dermal grafts were ready for transplantation.
Autologouskeratinocytes Human keratinocytes were isolated from the same 2x2 em skin biopsies. After incubation at 37°C for I hour, the epidermal layer was gently peeled off from the dermis and the epidermal sheets were treated with 0,25% trypsin for 15 minutes at 37°C under gentle stirring. Cell suspensions were seeded at a density of2,0 x 104 keratinocytes/cm' on a feeder layer of lethally irradiated 3T3 mouse fibroblasts. Culture medium was composed 00 parts ofDMEM, I part of Ham's FI2 supplemented with 10% FCS, Sug/ml insulin, 10-10 M cholera toxin. 0,4 ug/ml hydrocortisone, and l Oug/ml rh-EGF (all reagents were purchased from Sigma Chemical Co. , S1. Louis, MO). Keratinocytes were used up to passage 4. When keratinocytes were seeded on the HYAFF@-laserperforated membrane, the following seeding densities were 4 employed: 2,0 x 10 keratinocytes/cnr', 2,5 x 104 feeder cells (size unit: 10 x 10 em), The medium was renewed every second day. Starting day 7 from seeding, all the microperforations were populated with colonies of basal cells and the membrane was ready for grafting. The epidermal sheets were rinsed in PBS several times in order to remove the FCS, transferred into suitable trays filled with nutritional FCS-free medium, sealed and double packaged under sterile conditions. Transplantation could take place the following day.
Patient history In patients with massive injuries, skin biopsies (2x2 cm) were taken during the first surgical session with the electric dermatome after the decision of performing dermal and epidermal cell transplantation had been made. Patient's or parent's consent declarations were obtained in written form. First the wounds were conditioned by regular surgical debridement prior to fibroblast-grafting. After approximately two weeks the cultured ,,neo-dermis" consisting of autologous fibroblasts grown on biocompatible threedimensional scaffolds made up of an benzylester ofhyaluronan (Hyalograft 3D"') was ready for grafting. One week after the initial fibroblast-transfer and ingrowth of the dermal substitute the transplantation of autologous keratinocytes on HYAFF@ membranes (Laserskin") was performed. Approximately 10 days after keratinocyte transplantation mesh-grafting (0.2 rom thin, ratio I :6) onto the formed epithelium was carried out. The described patient is a male, 65 years old; after a train accident he showed extensive traumatic soft tissue loss ofhis right arm from the axillar region down to the palm of his hand with comminuted fracture of the forearm, metacarpal fractures I-IV; at the local hospital he was treated with external fixator and plaster cast; massive superinfection with Pseudomonas Aeruginosa and Staphylococcus Aureus on arrival in our hospital was obvious (Fig. 3-8).
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Patient: male,65 years old, extensive soft tissue loss right ann from axilla down to the palm ofhis hand with comminuted fracture ofthe forearm and metacarpal fractures I-IV after train accident, treated with external fixator and plaster cast. Appearance ofthe previously superinfected tissue defect in our department after surgical debridement a. total right ann b. dorsal view of right forearm with external fixator
Fig.4a
Fig.4
Fig.Sa
Fig.S
Fig.4b Patient: day 20 after admission; 4 days after autologous fibroblast transplantation on Hyalograft 3DTM, first change of secondary dressings, appearance of dry scaffold surface a. total right ann b. right forearm, dorsal view, removed instable fixator
Fig.Sb Patient: day 26 after admission; 10 days after fibroblast transplantation; take rate of grafts approximately 95%, impressive equalization of levels between
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Application ofhyaluronan in tissue engineering
surrounding healthy skin and former tissue defect area; no adverse or immunogenic reactions; no signs of infection a. total right arm, complete integration of grafts b. right forearm, dorsal view, superficial rejection of two fibroblast-sheets, unknown reason
Fig.6a
Fig.6b
Fig.6
Patient: day 35 after admission; 5 days after autologous keratinocyte-grafting on Laserskinf-membranes, first change of secondary dressings, diminished fluidloss, signs of beginning epithelialization; almost complete biodegradation of hyaluronan sheet, no adverse effects, no rejection; manipulations possible without pain a. right arm, healthy, clean tissue appearance, thin re-epithelialization visible b. right forearm, same smooth tissue structure
Fig.7a
Fig.7b
Fig.7
Patient: day 45 after admission to our department, first change of secondary dressing 5 days after mesh-grafting (0.2 rom thin, mesh-ratio I :6), perfect take result, firm attachment ofgraft on underlying thin epithelium a. right arm, stable repair b. right forearm; even mechanically insufficient meshed skin shows strong adherence to underlying skin layer
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Fig.8b
Fig.8a Fig.8
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Patient: out-patient visit, 10 weeks after initial admission, 4 weeks after final mesh-grafting; complete tissue replacement with normo-elastic properties and good cosmetic result; shoulder-, elbow- and wrist-joints show good mobility a. right ann, dorsal view, tissue reconstruction complete b. right forearm; good final result
RESULTS Preparation of grafts Human fibroblasts obtained from the patient's skin biopsy proliferated during the primary culture phase which took approximately 8 days; subsequently seeded onto non-woven HYAFpGl'-11 fleeces where they were able to adhere and proliferate within the scaffold (Fig. I). Human epithelial celIs harvested from the same biopsy proliferated in medium during the primary culture phase which lasted approximately 8 days; they were subsequently seeded on the Laserskinf-membrane where they showed rapid proliferation (Fig. 2). After a few days the celIs reached a sub-confluent stage and were suitable for grafting. The composite keratinocyteLaserskinf-sheets remained stable throughout the culturing procedure and did not show any sign of contraction. The transparency of the membranes made cell observation using light microscopy possible at any time. Patients In the described patient clean and non-infected defect-areas were revealed after regular surgical debridement under sterile conditions in the operating theatre (Fig. 3). Skin specimen (2x2 ern; O,6mm thick) was taken with an electrical dermatome in one of the first surgical sessions without any problems after written permission was obtained. Hyalograft 3D""-transplantation was performed 16 days after initial hospital-admission. The performance of transplantation and handling of the grafts was very easy from a surgical point of view. To avoid shear- or pressure-forces we applied non-adhesive paraffin secondary dressings below ordinary sterile gauze dressings, put on careful elastic bandages and kept the patient in bed for the first three days. After covering the soft tissue defects with dermal equivalents, pain experienced in the treated regions was reduced. First dressing changes was performed 4-5 days after transplantation under sterile conditions in the operating theatre (Fig. 4). Firm adherence of the grafts onto the underlying tissue was remarkable in the patient. The most superficial layers had dried out due to reduced wound fluid-secretion after grafting. 8- IO days after transplantation, integration of the fibroblastseeded biomaterial was almost complete (Fig. 5). Equalization of the levels between healthy
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Application ofhyaluronan in tissue engineering
surrounding skin and regenerated defect-areas was visible. No signs of infection, allergic reactions or any other side-effects were evident. 14 days after transplantation of Hyalograft 3D"", Laserskinf-graffing was possible. Again handling of the grafts was uncomplicated. In order to prevent shear and pressure-forces, patients were immobilized until first change of secondary dressings during the critical period of cell-attachment. Afterwards intensive physiotherapeutic exercise was resumed. 5-10 days after keratinocyte transfer the membrane was biodegraded. Thin epithelialization with diminished wound fluid secretion was evident (Fig. 6). To achieve improved biomechanical tissue properties mesh-graft-transplantation (0,2 mm thick, ratio 1:6) was performed onto the layer of thin epithelium in the patient 10 days after Laserskinf-grafting (Fig. 7). Two weeks after mesh-graft application all the defects showed final and complete tissue regeneration with macroscopically normo-elastic properties. Mobility of underlying joints was not impaired. After patient dismissal, control visits were performed on an out-patient basis (Fig. 8). DISCUSSION Chronic skin ulcers and burn wounds as examples for acute skin defects represent the most promising clinical applications of soft-tissue engineering. By presenting our results, we would like to widen the indications for tissue engineering to the area of trauma and reconstructive surgery. The continuous search for improved methods of clinical application of grafts composed of autologous fibroblasts and keratinocytes as skin substitutes cultured in vitro is reflected in the intense search for innovative carrier systems, i.e, biomaterials that are able to support the growth of human skin cells [39]. Amongst the various materials like collagen [19-22], autologous [17,40-43] or allogeneic [44-50], fibronectin [23] and fibrin glue [6,24] hyaluronan seems to be a very promising substrate. HA has been proven to play a fundamental part in wound healing itself both in adult and fetal tissue [38,51,52]. In comparison with the other mentioned macromolecules HA derivatives are less immunogenic and reduce the potential risk of viral and prion infections [53,54]. Development of cultured dermis consisting of autologous fibroblasts on threedimensional HYAF~scaffolds for primary reconstruction of extensive soft tissue defects represents a promising innovation from a clinical as well as a morphological point of view. Autologous fibroblasts actively proliferated throughout the culturing procedure and as previously shown by Campoccia et aI. [55], morphological observations on paraffin-embedded specimens demonstrated that the cells migrate through the non-woven fleece, populate the inner space and both sides of the biomaterial to produce a fibrillar network. Furthermore, immunohistochemical examinations detected the production of extracellular matrix components such as collagen type IV, fibronectin and laminin [56]. Our report confirms the in vivo biocompatibility and complete incorporation of the living autologous dermal equivalent. In all patients there was excellent performance and near-total take rate of all the sheets. Very interesting findings were reduced pain sensation and clearly reduced loss of wound secretions which was evident by the appearance of dried upper surfaces of the neo-dermis already visible at first change of secondary dressings. Ten days after dermal grafting the deeply fissured and clefted tissue defects showed smooth equaIizing at the margins to healthy surrounding skin without any signs oflocal or general adverse effects. From our own point of view we can state that the described method of dermal replacement enables the creation of ideal preconditions for any subsequent epidermal grafting procedure. The important factor in the strategy of restoring damaged soft tissue in layers is the assumed concept that the dermal component constitutes a permissive and regulatory microenvironment for the growth and differentiation of cultured keratinocyte-grafts [26,55,57].
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The second step in building up normal soft tissue morphology in our patients was the transplantation of autologous keratinocytes, obtained from the same biopsy and cultured separately on Laserskin"-membranes. Positive results based on clinical as well as preclinical settings have been reported using keratinocytes cultured on laser-perforated HYAF~-membranes [11,12,58]. The microperforated structure of the sheet itself enables keratinocytes to populate the pores and colonies grow both above and beneath the membrane. Furthermore, the cells differentiate and exhibit features similar to physiological epidermis like the presence of hemidesmosomes, previously shown by Trabucchi and co-workers [59]. As soon as the keratinocytes are subconfluent, they are ready for grafting; time spent in culture can be reduced, earlier grafting is possible. Culturing keratinocytes to confluent stages causes transition from a highly proliferative state to one of irreversible growth arrest in the meaning of terminal differentiation [60]. If keratinocytes can be transplanted in the state of hyperproliferation, before differentiation is induced, the dermal wound bed has the possibility to function as a "culture system in vivo", allowing the cells to adhere, proliferate and later to differentiate and build up cornified layers. In the demonstrated tissue defect good vascularized stable tissue with proliferating fibroblasts and dermal extracellular matrix components was visible. The incorporation of dermal equivalents should create perfect conditions for taking of the autologous keratinocyte grafts. The Laserskinf-graft we used was peeled from the petri dish without dispase digestion and could be easily applied to the wounded sites. First change of secondary dressings revealed ongoing biodegradation of the membrane, thin epithelial coverage was visible 8-10 days following grafting. The lack of biomechanical stability ten days after the final cell-grafting procedure is not a problem in smaller or in chronic wounds. Our patients are mostly "healthy" human beings, who are violently tom from the community by trauma. The injuries have to be restored as quickly as possible with the aim of minimizing functional disabilities. Therefore we decided to follow a strategy that comprises extremely thin mesh-grafting with mesh-ratio I:6, to minimize the donor site lesion, 10 days after Laserskinf-transplantation. All patients have been discharged from the hospital within 8 weeks of the trauma. They are followed on a regular out-patient basis. All patients are reintegrated in their normal daily routine. CONCLUSION
The present study has shown that hyaluronan benzyl ester, both as non-woven fleece and as membrane structure, is easy to handle, biocompatible, biodegradable and non-immunogenic. The combined use of hyaluronan scaffolds, cultured autologous keratinocytes, fibroblasts and mesh grafts leads to rapid wound closure and to a mechanically stable tissue. In summary, first experiences with transfer of Hyalograft 3D'" with autologous fibroblasts in order to replace lost dermal tissue in deep and extensive soft tissue defects indicate that this procedure is a viable and very promising treatment option. However, due to the mechanical instability of autologous epithelial grafts, thin mesh grafts are still required. Further studies are needed to establish whether this form of treatment will be accepted as standard procedure.
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REFERENCES 1. Langer R, Vacanti JP, Tissue engineering, Science, 1993,260,920-6. 2. Rheinwald JG, Green H, Serial cultivation of strains of human epidermal keratinocytes: the formation ofkeratinizing colonies from single cells, Cell, 1975, 6, 331-344. 3. O'Connor NE, Mulliken JB, Banks-Schlegel S et al, Grafting of burns with cultured epithelium from autologous cells, The Lancet, 1981,1,75-78. 4. Compton CC. Current concepts in pediatric burn care: the biology of cultured epithelial autografts: an eight-year study in pediatric burn patients, Eur J Pediatr Surg., 1992, 2, 216-222. 5. Donnersmarck von GH, Millbauer W, Hefter E, Hartinger A, Die Verwendung von Keratinozytenkulturen in der Schwerbrandverletztenbehandlung: bisherige Erfahrungen, Ausblicke zur weiteren Entwicklung, Unfallchirurg., 1995, 98, 229-232. 6. Kaiser HW, Stark GB, Kopp J et al, Cultured autologous keratinocytes in fibrin glue suspension, exclusively and combined with STS-allograft (preliminary clinical and histological report ofa new technique), Burns, 1994,20,23-29. 7. Tamisani AM, Ferretti S, Sangiorgio L, Critical reflections on the use ofhuman cultured keratinocytes in children with burns, Eur J Pediatr Surg., 1992, 2, 223-226. 8. RennekampffHO, Kiessig V, Hansbrough JF, Current concepts in the development of cultured skin replacements, J Surg Res., 1996, 62, 288-295. 9. Whalen E, Donnelly TA, Naughton G, Rheins LA, The development ofthree-dimensional in vitro human tissue models, Hum Exp Tox., 1994, 13, 853-859. 10. Luyn van MJA, Verheul J, Wachem van PB, Regeneration of full-thickness wounds using collagen split grafts, J Biomed Mater Res., 1995,29, 1425-1436. 11. Hollander DA, Stein M, Bernd A, WindolfJ, Pannike A, Autologous keratinocytes cultured on benzylester hyaluronic acid membranes in the treatment of chronic fullthickness ulcers, J Wound Care., 1999, 8,351-355. 12. Meyers SR, Navsaria HA, Grady J et a~ A hyaluronic acid membrane delivery system for cultured keratinocyte auto grafts: clinical take rates in a porcine kerato-dermal model, Wound Rep Reg., 1995, 3, 390. 13. Yannas IV, Burke JF, Design ofan artificial skin, I: basic design principles, J Biomed Mater Res., 1980, 14,65-81. 14. Hansbrough JF, Current status of skin replacements for coverage of extensive burn wounds, ./Trauma, 1990, 30(suppl), 155-160. 15. Yannas IV, Hansbrough JF, Ehrlich HP, What criteria should be used for designing artificial skin replacements and how well do the current grafting materials meet these criteria? JTrauma, 1984,24,29-39. 16. Bell E, Ehrlich HP, Sher S et al, Development and use ofa living skin equivalent, Plast Reconstr Surg., 1981, 365, 77-94. 17. Tompkins RG, Burke JF, Progress in burn treatment and the use of artificial skin, World J Surg., 1990, 14,819-824. 18. Yannas IV, Burke JF, Gordon PL, Huang C, Rubenstein RH, Design ofan artificial skin, II: control of chemical composition, J Biomed Mater Res., 1980, 14, 107-131. 19. Bell E, Ehrlich HP, Buttle DJ, Nakatsuji T, Living tissue formed in vitro and accepted as skin equivalent tissue of full thickness, Science, 1981,211, 1052-1054. 20. Boyce S, Christianson D; Hansbrough JF, Structure ofa collagen-GAG skin substitute optimized for cultured human epidermal keratinocytes, J Biomed Mater Res., 1988,22, 933-938. 21. Hansbrough JF, Morgan J, Greenleaf G, Advances in wound coverage using cultured cell technology, Wounds, 1993, 5, 174-194.
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22. Lamme EN, De Vries HJC, Van Veen H, Gabbiani G, WesterhofW, Middelkoop E, Extracellular matrix characterization during healing of full-thickness wounds treated with collagen/elastin dermal substitute shows improved skin regeneration in pigs, J Histochem Cytochem., 1996,44,1311-1322. 23. Ejim OS, Blunn GW, Brown RA, Production of artificial-oriented mats and strands from plasma fibronectin: a morphological study, Biomaterials, 1993, 14,743-749. 24. Hundyadi J, Farkas B, Berteny C, Hugo NE, The effect offibrin glue on skin grafts in infected sites, Plast Reconstr Surg., 1988,89,268-272. 25. Prunieras M, Regnier M, Schlotter M, A new method to culture human epidermal cells on allogeneic or xenogeneic dermis: preparation of recombined grafts, Ann Chir Plast., 1979, 24,357-362. 26. Krejci NC, Cuono CB, Langdon RC, McGuire J, In vitro reconstruction of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis, J Invest Dermatol, 1991, 97, 843-848. 27. Koyano T, Minoura N, Nagura M, Kobayashi KI, Attachment and growth of cultured fibroblast cells on PVAlchitosan-blended hydrogeIs, J Biomed Mater Res., 1998, 39, 486490. 28. Cooper ML, Hansbrough JF, Spielvogel RL, Cohen R, Bartel R, Noughton G, In vivo optimization ofa living dermal substitute employing cultured human fibroblasts on a biodegradable polyglycolic acid or polyglactin mesh. Biomaterials, 1991, 12,243-249. 29. Andreassi L, Casini L, Trabucchi E et al, Human keratinocytes cultured on membranes composed of benzyl ester ofhyaluronic acid suitable for grafting, Wounds, 1991,3, 116126. 30. Toole BP. Proteoglycans and hyaluronan in morphogenesis and differentiation, In: Hay ED, editor. Cell biology ofextracellular matrix (2nd edition). New York: Plenum Press, 1991,305-341. 31. Toole BP, Hyaluronan in morphogenesis, J Intern Med., 1997, 242, 35-40. 32. Presti D, Scott J, Hyaluronan-mediated protective effect against cell damage caused by enzymatically produced hydroxyl radicals is dependent on hyaluronan molecular mass, Cell Biochem Function, 1994, 12,281-288. 33. Abatangelo G, O'Regan M, Hyaluronan: biological role and function in articular joints, Eur J Rheumatol Irif/ammation, 1995, 15(1), 9-16. 34. Abatangelo G, Martelli M, Vecchia RP, Healing of hyaluronic acid-enriched wounds: Histological observations, J Surg Res., 1983, 35, 410-416. 35. West DC, Hampson IN, Arnold F, Kumar S, Angiogenesis induced by degradation products of hyaluronic acid, Science, 1985,228, 1324-1326. 36. Burd DA, Siebert JW, Ehrlich HP, Garg HG, Human skin and post-burn scar hyaluronan: demonstration ofthe association with collagen and other proteins, Matrix Vol., 1989,9, 322-327. 37. Wiegel PH, Frost SJ, McGary CT, LeBoeufRD, The role of hyaluronic acid in inflammation and wound healing, Int J Tiss Reac., 1988, 10, 355-365. 38. Chen WYJ, Abatangelo G, Functions ofhyaluronan in wound repair, Wound Rep Reg., 1999, 7, 79-89. 39. Rue LW, Cioffi WG, McManus WF, Pruitt BA, Wound closure and outcome in extensively burned patients treated with cultured autologous keratinocytes, J Trauma, 1993, 34, 662-668. 40. Heimbach D, Luterman A, Burke J et al, Artificial dermis for major burns. A multi-center randomized clinical trial, Ann Surg., 1988,208, 313-320. 41. Murphy GF, Orgill DP, Yannas IV, Partial dermal regeneration is induced by biodegradable collagen-glycosaminoglycan grafts, Lab Invest., 1990,62,305-313.
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42. Hansbrough JF, Boyce ST, Cooper ML, Foreman TJ, Burn wound closure with cultured autologous keratinocytes and fibroblasts attached to a collagen-glycosaminoglycan
sub&rate,JAAdA,1989,262,2125-2130. 43. Boyce ST, Glatter R, Kittmiller WJ, Treatment of chronic wounds with cultured skin substitutes: a pilot study, Wounds, 1996, 7, 24-29. 44. Hull BE, Finley RK, Miller SF, Coverage of full-thickness burns with bilayered skin equivalents: a preliminary clinical trial, Surgery, 1990, 107, 496-502. 45. Nanchahal J, Otto WR, Dover R, Dhital SK, Cultured composite skin grafts: biological skin equivalents permitting massive expansion, The Lancet, 1989,2, 191-193. 46. Cuono C, Langdon R, McGuire J, Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury, The Lancet, 1986, 1, 1123-1124. 47. Cuono CB, Langdon R, Birchall Net al, Composite autologous-allogeneic skin replacement: development and clinical application, Plast Reconstr Surg, 1987, 80,626637. 48. Krant D, Eckhardt M, Patton ML, Combined simultaneous application of cultured epithelial autograft and Allodenn, Wounds, 1995, 7, 137-142. 49. Gentzkow GD, Iwasaki SD, Hershon KS et ai, Use of dermagraft, a cultured human dermis to treat diabetic foot ulcers, Diabetes Care, 1996, 19, 350-354. 50. Pollak RA, Edington H, Jensen JL, A human dermal replacement for the treatment of diabetic foot ulcers, Wounds, 1997, 9, 175-183. 51. Moriarty KP, Crombleholme TM, Gallivan EK, O'Donnal C, Hyaluronic acid-dependent pericellular matrices in fetal fibroblasts: implication for scar-free wound repair, Wound Rep Reg., 1996, 4, 346-352. 52. Siebert JW, Burd AR, McCarthy JG, Weinzweig J, Ehrlich HP, Fetal wound healing: a biochemical study of scarless healing, Plast Reconstr Surg., 1990, 85, 495-502. 53. Gallico GG, Biologic skin substitutes, Clin Plast Surg., 1990, 17,519-526. 54. Heck E, Bergstresser P, Baxter C, Composite skin graft: frozen dermal allografts support engraftment and expression ofautologous epidermis, J Trauma, 1985, 25, 106-112. 55. Campoccia D, Doherty P, Radice M, Brun P, Abatangelo G, Williams DF, Semisynthetic resorbable materials from hyaluronan esterification, Biomaterials, 1998, 19,2101-2127. 56. Zacchi V, Soranzo C, Cortivo R, Radice M, Brun P, Abatangelo G, In vitro engineering of human skin-like tissue, J Biomed Mater Res., 1998, 40, 187-194. 57. Coulomb B, Lebreton C, Dubertret L, Influence of human dermal fibroblasts on epidermalization, J Invest Dermatol., 1989, 92, 122-125. 58. Donati L, Marazzi M, Veronesi AM et al, Treatment of cutaneous wound with cultured human keratinocytes on hyaluronic acid membrane, Wound Rep Reg., 1995, 3, 363. 59. Trabucchi E, Andreassi L, Malcovati M et al, Ultramicroscopic observations of cultured epithelial sheets before and after grafting for major human burns, Wounds, 1991, 3, 83-88. 60. Poumay Y, Pittelkow MR, Cell density and culture factors regulate keratinocyte commitment to differentiation and expression of suprabasal KIIKIO keratins, J Invest Dermatol., 1995, 104(2),271-276.
HYALURONIC ACID SELF -ASSOCIATION IN THE PRESENCE AND ABSENCE OF SALTS Theresa M. Mcintire and David A. Brant* Department ofChemistry University of California Irvine, CA 92697-2025 USA
ABSTRACT
Carbohydrate polymers and oligomers are excellent candidates for molecular level storage of biological information through variations in the chemical composition, primary sequence, and branching pattern. Diversity of polysaccharide primary structure affords diversity in higher order structure, and information may be stored in the three dimensional spatial relationships of the sugar residues as well. 1 This allows carbohydrates to function as highly specific markers in biological recognition processes. 2 Carbohydrates operate at cell surfaces, where they often occur as components of glycoproteins, glycolipids, or proteoglycans anchored in the cell membrane. 3 Signaling and recognition usually involve carbohydrate-protein interactions. 4-5 Instances of carbohydrate-carbohydrate signaling interactions are likewise emerging. 6 Carbohydrate-carbohydrate interactions are also clearly important in stabilizing the three dimensional structures of many biologically active oligo- and polysaccharides. 7 Many polysaccharides, e.g., hyaluronic acid (HA), contain charged groups that can affect their conformation and thus their physicochemical and biological properties. The self-interactions these polymers undergo are clearly important in stabilizing the three dimensional structures that may be important for their function in signaling and recognition. In this report we show that the extent and nature of association of sodium hyaluronate (NaHA) in aqueous solution, observed using AFM, is sensitive to the presence of salts. KEYWORDS
Atomic force microscopy (AFM), hyaluronic acid (HA), scanning probe microscope (SPM), self-association INTRODUCTION
The scanning probe microscope (SPM) 8 has been a valuable tool for routine imaging of complex biological structures as well as individual molecules with nanometer-scale resolution. 9 Carbohydrate-carbohydrate interactions have been investigated for several polysaccharides using AFM. 10-15 In this paper we document an increase in the extent of chain association with increasing HA concentration and the formation of stiff HA fiber networks in the presence of added low molecular weight salts.
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MA TERIALS & METHODS
A culture broth of the bacterium Streptococcus equi was controlled to produce HA with a molecular weight of 2 x 106 g/moI. This high molecular weight HA was purified and heat degraded to produce a sample of lower molecular weight (NaHA, 3 x 105 g/mol). These NaHA samples were supplied by Vitrolife UK Ltd. The NaHA stock solutions were diluted with distilled water or a low molecular weight salt solution to a final polymer concentration of 1 - 30 ug/rnl., Aliquots of these diluted solutions were deposited by spraying a fine aerosol 10, 13, 16-18 onto freshly cleaved mica (Polysciences, Inc., Warrington, PA) and air dried. Mica is easily prepared by cleaving with tape and provides an atomically flat substrate free of artifacts found on other commonly used SPM substrates such as highly oriented pyrolytic graphite. After air drying for a few hours, samples were imaged by AFM. Specimens were examined using a ThermoMicroscopes AutoProbe ® CP Research (Sunnyvale, CA) scanning probe microscope equipped with an NCAFM probe head. A piezoelectric scanner with a range up to 50 urn was used for all images. The scanner was calibrated in the xy directions using a 1.0 urn grating, and in the z direction using several conventional height standards. 10, 12-13 The oscillation frequency (I) of the cantilever/tip was offset from (1)0 to higher frequencies by a few kHz. 10, 13, 19 The oscillation amplitude and z direction set point were adjusted to avoid tip-sample contact according to the operating procedures for noncontact mode imaging. All measurements were performed in air at ambient pressure and humidity. Images were stored as 256 x 256 point arrays and analyzed using AutoProbe® image processing software supplied by ThermoMicroscopes.
Figure I. Scale bar =200 nm.
Figure 2. Scale bar =500 nm.
Mesh-like networks were formed at a NaHA concentration of 10 Ilg/mL in aqueous solution in the absence of added salt, as seen in Figure I. The NaHA networks showed no molecular ends or tails. The mean thickness of these molecular sheets, 0.66 nm, is consistent with a monolayer of HA molecules, suggesting strong lateral association. Diluting the HA to 1-31lg/mL yields mostly single strand species as shown in Figure 2. In the presence of added low molecular weight salts various extents of chain association or aggregation were seen. With 0.10 M sodium acetate (Figure 3) or 0.10 M lithium acetate (Figure 4) stiff fiber aggregates were observed. The average height
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of the fibrillar polymer bundles measured normal to the mica plane is about 1.4 nm, suggesting that they are composed of an association of several chains. The lateral spacing between tightly packed fibrils is about 19 nm.
Figure 3. Scale bar =200 nm.
Figure 4. Scale bar = 250 nm.
Less lateral ordering was seen with higher sodium acetate concentrations of 1.5 M, as shown in Figure 5. The average height of the rod-like polymer bundles measured normal to the mica plane is greater than 1.7 nm. ACKNOWLEDGEMENTS The authors acknowledge financial support from the National Institutes of Health NIH Grant GM 33062 (DAB). HA samples were kindly supplied by Vitrolife UK Ltd. Scale bar = 250 nm. REFERENCES 1. Dwek, R. A., Glycobiology: Toward Understanding the Function of Sugars. Chemical Reviews, 1996, 96, 683-720. 2. Laine, R. A., The Information Storing Potential of the Sugar Code, in Glycosciences: Status and Perspectives, Gabius, H.-J., and Gabius, S., Eds., Chapman and Hall, London, 1997, pp. 1-14. 3. Varki, A., Biological Roles of OIigosaccharides: All of the Theories are Correct. Glycobiology, 1993,3,97-130. 4. Ryan, C. A., and Farmer, E. E., Oligosaccharide Signals in Plants: A Current Assessment. Annual Review of Plant Physiology and Plant Molecular Biology, 1991,42,651-674. 5. Jackson, R. L., Busch, S. J., and Cardin, A. D., Glycosaminoglycans: Molecular Properties, Protein Interactions, and Role in Physiological Process. Physiological
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Characterisation and solutionproperties ofhyaluronan
Reviews, 1991,71,481-539. 6. Bovin, N. V., Carbohydrate-Carbohydrate Interaction, in Glycosciences: Status and Perspectives, Gabius, H.-J., and Gabius, S., Eds., Chapman and Hall, London, 1997, pp. 277-289. 7. Brant, D. A., Shapes and Motions of Polysaccharide Chains. Pure and Applied Chemistry, 1997,69, 1885-1892. 8. Binnig, G., Quate, C. F., and Gerber, Ch., Atomic Force Microscope. Physical Review Letters, 1986, 56, 930-933. 9. Morris, V. J., Biological Applications of Scanning Probe Microscopies. Progress in Biophysics and Molecular Biology, 1994,61, 131-185. 10. McIntire, T. M., Penner, R. M., and Brant, D. A., Observations of a Circular, Triple-Helical Polysaccharide Using Noncontact Atomic Force Microscopy. Macromolecules, 1995,28,6375-6377. 11. Brant, D. A., and McIntire, T. M., Cyclic Polysaccharides, in Large Ring Molecules, Semlyen, J. A., Ed., Wiley, Chichester, England, 1996, pp. 113-154. 12. McIntire, T. M., and Brant, D. A., Imaging Carbohydrate Polymers With Noncontact Atomic Force Microscopy, in Techniques in Glycobiology, Townsend, R. R., and Hotchkiss, A. T., Eds., Marcel Dekker, New York, NY, 1997, pp. 187208. 13. McIntire, T. M., and Brant, D. A., Imaging of Individual Biopolymers and Supramolecular Assemblies Using Noncontact Atomic Force Microscopy. Biopolymers, 1997,42, 133-146. 14. McIntire, T. M., and Brant, D. A., Observations of the (l~3)-I3-D-Glucan Linear Triple Helix to Macrocycle Interconversion Using Noncontact Atomic Force Microscopy. Journal ofthe American Chemical Society, 1998, 120,6909-6919. 15. McIntire, T. M., and Brant, D. A., Imaging of Carrageenan Macrocycles and Amylose Using Noncontact Atomic Force Microscopy. International Journal of Biological Macromolecules, 1999,26,303-310. 16. Tyler, J. M., and Branton, D., Rotary Shadowing of Extended Molecules Dried from Glycerol. Journal of Ultrastructure Research, 1980, 71, 95-102. 17. Stokke, B. T., Elgseeter, A., and Smidsred, 0., Electron Microscopic Study of Single- and Double-Stranded Xanthan. International Journal of Biological Macromolecules, 1986,8,217-225. 18. Stokke, B. T., and Elgsa:ter, A., Conformation, Order-Disorder Conformational Transitions and Gelation of Non-Crystalline Polysaccharides Studied Using Electron Microscopy. Micron, 1994, 25, 469-491. 19. Braunstein, D., Imaging an F-Actin Structure With Noncontact Scanning Force Microscopy. Journal of Vacuum Science and Technology A, 1995, 13, 1733-1736.
EMAIL ADDRESSES
[email protected],
[email protected] COMPARISON OF THE REACTIVITY OF DIFFERENT REACTIVE OXIDATIVE SPECIES (ROS) TOWARDS HYALURONAN B.J. Parsons", S. AI-Assaf, S. Navaratnam & G.O. Phillips The North East Wales Institute, Free Radical Chemistry & Photochemistry Group, Wrexham, LLI I 2A W. UK. E-mail
[email protected] ABSTRACT
Hyaluronan is a linear biopolymer found in all connective tissues and amongst its many possible roles, it has the important functions of lubrication and shock absorbing. Its biological usefulness has been attributed to its molecular weight, shape and structure. In inflammatory diseases, oxygen-derived free radicals are produced which can participate in the degradation of hyaluronan. Different mechanisms have been proposed for the generation of reactive oxidative species (ROS) such as 'OH and peroxynitrite in a biological environment. Hyauronan has been found to be particularly susceptible to attack by ROS in comparison with other biopolymers. This paper describes the interaction of some ROS, including 'OR, Brz", Ch- and, peroxynitrite with hyaluronan by measuring molecular weight changes using gel permeation chromatography (GPC) coupled to multiangle laser light scattering techniques. Kinetic measurements using stopped-flow techniques have also been used to investigate the depolymerisation reactions of peroxynitrite with hyaluronan. KEYWORDS
Hyaluronan, degradation, ROS, hydroxyl radicals, peroxynitrite INTRODUCTION The role of hydroxyl radicals in the degradation of hyaluronan. One of the main secretory components of synovial fluid is hyaluronan (HA) which is produced by hyalocytes of the synovial membrane and is a major component of joint tissue. The concentration and molecular size of hyaluronan shows great variation between individuals' 4 have also been detected with poly-N-acetylglucosamine, poly-galacturonic acid, and chondroitin sulfate A, and assigned to rearranged radicals. If this interpretation is correct, the observation of identical species with each polymer implies a common fragmentation pathway for all three polysaccharides, The changes in the spectra, particularly in the increased yield of low-molecular-weight material, as the ligand is changed and the pH raised from 4 to 7, suggest that both pH-dependent (base-catalyzed) and pH-independent processes give rise to fragmentation (strand scission) 12. Further information on the radicals generated from these polysaccharides, has been obtained by enzymatic digestion of the spin-adducts 21. Incubation of the adducts detected with hyaluronic acid with either bovine testicular or leech hyaluronidase (which cleave the [~-I,4] bond between glucuronic acid and N-acetylglucosamine, and the [~-1,3] bond between N-acetylglucosamine and glucuronic acid, respectively) resulted in the loss of the features of the polymer-derived radicals and the detection of low-molecular-weight radicals. The species formed as a result of this treatment were similar to those observed with each monomer, confirming that attack occurs on both sugar rings 12. These data are consistent with an important role for acid- and base-catalyzed radical rearrangements in HO' 1M): Micro'Visc Plus (iVisc Plus) Healon GV
Medium Viscosity Dispersive OVDs (lOOK>Vo > 10K):
1.4% NaHa
7.9M
4.8M
1.4% NaHa
5.0M
2.0M
Viscoat
3.0% NaHa 4.0% CDS
500K 25K
50K
Cellugel
2.0% chemically modified HPMC
lOOK
40K
3.0% Ha
500K
25K
Viscous-Cohesive OVDs (lM>Vo > lOOK): Vitrax MieroVisc (iVisc)
1.4% NaHa
6.IM
1.0M
Allervisc Plus (Viscorneal Plus) Provise
1.4% NaHa
5.IM
500K
1.0% NaHa
2.0M
280K
Healon
1.0% Nalla
4.0M
230K
Biolon
1.0% NaJ-Ia
3.0M
215K
Allervisc (Viscomeal) Amvisc
1.0% NaHa
5.IM
200K
1.2% NaHa
I.OM
lOOK
AmviscPlus
1.6% NaHa
1.0M
lOOK
Very Low Viscosity Dispersive OVDs (IOK>V o> lK):
MW(D) - molecular weight (Daltons) Vo(mPs) = zero shear viscosity (milli Pascal-seconds) M = million,' K = thousand
I-Cel
2.0% HPMC
90K
6.0K
Ocuvis
2.0% HPMC
90K
4.3K
Occucoat
2.0% HPMC
86K
4K
Hymccel
2.0%HPMC
86K
4K
Adatocel
2.0% HPMC
86K
4K
Visilon
2.0%HPMC
86K
4K
NaHa = sodium hyaluronate HPMC = hydroxypropylmethylcellulose CDS = chondroitin sulfate
However, lower viscosity dispersive OVDs do not maintain surgical spaces very well, they break up and cause irregular fracture boundaries (which obscure the surgeons view of the operative field), and require much more time, effort and irrigation to remove at the end of the surgical procedure. Many question whether the benefits derived from their routine use outweigh the drawbacks of reduced visibility and possible corneal endothelial damage caused by the increased irrigation required for their removal. The classification system for OVDs, and the subsequent analysis of the best uses and drawbacks of each group, made it clear that no OVD had lived up to its manufacturer's hopes of becoming "the best" for all circumstances occurring in cataract surgery. Surgeons were therefore faced with the dilemma that the OVD they chose for a surgical case may be excellent tor some steps of phaco, but had unavoidable drawbacks for others. We were forced to identify the surgical steps that caused each of us the most difficulty, and choose as our routine OVD, one that offered considerable assistance in the performance of that task, while accepting the fact that it may make other parts ofthe procedure more difficult.
Ophthalmic viscosurgical devices
125
The Viscoelastic Soft-Shell Technique It was into the above scenario that I introduced the concept of the "Dispersive-cohesive viscoelastic soft-shell technique". It is a programmed method of using a dispersive and a cohesive OVD together, to take advanta~e of the optimal properties of each class and minimize their corresponding drawbacks'!' 2. As long as two OVDs do not mix, different physical effects can be achieved in adjacent areas of the AC simultaneously. The Soft-shell technique is applicable to all facets of cataract surgery, but is particularly useful in managing complications, which previously were not well handled by other surgical techniques'0.05, Fig. 3).
12
~
0
•
PBS 1"'/0 Na-HA
'" u
-S!_
"NE
~
~.s ~ "0
4
C W
** 0
5 days
Figure 3.
3 weeks
The size of the endothelial defect area 5 days and 3 weeks after alkali wounding. Data are plotted as the mean and 95% confidence intervals in each group. ** p 1.5 X 106) some difficulties appear during the filtration even through 0.45 um filters. Moreover, it is necessary to work at very low shear rates to reach the Newtonian plateau ( < 100 S·l). The Huggins constant k', as well as the product ofC and intrinsic viscosity (C*['Il]) characterizing the end of the linearity of the reduced viscosity against polymer concentration plots are lower than the value found for HE and HA (Table 1). Table 1. Values of the intrinsic viscosity, Huggins constant k' and the critical overlap parameter C*[ '11] over which the Huggins law no longer applies. Sample Rylan 49 Healon Hyaluronan
(t)) (mUg) 8100 6400
k' 0.2 0.4 0.4
C*(!))
1.2 3.5 3.8
In Table 1 the values of k' and C*[Tl] obtained for HA, HE and HY are compared. There are significant differences between HA, HE and HY. For HY both k' and C*[Tl] values are considerably lower and can be attributed to the presence of large size aggregates and cross-link points in the high molecular weight hylan. These differences cannot be detected from a log-log plot of the relative viscosity versus C['11] due to the lack of sensitivity in this representation. The values ofk' and C*[Tl] have been directly deduced from reduced viscosity versus C['11] curves in a linear representation. Molecular weight determination has been performed using multiangle laser light 6 scattering. For hylan samples with molecular weight higher than about 1.5 x 10 , the weight average molecular weight (Mw) depends on the pore size ofthe filter used to treat the solution. Large aggregate retention on the filters and pressure disaggregation during 6 the filtration process could be factors for the decrease ofMw from 10 x 10 to ca 1.5 x 106 . From ['11] and M w measurements, the Mark-Houwink parameters have been determined for the hylan samples from unfiltered solutions. We have confirmed the K and a values of 0.033 ern' g" and 0.77 which were found previously 29. These values are very close to K and a values of 0.0336 and 0.79 found for linear hyaluronan 13. The small difference in the 'a' parameter may be explained by the difference in the solvent concentration used (0.15 M NaCI for HY and 0.1 M for HA). Filtration, despite reducing Mw, exerted no significant effect on the intrinsic viscosity. This shows the presence of high molecular weight material, such as large compact aggregates, which did not contribute to the intrinsic viscosity but did do so to light scattering. This is not the
Behaviour in aqueous solutions
185
case for samples containing large viscoelastic aggregates and / or high molecular weight molecules such as hylan 49. We have previously reported the presence of such aggregates using atomic force microscopy 27. The comparative behaviour in extensional viscosity has also been compared for both HA and HY 30. When these parameters are applied to the results from the viscometric measurements at low shear rate using the Contraves rheometer, the M w ofHY (hylan 49) corresponds to 8 to 10 X 106 in good agreement with the value estimated from light scattering (Table 2). This also confirms the high molecular weight values found by light scattering for HY. Thus the special cross-linked structural features of HY, while involving networks provide a viscometric behaviour associated with a HA equivalent to Mw ~ lOx 106 in dimensions. Larger or smaller molecular sizes of HY can be produced showing the same overall characteristics and equivalent molecular weights from both methods. In the same way, the intrinsic viscosity obtained for HE, 6400 mUg at 250C in O.IM NaCI corresponds to an equivalent weight average molecular weight of 4.8 ± 0.3 x 106 determined using the Mark-Rouwink equation and a value of 4.7 ± 0.2 x 106 determined by static light scattering. Table 2. Weight average molecular weight (Mw x 106 ) for hylan under different filtration regimes. Samples Rylan (1065) Rylan (49) Rylan (50) Rylan (20) Hylan (50301)
10 10 12.5 1.9 1.78
0.45 1lm 3.1 3.0 3.0 1.6 1.47
0.2 J.1m 2.4
1.34
Shear flow measurements The shear flow behaviour of different hylan samples of different molecular weights have been compared with that of linear hyaluronans and Healon, For example, Figure Ia shows the viscosity of hylan and Figure I b for Healon solutions as a fimftion of the shear rate. It can be clearly seen that even at relatively high viscosities (> 10 mPa.s) the -2 -I Newtonian plateau is reached at lower than lOs . Knowing the respective intrinsic viscosities we are able to compare the behaviour of HA, HE and HY using a master curve representing the relative viscosities as a function of the overlap parameter C[TIl (Figure 2). From Figure 2 it can be seen that HY 49 lies on this master curve and the Newtonian viscosity for HY and its variation with the concentration corresponds to that expected for a linear hyaluronan having an intrinsic viscosity equal to that of hylan. The Newtonian viscosity, 110, equals to about 5 x 105 mPas for HE solution at 10gIL in O.IM NaCI at 25°C (Figure Ib). Then, from the master curve (Figure 2) this value corresponds to C[ TIl of 64 which characterises the HE solution. Consequently, Newtonian viscosities ofHA, HE and HY solutions are directly related to their apparent intrinsic viscosity irrespective of the presence of aggregate structures. 4 1.and the viscometric activation In the hylan semi-dilute regime, Tlsp scales as C . energy, E. ,(TI = A expEJRT), after reaching a maximum, decreases with concentration (C) in a similar manner to that observed for HA. This decrease was interpreted for HA as
186
Rheological behaviour ofhyaluronan
an increase in the stiffness of the chain, corresponding to a chain expansion 31,32. This can explain the larger viscosity exponent 4.1 found in this domain compared with the theoretical predictions, as for example in the reptation model of de Gennes, which scales as C3.75 in a good solvent 33,34. At larger concentrations when E. tends towards a plateau for hyaluronans 31, the viscosity exponent decreases to about 3.6 in good agreement with the de Gennes predictions. a
•
Q
90
~
85
.....
-E=
...
~
~
80
75
70
% bylan B gel slurry Figure 4.
The percent strain recovery of hylan A solution and hylan B gel slurry mixtures. The stress was held constant at 0.5 Pa during the retardation step, then released at the start of the recovery step. The percent strain recovery is calculated at 180 seconds after the release of the strain.
100
Creep experiments Table 1.
199
Viscosity Results of Creep Experiments For Mixtures of Hylan A Solution (1% polymer) and Hylan B Gel Slurry (0.5% polymer).
Average % Increase Mixture Comoosition Zero Shear in Viscosity hylanA hylan B over 1 % Viscosity* solution gel slurry hylan A (Pa.s) (1 % polymer) (0.5% polymer) (%) (volume %) (volume %)
100 80
0 20
1200 + 600 1850±800
50 20
50 80
3200+ 800 7000± 1700 29,OOO± 10,000
-
50
Medical Applications
Synvisc" for viscosupplementation
160 460
Hylagel"" Neuro for viscoseparation
Hylagel(g) Uro Hylaform@ for vtscoausmentatlon *The zero shear viscosity is calculated from the slope of the compliance versus time curve generated during the retardation step.
0
Table 2.
100
2200
Elasticity Results of Creep Experiments For Mixtures of Hylan A Solution (1 % polymer) and Hylan B Gel Slurry (0.5% polymer).
Mixture Comnesltien hylan A hylan B solution gel slurry (1 % polymer) (0.5% polymer) (volume %) (% volume)
Average % Increase in Percent Elasticity over Strain 1% Hylan A (%) Recovery*
Medical Applications
(%)
-
100 80
0 20
83+7 89±.5
8
50 20
50 80
95+2 98±1
15 18
Synvisc@ for viseosunnlementation Hylagel(g) Neuro for
viscesenaration
Hylagef8'Uro Hylaform@ for viscoauzmentatien *The higher the percent strain recovery, the more elastic solid-like IS the material. An ideal elastic material will have a 100% strain recovery. An ideal viscous liquid will have 0% strain recovery.
0
100
99±0.3
21
200
Rheological behaviour of hyaluronan
1. 50% hylan A + 50% hylan B 2. 20% hylan A + 80% hylan B 3.80% hylan A + 20% hylan B 1.5000
4. hylan A solution (1 % polymer) 5. hylan B gel slurry (0.5% polymer)
1.2500
";=
-.
1.0000
r:J:J 0.75000
~
0.50000
Figure 5.
Time (s) The characteristic nngmg effect observed when 0.5 Pa stress is initially applied during the retardation step.
CONCLUSIONS These creep experiments using very low shearing stress demonstrated the rheological efficacy of mixing hylan B gel slurry and hylan A solution to obtain a material which is both more elastic and viscous than hylan A alone, even at a 20% hylan B + 80% hylan A ratio. Much higher zero shear viscosity values were extrapolated using this method for these high MW materials than previously calculated using controlled strain rheometers. The addition of hylan B gel slurry to hylan A solution in such therapeutic devices as Synvisc" (hylan G-F 20) and HylagelfNuro (hylan G-P 80) substantially increases the viscous and elastic properties of these products.
REFERENCES I. E.A. Balazs, & E. A. Leshchiner, Hyaluronan, its crosslinked derivative -hylan- and their medical applications, In: Cellulosics Utilization: Research and Rewards in Cellulosics (Proceedings of Nisshinbo International Conference on Cellulosics Utilization in the Near Future), H. Inagaki & G.O. Phillips (eds.), Elsevier Applied Science, New York, 1989, pp 233-241. 2. Advantage Software Help Files, TA Instruments, Inc., New Castle, DE, PIN 92571O.0TOI Version 1.1.
FUNCTIONS OF HYALURONAN IN WOUND REPAIR W. Y. John Chen ConvaTec Wound Healing Research Institute, First Avenue, Deeside Industrial Park, Flintshire, United Kingdom, CH52NU
ABSTRACT Hyaluronan (HA), a major extracellular matrix macromolecule, has a repeated disaccharide structure that is completely conserved throughout a large span of evolution, indicating a fundamental biological importance. It has unique hygroscopic, rheological and viscoelastic properties, binds to many other extracellular matrix molecules, to body cells through cell surface receptors, and has a unique mode of synthesis in which the molecule is extruded immediately into the extracellular space upon formation. HA has many roles in biology, including skin wound healing. HA and its various chemical derivatives have already been used in wound healing or related tissue repair applications. This article aims to review how HA may function in wound healing through the utilisation of its general physicochemical and biological properties. The challenges of elucidating how the many functions of HA interact in tissue repair, and the possible functions of externally applied HA in modulating the wound healing response, are also discussed.
INTRODUCTION HA is a major component of the extracellular matrix. Most cells in the body have the capability to synthesise HA during some points of their cell cycles, implicating its function in some fundamental biological processes. It is generally accepted that HA is associated with the tissue repair process, as first elucidated by studies in morphogenesis and oncology [1-3]. Although HA may participate in tissue repair processes, on the whole, the detailed mechanisms of how it functions are not entirely clear and are only beginning to be elucidated. Some of these functions may be attributed to its role as an integral part of the extracellular matrix. Because of its unique hygroscopic, rheological and viscoelastic properties, HA may also affect cellular behaviour by affecting the extracellular macro- and microenvironment through its complex interactions with cells and other connective tissue components. HA and its oligosaccharides may also directly affect cell function through receptor binding events that directly lead to alteration of specific gene expression. Because of its unique physicochemical properties, and in particular its nonimmunogenicity, HA has found medical applications for many years, primarily in ocular and joint surgery [4-6]. More recently, the reported benefits of exogenously applied HA in tissue repair have resulted in HA-based biomaterials being developed for wound healing purposes.
148
The function and use of hyaluronan in wound healing
EXPERIMENTAL STUDIES OF HYALURONAN IN WOUND HEALING The function of HA in tissue repair is complex. It is recognised that HA has a dynamic role in connective tissue activation and inflammation [7]. The role of HA in tissue homeostasis can also facilitate the many biological functions that contribute to tissue repair. For a comprehensive review of the functions of HA in wound healing, please refer to the recent review of Chen & Abatangelo [8]. Many reports have attested to the effects of exogenous HA in influencing a beneficial wound healing outcome. In animal experiments, topically applied HA has been shown to accelerate skin wound healing in rats [9,10] and hamsters [11]. Similar results have been observed in the healing of perforated tympanic membranes in rats [12]. Corneal epithelial wound healing is also reported to be stimulated by applied HA [13]. In a study using the porcine dermal wound model, Navsaria et al. [14] showed that a benzyl alcohol ester of HA promotes healing and resulted in a better-organised wound bed, in comparison to the placebo group. HA has also been reported to affect beneficially the quality of tissue repair. The lack of fibrous scarring in foetal wound healing has been attributed, at least in part, to HA, the levels of which remain high for longer periods than in adult wounds. This leads to the suggestion that HA may, at least in part, reduce collagen deposition and therefore results in reduced scarring [4,15,16]. HA may also have a protective effect on chronic wounds, many of which have been shown to be highly inflammatory [17-21]. Foschi et al. [10] showed that HA prevents free radical damage to granulation tissue in rats. Ialenti & Di Rosa [22] also demonstrated the inflammation-moderating effect ofHA.
CLINICAL STUDIES OF HYALURONAN IN WOUND HEALING Exogenous HA has been used successfully for many years in ophthalmologic applications, joint conditions and post-surgical wounds [4-6]. An early paper by Vilesov et al. [23] has described the use of HA in burn wound bed preparation. Trabucchi et at. [24] carried out a clinical study, using topical treatment with HA, through the drains of the laparotomy suture. HA treatment reduced the incidence and degree of dehiscence macroscopically, increased the maturation of granulation tissue during the first post-operation days, and stimulated fibroblasts to synthesise procollagen shortly after the operation. Ortonne [25] reported a multicentre controlled clinical study on 50 patients with venous leg ulceration. The efficacy and safety of HA in comparison to Dextranomer, the product of choice for this indication in France, was evaluated. Both groups recorded significant wound improvements, but there was a faster and greater reduction in ulcer dimensions following treatment with HA, as well as significant reduction of oedema compared to the control group. Edmonds and Foster [26] reported encouraging results using a benzyl ester of HA for treatment of diabetic foot ulcers when used as an adjunct to the standard treatment that consists of sharp debridement, pressure relief and infection control. This was in comparison to the control group of patients who received the standard treatment only. Overall, the amount of experimental and clinical data available on the beneficial effects of HA is limited. However, the available data, although anecdotal, has already stimulated the exploration of HA and HA-derived materials as wound healing products. Some of these are already available for clinical practice.
functions of hyaluronan in wound repair
149
Table 1: A summary of tissue repair events involving hyaluronan. Stase Process Inflammatory phase lnllammation Activation
Granulation phase
Reepithelisation
Remodelling
Mechanism • Enhancement of cell infiltration • Increase of proinflammatory cytokines TNF-<X. IL- II} and IL-8 via a CD44-mediated mechanism • Facilitates primary adhesion of cytokine-activated lymphocytes to endothelium Inflammation • Free radical scavenging and antioxidant properties Moderation • TSG-6 and lui mediated inhibition of inflammatory proteinases Cell prnlilcralion • Hyaillfonan synthesis facilitates cell detachment and mitosis Cell migration • Increased hyaluronan synthesis • Hyaluronan-rich granulation tissue provides open. hydrated matrix that facilitates cell migration • Receptor mediated cell migration, e.g. CD44, RHAMM Angiogenesis • Angiogenic properties of low molecular weight byaluronan 01igosuccharidcs Kcratinocyte • Hyaluronan-rlch matrix is associated with proliferating functions basal keratinocytes • Facilitates keratinocyte migration via a CD44-mediated mechanism Scarring • Hyaluronan-rich matrix may reduce collagen deposition leading to reduced scarring as seen in foetal wound healing
Reference 29 30 31 47-49 50 44-46 35.39·41 2 32,33 54-61 28
64 4.15.16
ROLE OF HYALURONAN IN WOUND HEALING PROCESSES Following injury, wound healing follows a series of tightly regulated sequential events. These are inflammation, granulation, reepithelialisation and remodelling. HA is likely to have a multi-faceted role in mediation of these cellular and matrix events. This is summarised in Table I. The putative roles of HA in this sequence of wound healing events are described in more detail in the following sections. Inflammation Inflammation is the important first step that occurs shortly after injury. Inflammation cleanses the wound of damaged tissue, combats infection and recruits the necessary cell populations to rebuild the tissue. Wound tissue in the early inflammatory phase of wound repair is rich in HA, probably reflecting increased synthesis [27,28]. HA can act as a promoter of early inflammation. HA has been shown to enhance cellular infiltration into sites of inflammation [29]. Kobayashi & Terao [30] have shown a HA-dose-dependent increase of the proinflammatory cytokines TNF-a, IL-I~ and lL-8 production by human uterine fibroblasts, via a CD44 mediated mechanism. Microvascular endothelial cells, in response to inflammatory cytokines such as TNF-a and IL-l~, and bacterial lipopolysaccharide, also synthesise HA and this has been shown to facilitate primary adhesion of cytokine-activated lymphocytes expressing the HAbinding variants of CD44 [31] under laminar and static flow conditions. In a somewhat contradictory role, HA can also be a moderator of inflammation. This may contribute to the stabilisation of granulation tissue matrix. Granulation and organisation of the granulation tissue matrix Granulation tissue matrix is rich in HA [27,28] and may contribute in a variety of ways to essential tissue repair functions. HA and cell migration • Cell migration into the wound site is essential for the
150
The function and use ofhyaluronan in wound healing
formation of granulation tissue. An HA-rich extracellular matrix, characteristic of early granulation tissue, is regarded as a conducive environment for cell migration into this provisional wound matrix because of an open, hydrated matrix [2]. At the same time, via cell surface HA receptors, directed migration and control of the cell locomotory mechanisms can be mediated. The principal HA receptors include CD44, ICAM-I and RHAMM. RHAMM, in particular, forms links with several protein kinases associated with cell locomotion [32,33]. During foetal development, the migration path through which neural crest cells migrate is rich in HA [1]. Increased cell movement in response to HA can also be demonstrated experimentally in other cell types [32,33], whereas cell movement can be inhibited, at least partially, by HA degradation or blocking HA receptor occupancy [34-38]. HA synthesis has also been shown to correlate with cell migration [35,39-41]. HA synthesis may itself provide the dynamic force to facilitate cell migration, as it is synthesised at the plasma membrane and released directly into the extracellular environment [42,43]. This may provide the hydrated microenvironment, at sites of synthesis, to facilitate cell detachment essential for cell migration. Cell proliferation - cell proliferation is also an essential part of tissue repair. It has been shown that increased HA occurs, and is essential for fibroblast detachment from the matrix and mitosis [44]. HA has been shown to facilitate cell detachment [45,46], but has not displayed any direct mitogenic activity. However, through facilitating cell mitosis in response to mitogenic factors which are abundant during the early phases of tissue repair, HA may also have an important, albeit indirect, role in cell proliferation. HA and inflammation - Although inflammation is an integral part of granulation tissue formation, for normal tissue repair to proceed, inflammation needs to be moderated. The initial granulation tissue formed is highly inflammatory, with a high rate of tissue turnover mediated by matrix degrading enzymes and reactive oxygen metabolites that are products of inflammatory cells. Inflammation needs to be moderated in order to allow stabilisation of the granulation tissue matrix. HA protects against free-radical damage to cells [47-49]. This is probably mediated through a free-radical scavenging property. In a rat model of free-radical-induced inflammation, HA has been shown to reduce damage to the granulation tissue [10]. In addition to the free-radical scavenging role, HA may also function in the negative feedback loop of inflammatory activation. For example, HA binding to tumour necrosis factor stimulated gene-6 protein (TSG-6) and inter-n-inhibitor (IaI) has been reported to form a potent proteinase inhibitor complex that may attenuate the high levels of proteinase activity associated with inflammation [50J. As shown in a murine air pouch model of inflammation, where HA has been shown to have a proinflammatory property, administration of TSG-6 results in reduction of inflammation comparable with systemic dexamethasone treatment [51]. Angiogenesis - HA may also have a role in the control of angiogenesis. High molecular weight HA has been shown to inhibit angiogenesis [52,53], but low molecular weight HA oligosaccharides have been shown to promote angiogenesis in several experimental models [54-56], and to enhance the production of collagens by endothelial cells [57]. This phenomenon may be related to HA oligosaccharides inducing expression of several inflammatory genes including TNF-a. and IL-113 [58-61J. Observations of angiogenesis coinciding with increase of hyaluronidase and degradation of matrix HA have been made in several in vivo systems [53,62]. Hyaluronidase digestion of foetal wound HA leading to fibroplasia and capillary formation [63] are in general agreement with the hypothesis of a physiological role HA and its oligosaccharides have in the control of angiogenesis.
Functions of hyaluronan in wound repair
151
Reepithelialisation HA has important functions as an integral patt of the extracellular matrix of basal keratinocytes. Its free-radical scavenging function suggests an important role in normal epidermal biology, whereas its role in keratinocyte proliferation and migration strongly implies an important role in the reepithelialisation process. In wound healing, epidermal HA is particularly implicated in the control of keratinocyte proliferation. In healing wounds, HA is expressed in the wound margin, in the connective tissue matrix, and co-locates with CD44 expression in migrating keratinocytes [28]. Kaya et at. [64] showed that suppression of CD44 expression by an epidermis-specific antisense transgene resulted in animals with defective hyaluronate accumulation in the superficial dermis, accompanied by distinct morphologic alterations in basal keratinocytes and defective keratinocyte proliferation in response to mitogen and growth factors. Also observed are decrease in skin elasticity, and impaired local inflammatory response and tissue repair. These observations are strongly supportive of the important roles HA and CD44 have in skin physiology and tissue repair.
Foetal wound healing and scarring Foetal wound healing is characterised by lack of fibrous scarring. HA content in foetal wounds remains high for longer periods than in adult wounds, leading to the suggestion that HA may, at least in part, reduce collagen deposition and therefore reduce scarring [15,16]. These suggestions are in agreement with the data which showed that applied HA resulted in reduced scarring of healed tympanic membranes that had been perforated [12], and with Balasz & Denlinger [4] who hypothesised that aHA-rich environment inhibits the matrix cells responsible for fibrous scars. In a recent paper, West et at. [16] showed that in adult and late gestation foetal wound healing, removal of HA results in fibrotic scarring. As HA is a multi-functional molecule, it has many other functions that may well also contribute to the scarless healing quality of foetal wounds. A persistent HA-rich environment may affect cell-cell and cell-matrix interactions differently from that of an environment in which elevated HA is transient, such as that of the post-natal wound environment. This may lead to different activation and control of various cell populations in comparison to the post-natal wound environment.
CHRONIC WOUND PATHOLOGY AND HEALING CHALLENGES Much of the information on the role of HA in wound repair has been elucidated from the healing of acute wounds. With recent advances in the study of chronic wounds, such as venous leg ulcers, diabetic foot ulcers and pressure ulcers, it is becoming clear that the chronic wound pathology is quite different from that of acute wounds [17-21]. Circulatory abnormalities, leading to chronic inflammation, arc the main features of these wounds (Figure I). The main causes for generation of these wounds and the impairment of their healing are likely to be the excessive tissue breakdown processes that are characteristic of severe inflammation. These are primarily reactive oxygen metabolites and matrix degrading enzymes, which, working in concert, can lead to very rapid tissue turnover, further activation of matrix proteinases, impairment of proteinase inhibitor functions and continuous promotion of inflammation. Whether HA has any function in chronic wound pathology is at present not known.
152
The function and usc ofhyaluronan in wound healing
Ischaemia-reperfusion _ . . . . cycles
Tissue Ischaemia
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,
~
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Figure 1: Schematic diagram showing the pathological events that are likely to occur during pathogenesis of the common chronic wounds - venous ulceration, pressure ulceration and diabetic foot ulceration. Recent clinical studies have reported some benefits of externally applied HA or HAbased materials on the healing of chronic wounds [25,26]. Even though these results are from small studies and should be considered to be anecdotal, they nevertheless raised the interest as to whether, and to what extent, HA may playa role in the modulation of the pathology of chronic wounds. In particular, it is interesting to speculate whether the inflammation modulation role, and angiogenic property of HA oligosaccharides, may function in promoting chronic wounds to heal. CONCLUSIONS It is clear that HA has many functions in biology. Some of its functions may be attributable to its biophysical properties and some to its biological properties as an extracellular matrix molecule as well as a biological signalling molecule. Much work still needs to be done in order to elucidate the biological mechanisms of HA in tissue processes. Although many of the HA functions are known individually, how these function interact with each other, and how they are related to other tissue factors to mediate the complex biological processes that are necessary for wound repair, remains poorly understood. Elucidating these complex mechanisms will undoubtedly be as a scientific challenge for the future. Because of its unique physicochemical properties, HA has already seen biomedical applications. In tissue repair, its physicochemical properties, the promising results shown in in vivo experimental studies and early clinical studies, and its known biological properties, strongly indicate applications in mediation of the wound healing process as well as a biomaterial for bioengineering purposes. Already in this field, products have been developed for anti-adhesion, wound healing, tissue implants and for
Functions ofhyaluronan in wound repair
153
moisturising purposes [4,5]. These products are either based on pure HA, or derivatives using various chemical modification techniques to improve their physical handling and stability characteristics. Currently, HA-based medical products are mostly classified as medical devices, utilising the physical attributes of HA to achieve their intended functions. No doubt when the biology of HA becomes better known, applications will also be developed to utilise its biological functions.
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C. L. Hall, L. A. Lange, D. A. Prober, S. Zhang & E. A. Turley, pp60 c, src is required for cell locomotion regulated by the hyaluronan receptor RHAMM. Oncogene, 1996, 13,2213-24. G. M. Morriss-Kay, F. Tuckett & F. Solursh, The effect of Streptomyces hyaluronidase on tissue organization and cell cycle times in rat embryos. J. Embryol. Exp. Morph. 1986,98,59-70. S. L. Schor, A. M. Schor, A. M. Grey, W. Y. J. Chen, G. Rushton, M. E. Grant & I. Ellis, Mechanism of action of the migration stimulating factor produced by fetal and cancer patient fibroblasts: effect on hyaluronic acid synthesis. In Vitro Cell. Dev. BioI. 1989,25, 737-746. S. D. Banerjee & B. P. Toole, Hyaluronan-binding protein in endothelial cell morphogenesis. 1. Cell Bioi. 1992, 119,643-652. S. K. Samuel, R. A. Hurta, M. A. Spearman, J. A. Wright, E. A. Turley & A. H. Greenberg, TGF-~ 1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan. J. Cell BioI. 1993, 123, 749-58. E. A. Turley, L. Austen, D. Moore & K. Hoare, ras-transformed cells express both CD44 and RHAMM hyaluronan receptors: only RHAMM is essential for hyaluronan promoted locomotion. Exp. Cell Res. 1993,207,277-82. W. Y. J. Chen, M. E. Grant, A. M. Schor & S. L. Schor, Differences between adult and foetal fibroblasts in the regulation of hyaluronate synthesis: correlation with migratory activity. J. Cell Sci. 1989,94, 577-584. I. Ellis, J. Banyard & S. L. Schor, Differential response of fetal and adult fibroblasts to cytokines: cell migration and hyaluronan synthesis. Dev. 1997, 124, 1593-1600. I. R. Ellis & S. L. Schor, Differential effects of TGF-~l on hyaluronan synthesis by fetal and adult skin fibroblasts: implications for cell migration and wound healing. Exp. Cell Res. 1996,228, 326-33. P. Prehm, Synthesis of hyaluronate in differentiated teratocarcinoma cells: mechanism of chain growth. Biochem. J. 1983, 211, 191-198. N. Mian, Characterization of a high-Mr plasma-membrane-bound protein and assessment of its role as a constituent of hyaluronate synthase complex. Biochem. J. 1986,237,343-357. M. Brecht, U. Mayer, E. Schlosser & P. Prehrn, Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem. J. 1986,239,445-450. G. Abatangelo, R. Cortivo, M. Martelli & P. Vecchia, Cell detachment mediated by hyaluronic acid. Exp. Cell Res. 1982, 137,73-78. B. J. Barnhart, S. H. Cox & P. M. Kraemer, Detachment variants of Chinese hamster cells. Hyaluronic acid as a modulator of cell detachment. Exp. Cell Res. 1979, 119, 327-332. D. Presti & J. E. Scott, Hyaluronan-mediated protective effect against cell damage caused by enzymatically produced hydroxyl (OH·) radicals is dependent on hyaluronan molecular mass. Cell Biochem. Funct. 1994, 12, 281-288. B. J. Kvam, E. Fragonas, A. Degrassi, C. Kvam, M. Matulova, P. Pollesello, F. Zanetti & F. Vittur, Oxygen-derived free radical (ODFR) action on hyaluronan (HA), on two HA ester derivatives, and on the metabolism of articular chrondrocytes. Exp. Cell Res. 1995,218,79-86. K. Fukuda, S. Tanaka, F. Kumano, S. Asada, M. Oh, M. Ucno & M. Takayama, Hyaluronic acid inhibits interleukin-l-induced superoxide anion in bovine chondrocytes.lnflamm. Res. 1997,46,114-117.
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The function and use of hyaluronan in wound healing H. G. Wisniewski & J. Vilcek, TSG-6: an IL-l/TNF-inducible protein with antiinflammatory activity. Cytokine Growth Factor Rev. 1997,8, 143-56. H. G. Wisniewski, J. C. Hua, D. M. Poppers, D. Nairne, J. Vilcek & B. N. Cronstein, TNF/IL-I-inducible protein TSG-6 potentiates plasmin inhibition by inter-alpha-inhibitor and exerts a strong anti-inflammatory effect in vivo. J. Immunol. 1996, 156, 1609-1615. H. F. Dvorak, V. S. Harvey, P. Estrella, L. F. Brown, J. McDonagh & A. M. Dvorak, Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing. Lab. Invest. 1987,57,673-86. D. C. West & S. Kumar, The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity. Exp. Cell Res. 1989, 183, 179-196. V. C. Lees, T. P. Fan & D. C. West, Angiogenesis in a delayed revascularization model is accelerated by angiogenic oligosaccharides of hyaluronan. Lab. Invest. 1995, 73, 259-66. Sattar, P. Rooney, S. Kumar, D. Pye, D. C. West, 1. Scott & P. Ledger, Application of angiogenic oligosaccharides of hyaluronan increases blood vessel numbers in rat skin. J. Invest. Derm. 1994, 103, 576-579. D. C. West & D. M. Shaw, Tumour hyaluronan in relation to angiogenesis and metastasis. In: The Chemistry, Biology and Medical Applications (Jj' Hyaluronan and its Derivatives. T. C. Laurent (ed.) London: Portland Press, 1998, pp 227-33. P. Rooney, M. Wang, P. Kumar & S. Kumar, Angiogenic oligosaccharides of hyaluronan enhance the production of collagens by endothelial cells. J. Cell Sci.
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P. W. Noble, F. R. Lake, P. M. Henson & D. W. H. Riches, Hyaluronate activation of CD44 induces insulin-like growth factor-I expression by a tumor necrosis factor-a-dependent mechanism in murine macrophages. .1. Clin. Invest. 1993,91, 2368-2377. C. M. McKee, M. B. Penno, M. Cowman, M. D. Burdick, R. M. Strieter, C. Bao & P. W. Noble, Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44. J. Clin. Invest. 1996, 15, 2403-13. P. W. Noble, C. M. McKee, M. Cowman & H. S. Shin, Hyaluronan fragments activate an NFKB/IKBa autoregulatory loop in murine maerophages. J. Exp. Med. 1996, 183,2373-8. P. W. Noble, C. M. McKee & M. R. Horton, Induction of inflammatory gene expression by low-molecular-weight hyaluronan fragments in macrophages. In: The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives, T. C. Laurent (ed.), Wenner-Gren International Series 72, Portland Press, London, 1998, pp 219-225. D. C. Liu, E. Pearlman, E. Diaconu, K. Guo, H. Mori, T. Haqqi, S. Markowitz, J. Willson & M. S. Sy, Expression of hyaluronidase by tumor cells induces angiogenesis in vivo. Proc. Natl. Acad. Sci. USA, 1996,93,7832-7837. B. A. Mast, R. F. Diegelmann, T. M. Krummel & 1. K. Cohen, Hyaluronic acid modulates proliferation, collagen and protein synthesis of cultured fetal fibroblasts. Matrix, 1993, 13,441-446. G. Kaya, 1. Stamenkovie, P. Vassalli, 1. L. Jorcano & 1. Rodriguez, Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 trans gene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation. Genes Dev. 1997, 15,996-1007.
THE ROLE OF PERICELLULAR MATRIX FORMATION DURING WOUND HEALING IN RENAL STONE DISEASE Marieke SJ Schepers", Burt G vd Boom & Carl F Verkoelen Department of Urology, Erasmus University Rotterdam. iN! Be330, P. a.Box 1738, 3000 DR Rotterdam, The Netherlands.
ABSTRACT The adherence of crystals to the surface of renal tubular cells is considered one of the earliest events in kidney stone formation. Since crystal-cell interaction is difficult to study in vivo, we developed a model to study crystal retention using Madin Darby Canine Kidney cells (MDCK). Cultured on permeable supports MDCK cells form functional epithelial monolayers with a high transepithelial electrical resistance (TER>5000 Q"'cm2) that are non-adherent to calcium oxalate (CaOx) crystals. The epithelium becomes susceptible to crystal adherence, however, during its recovery from mechanical created wounds. During repair the cells produce a cell coat enriched with hyaluronan. This polysaccharide in the pericellular matrix of mobile cells has been identified as a binding molecule for CaOx crystals. The association of the hyaluronan enriched cell coat is mediated by specific receptors. The main cell surface receptor for hyaluronan is CD44. It is possible that CD44 also serves as hyaluronan receptor at the surface of MDCK cells. Studies with anti CD44 antibodies demonstrated the co-localisation of CD44 and hyaluronan at the surface of mobile cells. Pericellular matrices are structurally stabilised by hyaluronan binding proteins (HASP), like members of the inter-u-trypsin inhibitor (IT!) family of proteins. Studies with specific antibodies showed that ITI-related proteins are indeed present in the hyaluronan-rich cell coat surrounding proliferating MDCK cells. The possibility that the association of crystals with pericellular matrix constituents along the urinary tract somehow is involved in the pathophysiology of renal stone disease is supported by the fact that the organic kidney stone matrix is enriched with hyaluronan and IT! proteins and by the finding that urine of stone forming men is enriched with IT! proteins. Collectively these observations suggest that tissue repair plays an important role in the aetiology of nephrolithiasis.
KEYWORDS Nephrolithiasis, MDCK strain I, crystal-cell interaction, pericellular matrix, inter-a -trypsin-inhibitor, hyaluronan, tissue repair.
INTRODUCTION The accumulation of crystalline material in the kidney sooner or later leads to stone formation. Calcium crystals that are occasionally present in anyone's urine normally are eliminated unhindered with the urine. Binding to renal tubular epithelium may turn harmless urine crystals into a stone nidus l •3 , Considering the potential hazard of crystal attachment it is reasonable to assume that physiological mechanisms exist to prevent
158
The function and use of hyaluronan in wound healing
it. Evidence has been provided that, at sites in the urinary tract where crystals can be formed, the tissue indeed is resistant to crystal adherence', Tissue injury triggers an inflammatory response followed by re-epithelization and remodelling. Cell culture studies showed that calcium oxalate crystals adhered to the hyaluronan-rich pericellular matrix expressed by proliferating and migrating cells in the wound. The present study was undertaken to study the role of tissue repair in crystal retention more in detail.
MATERIALS & METHODS Cell culture, Preparation of CaOx crystals, Crystal binding, Wounds made in confluent monolayers These studies were performed with MDCK strain I cells. All these methods are described in detail elsewhere'
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Crystal retention. Crystals adhering to hyaluronan-rich pericellular matrix formed by mobile MDCK cells.
Possible mechanisms of stone formation in the kidney Although many risk factors have been identified, it is still difficult to outline the exact sequence of events leading to stone formation in the kidney. The finding that crystals adhere to the HA-enriched PCM surrounding wounds suggests that tissue damage in the kidney precede crystal retention and stone formation. It is conceivable that relatively small areas in the renal tubules are frequently subjected to injury-repair. Crystals passing these areas are continuously retained and released again into the tubular fluid after the wounds are healed. During this process the crystals are provided with a coat of sticky PCM which further retards their transit through the nephrons. Stones are cumulating crystalline/PCM material in corners that are not directly subjected to the force of the rapidly flowing fluid. Because of the limited and local nature of the epithelial damage this process may often proceed unnoticed. On the other hand, it is remarkable that the highest levels of nephrolithiasis-related proteins are found in urine of patients that actually actively are forming stones, suggesting that the inflammatory response is provoked by the stones themselves. In this scheme it is conceivable that newly formed crystals in the tubular fluid adhere to areas of injuryinduced reorganizing epithelia surrounding an existing stone leading to further stone
162
The function and use ofhyaluronan in wound healing
enlargement. In the latter situation, the stone-induced renal lesions may accelerate the stone forming process. Finally, it is possible that epithelial injury-repair processes in the kidney not only initiate but also aggravate stone formation.
REFERENCES 1. 2. 3. 4.
5.
6. 7.
8. 9. 10. 11.
12.
13.
Kok DJ, Khan SR. 'Calcium oxalate nephrolithiasis, a free or fixed particle disease'. Kidney Int 1994;46:847-854. Mandel N. 'Mechanism of stone formation'. Semin NephrolI996;16:364-374. Lieske JC, Toback FG. 'Interaction of urinary crystals with renal epithelial cells in the pathogenesis of nephrolithiasis'. Semin Nephrol1996; 16:458-473. Verkoelen C, Boom B vd, Romijn 1. 'Identification of hyaluronan as binding molecule for calcium oxalate crystals at the surface of mobile renal tubular cells in culture'. Kidney Int 2000;58:1045-1054. Verkoelen CF, van der Boom BG, Houtsmuller AB, Schroder FH, Romijn JC. 'Increased calcium oxalate monohydrate crystal binding to injured renal tubular epithelial cells in culture'. Am J PhysiolI998;274:F958-965. Verkoelen CF, Van Der Boom BG, Kok OJ, Schroder FH, Romijn JC. 'Attachment sites for particles in the urinary tract'. J Am Soc Nephrol1999; 10 Suppl 14:S430-435. Verkoelen CF, van der Boom BG, Kok DJ, Houtsmuller AB, Visser P, Schroder FH, Romijn JC. 'Cell type-specific acquired protection from crystal adherence by renal tubule cells in culture'. Kidney Int 1999;55:1426-1433. Verkoelen CF, van der Boom BG, Kok OJ, Romijn JC. 'Sialic acid and crystal binding' . Kidney Int 2000;57:1072-1082. Knudson CB, Toole BP. 'Changes in the pericellular matrix during differentiation of limb bud mesoderm'. Dev BiolI985;112:308-318. Atmani P, Khan SR. 'Role of urinary bikunin in the inhibition of calcium oxalate crystallization'. JAm Soc Nephroll999;10 Suppl 14:S385-388. Medetognon-Benissan J, Tardivel S, Hennequin C, Daudon M, Drueke T, Lacour B. 'Inhibitory effect of bikunin on calcium oxalate crystallization in vitro and urinary bikunin decrease in renal stone formers'. Urol Res 1999;27:69-75. Marengo SR, Resnick MI, Yang L, Chung JY. 'Differential expression of urinary inter-alpha-trypsin inhibitor trimers and dimers in normal compared to active calcium oxalate stone forming men'. J UrolI998;159: 1444-1450. Atmani P, Glenton PA, Khan SR. 'Role of inter-alpha-inhibitor and its related proteins in experimentally induced calcium oxalate urolithiasis. Localization of proteins and expression of bikunin gene in the rat kidney' Urol Res 1999;27:63-67.
RHEOLOGY OFHYALURONAN PRODUCTS Ove Wik, Bengt Agerup and Hege Bothner Wik Q-Med AB. Seminariegatan 21. S-752 28 Uppsala, Sweden
ABSTRACT
Various modified (stabilized or cross-linked) hyaluronan products used for tissue augmentation were examined by rheometry. Five products - Restylane Fine Lines, Restylane, Perlane, Hylaform and Dermalive - exhibited typical gel-like behaviour to varying degree after examination of the viscoelastic response as a function of frequency. This suggests that all products contain hyaluronan with permanent linkages between polysaccharide chains. One product (Rofilan) claimed to be a 'Hylangel' containing 'cross-linked' hyaluronan at a concentration of 20 mg/ml exhibited a behaviour typical of hyaluronan solutions. The results demonstrate that this product contains free hyaluronan chains with a molecular weight of 2 million. KEYWORDS
Rheology, viscoelasticity, cross-link, tissue augmentation. INTRODUCTION
Hyaluronan is intimately linked with rheology. The remarkable viscous and elastic behaviour of hyaluronan solutions in general and of body fluids such as synovial fluid in particular has been studied for decades. After the pioneering work by Balazs 1-2, modem rheometers were utilised for the subsequent development of hyaluronan products for use in e.g. ophthalmology and joint disorders. Fittingly, the multifaceted use ofhyaluronan in medicine has been described by Balazs as "viscosurgery" 3. In recent years hyaluronan has been modified by means of various types and varying degree of cross-linking 4-6. As a consequence of these modifications, products with quite different rheological behaviour have been marketed. We have performed basic rheological studies on some commercial products containing modified, gel-like hyaluronan derivatives, and report data on the viscous and elastic properties as a function of frequency. MATERIALS AND METHODS Samples
The following samples of modified hyaluronan products used in tissue augmentation were used. Lot numbers are given in parenthesis. Restylane Fine Lines (6083), Restylane (5922) and Perlane (6106) were obtained from Q-Med AB, Uppsala, Sweden. Hylaform (A709) was obtained from Biomatrix Inc., Ridgefield NJ, USA. Dermalive (VR26060) was obtained from Dermatech, Paris, France. Rofilan (4991) was obtained from Rofil Medical Nederland B.V., Breda, Netherlands. The products manufactured by Q-Med AB (Restylane Fine Lines, Restylane and Perlane) contain non-animal stabilised hyaluronan (NASHA) at a concentration of 20
202
Rheological behaviourofhyaluronan
mg/m!. These products are specifically designed for tissue augmentation in different layers of the skin. Information on the other products was obtained from the packaging inserts. Hylaform contains Rylan B at a concentration of5.5 mg/m!. Dermalive is a suspension of non resorbable fragments of acrylic hydrogel and a solution of slightly cross-linked hyaluronan. The product contain 200 mg/ml of acrylic fragments and 14.4 mg/ml ofhyaluronan. Rofilan is stated to be a 'Hylangel' containing 'cross-linked' hyaluronan at a concentration of 20 mg/m!. Rheological characterisation Bohlin VOR Rheometer System (Bohlin Reologi AB, Sjobo, Sweden) with the Windows compatible Millenium software was used for rheological characterisation. The samples were more or less gel-like and therefore all samples were studied in the oscillation mode by recording the response to varying frequency and strain. All experiments were performed at 25°C. When sufficient amount of sample (about 3 m!) was available the cup and bob measurement system C14 was used. Otherwise the cone and plate system CP5/30 (diameter 30 mm, cone angle 5°) was used. Precautions were taken to exclude possible effects of the formation of a dry hyaluronan film on surface layers. The strain-dependent response was recorded to ascertain determination of the viscoelastic response within the linear region. RESULTS AND DISCUSSION The viscoelastic response is shown in Fig. 1 where the elastic modulus (0') and viscous modulus (0") are plotted as function of frequency. All data were recorded at low enough strain to ascertain recordings in the linear region. Elastic modulus, G' (Pa) Viscous modulus, G" (Pa) ••••••.
Elastic modulus, G' (Pa) - - Viscous modulus, G" (Pa) ••• _••• 1000,......---------------,
1000.,......---------------,
-4I~~~~
100L_ _- - , - - -........
100
• :0:: : ~: ~ : ~ : : e:=0 =:0:: : .-"
.
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•• 10
10
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• Reslyl.ne Fine Lines • Reslyl.ne o Perlane
.001
.01
.1
Frequency (Hz)
Figure 1.
1
10
o
.001
.01
.1
1
Frequency (Hz)
The elastic and viscous moduli as a function of frequency plotted on log-log scale.
10
Rheology ofhyaluronan products
203
The classical fashion of describing the viscoelastic properties shown in Figure 1 is, though proper from a rheological point of view, somewhat complicated when discussing the rheological properties with the end user of products. Therefore, EA Balazs introduced a simple, yet very illustrious way to present and describe the viscoelastic properties of hyaluronate solutions and products. In most instances the interesting aspect of the viscoelastic properties as shown in Figure 1 is the proportion between elasticity and viscosity. A simple relationship describing the viscoelastic properties is obtained by calculation the percentage elasticity (Elasticity, % in graphs below) as follows: Elastic modulus· 100 G' Elasticity ('Yo) = Elastic modulus + Viscous modulus = G' + G" • 100
The same information is, of course, also obtained from the frequency dependence of the phase angle. However, the response for a viscoelastic sample changing from viscous to elastic gives a change in the phase angle from 90° to 0°, whereas the introduced parameter Elasticity (%) changes from 0 to 100. These data are plotted in Fig. 2 demonstrating that most products are predominantly elastic at all frequencies, whereas Restylane Fine Lines change behaviour in a somewhat complicated fashion and Rofilan is viscous at low frequencies and elastic at high frequencies. From the viscoelastic response the dynamic viscosity - 11' - may be calculated. The frequency dependence of the dynamic viscosity coincides with the shear rate dependence of the shear viscosity according to the Cox-Merz rule (Fig. 3). The results demonstrate a gel-like behaviour with a continuously increasing viscosity even at low Elasticity ("!o)
Dynamic viscosity, Il' (Pa-s]
100r----------------,
o
10000
Perlane
• Restylane • Restylane Fine Lines o Oermalive
80
• Hylaform .... Rofilan
60
40 • Hylaform 10
• Restylane
20
o
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• Restylane Fine Lines ....Rofilan
.01
.1
1
10
Frequency (Hz)
Figure 2. The percentage elasticity (see formula in text) as a function of frequency plotted on lin-log scale
.01
.1
1
Frequency (Hz)
Figure 3. The dynamic viscosity as a function of frequency plotted on log-log scale
10
204
Rheological behaviourofhyaluronan
frequencies for all samples except Rofilan. For the latter product a Newtonian, constant zero shear viscosity at low frequencies was recorded. The zero shear viscosity of hyaluronan is dependent on the concentration and molecular weight 7. Using the published formulas Rofilan was found to contain hyaluronan with a molecular weight of 2 million using the zero shear viscosity obtained (80 000 Pa • s) and the concentration (20 mg/m1) stated. CONCLUSIONS
The viscoelastic data obtained demonstrated that most products exhibit a gel or gellike behaviour to varying degree with an almost constant response independent on frequency. A slight variation in response was observed for Hylaform. Restylane Fine Lines showed a mixed behaviour changing from gel-like to solution-like indicating that the product is a pseudo-gel with a complicated mixture of solution- and gel-like response. Rofilan changed behaviour from predominantly viscous at low frequencies to elastic at high frequencies typical of a solution containing non-modified, separate hyaluronan molecules with molecular weight 2 million. REFERENCES
1.
D.A. Gibbs, E.W. Merrill, K.A. Smith & E.A. Balazs, The rheology of hyaluronic acid, Biopolymers, 1968,6,777-791.
2.
E.A. Balazs & D.A. Gibbs, D.A. The rheological properties and biological function of hyaluronic acid, In Chemistry and Molecular Biology of the Intercellular Matrix, E.A. Balazs (ed.), Academic Press, London and New York, 1970, pp. 1241-1254. E.A. Balazs & J.L. Denlinger, Clinical uses of hya1uronan, CIBA Foundation Symposium, 1989, 143,265-275.
3. 4.
E.A. Balazs et aI., Chemically modified hyaluronic acid preparation and method of recovery thereof from animal tissues, U.S. Patent No 4,713,448, 1987.
5.
B. Agerup, Polysaccharide gel composition, PCT/SE/96/00684, 1996.
6.
G.D. Prestwich et ai, Chemical modification of hyaluronic acid for drug delivery, biomaterials and biochemical probes. In The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives, T.e. Laurent (ed.) Portland Press, London and Miami, 1998, pp. 43-65.
7.
H. Bothner, Rheological studies of sodium hyaluronate in pharmaceutical preparations, Thesis, Uppsala University, 1991.
STRUCTURAL CHANGE IN HYDROGELATION OF HYALURONAN INDUCED BY ANNEALING THE SOLUTION IN SOL STATE Masato 'Takahashi', Takahiro Isekl', Hirotsugu Hattoril , Tatsuko Hatakeyama'' and Hyoe Hatakeyama' 'Department ofFine Materials Engineering, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, JAPAN 2Department of Textile Science, Faculty ofHome Economics, Otsuma Women's University, 12 Sanbancho, Chiyoda -ku; Tokyo 102-8357, JAPAN. 2Department ofApplied Physics and Chemistry, Faculty ofEngineering, Fukui University of Technology, 3 -6-1 Gakuen, Fukui 910-8505, JAPAN.
ABSTRACT Structural change of hyaluronan (HA) aqueous solutions in gelation induced by annealing in the sol state was investigated by differential scanning calorimetry (DSC). Melting enthalpy of water (.1 H m ) in the system was used as an index of structural change of HA molecules . .1 H m values remained constant when 3 wt% sol annealed at 60°C for 1 hr was maintained at the gelation temperature of 5 °C for 40 hours. On the other hand, .1Hm values fluctuated in an oscillatory manner during gelation at 5 °C when the solution was previously annealed at 60°C for 15 and 18 hr. This fluctuation (variation of .1 Hm) was not observed when the sol was annealed for 48 hours. The results suggests that a conformational change of HA molecules proceeds continuously during gelation when homogenization of the sol state is insufficient.
KEYWORDS Hyaluronan, gelation, annealing, differential scanning calorimetry
INTRODUCTION The gelation of polysaccharides is induced when aqueous solutions are annealed in the sol state [1-3]. By the annealing, hyaluronan (HA) and xanthan gum (XA), which are known as non-gelling polysaccharides, were found to form hydrogels [1-4]. It is thought that the molecular conformation of these polysaccharides changes in aqueous solution during annealing. The structural change of polysaccharides during the annealing process is revealed by the experimental results obtained by the falling ball method (FBM), differential scanning calorimetry (DSC), viscoelastic measurements and small angle x-ray scattering (SAXS) using a rotator anode and synchrotron radiation as the x-ray source [1-4]. When the junction zone formation of physical gels is investigated, not only the structural change of the polysaccharides in the sol state but also the structural change in the gelation process is an important subject to be considered. In this study, structural change of HAlwater systems in the hydrogelation process induced by annealing the
206
Rheological behaviour ofhyaluronan
solution in the sol state was investigated by differential scanning calorimetry (DSC). By DSC, the amount of bound water restrained by the saccharide network was estimated by calculating the melting enthalpy (f1Hm ) of water in the system. The annealing time dependency on the structure formation of HA aqueous solutions in the gelation process is discussed through variation of AHm • EXPERIMENTS
The HA used was supplied by Kibun Food Chemical Co. The nominal molecular weight was 2 x 106 • The glassware used in sample preparation was sterilized before use in order to avoid biological contamination. In DSC measurements, a Seiko DSC200 was used. The enthalpy of melting (f1Hm ) of water was measured at the heating rate 10°C/min. The experimental procedure is reported in detail elsewhere
[2,4]. RESULTS AND DISCUSSION
Annealing of solution As reported in our previous study [4], HA 3 wt % solutions formed gels when the aqueous solution was annealed at T = 60°C for more than 6 hr. Gelation was confirmed by FBM. It was revealed that the gel-sol transition temperature T g•s initially increases with the increase of annealing time. As shown in Figure 1, the enthalpy of melting f1Hm per 1 mg of water in the system measured by DSC showed oscillational change with repeated increases and decreases, and then approached a constant value in the annealing process. The final value of f1Hm was slightly smaller than that of the initial value. These experimental results suggested the following structural change occurs in the annealing and subsequent cooling processes. In the non-annealed solution, HA molecules form molecular assemblies and the solution is regarded as the emulsion or suspension of such assemblies. These assemblies seem to be metastable
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Figure 2. LJ.llm of 3 wt% sol annealed at 60°C for 1 hr. as a function oftime at 5°C.
Structuralchange in hydrogelation
207
and easily destroyed by annealing the solution in sol state. If ,
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RESULTS Promotor analysis of the murine Hyal-2 gene
The cDNA encoding the human Hyal-2 enzyme has also been used to clone the murine cDNA and the gene of the murine orthologue". It is a small gene encompassing without promotor about 3 kbp with two introns of 530 and 415 bp, respecively (Genebank Ace: AJ000059, AJ000060).
190
The role of hyaluronan in tissues
-- --
Recognitionsite for Mspl and Hpall:
Mspl
CCGG
99::>::> Hpall is blocked byCpG Methylation
Hpall embryo
adult
Figure 2: PCR analysis of methylated genomic DNA. DNA was prepared from newborn [1, 2] and adult [3, 4] mice. The putative CpG island was amplified by PCR in the presence of either MEl or ME2 together with ME3 primers (see also Fig. 1) using untreated (upper panel), MspI, or Hpall treated DNA as a template. In order to control DNA integrity after the restriction digestion, a region flanking the MspI/ Hpall recognition sequence was amplified by ME2/ME3 which gives rise to a fragment 331bps in length [1, 3]. ME1 used in lane 2 and 4 binds within the putative CpG island and amplifies together with ME3 a product of 7l8bps.
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. .Q. 1. genomlc~ DNA +o
mRNA
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+-
B
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Figure 3: A) Detection of Hyal-2 mRNA by Reverse-Transcription PCR using primer pair E3f (=0) and E4f (=i). B) Genomic DNA [1] and Hyal-2 cDNA [2] were ampified using E3f and E4r as primers. DBT cells [3] were treated with 1f!M 5-aza2'deoxycytidin [4], mRNA was prepared, subsequently reverse transcribed and amplified with E3f and E4r. Amplification of genomic DNA or the products obtained before [4] with E4f and E4r yieled a specific fragment of smaller size [5, 6]. Lane 7 shows a DNA molecular weight marker (pBR327/HinFI). cDNAs used in [3] and [4] were normalized for GAPDH transcripts [8, 9].
Transcriptional regulation of Hyal-2 hyaluronidase
191
Dot blot analysis of the murine and human genomic sequences, reveal long stretches of the 5' flanking region of the Hyal-2 gene of either species as highly homologous (Fig. I). The 5' flanking region, the first exon and intron is highly GC rich, a GC content of more than 65% and a ratio of expected versus observed CpG dinucleotides of 0.69 has been calculated. The 3' end of the island extends into part of the first intron. Moreover, the CpG content is also highly enriched upstream to the transcriptional start site of the human gene (%GC=69.9; -1630-400 bps). Cross and colleagues 10 have cloned hypermethylated DNA by affinity chromatography from adult human blood. Indeed, a partial sequence (222bps) has been reported to the DNA databases (Macdonald et al., 1995: Genbank: Z64462) which is derived from the human Hyal-2 gene. Transcriptional regulation of Hyal-2 Genomic DNA prepared from brains of newborn and adult mice was restricted with MspI or Hpall. Both endonucleases recognize the tetrameric sequence: CCGG. However, Hpall is blocked by CpG methylation. The Hpall and Mspl restricted DNAs were subjected to PCR analysis using specific primers which amplify either a DNA fragment surrounding Hpall/Mspi recognition sequences or, as a control, a region adjacent to the CCGG tetramers. In the case of DNA derived from adult brains, the portion investigated was inert to digestion by Hpall whereas DNA from newborn mice was cleaved with Hpall. Both DNAs were cleaved by MspI in the region of interest (Figure 2). This indicates that de novo methylation modifies the 5' region of mouse Hyal-2 gene. Therefore, mouse DBT cells which contain neither endogenous Hyal-2 transcripts nor hyaluronidase activity have been cultured in the presence of 5' -de-aza-cytidine which is known to inhibit CpG methylation II. mRNA was isolated and reversetranscribed. cDNA was amplified using a primer pair which binds within exon3 and 4 giving rise to a 337bp fragment. Genomic DNA containing the third intron results in a 797bp DNA product. As shown in Figure 3, clearly, the Hyal-2 gene is activated in cells treated with de-aza-cytidine. In addition, those cells exhibit hyaluronidase activity compareable to the enzymatic properties of human Hyal-2 (data not shown).
DISCUSSION In mice, the Hyal-2 mRNA is present in many different tissues. An interesting exception is the brain, which apparently does not contain this enzyme. Several ESTs encoding part of the Hyal-2 enzyme, however, have been isolated from infant human brain. The experimental data presented in this contribution suggest that transcription of Hyal-2 is generally regulated by de novo methylation of CpG island present in the promoter and 5' -untranslated region. Expression of Hyal-2 is thus detectable at embr~onic stages but is down-regulated after birth, and completely silenced in the adult brain. These results strongly suggest a role of Hyal-2 in developmental processes and possibly also in tumorigenesis and de-differentiation phenomena. Due to the wide distribution of Hyal-2 in animal tissues it seems likely that the product of this gene plays an important role in the catabolism of hyaluronan. Indeed, no Hyal-2 mutants have been described. A deficiency in Hyal-2 could prevent degradation of long chain HA as well as production of HA fragments that may have important physiological functions. As outlined above, we could provide the first biochemical evidence that in solution HA is not simply a random-coil but forms well-ordered structures depending on the length of the polysaccharide chain. Recently, we were also
192
The role or hyaluronan in tissues
able to show that HA-synthesizing enzymes Xhasl and 2 form products of different chain lengths 7. This clearly demonstrated that it is not irrelevant which "kind" ofI-IA is being synthesized or degraded in certain tissues, organs or body fluids. The steady-state of many physiological processes and/or the differentiation status of cells are certainly influenced by the highly complex extracellular network of proteins, proteoglycans and polysaccharides. It is evident that in many types of ECM, HA is not just a passive part of the network, but that it also exerts regulatory functions. It has clearly been shown that low-molecular HA is able to influence gene expression 12. In line with this, changes of the normal HA content of the ECM has often been observed during malignant aberration of cells or tissues. In particular, this phenomenon has been described in certain types of cancer 13. The factors controlling the invasive behavior of tumors are not well understood. Since there is evidence that HA plays an role in invasiveness of some tumors 14.15, we designed experiments aiming to test the role of Hyal-2 in process. We have transfected a cell line derived from an astrocytoma with Hyal-2. When inoculated in brain these cells formed tumors with enhanced growth rates, an effect not observed at subcutaneous sites 16. This actually may reflect different extracellular environments, with e.g. higher levels of HA being found in the brain than in the subcutaneous space. The transcription of Hyal-2 and consequently the production of intermediate-sized HA-chains are blocked in the adult brain. Due to their physico-chemical properties, these short chains may facilitate cell migration and invasiveness. We also found that the vascularization level is increased in these intracerebral tumors. Even though the developing embryo is rich in HA 17 very little has been found in the developing brain 18. This may be due to the fact that the level of expression of Hyal-2 is particularily high in the embryonic brain. In general, changes in either hyaluronan concentration and/or polymer length may regulate cellular responses during tissue and organ differentiation. Formation of hydrated pericellular matrices is known to facilitate cell-rounding during mitosis 19. Subsequently, hyaluronan concentration decreases and leads to a reduced volume of intercellular matrix and condensation of cells prior to differentiation. Thus the process may be divided into two stages: a primary I-lA-richphase in which undifferentiated stem cells proliferate and migrate, followed by removal of the HA and the onset of cellular differentiation and morphogenesis 20. These results are in agreement with a concept of exclusion of spherical macromolecules by a network of randomly associated rods 21. This hypothesis was specifically applied to polysaccharides 22 and later on tested experimentally for cells 23. According to this model of organogenesis 24, cells coated by a glycocalyx of a linear polysaccharide, should disperse and migrate down a viscosity gradient of matrix polymer. On the other hand, cells that internalize their surface coat should aggregate 25. For instance, during migration myoblasts retain a hyaluronan coat that prevents their fusion. Prior to the initiation of fusion this coat disappears 26. This indicates, that the exclusion of cells by a network of linear polymer is a function of its concentration and viscosity. Low molecular weight GAG such as chondroitin sulfate effectively promotes aggregation whereas highly viscous solutions of hyaluronan inhibit tins phenomenon. However, the bulk viscosity at the same concentration of hyaluronan decreases dramatically with the decrease of polymer lengths 27. Furthermore, no pericellular coats can be observed with low molecular weight polysaccharide. There is increasing evidence that hyaluronan indeed plays a crucial role in creating cell-free spaces 28, and in controlling cell proliferation 29, migration 30 and differentiation 31. In line with this, one can expect that the biosynthesis of HA-synthesizing and degrading enzymes are tightly regulated in space and time during embryogensis.
Transcriptional regulation ofllyal-2 hyaluronidase
193
ACKNOWLEDGEMENTS
The technical assistance of Anita Weber is gratefully acknowledged. The authors would like to express their gratitude to Christa Mollay for many fruitful discussions. This work was supported by grant 13001-Bio from the Austrian Science Foundation (FWF) . REFERENCES
1. Wei, M.H., F. Latif, S. Bader, V. Kashuba, J.Y. Chen, F.M. Duh, Y. Sekido, C.C. Lee, L. Geil, 1. Kuzmin, E. Zabarovsky, G. Klein, B. Zbar, ID. Minna&M.I. Lerman, Construction of a 600-kilobase cosmid clone contig and generation of a transcriptional map surrounding the lung cancer tumor suppressor gene (TSG) locus on human chromosome 3p21.3: progress toward the isolation of a lung cancer TSG, Cancer Res., 1996 56. 1487-92. 2. Daly, M.C., R.H., Xiang, D. Buchhagen, C.H. Hensel, D.K. Garcia, A.M. Killary, J.D. Minna&S.L. Naylor, A homozygous deletion on chromosome 3 in a small cell lung cancer cell line correlates with a region of tumor suppressor activity. Oncogene, 19938,1721-1729. 3. Jones, M.H., P.M. Davey, H. Aplin&N.A. Affara, Expression analysis, genomic structure, and mapping to 7q31 of the human sperm adhesion molecule gene SPAMI. Genomics, 199529,796-800. 4. De Maeyer Guignard, J., A. Cachard Thomas&E. De Maeyer, Linkage analysis of the murine Hyal-I locus on chromosome 9, J Exp. Zool., 1991 258,246-8. 5. Fiszer Szafarz, B.&E. De Maeyer, Hyal-l, a locus determining serum hyaluronidase polymorphism, on chromosome 9 in mice, Somat. Cell. Mol. Genet., 1989 15, 79-83. 6. Dc Maeyer, E.&J. De Maeyer-Guignard, The growth rate of two transplantable murine tumors, 3LL lung carcinoma and B l6F 10 melanoma, is influenced by Hyal1, a locus determining hyaluronidase levels and polymorphism. Int. .J. Cancer, 1992 51,657-60. 7. Lepperdinger, G., B. Strobl&G. Kreil, HYAL2, a human gene expressed in many cells, encodes a lysosomal hyaluronidase with a novel type of specificity. J BioI. Chem., 1998273,22466-70. 8. Lepperdinger, G., B. Strobl, A. Jilek, A. Weber, .I. Thalhamer, H. FlOckner&C. Mollay, The lipocalin Xlcpll expressed in the neural plate of Xenopus laevis is a secreted retinaldehyde binding protein. Prot. Sci., 19965,1250-1260. 9. Strobl, B., C. Wechselberger, D. Beier&G. Lepperdinger, Structural organization and chromosomal localization of Hyal2, a gene encoding a lysosomal hyaluronidase. Genomics, 1998 53, 214-219. 10.Cross, S.H., J.A. Charlton, X. Nan&A.P. Bird, Purification of CpG islands using a methylated DNA binding column. Nature Genetics, 19946,236-244. 11.Creusot, F., G. Acs&J.K. Christman, Inhibition of DNA rnethyltransferasc and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza2'-dcoxycytidinc, J BioI. Chem., 1982257,2041-2048. 12.Noble, P.W., C.M. McKee, M. Cowman&H.S. Shin, Hyaluronan fragments activate an NF-KB/I-KBa autoregulatory loop in murine macrophages, J Exp. Med., 1996 183,2373-2378.
194
The role of hyaluronan in tissues
13.Knudson, W., Hyaluronan in malignancies, in The Chemistry, Biology and Medical Applications ofHyaluronan and its Derivatives, T.C. Laurent, Editor. 1998, Portland Press Ltd: London. p. 161-169. 14.Culty, M., M. Shizari, E.W. Thompson&C.B. Underhill, Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: correlation with invasive potential..J Cell. Physiol., 1994 160,275-86. 15.zhang, H., G. Kelly, C. Zerillo, D.M. Jaworski&S. Hockfield, Expression of a cleaved brain-specific extracellular matrix protein mediates glioma cell invasion In vivo .J. Neurosci., 199818,2370-6. 16.Novak, U., 8.S. StyIIi, A.H. Kaye&G. Lepperdinger, Hyaluronidase-Z overexpression accelerates intracerebral but not subcutaneous tumor formation of murine astrocytoma cells. Cancer Res., 199959,6246-50. 17.Fenderson, B.A, 1. Stamenkovic&A Aruffo, Localization of hyaluronan in mouse embryos during implantation, gastrulation and organogenesis. Differentiation, 1993 54,85-98. I8.Koprunner, M., J. Miillegger&G. Lepperdinger, Synthesis of hyaluronan of distinctly different chain length is regulated by differential expression of Xhasl and 2 during early development of Xenopus laevis. Mech. Dev., 2000 90, 275-278. 19.Laurent, TC.&J.R. Fraser, Hyaluronan. Faseb L, 1992 6, 2397-404. 20.Toole, B.P., Proteoglycans and hyaluronan in morphogenesis and differentiation., in Cell Biology of Extracellular Matrix, E.D. Hay, Editor. 1991, Plenum Press: New York. 21.0gston, AG., The spaces in a uniform random suspencsion of fibers. Trans. Farady Soc., 1958 54,1754-1757. 22.Laurent, TC., The interaction between polysaccharides and other macromolecules. 9. The exclusion of molecules from hyaluronic acid gels and solutions. Biochem . .I, 196493,106-12. 23.Morris, J.E., Steric exclusion of cells. A mechanism of glycosaminoglycan-induced cell aggregation. Exp. Cell Res., 1979 120, 141-153. 24.Edwards, P.A W., Differential cell adhesion may result from nonspecific interactions between cell surface glycoproteins. Nature, 1978 271, 248-249. 25.Morris, J.E., Proteoglycans and the modulation of cell adhesion by steric exclusion. Dev Dyn, 1993 196, 246-51. 26.0rkin, R.W., W. Knudson&B.P. Toole, Loss of hyaluronate-dependent coat during myoblast fusion. Dev. Bioi., 1985107,527-30. 27.Bothner-Wik, H.&O. Wik, Rheology ofHyaluronan, in The Chemistry, Biology and Medical Applications ofHyaluronan and its Derivatives, TC. Laurent, Editor. 1998, Portland Press: London. 28.Comper, W., D, &T.C. Faurent, Physiological function of connective tissue polysaccharides. Physiol. Rev., 197858,255-315. 29.Brecht, M., U. Mayer, E. Schlosser&P. Prehm, Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem. .I, 1986239,445-50. 30.Poclmann, R.E., A.C. Gittenberger de Groot, M.M. Mentink, B. Delpech, N. Girard&B. Christ, The extracellular matrix during neural crest formation and migration in rat embryos. Anat. Embryol. Berl., 1990 182,29-39. 31.Toole, B.P.&J. Gross, The extracellular matrix of the regenerating newt limb: synthesis and removal of hyaluronate prior to differentiation. Dev. Bioi., 1971 25, 5777.
HYALURONAN SYNTHASE EXPRESSION IN HUMAN ENDOMETRIUM DURING THE MENSTRUAL CYCLE Marianne Tellbach 1 *, Lois A. Salamonsen", Gary Brownlee", Tracey Brown! & Marie-Paule Van Damme' J
Department of Biochemistry and Molecular Biology, PO Box 13D, Monash University, Clayton, Victoria 3800, Australia.
2
Prince Henry's Institute ofMedical Research, PO Box 5152, Clayton, Victoria 3168, Australia.
ABSTRACT The human endometrium undergoes extensive remodelling during the menstrual cycle in preparation of a favorable environment for implantation. Biphasic fluctuations of hyaluronan (HA) in the cycling endometrium have been demonstrated with peaks occurring during the early proliferative (days 6-8) and mid secretory (days 19-23) stages of the cycle. The second, mid secretory, HA peak encompasses the time of embryo implantation suggesting a possible role for HA in this process. The aim of the present study was to investigate expression patterns of the 3-hyaluronan synthase (HAS) isoforms: HAS-I, HAS-2 and HAS-3. In situ hybridization was carried out on formalin fixed endometrial tissue across the cycle to determine whether these enzymes were expressed in human endometrium and if so, their cellular source. Riboprobes were constructed for each HAS: HAS-l (64bp), HAS-2 (60bp) and HAS-3 (550bp) and labeled with digoxygenin (DIG). The HAS protein was also localized immunohistochemically using a polyclonal antibody against extracellular HAS peptides. All three forms of HAS mRNA were expressed in the human endometrium. Expression was observed in both stromal and glandular epithelial cells with greatest intensity in epithelial cells and considerable cyclical changes. In all cases expression of mRNA was low during menstruation (days 1-5). Levels increased throughout the proliferative stage (days 6-14) of the cycle and for HAS-2 and HAS-3 remained elevated until the mid secretory (days 19-23) / implantation phase. HAS-l expression temporarily decreased during the late proliferative stage (days 12-14). Following implantation expression of all HAS types decreased dramatically. HAS protein localization was demonstrated on the plasma membrane of both glandular epithelial and stromal cells. The partial correlation between HA and HAS supports a role for these enzymes in endometrial HA regulation during the menstrual cycle and also suggests tight control of enzyme expression.
KEYWORDS Endometrium, hyaluronan, hyaluronan synthases
238
Biosynthesis and biological degradation ofhyaluronan
INTRODUCTION During the menstrual cycle, the architecture of the endometrium undergoes dynamic remodelling in preparation of a favorable environment for blastocyst implantation. Ovarian steroid hormones have been assumed as the principal orchestrators of this event and are responsible for proliferation and differentiation of epithelial and stromal cells in the creation of a receptive endometrium. The human endometrium is normally a hostile environment for embryo implantation but for a few days in each menstrual cycle it attains a unique state of receptivity when, if the appropriate remodelling has occurred, the embryo will implant and pregnancy will be established. In the absence of implantation, tissue regression occurs followed by the shedding of the functionalis layer at menstruation (day 1-5) and regeneration of the denuded tissue. The basalis layer is responsible for regeneration, which is maximal prior to ovulation (day 14), during the proliferative stage of the cycle (days 6-14). Re-epithelialization, re-vascularization, numerous cell divisions and the production of extracellular matrix components characterize the proliferative stage I. Following ovulation, the cells differentiate, there is an increase in the degree of hydration of the tissue and the glands increase secretions into the lumenal cavity. This latter part of the cycle is termed the secretory phase (days 15-28). Throughout the course of the cycle the endometrium is transformed from a thin dense tissue into a thick, highly permeable secretory tissue. HA, a major component of most extracellular matrices, has been identified in the human endometrial stroma during the menstrual cycle. Biphasic fluctuations of HA occur throughout the cycle with peaks during the early proliferative and mid secretory phases 2. High HA levels are a common component of tissues with rapid cell proliferation 3 and this may explain the elevated HA levels during the early proliferative stage of the menstrual crcle. Furthermore high HA levels have been positively correlated with mitosis 4, while inhibiting cell differentiation 6.7 and consequently create an environment promoting cell proliferation. The hydrated matrix also encourages the diffusion of growth factors such as TGF-~ and promotes cytokine protection from proteolytic enzymes 8. The second, mid secretory, HA peak encompasses the time of implantation. It has been suggested, that a HA rich matrix may facilitate invasion of the stroma during this process 9. This data supports a role for HA in the preparation of the endometrium for embryo implantation, a critical step in the establishment of pregnancy. However, nothing is known about how the peaks of HA in the endometrium are regulated. The aim of the present study was to investigate expression patterns of HAS, the enzymes responsible for HA synthesis. HAS are a multigene family with three known members HAS-I, -2 and -3. Each HAS has distinct enzymatic properties in terms of enzyme stability, the elongation rate and chain lengths of the HA synthesized 10. MATERIALS AND METHODS Tissue collection Endometrial tissue was obtained at curettage from 84 cycling women (3 complete cycles) with no apparent endocrinological problems and normal endometrial histology. None of the patients had received steroid treatment during the past 12 months. Informed consent was obtained from each patient and approval was given by the Human Ethics Committee at Monash Medical Centre, Melbourne, Australia. Endometrial dating was confirmed by independent histological examination.
Synthase expression in human endometrium
239
RNA probes and in situ hybridization HAS-I (64bp) II, HAS-2 (60bp) 12 and HAS-3 (550bp) cDNA were cloned into the expression vector pGEM-T (Gary Brownlee, Monash University, Australia). HAS plasmids were cut with Not I or Nco I to generate sense and antisense probes which were then labeled by doxigenin (DIG) RNA-labeling 13. HAS mRNA was localized in serial sections (5 microns) of formalin fixed, paraffin embedded endometrial tissue by in situ hybridization as previously described 14. To aid in permeablization of the tissue, sections were incubated in proteinase K (12 l1g/ml) at 37°C for 30 min, and post fixed with 4% paraformaldehyde at 4 °C for 10 min. Sections were then treated with acetyl ate prior to prehybridization. Hybridization was performed overnight at 42°C for HAS-2, -3 and 48°C for HAS-l with probe concentrations of 20, 18, and 10 ng/111 respectively. Probes were diluted in hybridization buffer containing 10% dextran sulphate in addition to all components of the prehybridization solution. Sections underwent a number of stringency washes at the temperature of hybridization and, to reduce background, were incubated with RNase A (20 ug/ml) at 37°C for 30 min. Slides were blocked with 10% normal sheep serum, 10% fetal calf serum and 0.1 % triton X-loo for 30 min. Sections were incubated with Anti-DIG-AP (1 :750) in blocking solution at 4°C overnight. Nitroblue tetrazolium / 5bromo-4-chloro-3-indoyl phosphate was applied and sections were incubated in the dark for 1-5 hrs at room temperature. HAS mRNA was represented by the deposition of a blue / purple precipitate. Sections were not counterstained. Evaluation of in situ hybridization To determine the endometrial reactivity for HAS mRNA expression, the staining intensity of cells were evaluated. Two observers using a light microscope graded the relative intensity of the precipitant. The relative intensity was estimated semiquantitatively on a 5-level scale as follows: very weak, 1; weak, 2; moderate, 3; strong, 4 and very intense, 5. Immunohistochemistry Immunological detection of HAS in endometrial tissue was preformed using a polyclonal human antibody cross-reacting with external peptides of HAS-I, -2 and 3 (Tracey Brown, Monash University Australia). Hydrated 5 11m paraffin sections were pre-treated with 0.2% trypsin / PBS for lhr at 37°C. Heterophite proteins were blocked with 10% FCS / PBS followed by HAS detection with a HAS polyclonal antisera (1 :250) at 37°C for 1 hr. Endogenous peroxidase was eliminated by incubation with 0.6% hydrogen peroxide / methanol for 20 min. Final visualization was performed with an anti-sheep IgO / HRP (1:100) for I hr at 37°C, fol1owed by reaction with HAS protein localization was determined by the addition of the colourimetric substrate 3,3'diaminobenzidine tetrahydrochloride (DAB). Sections were counterstained with hematoxylin, dehydrated and mounted.
RESULTS AND DISCUSSION To identify which HAS isoforms the human endometrium expressed and their site, in situ hybridization of HAS-I, -2 and -3 was preformed (Fig 1). Formalyn sections were
240
Biosynthesis and biological degradation of hyaluronan
Figure 1. Cellular location of HAS mRNA in human endometrium during the mid proliferative phase (day 10) of the menstrual cycle. HAS-I, -2 and -3 mRNA (A, C and E respectively) were detected in the endometrium by in situ hybridization and localized predominantly in the glandular epithelial cells with less intense staining in the surrounding stroma. Serial control sections incubated with HAS-I, -2 and -3 sense riboprobes (B, D and F respectively) showed no specific staining. (Magnification X20)
Synthase expression in human endometrium
241
probed with DIG-labeled antisense RNA probes for each HAS and sense probes were used as controls for non-specific hybridization. All HAS types were expressed in the endometrium by both stromal and glandular epithelial cells however a greater expression was displayed in the glandular epithelium (Fig 1). This is somewhat surprising given the location of endometrial HA as predominantly stromal 2 and suggests that the HA produced by the glandular HAS may transverse the basement membrane. Immunohistochemistry was also conducted on the endometrial samples using a polyclonal antibody cross-reacting with HAS-I, -2 and -3 and revealed predominant HAS localization in the glandular epithelial cells, thus confirming the in situ localization studies (data not presented). Expression of HAS-I, -2 and -3 demonstrated considerable changes throughout the menstrual cycle (Fig 2). HAS-I levels in both the glandular epithelial and stromal cells were low during the menstrual phase (days 1-5) of the cycle. At this time tissue breakdown has occurred and the endometrial layer is shed. HAS-I levels increase during the early proliferative stage (day 6) in conjunction with an increase in HA probably due to the extensive proliferation required to rebuild the functional endometrial layer. During the mid proliferative stage (days 9-11) of the cycle HAS-1 expression remains elevated however HA levels have decreased. Around day 13 HAS-1 expression suddenly declines. The early secretory phase (days 15-18) of the cycle is marked by an increase in HAS-1 transcription, an event well correlated with the upcoming HA peak at implantation. HAS-1 levels decrease dramatically on day 19 and remain low throughout the remainder of the cycle. This seems appropriate given the events occurring in the endometrium at this stage. If the cycle is infertile, the endometrium regresses, probably due to loss of water from the tissue. HAS-2 and -3 expression during the menstrual cycle differ slightly to that of HAS-I. Low expression levels are apparent during menses (days 1-5) and the early proliferative phase (days 6-8). Expression does not increase until day 9, somewhat later than the increase in HAS-1 levels for this time of the cycle. Expression remains elevated until day 18 of the secretory phase (days 15-28), after which levels dramatically decrease. HAS expression is partially correlated with HA content during the menstrual cycle as demonstrated by the low HAS / HA levels during menses and the mid / late secretory phases. Between mensturation and mid secretion HAS levels remained elevated while HA levels revealed two peaks separated by a HA depression. This suggests that the endometrial HA is not solely regulated by HAS and suggest a possible role for the degradative hyaluronidases. A possible reason for elevated HAS levels during the HA depression could be related to the vascular remodeling and angiogenesis occurring within the endometrium. It has been found that partial degradation products of HA, between 4 and 25 dissacharides in length, induce angiogenic responses 15. Furthermore, angiogenesis in the endometrium is greatest during the mid to late proliferative phase and during the early secretory phase as determined by the levels of vascular endothelial growth factor, an angiogenic marker and its tyrosine kinase receptors, FIt-1 and KDRJFlk-1 16,17. CONCLUSION The results obtained support a role for all three HAS isoforms in endometrial HA production and a probable role for HAS in endometrial HA regulation although this does not appear to be the sole regulatory mechanism.
242
Biosynthesis and biological degradation ofhyaluronan
5l 4
1
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3
2
1 5 4
3
2
1 5
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4
3
2 1 1
4
7
10
13
16
19
22
25
28
STAGE OF CYCLE
Figure 2. HAS expression in the human endometrium during the menstrual cycle as determined by in situhybridization. Sections from eachday of the cycle(n=3) wereprobed with HAS-I (A), HAS-2 (B) and HAS-3 (C) DIG-labeled riboprobes. Expression was quantified visuallyin both the glandular epithelial ( ) and stromal (- - - -) cells. HA levels are also shown ( _ ) as determined by Salamonsen et al. 2 , to aid in correlation between HA and HAS.
Synthase expression in human endometrium
243
ACKNOWLEGMENTS The authors acknowledge financial support from Meditech Research Limited, Level 1, Sterling House, 8 Parliament Place, West Perth, 6005 Australia REFERENCES I. E. C. Wienke, F. Cavazos, D. G. Hall & F. V. Lucas, Ultrastructure of the human endometrial stroma cell during the menstrual cycle, Arn J Obstet GynecoL., 1968, 102(1),65-77. 2. L. A. Salamonsen, S Svetlana & R. Stem, Distribution of hyaluronan in human endometrium across the menstrual cycle, Cell Tissue Res., 2001, 306, 335-340. 3. B. P. Toole, Proteoglycans and hyaluronan in morphogenesis and differentiation, In: Cell biology of the extracellular matrix, E. D. Hay (eds.), Plenum Press, New York, 1991, pp 305-341. 4. M. Brecht, U. Mayer, E. Schlosser & P. Prehm, Increased hyaluronate synthesis is required for fibroblast detachment and mitosis, Biochem., 1986, 239, 445-450. 5. N. Mian, Analysis of cell-growth-phase-related variations in hyaluronate synthase activity of isolated plasma membrane fraction of cultured human skin fibroblasts, Biochem., 1986, 237:333-342. 6. M. J. Kujawa, D. G. Pechak, M. Y. Fiszman & A. I. Caplan, Hyaluronic acid bonded to cell culture surfaces inhibits the program of myogenesis, Develop. Biol., 1986,113, 10-16. 7. M. J. Kujawa & K. Tepperman, Culturing chick muscle cells on glycosaminoglycan substrates: attachment and differentiation, Dev. Biol. 1983,99,277-286. 8. T. Tanaka, T. Nakamura, H. Ikeya, T. Higuchi, A. Tanaka, A. Morikawa, Y.Saito, K. Takagaki & M. Endo, Hyaluronate depolymerization activity induced by progesterone in cultured fibroblasts derived from human uterine cervix, FEBS Lett., 1994, 347, 95-98. 9. D. D. Carson, A. Dutt & J-P. Tang, Glycoconjugate synthesis during early pregnancy: hyaluronate synthesis and function, Dev. Biol., 1987, 120,228-235. 10. N. Itano, T. Sawai, M. Yoshida, P. Lenas, Y. Yamada, M. Imagawa, T. Shinomura, M. Hamaguchi, Y. Yoshida, Y. Ohnuki, S. Miyauchi, A. P. Spicer, J. A. McDonald & K. Kimata, Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties, J. Biol. Chem., 1999, 274, 25085-25092. 11. A. M. Shyjan, P. Heldin, E. C. Butcher, T. Yoshino & M. J. Briskin, Functional cloning of the cDNA for a human hyaluronan synthase, J Biol Chem., 1996, 271(38),23395-9. 12. K. Watanabe & Y. Yamaguchi, Molecular identification of a putative human hyaluronan synthase, J Biol Chem., 1996,271 (38),22945-8. 13. P. Komminoth, Digoxigenin as an alternative probe labeling for in situ hybridization, Diagn. Mol. Pathol., 1992, 1, 142-150. 14. G- Y. Nie, Y. Li, H. Minoura, 1. K. Findlay & L. Solamonsen, Complex regulation of calcium binding protein D9K (Calbindin-D9K) in the mouse uterus during early pregnancy and at the site of embryo implantation, Biol. Reprod., 2000, 62, 27-36. 15. D. C. West, I. N. Hampson, F. Arnold & S. Kumar, Angiogenesis induced by degradation products of hyaluronic acid, Science, 1985, 228, 1324-1326.
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Biosynthesis and biological degradation ofhyaluronan
16. M. Perrot-Applanat, Hormonal regulation of vascular cell function: angiogenesis, In: Comprehensive vascular biology and pathology - an encyclopedic reference, A. Bikfalvi (eds.), Springer-Verlag, Heidelberg, Germany, 1999, in press. 17. G. Meduri, P. Bausero & M. Perrot-Applanat, Expression of VEGF receptors in the human endometrium: modulation during the menstrual cycle, Biol. Reprod., 2000, 62,439-447.
IN VIVO INVESTIGATION OF HYALURONAN SYNTHASE FUNCTION DURING VERTEBRATE EMBRYOGENESIS Janet Y. Lee* and Andrew P. Spicer, Rowe Program in Genetics, Department ofBiological Chemistry, University ofCalifornia Davis, School ofMedicine, Tupper Hall, Davis, California 95616, USA.
ABSTRACT Hyaluronan (HA) is synthesized at the eukaryotic plasma membrane by anyone of three members of the HA synthase (HAS) family. Northern analyses also revealed unique expression patterns; Hasl and Has3 are primarily expressed at early and later stages of embryogenesis, respectively. In contrast, Has2 is expressed throughout embryogenesis from as early as E7.5. This predominance of Has2 is also observed in loss-of-function studies; Hasl and Has3 null homozygotes are healthy and viable, while Has2 null embryos die at EIO.5. Thus, Has2 is the major HA synthase involved in normal embryo development. We are continuing our in vivo investigations into HA synthase function by expression analyses and by conditional gene targeting. Whole mount in situ hybridization studies revealed expression of Has2 in neural crest cellderived cardiac and craniofacial structures, in addition to the developing limb. In particular, Has2 may playa pivotal role in limb and joint development, as its expression pattern closely mimics that of growth factors necessary for limb and joint development. Has3 expression was detected at the developing vibrissae and hair follicles, which may indicate a role for Has3-dependent HA synthesis in the formation of whiskers, hair follicles and/or their associated sensory neurons. Our conditional gene targeting of Has2 will follow an allelogenic approach in order to establish mouse lines from which multiple variant lines will be derived through crosses with recombinase expressing transgenic mice. Through this approach, we will generate mice with hypomorphic phenotypes, as well as those with tissue-specific deficiencies in HA biosynthesis. KEYWORDS Hyaluronan synthase, HAS, Has2, gene targeting, expression INTRODUCTION In vertebrates, HA is synthesized by anyone of three hyaluronan synthases (HAS), designated, HASI, HAS2 and HAS3, respectively 1.2. During embryogenesis, large amounts of HA are synthesized by many cell types particularly those that are proliferating and migrating. Through specific inactivation of the mouse Has2 gene 3, we have recently shown that HA is critical for embryogenesis and is required for the maintenance and possibly the creation of extracellular matrix-defined spaces throughout the embryo, in addition to the migration of endocardial cushion cells. We rely upon the mouse as our model system for investigation of HA biosynthesis and function during vertebrate embryogenesis. Our most recent strategy employs sitespecific recombinases to create conditional or tissue-specific deficiencies of Has2
246
Biosynthesis and biological degradation ofhyaluronan
function during embryogenesis 4. However, before embarking upon this approach, we reasoned that it would be extremely important to document the normal expression pattern of the mouse Has genes. Why is this an important consideration? Conditional gene targeting relies upon the use of cell-type or tissue-specific promoter/enhancer sequences to restrict expression ofa Cre recombinase transgene, the activity of which is required for inactivation of the gene of interest. Numerous transgenic mice have now been derived in which Cre recombinase is expressed in a cell-type or tissue specific manner. Additional promoter/enhancer sequences are continually being characterized in an effort to derive transgenic mouse lines expressing Cre in essentially any spatial or temporal pattern. In order to investigate Has2 function in the tissue of interest, the Cre transgene must be expressed in either the cell that expresses Has2 or its precursor. Thus, it is of paramount importance that we know which cells express Has2 under normal circumstances during embryogenesis. Furthermore, it is also important to know if any of these cells, or their neighbouring cells, also express Hasl and/or Has3. As HA is released into the extracellular matrix of synthesizing cells, it may clearly act in a noncell-autonomous fashion, rescuing any defects that may result from restricted inactivation ofHas2 in a single cell. Herein, we present our strategy to create a Has2 conditional knockout in the mouse, in addition to preliminary data on the expression patterns for the Has gene family during mouse embryogenesis. MATERIALS AND METHODS Creation of the mouse Has2 allelogenic gene targeting vector
Three contiguous mouse Has2 genomic DNA fragments of 4.7kb, 1.6kb and 3.3.kb were cloned into a vector designed for allelogenic gene targeting. The 4.7 kb fragment represents the 5' arm of homology, the 1.6 kb fragment was cloned between two 10xP sites and the 3.3 kb fragment, representing the 3' arm of homology was cloned downstream of the selectable PGKneo cassette, which is flanked by two fit sites. The structure of a targeted allele (Has2 hy'j is illustrated in figure 1. Whole-mount in situ hybridisation
Staged embryos [E7.5 - El5.5] were isolated from timed pregnant mice, fixed in 4% paraformaldehyde at 4°C for 2-12h, and subsequently dehydrated in methanol. Digoxigenin riboprobes were generated from plasmids containing the mouse Has2 or Has3 open-reading frames according to the manufacturer's instructions (http://biochem.roche.com). The procedure for whole-mount in situ hybridisation was performed essentially as previously described 5, with modifications. RESULTS AND DISCUSSION Generation of the mouse Has2 allelic series
A hypomorphic Has2 allele (HasiIY'j is predicted to result from insertion of the PGKneo cassette into intron 3. Through crossing with transgenic mice expressing Flp recombinase, the neo cassette will be deleted, resulting in a functionally wild-type Has2 allele (Has2Llneo ) 6. By a similar strategy, crossing with transgenic mice expressing Cre
Synthase function during vertebrate embryogenesis
247
recombinase will delete exon 3, converting the targeted allele or its Flp-recornbined descendants into a functionally null allele (Has2 L!ex3). Has2 Allelic Series HHH
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Design of the allelogenic targeting vector for mouse Has2. The basic structure of the wild-type Has2locus is illustrated at the top. Exon 1 (not shown) is located 12.5 kb upstream of exon 2 and contains the entire 5' UTR. The black filled boxes represent the open-reading frame. Arrowheads and ovals represent 34bp loxP and frt sites, respectively.
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Figure 2.
Expression of Has2 and Has3 during aspects of craniofacial morphogenesis. (A) Has2 is expressed in regions of precartilage formation, as well as around the developing vibrissae. (B) A high level ofHas3 expression is also apparent in the developing vibrissae.
248
Biosynthesis and biological degradation ofhyaluronan
Whole-mount in situ hybridisation analyses of Has2 and Has3 During vertebrate development, Has2 is expressed in tissues where hyaluronan is abundant, including the developing limb and endocardial cushions ofthe heart (data not shown). In contrast, Has3 expression is restricted to specialized tissues such as tooth and hair.
CONCLUSIONS Has2 is the major HA synthase involved in HA biosynthesis during vertebrate embryogenesis. Has2 is expressed in multiple tissues in a distinct temporal and spatial pattern, which correlates closely with tissues that are undergoing rapid expansion, cell migration and/or proliferation. Conversely, Has3 is expressed in a restricted number of tissues, although its expression overlaps, in part, with that ofHas2. The allelogenic targeting approach would provide a particularly powerful tool in dissecting the role of Has2-dependent HA function during embryonic development and potentially through adult maturation.
ACKNOWLEDGEMENTS This work was supported by research grants from the American Heart Association National Office AHA 0030184N and the March of Dimes Birth Defects Foundation #1FYOO-361 to APS.
REFERENCES I. 2.
3.
4. 5.
6.
A. P. Spicer & 1. A. McDonald, 'Characterization and molecular evolution of a vertebrate hyaluronan synthase (HAS) gene family', J. Bioi. Chem., 1998, 273, 1923-1932. N. Itano, T. Sawai, M. Yoshida., P. Lenas, Y. Yamada., M. Imagawa., T. Shinomura, M. Hamaguchi, Y. Yoshida., Y. Ohnuki, S. Miyauchi, A. P. Spicer, 1. A. McDonald & K. Kimata., 'Three isoforrns of mammalian hyaluronan synthases have distinct eniymatic properties', J. Bioi. Chem., 1999,274,25085-25092. T. D. Camenisch, A. P. Spicer, T. Brehm-Gibson, J. Biesterfeldt, M. L. Augustine, A. Calabro, Jr., S. Kubalak, S. E. Klewer & J. A. McDonald, 'Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme. J. Clin. Invest., 2000, 106,349-360. E. N. Meyers, M. Lewandoski & G. R. Martin, 'An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination', Nature Genetics, 1998, 18, 136-141. R. A. Conlon & 1. Rossant, 'Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo', Development, 1992, 116,357368. S. M. Dymecki, 'Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice', Proc. Natl. Acad: Sci. USA, 1996,93, 6191-6196.
INFLUENCE OF SUBSTRATE AND ENZYME CONCENTRATIONS ON HYALURONAN HYDROLYSIS KINETICS CATALYSED BY HYALURONIDASE T. Asterion, B. Deschrevel, F. Gonley,
J.e. Vincent*
Laboratoire "Polymeres. Biopolymeres, Membranes" UMR 6522 Universite de Rouen - CNRS 76821 Mont-Saint-Aignan cedex, France
ABSTRACT
It has been shown that both hyaluronan (HA) and hyaluronidase (HAase) are present at high levels in the extracellular matrix (ECM) of cancer tumours. As high molecular weight HA (h-HA) is anti-angiogenic and low molecular weight HA (l-HA) is angiogenic, HAase may play an important role in regulating the h-HA/I-HA balance. A detailed study of the HA hydrolysis kinetics catalysed by HAase may thus be useful to understand its implication in cancer development and should be examined in vitro through a model system. Kinetics was monitored by the Reissig method (improved in our laboratory) which estimates the number of reducing ends formed by hydrolysis of 13(1-4) glycosidic bonds. We carried out the effects of both substrate and enzyme concentrations and of ionic strength on the kinetics. The most original results concern the non-linear shape of the time course and the atypical behaviour of the initial rate versus both substrate and enzyme concentrations. The substrate dependence curve seems to be of the Michaelian type only for low concentrations. A significant decrease in the initial rate is observed for higher concentrations, which suggests inhibition phenomena. In order to explain our experimental results, we have to consider that enzymatic degradation of a polysaccharide is a particular case of enzymatic reactions because of the polymeric nature of the substrate. Elaboration of a kinetic modelling, in agreement with the experimental results, allows us to suggest a few assumptions about the reaction mechanism. KEYWORDS
Hyaluronan, hyaluronidase, enzymatic hydrolysis, kinetics, reducing ends assay, substrate-dependence, enzyme-dependence. INTRODUCTION
Hyaluronan (HA) is a linear negatively-charged polysaccharide composed of Dglucuronic acid-13(l,3)-N-acetyl-D-glucosamine disaccharide units linked 13(1,4). HA is present in both microorganisms and vertebrates and is one of the main component of the extracellular matrix (ECM) of higher animals. Its size ranges from a few hundred thousand to ten million Daltons according to its origin. HA is the main substrate of hydrolytic enzymes, called hyaluronidases (HAases). Both lysosomal and testicular HAases (E.C. 3.2.1.35) hydrolyze 13(1,4) glycosidic bonds producing HA fragments with a N-acetyl-D-glucosamine at the reducing extremity [1].
250
Biosynthesis and biological degradation ofhyaluronan
Several techniques [2-3] have been described to assay HAase activity: turbidimetry, viscosimetry, ELISA-like assay, HPLC-SEC chromatography and colorimetric assays [4-7]. The colorimetric method of Reissig et al. [7] has mostly been used to investigate HAase activity and most of the authors estimate the activity by measuring the total reducing ends produced by the reaction after a given incubation time ranging from 10 minutes to 2 hours [8-9]. However, due to the non-linear shape of the kinetics, the enzyme activity should be estimated by the initial rate of the reducing ends production. Here, we are interested in the kinetics of the HA-HAase reaction and the method of Reissig et al. is particularly suitable since it gives the rate of glycosidic bond cleavage directly. The determination of the initial reaction rate allows us to study the influence of enzyme and substrate concentrations. MATERIALS & METHODS
Bovine testicular HAase with a specific activity of 990 units per mg was obtained from Sigma (H 3884). Sodium hyaluronate (HA) from bovine trachea was purchased from Fluka (Nr. 366047/1). HA and HAase were used without any further purification. The Reissig method needs two solutions: i) a borate solution prepared by dissolving 4.94 g boric acid and 1.98 g potassium hydroxide in 100 mL Milli-Q water (Waters), and ii) a 0.1 giL DMAB solution prepared by dissolving 5 g DMAB (Sigma D 8904) in 6.25 mL hydrochloric acid 12 N completed with glacial acetic acid to a final volume of 50 mL. A 1110 dilution of this solution with glacial acetic acid was performed just before use. Absorbance was measured with an Uvikon 860 Kontron spectrophotometer equipped with a temperature-controlled chamber and connected to a Pc. Measurement of HAase activity is based on the Reissig method [7] which determines the concentration of liberated reducing ~-N-acetyl-D-glucosamine from HA. P-Nacetyl-D-glucosamine (Sigma A 8625) was used as a standard. The HA solution, containing 5 mM ammonium acetate pH 5, is placed in a reactor, adjusted to the chosen pH with either acetic acid or ammoniac, stirred and temperature controlled at 37°C. The reaction was started by adding concentrated HAase. At each time point, a 200 J.lL aliquot of the reacting mixture was removed from the reactor and added to 50 J.lL of borate solution in a glass tube. The tube is immediately vortexed, heated in a boiling water bath for exactly 3 minutes, then placed in a cold water bath (approximately 10°C) until 10 aliquots have been treated. Then, 1.5 mL of the diluted DMAB solution was added to each tube. The content is rapidly vortexed and placed at 37°C for exactly IS minutes. This was transferred to a plastic cuvette of I cm pathlength, immediately scanned between 400 and 700 nm and the spectrum saved on the computer. RESUL TS & DISCUSSION
By following the entire time course of the reducing ends concentration under various HA and HAase conditions, non-linear shapes were observed which have been fitted by a bi-exponential curve [10]. The HAase activity was then determined by measuring the reaction rate at time zero. Influence of the substrate concentration
The HAase activity was measured for different HA concentrations ranging from 0.1 to 3 mg/mL. An atypical substrate-dependence curve (fig. 1) was observed. At low HA
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concentrations, the HAase activity increases classically whereas at high HA concentration it falls down and stabilises at a low level. The kinetics does not obey the Michaelis-Menten mechanism. However, apparent constants Vm and Km could be determined for the low HA concentrations but only KIn (0.25 mg/mL) may have a certain signification. Concerning high HA concentrations, three hypotheses can be proposed: i) solution viscosity hinders enzyme diffusion, ii) changes in HA conformation penalise HA-HAase interactions, and iii) inhibition phenomena exist. Influence of the enzyme concentration The HAase activity was then measured for different HAase concentrations ranging from 0.25 to 5 mg/mL. As for the substrate-dependence curve, an atypical enzymedependence was obtained. A sigmcidal shape (fig. 2) was observed with a very low activity below lmg/ml HAase. This behaviour may be interpreted as a transition between a low level and a high level regimes which can be used for regulatory mechanisms by the ECM. These observations are in agreement with the hypotheses proposed above. CONCLUSION Whereas most of the authors estimate the enzyme activity by measuring the total reducing ends produced by the reaction after a given incubation time, we followed the entire time course of the reducing end production and deduced the HAase activity from the rate at time zero. The most original results concern the atypical behaviour of the initial rate versus both substrate and enzyme concentrations. The substrate-dependence seems to be of the Michaelian type only for low concentrations. A significant decrease in the initial rate is observed for higher concentrations. The sigmoidal enzymedependence may be interpreted as a transition between low level and high level regimes. The enzymatic hydrolysis of HA is a particular case of enzymatic reactions because of the polymeric nature of the substrate: i) the structure and conformation of the substrate may greatly influence the geometry of the active site of the enzyme, and ii) non-specific
252
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. However, long-term exposure to dust containing LPS, as occurs in grain handlers or pig farmers, results in pulmonary inflammation accompanied by respiratory symptoms, and is implicated in the development oflung diseasesv'. In vitro studies have demonstrated that LPS can induce HA production in different types ofcells, including lung fibroblasts and endothelial cells ofthe microvascular bed", The aim of the present study was to investigate the effect oflocal LPS exposure on HA deposition in the lung in vivo. To this end, we used a murine model of acute pulmonary inflammation, induced by a single intratracheal instillation of LPS. Neutrophil influx in the lungs was determined as a characteristic for acute inflammation. HA content was assessed by histolocalization using biotinylated HA binding protein (b-HABP).
MATERIALS & METHODS Experimental protocol Male Swiss mice (30-40 g) were obtained from Charles River Breeding Laboratories (Heidelberg, FRG). Animals were housed individually in standard laboratory cages and allowed food and water ad libitum throughout the experiments, which were carried out under a protocol approved by the Institutional Animal Care Committee of the Maastricht University, The Netherlands. Mice were instilled intratracheally (IT) by a non-surgical technique". Bromothymol blue dissolved in 50 1110.9% NaCI was instilled to check distribution ofthe solution in the lung. Macroscopic and microscopic analysis demonstrated that blue marker dye had spread throughout the whole lung. Mice (n=6 per group) were anaesthetised by intraperitoneal injection of 3 mg/kg xylazinc and 75 mg/kg ketamin (Nimatek, AUV Cuijk, the Netherlands). 5 ug of LPS (Escherichia coli, serotype 055:B5, Sigma, St. Louis, MO) dissolved in 50 IIIsterile 0.9% NaCl was instilled IT via a canule, followed by 0.1 5 mlofair. Sham mice were instilled IT with 50 IIILPS-free sterile 0.9% NaCl, whereas control mice received no treatment. After IT treatment, the mice were kept in an upright position for 10 minutes to allow the fluid to spread throughout the lungs. Mice were sacrificed at 4, 8, 24 or 72 hours after instillation. After thoracotomy, lungs were prepared for light microscopy and myeloperoxidase analysis.
Histology The left lung was inflated with 10% phosphate-buffered formalin (pH 7.4) at a pressure of 20 cm H 20 through the trachea for 15 min and subsequently fixed in formalin for 24 hours. After paraffin embedding, 4 11m sections were cut and stained with hematoxylin and eosin (HE) for histological analysis.
Determination of myeloperoxidase Myeloperoxidase (MPO) was isolated from snap frozen lung tissue of the right lung as previously described!", Enzymatic detection of MPO was performed in a 96-well plate (Greiner, Nurtingen, FRG) according to Daemen et al.!'. Briefly, assay mixtures consisted of 40 J..lI 0.75 roM H 202 in 80 mMPBS (pH 5.4) and 40 J..ll sample diluted in 50 mMPBS (pH 6.0), 0.5% hexa-l,6-bis-decyltrimethylammonium bromide (Sigma). The reaction was
Intratracheal instillation oflipopolysaccharide
2 I9
initiated by adding 20 III of 8 mM 3,3',5,5'-tetramethylbenzidine (TMB; Boehringer Mannheim, Mannheim, FRG) in dimethyl sulfoxide (Sigma) and stopped after 15 min by adding 100 Ill/well 1 M H 2S04 . Subsequently, optical density was determined at 450 nm. All samples were assayed in triplicate. MPO activity was calculated per mg lung tissue and corrected for wet/dry ratios. A titration curve of horseradish peroxidase was used for the calculation ofMPO activity, which is expressed in arbitrary units (mean ± SEM). Statistical analysis was performed by means ofMann-Whitney U test and probabilityvalues below 0.05 were considered to be statistically significant.
Histolocalization of HA Histolocalization of HA was determined on paraffin sections using biotinylated bovine nasal cartilage HABP, which was a kind gift from J. Melrose (University of Sidney, Australia). Sections were subjected to deparaffinization followed by rehydration. Sections were stained with b-HABP (50 ug/ml) at 4°C for 24 hours. After washing, the Vectastain avidin:biotinylated peroxidase complex (ABC) system was used according to the manufactor's instructions (Vector, Burlingame, CA). Enzymatic reactivity was visualised with 3-amino-9-ethylcarbazole. Sections were lightly counterstained with hematoxylin and mounted in faramount (DAKO, Glostrup, Denmark). No significant stainingwas detected in sections pre-treated with 50 U/mJ Streptomyces hyaluronidase (Calbiochem, San Diego, CA) at 37°C for 2 hours indicating that this HABP staining reaction was specific for HA.
RESULTS & DISCUSSION IT LPS instillation in mice results in a strong acute pulmonary inflammation Analysis of general inflammatory characteristics on HE stained paraffin sections demonstrated that IT LPS challenge resulted in a transient pulmonary inflammation. Strong infiltration of neutrophils into the alveolar area was observed, which was found to be timedependent. Presence of neutrophils in the alveolar spaces was evident from 8 hours after -,~ 60 C :l
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Figure 1. Quantification of pulmonary neutrophil infiltration after IT LPS instillation, MPO activity was measured in lung homogenates and expressed in arbitrary units (mean 'if SEM). * P, Nicole English" Vincent C. Hascall1 , Markku Tammr', Robert Hyman' /Cancer Biology Laboratory. The Salk Institute. P.O. Box 85800. San Diego, CA 92186. USA 2
Department of Biomedical Engineering. Cleveland Clinic Research Institute. 9500 Euclid Avenue. Cleveland. Ohio 44195. USA 3
Department ofAnatomy. University of Kuopio, FlN-70211 Kuopio, Finland
ABSTRACT The interaction of unlabeled HA preparations with cell surface CD44 was assayed by their ability to inhibit the binding of high M, fluorescein-conjugated HA (FL-HA). Using unlabeled low M, hyaluronan oligomers of defined sizes, we observed monovalent interaction of CD44 with oligomers between 6 and 18 sugar residues. Above 20 sugars, there was a 2-4-fold increase in blocking activity of the oligomers, suggesting that divalent binding was occurring. Pretreatment of the cells with IRAWBl4, a CD44specific inducing mAb, resulted in a more dramatic increase in blocking activity of HA oligomers of lengths above 18 sugars compared to their activity on non-induced cells, suggesting that the mAb greatly enhanced divalent binding and/or promoted more multivalent interactions. FL-HA fragments of 100 kDa dissociated rapidly from mAb induced cells in the presence of excess unlabeled HA. 3) In the absence of a large excess of unlabeled HA, bound FL-HA dissociated very slowly from both mAb induced and non-induced cells. These observations support the conclusion that CD44 mediated HA binding at the cell surface is the result of multiple weak and transient binding interactions. INTRODUCTION CD44 is a major cell-surface receptor for hyaluronan. It is involved in extra-cellular matrix assembly, in cell adhesion and migration, and in tumor cell invasion and metastasis 1-4. CD44 shares an -100 amino acid region of homology with other HA binding proteins termed a "link module" or "proteoglycan tandem repeat (PTR)" (see review"). Though CD44 uses a link module domain to bind HA, it differs in important ways from other HA binding proteins. While the link modules of TSG-6 and link protein are capable of binding HA on their own 5-7, HA binding by CD44 requires sequences outside the link module 8, is regulated by cell specific factors 1.2.9-12, and probably requires multiple CD44/HA engagements to achieve a functional avidity. Depending on the type and activation state of the cell in which it is expressed CD44 may be inactive (unable to bind HA), inducible (able to bind HA upon treatment with certain
342
Cell surfaces and hyaluronan receptors
CD44-specific mAb or inducers of cell activation such as phorbol ester) or constitutively active (able to bind HA without any treatment) 9. Our laboratory's study of CD44 has focused on the mechanisms regulating its hyaluronan binding function including the role of CD44 cytoplasmic and transmembrane domains, cell specific glycosylation of CD44 and receptor dimer-/oligomerization 9. 13-1S. The activation state seems to be determined, at least in part, by posttranslational modification (especially glycosylation) of the CD44 molecule itself, because CD44-Ig fusion proteins display the same activation phenotype as the cell surface CD44 of the cells in which they are made 10.11.15.16. The most significant feature that distinguishes CD44 from other HA binding proteins is that CD44 binding to HA takes place at the cell surface where multiple closely arrayed CD44-receptor molecules interact with the highly multivalent repeating disaccharide chain of HA. The affinity of a single CD44-HA binding domain for HA is likely to be very low. CD44-Ig fusion proteins (which are at least dimeric) were estimated to have K, in the range of 10,4 to 10-5 IS. Thus, binding of a CD44-positive cell to an HA substrate or of a soluble HA molecule to the surface of a CD44-positive cell involves multiple weak receptor-ligand interactions. These features of CD44 in conjunction with the unique properties of the HA ligand, which presents a very long, repeating chain of linked CD44 binding sites, create a system that is highly dependent on multiple cooperating interactions in order to achieve functional binding.
RESULTS Binding of HA to a CD44+ cell requires a critical density of CD44 molecules
Evidence for cooperativity from the CD44 side includes the observations that: 1) A 'threshold' level of CD44 expression is required for detectable binding of FL-HA (or adhesion to an HA substrate), and once that density is reached, binding activity increases with increasing CD44 expression 13-15. 2) Clustering of CD44 on the cell surface, either as covalent dimers or larger aggregates, increases the HA binding activity without an increase in the number of CD44 molecules 13.14.17. CD44 clustering lowers the threshold at which HA binding can be detected. Length Requirements for HA binding to cell surface CD44
What are the requirements on the HA side of the binding reaction? For example, what length of HA chain is needed to: 1) occupy a single CD44 binding site? ; 2) .. .link two CD44 receptors in a dimeric CD44 complex with HA, and 3) ... achieve stable binding of HA at the cell surface? To explore some of these questions we have looked at the interaction of HA oligomers of defined sizes with cell surface CD44. HA binding was detected by flow cytometry using a fluorescein-conjugated HA probe (FL-HA) made from rooster comb HA with a M, of about 106 • For most binding studies we used a CD44-negative cell line that was transfected with wild type CD44H and selected for high levels of CD44 expression. CD44 expressed in this line is binds HA constitutively. Binding of small, unlabeled HA oligosaccharides of defined M. (prepared as described in 18) was detected by their ability to block binding of FL-HA. Cells that had been pre-incubated with serial dilutions of unlabeled oligomers were subsequently labeled with a standard dilution of FL-HA (still in the presence of the unlabeled competitor) and assayed for FL-HA binding by flow cytometry 17. Sets of blocking
Binding by cell surface CD44
343
curves were used to determine the concentration of unlabeled HA giving 50% inhibition of FL-HA binding as a measure of the relative avidity of the unlabeled HA for CD44. About 5x more HA4 was required to achieve 50% blocking than HA6 or HAs. Therefore, it appeared that 6 sugars was the minimum size of HA chain to occupy the CD44 binding site. Chains of 6 and 8 sugars blocked almost equally, but there was an increase in blocking efficiency with oligomers between 10 and 18 sugars. This difference was small, but was seen repeatedly. We suggest that six sugars is the minimal size to occupy the CD44 binding site, in agreement with previous studies of Underhill et al and Knudson et aI 19•2o, but that an oligomer of at least 10 sugars is optimal. A series of larger oligomers was used to look for an HA chain length that would engage two CD44 molecules on the cell surface. We expected that there would be an increase in blocking activity at this point, because binding to two CD44s would reduce the probability of dissociation of the sugar chain. Each preparation of > 18 sugars was a mixture of oligomers of several sizes with mean sizes of 22, 26, 30, 34, and 38 sugars. All together they encompassed a range between 20 and over 40 mono-saccharides. The micromolar concentration required for 50% inhibition of FL-HA binding was plotted against oligomer chain Iength!". Between HAlO and HA 1S ' there was little change in the concentration needed for 50% inhibition with increasing oligomer size. Above HA ,s, there was a 2-4-fold increase in blocking efficiency. The increase in avidity at HA_20 suggests that divalent binding occurs at this chain length. It is noteworthy that there was no evidence for a further increase in avidity between oligomer preparations beyond HA20 up to over 40 sugar chains with non-induced cells. The significance of this observation is unclear. If linked binding of 3 or 4 CD44 molecules were occurring, one would expect some increase in binding avidity with the larger oligomer preparations. One possible explanation is that CD44 has an inherent tendency to spontaneously dimerize in the membrane and thus allow dimeric binding. (The possibility of CD44 dimerization has been suggested in several publications 2 1•23 ) . Higher order aggregation may not be favorable without mAb induction (see below). If the cells were pretreated with the inducing mAb IRAWB 14, which enhances FLHA binding, the blocking curves with lower M, oligomers, below HA 20 , were very similar to the curves with non-induced cells. But with oligomers of 20 sugars and above, there was a much more dramatic increase in blocking efficiency than with non-induced cells, and there was a steady increase in blocking avidity (decrease in the concentration needed to achieve 50% blocking) with each increase in HA chain length. This suggested that the mAb specifically enhanced divalent (and multivalent?) CD44 binding to the longer HA chains. IRAWB 14 mAb may promote larger aggregations of CD44 and/or orient neighboring CD44 molecules in a configuration that optimizes the possibility of dimeric or more multivalent binding. Though we observed quite efficient blocking of FL-HA binding to IRAWB 14 induced cells with oligomers containing chains of 30-40 sugars, we did not obtain detectable binding with AMAC- conjugated oligosaccharides of -22 or -38 sugars by microscopy or flow cytometry (Lesley, Tammi and Hascall, unpublished). It was concluded that binding of these small oligomers was too weak and too transient to detect by direct binding methods. In order to determine what chain length was needed to obtain stable binding, we digested high M, FL-HA with Streptomyces hyaluronidase (Fluka). Digests were then passed through Centricon filters (Millipore) of different molecular weight cut off (MWCO) to estimate their sizes. Results of digestion and filtration are shown in Figure 1. Sample D, which was incubated along with the others, but with no hyaluronidase added, was retained above the 100 kDa MWCO filter, while
344
Cell surfacesand hyaluronan receptors
all the digested samples passed through. Sample A, digested with the highest concentration of hyaluronidase (1.25 TRU/ml final concentration) readily passed through 50 kDa and 30 kDa MWCO filters, but was mostly retained above the 10 kDa MWCO filter. Digests Band C, two- and four- fold dilutions, respectively, of the enzyme concentration used in A, both concentrated between the 30 and 50 kDa MWCO filters, but B, digested with the higher hyaluronidase concentration, had more material below 30 kDa, while C had more material above 50 kDa. Hence, we have assigned estimated sizes of -30 kDa and -50 kDa to B and C, respectively. A is assigned a size of >30 kDa. Significantly, none of the digested samples bound to non-induced CD44+ cells, while all bound to IRAW14 treated cells. Table 1 summarizes the size estimates and binding properties of the FL-HA fragments. FL·HA digests separated by Centricon Filtration 80
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A, 100 kD
30-50 kD
50-100 kD
10-30 kD
500
>25
Binding to Non-induced cells
Binding to IRAWBI4 induced cells
+
+ + + +
Binding by cell surface CD44
345
Dissociation of FL-HA from Induced CD44+ cells -0-
___ -
100
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High MW FL-HA -50 kDa FL-HA -30 kDa FL·HA T,,1I2
80
60
Cl
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'0 C, reaching a plateau after 3h. The mechanism of LYVE-I mediated HA-uptake, like that for CD44-mediated uptake is currently unclear. In common with CD44, the LYVE-I molecule does not contain motifs for endocytosis via the clathrin-mediated pathway (ie. the clathrin and AP-2 clathrin adapter binding motifs NPXY and YXX<j> respectively, see ref. 27). Indeed LYVEI also lacks the di-hydrophobic LV motif present within the CD44 cytoplasmic tail that mediates basolateral sorting in polarized epithelial cells 28. Ongoing experiments in our laboratory are currently directed towards characterizing the apparently unusual endocytic mechanism by which LYVE-I mediates the uptake of hyaluronan. What is the physiological role of LYVE·l ? The lymphatic system forms an extensive network of vessels, facilitating the migration
362
Cell surfaces and hyaluronan receptors
DClTUMOUR • LYVE·l CD44
\1'
HA
Figure 5. Possible roles for LYVE-l in lymphatic vessel trafficking. The cartoon depicts a small lymphatic capillary underlying the dermis in skin. LYVE-l on the luminal and abluminal faces of lymphatic vessel endothelial cells is speculated to function as a receptor for the transport of HA and I or the migration of leukocytes or metastasizing tumour cells (expressing CD44) into the vessel lumen.
of leukocytes from the peripheral tissues to the lymph nodes for the purpose of immune surveillance. Moreover, the lymphatic system is known to be a major conduit for the transport and degradation of hyaluronan 7,8. During this process newly turned-over HA originating from the tissues enters the afferent lymphatics, is degraded in lymph nodes and exits to the vasculature via the thoracic duct, for uptake and terminal hydrolysis in the liver 8. Currently it is not known which receptor(s) are involved in HA-uptake and degradation in lymph node or in binding I transport of HA in lymph capillaries and vessels. Clearly, LYVE-l is a candidate for one or more of these processes. Given the finding that LYVE-l can mediate HA-uptake in vitro, and that LYVE-l appears to be expressed on both the luminal and ablurninal faces of the endothelium 15,24, it is tempting to speculate that LYVE-l plays a role in the transcytosis of tissue HA into the lumena of draining lymphatic vessels in vivo. Alternatively, LYVE-I through its capacity to bind HA might provide an adhesive surface for migration of cells bearing CD44 or other HA-receptors into the lymphatics. Examples might include dendritic cells such as epidermal Langerhans cells, macrophages or metastasizing tumour cells (see Figure 5). The availability of appropriate experimental models should allow these hypotheses to be tested in the near future.
FEL-l and FEL-2. As outlined above, EST database searches identified two further candidate HA receptors PEL-I and FEL-2 in addition to LYVE-I (see Figure 1). These encode large multidomain receptors containing a single Link module at the C-terminal end of the predicted extra- cellular domain - an unusual location for this module. The sequences of the PEL-I and 2 Link modules (not shown) correspond to those recently reported for two protein fragments termed WF-HABP and BM-HABP respectively 29 and are more similar to TSG-6 Link 30 than either LYVE-I or CD44 Link. In addition both the FEL-l and FEL-2 ectodomains contain ROD putative integrin-binding motifs and multiple EGF repeats together with tandem repeats of a protein module contained in the human TOF(3-
Novel Endothelial Hyaluronan Receptors
363
inducible matrix protein beta-Igh3 31 and the Drosophila cell surface Fasciclin I protein 32 - implicated in cell adhesion. Northern blot hybridization with cDNA probes for FEL-l and FEL-2 indicate both receptors are subject to extensive alternative splicing and are expressed in a variety of cells including both leukocytes and vascular endothelia. These properties raise the exciting possibility that FEL-l and/or FEL-2 may have some functional overlap with the CD44 molecule. However experiments with truncated ectodomain Fc fusion constructs in HAbinding ELISAs so far indicate the FEL Link modules are not constitutively functional. We
CONCLUSIONS. Searching the EST database for proteins containing the HA-binding Link module has yielded three novel receptors, LYVE-l, FEL-l and FEL-2. LYVE-l is expressed almost exclusively in the endothelium lining lymphatic vessels where it may play a role in the transport of HA and the migration of immigrant leukocytes. LYVE-l is also a powerful new marker for lymphatics that is currently being used to study tumour-associated lymphangiogenesis in human cancers and transgenic mouse models of neoplasia. FEL-l and FEL-2 are large multidomain integral membrane molecules likely to play roles in cell and extracellular matrix adhesion. Preliminary data suggest they are expressed in both leukocytes and endothelial cells and that they are not constitutively active as HAbinding molecules. Research in our laboratory is currently directed towards understanding the physiological functions of each of these molecules.
ACKNOWLEDGEMENTS. This work was funded by grants from the MRC, Cancer Research Campaign and The A.I.C.R. to Dr David Jackson. Dr.Suneale Banerji is a post-Doctoral Research associate and Remko Prevo a D.Phil. student in the MRC Human Immunology Unit, Oxford.
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P. J. Neame & F. P. Barry, 'The Link proteins', Experientia , 1993, 49, 393-402. A. J. Day, 'The structure and regulation of HA-binding proteins.', Biochem. Soc. Trans. , 1999, 27, 115-121. D. Kohda, C. J. Morton, A. A. Parkar, H. Hatanaka, F. M. Inagaki, 1. D. Campbell & A. J. Day, 'Solution structure of the link module: a hyaluronan binding domain involved in extracellular matrix stability and cell migration.', Cell, 1996,86,767-775. S. Banerji, J. Ni, S.-X. Wang, S. Clasper, J. Su, R. Tammi, M. Jones & D. G. Jackson, 'LYVE-l, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.', J. Cell Bioi. ,1999, 144, 789-801. Prevo, R., Banerji, S., Ferguson, D.J.P., Clasper, S. & Jackson, D.G. 'Mouse LYVE-l is an endocytic receptor for hyaluronan in lymphatic endothelium.' J. Bioi. Chern. 2001,276, 19420-19430. A. N. de Belder & K. O. Wik, 'Preparation and properties of fluorescein-labelled hyaluronate.', Carbohydrate Res. ,1975,44,251-257. S. Banerji, A. J. Day, J. D. Kahmann & D. G. Jackson, 'Characterization of a functional hyaluronan-binding domain from the human CD44 molecule expressed in Escherichia coli.', Protein Exp. Purification. , 1998, 14, 371-381. R. J. Peach, D. Hollenbaugh,!. Stamenkovic & A. Aruffo, 'Identification of hyaluronic acid binding sites in the extracellular domain of CD44.', J. Cell Bioi. , 1993, 122, 257-264. J. Bajorath, B. Greenfield, S. B. Munro, A. J. Day & A. Aruffo, 'Identification of CD44 residues important for hyaluronan binding and delineation of the binding site.', J. Bioi. Chern. ,1998, 273, 338-343. D. Liu & M. S. Sy, 'A cysteine residue located in the transmembrane domain of CD44 is important in binding of CD44 to hyaluronic acid.', J. Exp. Med. , 1996, 183, 1987-1994. C. T. McGary, R. H. Raja & P. H. Weigel, 'Endocytosis of hyaluronic acid by rat liver endothelial cells.', Biochem. J. , 1989, 257, 875-884. P. W. Kincade, Z. Zheng, S. Katoh & L. Hanson, 'The importance of cellular environment to function of the CD44 matrix receptor.', Current Opin. Cell Bioi. , 1997, 9, 635-642. Mandriota, S. Jussila, L., Jeltsch, M., Compagni, A., Baetens, D., Prevo, R., Banerji, S., Huarte, J., Montesano, R., Jackson, D., Orci, L., Alitalo, K., Christofori, G. & Pepper, M.S. 'Vascular endothelial growth factor-Cvmediated Iymphangiogenesis promotes tumour metastasis.' EMBO. J. 2001, 20, 672-682. Stacker, S.A. Caesar, c., Baldwin, M.E., Thornton, G.E., Williams, R.A., Prevo, R., Jackson, D.G., Mishikawa, S., .Kubo, H., & Achen, M.G. 'VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.' Nature Med. 2001, 7, 186-191. Skobe, M, Hawighorst, T., Jackson, D., Prevo, R., Janes, L., Velasco, P., Riccardi, L., Alitalo, K., Claffey, K. & Detrnar, M. 'Induction of tumour Iymphangiogenesis by VEGF-C promotes breast cancer metastasis.' Nature Med. 2001,7, 192-198. J. S. Bonifacino & E. C. Dell'Angelica, 'Molecular bases for the recognition of tyrosine-based sorting signals.', J. Cell Bioi. ,1999, 145, 923-926. H. Sheikh & C. M. Isacke, 'A di-hydrophobic Leu-Val motif regulates the basolateral localization of CD44 in polarized Madin-Darby canine kidney epithelial cells.', J. Bioi. Chern. , 1996, 271., 12185-12190. E. Tsifrina, N. M. Ananyeva, G. Hastings & G. Liau, 'Identification and characterization of three cDNAs that encode putative novel hyaluronan-binding proteins, including an endothelial cell-specific hyaluronan receptor.', Am. J. Pathol. 1999, 155, 1625-1633,. T. H. Lee, H. G. Wisniewski & J. Vilcek, 'A novel secretory tumor necrosis factorinducible protein (TSG-6) is a member of the family of hyaluronate binding proteins, closely related to CD44', J Cell Bioi, 1992, 116, 545-557. J. Skonier, M. Neubauer, L. Madisen, K. Bennett, G. D. Plowman & A. F. Purchio, 'eDNA cloning and sequence analysis of beta ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factorbeta.', DNA Cell Bioi. ,1992, 11,511-522. K. Zinn, L. McAllister & C. S. Goodman, 'Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drosophila.', Cell, 1998, 53, 577-587.
MUTUALLY EXCLUSIVE DISTRIBUTION OF LINK PROTEIN AND HYALURONECTIN DURING CARTILAGE MORPHOGENESIS: A ROLE FOR FREE HYALURONAN P. Rooney'>, N. Girard 2, B. Delpeejr', .J. Ponting'' & S. Kumar'' JDepartment
ofBasic Dental Science, Dental School, University of Wales College ofMedicine, Heath Park, Cardiff, CF14 4XY, UK.
2Laboratoire d'Oncologie Moleculaire, Centre Henri-Becquerel, Rauen, France. 3Department
ofPathological Science, University ofManchester, Oxford Road, Manchester, MI3 9PT,
UK
ABSTRACT
Cartilage morphogenesis is a pre-requisite for skeletal development and maintenance and involves a combination of cell division, cell hypertrophy and extracellular matrix secretion. Matrix secretion can account for up to 57% of the increase in volume of a cartilage long bone rudiment, however, the shape is regulated via the differential structure of the perichondrium where a multilayered, "tight" structure is found around the central hypertrophic zone and an overlapping, "loose" structure, which merges with the mesenchyme, is seen around the rounded zones. Hyaluronan (HA) is found within cartilage extracellular matrix as a backbone for aggregating proteoglycans (PG) and as a free glycosarninoglycan within a pericellular coat. In this study, the distribution ofPG-bound-HA has been determined in developing rat cartilage by a combination of antibodies to the HA binding proteins, link protein (LP) and hyaluronectin (HN) and PG-free-HA via enzyme-linked, sheep-brain HN as a probe. A mutually exclusive pattern of staining was observed for LP and HN with intense LP staining within the cartilage matrix but not in the perichondrium or the surrounding mesenchyme. LP was most intense in the hypertrophic zone but little staining was observed pericellularly around hypertrophic cells. In contrast, HN and PG-free-HA was detected within the pericellular region, even during cartilage resorption, and was also present in both the perichondrium, particularly surrounding rounded zones and within the adjacent mesenchyme. At later stages, HA staining was detected at presumptive joint regions, separating the rudiments. We suggest that the LP antibody detects HA bound to the PO aggrecan, whereas the HN antibody and probe detects aggrecan-free-HA. PG-bound-HA is constrained within the extracellular matrix whereas free-HA would be capable of utilising hydrodynamic forces to expand and produce space which enlarging rounded zones can occupy. The observation that overlapping perichondrial cells are rich in free-HA indicates that these cells may regulate cartilage morphogenesis by synthesising free-HA, increasing the width ofthe zone and allowing expansion to occur. KEYWORDS
Cartilage development, hyaluronan binding proteins, perichondrium, free-hyaluronan
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Aspects of hyaluronan in joints
INTRODUCTION Cartilage morphogenesis appears to follow a similar pattern in every vertebrate species. The vertebrate limb develops as a outgrowth from the lateral body wall and consists of an ectodermal pouch filled with underlying mesoderm. I Chondrogenesis begins as a condensation of mesenchymal cells which, in the case of long bones is usually a sausage shape.' Cells in the centre of the condensation secrete cartilage extracellular matrix and the cells begin to be pushed apart, however, at this stage, the early cartilage rudiment consists of only two cell types arranged into three cellular zones along the proximal-distal axis - rounded, flattened and rounded.' Flattened chondrocytes rapidly hypertrophy and this, accompanied by the accumulation of extracellular matrix causes cells at the periphery of the central region to become constrained and aligned perpendicularly. At the light microscope level, this early perichondrium can be seen to be directly derived from what were flattened chondrocytes, e.g., in the developing chick ulna, at the stage where cartilage extracellular matrix secretion begins, the sausage shaped rudiment consists of 25 flattened cells across its short axis but when cell hypertrophy begins, the hypertrophic zone contains 17 cells across its short axis and the presumptive perichondrium is four At this stage the rudiment consists of three types of cells thick on each side.3,4 chondrocytes arranged into five identifiable zones - rounded, flattened, hypertrophic, flattened and rounded. As the rudiment continues to develop, the perichondrium expands to cover more than the central hypertrophic zone and eventually surrounds the entire rudiment. The perichondrium is a variable structure which is multilayered and "tight" around the hypertrophic zone with many cell-cell contacts, overlapping around the flattened zone and "loose" with little or no cell-cell contact at the rounded zone." From this stage onwards, the rudiment increases in length more than width with radial expansion occurring primarily in the rounded and flattened zones, we have suggested that this shape is regulated bi the structure of the perichondrium, a process called "directed dilation" by Wolpert ..5 Although the perichondrium can influence the shape of the cartilage rudiment, it can only act on the intrinsic factors and pressures produced by the cartilage. Cartilage morphogenesis is associated with cartilage growth and involves cell proliferation, cell hypertrophy and extracellular matrix secretion. Extracellular matrix secretion is by far the largest factor involved in cartilage growth, accounting for 57% in developing chick cartilage. In contrast, cell proliferation accounts for 6% and cell hypertrophy, where the volume of the cell increases eight-fold, accounts for 37%.3 The components of cartilage extracellular matrix have been extensively studied, 6,7 the major macromolecules being collagens (primarily types II, VI, IX, X, XI and other minor collagens) and proteoglycans (PO - primarily aggrecan but also some nonaggregating PO) together with growth factors and glycoproteins. Large aggregating PO consist of many PO monomers, each bound to a hyaluronan (RA) backbone via the HAbinding protein, link protein (LP). However, cartilage extracellular matrix also contains the RA as a non-PG bound free glycosarninoclycan, located within the pericellular coat surrounding chondrocytes." HA is a non-sulphated glycosarninoglycan composed of a repeating disaccharide unit of D-glucuronic acid and N-acetyl-D-glucosarnine which has long been linked with morphogenesis and differentiation of several cell types,"!' where it is thought to playa role either by its hydrodynamic properties or by cell-matrix interactions. 12.13
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In this study, we have investigated the distribution of HA in developing rat cartilage with a view to determining if HA plays a role in the morphogenesis of the tissue. Our specific aim has been to localise and evaluate the role of PG-free-HA using a combination of antibodies to the HA-binding proteins LP and hyaluronectin (HN - a glycoprotein isolated from sheep and human brain and thought to be related to the PG versican I4•17) and an enzyme-linked HN probe designed to bind PG_free_HA. 18 . The results indicate that LP and HN distribution are mutually exclusive and that PG-free-HA plays a role both in the increase in volume observed during cell hypertrophy and also in the radial expansion of rounded cell zones. PG-free-HA is synthesised by cells of the perichondriwn, once again implicating this tissue in morphogenesis. MATERIALS AND METHODS Tissues Embryos were removed from pregnant Sprague-Dawley rats at days 16, 17 and 18 of gestation. Individual fore and hind limbs were removed and the entire limbs were fixed and processed for wax histology. 5 urn sections were cut through the whole limb along the long axis. Sections were stained with Haernatoxylin and Eosin for visualisation and serial sections were utilised for immunohistochemistry and enzyme-linked histochemistry. Immunohistochemistry Following de-waxing, sections were pre-treated with H202 and exposed to the primary antibodies, polyclonal anti-mouse LP antibody, 8-A-4, (kindly donated by Professor B. Caterson, Cardiff University, Wales) or porclonal anti-rabbit human HN purified by immunoadsorption as previously described. 1 Peroxidase anti-mouse and anti-rabbit antibodies were used as secondary antibodies and visualisation was observed following DAB treatment. Specific y globulins absorbed out on insolubilised HN were used as a control. Enzyme-linked histochemistry HA localisation was performed by an affinity immunological technique with alkaline phosphatase - linked sheep brain HN as previously described." Enzymatic activity was detected with Fast Red in the presence of Naphtol -As-Mx as substrate and levamisole as endogenous phosphatase inhibitor. Nuclear counterstaining was performed with haernatoxylin. Controls were obtained by two pre-incubations with Streptomyces hyaluronidase, 20 TRU/ml for 2 hours at 37°C in a humidified chamber. RESULTS The results from this study show that the distribution of LP and lIN are mutually exclusive in developing cartilage (Figs. 1 and 3). At all stages of development, LP is restricted to cartilaginous regions with negative staining in the perichondrium and mesenchyme (Fig. 1). The most intense staining was observed in the hypertrophic cell zone but little LP was detected within the pericellular region (not shown). In contrast, HN was detected within both the perichondrium and mesenchyme adjacent to the
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Figure 1. Link protein restricted to cartilaginous regions. 16 day embryo. DAB/peroxidase staining.
Figure 3. HN staining of Figure 1 showing opposite distribution as LP. DAB/peroxidase staining.
Figure 2. HN staining around expanding cartilage zone and extending into the perichondrium. DAB/peroxidase staining.
Figure 4. HN staining in presumptive joint region of 18 day embryo. DAB/peroxidase staining.
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rounded cell zone (Figs. 2 and 3). Each limb contained rudiments of varying developmental age along the proximal-distal axis and consequently the distribution of HN at differing developmental stages could be assessed. In the most distal digits (i.e. the youngest stages), HN was found along the entire length of the perichondrium but was still most intense at the rounded zone perichondrium (Fig. 2). At later stages, HN was restricted to rounded zone perichondrium (Fig. 3) and in developmentally older rudiments, HN was only observed in the presumptive joint regions separating the rudiments (Figure 4). Within the hypertrophic cell zone, intense HN staining was observed at the periphery of hypertrophic cell lacunae and also in the immediate pericellular region, extracellular matrix outside the pericellular region was negative for HN (Fig. 5). When an enzyme-linked HN probe was used to detect PG-free-HA, it was found to co-localise with HN but was more extensive. PG-free-HA was present in the perichondrium and adjacent mesenchyme of embryos of all stages but was also present throughout the extracellular matrix of hypertrophic cells (Figs. 6, 7). Pre-treatment of the sections with hyaluronidase completely removed any positive staining indicating that HA was being detected by the probe (Fig. 8). DISCUSSION Due to the glycosaminoglycan nature of HA, direct immunolocalisation is difficult, consequently, in this report, HA localisation was assessed by the localisation of HA binding proteins and the idea that binding proteins indicate the presence ofHA. LP is a HA binding protein which is known to bind the aggregating PG aggrecan to its HA backbone, therefore, the restriction of LP to cartilage extracellular matrix is not unexpected (Fig. 1). lIN is also a HA binding protein which is thought to bind to HA at the same binding site and in the same manner as LP, 10 the binding regions both having two link modules arranged in tandem array;" consequently the ability of the HN antibody to detect HN suggests that the HA it is detecting is not bound to aggrecan. HN has a molecular weight of 40 - 70 kDa, it is considered to be a glycoprotein whose primary structure contains sequences which are identical with portions of the N-terminal domain of the PG versican and HN is often considered to be a breakdown product of versican splice variant Vl. 16•19 However, although versican is present in prechondrogenic tissues within developing limbs, it is believed to be absent in cartilage where it is replaced by aggrecan." The pericellular coat of chondrocytes is rich in aggrecan-free-HA, which is believed to be tethered at the cell surface by the HA receptor CD44. 8,20 Our data indicate that some of the aggrecan-free-HA is as a free glycosaminoglycan but some of it is bound to HN (Fig. 5). In addition, hypertrophic lacunae are also known to be rich in HA,21 our results suggest that once again, this HA is part free glycosarninoglycan and part bound to HN. Since hypertrophic chondrocytes increase in volume by up to eightfold, these observations suggest that HN-bound HA may still be able to swell in size and aid the increase in volume. The perichondrial region surrounding rounded cartilage zones demonstrated some of the most intense staining for both HN antibody and HN probe (Figs. 2, 3 and 7). We have already demonstrated that the perichondrium is this region consists of overlapping cells which offer little resistance to swelling pressure," We would suggest that perichondrial cells in these regions synthesise PG-free-HA and this HA swells allowing the rounded zone to expand. Once a tight perichondrium develops around the central
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Figure 5. HN staining within the hypertrophic zone of 17 day embryo. Intense staining is observed at the periphery of the lacunae and immediately pericellularly. DAB/peroxidase staining.
Figure 7. HN-detected free HA at perichondrium of 18 day rounded cartilage zone. Alkaline phosphatase detection.
Figure 6. HN-detected free-HA distribution in 17 day cartilage. Intense staining is observed in the hypertrophic region and in the perichondrium surrounding expanding cartilage. Alkaline phosphatase detection.
Figure 8. Hyaluronidase pretreatment totally removes any positive HN-detected free HA from 18 day embryos.
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hypertrophic zone, the cartilage rudiment increases in radial width mostly at the rounded cell zones.' Increase in width around the central zone is due mainly to appositional deposition of bone layers.' The presence of PG-free-HA around hypertrophic chondrocytes may also aid in the process of endochondral ossification where the hypertrophic cartilage zone is invaded by blood vessels, resorbed and replaced by bone. Breakdown products of HA, 3 - 10 disacccharide units in length are known to be angiogenic and stimulate the expression of transcription factors for metalloproteinases.J' It is conceivable that PG-free-HA may be more susceptible to degradation and thus allow/stimulate cartilage vascularisation.
CONCLUSION HA within developing rat cartilage appears to be present in three states, i) linked to the PG aggrecan via LP, ii) linked to the glycoprotein HN, iii) as a free glycosarninoglycan. lIN-linked HA and free HA are located in regions where expansion in chondrocyte or cartilage zone volume is observed. lIN-linked and freeHA utilise the ability of HA to swell via hydrodynamic forces to increase in volume and thus influence morphogenesis.
REFERENCES
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J. P. Iannotti, S. Goldstein, J. Kuhn, L. Lipiello, F. S. Kaplan & D. 1. Zaleske, The formation and growth of skeletal tissues, In: Orthopaedic Basic Science: Biology and Biomechanics of the Musculoskeletal System, Amer. Acad. of Orthopaedic Surgeons, J. A Buckwalter (ed.), 2000, pp 77 - 109. C. W. Archer, P. Rooney & L. Wolpert, The early growth and morphogenesis of limb cartilage. Prog. in Clin., Biol. Res., 1983, 11OA, 267-278. P. Rooney, C. W. Archer, & L. Wolpert, Morphogenesis of cartilaginous long bone rudiments, In: The role ofextracellular matrix in development, R. L. Trelsted (ed.), Alan R. Liss, New York, 1984, pp 305-322. P. Rooney & C. W. Archer, The development of the perichondrium in the avian ulna,1. Anatomy, 1992, 181,393-401. L. Wolpert, Cartilage morphogenesis, In: Cell Behaviour, R. Bellairs, A S. G. Curtis, & G. Dunn, 1982, Cambridge University Press, pp 359-372. A Serafini-Fracasini & J. W. Smith, The Structure and Biochemistry ofCartilage, Churchill-Livingstone, Edinburgh, 1974, pps 354. M.E. Grant, A P. L. Kwan, G. P. Bates, 1. T. Thomas & 1. McClure, The structure and synthesis of cartilage collagens, In: The Control of Tissue Damage, AM. Glauert (ed.), Elsevier Science Publishing Co. Inc., 1988, pp 3-28. C. B. Knudson & B. P. Toole, Changes in the pericellular matrix during differentiation of limb bud mesenchyme, Dev. Biol., 1985, 112:308-318. B. P. Toole, Proteoglycans and hyaluronan in morphogenesis and differentiation In: Cell Biology ofthe Extracellular Matrix, 2nd edn., E. Hay (ed.), Plenum Press, New York, 1991, pp 305-341. B.P. Toole, Hyaluronan, In: Proteoglycans: Structure, Biology and Molecular Interactions, R. N. Iozzo (ed.), Marcel Decker Inc, 2000, pp 61-92. P. Rooney & S. Kumar, Inverse relationship between collagens and hyaluronan in development and angiogenesis, Differentiation, 1993, 3-9.
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12. M. Brecht, U. Mayer, E. Schlosser & P. Prehm, Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochemical J.. 1986, 239: 445450 13. E. Ruoslahti & Y. Yamaguchi, Proteoglycans as modulators of growth factor activities, Cell, 1991, 64: 867-869. 14. B. Delpech & C. Halavent, Characterisation and purification from human brain of a hyaluronic acid-binding glycoprotein, hyaluronectin. 1. Neurochem 1981, 36: 855-859. 15. B. Delpech, Immunochemical characterisation ofthe hyaluronic acidhyaluronectin interaction, 1. Neurochem., 1982,38: 978-984. 16. B. Delpech, C. Maingonnat, A. Delpech, P. Maes, N. Girard, P. Bertrand, Characterisation of a hyaluronic acid-binding protein from sheep brain: Comparison with human brain hyaluronectin. Int. J. Biochem., 1991,23: 329-337. 17. D. R Zimmermann, Versican, In: Proteoglycans: Structure, Biology and Molecular Interactions, R.N. Iozzo (ed.), Marcel Decker Inc, 2000, pp327-342. 18. B. Delpech, Enzyme-linked hyaluronectin: A unique reagent for hyaluronan assay and tissue location and for hyaluronidase activity detection, Anal. Biochem., 1995, 229: 35-41. 19. R. V. Iozzo, Matrix proteoglycans: from molecular design to cellular function, Ann. Rev. Biochem., 1998, 67: 609-652. 20. W. Knudson, D. 1. Aguiar, Q. Hua & C. B. Knudson, CD44-anchored hyaluronan-rich pericellular matrices: An ultrastructural and biochemical analysis, Exp. Cell Res., 1996, 228:216-228. 21. P. Pavasant, T. Shizari, & C. B. Underhill, Hyaluronan contributes to the enlargement of hypertrophic lacunae in the growth plate, 1. Cell Sci., 1996, 109:327-334.
MAINTENANCE OF CARTILAGE EXTRACELLULAR MATRIX: THE PARTICIPATION OF HAS-2 AND CD44 Warren Knudson', Yoshihiro Nishida 2 & Richard S. Peterson! J
J
Department ofBiochemistry. Rush Medical College. Rush-Presbyterian-St. Luke's Medical Center, Chicago. IL 60612, USA
Department ofOrthopedic Surgery. Nagoya University School ofMedicine. Nagoya 466-8550, Japan
ABSTRACT
Cartilage is a tissue whose function is highly dependent on the maintenance of its extracellular matrix. Hyaluronan (HA) provides a unique role in cartilage, serving to sequester and retain proteoglycan within the tissue. A fraction of the HA-proteoglycan rich matrix remains anchored to the chondrocytes via interactions with the HA receptor CD44. We have also determined that chondrocytes utilize the same CD44 receptor to internalize HA, thus providing one mechanism for HA catabolism in cartilage. To better understand these processes, the expression of HA synthases (HAS) and CD44 was examined. Using quantitative-competitive RT-PCR we determined that human as well as bovine articular chondrocytes express primarily HAS-2, substantially less HAS-3 and no HAS-I. Antisense oligonucleotides directed against HAS-2 inhibited chondrocyte HA production in proportion to the level of inhibition of HAS-2 mRNA. It was therefore concluded that HAS-2 is the predominant gene involved in HA synthesis by articular chondrocytes. Incubation of chondrocytes with an anabolic cellular mediator, osteogenic protein-l , an agent that stimulates collagen type II and aggrecan production, resulted in a substantial increase in HAS-2 and CD44 mRNA copy numbers and, a pronounced accumulation of pericellular HA. No change in HAS-3 mRNA was observed. Interestingly, treatment of chondrocytes with the catabolic cytokine IL-la also resulted in an increase in HAS-2 and CD44. However in this case, less accumulation of HA within the pericellular matrix was observed. Visualization of intracellular HA suggested that the increased synthesis of HA due to IL-l was offset by enhanced CD44-mediated HA endocytosis. Thus, chondrocytes maintain their extracellular matrix composition, in part, by coordinating the expression of two components critically necessary for retention of proteoglycan, namely HAS-2 and CD44. KEYWORDS
Hyaluronan, HAS, CD44, antisense, chondrocytes, cartilage INTRODUCTION
The abundant extracellular matrix of cartilage is composed predominately of collagen type II and aggrccan proteoglycan (PG) 1. Aggrecan can be visualized within slices of normal human articular cartilage by staining of the tissue with the basic dye safranin O. Healthy human articular cartilage exhibits intense red staining throughout all the layers of cartilage with the exception of the uppermost layer of cells and matrix as shown in Figure
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l A, However, on some occasions we have received samples of normal human donor cartilages in which there is extensive loss of PG-with loss progressing in gradient fashion from the superficial to deeper layers of the tissue (Figure IB). Further, the loss of PG staining appears to originate within the pericellular matrix, the matrix closest in proximity to the cells of cartilage, the chondrocytes. Does this represent early osteoarthritis? The answer is as yet unknown. The tissue was derived from a donor with no known history of arthritis or joint pain and, by gross examination, the surface properties of the tissue were normal. However, osteoarthritis is characterized by an extensive loss ofPG. Further, the degenerative disease is thought to be due to inherent changes in the metabolism of the resident chondrocytes, a process termed chondrocytic chondrolysis 2. Therefore, the changes observed in Figure IB represent what one might expect to observe during the early stages of osteoarthritis. In other words, the cell-associated matrix of cartilage chondrocytes is where metabolism is most active and where changes will first be witnessed. How are PGs retained within cartilage? It has long been known that the principal PG of cartilage, aggrecan, forms strong, link protein stabilized interactions with filaments of another glycosaminoglycan, hyaluronan (HA) 3-6. Chondrocytic chondrolysis is thought to involve proteolytic cleavage of the core protein ofaggrecan and release of the PG from the tissue 2. We have determined that PG bound to filaments of HA are, in turn, bound to the plasma membrane of chondrocytes via the interaction of the HA with the HA receptor CD44 7-10. Chondrocytes removed from the tissue and grown in culture exhibit a prominent cell-associated matrix that we visualize using a particle exclusion assay (Figure 2A). This cell-associated matrix is rich in aggrecan yet sensitive to treatment with dilute Streptomyces hyaluronidase. The gel-like coat is retained even during centrifugation of the cells and as the assay suggests, able to resist the intrusion of small particles (in this case, glutaraldehyde-fixed red blood cells). Nonetheless, these cell-associated matrices can be readily released by the addition of HA hexasaccharides, oligosaccharides that compete for the binding of HA to the HA receptor, CD44. Further, when the coats are released by hyaluronidase treatment, the matrices are re-synthesized and re-assembled within 24 hours. However, re-assembly can be inhibited by the presence of HA hexasaccharides, nonsulfated chondroitin, or anti-CD44 antibodies 8-10. This, and other experiments have led us to develop a model for cartilage depicted in Figure 2B. Part of our long term goal is to determine how retention of HA and PG within the cell-associated matrix of chondrocytes participates in overall maintenance of healthy cartilage tissue. Although these cell associated coats can be displaced from chondrocytes via the addition of HA hexasaccharides, this does not mean that all of the HA and PG is displaced from the surface. Using 3H-acetate labeling, following hyaluronidase treatment, 3H-HA re-accumulates at the cell surface reaching semi-saturation between 4-6 hours. 3H-HA appears in the media after a short lag phase. So, of this cell surface bound HA, how much is displaceable? In separate studies by C. Knudson, >90% of the HA at the 2 and 4 hour time points was not displaceable 11. However, between 4 to 6 hours there was a switch. Now, at the 6 hour time point, 80% of the 3H-HA is displaceable. Similar percentages are observed for the retention ofPG. Thus with chondrocytes, a portion of the cell-associated matrix is represented by non-displaceable HA and PG. However, this residual matrix is not sufficient to support the assembly of a cell-associated matrix that can exclude particles as in Figure 2A. Most of the residual non-displaced PG of these chondrocytes can be removed by treatment with Streptomyces hyaluronidase and thus likely represents aggrecan bound to non-displaceable HA. What is this non-displaceable HA? We have always
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assumed that this represented synthase-bound HA (Figure 2B) and, that somewhere between 4-6 hours there is a transfer from the HA synthase to CD44. In some ceIls, coats are established that are non-displaceable with HA oligosaccharides and thus presumably represent a matrix assembled on a synthase-bound HA scaffold 12. Transmission electron micrographs of HA hexasaccharide-treated chondrocytcs stained with ruthidium hexamine trichloride to detect PGs reveal small patches of PG granuoles tethered to the ceIl surface 10. It is intriguing to speculate that these granuoles represent the PG bound to synthasebound HA as illustrated in Figure 2B. This cartoon figure illustrates that two components, namely CD44 and HA synthase, participate in the retention of HA and PG. As will be discussed below, these two proteins also share responsibility for the overaIl metabolism ofHA. Up until the last few years the nature and identity of the HA synthase (HAS) has been a mystery. Now it is known that a single protein is responsible for the synthesis ofHA 13. It is also now known that eukaryotic cells exhibit at least three separate but highly homologous HAS genes, each present on three different chromosomes 14. The HAS genes have been designated has-I, has-2 and has-3 13. HAS-I, HAS-2 and HAS-3 are purported to exhibit different elongation rates and synthesize HA of differing sizes 15. The HAS responsible for the synthesis of HA in cartilage was unknown and as such, represents the primary focus of this discussion. Three approaches were taken to determine the primary HAS gene used by chondrocytes. As anti-HAS antibodies were not readily available, the first approach was to document HAS mRNA expression levels. A second approach was to transfect chondrocytes or cartilage tissue slices with antisense phosphorothiate oligonucleotides directed against HAS mRNA and determine the effect on the synthesis of HA as well as other HA-mediated functions (e.g., periceIlular matrix assembly). The third approach was to examine changes in HAS mRNA expression that occur in response to ceIlular mediators/growth factors known to alter HA production. From these studies we determined that HAS-2 is likely the major HAS used by chondrocytes 16-19.
Figure 1. Human articular cartilage. Panels depict safranin 0 stained sections of normal articular cartilage derived from the talocrural joint of two human donors.
Figure 2. HAlPG cell-associated matrix of cartilage chondrocytes. Panel A depicts the cell-associated matrix as visualized by the use of a particle exclusion assay. Panel B is an illustration of the molecular components likely responsible for the matrix assembly shown in panel A.
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METHODS Tissue acquisition and culture Human articular cartilage was obtained from the talocrural ankle joint of normal human donors. Tissue was obtained within 24 hours of death through the Regional Organ Bank of Illinois (ROBI), and all donors were documented as having no known history of joint disease. Donors ranged in age from 33 to 58 years. Bovine articular cartilage was obtained from the metacarpophalangeal joints from 18-month-old steers obtained from a local slaughterhouse. Full thickness slices of articular cartilage were dissected under aseptic conditions and subjected to sequential pronase and collagenase digestion to liberate chondrocytes from the tissue 20. Isolated chondrocytes were cultured for 5 days in alginate beads with daily medium changes 20. In some experiments, the chondrocytes were treated with a cellular mediator for up to 14 days prior to release from the alginate using 55 mM sodium citrate 21. In other experiments, after 5 days of recovery, the chondrocytes were released from the alginate beads and used as monolayer cultures prior to analysis. For culture of intact cartilage tissue, full thickness slices (~I xlOxI0 mm) of human or bovine articular cartilage were cultured directly in 1.0 ml of DMEM-4.5 containing 10% fetal bovine serum (FBS). Typically, the slices were used in an experiment following I day of culture for recovery. At the end of the experiment, the slices were either (I) frozen in liquid nitrogen, ground to a powder and extracted for total RNA using the Trizol reagents 18, (2) resuspended in 100 mM Nf4Acetate, 0.0005% phenol red, pH 7.0 at 100 mg tissue wet weight per ml and incubated with 125 ug/ml Protease K for fluorophore-assisted carbohydrate electrophoresis (FACE) analysis 18 or; (3) embedded in Histo PrepTM freezing medium for cryostat sectioning (8.0 urn) for histochemical / immunohistochemical staining for PG, HA and CD44. For HA staining, the tissue sections were pretreated with 2 units of chondroitinase ABC (at pH 8.0) for 2 hour at 37°C for unmasking. Following this treatment the tissues were incubated with 2.0 ug/ml of a biotinylated HA binding protein (HABP) probe for 2 hour at room temperature followed by streptavidin peroxidase and color reagents for the detection of HA. In other experiments, human cartilage tissue was dissected into two layers (superficial/uppermiddle and lower-middle/deep layers) 22. Each of the dissected cartilage slices was cultured separately in the presence or absence of IL-I a. prior to analysis. RESULTS AND DISCUSSION Detection and quantification of chondrocyte HAS-2 and HAS-3 mRNA A quantitative competitive RT-PCR approach was taken to characterize HAS expression by human and bovine articular chondrocytes 16, Using this approach it was determined that human as well as bovine articular chondrocytes express both HAS-2 and HAS-3. However, the level of HAS-2 expression is -40 times greater than HAS-3 16. Neither bovine nor human chondrocytes expressed any detectable levels of HAS-I even at greater than 35 cycles ofRT-PCR. The same primers were able to amplify HAS-I mRNA derived from human dermal fibroblasts (positive control). Nonetheless, the possibility exists that HAS-I mRNA is expressed but at very low levels or only expressed under certain pathological conditions. However, given that HAS-2 and HAS-3 are readily detectable, it is not likely that a low abundance of HAS-I, if it exists, is physiologically
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relevant. The copy number for HAS-2 and HAS-3 varied slightly in human cartilage samples from donor to donor but the ratio of HAS-2 to HAS-3 remained quite constant. In all cases, HAS-2 represented the predominant enzyme. When total RNA was isolated [rom intact human cartilage slices, similar values for HAS-2 versus HAS-3 mRNA expression were observed 18-19. This 25-50 fold difference in copy numbers suggests that HAS-2 is the predominate HAS message expressed by articular chondrocytes.
The effect of HAS-2 antisense oligonucleotides on chondrocyte HA production Given the predominance of HAS-2 mRNA in chondrocytes, phosphorothioate oligonucleotides (16-mers) were prepared directed against sequence that included the translation start site of human HAS-2 mRNA 16. Antisense, sense and reverse sequence oligonucleotides were prepared and transfected into human chondrocytes using lipofectamine as a carrier. The protocol used for cultured chondrocytes used one transfection pulse of oligonucleotide for 5 hours under serum-free conditions followed by the addition of fresh medium containing fetal bovine serum. Total RNA was isolated from the cells at various times following transfection and characterized by quantitative competitive RT-PCR. HAS-2 mRNA was inhibited by -30% 8 hours, subsequently reaching a maximum inhibition of 60% by 24 hours as compared to cells treated with the sense control oligonucleotide. Forty to 48 hours following transfection, the levels of HAS-2 began to return to pre-transfection levels. The HAS-2 antisense or control oligonucleotides had no effect on the mRNA expression of HAS-3, aggrecan or GAPDH. Thus, the effect of the HAS-2 antisense oligonucleotide appeared to be specific but what about HAS-2 protein? HAS-2 antibodies were not available to determine changes in protein levels. So instead, changes in the levels ofHA accumulation were examined. One function of chondrocyte HA discussed above is to participate in cell-associated matrix assembly. At 24 hours post-transfection with HAS-2 antisense oligonucleotides, no coats were observed surrounding the chondrocytes. On the other hand, chondrocytes transfected with reverse and sense HAS-2 oligonucleotides displayed coats similar to control, untreated cells. At 48 hours post-transfection, small coats surrounding the chondrocytes began to reappear. To detect HA production directly, the transfected chondrocytes were incubated with a biotinylated HABP probe followed by peroxidase conjugated streptavidin and color reagents. Intense staining for HA was observed surrounding the sense HAS-2 treated chondrocytes with staining extending well beyond the limits of the plasma membrane. HABP staining intensity and extent were significantly reduced in the antisense treated cells. Quantification by image analysis suggested an -60% reduction in HABP staining intensity of antisense treated cells as compared to control cells. Thus, the change in HA staining matched the level of HAS-2 mRNA inhibition. Antisense or sense oligonucleotide-treated cells were also labeled with 35S-S04 and incorporation into PG determined. The total level of 35S_PG was equivalent in the sense and antisense treated chondrocytes. This was expected as no effect on aggrecan mRNA due to HAS-2 antisense oligonucleotide was observed. However, the antisense transfected cells exhibited a 30% reduction in the amount of cell-associated PG with a concomitant increase in mediumlocalized PG as compared to control, sense treated cells. This again confirms that the amount of PG retained by chondrocytes is proportional to the amount of cell-associated HA. In pilot studies we determined using rhodamine-tagged phosphorothioate oligonucleotides that these nucleotide probes could readily penetrate into intact cartilage tissue slices and accumulate within chondrocyte lacunae (likely within chondrocytes).
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We also found that this penetration and accumulation was facilitated by lipofectamine, just as it was for transfection of cultured chondrocytes. Therefore, human cartilage slices were incubated for 7 days with HAS-2 sense or antisense oligonucleotides (with daily medium changes) and then evaluated for accumulation of HA within the tissue. As with the cultured chondrocytes the antisense-treated slices displayed a substantially lower expression ofHA, throughout the matrix, as compared to sense oligonucleotide slices. The effect of cellular mediators I cytokines on HAS and CD44 expression. The bone morphogenic protein-7 (BMP-7) also known as osteogenic protein-I (OP-I) has been shown to stimulate aggrecan synthesis in cultured bovine and porcine articular chondrocytes as well as cartilage explants 23-24. Flechtenmacher et aI., also demonstrated stimulation of collagen type II as well as aggrecan in human articular chondrocytes 25. Since matrix retention also depends on HA and the HA receptors, the effect of BMP-7 on HAS and CD44 was examined. Treatment of bovine or human articular chondrocytes with BMP-7, resulted in a substantial increase in the exclusion size of the pericellular matrix 17-19. Many of the matrices grew to over one-cell-diameter-representing some of the largest coats we have visualized to date (larger than coats present on rat chondrosarcoma cells). Staining of these cells for HA revealed an increase in staining intensity and extent. Flow cytometry analysis demonstrated increases in CD44 as well 17. So, given that HA accumulation was dramatically increased under these conditions, what about HAS mRNA expression? Using the same quantitative RT-PCR approach described above, BMP-7 treatment resulted in 1.5 fold increases in HAS-2 and CD44 mRNA increasing to 2.0 fold by 24 hours. The ratio of HAS-2 copy numbers appears to peak at 3 days of BMP-7 treatment, ~3.7 times the level expressed by control cultures. Aggrecan mRNA copy numbers showed a significant upregulation at 3 days of treatment (2.3 fold difference) increasing to 4.2 fold by 7 days of treatment. No changes in HAS-3 or GAPDH were noted over the same time period. Slices of bovine articular cartilage were also treated BMP-7. Biotinylated HABP staining for HA revealed that control untreated cartilage slices were positive for HA, with definitive staining throughout the extracellular matrix. However, in the presence ofBMP7, the staining for HA was substantially increased, elevated both within the matrix and surrounding the chondrocytes. When the tissue slices were analyzed for mRNA expression, a 3.4 fold increase in copy number for HAS-2 was observed. As with the isolated chondrocytes, there was no change in HAS-3 copy number 17. The inflammatory cytokine IL-I has been shown to enhance chondrocyte catabolism inhibit aggrecan PG expression 27 yet induce an increase in CD44 expression 21. The effect on chondrocyte HA synthesis was unknown although D'Souza et al., had reported increases in 3H-HA in bovine articular chondrocytes due to IL-I 28. Following only two days of treatment with IL-Ia., human articular chondrocytes displayed 2.5 fold drop in copy number for aggrecan but no change in either GAPDH or HAS-3 mRNA. CD44 copy number as expected, was 4 fold higher than control, untreated cells. Interestingly, HAS-2 mRNA copy numbers were also -3 fold higher than control chondrocytes 18. Again, changes in HA expression are reflected by changes in HAS-2 mRNA with little effect on HAS-3. 26,
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Human articular cartilage slices were also treated with IL-la. Safranin 0 staining for PG was lost from the superficial to upper middle layers of the tissue. Given the increase in HAS-2 rnRNA in the chondrocytes, it was expected that HABP staining for HA would be enhanced in the IL-l treated slices. However, little to no change in HA was observed in the middle / deep layers. Further, the superficial/upper middle layers appeared devoid of HA staining. To determine if this was merely a staining artifact, the cartilage slices were carefully dissected (divided into -upper 1/3 and deeper 2/3), cultured separated with or without IL-la and then analyzed using FACE analysis. As with the staining, there was little to no change in the middle / deep layers but a substantial loss of HA disaccharide recovered from the superficial/upper middle layers of the IL-l treated tissue. To investigate whether the expression of HAS was different in the tissue or tissue layers as compared to isolated cells, RNA was extracted from the dissected tissue layers. HAS-2 mRNA copy number was still elevated 3 fold in both the upper and lower layers of cartilage. CD44 copy number was also elevated in the tissue, 5 fold in the upper layers and 3 fold in the deeper. One possible mechanism to explain this data was that HA synthesis is upregulated but, the increased CD44 induced by IL-l was now participating more in HA catabolism 21-29 rather than matrix retention. To investigate this aspect, we went back to the human articular chondrocyte culture system. In the IL-l treated cells, the size of the particle exclusion pericellular matrix was visibly reduced as compared to control cells. This however could be due to decreased PG expression (observed by mRNA as well as alcian blue staining) and/or a change in CD44 function. HABP staining for HA was more intense on the IL-l treated chondrocytes. To investigate HA internalization, the chondrocytes were trypsinized extensively, permeabilizcd and then stained using the biotinylated HABP probe. Both control and treated chondrocytes displayed positive staining, but staining was sequestered into round inclusions (i.e., vesicles). In the IL-l treated chondrocytes, the staining for intracellular HA was intense-substantially more than in the control untreated cells. Thus, it would appear that, by whatever mechanism, IL-I treated chondrocytes have an increased capacity to internalize HA. CD44-mediated HA internalization
The mechanism for the accumulation of endogenous intracellular HA at present is unknown. Does this represent HA synthesized from an intracellular site? Possibly, but not likely given the localization of HAS enzymes. Control experiments rule out the expression of intracellular endogenous biotin-containing proteins. Does this represent HA internalized via CD44? This, is at present also unknown. However, the appearance of intracellular HA within perinuclear vesicles looks identical to exogenous fluorescein- or 3H-labeled HA that we find is internalized by chondrocytes via a CD44-dependent mechanism 21, 29. The internalization of fluorescein-H'A or 3H-HA can be blocked by addition of unlabeled HA, HA oligosaccharides, or pre-incubation of the chondrocytes with anti-CD44 monoclonal antibodies. Fluorescein-labeled dextran was not internalized ruling out fluid phase pinocytosis for internalization ofHA. Internalized 3H-HA could be isolated in two pools, as 3H-counts voided on a Sepharose CL-2B column and as 3I-I-counts included on a Sephadex G-50 column. We speculated that these pools represent internalized HA within endosomes and within lysosomes. Further, the generation of small fragments of 3H-HA could blocked by the addition of the lysosomotropic agent chloroquine. Thus, exogenous, labeled HA can be bound to the cell surface of chondrocytes via CD44, internalized into
326
Aspects ofhyaluronan injoints
the cells and degraded to small fragments. We have also shown that IL-l treated chondrocytes, that exhibit a 5-fold increase in CD44 expression, accumulate 3-times as much fluorescein-labeled HA during a 24 hour incubation as compared to control untreated cells. All these data validates the potential that increased CD44-mediated internalization and catabolism of HA could be responsible for the lack of HA accumulation observed in IL-l treated cartilage slices. If anabolic agents such as BMP-7 increase CD44 expression and catabolic mediators increase CD44 expression, how do chondrocytes regulate thefunction ofCD44? We have begun to address this issue by a variety of approaches. We have shown previously that the transfection of COS-7 cells with human CD44 containing overexpression constructs led a capacity to assemble chondrocyte-like pericellular matrices in the presence of exogenous purified aggrecan and HA 9. In new preliminary work, we have found that these same CD44-transfected COS-7 cells have the capacity to bind and internalize fluorescein-labeled HA. This has allowed us to address whether interactions of different regions within the intracellular tail domain of chondrocytes are responsible for HA matrix retention and assembly versus HA internalization. Some intracellular domain truncation mutants of CD44 result in no coats, no fluorescein-HA binding and consequently, no fluorescein-HA internalization. However, we have found one truncation mutant, CD44H~54, in which 54 of the 70 amino acids ofthe CD44 intracellular domain have been deleted, has the capacity to bind fluorescein-HA to the cell surface (similar to control CD44Hwt-transfected cells) but, lacks the capacity to internalize the HA. This suggests that chondrocytes may use a similar mechanism that includes differential interactions with the proximal versus distal portions of the CD44 cytoplasmic tail, to regulate the function of various functions. The nature of these interactions are, at present, speculative. The interactions may include selective differential binding to different actin-binding-proteins or different interactions of CD44 with itself. In summary, our work suggests that two proteins in particular, HAS-2 and CD44, control much of the metabolism, retention and function of HA that occurs within cartilage tissues. Acknowledgements: Collaboration with the laboratory of Cheryl B. Knudson, Ph.D. as well as Allan Valdellon, M.D. of the Regional Organ Bank of Illinois and his staff; are gratefully acknowledged. Special thanks to Susan Chubinskaya, Ph.D., co-director of the in situ hybridization/histochemistry core of the Department of Biochemistry at Rush for use of the safranin 0 stained human donor tissue shown in Figure IB. This work was supported in part by NIH grants and ROI-AR43384 and P50-AR39239 as well as a grant from the National Arthritis Foundation.
REFERENCES 1. 2. 3.
W. Knudson & K. E. Kuettner, In: Primer on the Rheumatic Diseases, u" ed., R. L. Wortmann, ed., Arthritis Foundation, Atlanta, 1997,33-38. K. E. Kuettner, In: Rheumatology, J. H. Klippel and P. A. Dieppe, eds., Mosby-Year Book Europe Limited, St. Louis, MO, 1994, 6.1-6.16. D. Heinegard & V. C. Hascall, 'Aggregation of cartilage proteoglycans. III. characteristics of the proteins isolated from trypsin digests of aggregates', J Bio!. Chem., 1974, 249, 4250-4256.
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1. A. Buckwalter & L. C. Rosenberg, 'Electron micrographic studies of cartilage proteoglycans', J. Biol. Chem., 1982, 257, 9830-9839. 5. V. C. Hascall, T. R. Oegema, M. Brown & A. I. Caplan, 'Isolation and characterization ofproteoglycans from chick limb chondrocytes grown in vitro', J. Bioi. Chem., 1976, 251,3511-3519. 6. J. H. Kimura, T. E. Hardingham, V. C. Hascall & M. Solursh, 'Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma', J. Bioi. Chem., 1979,254,2600-2609. 7. W. Knudson & C. B. Knudson, 'Assembly ofa chondrocyte-like pericellular matrix on non- chondrogenic cells', J. Cell Sci., 1991, 99, 227-235. 8. C. B. Knudson, 'Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix', J. Cell BioI., 1993, 120,825-834. 9. W. Knudson, E. Bartnik & C. B. Knudson, 'Assembly of pericellular matrices by COS-7 cells transfected with CD44 homing receptor genes', Proc. Nat!. Acad. Sci. USA, 1993, 90, 4003-4007. 10. W. Knudson, D. J. Aguiar, Q. Hua & C. B. Knudson, 'CD44-anchored hyaluronan-rich pericellular matrices: An ultrastructural and biochemical analysis', Exp. Cell Res., 1996,228,216-228. 11. C. B. Knudson, In: The Chemistry, biology and medical applications of hyaluronan and its derivatives, T. C. Laurent, ed., Portland Press, London, 1998,216-228. 12. P. Heldin & H. Pertoft, 'Synthesis and assembly of the hyaluronan-containing coats around normal human mesothelial cells', Exp. Cell Res., 1993, 208, 422-429. 13. P. H. Weigel, V. C. Hascall & M. Tammi, 'Hyaluronan synthases', J. Bioi. Chem., 1997,272,13997-14000. 14. A. P. Spicer, M. F. Seldin, A. S. Olsen, N. Brown, D. E. Wells, N. A. Doggett, N. Itano, K. Kimata, J. Inazawa & J. A. McDonald, 'Chromosomal localization of the human and mouse hyaluronan synthase genes', Genomics, 1997,41,493-497. 15. N. Itano, T. Sawai, M. Yoshida, P. Lenas, Y. Yamada, M. Imagawa, T. Shinomura, M. Hamaguchi, Y. Yoshida, Y. Ohnuki, S. Miyauchi, A. P. Spicer, J. A. McDonald & K. Kimada, 'Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties', J. Bioi. Chem., 1999,274,25085-25092. 16. Y. Nishida, C. B. Knudson, J. J. Nietfeld, A. Margulis & W. Knudson, 'Antisense inhibition of hyaluronan synthase-2 in human articular chondrocytes inhibits proteoglycan retention and matrix assembly', J. Biol. Chem., 1999,274,21893-21899. 17. Y. Nishida, C. B. Knudson, W. Eger, K. E. Kuettner & W. Knudson, 'Osteogenic protein-l stimulates cell-associated matrix assembly by normal human articular chondrocytes', Arthritis Rheum., 2000, 43, 206-214. 18. Y. Nishida, A. L. D'Souza, J. M. A. Thonar & W. Knudson, 'IL-la stimulates hyaluronan metabolism in human articular cartilage', Arthritis Rheum., 2000, 43, 1315-1326. 19. Y. Nishida, C. B. Knudson, K. E. Kuettner & W. Knudson, 'Osteogenic protein-l promotes the synthesis and retention of extracellular matrix within bovine articular cartilage and chondrocyte cultures', Osteoarthritis Cartilage, 2000, 8, 127-136. 20. H. J. Hauselmann, M. B. Aydelotte, B. L. Schumacher, K. E. Kuettner, S. H. Gitelis & E. J.-M. A. Thonar, 'Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads', Matrix, 1992, 12, 130-136. 21. G. Chow, C. B. Knudson, G. Homandberg & W. Knudson, 'Increased CD44 expression in bovine articular chondrocytes by catabolic cellular mediators', J. Biol. Chem., 1995, 270, 27734-27741.
4.
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22. M. B. Aydelotte, B. L. Schumacher & K. E. Kuettner, In: Articular Cartilage and Osteoarthritis, K. E. Kuettner, R. Schleyerbach, J. G. Peyron and V. C. Hascall, eds., Raven Press, New York, 1992,237-250. 23. P. Chen, S. Vukicevic, T. K. Sampath & F. P. Luyten, 'Osteogenic protein-I promotes growth and maturation of chick sternal chondrocytes in serum-free cultures', J. Cell. Sci, 1995, 108, 105-114. 24. S. A. Lietman, M. Yanagishita, T. K. Sampath & A. H. Reddi, 'Stimulation of proteoglycan synthesis in explants of porcine articular cartilage by recombinant osteogenic protein-I (bone morphogenetic protein-Z)', J Bone and Joint Surg, 1997, 79-A, 1132-1137. 25. J. Flechtenmacher, K. Huch, E. I. M. A. Thonar, I. Mollenhauer, S. R. Davies, T. M. Schmid, W. Puhl, T. K. Sampath, M. B. Aydelotte & K. E. Kuettner, 'Recombinant human osteogenic protein 1 is a potent stimulator of the synthesis of cartilage proteoglycans and collagens by human articular chondrocytes', Arthritis Rheum., 1996,39,478-488. 26. J. A. Tyler, S. Bolis, J. T. Dingle & J. F. S. Middleson, In: Articular Cartilage and Osteoarthritis, K. E. Kuettner, R. Schleyerbach, I. G. Peyron and V. C. Hascall, eds., Raven Press, New York, 1992,251-264. 27. M. B. Aydelotte, R. X. Raiss, R. Schleyerbach & K. E. Kuettner, 'Effects of Interleukin-l on metabolism of proteoglycans by cultured bovine articular chondrocytes', Trans. Ortho. Res. Soc., 1988, 13,247. 28. A. L. D'Souza, K. Masuda, L. Otten, S. Momohara, L. Wang & E.-I. M. A. Thonar, 'Effects of IL-l a on the metabolism of hyaluronan in different compartments of the matrix formed by adult articular chondrocytes in vitro', Trans. Ortho. Res. Soc., 1997, 22,470. 29. Q. Hua, C. B. Knudson & W. Knudson, 'Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis', J. Cell Sci., 1993, 106, 365375.
AN INSIGHT INTO CELLULAR SIGNALLING MEDIATED BY HYALURONAN BINDING PROTEIN (HABPl) T. B. Deb, M. Majumdar, A. Bharadwaj, B.K. Jha & K. Datta' School a/Environmental Sciences. Jawaharlal Nehru University. New Delhi-1 10067. India
ABSTRACT We have reported the characterization of a cell surface glycoprotein of 34 kDa on SDS-PAGE, having specific affinity for hyaluronan and this protein has been termed as HABPI by GDB (Ac. No. 9786126). The role of HABPI in cell adhesion and tumor invasion has also been confirmed. In continuation, the gene encoding hyaluronanbinding protein from human fibroblast was isolated and its localization on human chromosome 17p12-p13 has been reported. Sequence analysis shows the identity of HABPI with other proteins P-32, a protein co-purifying with splicing factor SF2; and gCIqR, the receptor for the globular head of C1q, indicating its multifunctional nature. Several reports suggest differential localization of this protein in various cell types. To confirm its role in signalling, the enhanced phosphorylation of HABPI is being reported in mitogen activated cells and in sperms induced with progesterone and Calyculin A, the acrosome reaction and capacitation inducers. Another interesting observation is oligomerization of HABPI, which enhances its affinity for hyaluronan, highlighting its regulatory role in hyaluronan mediated signalling. Hexasaccharide of hyaluronan is the minimum chain length required for interaction with HABPI. Attempts are being further made to address the intricate mechanism by which this protein is phosphorylated and to understand how HABPI phosphorylation is related to the signalling pathway and if there occurs any nuclear translocation after phosphorylation. INTRODUCTION Hyaluronan, the ubiquitous glycosaminoglycan present in extracellular matrix and pericellular matrixes is involved in structural organization of extracellular matrix and its level is regulated during rapid tissue proliferation and regeneration'. To establish the mode of cellular interaction, a number of ECM and cell surface hyaluronan binding proteins have been identified and grouped as the family of "hyaladherins'", since they share the common hyaluronan-binding motif. In our laboratory, we have been working on a novel member ofhyaladherin family, named HABPI 3 . We confmned its role in cell adhesion & tumor invasion" and sperm maturation & motility':", This protein is ubiquitously present in different cell types and is associated with diverse cellular signalling, as evident by the inhibition of HA-binding to lymphocytes and HA mediated lymphocyte aggregation by pretreatment of cells with anti-HABPI antibodies. Its role in sperm-oocyte interaction has also been established, since the blocking of sperm surface HABPI by its antibody inhibits zona binding'. Its role in cellular signalling is established by the observation on enhanced phosphorylation at serine/threonine residues of HABPI and increased IP3 and PLC-y formation in transformed and HA supplemented cells", which is inhibited by pretreatment of the cells with antibodies against HABPI 8. Enhanced phosphorylation of HABPI in HA supplemented motile sperm is also reported by our group", To further
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Cell surfaces and hyaluronan receptors
analyze the role of this protein in cellular functions, the cDNA encoding HABPI from human skin fibroblast was cloned and sequenced. The presence ofHA-binding motif was confirmed and subsequently, the recombinant protein has been shown to bind to HA9• The gene encoding this protein has been shown to localized on human chromosome 17pI2-p13 lO• Computer search analysis of the sequence encoding HABPI revealed its multifunctional characteristics11, since it was found to be identical with SF2/P-32, a protein copurified with splicing factor SF212 and gClqR, the receptor of complement subcomponent". Sequence analysis further supports the role of this protein in cellular signalling as it has five casein kinase II (CKII) phosphorylation sites, and one extracellular signal regulated kinase (ERK) site. Protein phosphorylation plays a major role in molecular signalling event in cellular proliferation in which phosphorylation by CKII is also involved". Rapidly transient increased activity of cytoplasmic CKII was reported by the addition of serum to cultured cells followed by a change in the properties of growth regulating proteins including oncogenes and transcription factors". Thus, in order to correlate phosphorylation of HABPI with cellular signalling, we made an attempt to investigate the in vivo phosphorylation of HABPI in sperms, treated with either progesterone or Calyculin A, the acrosome reaction and capacitation inducers respectively. Simultaneously we intend to examine whether HABPI acts as substrate for CKII in vitro. MATERIALS & METHODS Materials
Chemicals used in the study were purchased from Sigma Chemicals Co., S1. Louis, USA, unless otherwise mentioned. [y_32 p] ATP was purchased from BARC, India. Recombinant casein kinase II was purchased from Boehringer Mannheim, Germany. Carbobind plates were purchased from Corning Costar, Netherlands. HAhexasaccharide was a kind gift from Dr. B. P.Toole. Purification of recombinant 34 kDa HABPI
The E. coli BL21 (DE3) was transformed with expression vector construct containing the mature HABPIIP-32 cDNA insert and protein expression was induced by the addition of IPTG. The overexpressed protein was purified by HA-Sepharose-4B chromatography as described earlier" Differential affinity of HA-oligosaccharides with HABPI
The various oligosaccharides at different concentrations were covalently linked with carbobind costar plates according to the supplier's instruction. After coating, the solution was removed and the wells were rinsed with PBS containing 0.05% Tween-20 and blocked with 5% non-fat milk at room temperature. The plates were incubated with 100 III of biotinylated HABPI (10 ug/ml) at room temperature for I hour and probed with Extravidin-HRP and detected with ABTS. Optical density was measured at 405 run. Immunoprecipitation of hyaluronan-binding protein
e
I x 106 intact spermatozoa were incubated with I mCi of 2 p], in phosphate free Biggers-Whitten-Whittingham medium for 45 min at 37°C with and without the exogenous substances, Calyculin A, orthovanadate and progesterone. After labeling,
Cellular signalling
367
the sperm pellets were rinsed with ice-cold PBS and solubilized with radio-immuno precipitation (RIPA) buffer. The lysates were centrifuged at 15,000 x g for 3 min at 4°C and the supernatant was incubated with 1.5 mg of swollen protein A-Sepharose-4B conjugated to anti-HABPI antibodies overnight at 4°C with continuous rocking. Precipitates were washed thoroughly, three times with lysis buffer, twice with 0.15M NaCl and twice with 0.1% SDS, boiled in 1 x Laemmli sample buffer and resolved on 10% SDS-PAGE. For visualizing the labeled protein, the gel was stained with CBB, dried and analysed on a phosphoimage analyzer (Bio-Rad, USA). In vitro liver kinase assay
Liver extract was prepared by homogenizing the tissue in sucrose and then centrifuged and the extract was used as a source of protein kinases. Phosphorylation of recombinant HABPI/P-32 was carried out with the liver extract. The reaction mixture (60 fJ.l) contained 20 mM HEPES, pH 7.4; 3 mM MnCh, 5 fJ.Ci [y_32 p ] ATP, 50 fJ.M ATP and 5 ul of liver kinase (containing 5 ug total protein) and the reaction was carried at 37°C for 30 minutes. The reaction was stopped by the addition of Laemmli sample buffer, boiled, resolved on 4-20% gradient SDS-PAGE and analysed by autoradiography. In vitro casein kinase II assay
Casein kinase II in vitro kinase assay was performed according to the supplier kit protocol (Boehringer Mannheim). The reaction assay (50 ul) included 50 mM TrisHCI, pH 6.9; 130 mM KCI, 10 mM MgCh, 4.8 mM dithiothreitol, 5 fJ.Ci [y_32 p ] ATP (specific activity: 3000 Ci/mmole) and different amount of recombinant HABPI/P-32 in the presence ofCKII stimulator (spermidine) or inhibitor (heparin). The reaction was initiated with the addition of I fJ.I of diluted recombinant CKII (corresponding to 0.1 mU) and the reaction was performed at 37°C for 30 min. The reaction was terminated by 10% TCA (final concentration) and proteins were precipitated with 40 ug of carrier protein BSA. Pellet was washed with 5% TCA and ethanol.diethyl ether (1:1), solubilized in SDS-PAGE sample buffer and analyzed in 12.5% SDS-PAGE. For dephosphorylation of proteins, the dried pellet after ethanol:ether (1:1) wash was solubilized in a reaction mixture containing 50 mM Tris-HCI, pH 7.5; 50 mM MgCh and incubated with 7.3 units of alkaline phosphatase at 37°C for 15 min. Reaction was stopped by Laemmli buffer and analyzed on a 12.5% SDS-PAGE. The in vitro CKII assay was also done using tissue purified CKII enzyme. The reaction was performed in the same way as done with recombinant CKII (described above). Tissue purified CKII was used as kinase (I :20 dilution) and HA (5 ug/assay) was used in one ofthe reactions.
RESULTS Comparative affinity of HABPI to HA-hexasaccharide and HA-polymer
In continuation of our earlier studies, we report here that HABPI exhibits almost similar affinity towards HA-hexasaccharide as that shown for HA- polymer (Fig. 1). However, it does not interact with HA disaccharide suggesting a requirement for a critical chain length for HA-HABPI interaction.
368
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Hyperphosphorylation of HABPI with protein phosphorylation modulators To see the phosphorylation status ofHABPl, rat sperms were metabolically labeled with ezp] orthophosphate under stimulation with progesterone and Calyculin A and subsequently, immunoprecipitated with anti-HABPl antibody and protein Avsepharose 0.9 , . . . - - - - - - - - - - - - - - - - ,
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under the stimulation of Calyculin A (lane 1), orthovanadate (lane 2) and progesterone (lane 3) as compared to the control (lane 5). Pre-immune serum (PIS) control is seen in lane 4. This observation suggests the hyperphosphorylation of HABPI in the presence of progesterone (tyrosine kinase stimulator) and Calyculin A (serine/threonine phosphatase inhibitor), acrosome reaction and capacitation inducers, respectively and Na3V04 (tyrosine phosphatase inhibitor). Evidence for HABPI as a substrate of CKII The in vitro phosphorylation of recombinant HABPIIP-32 was carried out in three successive steps using kinase from different sources: (a) crude rat liver extract known to contain a variety in kinases including CKlI, (b) tissue purified CKlI and (c) commercial recombinant CKlI, free from any contaminating kinase. These experiments clearly established the 34 kDa HABP 1 as a substrate of CKlI. Initially, the crude liver extract was used to phosphorylate the recombinant HABPl. As shown in Fig.3A, recombinant HABPI did not undergo any autophosphorylation in vitro (lane 1), but was phosphorylated only when incubated in the presence of rat liver kinase (lane 3), raising the possibility of rHABPI being a substrate of protein kinase. Furthermore, the phosphorylation ofrHABPI was found to be concentration dependent as seen by an increase in its phosphorylation with an increase in concentration (Fig. 3B). The observation on the phosphorylation ofHABPl by crude liver kinase, suggested CKlI as one of the probable kinases in liver extract, responsible for the in vitro phosphorylation of HABPIIP-32. As shown in Fig. 4A, tissue purified CKlI also phosphorylated rHABPI (lane 6) which was significantly enhanced by the addition of
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Phosphorylation of purified rHABPl/P-32 protein by liver kinases in vitro. Purified rHABPI (10 ug) was incubated under standard assay conditions with [y_ 32p]_ATP (5 ~Ci/assay). rHABPI (lane 1, 10 ug) only, crude liver kinases (lane 2, 10 ug total protein), rHABPI (lane 3, 10 ug) in the presence ofliver kinases (10 ug total protein). B. Phosphorylation of rHABP I was carried out in a concentration dependent manner in the presence of crude liver kinases (10 ug total protein), 5 ug ofrHABPI (lane 1), 10 Ilg of rHABP I (lane 2),15 ug of rHABPl (lane 3) and 20 ug ofrHABPl (lane 4). Proteins were
separated in 4-20% gradient SDS-PAGE, dried and autoradiographed. Spermidine and heparin showed a stimulation (lane 5) and inhibition (lane 4) in HABPI phosphorylation by the tissue purified CKlI (Fig. 4A), substantiating the possibility of HABP 1 as the substrate ofCKII.
370
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rHABPI as CKII substrate was further confirmed by the use of commercially available recombinant CKII. As shown in Fig. 4B, recombinant CKII was shown to be autophosphorylated at the regulatory B subunit of 26 kDa and catalytic a. subunit of 42 kDa (lane 4) which was stimulated by spermidine (lane 3) and inhibited by heparin (lane 2), the known stimulator and inhibitor of CKII respectively. Substrate phosphorylation by CKII using histone as substrate was also studied (lane l ). Interestingly, CKII was shown to phosphorylate rHABP} at the concentration of 0.5 Ilg (lane 8) and I ug (lane 7) along with autophosphorylation of CKII subunits. However, higher concentrations of rHABP} at the level of 5 Ilg (lane 6) and 10 Ilg (lane 5) inhibited autophosphorylation of both the regulatory and catalytic subunit of CKII and
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354
Aspects of hyaluronan in joints
The 500-730 kDa hyaluronan (Hyalgan®, Fidia, Italy) is a preparation widely used for La. treatment of OA and a large body of clinical studies exists demonstrating the efficacy on OA symptoms of this specific HA fraction and the long duration of the effect 7. Recently, combined arthroscopic and histomorphometric studies on the structural features of human joint tissues 8-10 provided some evidence that Hyalgan® treatment was effective in reducing structural alterations typical ofOA. The results obtained in these studies are reviewed in the present paper and discussed with particular reference to the mechanism of action of the 500-730 kDa hyaluronan. PATIENTS AND METHODS Patients This paper describes data coming from two combined arthroscopic and histomorphometric clinical studies 8-10 designed to study the effect of 500-730 kDa hyaluronan (Hyalgan®, Fidia, Italy) on joint morphology in patients fulfilling the criteria of the American College of Rheumatology for the diagnosis of knee OA. The first trial was a 6-month open study carried out on 40 patients, while the second one was a randomized controlled study on 99 patients comparing the effect of HA and methylprednisolone (MP) 6 month following the treatment. Study design At the admission visit, after a check of the selection criteria and an evaluation of the clinical and radiological severity of OA (Kellgren-Lawrence), patients judged uncontrollable or unreliable, those with severe concomitant diseases making assessment of the result difficult, those receiving therapies during the last six months (apart from FANS), women in pregnancy or breast feeding were excluded from the trial. Patients judged eligible for the study underwent a first knee arthroscopy during which biopsies of cartilage and synovial membrane were obtained. After 15-20 days from the arthroscopic inspection, all the patients had a baseline visit with collection of blood and urine samples for routine laboratory assessments. Patients included in the first study then started on a course of i.a. injections of 2 ml Hyalgan® (10 mg/ml 500-730 hyaluronan in saline) once a week for 5 weeks. In the second trial this treatment was administered to half of the patients, while half of them were treated with I ml MP (Depo-Medrol®, Pharmacia & Upjohn, 40 mg/ml 6methylprednisolone acetate in saline) once a week for 3 weeks. In both studies on day 180 each patient underwent a second arthroscopic examination and a second biopsy was performed. During the second trial a normal control group of tissue samples was also obtained from 19 subjects who underwent arthroscopy for pain, but did not reveal any sign of either OA or rheumatoid arthritis. Arthroscopic examination In both studies arthroscopic and rnicroarthroscopic assessments performed under local anaesthesia used Hamou-Storz and Microview-Wolf arthroscopes. Entry was always effectuated antero-laterally with the knee flexed at an angle of 30°. Intermittent irrigation with Ringer acetate was used to optimize the degree of distension of the joint
Intra-articulartreatment
355
cavity. Microartrhroscopy of the synovial membrane was carried out following the injection of a 1% aqueous solution of methylene blue (3 ml, pH 4.5), followed after 5 minutes by a saline wash. The entire arthroscopic examination was always performed by the same investigator and documented using a video camera. All the videotapes were analysed by a second investigator using a blinded procedure in which the investigator was unaware of patient identity and chronology. To assess the degree and extension of cartilage damage for each compartment of the knee Outerbridge and Noyes scales 11-12 were used. The scoring system proposed by Pasquali Ronchetti et at. 13 was used for the grading of synovitis. Biopsy sampling During arthroscopy biopsies of both synovial membrane and cartilage were taken according to a series of standardized procedures described by Frizziero et at. 8. Briefly, the synovial membrane was sampled in the suprapatellar pouch and in the antero-medial compartment. Biopsies of articular cartilage were only taken from those patients with arthroscopic grade II lesions (i.e. cartilage marked by fibrillation, fissures, and a velvety aspect). Samples were taken from the edge of the lesions, which was easily identified by the more intense staining with methylene blue of the damaged area compared to healthy tissue. Since the same tissue region had to be sampled again during the final arthroscopic examination on day 180, a graded needleprobe was inserted under continuous arthroscopic control, using a second route of access,depending on the tissue compartment involved, to determine the precise sampling area. After excision biopsy samples from both synovial membrane and articular cartilage were immediately divided into two blocks and fixed for light and trasmission electron microscopy respectively according to standard procedures. Histological evaluations In both studies the histological evaluation of the synovial membrane was carried out estimating, by using established criteria and scores, the following parameters: lining cell arrangement, cell appearance (size, shape, vacuolization) and type, matrix features (fibrosis, oedema, necrosis), vessel number and appearance. In the second study a semiquantitative description of the synovial cell ultrastructure was also reported. A global assessment of cartilage damage was obtained in the first study using the well known Mankin scale. A quantitative morphometric approach was followed in the second study by using a computer assisted image analysis system (IBAS, Kontron, Germany) to estimate a set of widely accepted parameters in literature: surface roughness I~, chondrocyte density 15 and ultrastructure 16 . The mean thickness of the 'superficial amorphous layer were also measured and its appearance estimated by a fourpoint scale: O=absent; I=fragmented; 2=discontinuous; 3=compact. All of the evaluations were performed under blind conditions.
RESULTS Arthroscopy In the open study 8 at the final control (day 180), the mean (±SD) total score for the synovitis grading was 48.5±13.4 compared with 62.3±9.7 at baseline. This was equivalent to an improvement of 22%, the difference being highly significant (p 0.4). For example, LNCaP prostate cancer cells respond to HA in the absence of detectable amounts of CD44 at the cell surface, while T-24 and UMUC-3, which express high levels of CD44 at the cell surface, respond marginally to HA. However, since over ten isoforms of CD44 has been described", we can not exclude the possibility that a discrete CD44 isoform could be associated with the anti-proliferative activity observed with HA. This anti-proliferative activity could also be associated with other HA receptors. In particular, RHAMM and its isoforms have been implicated in the regulation of cell cycle progression 13. For example, overexpression of RHAMM by transfection into non-malignant fibroblasts has been shown to transform these cells into a fully metastatic fibrosarcoma".
HA potentiate the activity of anti-cancer agents based on DNA MCC, a mycobacterial cell wall preparation where mycobacterial DNA is preserved and complexed to the cell wall, and M phlei DNA possess anti-cancer activity against various cancer types':". More particularly, MCC has been shown to directly induce apoptosis in human bladder cancer cells. M ph lei DNA associated with MCC is responsible for its pro-apoptotic activity". MCC appears to mediate its anti-cancer activity
424
The action of hyaluronan in cells
by modulating the expression of a number of oncogenes, cell-cycle-related proteins and genes regulating apoptosis". We have tested whether HA can potentiate the activity of MCC or M. phlei DNA against bladder and prostate cancer cells. We have found that HA at various concentrations can act synergistically with MCC or M. phlei DNA As illustrated in figure 2, HA at 0.8 ug/ml potentiates the activity ofMCC and M. phlei DNA for both PC-3 prostate cancer cells (figure 2a) and RT-4 bladder cancer cells (figure 2b). Experiments are underway to understand why HA acts synergistically with MCC and M. phlei DNA. One possible explanation for this synergistic activity is the protection of the DNA from degradation by deoxyribonucleases present in the milieu (manuscript in preparation). Another possibility is the differential action of DNA and HA on cell cycle progression. HA has been shown to be present in the nucleus of cells, suggesting that HA may be involved in nucleolar function, chromosomal rearrangement, or other events in proliferating cells". HA potentiate the activity of anti-cancer drugs We have determined whether HA can also potentiate the activity ofchemotherapeutic drugs. These studies were carried out at an HA concentration which not have inhibitory activity. As shown in figure 3, we found that HA at 0.008 ug/ml can potentiate the activity ofcisplatin, an alkylating agent, and 5-fluorouracil, a DNA/RNA antimetabolite, against RT-4 bladder cancer cells. We have also tested the activity ofHA in combination with tamoxifen, an anticancer drug used in the treatment ofbreast cancer. Tamoxifen, an oestrogen receptor antagonist, can also directly induces apoptosis in breast cancer cells 17. We have found that HA at 0.008 ug/ml interacts synergistically with tamoxifen against MCF-7 cancer cells (figure 3). Our data suggest that the synergistic activity observed between HA and a number of anti-cancer drugs (MCC, M. phlei DNA, cisplatin, 5fluorouracil and tamoxifen) seems to be independent of their mechanism of action. Furthermore, it has been shown recently that the covalent linkage of HA to sodium butyrate, an anti-cancer compound, improves the anti-proliferative activity of butyrate towards MCF-7 breast cancer cells 18. CONCLUSIONS Our data show that HA purified from Streptococci sp. and having a molecular mass of 5.0-7.5 x 105 Da has a direct anti-proliferative activity on a number of cancer cells at low concentrations (0.8-80 ug/ml). In a number ofthe cancer cell lines tested, inhibition of proliferation by HA was significantly less at the highest concentration used. Cancer cells originating from bone marrow appear to be insensitive to HA at the concentrations tested. The activity of HA appeared to be independent of the presence of a number of escape mechanisms associated with cancer progression and of the presence of CD44 at the cell surface. HA also potentiated the activity of a number cancer drugs having different mechanisms of action. Our data indicates that HA having a molecular mass of 5.0-7.5 x 105 Da has considerable potential for development as a chemotherapeutic agent or as an adjunct to anti-cancer agents.
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PC-3 cells (a) or RT-4 cells (b) were incubated at I x l O'cells/ml with 0.8 ug/ml of HA alone or in combination with increasing concentrations of MCC or M phlei DNA for 48 h at 37°C, 5% CO 2, Cellular proliferation was determined by MTT reduction as described in the Materials & Methods. The results shown are the means of three independent experiments. S.D. were less than 10% and are not shown.
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REFERENCES 1.
2.
3.
4. 5.
J. Entwistle, C.L. Hall & E.A. Turley, HA receptors: regulators of signalling to the cytoskeleton, 1. Cell. Biochem., 1996,61,569-577. M. Slevin, J. Krupinski, S. Kumar & J. Gaffney, Angiogenic oligosaccharides of hyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplasmic signal transduction pathway resulting in proliferation, Lab. Invest., 1998,78,987-1003. D.C. West & S. Kumar, The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity, Exp. Cell Res., 1989, 183, 179196. R.M. Greco, J.A. Iocono & H.P. Ehrlih, Hyaluronic acid stimulates human fibroblast proliferation within a collagen matrix, 1. Cell. Physiol., 1998, 177,465-473. M. Inoue & C. Katakami, The effect of hyaluronic acid on corneal epithelial cell proliferation,Invest.Ophtamol. Vis. Sci., 1993,34,2313-2315.
Anti-cancer activity 6. 7. 8.
9. 10.
11.
12.
13.
14.
15.
16. 17. 18.
427
R.L. Goldberg & B.P. Toole, Hyaluronate inhibition of cell proliferation, Arthritis Rheum., 1987,30,769-778 C. Zeng, B.P. Toole, S.D. Kinney, I-W Kuo & I. Stamenkovic, Inhibition of tumor growth in vivo by hyaluronan oligomers, Int. J. Cancer, 1998, 77, 396-401. M.e. Filion, P. Lepicier, A Morales & N.e. Phillips, Mycobacterium phlei cell wall complex directly induces apoptosis in human bladder cancer cells, Br. J. Cancer. 1999, 79,229-235. D. Naot, R.V. Sionov & D. Ish-Shalom, CD44: structure, function, and association with the malignant process, Adv. Cancer Res., 1997, 71,241-319. Q. Yu, B.P. Toole & I. Stamenkovic, Induction ofapoptosis ofmetastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function, J. Exp. Med., 1997,186,1986-1996. N. Ohta, H. Saito, T. Kuzumaki, M.M. Ito, T. Saito, K. Nakahara & M. Hiroi, Expression of CD44 in human cumulus and mural granulosa cells of individual patients in in vitro fertilization programmes, Mol. Hum. Reprod., 1999, 5, 22-28. e.R. Mackay, H.J. Terpe, R. Stauder, W.L. Martson, H. Stark & U. Gunthert, Expression and modulation ofCD44 variant isoforms in humans, J. Cell. Bioi., 1994, 124,71-82. S. Mohapatra, X. Yang, I.A Wright, E.A Turley & AH. Greenberg, Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin Bl expression, J. Exp. Med., 1996,183,1663-1668. e.L. Hall, B. Yang, X. Yang, S. Zhang, M. Turley, S. Samuel, L. Lange, e. Wang, G.D. Curpen, R. Savani et al, Overexpression ofthe hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation, Cell, 1995, 82,19-28. S. Reader, M. C. Filion, V. Marie, B. Filion & N. e. Phillips, Mycobacterial cell wall-DNA complex (MCC) inhibits proliferation and induces apoptosis in androgendependent and independent human prostate cancer cells, Br. J. Cancer, 1999, 80S2, 76. S.P. Evenko & T.N. Wight, Intracellular localization ofhyaluronan in proliferating cells, J. Histochem. Cytochem., 1999,47, 1331-1342. R.E. Favoni & A. de Cupis, Steroidal and nonsteroidal oestrogen antagonists in breast cancer: basic and clinical appraisal, Trends in Pharm. Sci., 1998, 19,406-415. D. Coradini, e. Pellizzaro, G. Miglierini, M.G. Daidone & A Perbellini, Hyaluronic acid as drug delivery for sodium butyrate: improvment of the anti-proliferative activity on a breast-cancer cell line, Int. J. Cancer, 1999, 81, 411-416.
CONTROL OF HYALURONAN SECRETION INTO JOINT FLUID IN VIVO: ROLE OF PROTEIN KINASE C (PKC) C. L. Anggiansah', D. Scotr', A. Pout, P. J. Coleman", J. James', A. Houston', R. M. Mason z & J. R. Levick,,1 I Department ofPhysiology,
St George's Hospital Medical School, London SWI7 ORE, U. K.
2Molecular Pathology, Division ofBiomedical Sciences, Imperial College School ofMedicine, London W68RF, U. K.
ABSTRACT The rate of secretion of hyaluronan into the joint cavity of rabbit knees was measured in vivo over 6 h using a washout method and hyaluronan analysis by HPLC. Hyaluronan secretion in vivo was stimulated by joint distension, indicating the existence of a mechanosensitive regulatory pathway. The hyaluronan secretion rate increased >3-fold upon activation of protein kinase C (pKC) by phorbol ester. This effect was partially inhibited by cycloheximide, indicating that part of the response to PKC involves new protein synthesis.
KEYWORDS Synovium, secretion rate, stretch, protein kinase C, phorbol ester, cycloheximide.
BACKGROUND; HYALURONAN SECRETION INTO JOINTS IN VIVO Hyaluronan is secreted by synoviocytes into synovial fluid, where it not only serves as a lubricant but also has a profound buffering effect on fluid loss from the joint cavity during periods of raised pressure, as described by Levick et al. in this volume. The concentration of hyaluronan in the joint fluid is an important factor governing its actions'. Because joint fluid is continually being replaced by capillary filtration and lymphatic drainage', hyaluronan slowly drains out of the joint cavity', despite partial reflection of hyaluronan molecules by the synovial lining". As a result, the hyaluronan concentration can only be maintained by continuous secretion by the lining synoviocytes. Because reflection helps to retain hyaluronan in the cavity, the intraarticular half-life of hyaluronan is long, around 1Yz days in rabbit knees (cf. 2h for albumin or water), and a low secretion rate (3-5 ug hOi) suffices to maintain the physiological concentration 00.6 mg mr l . Since intra-articular concentration is stable, it is probable that hyaluronan secretion rate is a regulated variable and subject to physiological control processes. One such regulatory influence, identified recently in vivo, is stretch". As Figure 1 shows, the hyaluronan secretion rate into the cavity of a rabbit knee increase by ~20% within hours of distension of the joint lining by an acute volume expansion of 2 ml endotoxin-free physiological electrolyte solution. Control studies show that the increase is related to the joint expansion, not intra-articular cannulation. Thus the physiological secretion rate
370
Aspects of hyaluronan in joints 9
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appears to be mechano-sensitive in vivo. This may also be relevant to the relation between joint embryogenesis and limb bud movement. Work by other groups has concentrated on stimulation of hyaluronan secretion by cultured cells in vitro, rather than in vivo, in response to growth factors and cytokinesr". In view of the findings in Figure 1 we asked 'What intracellular pathways might be involved in regulating h~aluronan secretion rate in vivo?' Studies in vitro by the groups of Heldin 7, 9 and Prehm show that protein kinase C (pKC) is often a key mediator of increased hyaluronan secretion. Our objective, therefore, was to test whether activation of conventional, phorbol ester-sensitive PKC isotypes could increase hyaluronan secretion rate into the joint cavity of rabbit knees in vivo. METHODS
New Zealand rabbits (2Y:z kg) were anaesthetized and a sterile cannula inserted into the cavity of both knee joints. The native synovial fluid hyaluronan was removed from the cavity by twenty 1 ml washes. This ensures complete removal of endogenous fluidphase hyaluronan (Figure 2). One joint was then injected with 1 ml of 200 ng mr' PMA (phorbol 12-myristate 13-acetate) to activate classical PKC isotypes. The contralateral joint received 1 ml sterile electrolyte solution as a control. Because PMA is a small solute that is quickly cleared by the fenestrated synovial capillaries (see later), 0.3 ml was aspirated from the cavity every half-hour and replaced by an equal 'top-up' volume of fresh PMA solution. The same procedure was followed on the control side using a sterile electrolyte solution. The joints were allowed to secrete hyaluronan into the cavity for 6 hours. The new hyaluronan is formed de novo, not by leaching from surrounding tissue, as shown by its
Control of secretion intojoint fluid
371
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Washrunber Figure 2. Initial washout of endogenous synovial fluid hyaluronan from rabbit knees. The recovered hyaluronan in this series of22 joints averaged 230±15 f..lg (s.e.m.). dependence on ceIlular energy metabolism (Figure 1). After 6 hours each joint was washed out using the 20 x 1 ml procedure to recover the newly synthesised hyaluronan. The amount and weight-average molecular masses of hyaluronan in the initial and 6h washouts (plus aspirates) were determined by size-exclusion high-performance liquid chromatography at 206 nm as described by Coleman et al.s. Subsidiary experiments assessed the intra-articular half-lives of three small, easily assayed solutes of comparable molecular size to PMA (acridine orange, patent blue and Evans blue). After an intra-articular injection of 1 ml solution of known concentration, sequential, aliquots were aspirated at intervals for colorimetric assay.
RESULTS Stimulation of hyaluronan secretion rate by PKC The basal rate of hyaluronan secretion into the cavities of the control joints over the 6 hour period was 2.7 ± 0.5 ug per hour (n=5, mean ± s.e.m.). In joints treated with 200 ng mr l PMA the rate of hyaluronan secretion increased to almost 4 times the control value (p 10-times the concentration that totally blocks protein synthesis by cultured chondrocytes'"). In 3 successful paired experiments to date, CHX reduced hyaluronan secretion in every case, from 10.6 ± 4.2 IJ.g h-I in PMA-stimulated joints to 7.0 ± 4.3 IJ.g h-I in PMA+CHX treated joints. DISCUSSION The results show that PKC has a potential role in the pathways regulating hyaluronan secretion into joint fluid. Similarly, HAS gene transcription is increased by PKC in vitro:", Although the CHX results need further confirmation, they indicate that CHX only partially inhibits hyaluronan secretion in vivo, namely by 34%. Ifso, it follows that 1] synthesis de novo of new HAS-I/2/3 or an upstream, rate-limiting enzyme ll contributes to the upregulation of joint hyaluronan secretion in hours; and 2] some additional regulatory mechanism may exist, since CHX only partially blocked the stimulatory effect of PKC despite a very high CHX concentration. The possible existence of a regulatory membrane complex" or supply-limitation by uridine diphosphoglucose dehydrogenase (UDPDG)lI require clarification. A speculative transduction system for the stimulation of hyaluronan secretion by synovial stretch must begin with a mechanosensor, for which synoviocyte integrins and/or stretch activated ion channels are candidates, activating PKC throu~h the phosphoinositide cascade. PKC activation of the MAP kinase cascade is known' . This could lead to nuclear transcription of HASIUDPDG and, to account for the CHXresistant secretory component, possibly phosphorylation of other regulatory factors.
374
Aspects ofhyaluronan in joints
It is concluded that PKC can control HA secretion into the synovial fluid of rabbit knee joints in vivo; and that its action is partially dependent on new protein synthesis. ACKNOWLEDGEMENTS The research was funded by Wellcome Trust grants 039033/Z, 056983/Z and European Community Training & Mobility of Researchers grant ERBFMRXCT980219. REFERENCES 1. D. Scott, P. 1. Coleman, R. M. Mason & 1. R. Levick, 'Concentrationdependence of interstitial flow buffering by hyaluronan in joints', Microvasc. Res., 2000, 59, 345-353. 2. 1. R. Levick, R. M. Mason, P. 1. Coleman & D. Scott. In: Biology of the Synovial Joint. C. W. Archer, M. Benjamin, B. Caterson & 1. R. Ralphs (eds), 1999. Harwood Academic Publishers, London. pp. 235-252. 3. 1. R. E. Fraser, W. G. Kimpton, B. K. Pierscionek & R. N. P. Cahill, 'The kinetics of hyaluronan in normal and acutely inflamed synovial joints: observations with experimental arthritis in sheep', Seminars in Arthritis & Rheumatol., 1993,22 Suppl. 1,9-17. 4. D. Scott, P. 1. Coleman, R. M. Mason & 1. R. Levick, 'Direct evidence for the partial reflection of hyaluronan molecules by the lining of joints during transsynovial flow', 1. Physiol., 1998, 508, 610-623. 5. P. 1. Coleman, D. Scott, 1. Ray, R. M. Mason & 1. R. Levick, 'Hyaluronan secretion into the synovial cavity of rabbit knees and comparison with albumin turnover', J. Physiol., 1997,503,645-656. 6. A. P. Spicer & T. K. Nguyen, 'Mammalian hyaluronan synthase:investigation of functional relationships in vivo', Biochem. Soc. Trans. 1999,27, 109-115. 7. P. Heldin, T. Asplund, D. Ytterberg, S. Thelin & T. C. Laurent, 'Characterization of the molecular mechanism involved in the activation of hyaluronan synthetase by PDGF in human mesothelial cells', Biochem. J., 1992, 283, 165-170. 8. L. Klewes & P. Prehm, 'Intracellular signal transduction for serum activation of hyaluronan synthase in eukaryotic cells', 1. Cell. Physiol., 1994, 160,539-544. 9. M. Suzuki, T. Asplund, H. Yamashita, C-H Heldin & P. Heldin, 'Stimulation of hyaluronan biosynthesis by platelet derived growth factor BB and TGF -~ 1 involves activation of protein kinase C', Biochem. 1.,1995,307,817-821. 10. M. K. Bansal & R. M. Mason, 'Evidence for rapid metabolic turnover of hyaluronate synthetase in swarm rat chondrosarcoma chondrocytes', Biochem. 1., 1986,236,515-519. 11. A. A. Pitsillides, L. S. Wilkinson, S. Meydizadeh, M. T. Bayliss & J. C. W. Edwards, 'Uridine diphosphoglucose dehydrogenase activity in normal and rheumatoid synovium', 1. Exp. Path., 1993,74,27-34. 12. N. Mian, 'Characterisation of a high-Mr plasm-membrane-bound protein and assessment of its role as a constituent of hyaluronate synthase complex', Biochem. J., 1986,237, 343-357. 13. D. C. Schonwasser, R. M. Marias, C. 1. Marshall & P. J. Parker, 'Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel and atypical protein kinase C isotypes', Mol. Cell. BioI., 1998, 18, 790-798.
HYALURONAN METABOLISM IN RESPONSE TO MECHANICAL STRAIN IS MODULATED BY MATRIX DEPLETION. G. P. Dowthwaite, S.Thomas, C. R. Flannery, A. A. Pitsillides 1 & C.W. Archer*. Connective Tissue Biology Laboratory, School ofBiosciences, CardiffUniversity, Cardiff, CFlO 3US. JDepartment
of Veterinary Basic Sciences, The Royal Veterinary Col/ege, University ofLondon, Royal Col/ege Street, London, NWI Oro, U.K
ABSTRACT Many studies have highlighted the importance of movement-induced mechanical stimuli in the development of functional synovial joints. However, such phenomenological results have failed to provide a full explanation of the mechanism essential for the morphogenesis of fluid-filled joint cavities. We have previously demonstrated that the large glycosaminoglycan hyaluronan (HA) in association with its principal cell surface receptor CD44, playa major role during the morphogenesis of chick joints (see Osbourne et aI., in this volume). Here, we have taken cells from the surface of recently cavitated joints and subjected them to a brief period of dynamic mechanical strain (3800 f.lE for 10 minutes) and measured changes in HA synthesis/release and HA synthase gene expression. In addition, we have subjected cells to matrix-depletion prior to the application of mechanical strain in order to examine any potential modulatory function of the extracellular matrix during the cell's response to strain. Removal of the cell-associated HA-containing matrix with hyaluronidase significantly increases the release of HA into tissue culture media over 24 hours and is associated with alterations in HA synthase gene expression. Such changes in HA release are shown to be synergistically enhanced by the application of dynamic mechanical strain. These results show that cell-matrix interactions modify the response of embryonic cells to mechanical strain and provide further insight into mechanodependent mechanisms ofjoint cavity morphogenesis. KEYWORDS Hyaluronan, hyaluronan synthase, articular fibrocartilage, mechanical stimulation, matrix depletion. INTRODUCTION The role of movement-induced mechanical stimuli in joint morphogenesis has long been recognised using paralysis or in vitro culture 1-4. In particular, these immoblisationJparalysis studies directly showed that movement was required for joint cavitation between cartilage anlagen and for the maintenance of previously cavitated joints, thus indirectly implicating mechanical stimuli in the process. More recent efforts have centred on providing a cellular basis to the phenomenological experiments mentioned above. Previous work by us identified
376
Aspects ofhyaluronan injoints
hyaluronan (HA) as a major constituent of the early coalescing extracellular vesicles that merge to form the cavity' and the association of uridine diphospo-glucose dehydrogenase (UDPGD), an enzyme involved in HA slnthesis, with the fibrocartilaginous articular cells of the presumptive joint line 6- • Later studies also demonstrated the involvement of the HA receptors CD44, IVD4 together with the actin capping protein moesin within these fibrocartilage cells. Furthermore, the importance of functional binding of HA to its respective cell receptors was demonstrated by the intraarticular injection of oligosaccharides of HA that displaced nascent HA with a consequent inhibition of cavitation (as assessed by reduction in HA accumulation) and a down-regulation of the recerotor expression 9,10. Similarly, paralysis of embryos in ovo showed very similar results 0. In an attempt to defme the effects of mechanical strain on the relevant fibrocartilage cells, we have isolated them in culture, and subjected them to a known strain regime. In brief, cells were placed on a 4-point bending jig that subjected cells to 3,800 f.!E at 1 Hz for 10 minutes. Strain enhanced the accumulation ofHA in the medium by 10 fold at 24 hours post stimulation when compared with static controls. (For more details of these experiments see the chapter by Osbourne et al., in this volume). Thus, for the first time, we were able to demonstrate a direct effect of mechanical stimulation on the accumulation ofHA by fibrocartilaginous articular cells in culture, an effect that can be correlated with an in vivo morphogenetic event. MATERIALS & METHODS Fibrocartilage cell isolation and culture Articular fibrocartilage was aseptically excised from the tibiotarsal joints of stage 42 11 White Leghorn chicken embryos and fibrocartilage cells isolated as described previously 12. Briefly, cells were isolated from diced fibrocartilage by digestion in 300 units mI,l collagenase (type I; Sigma, UK) for 1 hour at 37°C in sterile PBS. Cells were centrifuged and resuspended in Dulbeccos modified eagle medium containing 2 mM Lglutamine, 50 ug mI- 1 Gentamycin, 50 f.!g ml' ascorbic acid, 1 mg ml" D-glucose and 5% chick serum (DMEM(+); Life Technologies Ltd. UK). Cells were then either seeded into 75 cm2 tissue culture flasks and expanded or plated out directly. In all experiments, only primary or passage I (PI) cells were used. Matrix depletion using hyaluronidase Fibrocartilage cells (I x 105 mI- 1 in 35 mm dishes) were incubated overnight in Thereafter, media were removed and DMEM without chick serum (DMEM( Streptomyces hyaluronidase (Sigma, UK) added at 25 ill ml" and incubated at 37°C for 60 minutes. Cells were then washed 3 times with PBS (with gentle agitation for 5minutes) and fresh DMEM(-) added to the dishes. In addition, some experiments involved supplementation of such matrix-depleted cells with 200 ug mI-\ hyaluronan (Sigma) diluted in fresh DMEM(-) which was added immediately after the final wash in PBS. Controls for these experiments included cells that were not treated with hyaluronidase but were supplemented with media containing 200l1g mI-] HA (HA only control), cells treated with hyaluronidase alone (HAase only control) and cells cultured in serum free media alone (no treatment control).
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Metabolism modulated by matrix depletion
377
Application of mechanical strain to cells in vitro
Application of controlled, dynamic mechanical strain was achieved by subjecting plastic strips (6 x 2 ern) on which the cells were plated to four point bending in a specially designed and calibrated jig 12,13. Briefly, primary or first passage cells were plated onto plastic strips and grown to confluence (2 x 105 cells/strip) in DMEM(+) in a humidified atmosphere of 95% air/5% CO2 at 37°C. Confluent cells were serumdeprived in DMEM(-) overnight and the strips placed in the jig in 12 mI of fresh DMEM(-) and allowed to equilibrate for 30 minutes. Strips were then dynamically strained using 4 point bending to engender 3800 IlE at 1Hz for 10 minutes (total of600 cycles). Controls comprised strips carrying cells subjected to a similar cyclic perturbation of the medium as strained cells but without the applied strain (flow), and strips that were unperturbed (static). Medium was sampled (400 Ill) immediately prior to the application of strain and at various time points up to 24 hours. In addition, some experiments incorporated pre-treatment of cells with 25 ill ml" hyaluronidase (as descried above) followed by 10 minutes of strain, and others examined the effect of exogenous HA supplementation of media after HAase treatment. Determination of media HA concentration.
Duplicate samples of undiluted medium were assessed for HA concentration uSin either a competitive ELISA plate-based assay 14 or a modified uronic acid assay I f• Comparison of the 2 assays revealed no significant differences in HA concentration from duplicate samples (data not shown). cDNA synthesis and polymerase chain reaction
Total RNA was isolated from hyaluronidase-treated and control cells (1 x 105 cells ml") at 0, 2, 6 and 24 hours after treatment using Tri reagent (Sigma; manufacturers instructions) and incubated with 10 units of DNase (Calbiochem, U.K.) at 37°C for 30 minutes. RNA was reverse transcribed into first strand cDNA using the Gene Amp PCR core kit (Perkin Elmer, UK) following the manufacturers instructions. Reverse transcribed mRNA was then amplified using the polymerase chain reaction (PCR) with gene-specific primers identified from partial DNA sequences of the chick HAS 2 and 3 genes 16: HAS 2 sense: 5'-ACC CGC TGG AGT AAA TCG TAT-3'; HAS 2 anti-sense: 5'-TAA GGA AGA AAG GAA AGA ATC-3'; HAS 3 sense; 5'-CCT ACT TTG GCT GTG TGC-3' and HAS 3 anti-sense: 5'-GCG GGT CTG ITG GTT GAG-3'. Conditions for 'hot start' PCR were as follows: a lower mixture containing 1.25 III lOx PCR buffer, 3 III MgCh (final concentration 1.5 mM), 4 III of dNTP (mix fmal concentration 200 1lM), I III of each primer and an Ampliwax Gem 100 (Perkin Elmer) bead were incubated at 70°C for 90 seconds and cooled to 20°C. An upper mixture containing 5 III lOx PCR buffer, 0.3 III Taq polymerase (final concentration 1.5 units), 0.1 ug mRNA and sterile water to a final volume of 50 III was laid over the solidified wax pellet. Samples were initially denatured at 94°C for 60s followed by 40 cycles of 95°C 30s, 48°C 45 s for HAS 2, 57.6°C 45s for HAS 3, noc 30s and a fmal extension step at ire: for 5 minutes. PCR products (10 Ill) were run on 3% agarose gels containing 1 ug mI -I ethidium bromide at 100 volts for 30-45 minutes and visualised under UV light. PCR products of the expected size were then purified using a Wizard PCR Prep kit (Promega, UK) following the manufacturers instructions. Purified PCR
378
Aspects of'hyaluronan in joints
products were then sequenced using the d-rhodamine dNTP cycle sequencing kit (ABI Prism) following the manufacturers instructions and sequenced using an ABI Prism 377 automated sequencer. Sequence homologies to published HAS gene sequences were confirmed using the BLAST search protocol 17. RESULTS & DISCUSSION
Effect of mechanical stimulation on hyaluronidase pre-treated fibrocartilage cells. A. Strain-related changes in medium HA concentration In order to determine the specific increases in HA concentration in the medium of cells exposed to a short (lOminute) period of dynamic mechanical strain or medium flow, with or without prior treatment with HAase, data have been converted to a logarithmic scale (1oglO). In addition to equalising variability, this allows simple additive, or more complex multiplicative (synergistic), models of interaction between two factors (mechanical stimuli and matrix involvement) to be examined. The statistical significance ofthese results was evaluated using ANOV A and r-tests. Without prior HAase treatment of cells, both 'flow-specific' and 'strain-specific' increases (p2.0xl0"
Rooster comb
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HA preparations were provided by or purchased from several manufacturers. Suplasyn® was from Bioniche Life Sciences Inc., London, Ontario, Canada.
The ability of high molecular weight HA to induce the synthesis of IL-12 and TNF-a. by monocytes suggested that contaminating molecules could be responsible for the induction of these cytokines. Endotoxin contamination has been shown to be able to induce pro-
432
The action of hyaluronan in cells
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Monocytes were incubated at 1 x l06cellsJml for 48 h at 37°C, 5% CO 2 with (a) different concentrations ofHA preparation "D" and "F", and (b) with 10 ug/ml ofHA preparations "D" and "F" or with 10 ug/ml ofHA preparations "D" and "F" treated with 10 U of DNAse I as described in the Materials & Methods. IL-12 and TNF-ex levels in the supernatant were measured after 48 h by ELISA. Data are expressed as the mean +/- S.D. of three independent experiments.
Pro-intlammatory activity of DNA
433
inflammatory cytokines such as IL-12 and TNF -a by monocytes and to cause septic shock in mammals!", However, none of the HA preparations evaluated contained detectable endotoxin or protein. DNA from numerous species has been shown to induce pro-inflammatory cytokines including IL-12 and TNF-a I 5 • In vivo, DNA purified from either Gram-positive or Gramnegative bacteria has been shown to cause septic shock in D-galactosamine-sensitized mice". Administration ofbacterial DNA as well as synthetic oligonucleotides into the lungs ofmice has been found to cause severe inflammation in the lower respiratory tract", Furthermore, intra-articularly injection ofDNA containing CpG motifs has been shown to induce arthritis in mice". We therefore determined whether the different HA preparations contained DNA. We found that 3 out of the 7 HA preparations tested contained significant levels of DNA with molecular weights ranging from 500 b.p. to> 20 000 b.p.; HA preparation "A": 5% , HA preparation "D": 3% and HA preparation "F": 15%. Treatment ofthe HA preparations with DNase I, an endonuclease which digests both single- and double- stranded DNA molecules to small oligodeoxyribonucleotides, resulted in the abolition ofIL-12 and TNF-a induction by HA preparation "D" and in a reduction ofIL-12 and TNF-a synthesis by HA preparation "F", with reductions of 77% and 32% respectively (Figure Ib). The results obtained with the preparation "F" correlated with an incomplete digestion ofthe DNA by the DNAse I (data not shown). Inactivated DNase I had no effect on the cytokine-inducing activity ofHA preparations "D" and "F" (data not shown). The DNase I used in these studies does not possess hyaluronidase activity. The presence ofDNA in HA preparations could be explained by association or contamination during the isolation and purification ofHA from rooster comb or from bovine trachea. One HA preparation, HA preparation "A", isolated from streptococci sp. also contained DNA but did not elicited a cytokine response from monocytes. It would therefore appear that the nature of the DNA is important. Experiments are underway to determine the sequence motifs present in DNA associated with HA which are responsible for the induction of cytokines.
CONCLUSIONS In summary, we found that two out of seven HA preparations examined had the ability to induce pro-inflammatory cytokines. The ability ofHA to induce these cytokines was not related to the presence of low molecular weight HA fragments, but rather to the presence of contaminating DNA. It is perhaps advisable to determine whether pro-inflammatory DNA is present in HA preparations before their use in the treatment ofpatients with inflammatory disorders as well as the potential modulation ofpro-inflammatory cytokines or inflammation in animal models.
REFERENCES
2.
A. Lussier, A. Cividino, C. A. McFarlane, W. P. Olszynski, W. 1. Potashner & R. De Medicis, Viscosupplementation with Hylan for the treatment of osteoarthritis: findings from clinical practice in Canada, J. Rheumato/., 1996, 23, 1579-1585. H. Matsuno, K. Yudoh, M. Kondo, M. Goto & T. Kimura, T. Biochemical effect of
434
3. 4.
5. 6. 7. 8.
9.
10.
11.
12. 13. 14.
15.
16. 17.
18.
The action ofhyaluronan in cells intra-articular injections of high molecular weight hyaluronate in rhematoid arthritis patients, Inflamm. Res., 1999,48, 154-159. A. Morales, L. Emerson, J. Curtis Nickel & M. Lundie, Intravesical hyaluronic acid in the treatment of refractory interstitial cystitis. J. Urol., 1996, 156,45-48. M. P. Puttick, J. P. Wade, A. Chalmers, D. G. Connell & K. K. Rangno, Acute local reactions after intraarticular hylan for osteoarthritis ofthe knee. J. Rheumato/., 1995,22, 1331-1314. M. E. Adams, Acute local reactions after intraarticular hylan for osteoarthritis of the knee. J. Rheumato/., 1996,23,944-945. D. O'Hanlon, Acute local reactions after intraarticular hylan for osteoarthritis of the knee. J. Rheumato/., 1996,23,945-946. J. R. Kirwan & E. Rankin, Intra-articular therapy in osteoarthritis. Bail/ieres Clin. Rheumatol., 1997,11,769-794. C. M. McKee, M. B. Penno, M. Cowman, M. D. Burdick, R. M. Strieter, C. Bao & P. W. Noble, Hyalronan (HA) fragments induce chemokine gene expression in alveolar macrophages: the role ofHA size and CD44. J. Clin. Invest., 1996, 98, 2403-2413. J. Hodge-Dufour, P. W. Noble, M. R. Horton, C. Bao, M. Wysoka, M. D. Burdick, R. M. Strieter, G. Trinchieri & E. Pure, Induction ofIL-12 and chemokines by hyaluronan requires adhesion-dependent priming of resident but not elicited macrophages. J. Immuno/., 1997, 159, 2492-2500. R. A. Dodds, J. R. Connor, F.H. Drake & M. Gowen, Expression of cathepsin K messanger RNA in giant cells and their precursors in human ostoarthritic synovial tissues. Arthritis Rheum., 1999,42, 1588-1593. F. Liote, B. Boval-Boizard, D. Weill, D. Kuntz & J. L. Wautier, Blood monocyte activation in rheumatoid arthritis: increased monocyte adhesiveness, integrin expression and cytokine release. Clin. Exp. Immunol., 1996, 106, 13-19. T. J. Christmas, & G. F. Bottazzo, Abnormal urothelial HLA-DR expression in interstitial cystitis. Clin. Exp. Immunol., 1992, 87,450-454. H. G. Lee & M. Cowman, An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution. Anal. Biochem., 1994,219,278-287. M. Astiz, D. Saha, D. Lustbader, R. Lin & E. Rackow, Monocyte response to bacterial toxins, expression of cell surface receptors, and release of anti-inflammatory cytokines during sepsis. J. Lab. Clin. Med., 1996, 128,594-600. D. M. Klinman, A-K. Vi, S. L. Beaucage, J. Conover & A. M. Krieg, A. M., CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon. Proc. Natl. Acad. Sci. USA, 1996,93,2879-2883. T. Sparwasser, T. Miethke, G. Lipford, K. Borschert , H. Hacker, K. Heeg & H. Wagner, Bacterial DNA causes septic shock. Nature, 1997, 386, 336-337. D. A. Schwartz, T. J. Quinn, P. S. Thome, S. Sayeed, A-K, Yi & A. M. Krieg, CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract. J. Clin. Invest., 1997, 100, 68-73. G-M. Deng, I-M. Nilsson, M. Verdrengh, L. V. Collins & A. Tarkowski, Intraarticularly localized bacterial DNA containing CpG motifs induces arthritis. Nature Med., 1999,5,702-705.
EFFECT OF HYALURONAN OLIGOSACCHARIDES ON THE EXPRESSION OF HEAT SHOCK PROTEIN 72 Heping xu·,J, Tomomi Ito" Akira Taweda', Hiroshi Maeda" Hiroko Yamanokuchi" Kyoko Isahara', Keiichi Yoshida" Yasuo Uchiyama", Akira Asari t 'Seikagaku Corporation. Tateno 3-1253, Higashiyamato-shi, Tokyo 207-0021 Japan. 2
Department of Cell Biology and Anatomy. Osaka University Medical School. Suita-shi, Osaka. Japan. 3
Present address: Department ofOphthalmology, University ofAberdeen Medical School. Foresterhill, Aberdeen AB25 2ZD. UK.
ABSTRACT We have previously shown that intra-articular treatment with a hyaluronan preparation (849 kDa), HA84 up-regulates heat shock protein 72 (Hsp72) expression and suppresses degeneration of synovial cells in an arthritis model. In that study, the HA administered was degraded into HA oligosaccharides in the synovial tissue, suggesting that HA84 or degradation products of HA may up-regulate Hsp72 expression. Thus, in the present study, we examined the effects of HA oligosaccharides of various molecular sizes on Hsp72 expression and/or cell death in stressed cells. Western blotting analysis showed that treatment of K562 cells with HA tetrasaccharides up-regulated Hsp72 expression after exposure to hyperthermia. On the other hand, treatment of the cells with HA of other sizes (di-, hexa-, deca-, dodeca-saccharides), HA84 or tetrasaccharides of keratan sulfate did not elicit any change in expression of the Hsp72 protein. Treatment of the cells with tetrasaccharides of HA up-regulated not only expression of the Hsp72 protein but also Hsp72 mRNA expression, and enhanced activation of HSFl, a transcription factor controlling Hsp72 expression, after exposure to hyperthermia. Since the level of Hsp72 protein was not affected by tetrasaccharides of HA when the K562 cells were kept at 37°C without any stress, it is evident that tetrasaccharides of HA did not act as a stress factor. In addition, tetrasaccharides of HA suppressed cell death in the case of K562 cells exposed to hyperthermia and of PC12 cells under serum deprivation. These results suggest that certain types of oligosaccharides i.e., the tetrasaccharides of HA, up-regulate Hsp72 expression by enhancing the activation of HSFI under stress conditions. KEYWORDS
436
The action of hyaluronan in cells
Cell death, heat shock factor I, heat shock protein 72, hyaluronan oligosaccharides
INTRODUCTION Heat shock proteins (Hsps) are induced to suppress cell damage when cells are exposed to environmental insult'. Hsp70 suppresses apoptosis by preventing processing of caspase 3 2• We have previously shown that intra-articular treatment with an HA preparation (840 kDa), HA84, suppresses degeneration of synovial cells in a canine arthritis model and up-regulates Hsp72 expressiorr'", In that study, we also examined the kinetics of HA84 degradation in synovial tissues by injecting fluorescent-labeled HA84 and found that some labeled HA particles could not be detected by means of an HA-binding protein that binds specifically to HA molecules larger than decasaccharides". These observations suggested that HA oligosaccharides formed through degradation of HA84 in the tissue may suppress cell damage by up-regulating Hsp72 expression. In the present study, we prepared HA oligosaccharides of various molecular sizes and treated cultured cells with them under stress conditions in an effort to determine the appropriate size of HA oligosaccharides required to up-regulate Hsp72 expression or to suppress cell death. Effects of HA molecules on Hsp72 expression were investigated by examining Hsp72 protein levels and Hsp72 mRNA levels, and the activation of heat shock factor 1 (HSFl), a transcription factor controlling Hsp72 expression, in K562 cells exposed to the stress of hyperthermia. HSFI is known to be transferred to the nucleus from the cytoplasm and it binds to a heat shock element in the DNA 5 ,6. Moreover, HSFI is phosphorylated, and its molecular weight thereby increases when activated, soon after heat shock treatment", In addition to Hsp72 expression and HSFI activation, the effects of HA molecules on cell death were examined using 1.
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P3 million) elastoviscous hyaluronan solution (Healon~ as a viscosurgical tool, to protect and manipulate tissues and to create tissue space in surgical procedures is established in animal studies and in clinical practice. The first international meeting on the use of Healon@ in eye surgery is held in Germanyll.1S. 1983: The first combination viscosurgical product made of 4% chondroitin sulfate (MW 25,000) and 3% hyaluronan (average MW 0.5 million) called Viscoat'" was mtroduced!" 1989: The first use of Healon@ to promote healing of chronic tympanic membrane perforation in ear surgeryt7·19. 1992: The use of Healon@ as a viscosurgical tool to facilitate the insertion of inner ear electrodes in cochlear implant surgery", 1992 - 1996: A great variety of viscoelastics mostly made of hyaluronan are available for ophthalmic viscosurgery. They represent a broad spectrum of rheological properties. The rapid development of new surgical procedures for lens extraction and intraocular plastic lens implantation often requires the use of two viscoelastics with different rheological properties during the surgery. The concepts of "cohesive" and "dispersive" viscoelastics are introduced21~2. Today, there are many hyaluronan solutions on the market for ophthalmic viscosurgery. They contain Na-hyaluronan of various average molecular weights (0.5 MW to > 4 million MW) and in various concentrations (1-2.3%). Consequently the rheological properties (elasticity and viscosity) of these fluids varies considerably. One preparation, in addition to low average molecular weight hyaluronan also contains chondroitin sulfate (Viscoat'").
Viscosurgery: a historical perspective
463
Shear Viscosity And Pseudoplasticity or Some Major Viscoela~tics1J
Product
Healon"' 5 Healon'"GV Healon'" AmviscW Amvisc"'Plus Viscoat" Proviso" MicroviscW Microvisc4l> Plus
Distributor and Manufacturer
Pharmacia Bausch & Lomb and Anika Alcon and Genzyme (-MHO Pharmaand Bohus Bio. Tech
Zero Shear Viscosity* PaS 5500 2500 240 60 100 60 200 1200 3700
Pseudoplasticity**
253 241 111 8 40 10 70 389 549
* Zero shear viscosity in physiological buffer solutions depends on the average molecular weight and concentration of hyaluronan.
** Defined as the quotient of shear viscous at O.lscc·' and 100 sec"
RHEOLOGICAL PROPERTIES OF VISCOELASTICS The elastoviscosity of hyaluronan solution depends on the average molecular weight and the concentration of the molecule dissolved in the physiological buffer solutions. Greater concentration and higher molecular weight yields solution with greater viscosity and elasticity. Molecular interactions and the presence of low molecular weight glycosaminoglycans can also influence pseudoplasticity and elasticity of the solution. The elastoviscous behavior is also greatly dependent on the shear rate or frequency at 'which it is measured. (Fig. 1 & 2)24. Low molecular weight polysaccharides, like chondroitin sulfate (MW 25,000) in relatively large concentration (3% or more) have low viscosity and they also exhibit "stickiness". When these "sticky" solutions are mixed with viscous low molecular weight hyaluronan (average MW 2000 kDa). The data demonstrate a rapid uptake and lysosomal degradation of IC-HA in REKs via a receptor dependent route separate from coated pits and caveolae, that involves a receptor with HA 10 specificity, identified as CD44 or functionally dependent on CD44. The IC-HA consists of small HA fragments which may have a specific role in cellular homeostasis.
KEYWORDS Hyaluronan, epidermis, keratinocyte, endocytosis, CD44, hyaluronan oligosaccharides, coated pits, caveolae
INTRODUCTION Hyaluronan is the major extracellular matrix component in epidermis I. The estimated concentration of hyaluronan between keratinocytes exceeds one rng/rnl, while its half life is only about one day 2, 3. Obviously, there must be an efficient catabolic mechanism which balances the rapid synthesis of HA, and maintains the hyaluronan concentration in a range that supports the normal cell-cell interactions in stratification and differentiation. Partial degradation of epidermal hyaluronan in organ cultures is also indicated by the decrease in the molecular weight of newly synthesized hyaluronan molecules (> 4x10 6 Da) to Ix106 and 0.5x106 Da after 24 and 48 h chases, respectively 3. Experiments in rat epidermal keratinocytes in organotypic cultures also demonstrated hyaluronan catabolism by keratinocytes 4. Keratinocytes express a high level of CD44 5, a receptor
518
Keratinocytes and hyaluronan
responsible for hyaluronan uptake in other cells 6, 7 and mice with blocked epidermal CD44 expression accumulate hyaluronan in skin 8, These results collectively suggest that hyaluronan uptake involves the CD44 receptor on keratinocytes. We previously found that rat epidermal keratinocytes (REKs) contain a pool of hyaluronan resistant to trypsin and Streptomyces hyaluronidase treatments 9, suggesting an intracellular location. The present study characterizes this pool in more detail, and shows that even in monolayer cultures, REKs actively internalize and metabolize hyaluronan in a receptor-mediated fashion via an endocytic route that is not dependent on coated pits or caveolae, but involves CD44. Increased amounts of intracellular HA are seen in proliferating and migrating keratinocytes. This is particularly interesting because it has been recently suggested that intracellular hyaluronan is involved in novel cell functions beyond those involved in extracellular matrix organization and regulation 10. 11. MATERIALS & METHODS
Cell culture A newborn rat epidermal keratinocyte line (REK) was developed by MacCallum and Lillie 12. REKs were cultured in Dulbecco's MEM (low glucose, Life Technologies, Paisley, UK) with 10% fetal bovine serum (HyClone, Logan, UT). For biochemical assays and radiolabeling, the cells were seeded at 100.000/ml and grown close to confluency in 6-well plates (Costar Corp., Cambridge, MA). For microscopic studies, the cells were plated in 8-well chamber slides (Lab-Tek, Nalge Nunc Int., Naperville, IL).
Modification of hyaluronan uptake and degradation To study the receptor mediated uptake of hyaluronan, we treated recently confluent REK cultures with hyaluronan oligosaccharides 9 or with chondroitin, chondroitin sulfate A, chondroitin sulfate C or heparan sulfate (Seikagaku Kogyo Co., Tokyo, Japan). Other cultures were incubated with anti-CD44 mabs; Ox 50 (Biosource Int., Camarillo, CA) or Hermes 3 (a gift from Dr. Sirpa Jalkanen, Turku) or with non-immune mouse IgG (Sigma). To block endocytosis through clathrin coated pits, we treated cells with chlorpromazine (Sigma), or hypertonic serum free medium with 0.4 M sucrose as described 13. To block uptake via caveolae, we treated cells with filipin (Sigma) or nystatin (Sigma) 14, To inhibit macropinocytosis, we treated cells with arniloride IS, The function of lysosomes was perturbed by using either ammonium chloride or chloroquine (Sigma) 16. Receptor recycling was inhibited by adding monensin (Sigma) to the culture medium.
Isolation of intracellular hyaluronan and assay of hyaluronan and chondroitin sulfate disaccharides The REK cultures were radio labeled with 20 and 100 MCilml of [3H]glucosamine and [35S]sulfate (Amersham, Little Chalfont, UK), Cell surface associated hyaluronan was removed by incubating the cells with trypsin (0.2 % trypsin-a. I % EDT A, Sigma) either alone or followed by incubation with Streptomyces hyaluronidase (15 TRU/ rnl in HBSS) for 4-12 h at 4°C. Hyaluronan remaining in the cell fraction after trypsin and extracellular hyaluronidase treatment was designated "intracellular". The samples were digested with proteinase K or papain, and hyaluronan and other glycosaminoglycans were precipitated with cold ethanol, followed by precipitation with CPC 9. Samples were subjected to the analysis of specific disaccharides as described previously 9. 4. Samples were digested with Streptococcus hyaluronidase and chondroitinase ABC (Seikagaku) and injected onto a Superdex Peptide column. The eluent was monitored at 232 nrn, and the fractions were counted for 3H and 35S activities. The carrier hyaluronan produced a disaccharide peak quatitated by absorbance at 232 nm,
Epidermal keratinocytes
519
which was used to monitor the recovery (about 80%). The chemical content of newly synthesized hyaluronan was calculated from the dual labeling data as described in detail previously 4.
Hyaluronan size assay The samples were digested with proteinase K, ethanol precipitated and chromatographed on Sephacryl S-1OOO (Pharmacia, Uppsala, Sweden). Healon (4Ilg) was added as a carrier to each fraction, followed by precipitation with ethanol at -20°C. The precipitates were analyzed for labeled hyaluronan using Superdex chromatography of specific disaccharides as described above.
Staining for endogenous intracellular hyaluronan and markers of cellular compartments The staining protocol was essentially as decribed before 9. The pericellular hyaluronan was removed by treating paraformaldehyde fixed cells with Streptomyces hyaluronidase (10 TRU/rnl for 20 min). Thereafter, the cells were permeabilized in 0.1 % Triton-X 100, and incubated with a biotinylated complex of hyaluronan binding region of cartilage proteoglycan and link protein (bHABC) 17. The bHABC specifically bound to HA was visualized either using avidin-biotin peroxidase (Vector Laboratories, Inc., Burlingame, CA) and DAB (Sigma), or by Texas Red streptavidin or by FITC-avidin D (Vector) . Optical densities of DAB-stained cells were measured as described 9. In dual staining protocols, monoclonal antibodies (against transferrin receptor, cathepsin D, caveolin I, and CD44) were mixed with bHABC. Texas Red-labeled antimouse antibody combined with FITC-avidin was used to visualize the mabs. For electron microscopy REKs were stained for HA as described above and postfixed with reduced osmium tetroxide. For dual staining of HA and CD44, antimouse secondary antibody, conjugated to 5 nm gold particles (Amersham, Little Chalfont, UK) was applied. Fluid phase and coated pit uptake were visualized by incubating REKs in the presence of lysine fixable, Texas Red-labeled dextran (MW 10,000, Molecular Probes, Oregon, USA) and FITC-Iabeled transferrin (Molecular Probes). Fluorescein-labeled hyaluronan preparations were gifts from Drs. Ronald Midura and Jayne Lesley.
RESULTS Hyaluronan in cytoplasmic vesicles bHABC localized most of the HA on plasma membranes of REKs. However, some was also located in vesicle-like structures close to the nucleus, suggesting an intracellular location 9. Treatment of living REK cultures or fixed cells with Streptomyces hyaluronidase before permeabilization and staining for hyaluronan did not remove this perinuclear staining. Confocal microscopy of cells pretreated with hyaluronidase exhibited a vesicle-like hyaluronan signal mostly in a perinuclear position. Transmission electron microsopy showed that the HA was localized in membrane bound vesicles.
Content of intracellular hyaluronan REKs contain a pool of hyaluronan that is resistant to trypsin digestion, a treatment that removes virtually all hyaluronan from the cell surfaces. The size of this trypsinresistant hyaluronan pool was larger in low cell density cultures, as demonstrated by analysis of parallel cultures seeded at different densities and metabolically labeled on the next day. Cultures with fewer REKs (14 x 104 cells/cm 2) exhibited a tenfold higher intracellular hyaluronan content than REKs seeded at 88 x 104 cells/cmz (see Fig. 1).
520
Keratinocytes and hyaluronan ;:::;-12 ......- - - - - - - .
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gel) was tested in abdominal and gynecological surgery both in laparoscopy and laparotomy. The synthetic HA plasmacoated devices were tested in a standardized model oflarge abdominal wall defect repair. In all experiments, the animals were provided with standard chow and water ad libitum. Animal care and surgery were performed under the National Guidelines of the Ministry of Health # 116/92. Abdominal surgery Forty New Zealand female white rabbits weighing 3-4 Kgs were randomly allocated into two treatment groups (n=20): no treatment (control group) and RYALOBARRlERlI> gel (treated group). At the time of laparotomy for adhesion evaluation, observers were blinded to the treatment applied. The animals were kept fasting for at least 24 hours before surgery, and anesthetized by intramuscular injection with SO mg/kg of Ketamine hydrochloride and 1.6 mg/kg Xylazine hydrochloride. They were then placed in supine position, shaved on the abdomen and prepped. After a 10 ern mid line laparotomy the parietal peritoneum of the right side was exposed and a 10 em" defect including both parietal peritoneum and the muscular fascia was created by sharp dissection. The cecum was then exposed, and abraded starting from the Sth to the io- haustrum distally from the ileocecal junction until bleeding appeared. The cecum was then returned to its normal anatomical position. The abdominal cavity was washed with Ringer's lactate solution and accurate hemostasis was performed. Ten weeks after surgery, the animals were euthanized and the grade and severity of adhesions were blindly evaluated applying a 0-11 adhesion score", The incidence of adhesion-free animals was also evaluated. Gynecological surgery Two different experimental standardized protocols were followed in this type of surgery: HYALOBARRIER iIll gel was tested in 64 female rabbits subjected to laparoscopy and 69 animals which received laparotomic surgery. In laparoscopy, adhesions (protocol 1) were induced by means of two different surgical trauma: abrading the uterine horns and denuding a delimited area of the adjacent peritoneum. Following surgery accurate hemostasis was achieved by means of an electrocoagulator and the animals were randomly assigned to HYALOBARRIER iIll gel treatment (n=22), TC7 Interceed'", a commercially available antiadhesive barrier (n=20), or no treatment (n=22). Treatments were applied uniformly over the raw surfaces. Six weeks after surgery, adhesions were blindly evaluated applying a
494
Clinical applications ofhyaluronan
scoring system (0-4 scale)" and determining the percentage of animals with severe adhesions. In laparotomic surgery (protocol 2) the four major blood vessels of each uterine hom ligament were excised until abundant bleeding appeared. No hemostasis or cautery was performed. Following surgery, the animals were randomly assigned to HYALOBARRIER
gel (n=20) treatment, Intergelf" (n=12), Seprafilm" (n=17) (two hyaluronan based devices), or no treatment. The materials were applied in order to cover completely all injured anatomical sites. Two weeks after surgery, the animals were euthanized and the adhesions were graded using a 0-4 score" and comparing the percent of adhesion-free animals in each treatment group. Abdominal surgery (large defect). Two different surgical meshes based on ePTFE and polyester mesh used in clinical practice for incisional hernia repair, were surface modified by hyaluronan plasma treatment. The biomedical devices were steam-sterilized. Seventy-two rabbits were anesthetized with ketamine/xylazine i.m. injection and subjected to the following surgical procedures: a 12 em' full-thickness parietal defect was created and the omentum was removed. The animals were randomly divided into four groups and the surgical meshes for abdominal repair were fixed to the abdomen after isolation of the muscular plane by means of non-absorbable sutures. Two groups received either ePTFE or polyester unmodified meshes (controls), the remaining two groups received the same devices subjected to HA-plasma coating treatment (treatments). Two months after surgery, a secondlook laparotomy was performed, the adhesions were blindly assessed applying a site-specific adhesion score", and the incidence of adhesion-free animals was calculated. The stability of the implanted meshes was evaluated, as was the presence of inflammatory reaction by means of histological observations. Clinical trials. Various clinical trials have been designed to demonstrate the safety and efficacy of hyaluronan derivatives: the crosslinked hyaluronan derivative HYALOBARRIER gel was tested in abdominal and gynecological surgery, whereas the benzylic ester derivative HYAFFTM 11 in non-woven form (commercial name Merogel") was tested in E.N.T. surgery. In abdominal surgery, 20 patients with ulcerative colitis and familial polyposis who were scheduled for colectomy and ileal pouch-anal anastomosis with diverting-loop ileostomy were enrolled in a pilot trial. Before abdominal closure, the patients were randomly assigned to treatment or control (untreated). The patients in the treated group received the gel uniformly distributed on the intestinal loops. second look laparoscopy for adhesion evaluation was programmed when patients returned for ileostomy closure. In gynecological surgery, three different trials were designed involving the following surgical procedures: laparoscopic surgery (study 1), hysteroscopic surgery (study 2) and laparotomy (study 3). In the first multicenter controlled clinical trial, eighty patients subjected to myomectomy, ovarian cysts removal, adhesiolysis, conservative treatment of ectopic pregnancy and endometriosis removal, randomly received HYALOBARRlER gel (n=40) or no treatment (n=40). In the second trial, sixty patients subjected to hysteroscopic surgery for septatae uterus, intrauterine adhesions, endometrial ablation, endometrial polyps and submucosal myoma randomly received the gel (n=30) or no treatment (n=30). Patients subjected to laparotomic surgery for myomectomy were enrolled in the third multicenter study and randomly assigned to a HYALOBARRIER treatment group or untreated control group. All the study protocols were approved by ethical review and all patients gave written informed consent. For ethical considerations, in the gynecological
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surgery studies, the second look laparoscopy required for adhesion assessment (6-12 months after surgery) was envisaged in the protocol at the discretion of the principal investigators. In the E.N.T. trial, inclusion criteria involved patients undergoing functional endoscopic sinus surgery (FESS) for bilateral or unilateral chronic sinusitis. Patients randomly received Merogel" in the treated group and FESS with standard postoperative treatment without application of the product in the control group. The safety of the hyaluronan-based device was evaluated recording the occurrence of adverse events at each control visit 1, 14, 30, 60 and 90 days after surgery. Its effectiveness was evaluated at 14, 30 and 90 days following initial surgery recording the presence of adhesions in the cavities by video sinus endoscopy. RESULTS
Preclinical Experiments Abdominal surgery At the time of second look laparotomy the gel had been completely absorbed. No abdominal hernias, infection, or other adverse events were recorded during postsurgical monitoring. The mean adhesion scores were respectively 0.65±0.45 and 7.73±0.83 for the HY ALOBARRIER gel and untreated control groups. In this case, significant differences were seen between the two groups (P = 0.0001 non parametric Kruskal-Wallis test). The percentage of adhesion-free animals was significantly greater in the group treated with ACP gel than in the untreated control group: 90% HY ALOBARRIER gel vs. 15% control (P = 0.0001 Chi-square test).
Gynecological surgery In the laparoscopic protocol, six weeks after surgery at the second look observation when Blauer's scoring system of adhesions (0-4 scale) was applied, the HYALOBARRIER gel treatment showed the lowest grade of postsurgical adhesions (1.25±0.28) compared to TC7 Interceed" (2.45±0.22) and the untreated group (2.24±O.26). Significant differences were found between the HYALOBARRIER gel vs. TC7 Interceed''" and untreated control groups (P < 0.05 Kruskall-Wallis non-parametric test). The HY ALOBARRIER gel treatment group showed the highest percentage of animals with severe adhesions (35%) compared to the TC7 Interceed" (85%) and to the untreated group (66%). In the laparotomic protocol (protocol 2) the group of animals treated with HY ALOBARRIER gel presented a low adhesion grade (1.15±0.32) and a percentage of adhesion-free animals corresponding to 55%. The other treatments also gave a low adhesion grade (Intergel" 1.17±O.46, Seprafilm" 2.29±O.44) and a percentage of adhesion-free animals corresponding to 58% in the Intergel" and 35% in the Seprafilm" groups. Statistically significant differences were found in all treatments vs. untreated control (P < 0.05 Kruskal-Wallis non-parametric test and Chi-square tests) but no differences were found between the treatment groups.
Abdominal surgery (large defect) At the time of second-look laparotomy, the new-grown mesothelial layer in the surgical defect was incorporated into all prosthesis, no evidence of seroma or intra-abdominal abscess was present. The HA-coated synthetic membranes gave lower adhesion score meanS±sem than the unmodified surgical devices. The HA-treated ePTFE group developed less severe adhesions
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(filmy and avascular adhesions) between the biomaterials and the intestinal loops or liver then the other treatments. The resulting adhesion grade was 0.44:1:0.30 in the HA-plasmacoated ePTFE group and 2.72±0.97 in the corresponding uncoated controls. The scores of HA-coated polyester meshes and the uncoated biomaterial were respectively 3.44±1.06 and 6.56±l.lS, a statistically significant difference was seen by applying the Kruskal-Wallis nonparametric test (P < 0.05). The same trend was seen by evaluating the incidence of adhesionfree animals in each treatment group: 66.6% of animals in the HA-ePTFE treated group showed absence of adhesions vs. S9.9% of the uncoated ePTFE group. A decrease in the percentage of adhesion-free animals was seen in the group treated with polyester-based devices: the percentages of animals with no adhesions were respectively 50% in the polyester-coated mesh group vs. 22% in the uncoated control group (P < 0.05, Chi-square test) that showed strong and widespread adhesions between mesh and bowel and liver. On histological observation, the thickness of the muscle-mesh interface did not differ between HA treated membrane groups and uncoated controls. Similarly, no inflammatory reaction or fibrosis at the muscle-mesh interface was seen.
Clinical Trials To date, 20120 patients have been enrolled in the pilot abdominal surgery study. Postoperatively, no complications or adverse events in the short or long term occurred in treated and untreated groups. Adhesion assessment by second look laparoscopy is currently ongoing. SO/SO patients in the gynecological laparoscopy and 60160 in hysteroscopy were enrolled and they received an average of20 and 10 ml of gel respectively. Clinical data recorded during the postoperative course indicate the absence of any serious adverse events. No statistically significant differences were observed in the incidence of the most frequent adverse events, fever and pelvic pain, which normally occurred in the postoperative period. This data confirmed the good tolerability of HYALOBARRIERiI> gel. Hospitalization time was no longer for the treated patients than for the untreated control group. Adhesion evaluation by second look laparoscopy is still ongoing. The gynecological trial in laparotomy is currently ongoing, to date 50/100 patients have been enrolled and no complications or serious adverse events have been observed. 66 patients were enrolled in the E.N.T. trial for a total of 117 nasal cavities subjected to surgery. The two study groups were statistically comparable for demographic characteristics. No serious adverse events occurred in the treated (Merogel") and control groups. During the control visit, clinical evaluation by means of video-endoscopy demonstrated a significant reduction (P < 0.05) in the percentage of sinuses with adhesions 30 and 90 days after surgery in the Merogel" group in comparison to the control (1.9% vs. 14.9% and 3.6% vs. 23.7% respectively). DISCUSSION Various methods of adhesion prophylaxis have been used during experimental and clinical trials. Practicing an appropriate surgical technique is the most effective strategy for reducing the risk of adhesion formation. The reduction of incisional areas by means of miniinvasive surgery, limiting trauma, avoiding introducing foreign bodies into the peritoneal cavity and achieving an adequate hemostasis, are surgical procedures that every surgeon should adopt. However, improvements in surgical technique alone have not satisfactorily reduced the incidence of adhesion formation. The barrier methods based on hyaluronan and its derivatives seem to be promising because of their efficacy and absence of serious side effects. The devices are used as a barrier to
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separate adjacent surfaces while the wound healing process occurs. This method was demonstrated to be effective because it produces an intraperitoneal "flotation" during peritoneal healing, avoiding fibrin bridge formation between adjacent organs. However, in many cases the solution's low viscosity and consequent rapid clearance represent a critical point, reducing the antiadhesive efficacy of the device. The hyaluronic acid derivatives HYALOBARRIER@ gel and HY AFFTM 11 (Merogel" are new, absorbable biomaterials, highly biocompatible as previously demonstrated in a series of toxicological studies, and effective barriers which can stay in place for an adequate time period prior to degradation. The results of our experimental studies showed that HYALOBARRlERII!> gel used intraperitoneally in an amount sufficient to cover the injured surfaces inhibits adhesion formation in different surgical procedures even in presence of critical conditions such as bleeding or inadequate hemostasis. Moreover, in clinical trials both in abdominal and gynecological surgery HYALOBARRIERII!> gel showed good tolerability without any adverse events attributable to the use of the device. Clinical results in the E.N.T. trial show that a hyaluronan benzylic ester derivative in nonwoven form (Merogel" can limit the incidence of postoperative adhesions compared to the standard surgical procedure. This clinical study suggested that hyaluronan-based devices may act as a barrier between the damaged surfaces interacting favorably in the postoperative healing process of sinus mucosa. In abdominal surgery, particularly in the large defects, the best preventive measure is peritoneum interpositioning. However, the peritoneal layer is not always present, consequently the use of a synthetic mesh is often required. The direct contact of the surgical mesh with underlying viscera can increase the risk of adhesions and fistula formation. In an experimental trial we demonstrated that HA applied to a synthetic device using a plasma coating process, can improve the biocompatibility properties reducing significantly the risk of adhesions without compromising the tensile strength characteristics of the devices. In conclusion, the Hyaluronan plasma-coating process appears promising, and should become a valuable tool in enhancing the tolerability of a synthetic material. Further clinical trials will be designed to confirm the experimental data.
CONCLUSIONS To date, preclinical studies, clinical safety and efficacy data, have indicated that Fab. hyaluronan derivative-based devices are effective in reducing adhesions in different kinds of surgery. The devices are non-toxic, non-antigenic, biocompatible and well tolerated and therefore represent promising devices for the prevention of postoperative adhesions.
REFERENCES 1. G.S. diZerega. The cause and prevention of postsurgical adhesions: a contemporary update. In: Gynecological Surgery and Adhesion Prevention. M.P. Diamond, G. diZerega, c.B. Linsky & R.L. Reid (eds.), Wiley-Liss Inc., New York, 1995, pp. 27-37. 2. H. Ellis, 'The Clinical Significance of Adhesions: Focus on Intestinal Obstruction', Eur. J. Surg. 1997; Suppl. 577:5-9. 3. J.F. Steege & A.L. Stout, 'Resolution of Chronic Pelvic Pain After Laparoscopic Lysis of Adhesions', Am J Obstet Gynecol 1991, 165, 278-283. 4. M.G.R. Hull, C.M.A. Glazener, N.J. Kelly, D.l. Conway, P.A. Foster et aI, 'Population study of causes, treatment and outcome of infertility' ,Br. Med. J., 1985,291, 1693-1697.
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5. M.P. Diamond & A.H. De Cherney, 'Pathogenesis of Adhesion Formation/Reformation: Application to Reproductive Pelvic Surgery', Microsurgery, 1987,8,103-107. 6. M.P. Diamond, 'Incidence of postsurgical adhesions, In: Peritoneal Surgery. G.S. diZerega (ed.), Springer-Verlag, New York:, 1999:217-220. 7. D. Menzies & H. Ellis, 'Intestinal obstruction from adhesions - how big is the problem?', Ann. Royal ColI. Surg. Engl., 1990,72,60-63. 8. N.F. Ray, W.G. Danton, M. Thamer, S.C. Henderson & S. Perry, 'Abdominal adhesiolysis: impatient care and expenditures in the United States in 1994', 1. Am. CoIl. Surg., 1998, 186, 1-9. 9. S.D. Wexner, 'Prevention and treatment of adhesive small bowel obstruction', In: Proceedings of the 90th Congress of the Italian Society of Surgery, L. Pozzi (ed.), Padua October 19th_ 22th, 1997, pp. 248-252. 10. J.F.Steege & a.L.Stout 'Resolution of chronic pelvic pain after laparoscopic lysis of adhesions' Am. J. Obstet. Gynecol., 1991, 165,278-283. 11. A Hershlag, M.P. Diamond & A.H. DeCherney, 'Adhesiolysis. Clin. Obstet. Gynecol., 1991, 34, 395-402. 12. Operative Laparoscopy Study Group, 'Postoperative adhesion development after operative laparoscopy: evaluation at early second-look procedures', Fertil. SteriI., 1991, 55,700-704. 13. R. Marana, A.A Luciano, L. Muzii, V.E. Marendino & S. Mancuso, 'Laparoscopy versus laparotomy for ovarian conservative surgery: a randomized trial in the rabbit model', Am. J. Obstet. Gynecol., 1994, 171,861-864. 14. A.M. Kappas, G.H. Barsoum, 1.B. Ortiz, M.R.B. Keighley, 'Prevention of peritoneal adhesions in rats with Verapamil, Hydrocortisone Sodium Succinate, and Phosphatidylcholine', Eur J Surg., 1992, 158,33-35. 15. AG. Turcapar, C. Ozarslan, E. Erdem, C. Bumin, N. Erverdi et aI., 'The effectiveness of low molecular weight heparin on adhesion formation in experimental rat model', Int. Surg. 1995,80,92-94. 16. M.P. Diamond, C.B. Linsky, T. Cunningham, L. Kamp, E. Pines, A.H. DeCherney & G.S. diZerega, 'Adhesion Reformation: Reduction by the Use of Interceed (TC7) Plus Heparin. 1. Gynecol. Surg., 1991,7,1-6. 17. R.L. Reid, Ha P.M. Hn, J.E.H Spence, T. Tulandi, AA. Yuzpe, D.M. Wiseman, 'A randomized clinical trial of oxidized regenerated cellulose adhesion barrier (lnterceed, TC7) alone or in combination with heparin', Fertil. Steril. 1997,67,23-29. 18. P.K. Amid, AG. Shulman, I.L. Lichtenstein, S. Sostrin, J. Young, M. Hakakha, 'Experimental evaluation of a new composite mesh with selective property of incorporation to the abdominal wall without adhering to the intestines', J. Biomed. Mater.
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Res., 1994,28,373-375. 19. U.K. Dasika, W.D. Widmann, 'Does lining Polypropylene with Polyglactin mesh reduce intraperitoneal adhesions?', Am. Surg. 1998,64,817-820. 20. J.W. Bums, K. Skinner, J. Colt et al., 'Prevention of Tissue Injury and Postsurgical Adhesions by Precoating Tissues with Hyaluronic Acid Solutions', 1. Surg. Res., 1995, 59, 644-652. 21. D.A Grainger, W.R. Meyer, AH. De Cherney et a!., 'The Use of Hyaluronic Acid Polymers to Reduce Postoperative Adhesions. J. Gynecol. Surg., 1991,7,97-101. 22. J.W. Bums, K. Skinner, J. Colt, L. Burgess, R. Rose & M.P. Diamond, 'A hyaluronate based gel for the prevention of postsurgical adhesions: evaluation in two animal species', Fertil, Steril., 1996,66,814-821. 23. M. Mensitieri, L. Ambrosio, L. Nicolais, D. Bellini & M. O'Regan, 'Viscoelastic properties modulation of a novel autocrosslinked hyaluronic acid polymer', 1. Mater. Sci. Mater. Med., 1996,7,695-698. 24. A Pavesio, D. Renier, C. Cassinelli & M. Morra, 'Anti-adhesive surface through Hyaluronan coatings', Medical Device Technology, 1997,8,20-27. 25. P. Narayanan, 'Surface functionalization by RF plasma treatment of polymers for immobilization of bioactive-molecules', J. Biomater. Sci. Polymer ed., 1994,6, (2), 181193. 26. S.P. Boyers, M.P. Diamond & AH. De Cherney, 'Reduction of postoperative pelvic adhesions in the rabbit with Gore-Tex surgical membrane', Fertil. Steril., 1988,49, 10661070. 27. K.L Blauer & RL. Collins, 'The effect of intraperitoneal progesterone on postoperative adhesion formation in rabbits', Fertil. Steril. 1988,49,144-149. 28. L. Hagberg, O. Wik & B. Gerdin, 'Determination of biomechanical characteristics of restrictive adhesions and of functional impairment after flexor tendon surgery: a methodological study of rabbits', J. Biomechanics, 1991,24,935-942.
EFFECT OF HYALURONAN ON MATRIX METALLOPROTEASE EXPRESSION IN FIBROBLASTS AND KERA TOCYTES. Isnard N., Legeais J·M., Renard G., Robert L. Laboratoire de Recherche en Ophtalmologie, Esc B3 6eme etage, Hotel Dieu, I place du Parvis Notre-Dame. 75004 Paris. France.
[email protected]
ABSTRACT Both hyaluronan and matrix metalloproteases (MMPs) are thought to be involved in tissue remodelling in a variety of physiological and pathological processes such as embryonic development, morphogenesis, wound healing or tumor progression. Several cytokines and growth factors are involved in the regulation of the biosynthesis of hyaluronan and also of MMP-s. In order to explore the possible relationship between these processes we studied the effect of hyaluronan on MMP-s expression (biosynthesis and activation) in culture of human skin fibroblasts and corneal keratocytes (explant cultures and cell cultures). These cells were shown to exhibit distinct phenotypes as far as matrix biosynthesis is concerned. Using a synthetic substrate: N-Suc(ala)3pNA we measured elastase-type endopeptidase activity produced by fibroblasts and keratocytes and characterised the MMPs by zymography. Hyaluronan added to fibroblast as well as keratocytes cultures stimulated the membrane bound elastase type endopeptidase activity in a dose dependant fashion. In presence of 1 mglml of hyaluronan there was an increase of MMPs expression and also an activation of these MMPs both by fibroblasts and keratocytes. KEYWORDS Hyaluronan, MMP-s, Fibroblasts, Keratocytes. INTRODUCTION Matrix metalloproteinases (MMP-s) are a family of extracellular matrix degrading enzymes that are thought to play a crucial role in tissue remodelling. MMP-s are involved in a variety of physiological and pathological processes such as embryonic development, morphogenesis, wound healing or tumor progression (1). Several MMP-s are expressed at low levels in normal tissues and are upregulated during pathological processes and tissue remodelling. Hyaluronan an extracellular polysaccharide plays an important role in a variety of physiological and pathological processes (2, 3,4). In wound healing and/or inflammation MMP-s and hyaluronan are both upregulated. Several cytokines and growth factors are involved in the regulation of the biosynthesis of hyaluronan (5) and also of matrix metalloproteases (6). We wanted to study the
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correlation between these processes both in human skin fibroblasts and corneal keratocytes. MATERIALS & METHODS Explant cultures : Cornea and skin were obtained from healthy donors and came from surgery departments of our hospital. Corneal rims remaining after grafting were cut in fragments of 1 mrrr' and cultivated in DMEM with or without hyaluronan (1 mg/ml) for 48 hours at 37°C with shaking. Dermal fragments were cultivated using the same protocol. The culture media were kept and the tissue extracts were obtained after incubation by homogenising, skin or corneal fragments in 0.1 M Tris-HCI pH8 buffer containing 1 roM cacr, 1 roM MgClz, 1 roM zinc acetate, 0.01 % Hrij 35 and 0.01 % NaN3, using an Ultraturax. Cell cultures : Corneal keratocytes and skin fibroblasts were cultured for 24 hours in DMEM without fetal calf serumwith or without hyaluronan (l mg/ml). Culture media were kept and the cells were sonicated in 0.1 M Tris-HCI pH8 buffer containing 1 roM CaClz, I roM MgCl z, 1 roM zinc acetate, 0.01 % Hrij 35 and 0.01 % NaN3. The cell extracts correspond to the intra and pericellular fractions. Elastase type endopeptidase activity: In order to determine elastase type endopeptidase activity we used a synthetic substrat : N-Suc(ala)3pNA as described (7). Gelatin zymography : Explant and cell culture media and cell or tissue extracts were studied by gelatin zymography as described (8). They were electrophoresed at 150 Volts on 10 % acrylamid gels containing 1 mg/ml gelatin. The gels were first incubated in a 2.5 % triton X-IOO in order to eliminate SDS and then in 0.1 M Tris buffer containing 0.1 M CaCl z and I roM Zinc acetate. After 24 hours of incubation at 37°C the gels were stained with Coomassie blue and destained in water. The activities of both active or inactive forms of MMP-s were quantified by densitometry using a morphometric software (Visiolab©). RESULTS & DISCUSSION Keratocyte and fibroblast cell cultures : As shown on the zymograms of Fig.I, MMP-2 is expressed, in its inactive pro-form by both cell types but MMP-9 in its inactive pro-form only by fibroblasts. In presence of hyaluronan, MMP-2 was increased by both cell types as shown by densitometric scanning of zymograms by about 20 %.
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B MMP·9~
MMP·2
Figure 1 : Gelatin zymography of culture MMP·2~ media of keratocyte (A) and skin fibroblast (B) Lane I : culture medium, control Lane 2 : culture medium, with I mg/ml hyaluronan
Cornea explant culture :
MMp·9
MMP·2
45kDA
Figure 2 : Gelatin zymography of tissue extracts (lanes 1 and 2) and culture media (lanes 3 and 4) of corneas cultured with (lanes 2 and 4) or without (lanes I and 3) hyaluronan. As shown on Fig.2, in cornea explant culture both MMP-2 and MMP-9 are expressed in their active and inactive forms. Both activities were higher in the culture medium than in the tissue homogenates showing that both enzymes are excreted by the cells during incubation. The activity of MMP-2 was somewhat more important than that of MMP-9. In presence of hyaluronan MMP-activity was slightly increased, by about 15 %. The major effect of hyaluronan was the activation of latent MMP-9 to its active form. The ratio of active to inactive form of MMP-9 increased in presence of hyaluronan from 0.75 to 3.4 in the corneal extract. The same increase of the ratio of active to inactive form of MMP-9 was even higher in the culture medium where it increased from 0.9 without hyaluronan to 4.6 in presence of hyaluronan.
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Skin explant cultures :
MMP·9
MMP-2
Figure 3 : Gelatin zymography of extracts (lanes 1 and 2) and extracellular media (lanes 3 and 4) of skin explant cultures, with (lanes 2 and 4) or without (lanes I and 3) hyaluronan. Fig.3 shows similar experiment carried out on skin explant cultures. Both MMP-2 and MMP-9 are expressed both in the culture medium and in the tissue homogenates. In the extracellular medium, the activity of MMP-9 was somewhat higher than the activity of MMP-2. The addition of hyaluronan increased here also the conversion of the inactive form of these enzymes to their active form. This ratio increased from 0.85 to 1.2 for MMP-2 and from 5.9 to 6.7 for MMP-9 in the culture media. Elastase type activity : The titration of elastase type endopeptidase activity with the synthetic substrate showed a dose dependant increase for skin fibroblasts at 1 mg/ml (+ 30 %, P < 0.05) and at 2 mg/ml (+ 65 %, P < 0,001) hyaluronan (7). In keratocytes, the increase was of the same order as in fibroblasts at 1 mg/ml hyaluronan (+ 52 %, P < 0.01) and no further increase was seen at 2 mg/ml hyaluronan. These results confirm and extend previous studies carried out with the same synthetic substrate which showed also an increase of the elastase-type endopeptidase activity of human skin fibroblasts in presence of 1 mg/ml hyaluronan (9). CONCLUSIONS We compared the expression of the latent and active forms of MMP-s in human skin fibroblasts and corneal keratocytes as well in skin and corneal explant cultures as well as the action of hyaluronan on these activities. The comparison of cell cultures and explant culture show that the presence of the extracellular matrix influences the expression of MMP-2 and MMP-9 as well as the conversion of the inactive pro-form to the active form. The ratio of active to the inactive form was different in cornea and skin explant cultures. For both cell-types in explant cultures a large part of both MMP-2 and MMP-9 activities was excreted in the culture medium.
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The addition of hyaluronan increased the conversion of the inactive to the active fOlTIlS, in the medium, for both tissues. For cornea explant cultures this ratio increased in presence of I mg/ml hyaluronan from 0.9 to 4.6 for MMP-9. For skin explant cultures the increase was from 0.85 to 1.2 for MMP-2 and from 5.9 to 6.7 for MMP-9. Both fibroblast and keratocyte cell cultures, at the 3rd passage expressed MMP-2 in its inactive form, but only small amounts of MMP-9 could be detected in the extracellular media of skin fibroblasts. MMP-2 and MMP-9 activities taken together were lower in cell cultures than in explant cultures. Hyaluronan at 1 and 2 mg/ml produced a dose dependent increase of elastase-type endopeptidase activity in fibroblasts. With keratocytes, an increase of 52 % was seen at 1 mg/mI hyaluronan and no further increase at 2 mg/mI. These results confirm and extend our previous observations concerning the increase of elastase type endopeptidase activity of hyaluronan (9). The main action of hyaluronan appears to be an increase of the conversion of latent forms of these enzymes to their active form. It appears that this activation concerns both MMP-2 and MMP-9. The differences noticed between corneal and skin cells confirm our previous conclusions based on the study of glycosaminoglycan biosynthesis, concerning the difference between the phenotypes of keratocytes and fibroblasts. (10, 11)
REFERENCES 1. Shapiro S. Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Current Opinion in cell Biology, 1998, 10 (5) : 602-608. 2. Kielty CM., Whittaker SP., Grant ME., Shuttleworth CA. Type VI collagen microfibrils : evidence for a structural association with hyaluronan. J Cell BioI., 1992, 118 (4) : 979-990. 3. Toole B. Hyaluronan in morphogenesis. J. of Internal Medicine, 1997, 242 : 35-40. 4. Inoue M., Katami C. The effect of hyaluronic acid on corneal epithelial cell proliferation. Invest. Ophthalmol. Vis. Sci., 1993, 34 (7) : 2313-23-15. 5. Heldin P., Asplund T., Ytterberg D., Thelin S., Laurent T. Characterization of the molecular mechanism involved in the activation of hyaluronan synthetase by plateletderived growth factor in human mesothelial cells. Biochem. J., 1992,283 : 165-170. 6. Kossakowska A., Edward D., Prusinkiewicz C., Zhang M., Guo D., Urbanski S., Grogan T., Marquez L., Janowska-Wieczorek A. Interleukin-6 regulation of matrix metalloproteinase (MMP-2, MMP-9) and tissue inhibitor of metalloproteinase (TIMP-l) expression in malignant non-Hodgkin's lymphoma. Blood, 1999,94 (6) : 2080-2089. 7. Isnard N., Legeais J-M., Renard G., Robert L. Effect of hyaluronan on MMPexpression and activation. Cell Bioi Int , 2001, in press. 8. Kleiner D., Stetler-Stevenson W. Quantitative zymography : detection of picogram quantities of gelatinases. Analytical Biochemestry., 1994,218 : 325-329. 9. Bernard E., Hornebeck W., Robert L. Effect of hyaluronan on the elastase-type activity of human skin fibroblasts. Cell BioI. Int., 1994, 18 (10) : 967-971. 10. Fodil-Bourahla I., Drubaix I. and Robert L. Effect of in vitro aging on the biosynthesis of glycosaminoglycans by human skin fibroblasts. Modulation by the elastin-larninin receptor. Mechanisms of Ageing and Development, 1999, 106: 241-260. 11. Dupuy F, Peterszegi G., Legeais J-M., Robert A-M., Robert L. and Renard G. Stabiblityof glycosaminoglycans during in vitro aging of human keratocytes. In preparation.
AGING AND REGULATION OF HYALURONAN BIOSYNTHESIS. COMPARATIVE STUDIES ON HUMAN SKIN FIBROBLASTS AND CORNEAL KERA TOCYTES. Robert L.*, Fodil I., Isnard N., Dupuy F., Robert A.M., Renard G. Laboratoire de Recherche en Ophtalmologie, Esc B3 6eme etage, Hotel Dieu, 1 place du Parvis Notre-Dame. 75004 Paris, France.
[email protected]
ABSTRACT Human skin fibroblasts and corneal keratocytes were compared for their GAGbiosynthetic activity as a function of passage number (in vitro replicative senescence) by comparing the fraction of total label eH-glucosamine) incorporated in total glycoconjugates and individual GAG-s. It appeared that keratocytes synthesised about as much non-GAG glycoconjugates (glycoproteins) as fibroblasts. Incorporation in GAG-s increased with passage number for fibroblasts and remained constant for keratocytes. Hyaluronan as % of total GAG-s was somewhat higher in fibroblasts than in keratocytes. A strong increase of incorporation was seen at the latest passage (15th) close to replicative senescence of fibroblasts but titration with hyaluronectin showed a decrease of hyaluronan concentration, interpreted as a sign of post-synthetic degradation of hyaluronan in pre-senescent fibroblasts. Regulation of GAG-biosynthesis as a function of passage number permitted thus to differentiate fibroblast and keratocyte phenotypes.
KEYWORDS Hyaluronan, GAG-s, fibroblasts, keratocytes, replicative senescence.
INTRODUCTION Regulation of glycosaminoglycan (GAG) biosynthesis is part of the phenotypic expression of extracellular matrix (ECM) biosynthesis by mesenchymal cells. Their phenotype can best be characterised by their "program" of matrix biosynthesis 1-3. This "program" comprises the "choice" of genes coding for matrix components to be expressed (qualitative aspect) and for the quantitative regulation (up or down) of the expression of the chosen genes. This "program" of regulated matrix biosynthesis starts during embryonal development and continues during maturation and aging. The structure and function of tissues, and their susceptibility to pathological modifications are largely dependent on the correct execution of this program and also on its modifications during aging. Regulation of GAG-biosynthesis represents an important part of the above outlined "program". The biological properties and role of individual GAG-s in both tissues studied are sufficiently well known to render useless any repetition in a short article. With these premises in mind we shall describe our studies on the expression of GAG-s
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Keratinocytes and hyaluronan
in skin fibroblasts and corneal keratocytes, both of human origin, in explant and cell culture and as a function of passage number (in vitro aging) 4.
MATERIALS & METHODS Cells Fibroblasts were cultured from skin samples obtained from plastic surgery, using standardised procedures 5, Corneas were obtained from the French Eye Bank after preservation in explant culture conditions as described 6, Cell cultures were derived from the corneas as described 7 and passaged in Dulbecco modified Eagle's minimal medium (DMEM) with 10 % foetal bovine serum and antibiotics. Biosynthesis of GAG-s was studied by adding 3H-glucosamine to the cultures (1 /lCi/ml) for a further incubation for 24 to 48 hours. Cells and medium were separated and individual GAG-s determined after selective degradation by specific enzymes, enabling the determination of incorporated radioactivity. For hyaluronan the Streptomyces hyaluronidase was used, for chondroitin 4 and 6 sulfate chondroitinase AC or ABC of Proteus vulgaris, for dermatan sulfate the chondroitin sulfate B-Iyase of Flavobacterium heparinum, for keratan sulfate, keratanase I from Pseudomonas, all from Sigma (St. Louis, MO.). For heparan sulfate, nitrous acid degradation was used 8. Digested GAG-s were separated from undigested macromolecules by gel filtration after precipitation of the undigested fraction by absolute ethanol containing 1 % potassium acetate. Hyaluronan concentration was determined by the titration method described by Delpech et al. 9 using hyaluronectin purified from sheep brain as described by Delpech and Halevent 10, Statistical significance was calculated using the non-parametric method of Mann and Whitney,
RESULTS & DISCUSSION Fibroblasts As shown on table I, skin fibroblasts incorporated actively 3H-glucosamine in total glycoconjugates. Incorporation in total glycoconjugates increased from the 5th passage (relatively "young" cells) to the 15th passage (cells near the end of their replicative lifespan) by about 45 %. Part of this incorporation concerned non-GAG glycoconjugates, essentially glycoproteins. This fraction was of the order of 28 % of total incorporation at the 5th passage and decreased to 14 % of total incorporation at the 15th passage. The relative amount of GAG-s synthesised appears thus to increase with passage number. Hyaluronan represented about 40 % of total GAG biosynthesis at the 5 th passage and somewhat less, about 35 % at the 15th passage. The relative amount of the other GAG-s, chondroitin sulfates and heparan sulfate changed less. A large proportion of the neosynthesised GAG-s was secreted in the culture medium (figures in parenthesis in table I), This is surprising for heparan sulfate, supposed to be localised mainly on cell membranes. Fig. I shows the increase of incorporation of the tracer with passage number for chondroitin 4 and 6 sulfates and dermatan-sulfate as well as for heparan sulfate. Little change was noticed between the 5th and 10t h passages, but a strong (and significant) increase is seen at the 15th passage, mainly for dermatan sulfate and heparan sulfate. No significant change was seen for chondroitin 4 and 6 sulfates.
539
Aging and regulation of biosynthesis. Table I.
Incorporation of 3H-glucosamine in total glycoconjugates and GAG-s by human skin fibroblasts in cell culture at the 5th and 15' h passages. The figures in parenthesis represent the % of total incorporated radioactivity recovered in the culture medium. (Recalculated from Table I of FodilBourahla et al., 1999). Hyal : hyaluronan, CSABC : total chondroitin-sulfates, HS : heparan sulfate. JH-GlcN Incorporation in non GAGPassage incorporated in glycoconjugates number total glycoconjugates cpmll06 cells 6 cpmll0 cells. 5
91278 ± 13123
25450
15
132871 ± 6 951
18636
% of total incorporation
in individual GAG-s
Hyal. 40 (92) 35 (88)
CSABC 29 (77)
28 (86)
HS 31 (70) 37 (76)
Fig. 2 shows the incorporation of the tracer in hyaluronan (upper graph) with a quite important increase between passage 10 and 15, as for the sulfated GAG-s (Fig. 1). When however the titrable concentration of hyaluronan was determined using hyaluronectin as the reagent there was a progressive decrease with passages (lower graph of Fig. 2). This discrepancy between the two methods might be due to a rapid post-synthetic degradation of newly synthesised hyaluronan.
50000 [J
o
Chondrctttn-sulfates A,C Dermatan sulfate
[J Heparan sulfate
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1l ~ =
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i
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Figure 1.
3H-glucosamine incorporation in chondroitin sulfates A and C, in dermatan sulfate and in heparan sulfate. The values are expressed as cpm per 106 cells.
540
Keratinocytes and hyaluronan
A 50000
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40000
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~=.. 20000 '"
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Figure 2.
A : Incorporation of 3H-glucosamine in hyaluronan by fibroblasts at increasing passages. B : Titration of the concentration of hyaluronan present in the culture media with hyaluronectin.
Aging and regulation of biosynthesis.
541
Cornea and keratocytes Table II shows the results of 3H-glucosamine incorporation in cornea explant cultures. The fraction incorporated in stromal glycoproteins was comparable to the figures found for fibroblasts, the major fraction of the label was found in secreted or stroma - bound GAG-s. Hyaluronan represented 32 % (tissue bound) to 38 % (secreted) of total GAG synthesis.
Table II.
Incorporation of 3H-glucosamine in GAGs in human cornea during a 48 hours explant culture. Results expressed as % of total GAGs incorporation. Average values ± S.D. 6 parallel experiments.
% of total GAG-s
Retained in corneal tissue
Secreted in the medium
Hyaluronan
32.1 ± 3.0
38.1 ± 4.4
Keratan sulfate
18.6 ± 1.9
13.8 ± 1.8
Dermatan sulfate
20.9 ± 1.3
12.9 ± 2.0
Chondroitin sulfates (A+B +C)
41.4 ± 2.6
38.7 ± 2.0
Heparan sulfate
7.7 ± 0.7
9.4 ± 1.3
When keratocytes were cultured through 10 passages total incorporation in GAG-s remained relatively stable. Incorporation in total GAG-s did not show any significant variation with increasing passage number. About 75 % of the neosynthesised GAG-s were secreted in the culture medium. As shown on Fig. 3 this remarkable stability of GAG-biosynthesis was seen for every individual GAG-s synthesised between the 4 th and 10th passages. The presented results clearly show that the fibroblast and keratocyte phenotype are quite different as far as GAG-biosynthesis is concerned. Keratocytes synthesised about as much non-GAG glycoconjugates as fibroblasts. They also exhibited an increase of incorporation of 3H-glucosamine with increasing passage number in hyaluronan as well as in sulfated GAG-s (Figs. I and 2). Keratocytes synthesised comparable proprtions of hyaluronan (32 to 38 % of total GAG-s) as fibroblasts (35 to 40 % of total GAG-s). GAG-synthesis by keratocytes did not show the passage dependent shift seen with fibroblasts. Keratocyte phenotype characterised by GAG-biosynthesis appears therefore more stable than fibroblast phenotype at least as far as shown by the study of replicative senescence.
542
Keratinocytes and hyaluronan
A
60.0 50.0
I.'l
~
iCP4 ' ICP6 I
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,$ 30.0
] ~
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Chondroitin sulfate A,B,C
Derrnatan sulfate
Heparan sulfate
40,0 35.0
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Figure 4.
Keratan sulfate
Chondroitin sulfate A,B,C
Dennatan sulfate
Heparan sulfate
Incorporation of 3H-glucosamine in individual GAG-s secreted (A) and retained in cells (B) at increasing passages of keratocytes expressed as % of incorporation in total GAG-s. Average of results from 5 different donors ± SD. Dermatan sulfate was counted in the sum of chondroitin sulfate, resulting in a sum of sulfated GAG-s above 100 %.
CONCLUSIONS
These experiments clearly establish a phenotype difference between human skin fibroblasts and corneal keratocytes judged by the relative amount of GAG-s synthetised during successive passages (in vitro aging).
REFERENCES 1. Robert L., Le fibroblaste, definition de son phenotype par son "programme" de biosynthese de la matrice extracellulaire. Pathol. Biol., 1992, 40, 851-858. 2. Robert L., Mechanisms of aging of the extracellular matrix. Role of the elastinlaminin receptor, Novartis Price Lecture, Gerontology, 1998,44,307-317. 3. Robert L., Le vieillissement, , Belin-CNRS Ed., Paris,1994.
Aging and regulation of biosynthesis.
543
4. Hayflick L., The cellular basis for biological aging. In • Handbook of the biology of aging.: Finch C.E., Hayflick L. (eds.), Van Nostrand Reinhold Company, New York, 1977, pp 159-186. 5. Fodil-Bourahla 1., Drubaix 1., Robert L., Effect of in vitro aging on the biosynthesis of glycosaminoglycans by human skin fibroblasts. Modulation by the elastinlaminin receptor, Mech. Ageing. Develop .. 1999, 106,241-260. 6. Isnard N., Legeais J.M., Renard G., Robert L., Effect of hyaluronan on MMPexpression and activation. Cell BioL. lnt, 2001, 25 (8), 735-739. 7. Dupuy F., Savoldelli M., Tixier J.M., Robert A.M., Robert L., Legeais J.M., Renard G., Chemotactic penetration of keratocytes in ePTFE polymer in vitro, J. Biomedical Materials Research, 2000, 56,487-493. 8. Shively J.E., Conrad H.E., Formation of Anhydrosugars in the chemical depolyperisation of heparin. Biochemistry, 1976, 15, 3932-3942. 9. Delpech B., Bertrand P., Maingonnat C., Girard N., Chauzy C., Enzyme-linked hyaluronectin : A unique reagent for hyaluronan assay and tissue location and for hyaluronidase activity detection. AnaL. Biochem.,1995, 229, 35-41. 10. Delpech B., Halevent c., Characterization and purification from human brain of a hyaluronic acid-binding glycoprotein, hyaluronectin, , J. Neurochem., 1981,36(3), 855-859.
EFFECTS OF KGF AND TGF-8 ON HYALURONAN SYNTHESIS AND DISTRIBUTION IN EXTRA-, PERI·, AND INTRACELLULAR COMPARTMENTS OF EPIDERMAL KERATINOCYTES Karvinen S.,* Tammi M., Tammi R. Dept. ofAnatomy, University ofKuopio r.o.so» 1627, 70211 Kuopio, Finland
ABSTRACT The aim of this study was to examine the effects ofKGF and TGF-13 on HA synthesis and proliferation of rat epidermal keratinocytes. KGF stimulated proliferation at small doses (I and 10 ng/ml). TGF-13 inhibited keratinocyte proliferation at 10 ng/ml. Both growth factors increased the migration of the cells. HA localization was studied with a histochemical staining assay using biotinylated aggrecan Gl domain link-protein complex. In a part of the samples extracellular HA was enzymatically digested prior to permeabilization of the membranes to specifically examine intracellular HA. A five to sixfold increase of intracellular HA staining intensity as compared to control was observed with both growth factors at 24 hours. A twofold increase in pericellular HA was also found with KGF, but no marked effect was seen with TGF-I3. Metabolic incorporation of eH] glucosamine and 35S04 into labelled hyaluronan and chondroitin disaccharides from medium, trypsinate and cell samples revealed a sixfold increase of intracellular HA with KGF at 100 ng/ml. A twofold increase of HA in medium samples was also seen with KGF, but it had no effect on trypsinate samples. TGF-13 at 10 ng/ml had no significant effect on extracellular HA, but induced a twofold increase of intraand pericellular HA. KEYWORDS Hyaluronan, keratinocytes, KGF, TGF-I3, proliferation, migration INTRODUCTION Hyaluronan (HA) is a large, linear polysaccharide that forms the main component of the extracellular matrix in the epidermis of skin7.8. It forms a loose, gel-like matrix, which enhances the exchange of molecules between cells and the bloodstream", and creates an environment that favors the migration of cells". Keratinocyte growth factor (KGF), is a powerful mitogenic agent. secreted by stromal fibroblasts, that influences only epithelial cells'. In epidermis. KGF receptors are primarily expressed in the spinous cell layer in which the differentiation of keratinocytes takes place. KGFR can also be found in the basal cell layer, but not in the granular- or clear cell layers". No previous results have been published linking KGF with hyaluronan. Transforming growth factor beta (TGF-I3) is encoded by three different genes as three isoforms (TGF-J3 1,2 and 3). It controls cell proliferation and differentation in several cell types and also balances the effects of other growth factors either by enhancing their
546
Keratinocytes and hyaluronan
effects or inhibiting them". TGF-13 signaling requires the binding of TGF-13 to type II receptor and the complex binding to type I receptor!'. TGF -13 has been found to stimulate HA synthesis in fibroblasts". MATERIALS AND METHODS Cell proliferation
Newborn rat epidermal keratinocytes were seeded into 24 well plates at 60.000 cells /well. The following day, KGF (0,1,10 and 100ng/ml, Sigma, USA) or TGF -13 (0,1,5 and 10ng/ml, Gibco, Scotland) was added. The cells were incubated at 37°C, and the cell number counted on the three following days (at 24, 48 and 72). The growth medium (MEM, Gibco) was removed, the cells washed with HBSS (EuroClone) and incubated with 0.05%trypsin-0.02% EDTA until detached. The cells were harvested and wells washed with HBSS. The cells were counted using a hemacytometer on Olympus CK2 inverted phase contrast microscope. Migration
The cells were seeded at 600.000 cells/well on 6 well plates and grown for 24 h. A cell-free area was introduced by scraping the monolayer crosswise with a sterile I000111 pipette tip, washed with HBSS and fresh medium was added. The width of the cell-free area was approximately 1000 urn, TGF-B at 5 and 10 ng/ml or KGF at 10 and 100 ng/ml was added to two wells each, 2 wells on each 6-well plate remained as contro!. The area covered by the cells in 8 crossing areas in duplicate wells of each growth factor concentration was measured immediately after scraping and 24 h later using a phase conrast microscope, videocamera and NIH-image® software. The distance the cells had migrated was calculated as follows: (-.Jb--.Ja)/2, where a= area covered by the cells at Oh and b= area covered by the cells after 24 h. The results (pixels) were converted to micrometers. Staining of HA
The cells were seeded into 8-well chamber slides at 20.000 cells/well, and cultured for 48 h at 37°C. Fresh medium was added with KGF at 100 ng/ml, or TGF-13 at 10 ng/ml for 4, 6, and 24 h. The cells were fixed with 2% paraformaldehyde 0.05% glutaraldehyde in sodium phosphate buffer, pH 7.4 (PB) for 20 min, washed with PB, permeabilized with Triton X-IOO, and incubated with a specific hyaluronan binding probe" overnight at 4°C, followed by avidin-biotin-complex (Vector, CA) and diaminobenzidine with 0.005% hydrogen peroxide. Optical densities were analysed by a light microscope and digital camera. In part of the slides, extracellular hyaluronan was digested prior to the permeabilisation of the membranes with Streptomyces hyaluronidase (Seikagaku). Metabolic labeling assay
The cells were seeded into 6 well plates at 200.000 cells/well and cultured until subconfluent. Fresh medium containing CH] glucosamine (2011Ci/ml) and 35 S04 (10 llCi/ml) was added for 24 h. The medium was harvested and wells washed with precooled HBSS which was collected with the medium. The cells were trypsinized, and 50
Effects of KOF and TOF-13 on synthesis
547
III of the cell suspension was taken aside to count the cell number. The wells were washed twice with HBSS, the cells sentrifuged in the cold (5 min at 4000 rpm) and washed once with 250111 cool HBSS containing 10% fetal bovine serum. All washes were combined with the trypsin solution and the cells digested overnight at 4°C with HBSS containing 41lg HA (Healon, Pharmacia) and 20 TRU Streptomyces hyaluronidase, and washed with cool HBSS. All samples were kept in -20°C prior to analysis. The analysis was done as described in Tammi et ai, 1998, except that the CPC and ethanol precipitations were replaced by dialysis with a 10 kDa cut off. RESULTS AND DISCUSSION The effects ofKGF and TGF-8 on keratinocyte proliferation
The cells were cultured in the presence of the growth factors, and counted. Neither KGF nor TGF-13 influenced cell numbers at 24 h. The number of cells in wells containing TGF-13 was slightly lower than in control cells at 48 h (Fig.1). At 72 h TGF-13 at 10 ng/ml had decreased proliferation while the lesser amounts had no significant effect. At 48 and 72 h, KGF at 10 and 1 ng/ml caused an increase in proliferation, but 100 ng/ml showed no effect (Fig.I). It has been previously reported that KGF does not increase keratinocyte proliferation until the cells are subconfluenr'. The cell density at 24 h culture may therefore have been too low to bring about the full stimulatory effect of KGF. TGF-13 has been reported to inhibit keratinocyte proliferation as revieved by Sporn et a1 5 . The reason why the lesser amounts did the opposite is not known. 5 10 0
o control o Y.GF 1 llglml
o control
o TOF·IJ 1 llghnl
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:!
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7'!
Time in TGF·8 (h)
Effect of KGF and TGF-f3 on keratinocyte proliferation. The bars show the range of 4 parallel cultures.
TGF -8 and KGF both increase the migration of rat epidermal keratinocytes
To investigate the effects on migration we cultured cells with different concentrations of the growth factors for 24 hours. The keratinocytes with no added growth factors, migrated approximately 150 11m in 24 hours. TGF-13 increased migration at both doses with slightly higher response at higher concentration. The increase was 30-40 %. KGF
548
Keratinocytes and hyaluronan
also caused an increase in migration, approximately 25-30% to control. As with TGF-B, the higher concentration showed a more marked response.
]:
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--
0.15
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The effects of KGF and TGF-B on the migration of REK cells. The keratinocyte migration was analyzed using a scratch assay with 24 h follow up. The average migration distance of 6 experiments ± SE is shown.
= '0
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The wounding-induced migration assay. The columns indicate the average movement of the migration front. M=mock transfection, Aeantisense, Sesense cell lines. Error bars = S.E. from 3-5 separate experiments.
560
Keratinocytes and hyaluronan
CONCLUSIONS This study indicates that Has2 directed hyaluronan synthesis represents an important regulatory control of the migration of epidermal keratinocytes. The expression levels of Has2 also affected their phenotype in other aspects, including general cell spreading, f?rmation of lamellipodia, and the onset of proliferation. These findings are completely in line with the abundance of hyaluronan in the epidermis and with its rapid turnover during the migration and spreading of differentiating keratinocytes, and in their role in wound healing.
ACKNOWLEDGEMENTS This work was supported by Academy of Finland, grant # 40807, Finnish Cancer Foundation, EVO funds of Kuopio University Hospital and Kuopio University Biotechnology Funds.
REFERENCES T. C. Laurent and J. R. E. Fraser, Hyaluronan, Faseb J. , 1992, 6, 2397-2404. W. Y. Chen and G. Abatangelo, Functions of hyaluronan in wound repair, Wound Repair Regen ., 1999,7, 2, 79-89. 3. R. Tammi, 1. A. Ripellino, R. U. Margolis and M. Tammi, Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe, J. Invest. Dermatol., 1988,90,412-414. 4. R. Tammi, A.-M. Saamanen, H. 1. Maibach and M. Tammi, Degradation of newly synthesized high molecular mass hyaluronan in the epidermal and dermal compartments of human skin in organ culture, J. Invest. Dermatol., 1991,97, 126130. 5. R. Tammi, M. Tammi, V. C. Hascall, M. Hogg, S. Pasonen and D. K. MacCallum, Collagen substrates surfaced with a pre-formed basal lamina alter the metabolism and distribution of hyaluronan in epidermal keratinocyte "organotypic" cultures, Histochern. Cell. Biol., 2000, in press. 6. P. H. Weigel, V. C. Hascall and M. Tammi, Hyaluronan synthases, J. BioI. Chem., 1997,272,13997-14000. 7. J. Brinck and P. Heldin, Expression of recombinant hyaluronan synthase (HAS) isoforms in CHO cells reduces cell migration and cell surface CD44, Exp. Cell Res. , 1999,252,2,342-351. 8. N.ltano, T. Sawai, M. Yoshida, P. Lenas, Y. Yamada, M. Imagawa, T. Shinomura, M . Hamaguchi, Y. Yoshida, Y. Ohnuki, S. Miyauchi, A. P. Spicer, J. A. McDonald and K. Kimata, Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties, J. Biol. Chern. ,1999,274,35,25085-25092. 9. S. L. Schor, A. M. Schor, A. M. Grey, J. Chen, G. Rushton, M. E. Grant and 1. Ellis, Mechanism of action of the migration stimulating factor produced by fetal and cancer patient fibroblasts: effect on hyaluronic and synthesis, In Vitro Cell Dev. B io I., 1989,25,8,737-746. 10. 1. R. Ellis and S. L. Schor, Differential effects ofTGF-betal on hyaluronan synthesis by fetal and adult skin fibroblasts: implications for cell migration and wound healing, Exp. Cell Res., 1996,228,2,326-333. 11. H. P. Baden and J. Kubilus, The growth and differentiation of cultured newborn rat keratinocytes, J.lnvest. Dermatol., 1983, 80, 2,124-130. 1. 2.
EGF REGULATES HAS2 EXPRESSION, CONTROLS EPIDERMAL THICKNESS AND STIMULATES KERATINOCYTE MIGRATION Markku I. Tammi l *, Juha-Pekka Pienimakil , Kirsi RiUal , Csaba Fiilop2, Mikko J. Lammi), Reijo Slronent, Ronald Midura 2, Vincent C. Hasca1l2, Merja Luukkonent, Kari Torronen l , TUna Lehtot, & Raija Tammi! I
Department of Anatomy. University of Kuopio, P.O.B. 1627. 70211 Kuopio, Finland
2Department ofBiomedical Engineering. Lerner Research Institute. Cleveland Clinic Foundation. Cleveland. OH 44195. USA.
ABSTRACT High concentrations of hyaluronan reside in the small space between the vital kertinocyte layers of human and animal epidermis and influence keratinocyte interactions, including growth, mobility and differentiation. We have previously found that the content of epidermal hyaluronan in human skin organ cultures is decreased and increased by cortisol and retinoic acid, and associated with enhanced and retarded terminal differentiation, respectively. To further substantiate this idea, we incubated epidermal keratinocytes with epidermal growth factor (EGF), and found a marked increase in hyaluronan synthesis which correlated with faster migration in an in vitro wounding assay of keratinocyte monolayers. EGF increased hyaluronan also in stratified, differentiated organotypic cultures, and increased the height of vital epidermis and reduced the thickness of the cornified layers, findings in line with an inhibition of terminal differentiation of keratinocytes. The stimulation of hyaluronan synthesis by EGF was due to upregulation of hyaluronan synthase 2 (HAS2) but not HAS 1 or HAS3. A part of the EGF influence on the structure of epidermis, and on skin wound healing, is thus mediated through its control of HAS2 expression.
KEYWORDS Hyaluronan, keratinocytes, epidermis, migration, wound healing, epidermal growth factor.
INTRODUCTION Skin epidermis and other stratifying epithelia contain abundant hyaluronan in a narrow extracellular space surrounding the epithelial cells 1. The half life of labelled epidermal hyaluronan in human skin organ culture is -1 day 2, indicating fast local turnover by keratinocytes. The importance of hyaluronan in the multilayered squamous epithelia is not completely understood, but we have hypothesized that the high concentration of hyaluronan is necessary to maintain an extracellular space for the nutritional needs of the more superficial cell layers, while the fast turnover allows the modulation of cell shape that occurs during differentiation, and the migration of kertinocytes in wound healing 3. In the epidermis of human skin organ cultures, hyaluronan synthesis and concentration is increased by retinoic acid 1, a widely used drug in skin diseases. At the same time,
562
Keratinocytes and hyaluronan
retinoic acid retards the differentiation of keratinocytes in epidermis. Glucocorticoids have also extensive use as a drug in skin diseases, and inhibit hyaluronan synthesis when applied in pharmacological doses 4. Glucocorticoids promote normal differentiation and decrease the thickness of the vital layers of epidermis. It thus appears that enhanced hyaluronan metabolism is associated with faster growth and remodelling of the vital part of epidermis, while decreased hyaluronan synthesis allows an earlier terminal differentiation of the upper epidermal cells. Whether the above mentioned correlation between hyaluronan metabolism and keratinocyte differentiation can be extended to other factors that modulate epidermal differentiation was examined in the present studies on the effect of epidermal growth factor (EGF). EGF is one of the most powerful agents that influences the behavior of keratinocytes. It transmits its information through the EGF-receptor (EGF-R), which belongs to the erbB-receptor tyrosine kinase family 5.6. In other cells, signaling through EGF-R regulates many cellular processes, including cell adhesion, expression of matrix degrading proteinases, and cell locomotion; these phenomena are all important e.g. in skin wound healing. Keratinocyte EGF-R expression is transiently elevated after wounding, and is important to keratinocyte proliferation and migration during reepithelization (for review, see 7). Exogenously added EGF and overexpression of EGF-R result in enhanced ligand-mediated migration ofkeratinocytes and faster reepithelialization 8.
MATERIALS AND METHODS
Cell culture A newborn rat epidermal keratinocyte (REK) cell line 9 was cultured in Dulbecco' s MEM (Gibco, Grand Island, NY) with 10 % fetal bovine serum (PBS) (HyClone, Logan, UT), streptomycin (50 J.Ig1ml), penicillin (50 Ulml) and 1-2 mM L-glutamine. Cells were trypsinized when they reached confluency using 0.02 % EDTA (w/v)/0.025 % trypsin (w/v) (Sigma, St. Louis, MO). For biochemical assays and radiolabeling, the cells were grown close to confluency in 6-well plates and incubated in the presence of 20 J.ICi/ml of [3H]glucosamine, and 100 to 200 J.ICi/ml [35S]sulfate (Amersham, Little Chalfont, UK). Organotypic cultures at air-liquid interface were performed as described 10.
Isolation of radiolabeled glycosaminoglycans After labeling in 6-well plates (9.6 cm 2/well), medium was removed and cell layer washed with 400 J.Il of Hank's solution (HyClone). For each culture, the medium and wash were combined and designated as "medium". Each cell layer was trypsinized, the trypsin solution and washes of the well and centrifuged cell pellet with MEM were combined and designated as "trypsinate". The resulting cell pellet was designated as the "intracellular" fraction. Carrier (6 J.Ig hyaluronan, Healonts, Pharmacia Uppsala, Sweden) was added to each sample to improve the recovery of radiolabeled glycosaminoglycans during the purification procedures and analysis. Medium, trypsinate and intracellular fraction were each digested for 1.5 h in 50 mM sodium-acetate, containing 200 J.Ig1ml papain (Sigma), roM EDTA and 5 roM cysteine, pH 6. Papain was inactivated in a boiling water bath and glycosaminoglycans precipitated with 1 % cetylpyridinium chloride, centrifuged, supernatant removed by aspiration and discarded. The precipitates were washed with water, dissolved in 4 M guanidine-HCl, and precipitated with ethanol. Details of the
EGF regulates HAS2 expression
isolation and analysis have been published previously
563
10.11.
Hyaluronan assay with double labeling Each purified sample was digested in 50 mM ammonium acetate, pH 7.0, with 25 mU chondroitinase ABC and 1 mU of Streptococcus hyaluronidase for 3 h at 37°C (both undigested material (heparan sulfate) and digestion products were analyzed on a Superdex Peptide column (Pharmacia) monitored for 3H and 35$ radioactivity, and for carrier hyaluronan at 232 nm 11. Incorporation of 35S04 provides a measure of the amount of the chondroitinldermatan sulfate synthesized during the labeling period. The [3H]galactosamine, derived from [3H]glucosamine, incorporated into the same chondroitinldermatan sulfate disaccharides provides an estimate of the effective specific activity of the UDP-N-acetylhexosamine precursor pool, and hence can be used to determine the chemical content of newly synthesized 3H-labelled hyaluronan 10. RT -rca with Hast, Has2, Has3 and GAPDH primers Keratinocytes were cultured in -28 cm 2 dishes until confluency and scraped into TRlzol®-reagent (Gibco) for total RNA isolation according to the instructions of the manufacturer. The RT-PCR reactions of Dnase treated samples were done with the RNA PCR Core Kit (Perkin Elmer, Branchburg, NJ). To obtain rat Hasl and Has3 specific primers, cDNA sequences were amplified from rat keratinocyte RNA with mouse Hasl and Has3 specific primers using RT-PCR. The PCR products were cloned into a pSportl (Gibco) plasmid and sequenced. Primers for Hasl and Has3 were 5'-GCT CTA TGG GGC GTT CCT C-3' and 5'-CAC ACA TAA GTG GCA GOO TCC-3', 5'-ACT CTG CAT CGC TGC CTA CC-3' and 5'-ACA TGA CIT CAC GCT TGC CC-3', respectively. Rat Has2 and GAPDH specific primers, 5'-TCG GAA CCA CAC TGT TTG GAG TG-3' and 5'-CCA GAT GTA AGT GAC TGA TIT GTC CC-3', and 5'TGA TGC TGG TGC TGA GTA TG-3' AND 5'-GGT GGA AGA ATG GGA GIT GC-3', were designed from GeneBank sequenced AFOO820I and MI770I, respectively. For quantitation of Has2 mRNA, a shortened (internal standard) Has2 cDNA containing the primer binding sites identical to those in the wild type Has2 cDNA was prepared by PCR. After in vitro transcription, the shortened Has2 RNA strand was purified and quantitated at 260 nm. RT-PCR was performed with constant amounts of the wild type and different concentrations of the shortened Has2 RNAs. The resulting products were run on an agarose-gel and quantitated by EtBr fluorescence by digital image analysis. Assay of keratinocyte migration Keratinocyte cultures approaching confluency were wounded by two perpendicular lines(-1 mm wide) scraped with a disposable pipette tip. The cleared areas were measured by an inverted microscope again 18 h after the treatment. The change in the area was measured and converted to mean migration distance (1JIll) of the cell front. Hyaluronan microscopy Cell layers were washed with Hank's balanced salt solution and fixed at room temperature for 20 min in PBS with 2 % paraformaldehyde with or without 0.5 %
564
Keratinocytes and hyaluronan
glutaraldehyde. The fixed cells were washed with 0.1 M sodium phosphate buffer, pH 7.4 (PB), and then blocked in 1 % BSA (w/v) containing 0.1 % Triton-Xl00 (v/v) in PB. Hyaluronan staining was done with biotinylated hyaluronan binding complex (bHABC), purified from a 4 M guanidine-HCl extract of bovine articular cartilage after dialysis and trypsin digestion, as described previously 11. The probe is a purified mixture of biotinylated G 1 domain of aggrecan and link protein. The bHABC probe, diluted to 5 lJg/ml in 3 % BSA (w/v), was added to the fixed cells, and incubated overnight at 4°C. After washing, avidin-biotin peroxidase (ABC-standard kit, Vector Laboratories, Inc., Burlingame, CA) was added for I h. The color was developed using 3,3'-diaminobenzidine (DAB) (0.05 %) and H202 (0.03 %). Counterstaining was done with hematoxylin for 2 min before mounting in Aquamount (BDH Laboratory Supplies, Poole, England). For confocal analysys Texas Red-labelled streptavidin (dilution 1:1000) was used instead of ABC. The cells were mounted with Vectashield (Vector), and viewed with a Perkin Elmer UltraVIEW confocal microscope (Wallac-LSR, Oxford, UK). Optical densities of the DAB stained cultures were measured as described previously 11. The specificity of the staining was confirmed by the lack of signal after pre-digesting fixed cultures with Streptomyces hyaluronidase or preincubating the bHABC probe with HA oligosaccharides. RESULTS Specific stimulation of keratinocyte hyaluronan synthesis by EGF The amounts of newly synthesized 3H-hyaluronan, 3H135S-chondroitin sulfate, and chondroitinase resistant glycosaminoglycans (mainly heparan sulfate) were determined using the double label method. Hyaluronan synthesis rate was highest with 20 ng/ml EGF, a concentration routinely used in subsequent experiments. The synthesis of other glycosarninoglycans (heparan sulfate and chondroitin sulfate) was not altered appreciably by EGF treatment. Since serum also stimulates hyaluronan synthesis, we examined the interactive effects of EGF and serum. Approximately similar increase of hyaluronan synthesis was seen whether the basal medium contained 0.5 % or 10 % serum, suggesting that factors in serum. such as IGF-l and PDGF that might contribute to increased synthesis, are additive with EGF. Since the vitality of cells may suffer in extended cultures with low serum concentration, 10 % serum was used in later experiments. Time course of EGF-induced hyaluronan synthesis Small aliquots of the radiolabeled precursors were added into the medium at different times following introduction of EGF, to monitor the time dependence of hyaluronan synthesis in 6 h labeling windows (Fig. 1). The medium change alone caused a marked increase of total hyaluronan synthesis. Nevertheless, EGF-treatment showed an additional -2-5 fold increase of newly synthesized total hyaluronan above the control at all time windows. The majority of hyaluronan synthesized in extended labeling periods was found in the culture medium, but a significant fraction remained associated with the cell layer in the 6 h labeling windows, either with the "trypsinate" or the "intracellular" fractions (Fig. 1). The newly synthesized hyaluronan in these fractions was substantially increased by EGF, peaking at the 0-6 and 6-12 h labeling windows. The "trypsinate" hyaluronan
EGF regulates HAS2 expression
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Hyaluronan synthesis rate after the addition of EGF. The 0 h time point represent control cultures before the change of fresh medium containing EGF, 2 days after the previous change. The amount of newly synthesized hyaluronan in growth medium and that associated with the trypsinate and the intracellular compartment are shown. Each point shows the synthesis during the preceding 6 h labeling period. The vertical bars show the range of duplicate cultures.
represents molecules bound to the receptors, mainly CD44, while most of the "intracellular" hyaluronan probably represents endocytosed material destined for lysosomal degradation. The EGF-induced increase of hyaluronan associated with the cell layer was also seen by staining the keratinocyte cultures with the hyaluronan specific probe (bHABC) (Fig. 2) and confirmed by determination of the total optical densities. The EGF-induced increase of intracellular hyaluronan was specifically measured in cultures digested with Streptomyces hyaluronidase to remove cell surface hyaluronan, then permeabilized and stained with bHABC. Morphological changes of keratinocytes and organotypic keratinocyte cultures
Shortly after introduction of EGF, the subconfluent, flattened keratinocytes began to round up and increase their membrane ruffles and microspikes, which was followed by cell elongation and the appearance of lamellipodia. These changes in morphology were apparent in a few of the cells even after 1 h, and most cells showed the altered morphology
566
Keratinocytes and hyaturonan
A
Figure 2.
Keratinocyte morphology and hyaluronan distribution in the presence of EGF. A control culture is shown in (A) and one treated with EGF for 18 h in (B). Hyaluronan is present on membrane patches and ruffles of the wellspread, non-polar control cells, while in the elongated, apparently migrating EGF-treated cells hyaluronan signal is generally enhanced and most abundant in the pericellular region.
at 6 h and thereafter (Fig. 2). REKs cultured at the air-liquid interphase form 3-4 vital cell layers on top of which there are several fully differentiated comeocytes. Hyaluronan is histologically visible between all the vital cell layers and treatment with EGF dramatically increases its staining intensity. At the same time, the height and number of the vital cell layers increases, suggesting delayed terminal differentiation of the keratinocytes. Localization of the cell-associated hyaluronan Most of the hyaluronan in control cultures resided in plasma membrane patches II. In EGF-treated cells, the hyaluronan signal intensity was generally increased. also covering the membrane ruffles and microspikes in cells undergoing rounding. In the cells elongated by EGF, the mid-portion (around the nucleus) and the trailing edge showed an intense hyaluronan signal (Fig. 2). The amount of hyaluronan that accumulated in response to EGF treatment was sufficient to exclude sedimenting red blood corpuscles on keratinocyte surfaces, a frequently used test of hyaluronan coat formation and hyaluronan synthesis. Confocal images showed more hyaluronan also at the underside of the EGF-treated cells than in control cells. The intracellular hyaluronan signal was present in structures conforming to cytoplasmic vesicles which were generally larger and markedly more abundant in EGFtreated cultures. No nuclear hyaluronan signal was found. The accumulation of intracellular hyaluronan in response to EGF was most conspicuous in rounding cells, while cells retaining a more flattened morphology contained less. Stimulation of migration by EGF treatment The cells became elongated in EGF-treated cultures, a common finding in cells with enhanced motility. A dose dependent stimulation in the migration of the keratinocytes by EGF was confmned by artificial wounding of the cell layer, and quantitation of the speed at which the cells migrated into the cleared area (Fig. 3). The migratory activity peaked at the same EGF concentration as for the synthesis of hyaluronan. The cell number almost
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Hyaluronan synthase mRNA expression in EGF-treated cultures. (A) Total RNAs isolated from equal numbers of keratinocytes 3 h after change into fresh medium with or without EGF were reverse transcribed and amplified with 35 peR cycles for the different Has types and GAPDH. (B) Assay of Has2 mRNA at different time points following change into EGF containing (open circles) and control medium (closed circles), utilizing the mRNA internal standards.
While it is obvious that the synthesis rate of hyaluronan in various cell types is controlled by cytokines and growth factors, including EGF, the contribution of the three known Has genes in this regulation remains uncertain. In particular, the interactions of different hormonal and growth factor effectors need further characterization, as indicated by an earlier report showing upregulation of Hasl mRNA in keratinocytes treated by TGF~ in the presence of hydrocortisone 12, a strong suppressor of Has2 13. The intracellular accumulation of endogenous hyaluronan in perinuclear vesicles by EGF treatment was quite striking and unexpected, likely a result of enhanced receptormediated uptake, since our unpublished data show that the intracellular hyaluronan staining enhanced by EGF is reduced by incubation with hyaluronan decasaccharides, known to displace hyaluronan from keratinocyte surface receptors II. Our preliminary data in EGF-treated cultures also indicate less than expected accumulation of hyaluronan in 24 h labelings, as compared to values integrated from shorter labeling windows. Rapid catabolism has not been demonstrated in other cell cultures, but hyaluronan is known to have a very short half life in the epidermis of human skin organ cultures 2 and in organotypic keratinocyte cultures 10. While the biological importance of the hyaluronan turnover in keratinocytes is not currently known, its association with the enhanced motility of the cells seems most plausible. The data in this study establish that hyaluronan, a major extracellular matrix molecule in stratified epithelia like epidermis, is specifically increased in keratinocytes by EGF upregulation of Has2, and that Has2 regulation can induce rapid and dramatic changes in their cellular environment. considering the small extracellular space into which hyaluronan is synthesized. The biological consequences include enhanced keratinocyte migration during wound healing and thickened vital epidermis likely due to delayed differentiation. The direct influence of Has2 expression on keratinocyte migration was demonstrated by transfection of Has2 genes in sense and antisense orientations (see Rilla et al., in this volume). The ability of epidermal keratinocytes to rapidly cover an open wound is a biologically
EGF regulates HAS2 expression
569
crucial motility response of keratinocytes. Healing of skin wounds involves transient upregulation of EGF-receptors 14, and is aided by EGF-like growth factors 8. These studies suggest that stimulated EGF-receptor signaling and enhanced hyaluronan metabolism are intimately connected with each other and with the epithelial wound healing process.
ACKNOWLEDGEMENTS Technical help of MS Arja Venalainen, MS Riikka Tiihonen and MS Paivi Perttula is gratefully acknowledged. Financial support was received from the Academy of Finland, Finnish Cancer Foundation and EVa Funds of Kuopio University Hospital.
REFERENCES R. Tammi, J. A. Ripellino, R. U. Margolis, H.I. Maibach & M. Tammi, 'Hyaluronate accumulation in human epidermis treated with retinoic acid in skin organ culture', J. Invest. Dermatol., 1989, 92, 326-332. 2. R. Tammi, A. M. Saamanen, H. I. Maibach & M. Tammi, 'Degradation of newly synthesized high molecular mass hyaluronan in the epidermal and dermal compartments of human skin in organ culture', J. Invest. Dermatol., 1991, 97, 126130. 3. M. Tammi & R. Tammi, Hyaluronan in the epidermis. in Science ofHyaluronan Today, V. Hascall and M. Yanagishita, Editors. 1999. Http://www.glycoforum.gIjp/science/hyaluronan/hyaluronanE.html 4. U. M. Agren, M. Tammi & R. Tammi,'Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture', J. Cell Physiol., 1995, 164,240-248. 5. E. Tzahar & Y. Yarden, 'The ErbB-2IHER2 oncogenic receptor fo adenocarcinomas: from orphanhood to multiple stromal ligands' , Biochim. Biophys. Acta, 1998,1377, M25-37. 6. K. L. Carraway 3rd & L. C. Cantley, 'A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerization in growth signaling', Cell, 1994, 78, 5-8. 7. L. G. Hudson & L. J. McCawley, 'Contributions of the epidermal growth factor receptor to keratinocyte motility, Microsc. Res. Tech., 1998,43,444-455. 8. L. J. McCawley, P. O'Brien & L. G. Hudson, 'Overexpression of the epidermal growth factor receptor contributes to enhanced ligand-mediated motility in keratinocyte cell lines' ,Endocrinology, 1997, 138, 121-127. 9. D. K. MacCallum & J. H. Lillie, 'Evidence for autoregulation of cell division and cell transit in keratinocytes grown on collagen at an air-liquid interface', Skin Pharmacol., 1990, 3, 86-96. 10. R. H. Tammi, M. I. Tammi, V. C. Hascall, M. Hogg, S. Pasonen & D. K. MacCallum, 'A pre-formed basal lamina alters the metabolism and distribution of hyaluronan in epidermal keratinocyte "organotypic" cultures grown on collagen matrices', Histochem. Cell Bioi., 2000, 113,265-277. 11. R. Tammi, D. MacCallum, V. C. Hascall, J. P. Pienimaki, M. Hyttinen & M. Tammi, 'Hyaluronan bound to CD44 on keratinocytes in displaced by hyaluronan decasaccharides and not hexasaccharides', J. Bioi. Chem., 1998,273,28878-28888. 12. Y. Sugiyama, A. Shimada, T. Sayo, S. Sakai & S. Inoue, 'Putative hyaluronan synthase mRNA are expressed in mouse skin and TGF- beta upregulates their expression in cultured human skin cells' , J. Invest. Dermatoi., 1998, 110, 116-121. 1.
570
Keratinocytes and hyaluronan
13. W. Zhang, C. E. Watson, C. Liu, K. J. Williams & W. P. Werth, 'Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in dermal fibroblasts and in osteoblasts: a molecular mechanism contributing to organ atrophy', Biochem. J., 2000,349,91-97. 14. C. M. Stoscheck, L. B. Nanney & L. E. King, Jr., 'Quantitative determination of EGF-R during epidermal wound healing', J. Invest. Dermatol., 1992, 99, 645-649.
INDEX activation energy 185 adhesion assays 444 adhesive anti- 67-68 adhesive bio- 68 adhesive force 72 aggrecan 332, 358, 497 albumin adsorption 293-304 alginate fibre 280 amide derivatives of hyaluronan 261 262 ' amidyl radical 157 anchorage-independent growth 349353 angiogenesis 469 annealing 206 antibodies 437 anti-cancer activity 419-426 anti-inflammatory 501 anti-proliferative activity 419-426 antisense probes 241 apoptosis 489-494 Arrhenius plots 120,121 articular chondrocytes 334 atomic force microscopy 67-74, 109-116,137-139 bacterial hyaluronan 37 basal lamina 511-516 Benoit-Doty relation 39 binding activity 342-347 binding affinity 368 binding assays 356 binding crystal 411- 416 bio compatibility 269 bio-adhesive 68 biocompatible gel 285-292 biodegradability 269 biological activity 306 biomaterials 261-266, 269-275 biostability 277-284 biosynthesis 228-236 biosynthesis glycosaminoglycan 537-542 birefringence 214,215,216 bladder cancer cells 419, 424 bone marrow 459 bowel disease 381
breast cancer cell lines 443-445 brevican 358 bromine free radical 143-150 calcium oxalate 411-416 cancer cells 419-426, 443-445 cancer, skin 525 cartilage 332 casein kinase II assay 367 caverolae 517 CD44 331-339,341-347,350-353, 381-388,396-398,408,419-426, 457-466,517-524 CD44 expression 443-445 CD44 hyaluronan receptor 355-364 CD44 overexpression of 350 cell activation 342 cell adhesion 341, 443 cell biological role of hyaluronan cell culture 332,390-398,412,473, 482,518,522,562 cell death 438-441 cell growth 484 cell hyaluronan interactions 349-354 cell migration 444 cell proliferation 451-455, 470 545550 ' cell proliferation assay 445 cell surface 374 cell surface CD 44 341-347 cell surface expression 415 cell surface receptors 408 cell-associated hyaluronan 566 cellular proliferation 421 cellular signalling 301-304,361 chain-chain association 123-134 characteristic ratio 76 chimeric glycosaminoglycan 233 chloramides 156 chlorine free radical 143-150 chondrocyte 331-339 chondroitin 233 chondroitin sulphate 233,518 Chrohn's disease 382 clinical applications of hyaluronan cloning 356 coated pits 521 cold crystallisation 324 collagen 511-516 colon 382 compliance 197
572
Index
confocal micrograph 385-388 confocal miscroscopy 412,438 confocal-FRAP 123-134 conformational properties 89-97, 99102 corneal keratinocytes 532-535, 537542 creep experiments 195-200 critical overlap parameter 181-188 critical shear rate 95, 187 crosslinked derivatives 261, 264268, 269-275 crosslinked hyaluronan 182-191, 201-204 crosslinking process 277-284 crystal binding 411-416 cummulus cell-oocyte complex 489494 cyclo-oxygenase 501-504 cystoskeleton 331-339 cystosplasmic vesicles 519 cytokines 305, 429-434, 473-478 cytoplasmic cell fractions 470 DEAC ion exchange resin 99-102 deacetylated derivative of hyaluronan 261, 263 degradation 455 degradation of hyaluronan 141-149, 401 dendritic cells 457-466 derivatisation of hyaluronan 278 diabetic renal disease 473 differential scanning calorimetry 205, 208,316-321,324-327 diffusion coefficients 270 dihydroxyalkyl radical 153 DNA 429-434 DNA bioconjugate 305-310 DNA synthesis 526-530 Dorfman, Albert 29-33 drug delivery 265 dynamic light scattering 43 dynamic measurements 188 dynamic viscosity 203 eicosanoids 501 elastase 532 elastic modulus 202, 203 elastic modulus of hyaluronan gels 296 elasticity 201-204
electron microscopy 513 electron microscopy of combined hyaluronan 269-275 electrophoresis 254 electrophoretic mobility 305-308 embryogenesis 245-248 endocytosis 401-409, 522 endometrial tissue 238 endometrium 237-244 endopeptidase activity 532 endothelial cells 302,303,401-409, 419-426,469-472 endothelial hyaluronan receptors 353-364 enthalpy of crystallisation 316-321 , 324-327 enthalpy of melting 206-208,316321, 324-327 enzymatic hydrolysis 249-252 enzyme assays 229 enzyme treatment 412 epichlorohydrin 279 epidermal growth factor 561-569 epidermal keratinocytes 545-550, 557-560, 561-569 epidermis 511-516,517-524 epithelial cells 473-478,561 EPR experiments 152-159 erk kinase 373-377 erythrocytes 390, 391 esterified hyaluronan 278 esters of hyaluronan 261, 264 ethidium bromide 307 eukaryotic plasma membrane 245248 excluded volume 77 exclusion, 27 expression of hyaluronan 414, 415 extensional flow 209-218 extracellular hyaluronan 447 extracellular matrix 332, 498-500, 511 fertility 489-495 fibrinogen, adsorption 293-304 fibroblasts 531-535 field flow fractionation of hyaluronan 55-64 first order rate constant 146,147 flow cytometric analysis 440 flow cytometry 333, 342, 444
Index fluorescein labelled hyaluronan 452455 fluorescent-labelled HA 436 force measurements 67-74 force-separation curves 213, 217 formaldelyde 182 fragmentation, polysaccharide 151159 free radical 141-150 free radicals, resistance to 281-283 galactosidase gene 305, 306 gamma ray crosslinked hyaluronan 266,267 Gaussian cloud 80 gel biocompatible 285-292 gel filtration chromatography - see gel permeation chromatography gel mobility shift assay 552 gel permeation chromatography, of hyaluronan 47-54,55-64,100102,127,230,286 gel slurry 195-200 gelatin zymography 532-535 gelation of hyaluronan 205-208 gels derivatised hyaluronan for 293304 gel-sol transition temperature 206 gene targeting 246-248 gene transfer 305-310 glass transition 316-321,324-327 glucosamine (3 H labelled) 538-542 glutaraldehyde 261,265 glycoconjugates 538-542 glycosaminoglycan biosynthesis 514, 537-542 glycosaminoglycans 231-256 glycosaminoglycans, radiolabelled 562 glycosyl transferase 227-236 glycosylation sites 253-258 grafting, polymer 233 HABPI 365-371 HABPS447 haptotaxis 444 HARE proteins 401-409 Healon 181-192,212-218 heat shock protein 72, 435-441 heparin 369 histochemical demonstration 513 histochemical staining 483
573
Huggin's constant 181,184 Huggin's equation 85, 92 hyaladherins 7, 162,365,373,407 hyaluronan anti-cancer activity 419426 hyaluronan behaviour in solution 3745 hyaluronan binding 341-347,360, 365-371, 373-377 hyaluronan binding sites 452 hyaluronan biological properties of 117-122 hyaluronan biosynthesis 288 hyaluronan biosynthesis of 8,31 hyaluronan cell biological role 7 hyaluronan chain characterisation 38,39 hyaluronan characterisation, 4, 5 25,26 hyaluronan chemical structure 41 hyaluronan clinical applications 9 hyaluronan confocal-FRAP 123-134 hyaluronan conformational properties 89-97, 99-012 hyaluronan covalently linked 67-74, 278-284 hyaluronan critical shear rate 95 hyaluronan crosslinked 99-102, 181192,201-204,261-268,269-275, 277-280 hyaluronan crystal binding 411-416 hyaluronan degradation 8,9, 141149,401,455,518 hyaluronan derivatives 261-266 hyaluronan DNA matrix 305-310 hyaluronan dynamic light scattering of 43 hyaluronan expression 414,415 hyaluronan fluorescein labelled 123134,341-347,451-455 hyaluronan force measurements 6774,109-116,137-139 hyaluronan from umbilical cords 1, 2 hyaluronan gel 285-292, 293-304 hyaluronan gel permeation chromatography of 47-54, 55-64, 91-97, 99-102, 127, 143-150 hyaluronan history of 1-10, 17-23 hyaluronan hydration of 45 hyaluronan hydrogelation 205-208,
574
Index
314 hyaluronan hydrolysis 249-252 hyaluronan in joint fluids 3 hyaluronan in rooster comb 3 hyaluronan in Streptocci 3 hyaluronan intracellular 447-449, 451-455 hyaluronan intrinsic persistence length 39 hyaluronan intrinsic viscosity 75-78, 82, 91-93, 105, 184 hyaluronan ion exchange of 99-102 hyaluronan isothermal titration calorimetry 161-171 hyaluronan light scattering on 91-97, 143-150 hyaluronan Mark-Houwink parameters 91, 184 hyaluronan metabolism 511-516 hyaluronan molecular mass 46-54, 55-64,91-97, 143-150,184,286, 431,521 hyaluronan molecular modelling of 39-44 hyaluronan networks of 6, 7 hyaluronan NMR 3 C) studies 117122,281 hyaluronan NMR 161-171 hyaluronan oligosaccharides 436441,457-466 hyaluronan pathology of 9 hyaluronan phase transition 323-327 hyaluronan physiological functions 26,27 hyaluronan production of 221-226 hyaluronan radius of gyration 25, 39, 4950,80,89,90, 127 hyaluronan receptors 355, 359, 396, 401-409 hyaluronan rheological properties 5, 6, 89-97, 103-108, 175-180, 195200, 201-204 hyaluronan secondary structure 118 hyaluronan self association 99, 123134,137-139 hyaluronan self diffusion 125-134 hyaluronan shear rate dependence 93 hyaluronan staining 412, 546 hyaluronan stimulation 555
e
hyaluronan structure 3, 4 hyaluronan sulphated derivative 293304 hyaluronan synthases 227-235,237244,245-248,473,481,557 hyaluronan synthesis 474,481-487 hyaluronan synthesis rate 565 hyaluronan tertiary structures 117122 hyaluronan turnover 401 hyaluronan viscoelasticity 89-97 hyaluronan viscometry of 47-54 hyaluronan x-ray diffraction of 44 hyaluronan zero shear viscosity 94, 95, 96 hyaluronan-binding proteins 7, 407, 447-449 hyaluronan-binding proteoglycans 497-500 hyaluronan-cell interactions 349-354 hyaluronan-protein interactions 161171, 293-304 hyaluronic acid-based polyurethane derivatives 313-322 hyaluronidase 230, 249-252, 253258,436,445 hyaluronidase Streptomyces 332, 390-398,497,538 hyaluronidase, resistance to 281-283 hybridisation 239,246-248 hydration of hyaluronan 46 hydrodynamic diameter 75-78 hydrogel hyaluronan 314 hydrogelation 205-208 hydrogels 293, 261, 265 hydrolysis 249-252 hydroxy radicals 141-150,151-159 Hylon 181-192, 195-200,209-218 hyperglycemia 473 hypochlorite 151-159 I CAM 1408 IHABP4447-449 immunohistochemistry 239 immunoblotting 553 immunocytochemistry 403-408 immunofluorescent antibody staining 357 immunohistochemical staining 361, 384 immunoperoxidase 357
Index immunoprecipitation 470 immunostaining 437-441 inflammation 381, 531 inflammatory disease 502 inflammatory disorders 429-434 integral-membrane proteins 357 intracellular HA 447-449,451-455 intrinsic viscosity 75-78,82,83,9193, 105, 106, 184 isocyanate 313 isothermal titration calorimetry 161171 joint protection 209-218 keratinocyte migration 563 keratinocytes 511-516, 517-524, 525-530,531-535,545-550,551555,557-560,561-568 kidney 412 kidney inflammatory disease 502 kinases 367, 471, 551 knee joint 210 Kratky plot 40 leucocyte adhesion 381-388 light scattering, of hyaluronan 47-54, 55-64,91-97, 143-150,286 link module 161-171, 341 link superfamily 357-362 liver cells 401-409 liver kinase assay 367 lyase 230 lymph node 408 lymphatic endothelium 359-363 lymphocytes 390-398 mammary cells 351-353 Mark-Houwink parameters 91,184 Martin equation 85, 86 MDCK cells 411-416 menstrual cycle 237-244 mesothelial cells 481-487 metabolic labelling 412-415,513 metabolic labelling assay 546 metal ion complex 300-304 metalloprotease expression 531-535 Meyer, Karl Alexander 3, 17-23 mitogen activated 551-555 mitosis 451-455 molecular mass of hyaluronan 4754, 55-64, 76, 91-97, 184 molecular motion 316 monoclonal antibodies 401-408
575
monocyte adhesion assay 383 mutagenesis 231 mycobacterial DNA 423 myeloperoxidase 151, 152 nanomechanically patterned surface 293-304 nephrolithiasis 411-416 neurocan 358 nitroso spin traps 154 NMR 161-171, 281, 283 NMR of crosslinked hyaluronan 269-275 Northern blotting analysis 437-441 nuclear protein extractor 552, 553 oligomers, hyaluronan 350 oligosaccharides of hyaluronan 436, 457-466,517-524 opposed jets 214 organ culture 332,511-516 oxidised hyaluronan 264 Pasteurella 227 pathology of hyaluronan PDGF 451-455 pericellular matrix 331-339,389-398 peristence length 39, 76, 77, 83 peritoneal mesothelial cells 481-487 peroxides 525-530 peroxynitrite 142-150 persistency test 287 phase diagram 318 phase transition 323-327 phase transition temperatures 316 phenotypic expression 537 phosphorylation 368-371 photoimmobilisation 295, 301 pinocytosis 521 plasma membrane 470 plasmid DNA 306 polymerase chain reaction 229 polymerisation of hyaluronan 229 polymorphic structure 317 polymorphism 253-258 polyurethane derivatives 313-322, 323-327 polyvinyl alcohol 279 porcine excisional wound model 307 progesterone 368 proliferating cells 497 prostate cancer 419, 424 protein extraction 553
576
Index
protein heat shock 435-441 protein hyaluronan binding 365-371, 407,447-449 protein-hyaluronan interactions 161171 proteins HARE 401-409 proteins integral-membrane 357 proteoglycans 231-256, 497-500 proximal tubular cells 473-478 radius of gyration 29, 39, 49, 50, 80, 89,90,127 receptors, hyaluronan 396, 401-409 REK cultures 517-524 relaxation times 188, 270 renal cells 473-478,501-504 renal tubule epithelium 412 reticulation 266 RHAMM 373-377,408,457-466 rheological analysis 294, 296 rheological behaviour 181-192, 201204 rheological creep experiments 195200 rheological flow profiles 175-180 rheological properties 89-97, 103108 rheumatoid synovial fluid 152 rhodamine 526 RNA extraction 483 rooster combs 99, 110 rubber elasticity theory 190 SANS 40 SAXS, of crosslinked hyaluronan 269-275 scanning electron microscopy 307 SEC - see gel permeation chromatography secondary structure 118 self association 99, 123-134, 137139 self diffusion 125-134 semi-dilute regime 185 semi-dilute solution 84 sense probes 241 serum electrophoretic pattern 254 shear flow measurements 185 shear flow of synovial fluid 209-218 shear rate dependence 93 shift factors 189 sialidase treatment 253, 255
signal transduction 469-472 signalling molecules 554 skin cancer 525 skin epidermis 561 skin fibroblasts 532-535, 537-542 specific viscosity 79, 81, 105 sperm 365-371 spermadine 369 spin trapping 154-159 spreading analysis 558 squeeze flow 209-218 stagnation point 215 staining of hyaluronan 412, 546 stratified epithelia 511, 561 Streptococcus bacteria 182, 221226,227 Streptococcus equi 90, 138 Streptomyces hyaluronidase 332, 390-398,497,538 subconfluent cultures 414 sulindac 476-478 sulphated derivative of HA 293-304 superoxide radical 141-150 sustained release 308 synovial cells 389-398 synovial fluid 117 synovial fluid 209-218 synthases, hyaluronan 217-235, 237-244, 245-248 T-cell stimulation 457-466 TEMPO - mediated oxidation 264, 265 tertiary structures 117-122 testicular hyaluronidase 230 thermal analysis 294, 297 thermal properties of hyaluronan derivatives 313-322 thromboxane 501-504 tissue augmentation 201, 202 tissue collection 238 tissue distribution 406 tissue engineering 293 tissue extraction 254 transcription factor 551-555 transfectants 350-353 transfected cells 356 transfections 306-310, 558-560 transmembrane 408 trypsin inhibitor 497 TSG-6161-171
Index
TSG-protein 358 tumour growth, inhibition of 349-353 TXA2502 Ugi's reaction product 265 umbilical cord 1, 2, 402 uniaxial compression 214 uridine diphosphoglucose dehydrogenase 483 urine 411 UV irradiation 525-530 versican 358,381-388,497-498 vesicles 519 vinyl sulfone 182 viscoelastic measurements 91-97 viscometric activation energy 185 viscometry of hyaluronan 47-54 viscosity of polymer solutions 79-87 viscosupplementation 195 viscous modulus 202, 203 water absorption capacity 281 water holding capability 323 water uptake 294 Western blotting 333, 439, 470 Wharton's jelly 117 wound assay 444 wound healing 481-486, 531, 561 wound repair 411-416 wounded cells 483 wounding-induced migration 559 Yamakawa and Fuji model 41 zero shear viscosity 94, 95, 96, 104, 197,204 zymogram technique 254
577
