Diabetic Retinopathy

Diabetic Retinopathy Developments in Ophthalmology Vol. 39 Series Editor W. Behrens-Baumann, Magdeburg Diabetic R...

Author: Gabriele E. Lang

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Diabetic Retinopathy

Developments in Ophthalmology Vol. 39

Series Editor

W. Behrens-Baumann, Magdeburg


Volume Editor

Gabriele E. Lang, Ulm

35 figures, 3 in color, and 25 tables, 2007

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

Gabriele E. Lang Universitätsklinikum Ulm Augenklinik Prittwitzstrasse 43 DE–89075 Ulm

Library of Congress Cataloging-in-Publication Data Diabetic retinopathy / volume editor, Gabriele E. Lang. p. ; cm. – (Developments in ophthalmology, ISSN 0250-3751 ; v. 39) Includes bibliographical references and index. ISBN-13: 978-3-8055-8243-8 (hardcover : alk. paper) ISBN-10: 3-8055-8243-9 (hardcover : alk. paper) 1. Diabetic retinopathy. I. Lang, Gabriele E. II. Series. [DNLM: 1. Diabetic Retinopathy–therapy. 2. Diabetic Retinopathy–diagnosis. W1 DE998NG v. 39 2007 / WK 835 D53626 2007] RE661.D5D52 2007 617.7⬘35–dc22 2006038094

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus. Disclaimer. The statements, options and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2007 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 0250–3751 ISBN-10: 3–8055–8243–9 ISBN-13: 978–3–8055–8243–8

Contents

VII List of Contributors IX Preface Lang, G.E. (Ulm) 1 Pathophysiology of Diabetic Macular Edema Joussen, A.M. (Duesseldorf/Cologne); Smyth, N. (Cologne/Southampton); Niessen, C. (Cologne) 13 Characterization and Relevance of Different Diabetic Retinopathy Phenotypes Cunha-Vaz, J. (Coimbra) 31 Optical Coherence Tomography Findings in Diabetic Retinopathy Lang, G.E. (Ulm) 48 Laser Treatment of Diabetic Retinopathy Lang, G.E. (Ulm) 69 Benefits and Limitations in Vitreoretinal Surgery for Proliferative Diabetic Retinopathy and Macular Edema Joussen, A.M. (Duesseldorf); Joeres, S. (Cologne) 88 Diffuse Diabetic Macular Edema: Pathology and Implications for Surgery Gandorfer, A. (München) 96 Intravitreal Triamcinolone Acetonide for Diabetic Retinopathy Jonas, J.B. (Heidelberg)

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111 Use of Long-Acting Somatostatin Analogue Treatment in Diabetic Retinopathy Boehm, B.O. (Ulm) 122 Vascular Endothelial Growth Factor and the Potential Therapeutic Use of Pegaptanib (Macugen®) in Diabetic Retinopathy Starita, C. (Sandwich); Patel, M.; Katz, B.; Adamis, A.P. (New York, N.Y.) 149 Pharmacologic Vitreolysis Gandorfer, A. (München) 157 Treatment of Diabetic Retinopathy with Protein Kinase C Subtype ␤ Inhibitor Lang, G.E. (Ulm) 166 Subject Index

Contents

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List of Contributors

Tony Adamis (OSI) Eyetech, Inc. 3 Times Square New York NY 10036 (USA) Bernhard O. Boehm Division of Endocrinology and Diabetes, Ulm University Robert-Koch-Strasse 8 DE–89081 Ulm (Germany) José Cunha-Vaz AIBILI Azinhaga Santa Comba, Celas PT–3000-548 Coimbra (Portugal) Arnd Gandorfer Vitreoretinal and Pathology Unit Augenklinik der LudwigMaximilians-Universität Mathildenstrasse 8 DE–80336 München (Germany) Sandra Joeres, MD Medical Retina Unit Doheny Image Reading Center Doheny Eye Institute

University of Southern California 1450 San Pablo Street DEI 3623 Los Angeles CA 90033 (USA) Jost B. Jonas Universitäts-Augenklinik Theodor-Kutzer-Ufer 1-3 DE–68167 Mannheim (Germany) Antonia M. Joussen Department of Ophthalmology Heinrich-Heine University Duesseldorf Moorenstraße 5 DE–40225 Duesseldorf Barrett Katz (OSI) Eyetech, Inc. 3 Times Square New York NY 10036 (USA) Gabriele E. Lang Universitätsklinikum Ulm, Augenklinik Prittwitzstrasse 43 DE–89075 Ulm (Germany)

VII

Dr. Carien Niessen Center for Molecular Medicine Cologne (CMMC) University of Cologne, LFI, 5, Room 59 Joseph Stelzmannstrasse 9 Manju Patel Pfizer Inc 50 Pequot Avenue MS 6025-B2234 New London CT 06320 (USA)

FList of Contributors

Neil Smyth Department of Developmental and Cell Biology School of Biological Sciences University of Southampton Southampton, Hampshire SO16 7PX (UK) Carla Starita Pfizer Global Research and Development, Building 508/1.75 IPC 613 Ramsgate Road, Sandwich CT13 9NJ Kent (UK)

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Preface

Over decades, there has been broad interest of ophthalmologists and diabetologists in diabetic retinopathy. Despite markedly improved prognosis for visual problems due to laser treatment and vitrectomy, diabetic retinopathy is still one of the leading causes of blindness worldwide. This book provides profound information about the newest developments in the diagnosis and treatment of diabetic retinopathy. The pathophysiology of diabetic macular edema is complex and not yet fully understood. The current knowledge of mechanisms of development and progression of diabetic macular edema is described. An innovative approach of multimedial mapping methods enables to differentiate between three diabetic retinopathy phenotypes, allowing personalized management strategies. Highresolution imaging by optical coherence tomography provides additional, new information about morphological findings in diabetic retinopathy. In this book, the standards and novel approaches of laser treatment of diabetic retinopathy are described, current surgical options for diabetic retinopathy and treatment techniques discussed, and the pathology of diffuse macular edema and implications for surgery proposed. Additionally, the treatment of diabetic retinopathy with triamcinolone and its complications, as well as the hypothesis for the use of somatostatin analogues are discussed. A new therapeutical approach is the use of vascular endothelial growth factor inhibitors in diabetic macular edema. Latest concepts of posterior vitreous detachment by pharmacological vitreolysis are described. An innovative pharmacological compound, the specific protein kinase C subtype ␤ inhibitor ruboxistaurin mesylate, significantly reduces the risk of visual loss in nonproliferative diabetic

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retinopathy and holds promise to improve the visual prognosis in patients with diabetic retinopathy. The book provides an update of new insights into the pathogenesis, diagnosis and especially the treatment of diabetic retinopathy and gives detailed information about latest research achievements. Therefore, it is suitable for general ophthalmologists, retina specialists and diabetologists. It provides a collection of latest findings in diabetic retinopathy by excellent authors, and therefore, deserves the attention of everyone who is interested in this subject. I want to thank all the coworkers for their great efforts in passing on their profound knowledge. The contents of the book will not only advance our understanding of diabetic retinopathy based on the provided knowledge, but also improve our diagnosis and treatment strategies in the permanent efforts to help the numerous patients who suffer from diabetic retinopathy and are threatened by visual problems. Prof. Dr. Gabriele E. Lang, Ulm

Preface

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Lang GE (ed): Diabetic Retinopathy. Dev Ophthalmol. Basel, Karger, 2007, vol 39, pp 1–12

Pathophysiology of Diabetic Macular Edema Antonia M. Joussena,b, Neil Smythb,c, Carien Niessenb a

Department of Ophthalmology, University of Duesseldorf, Duesseldorf, and Center for Molecular Medicine, University of Cologne, Cologne, Germany; c School of Biological Sciences, University of Southampton, Southampton, UK b

Abstract Diabetic maculopathy is the leading cause of visual loss in diabetic patients. The pathogenesis is not fully understood and a satisfactory therapy is currently not available. Malfunction of the blood-retinal barrier plays a central role in the disease and leads to retinal edema and secondary photoreceptor dysfunction. Diabetic vascular leakage and macular edema are regulated by a distinct combination of direct paracellular transport, alterations in endothelial intercellular junctions and endothelial cell death. The distribution and relevance of these three factors to diabetic maculopathy varies over the course of the disease. Cumulative endothelial cell death will become more relevant after prolonged diabetic conditions. This article reviews the current knowledge on the pathogenic mechanisms of diabetic macular edema. Copyright © 2007 S. Karger AG, Basel

The significant morbidity and mortality of diabetes mellitus predominantly results from its complications, among which the vascular dysfunction leading to macular edema resembles the most important vision-threatening complication. Hyperglycemia is the metabolic hallmark of diabetes and leads to widespread cellular damage. Endothelial cells are particularly vulnerable to hyperglycemia because they can poorly regulate intracellular glucose. An excess of glucose sets off a chain of metabolic events that culminate in overproduction of reactive oxygen species in the mitochondria and, in turn, leads to increased flux in the hexosamine and polyol pathways, increased formation of advanced glycation endproducts and activation of protein kinase C. These metabolic changes result in a plethora of tissue-specific functional defects with diabetes-associated vasculopathy as the central mediator of the pathophysiology of diabetic complications.

Early stages of vascular dysfunction are characterized by a breakdown of the blood-retinal barrier in both humans and rodent models of experimental diabetes. Breakdown of the blood-retinal barrier can be observed prior to latestage vascular alterations leading to proliferative diabetic retinopathy. Blood-retinal barrier breakdown contributes to macular edema, which occurs in over 25% of people with diabetes and correlates highly with visual impairment in people with diabetic retinopathy [1]. Treatment by laser coagulation is limited to focal edema, but is controversial in diffuse edema and proven to be ineffective in ischemic diabetic maculopathy.

Breakdown of the Blood-Retinal Barrier

Although changes to retinal blood flow may partially explain the extravasation of fluid, the most important mechanism is the breakdown of the bloodretinal barriers [2]. The movement of water through the blood-retinal barrier appears to have two dominant components: a passive (bidirectional) transport and an active transport directed from the retina to the blood. Theoretically, macular edema develops when the inflow of fluid into the retina exceeds the outflow. Passive transport (permeability) of fluorescein has been shown to increase in relation to the progression of retinopathy [3, 4]. In vitro studies of isolated retinal pigment epitheliumchoroid preparations showed that the outward active transport of fluorescein is substantially greater than the passive transport and that this transport is inhibited by metabolic (oubain) and competitive inhibitors (probenecid) [5–8]. Unlike active transport, passive permeability is related to the degree of retinopathy in that eyes with severe nonproliferative diabetic retinopathy have a passive permeability that is significantly increased compared with moderate retinopathy. The active resorptive functions of the blood-retinal barrier in diabetes are likely to be increased to counteract edema formation, although the increase is too little [9]. Besides the retinal pigment epithelium (outer blood-retinal barrier), the vascular endothelium (inner blood-retinal barrier) forms the main barrier against the passage of macromolecules and circulating cells from blood to the extracelluar space. In diabetes, the endothelial cell loss of the retinal vessels is likely to account for the majority of the early blood-retinal barrier breakdown and is the initial site of damage. Passive permeability through the endothelium can be increased by three general mechanisms: (1) increased transcellular transport, (2) dysfunction of the intercellular junctions, and (3) increased endothelial cell destruction. Although other factors such as impairment of the perivascular supporting cells might influence vascular permeability, the primary damage is likely to predominantly affect endothelial cells.

Joussen/Smyth/Niessen

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Transcellular Transport

Besides an increase in vascular permeability through endothelial cell death and alterations in the cell-cell junctions, direct transport via pinocytosis is potentially involved in the increased diabetic vascular leakage [10, 11]. Despite the fact that pinocytic transport is critically involved in transepithelial fluid exchange, its regulation in diabetic retinopathy has not been investigated in the context of the molecular factors involved, as outlined above. Distinct growth factors are causally related to neovascularization and/ or vascular leakage: the disruption of endothelial integrity leads to retinal ischemia and vascular endothelial growth factor (VEGF)-mediated iris and retinal neovascularization [12–14]. VEGF is 50,000 times more potent than histamine in causing vascular permeability [15–20]. Previous work has shown that retinal VEGF levels correlate with diabetic blood-retinal barrier breakdown in rodents [21, 22] and humans [23]. Flt-1(1-3Ig)Fc, a soluble VEGF receptor, reverses early diabetic blood-retinal barrier breakdown and diabetic leukostasis in a dose-dependent manner [14]. Early blood-retinal barrier breakdown localizes, in part, to retinal venules and capillaries of the superficial inner retinal circulation [24] and can be sufficiently reduced by VEGF inhibition. Although VEGF is only one of the molecules involved in the various cytokine cascades, it is likely to be one of the most efficient therapeutic targets. Ongoing clinical studies investigate the efficacy of VEGF inhibition on diabetic macular edema. Due to the current knowledge, VEGF causes vascular hyperpermeability by opening interendothelial junctions and induction of fenestrations and vesiculo-vacuolar organelles. As for the blood-retinal barrier endothelium, other cellular mechanisms may translate increased permeability caused by VEGF [25]. In these leaky blood vessels, the number of pinocytotic vesicles at the endothelial luminal membrane is significantly higher, and these pinocytotic vesicles transport plasma immunoglobulin G. By electron microscopy, no fenestrations or vesicles were found in the endothelial cells of the VEGFaffected eyes.

Intercellular Junctions and Their Alterations in Diabetic Retinopathy

Endothelial cells are important constituents of the vasculature and essential for the separation of blood from the surrounding tissues. They also control the passage of proteins and cells from the blood stream into these tissues, either by using a specialized transcellular vesicle transport system or by selective opening and closing of intercellular junctions.

Pathophysiology of Diabetic Edema

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It is likely that inflammatory agents increase permeability by binding to specific receptors that transduce intercellular signals, which in turn cause cytoskeletal reorganization and coordinated widening of the interendothelial contacts. Endothelial junctions also regulate leukocyte extravasation upon inflammatory stimuli. Once leukocytes have adhered to the endothelium, a coordinated opening of interendothelial cell junctions occurs without the loss of its barrier function. Under diabetic conditions, inflammatory mediators may cause aberrant opening of intercellular contacts, now also resulting in loss of barrier function and thus vascular leakage. However, both the regulation and the structural composition of retinal endothelial junctions and their alterations in diabetic retinopathy are largely unknown. Composition of Intercellular Junctions in the Retina Intercellular junctions of vascular endothelial cells consist of tight junctions, adherens junctions and gap junctions. Depending on the type of vessel, there is a fourth structure called ‘complexus adherents’ or ‘syndesmosome’ consisting of a mixture of adherens junction components and desmosomal components [26]. Although endothelial cells are polarized, tight junctions are not only found at the interface between the apical and basolateral membrane domains, as observed in simple epithelia, but are often intermingled with the adherens junctions all along the cleft [27]. The molecular composition and complexity of intercellular junctions varies along the vasculature. More complex and distinct structures are formed in those cells with an increased barrier function, such as the blood-retinal barrier [28]. Both tight junctions and adherens junctions consist of transmembrane molecules, which connect cells with each other and are linked to cytoskeletal linker molecules [29, 30]. In addition, a variety of regulatory molecules are also found at these sites. These are most likely important for the regulated interaction with the cytoskeleton and for communicating alterations in adhesion. Alterations in Intercellular Junctions in Diabetic Retinopathy One of the first clinical manifestations in diabetic retinopathy is vascular leakage, indicating that disturbance of the blood-retinal barrier is an early event. Even though tracer studies have shown a disturbance in the paracellular pathway [31], relatively little is known about how the molecular junctional components are affected. In the streptozotocin-induced diabetic rat model, occludin distribution and amounts are altered correlating with an increase in paracellular permeability [31]. In humans, there is one report on decreased expression of vascular endothelial (VE)-cadherin in the diabetic retina [32]. Alterations induced by diabetes have also been described in the placental vasculature where ZO-1,


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VE-cadherin, ␤-catenin and occludin showed reduced staining at the junctions correlating with an increase in tyrosine phosphorylation [33]. Regulation of Junctional Stability, Strength and Permeability Intercellular junctions are dynamic entities even under steady-state conditions and can exchange molecular components and interactions without losing cell-cell contact or barrier function. VEGF can induce phosphorylation of the tight junctional proteins occludin and ZO-1 [34]. In the diabetic retina, VEGF is strongly upregulated and has been implicated as a mediator of vascular leakage and neovascularization. At present, it is not known how the VE-cadherin/VEGF receptor interaction and its signaling pathway is altered in diabetic retinopathy. Diabetes is a consequence of insulin deficiency or insulin resistance. Thus, diabetic retinopathy may be caused directly by absent or aberrant insulin receptor signaling or it may result from secondary effects, since insulin signaling affects a diverse range of downstream pathways. Specific deletion of either the insulin receptor or its close relative the insulin-like growth factor receptor in vascular endothelial cells did not result in any obvious decrease in vascular integrity, nor did it seem to compromise the blood-brain barrier, arguing for the latter situation [35]. However, a direct effect cannot be ruled out because of overlapping functions of the insulin receptor and insulin-like growth factor receptor.

Matrix Changes Affect Formation of Edema in the Diabetic Retina

Degradation of the extracellular matrix affects endothelial cell function at many levels, causing endothelial cell liability which is required for cellular invasion and proliferation, or influencing the cellular resistance and therefore the vascular permeability. The degradation and modulation of the extracellular matrix is exerted by matrix metalloproteinases (MMPs), a family of zinc-binding, calcium-dependent enzymes [36, 37]. Elevated expression of MMP-9 and MMP-2 has been shown in diabetic neovascular membranes [38, 39], although a direct effect of glucose on MMP-9 expression in vascular endothelial cells could not be shown [40]. It is likely that MMPs participate at various stages during the course of the blood-retinal barrier dysfunction and breakdown. Their actions include early changes of the endothelial cell resistance that influence intercellular junction formation and function [41] and active participation in the endothelial and pericyte cell death [42] occurring late in the course of the disease.


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Extracellular matrix and basement membrane components are required for cellular adhesion, migration and differentiation. They also play a particular role in the delimiting of tissue boundaries and the formation of vascular and neural networks. Changes in the deposition of the extracellular matrix during diabetes mellitus are well established. However, the molecular composition of the basement membranes in the normal and diabetic retina is only poorly described. Furthermore, alterations in the repertoire of cellular receptors for basement membrane components and the effects these changes may have upon the cells of the retina have not been investigated. Basement membranes are specialized extracellular matrices found underlying all epithelia and endothelia and surround many mesenchymal cell types. Besides separating tissues, they have important roles in axonal guidance and neuronal migration and survival, as well as synapse formation [43], both by acting directly upon specific cell receptors and by acting as a reservoir for many growth factors, in particular the fibroblast growth factor and transforming growth factor-␤ family [44]. All basement membranes are formed by members of three ubiquitous protein families, i.e. laminins, nidogens and collagen IV, and by the proteoglycan perlecan. Basement membrane variability is derived from the fact that there are 15 laminin isoforms [45], 6 collagen IV chains and 2 members of the nidogen family. These proteins are often expressed in a highly regulated developmental and temporal manner and vary in their use of cellular receptors. Further basement membrane diversity is produced by the presence of more restricted proteins which may be integral basement membrane proteins, such as collagen XVIII [46], or associated with the basement membrane, such as matrilin-2 [47]. Basement membranes are found in three regions of the retina: in Bruch’s membrane underlying the pigment epithelium and separating it from the choroid, in the vitroretinal border as the inner limiting membrane, and in the endothelial basement membrane forming part of the blood-retinal barrier.

Cellular Interaction and Its Relevance to Vascular Leakage

Leukocyte infiltration of retinal tissue characterizes many inflammatory diseases such as diabetes, pars planitis or choroidal inflammatory diseases. In diabetes, activated leukocytes adhere to the retinal vascular endothelium [12, 48]. Increased leukostasis is one of the first histological changes in diabetic retinopathy and occurs prior to any apparent clinical pathology. Adherent leukocytes play a crucial role in diabetic retinopathy by directly inducing endothelial cell death in capillaries [49], causing vascular obstruction and vascular leakage. Endothelial cell death precedes the formation of acellular capillaries [48].


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However, over time, acellular capillaries prevail and become widespread. Although the mechanism of this destructive process remains elusive, it is clear that the interaction between altered leukocytes and endothelial cells and the subsequent endothelial damage represents a crucial pathogenic step [12, 49–51]. Previous studies of vascular casts in diabetic retinopathy have suggested that the loss of pericytes represents the earliest histologically visible alteration [52, 53]. Interaction between pericytes and endothelial cells is important in the maturation, remodeling and maintenance of the vascular system via the secretion of growth factors and/or modulation of the extracellular matrix [54]. There is also evidence that pericytes are involved in the transport across the bloodretinal barrier and the regulation of vascular permeability. Knowledge on vascular plasticity has greatly increased in the past years. It is likely that in the diabetic retina, repair mechanims take place as well. However, cell differentiation and recruitment to the vessel walls are likely to be altered under diabetic conditions. Adult bone marrow (BM) contains cells capable of differentiating along hematopoietic (Lin⫹) or nonhematopoietic (Lin⫺) lineages. Lin⫺ hematopoietic stem cells have recently been shown to contain a population of endothelial precursor cells (EPCs) with the capacity to form blood vessels [55, 56]. In a crucial set of experiments, adult mice were durably engrafted with hematopoietic stem cells isolated from transgenic mice expressing green fluorescent protein after which retinal ischemia was induced to promote neovascularization [57]. In this model, self-renewing adult hematopoietic stem cells had functional hemangioblast activity, i.e. they could clonally differentiate into all hematopoietic cell lineages as well as into endothelial cells that revascularize adult retina. Using green fluorescent protein chimeric mice, it was further demonstrated that laser injury of the choroidal vasculature was sufficient to induce stem cell recruitment and subsequent formation of choroidal neovascularization. Green fluorescent protein-positive cells formed part of the functional vasculature in the choroid as early as 1 week after injury and remained present during follow-up. Furthermore, it was shown that intravitreally injected Lin⫺ BM cells selectively target retinal astrocytes, cells that serve as a template for both developmental and injury-associated retinal angiogenesis. When Lin⫺ BM cells were injected into neonatal mouse eyes, they extensively and stably incorporated into forming retinal vasculature [58]. When EPC-enriched hematopoietic stem cells were injected into the eyes of neonatal rd/rd mice, whose vasculature ordinarily degenerates with age, they rescued and maintained a normal vasculature. In contrast, normal retinal angiogenesis was inhibited when EPCs expressing a potent angiostatic protein were injected.


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In diabetes, it was demonstrated that BM-derived EPCs are recruited to the pancreas in response to islet injury. EPC-mediated neovascularization of the pancreas could in principle be exploited to facilitate the recovery of nonterminally injured ␤-cells or to improve the survival and/or function of islet allografts [59]. Taken together, these studies emphasize the likelihood of an EPC involvement in repair mechanisms of the diabetic vasculature. Besides, in increased stem cell recruitment, these cells and their potential to differentiate might be altered in diabetes. Although likely, alterations in stem cell recruitment and differentiation in diabetes have not yet been investigated in detail.

Endothelial Cell Damage and Apoptosis in the Diabetic Retina

Blood-retinal barrier breakdown is at least in part due to endothelial cell damage and apoptosis. The proapoptotic molecule Fas ligand (FasL) induces apoptosis in cells that carry its receptor Fas (CD95) [60]. There is evidence that FasL is expressed on vascular endothelium where it functions to inhibit leukocyte extravasation. The expression of FasL on vascular endothelial cells might thus prevent detrimental inflammation by inducing apoptosis in leukocytes as they attempt to enter the vessel. In fact, during inflammation and ensuing tumor necrosis factor-␣ release, the endothelium not only upregulates several adhesion molecules [61], but also downregulates FasL and allows leukocyte adherence, survival and thus migration to sites of infection and wounding. In experimental diabetic retinopathy, inhibition of Fas-mediated apoptotic cell death reduces vascular leakage [50]. However, diabetic endothelial cell death, as to the cumulative damage during the diabetic course, might play an increasing role in diabetic vascular leakage, and thus, in diabetic maculopathy.

Conclusion

Diabetic macular disease is considered a structural alteration to the macula in any of the following manners: • collection of intraretinal fluid in the macula with or without exudates (lipids) and with or without cystoid changes; • nonperfusion of parafoveal capillaries with or without intraretinal fluid; • traction in the macula by fibrous tissue proliferation that is dragging the retinal tissue causing surface wrinkling, or detachment of the macula; • intraretinal or preretinal hemorrhage in the macula; • lamellar or full-thickness retinal hole formation; • a combination of the above.


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Early intervention in macular edema is undoubtedly advantageous, as the risk of ultrastructural alterations induced by a persistent macular edema increases with time. It is well known that with time, the central avascular zone and the areas of ischemia are likely to increase. The current hope to treat even ischemic maculopathy pharmacologically will largely depend on the long-term results with e.g. anti-VEGF therapies or intravitreal steroids. Currently, we are only at the edge of understanding diabetic macular edema at a molecular level, but it becomes clear that only a thorough investigation of the pathogenesis of diabetic retinal vascular leakage will help to identify new and potentially more efficient targets for intervention and prophylaxis of diabetic macular edema.

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Das A, McGuire PG, Eriqat C, Ober RR, DeJuan E Jr, Williams GA, McLamore A, Biswas J, Johnson DW: Human diabetic neovascular membranes contain high levels of urokinase and metalloproteinase enzymes. Invest Ophthalmol Vis Sci 1999;40:809–813. Salzmann J, Limb GA, Khaw PT, Gregor ZJ, Webster L, Chignell AH, Charteris DG: Matrix metalloproteinases and their natural inhibitors in fibrovascular membranes of proliferative diabetic retinopathy. Br J Ophthalmol 2000;84:1091–1096. Grant MB, Caballero S, Tarnuzzer RT, Bass KE, Ljubimov AV, Spoerri PE, Galardy RE: Matrix metalloproteinases expression in human retinal microvascular cells. Diabetes 1998;47:1311–1317. Fernandez-Patron C, Zouki C, Whittal R, Chan JSD, Davidge ST, Filep JG: Matrix metalloproteinases regulate neutrophil-endothelial cell adhesion through generation of endothelin-1. FASEB J 2001;15:2230–2240. Behzadian MA, Wang XL, Windsor LJ, Ghaly N, Caldwell RB: TGF-␤ increases retinal endothelial cell permeability by increasing MMP-9: possible role of glial cells in endothelial barrier function. Invest Ophthalmol Vis Sci 2001;42:853–859. Libby RT, Lavallee CR, Balkema GW, Brunken WJ, Hunter DD: Disruption of laminin ␤2 chain production causes alterations in morphology and function in the CNS. J Neurosci 1999;19:9399–9411. Lonai P: Epithelial mesenchymal interactions, the ECM and limb development. J Anat 2003;202: 43–50. Libby RT, Champliaud MF, Claudepierre T, Xu Y, Gibbons EP, Koch M, Burgeson RE, Hunter DD, Brunken WJ: Laminin expression in adult and developing retinae: evidence of two novel CNS laminins. J Neurosci 2000;20:6517–6528. Fukai N, Eklund L, Marneros AG, Oh SP, Keene DR, Tamarkin L, Niemela M, Ilves M, Li E, Pihlajaniemi T, Olsen BR: Lack of collagen XVIII/endostatin results in eye abnormalities. EMBO J 2002;21:1535–1544. Piecha D, Hartmann K, Kobbe B, Haase I, Mauch C, Krieg T, Paulsson M: Expression of matrilin2 in human skin. J Invest Dermatol 2002;119:38–43. Schröder S, Palinski W, Schmidt-Schönbein GW: Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy. Am J Pathol 1991;139:81–100. Joussen AM, Murata T, Tsujikawa A, Kirchhof B, Bursell SE, Adamis AP: Leukocyte-mediated endothelial cell injury and death in the diabetic retina. Am J Pathol 2001;158:147–152. Joussen AM, Poulaki V, Mitsiades N, Cai WY, Suzuma I, Pak J, Ju ST, Rook SL, Esser P, Mitsiades CS, Kirchhof B, Adamis AP, Aiello LP: Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocininduced diabetes. FASEB J 2003;17:76–78. Joussen AM, Poulaki V, Le ML, Koizumi K, Esser C, Janicki H, Schraermeyer U, Kociok N, Fauser S, Kirchhof B, Kern TS, Adamis AP: A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB J 2004;18:1450–1452. Hirschi KK, Rohovsky SA, D’Amore PA: PDGF, TGF-␤, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol 1998;141:805–814. Orlidge A, D’Amore PA: Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells. J Cell Biol 1997;105:1455–1462. Alt G, Lawrenson JG: Pericytes: cell biology and pathology. Cells Tissues Organs 2001;169:1–11. Hattori K, Heissig B, Wu Y, Dias S, Tejada R, Ferris B, Hicklin DJ, Zhu Z, Bohlen P, Witte L, Hendrikx J, Hackett NR, Crystal RG, Moore MA, Werb Z, Lyden D, Rafii S: Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(⫹) stem cells from bone-marrow microenvironment. Nat Med 2002;8:841–849. Rafii S, Lyden D, Benezra R, Hattori K, Heissig B: Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy? Nat Rev Cancer 2002;2:826–835. Grant MB, May WS, Caballero S, Brown GA, Guthrie SM, Mames RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW: Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 2002;8:607–612. Otani A, Kinder K, Ewalt K, Otero FJ, Schimmel P, Friedlander M: Bone marrow-derived stem cells target retinal astrocytes and can promote or inhibit retinal angiogenesis. Nat Med 2002;8: 1004–1010.


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Antonia M. Joussen Department of Ophthalmology Heinrich-Heine University Duesseldorf Moorenstraße 5 DE–40225 Duesseldorf Tel. ⫹49 0211 81 17321, Fax ⫹49 0211 81 16241, E-Mail [email protected]


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Characterization and Relevance of Different Diabetic Retinopathy Phenotypes José Cunha-Vaz Department of Ophthalmology, University Hospital of Coimbra, Centre of Ophthalmology, Institute of Biomedical Research on Light and Image, Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Abstract The natural history of initial lesions occurring in the diabetic retina has particular relevance for our understanding and management of diabetic retinal disease, one of the major causes of vision loss in the Western world. Diabetic retinal lesions are still reversible at the initial stage of mild nonproliferative diabetic retinopathy, opening real opportunities for effective intervention. Four main alterations characterize the early stages of diabetic retinopathy: microaneurysms/hemorrhages, alteration in the blood-retinal barrier, capillary closure, and alterations in the neuronal and glial cells of the retina. These alterations may be monitored by red dot counting on eye fundus images, by fluorescein methodologies and retinal thickness measurements. A combination of these methods through multimodal macula mapping has contributed to the identification of three different phenotypes, showing different patterns of evolution: pattern A, including eyes with reversible and relatively little abnormal fluorescein leakage, a slow rate of microaneurysm formation and normal foveal avascular zones (FAZ); pattern B, including eyes with persistently high leakage values, high rates of microaneurysm formation and normal FAZ; pattern C, including eyes with variable leakage, high rates of microaneurysm formation and abnormal FAZ. The identification of different phenotypes opens the door for genotype characterization, development of targeted treatments and personalized approaches in management strategy. Copyright © 2007 S. Karger AG, Basel

The natural history of initial lesions occurring in the diabetic retina has particular relevance for our understanding and management of diabetic retinal disease, one of the major causes of vision loss in the Western world. Four main alterations characterize the initial stages of diabetic retinopathy: the appearance of microaneurysms/hemorrhages, alteration in the blood-retinal

barrier (BRB) demonstrated by fluorescein leakage, capillary closure, and alterations in the neuronal and glial cells of the retina. These alterations may be monitored by a variety of methods, including retinal microaneurysm counting on eye fundus images, fluorescein leakage, retinal thickness measurements and psychophysical and electrophysiological testing. A combination of these methods using multimodal imaging has contributed to identifying different phenotypes of diabetic retinopathy. They show different types and rates of progression which suggest the involvement of different susceptibility genes. The identification of different phenotypes has opened the door for genotype characterization, different management strategies and targeted treatments. A new paradigm of diabetic retinopathy management is developing. Diabetic retinopathy must be detected and diagnosed earlier, and treatment must be commenced earlier. The ultimate goal should not be merely to prevent blindness, but to help patients enjoy their lives to their full potential, and to provide a clearer indication of when more active treatment, either systemic or local, or both, is justified. Diabetic retinopathy is a chronic retinal disorder that eventually develops, to some degree, in nearly all patients with diabetes mellitus. Diabetic retinopathy is characterized by gradually progressive alterations in the retinal microvasculature and is the leading cause of new cases of legal blindness among Americans between the ages of 20 and 74 years of age [1]. Diabetic retinopathy occurs in both type 1 (also known as juvenile-onset or insulin-dependent diabetes) and type 2 diabetes (also known as adult-onset or noninsulin-dependent diabetes). All the features of diabetic retinopathy may be found in both types of diabetes, but characteristically, the incidence of the main causes of vision loss, macular edema and retinal neovascularization is quite different for each type of diabetes [1]. Diabetic retinopathy in type 1 diabetes induces vision loss mainly due to the formation of new vessels in the eye fundus and development of proliferative retinopathy, whereas in type 2 diabetes, vision loss is most commonly due to macular edema, and proliferative retinopathy is relatively rare. It is apparent from the data available from a variety of large longitudinal studies that the evolution and progression of diabetic retinopathy vary according to the type of diabetes involved, showing dissimilarities among different patients even when belonging to the same type of diabetes, and that diabetic retinopathy does not necessarily progress in every patient to proliferative retinopathy. There is accumulated evidence indicating that only the nonproliferative stage of diabetic retinopathy (NPDR) is directly due to the systemic disease and associated hyperglycemia and other metabolic alterations. Proliferative

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Fig. 1. Diabetic retinopathy. Fundus photography of the posterior pole showing typical alterations, predominantly microaneurysms and hemorrhages.

retinopathy occurs in diabetic eyes only after the development of widespread ischemia due to capillary closure. Neovessels in the retina are a direct result of retinal ischemia and not influenced by the diabetic metabolic control. Its course and management are not different from other situations in the retina where there is abnormal new vessel formation. Following these concepts closely, we can state that in diabetes, a retinopathy develops that may ultimately result in extensive retinal ischemia. If that occurs, independently of diabetic metabolic control, neovascularization may develop. Proliferative retinopathy is, in fact, a complication of diabetic retinopathy, such as retinal detachment in diabetes is a complication of proliferative retinopathy. Both occur independently of the course of systemic diabetic disease and are not influenced by changes in metabolic control. Therefore, we will attempt to characterize diabetic retinopathy, i.e. the alterations occurring in the retina as a direct result of the systemic diabetic disease.

The Initial Alterations in Diabetic Retinopathy

The fundus abnormalities that are identified on clinical examination of mild to moderate NPDR include microaneurysms and/or hemorrhages, which appear as small red dots on the fundus images, and exudates (fig. 1).

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Fig. 2. Diabetic retinopathy. Fluorescein angiography showing fluorescent dots (microaneurysms) and diffusion of fluorescein around the lesions (fluorescein leakage).

Therefore, the initial stages of NPDR are characterized by the presence of microaneurysms and indirect signs of vascular hyperpermeability and capillary closure, i.e. both hard and soft exudates or cotton wool spots, respectively. It is particularly important to realize that the course and rates of progression of the retinopathy vary between patients. Microaneurysms, for example, may come and go. Once you get a microaneurysm you do not necessarily continue to have that microaneurysm. Microaneurysms may disappear due to vessel closure (fig. 2), which is an indication of worsening of the retinopathy because of progressive vascular closure [2]. Hemorrhages will obviously come and go as the body heals them. Clinical improvement may be apparent, but in reality, may mask the worsening of the disease. The initial pathological changes occurring in the diabetic retina are characteristically located in the small retinal vessels of the posterior pole of the retina, i.e. in the macular area. The structural changes in the small vessels include endothelial cell and pericyte damage and thickening of the basement membrane [3]. Pericyte damage has been reported as one of the earliest findings in diabetic retinal disease since the introduction of retinal digest studies [4]. However, pericyte apoptosis is more readily detectable than endothelial cell apoptosis, most probably because the pericytes are encased in the basement membrane and thus less accessible to clearing mechanisms, whereas apoptotic endothelial cells slough off into the capillary lumen and are cleared by blood flow. The alteration in the Blood-Retinal Barrier (BRB) demonstrated by fluorescein leakage is one of the earliest findings in diabetic retinal disease (fig. 3).

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Fig. 3. Diabetic retinopathy. Fluorescein angiography showing multiple microaneurysms and a few areas of capillary closure.

It appears to directly lead to clinically significant macular edema, which remains the most frequent cause of visual loss in diabetes. Altered autoregulation and progressively decreased retinal blood flow associated with retinal vascular alterations (endothelial cells and pericytes) facilitate the development of progressive capillary closure, a hallmark of progression of diabetic retinal disease. Capillary closure may be identified in the initial stages of NPDR by the presence of occluded capillaries surrounding the foveal vascular zone (FAZ). Finally, capillary closure leads to retinal ischemia, which creates the conditions for the development of the most dreaded complications of proliferative retinopathy. It is now generally accepted that at least three processes can contribute to retinal capillary occlusion and obliteration in diabetes: proinflammatory changes, microthrombosis and apoptosis [5].

Characterization of Retinopathy Phenotypes

It is well recognized that the duration of diabetes and the level of metabolic control are major risk factors for development of diabetic retinopathy. However, these risk factors do not explain the great variability that characterizes the evolution and rate of progression of the retinopathy in different diabetic individuals. There is clearly great individual variation in the presentation and course of diabetic retinopathy. There are many diabetic patients who


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after many years with diabetes never develop sight-threatening retinal changes, maintaining good visual acuity. However, there are also other patients that even after only a few years of diabetes show a retinopathy that progresses rapidly and may not even respond to laser photocoagulation treatment. We have recently performed a prospective 3-year follow-up study of the macular region in 14 patients with type 2 diabetes mellitus and mild nonproliferative retinopathy, using multimodal macula mapping [6]. In a span of 3 years, eyes with minimal changes at the start of the study (levels 20 and 35 of the Early Treatment Diabetic Retinopathy Study-Wisconsin grading) were followed at 6-month intervals in order to monitor progression of the retinal changes. The most frequent alterations observed, by decreasing order of frequency, were leaking sites [7], areas of increased retinal thickness and microaneurysms/ hemorrhages. Leaking sites were a very frequent finding and reached very high BRB permeability values in some eyes. These sites of alteration in the BRB, well identified in leakage maps, maintained, in most cases, the same location on successive examinations, but their BRB permeability values fluctuated greatly between examinations, indicating reversibility of this alteration. Areas of increased retinal thickness were another frequent finding. They were present in every eye at some time during the follow-up and were absent, at baseline, in only 2 of the 14 eyes. This confirms previous observations by our group [6] and by others [8]. The number of microaneurysms and small hemorrhages increased in most eyes during the 3-year follow-up period. This was particularly well demonstrated when the location of each microaneurysm was taken into consideration. This increase in the number of microaneuryms may be the most reliable indicator of retinal vascular damage and remodeling of the retinal circulation, particularly in the initial stages of diabetic retinopathy. Increased rates of microaneurysm accumulation were registered in eyes that had more microaneuryms at baseline and higher values of BRB permeability during the study. In summary, in this study, the rate of microaneurysm formation appears to have the potential to be a good indicator of retinopathy progression. We realized that by combining different imaging techniques, multimodal imaging of the macula made apparent three major patterns occurring during the follow-up period of 3 years. Pattern A included eyes with reversible and relatively little abnormal fluorescein leakage, a slow rate of microaneurysm formation and a normal FAZ (fig. 4a). This group appeared to represent eyes presenting slowly progressing retinal disease. Pattern B included eyes with persistently high leakage values, indicating an important alteration in the BRB, high rates of microaneurysm accumulation and a normal FAZ (fig. 4b).

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100 90

a

80 70 60 50 40

b

30 20 10

c Fig. 4. Multimodal images taken at 0-, 12-, 24- and 36-month visits (left to right) showing for each visit the FAZ (black contour), retinal leakage analyzer results and retinal thickness analyzer results. The retinal leakage analyzer color-coded maps of the BRB permeability indexes are shown; retinal thickness analyzer views show white dot density maps of the percent increases in retinal thickness. Pattern A: note the little amount of retinal leakage over the 4 represented visits and the normal FAZ contour. This patient showed a slow rate of microaneurysm formation. Pattern B: note the high retinal leakage showing a certain degree of reversibility and the normal FAZ contour. This patient showed a high rate of microaneurysm accumulation over the 3-year follow-up period. Pattern C: note the reversible retinal leakage and the development of an abnormal FAZ contour. This patient showed a high rate of microaneurysm formation.

All these features suggest a rapid and progressive form of the disease. This group may identify a ‘wet’ form of diabetic retinopathy. Pattern C included eyes with variable and reversible leakage and an abnormal FAZ (fig. 4c). This group is less well characterized considering the small number of eyes that showed an abnormal FAZ. It may be that abnormalities of the FAZ may occur as a late development of groups A and B or progress rapidly as a specific ‘ischemic’form (table 1). We have now extended our observations by following 57 patients with type 2 diabetes for 7 years; at the time of enrollment, all eyes presented mild NPDR.


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Table 1. Evolution of diabetic retinopathy

Pattern A (62%) Pattern B (20%) Pattern C (18%)

VF/RLA

Red dot formation rate

FA/FAZ

Phenotype

⬍4 ng/ml ⬎4 ng/ml ⬍4 ng/ml

⬍3/year ⬎3/year ⬎3/year

normal normal abnormal

slow progression leaky ischemic

FA ⫽ Fluorescein angiography; RLA ⫽ retinal leakage analyzer; VF ⫽ vitreous fluorometry.

In this larger study, the three different phenotypes were again clearly identified after an initial 2-year follow-up period. The discriminative markers of these phenotypes were: microaneurysm formation rate, measurements of fluorescein leakage, and signs of capillary closure in the capillaries surrounding the FAZ. After an average of 7 years of follow-up, 10 of these 57 eyes had developed clinically significant macular edema with clear indication for photocoagulation treatment. In this series of patients, after the initial 2-year follow-up period, 35 eyes (61% of the total) were identified as showing the characteristics of pattern A, i.e. slow progression, 12 (21%) were classified as presenting pattern B, and the other 10 (18%) had the characteristics of pattern C. Severe macular edema needing laser photocoagulation developed after 7 years of follow-up only in those eyes classified as belonging to patterns B and C. Of the 12 eyes classified as having pattern B, 5 (42%) developed severe macular edema. Similarly, of the 10 eyes identified with pattern C, 5 (50%) developed severe macular edema. None of the eyes classified as belonging to pattern A developed severe macular edema in the 7-year follow-up period. In summary, the slow progression type, pattern A, takes longer than 7 years to develop severe macular edema, one of the main complications of diabetic retinopathy, confirming that this subtype of diabetic retinopathy has a good prognosis. On the other hand, both other types of diabetic retinopathy progression, the leaky type, or pattern B, characterized initially by particularly high levels of leakage, i.e. alteration in the BRB, and the ischemic type, or pattern C, characterized by signs of capillary closure, much more frequently lead to the development of severe macular edema needing photocoagulation, with incidences at 7 years of 42% and 50%, respectively. If diabetic retinopathy is a multifactorial disease – in the sense that different factors or different pathways may predominate in different groups of cases with diabetic retinopathy – then it is crucial that these differences and the possible different phenotypes be identified [9]. The characterization of three

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different phenotypes of diabetic retinopathy, with different progression patterns, opens particularly interesting perspectives to gain more insight into the understanding and management of diabetic retinopathy. Diabetes mellitus is a familial metabolic disorder with strong genetic and environmental etiology. Familial aggregation is more common in type 2 than in type 1 diabetes. Rema et al. [10] reported that familial clustering of diabetic retinopathy was three times higher in siblings of type 2 subjects with diabetic retinopathy. Presence or absence of genetic factors may play a fundamental role in determining specific pathways of vascular disease and, as a consequence, different progression patterns of diabetic retinal disease. It could be that certain polymorphisms make the retinal circulation more susceptible to an early breakdown of the BRB (pattern B) or induce microthrombosis and capillary closure (pattern C). The absence of these specific genetic polymorphisms would lead to the pattern A. It is clear from this study and from previous large studies, such as from the Diabetes Control and Complications Trials group [11] and the UK Prospective Diabetes Study [12], that hyperglycemia plays a determinant role in the progression of retinopathy. It is interesting to note that hemoglobin A1C (HbA1C) levels are also largely genetically determined [13]. An interesting perspective of our observations, analyzed in the light of available literature, depicts diabetic retinopathy as a microvascular complication of diabetes mellitus conditioned in its progression and prognosis by a variety of different genetic polymorphisms, and modulated in its evolution by HbA1C levels, partly genetically determined and partly dependent on individual diabetes management. The interplay of these multiple factors and the duration of this interplay would finally characterize different clinical pictures or phenotypes of diabetic retinopathy. Therefore, the ultimate goal should be the characterization of relationships between genetic factors (represented by distinct genotypes) and their medically significant expression (distinct diabetic retinopathy phenotypes). Our observations of prospective studies on eyes with mild NPDR of patients with type 2 diabetes mellitus suggest three different phenotypes of diabetic retinopathy: a ‘wet’ or ‘leaky’ type, an ‘ischemic’ type, and finally, an apparently more common, slow progression type.

Progression of Retinopathy under Stabilized Metabolic Control

It must be realized that levels of hyperglycemia and duration of diabetes, i.e. exposure to hyperglycemia, influence the evolution and rate of progression classified by our group into three major clinical phenotypes of retinopathy progression.


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To determine the natural history of the initial alterations occurring in the retina in subjects with type 2 diabetes under a situation of stable metabolic control, our group performed a 2-year prospective study on eyes with mild nonproliferative retinopathy under intensive oral tritherapy [14]. In type 2 diabetes, very good levels of metabolic control may be achieved using only oral administration of drugs, in the absence of insulin therapy, by associating a sulphonylurea with a biguanide and an ␣-glucosidase inhibitor. Since this was a condition for inclusion in the study, only patients who accepted well the intensive oral tritherapy remained in the study. In this study, HbA1C levels were stabilized during the entire 2-year study period. Microaneurysm counts on fundus photographs and retinal thickness measurements were determined for each patient at 6-month intervals. The number of microaneurysms increased steadily throughout the 2-year study period in spite of the patients’ stabilized metabolic control, with more microaneurysms counted in the eyes of patients with worse glucose control. Microaneurysm formation rates during the 2-year study period varied widely among different patients. There appeared to be individual microaneurysm formation rates that may be genetically determined and are basically predetermined and independent of medical management, although influenced by metabolic control. It was interesting to note that higher values of microaneurysm formation rates were registered in patients with higher HbA1C levels both throughout the study and at baseline. Finally, retinopathy continued to progress under well-stabilized metabolic control indicating a role for genetic factors, but progression appears to accelerate in the eyes of patients under worse metabolic control.

Candidate Phenotype/Genotype Correlations

It is clear that hyperglycemia occurs in every patient with diabetes mellitus and is a fundamental factor for the development of diabetic complications. Several studies have provided evidence that good diabetes control is important to prevent progression of diabetic retinopathy, but showed that some patients develop a rapidly progressing retinopathy despite good control, while others escape the development of severe retinopathy despite poor control. The onset, intensity and progression of diabetic complications show large interindividual variations [6, 15]. There is, indeed, clear evidence from aggregation in families and specific ethnic groups, together with a lack of serious complications in some diabetic patients with poor metabolic control, that there is a genetic predisposition to develop some diabetic complications such as retinopathy [16].

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It is recognized that polymorphic variability in the genetic make-up of an individual can profoundly influence the expression of a gene and its response to environmental factors. As we predict that the impact of single common mutations on diabetic retinopathy development will be modest (increasing relative risk by 20–40% at most), the main issue of clinical relevance is whether the conferred risk of such a mutation is very much higher in some population subgroups. To be clinically useful in a risk algorithm we might require for any factor to have a relative risk of 2 or greater [17]. Such subgroups might be those carrying a second important mutation in another gene, and such individuals might be identified using conventional genetic strategies. Alternatively, one might identify individuals exposed to a given environment which amplifies the risk associated with that gene (i.e. geneenvironment interaction). Diabetic retinopathy shows familial aggregation and variation in disease severity which is not explained by environmental, biochemical or biological risk factors alone. There are, indeed, substantial variations in onset and severity of retinopathy in different patients which are independent of the duration of diabetes and level of glycemic control. One of the major problems is associated with poor characterization of different retinopathy phenotypes. It is fundamental before embarking on a search for candidate genes to define clinical phenotypes characterized by specific patterns of severity and progression of diabetic retinopathy. It is clear that it is necessary to first and well identify the diabetic retinopathy phenotypes that are associated with rapid progression of retinopathy to severe forms of the disease, such as macular edema and proliferative retinopathy. Only then studies on candidate genes are worth pursuing, involving appropriately well-defined subgroups of patients [16]. The situation of a complex and multifactorial disease such as diabetes favors the presence of gene-environment interactions. A key factor in the identification and study of gene-environment interaction is that an individual carrying such a mutation will develop the phenotype only if and when they enter the high-risk environment. Thus, the mutation will cause a specific retinal vascular alteration, i.e. an alteration in BRB or blood flow changes in the presence of a specific environmental challenge. This classical ‘lack of penetrance’ of a mutation will cause analytical problems and misphenotyping which will be particularly problematic with some sampling analytical designs. This ‘content dependency’ of a mutation (i.e. gene X environment effect) must be taken into consideration when analyzing associations between a candidate gene polymorphism and intermediate phenotypes. Most of the results published indicate the presence of genetic determinants for resistance or susceptibility to vascular complications. However, there is


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evidence of problems in replicating results, suggesting that the studies performed have been plagued with confounding factors. The results of our research group on the characterization of different phenotypes of diabetic retinopathy confirm that there are distinct morphological manifestations in diabetic retinopathy with different subjects presenting different rates of progression and different evolution patterns [6]. There is also evidence indicating that susceptibility to the late vascular complications of diabetes, such as retinopathy, depends, at least partly, on genetic factors [18]. It is clear that future studies should focus on the need to characterize more accurately different phenotypes with respect to retinopathy status. We agree entirely with Wharpea and Chakravarthy [16] when they state that agreed international standards for data collection, particularly agreement on a minimum data set for the phenotyping of retinopathy in subjects with diabetes, would permit the pooling of data from the many studies with enhanced power to detect associations.

Relevance for Clinical Management

It is accepted that in the initial stages of diabetic retinopathy when the fundus alterations detected by ophthalmoscopy or slit-lamp examination are limited to microaneurysms, small hemorrhages and hard or soft exudates, i.e. mild diabetic retinopathy or NPDR, an annual examination is indicated to every patient with 5 or more years of duration of their diabetes. This is the recommendation of the American Academy of Ophthalmology Guidelines for Diabetic Retinopathy [19]. Our observations and the identification of different diabetic retinopathy phenotypes in the initial stages of diabetic retinopathy, i.e. mild or moderate NPDR, characterized by different rates of progression of the retinopathy, suggest that specific approaches should be used when managing these different retinopathy phenotypes. A patient with mild or moderate NPDR, presenting retinopathy phenotype B (wet/leaky), characterized by a marked breakdown of the BRB, identified by increased values of fluorescein leakage and a high microaneurysm formation rate, registered during a period of 1–2 years of follow-up and indicating fast retinopathy progression, should be watched more closely and examined at least at 6-month intervals. Furthermore, blood pressure values and metabolic control should be closely monitored at least at 3-month intervals and medication given to keep HbA1C levels at ⬍7.1%, systolic blood pressure ⬍140 mm Hg and diastolic blood pressure ⬍85 mm Hg. Communication channels should be rapidly established between the ophthalmologist and the diabetologist, internist or general health care provider. Information should be given indicating that the chances of rapid retinopathy progression to more advanced stages of disease are

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in these patients relatively high, calling for immediate tighter control of both glycemia and blood pressure. A patient with mild or moderate NPDR presenting retinopathy phenotype C, the ischemic type, characterized by clear signs of capillary closure and variable microaneurysm formation rates, would similarly indicate the need for observation intervals less than 1 year, with particular attention to other systemic signs of microthrombosis. However, here, control of hyperglycemia and blood pressure must be addressed with some degree of caution. Improved metabolic and blood pressure control must be progressive and less aggressive than with phenotype B. It is realized that the ischemia that characterizes phenotype C may become even more apparent in eyes submitted to rapid changes in metabolic control, and rapidly lowering the blood pressure may increase the retinal damage associated with ischemia. Finally, a patient with mild or moderate NPDR, presenting phenotype A, identified by low levels of fluorescein leakage, no signs of capillary closure and low microaneurysm formation rates (all signs indicating a slowly progression type of diabetic retinopathy), may be followed at intervals longer than 1 year. If the examination performed at 2-year intervals confirms the initial phenotype characterization, the patient and his diabetologist, internist or general health care provider should be informed of the good prognosis associated with this retinopathy phenotype.

Targeted Treatments

It would be of great benefit to have a drug available which would prevent the need for photocoagulation and particularly one which may remove the other variables that remain a cause of concern. Thus, many patients remain poorly controlled and do not come to the doctor regularly, often losing their vision before they get medical attention in time for photocoagulation. The major large clinical trials have shown that tight glycemic control slows the development and progression of diabetic retinopathy. However, the constantly increasing incidence of type 2 diabetes and the evidence that retinal damage begins early on underscore the need for a medical treatment that is targeted to the initial retinal alterations and to specific phenotypes of the retinal diabetic disease. Several key pathways have been shown to be involved in the process of triggering diabetic retinal disease and they may play specific roles in the development of specific retinopathy phenotypes. Four candidates, the polyol pathway, nonenzymatic glucosylation, growth factors and protein kinase C, are considered to play leading roles in the development of diabetic retinal disease. The polyol pathway theory holds that increased glucose metabolism, through the enzyme aldose reductase interferes with sodium-potassium ATPase, damaging the retina [20].


25

The nonenzymatic glycosylation theory holds that the bonding of sugar molecules to other reactive molecules leads to critical retinal alterations and enhancement of processes of oxidative stress to the retina [21]. In the growth factor hypothesis, diabetes-induced damage promotes the liberation of growth factors that appear clearly as the best candidates to explain the developments of proliferative retinopathy. However, the potential role of growth factors in the initial stages and in nonproliferative retinopathy remains highly hypothetical [22]. Finally, many of the metabolic changes associated with hyperglycemiainduced oxidative stress, advanced glycosylation end products of diacylglycerol through the polyol pathway, ultimately activate protein kinase C. In the retina, there is evidence that activation of the ␤-isoform of protein kinase C is associated with retinal vasodilatation, leakage and alterations in retinal blood flow, thus making the ␤-isoform of protein kinase C an obvious target for intervention [23]. A role for inflammation has also been proposed, and inflammation mediators have been suggested to be responsible for the increased fluorescein leakage observed in the initial stages of diabetes by causing alterations in the tight junctions of the retinal vessels [24]. Leukocyte adhesion may play an important role in retinal microthrombosis and capillary closure [25]. It is possible that all these different mechanisms of disease play complementary roles in the progression of diabetic retinal disease. The identification of different retinopathy phenotypes, characterized by different rates of progression and different dominant retinal alterations, may indicate that different disease processes predominate in specific retinopathy phenotypes, probably determined by specific gene mutations. Individuals with a specific gene mutation which makes them more susceptible to the abnormal metabolic environment of diabetes will respond by developing a specific retinopathy phenotype. Identification of well-defined retinopathy phenotypes appears to be an essential step in the quest for a successful treatment of diabetic retinopathy. After the characterization of specific retinopathy phenotypes, the predominant disease mechanisms involved may be identified, and drugs directly targeted at the correction of these disease mechanisms may be used with greater chances of success (fig. 5).

Determining Risk in the Individual Patient: Preventative Ophthalmology

It is clear now that only a subset of patients with diabetes who develop some form of retinopathy is expected to lose functional vision during their lifetime.

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Evolution of diabetic retinopathy Diabetes metabolic dysfunction

Genetic factors promotiong vascular disease

HbA1C Vasodilatation

Endothelial/pericyte dysfunction

+blood pressure

Slow progressive type Pattern A

Capillary thrombi formation

+blood pressure

+blood pressure

Hyperpermeability alteration in BRB

Capillary closure

Wet/leaky type

Ischemic type

Pattern B

Pattern C

Fig. 5. Schematic development of NPDR leading to the three different patterns proposed: patterns A, B and C.

Identification of risk factors for progression to visual loss, precise calculations of the risk of progression to visual impairment in individual patients over given time periods, finally appears to be within attainable reach. This knowledge is crucial to decide which patients to treat, when to initiate treatment and how vigorously. A method of assessing the risk of progression for mild NPDR to severe macular edema and loss of functional vision for individual patients is clearly needed. A successful global risk assessment model that evaluates total disease risk based on the summation of major risk factors has been used for many years in the management of patients with cardiovascular disease. Eventually, a point system was established to facilitate assessment of an individual global risk of progression to an atherosclerotic cardiovascular event. More recently, Weinberg et al. [26] have initiated an attempt to establish estimates for glaucoma risk assessment. Diabetic retinopathy is a microvascular complication of diabetes mellitus that presents to the practitioner at various stages of a continuum that is characterized by accelerated retinal vascular changes involving also the neuronal and glial retinal tissue with eventual development of severe macular edema and/or abnormal retinal or optic disk neovascularization leading to irreversible functional visual loss.


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t

eas

Slow

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Fig. 6. Continuum of the progression of diabetic retinopathy. ERG ⫽ Electroretinography; FA ⫽ fluorescein angiography; RLA ⫽ retinal leakage analyzer.

The initial changes in the retina are often asymptomatic and undetectable with existing diagnostic tests. There is still no complete agreement on criteria for the diagnosis of early damage that predicts visual function loss. This suggests that waiting for overt signs of disease involves accepting some irreversible damage and probable progression. As the disease progresses, severe visual dysfunction and blindness will occur only in a small group of patients. Since many patients may be examined in the early stages of the disease, the goal of treatment must be to arrest delay or limit progression of predisposing retinal vascular damage to significant visual impairment. The continuum of diabetic retinopathy progression may be represented as depicted in figure 6, taking into consideration the three proposed diabetic retinopathy phenotypes: slow progression, wet/leaky and ischemic type. Different individuals with diabetes clearly have different rates of progression, and these must be identified and taken into account. The wealth of available epidemiological data, particularly the studies performed within the context of the Wisconsin Epidemiological Study of Diabetic Retinopathy should be looked at with the aim of determining the risk in the individual patient. Development of a method to assess patients based on summation of all the major risk factors will allow patients who are most likely to benefit from treatment to be identified and quantify the combined effect of the risk factors that practitioners should consider in making treatment decisions. In addition, the risks and benefits of various modalities should be considered in making such treatment decisions [26].

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Consolidation and analysis of cardiovascular risk factors from large patient data sets have led to the development of predictive algorithms that allow physicians to estimate individual patient risk of suffering an atherosclerotic cardiovascular event [27]. Similarly, an algorithm that would allow ophthalmologists to use a patient’s microaneurysm formation rate and other risk factors, such as retinal thickness progression and HbA1C levels, i.e. a so-called ‘risk calculator’, to estimate the risk of visual impairment for a diabetic patient, would certainly facilitate the standardization of treatment and help in determining appropriate treatment for individual patients. As in the cardiovascular model, a calculator would be a valuable adjunct to, and not a substitute for, experience and judgment of a well-trained physician. Finally, it is clear that identifying individual variations in disease progression by characterizing the diabetic retinopathy phenotype of each individual patient and other modulating risk factors such as HbA1C levels may open completely new perspectives for the management of diabetic retinal disease. If the patients with the greatest risk of progression and with the greatest potential to benefit from treatment can be identified by multivariate risk assessment, fewer patients will need to be treated to prevent 1 case of blindness. This is of extreme importance at a time where scarce resources must be focused and concentrated on the few individual cases that need close follow-up and timely treatment. References 1 2 3 4 5

6

7 8

9 10

Aiello LP, Gardner TW, King GL, Blankenship G, Cavallerano JD, Ferris FL 3rd, Kein R: Diabetic retinopathy. Diabetes Care 1998;21:143–156. Cunha-Vaz JG: Perspectives in the treatment of diabetic retinopathy. Diabetes Metab Rev 1992;8:105–116. Cunha-Vaz JG: Pathophysiology of diabetic retinopathy. Br J Ophthalmol 1978;62:351–355. Cogan DG, Kwabara T: Capillary shunts in the pathogenesis of diabetic retinopathy. Diabetes 1963;12:293–300. Gardiner TW, Aiello LP: Pathogenesis of diabetic retinopathy; in Flynn HW Jr, Smiddy WE (eds): Diabetes and Ocular Disease: Past, Present, and Future Therapies. AAO Monogr No 14. San Francisco, The Foundation of the American Academy of Ophthalmology, 2000, pp 1–17. Lobo CL, Bernardes RC, Figueira JP, Faria de Abreu JR, Cunha-Vaz JG. Three-year follow-up of blood-retinal barrier and retinal thickness alterations in patients with type 2 diabetes mellitus and mild nonproliferative diabetic retinopathy. Arch Ophthalmol 2004;122:211–217. Lobo CL, Bernardes RC, Santos FJ, Cunha-Vaz JG: Mapping retinal fluorescein leakage with confocal scanning laser fluorometry of the human vitreous. Arch Ophthalmol 1999;117:631–637. Fritsche P, Van der Heijde R, Suttorp-Schulten MSA, Pollack BC: Retinal thickness analysis (RTA). An objective method to assess and quantify the retinal thickness in healthy controls and diabetics without diabetic retinopathy. Retina 2002;22:768–771. Grange JD: Retinopathie Diabétique. Rapport à la Société Française d’Ophthalmologie. Paris, Masson, 1995. Rema M, Saravanan G, Deepa R, Mohan V: Familial clustering of diabetic retinopathy in South Indian Type 2 diabetic patients. Diabet Med 2002;19:910–916.


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11 12

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15 16 17

18 19 20 21 22

23

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Diabetes Control and Complications Trials Group: Effect of intensive therapy on the microvascular complications of type 1 diabetes mellitus. JAMA 2002;287:2563–2569. Stratton IM, Kohner EM, Aldington SJ, Turner RC, Holman RR, Manley SE, Matthews DR, the UKPDS Group: UKPDS 50: risk factors for incidence and progression of retinopathy in type II diabetes over 6 years from diagnosis. Diabetologia 2001;44:156–163. Snieder H, Sawtell PA, Ross L, Walker J, Spector TD, Leslie RDG: HbA1c levels are genetically determined even in type 1 diabetes. Evidence from healthy and diabetic twins. Diabetes 2001;50: 2858–2863. Ribeiro ML, Seres AI, Carneiro AM, Stur M, Zourdani A, Caillon P, Cunha-Vaz JG and on behalf of the DX-Retinopathy Study Group: Effect of calcium dobesilate on progression of early diabetic retinopathy: a randomised double-blind study. Grafe’s Arch Clin Exp Ophthalmol 2006; DOI 10.1007/s00417-006-0318-2. Rogus JJ, Warram JH, Krolewski AS: Genetic studies of late diabetic complications: the overlooked importance of diabetes duration before complication onset. Diabetes 2002;51:1655–1662. Warpeha KM, Chakravarthy U: Molecular genetics of microvascular disease in diabetic retinopathy. Eye 2003;17:305–311. Humphries SE, Talmud PH, Montgomery H: Gene-environment interaction: lipoprotein lipase and smoking and risk of CAD and the ACE and exercise-induced left ventricular hypertrophy as examples; in Malcom S, Gooship J (eds): Genotype to Phenotype. Oxford, Bios Scientific, 2001, pp 55–72. Wasgenknecht LE, Bowden DW, Carr JJ, Langefeld CD, Freedman BI, Rich SS: Familial aggregation of coronary artery calcium in families with type 2 diabetes. Diabetes 2001;50:861–866. Fong DS, Ferris F: Practical management of diabetic retinopathy. Focal Point 2003;21:1–17. Greene DA, Lattimer SA, Sima AAF: Sorbitol, phosphoinositides, and sodium-potassium-ATPase in the pathogenesis of diabetic complications. N Engl J Med 1987;316:599–606. Brownlee M: Glycation and diabetic complications (Lilly lecture 1993). Diabetes 1994;43:836–841. Clermont AC, Aiello LP, Mori F, Aiello LM, Bursell SE: Vascular endothelial growth factor and severity of nonproliferative diabetic retinopathy mediate retinal hemodynamics in vivo: a potential role for vascular endothelial growth factor in the progression of nonproliferative diabetic retinopathy. Am J Ophthalmol 1997;124:433–436. Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell SE, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL: Amelioration of vascular dysfunctions in diabetic rats by an oral PKC X inhibitor. Science 1996;272:728–731. Antonetti DA, Barber AJ, Khin S, Lieth E, Tarbell JM, Gardner TW, Penn State Retina Research Group: Vascular permeability in experimental diabetes is associated with reduced endothelial occluding content: vascular endothelial growth factor decreases occludin in retinal endothelial cells. Diabetes 1998;47:1953–1959. Adamis AP: Is diabetic retinopathy an inflammatory disease? Br J Ophthalmol 2002;86:363–365. Weinberg RN, Friedman DS, Fechtner RD, et al: Risk assessment in the management of patient with ocular hypertension. Am J Opthalmol 2004;138:458–467. Wilson PWF, D’Agostino RB, Levy D, Belanger AM, Silbershatz H, Kannel WB: Prediction of coronary heart disease using risk factor categories. Circulation 1998;97:1837–1847.

José Cunha-Vaz, MD, PhD AIBILI Azinhaga Santa Comba, Celas PT–3000-548 Coimbra (Portugal) Tel. ⫹351 239480100, Fax ⫹351 239480117, E-Mail [email protected]

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Optical Coherence Tomography Findings in Diabetic Retinopathy Gabriele E. Lang Augenklinik, Universitätsklinikum Ulm, Ulm, Germany

Abstract Ophthalmoscopy, fundus photography and fluorescein angiography are the common tools to diagnose diabetic retinopathy and diabetic macular edema. However, there is an increasing demand for high-resolution imaging of ocular tissues to improve the diagnosis and management of diabetic retinopathy. Optical coherence tomography (OCT) provides important additional information about the retina. It produces reliable, reproducible and objective retinal images especially in diabetic macular edema and provides information about vitreoretinal relationships that can clearly only be detected with OCT. It enhances the ability to exactly diagnose diabetic macular edema, epiretinal membranes, vitreomacular or vitroretinal traction. OCT also brings new insights into morphological changes of the retina in diabetic retinopathy. It demonstrates that macular edema is a complex clinical entity with various morphology. With the OCT, structural changes and quantitative assessment of macular edema have become feasible as determined with retinal thickness and volume. OCT is more sensitive to small changes in retinal thickness than slit-lamp biomicroscopy. Copyright © 2007 S. Karger AG, Basel

There is an increasing demand for high-resolution imaging of ocular tissues in the diagnosis and management of ocular diseases. Especially the diagnosis of retinal disorders has been dramatically improved by the introduction of optical coherence tomography (OCT). The Early Treatment Diabetic Retinopathy Study has defined the stages of diabetic retinopathy and diabetic macular edema on clinical grounds and by stereoscopic fundus photography. Fluorescein angiography provides important information about retinal perfusion, disturbances of the blood-retinal barrier and neovascularization. Recently, a new tool has been developed to gather additional information of the retina. OCT is a modern diagnostic imaging technique to examine living tissue noninvasively by means of high-resolution

Nerve fiber layer Inner plexiform layer Outer plexiform layer Inner photoreceptor layer Outer photoreceptor layer Retinal pigment epithelium Choroid

Fig. 1. OCT of a normal macula. Table 1. OCT potentials in image analysis OCT enables to detect Morphological changes Retinal thickness Retinal volume Surface area OCT allows image analysis Qualitative analysis Quantitative analysis Reflectivity Comparison of images obtained during subsequent examinations Follow the disease course Intervention studies

tomographic cross-sections of the retina. OCT measurements are similar to those of ultrasound B-mode examination. OCT can provide important information complementary to clinical examination and fluorescein angiography for certain findings in diabetic retinopathy. OCT Techniques and Principles

OCT is based on the analysis of the reflections of low coherence radiation from the tissue. The resolution with current clinically used instrumentation is 10 ␮m. It allows images to be obtained for the retinal, retinal pigment epithelial and choriocapillary layers (fig. 1). OCT potentials in image analysis enable to detect morphological changes, quantitative and qualitative analysis (table 1). With OCT qualitative analysis, one can differentiate between hyperreflectivity, hyporeflectivity and shadowing effects (table 2). The possibility to make repeatable, high-resolution measurements of the retina with good image quality is important for the diagnosis, follow-up and

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Table 2. OCT qualitative interpretation Hyperreflective Hard exudates Cotton wool spots Hyporeflective Intraretinal edema Exudative retinal detachment Cystoid macular edema Shadow effect Hemorrhages Exudates Retinal vessels

treatment of diabetic retinopathy. OCT of the posterior pole can be performed through a pupil as small as 3 mm in diameter. Mydriasis, however, makes the OCT examination easier. The OCT software, the latest version of Zeiss Stratus OCT is version 4, offers different scanning protocols. For diagnosis of diabetic maculopathy, fast macular thickness allows quantitative and qualitative analysis for the diagnosis and follow-up of diabetic retinopathy. The scans of the macular thickness mode require more time for the scan acquisition, but provide more detailed information of the six 6-mm-long radial scans. If the fixation is bad, an X-line mode can be chosen, because it can be taken in less than 2 s. For proliferative changes, single linear scans are recommended. While the scans are being taken, the position of the signal in the display window can be adjusted either automatically or manually to optimize the signal strength [1]. The images are displayed directly on the monitor in real time using a false color or gray scale that represents the degree of light reflected from tissues at different depths in the retina. The images are then saved and an analysis protocol can be selected. The software can map the thickness and volume of the macular region, based on six 6-mm-long radial scans. The scans are performed with intersections in the foveolar region. Each scan is rotated by 30⬚ in relation to the preceding one. At each of these locations, the signal is sampled longitudinally at 1,024 equal intervals over a depth of 2 mm. The macular retinal map divides the region into a central disc with a radius of 500 microns and 2 concentric rings divided into 4 quadrants. The normal retina in the macular region has a mean thickness of 200–250 microns, and the physiological foveal depression has a mean thickness of 170 microns. In the false color scale, blue is assigned to thickness between 150 and 210 microns, green to 210–270 microns, yellow to 270–320 microns, orange to 320–350 microns, red to 350–470 microns, and

Optical Coherence Tomography in Diabetic Retinopathy

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Table 3. Retinal thickness measured by OCT [2] Retinal thickness Fovea Normal Borderline Edema

150 ⫾ 20 ␮m 170–210 ␮m ⬎210 ␮m

Central zone (1.0 mm in diameter) Normal Borderline Edema

170 ⫾ 20 ␮m 190–230 ␮m ⬎230 ␮m

Perifoveal and peripheral areas Normal Borderline Edema

230 ⫾ 20 ␮m 250–290 ␮m ⬎290 ␮m

Volume Normal Boderline Abnormal

6.5 ⫾ 1 mm3 up to 8.0 mm3 ⬎8.0 mm3

white to over 470 microns. Panozzo et al. [2] have provided detailed thickness and volume measurements of a normal subject database (table 3). The normal foveal thickness is 150 ⫾ 20 ␮m. Retinal thickness can be classified as normal, borderline and edema [2]. When interpreting the OCT, one should always look at the original scans as well, in addition to the different image processing techniques. The alignment algorithm reduces the artifacts caused by axial movement of the eye during the scan acquisition phase. When using the algorithms, it is very important to take into account that the possibility of interpretation errors exists, because the software cannot always distinguish between the retinal variations resulting from ocular movements and the morphological variations. Consequently, there is always the possibility of misestimation when using the algorithms. The normalization algorithm eliminates the saturation points of the signal, redistributing the acquired values over the entire available range of false colors. This algorithm allows to compare scans with different signal strengths. Gaussian smoothing of the scales allows to evaluate data on a broad scale, at the expense of details. The median smoothing function applies the median value of points in the same 3 ⫻ 3 mm area, thereby eliminating background noise while minimizing the loss of important details. A normal subject database is available for macular retinal thickness [1].

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Table 4. OCT findings typical for macular edema Retinal thickening Cystoid macular edema Loss of foveal depression Detachment of the neurosensory retina Epiretinal membrane Pseudohole formation Vitreomacular and vitreoretinal traction Preretinal neovascularization Retinal thinning Secondary epiretinal membrane

OCT can be difficult or impossible to perform in patients with opacification of the cornea, lens or vitreous and in patients that cannot fixate. The analysis of the OCT has to be done in two steps, qualitative (morphology, reflectivity) and quantitative, and then results in the synthesis leading to the diagnosis together with the clinical and, if necessary, angiographic correlation. In the normal retina, the nerve fibers and retinal pigment epithelium are highly reflective, the plexiform and nuclear layers are medium reflective and the photoreceptors are low reflective (fig. 1). There is no significant difference in foveal thickness concerning age and right and left eye. However, men have a greater thickness than women (central area for men, 178 ⫾ 17 ␮m; central area for women, 165 ⫾ 17 ␮m) [3]. The retina is thinner in the temporal areas in comparison with the nasal, superior and inferior areas because of the arciform bunching of the optic nerve fibers. The information provided by OCT has markedly improved our understanding of diabetic retinopathy. OCT allows to detect macular edema, cystoid maculopathy, hard exudates, intra- and preretinal hemorrhages, cotton wool spots, epiretinal membranes (ERMs), and vitreomacular traction.

OCT Findings in Macular Edema

OCT makes it possible to detect, quantify and classify diabetic macular edema (table 4) and get additional important information to ophthalmoscopy and fluorescein angiography. Macular edema is a common cause of decreased vision in patients with diabetic retinopathy and can occur in any stage of the disease. The pathogenesis of diabetic macular edema is still not fully understood. Cytotoxic macular edema is initiated by intracytoplasmic swelling of Müller


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cells due to ischemia. It may progress to a vasogenic edema with the release of permeability factors such as prostaglandins and vascular endothelial growth factor [4]. The liquefaction necrosis of the Müller cells and adjacent neural cells due to persisting edema and ischemia leads to cystoid cavity formation predominantly in the outer retinal layer. The breakdown of the blood-retinal barrier leads to accumulation of fluid in the retinal cystoid spaces. Some edema may also result from abnormalities in the retinal pigment epithelium, which allows increased fluid from the choriocapillaris to pass through into the sensory retina. Vitreous traction can also play a role in the development of diabetic macular edema in some patients. Edema within 1 disc diameter of the center of the macula is found in about 9% of the diabetic population, 40% of whom have central macular involvement [5]. The proportion of patients with macular edema increases with the severity of overall retinopathy: 3% in mild nonproliferative diabetic retinopathy, 38% in moderate to severe nonproliferative diabetic retinopathy, and 71% in proliferative diabetic retinopathy. Older-onset diabetic patients are more likely to have visual impairment due to macular edema: 50% of older-onset compared with 20% of younger-onset diabetics [6]. Macular edema can be divided into focal and diffuse edema and cystoid maculopathy. In focal edema, OCT scans detect areas of thickened and hyporeflective retina (fig. 2). The edema can be located in the single scans and by retinal mapping and quantified by retinal thickness and volume mode. The map allows to locate the edema with great precision. OCT has been demonstrated to be more sensitive than biomicroscopy in detecting small changes in retinal thickness and morphology, especially in cases of mild cystoid macular edema [7]. Otani et al. [8] suggested three OCT patterns of diffuse diabetic macular edema: sponge-like swelling, cystoid macular edema and serous retinal detachment. In diffuse macular edema, the retina is becoming thicker and less reflective, with numerous small, irregular cavities reminiscent of spongy fabric (fig. 3). When the retina becomes thicker, the foveal depression finally disappears (fig. 4). If retinal edema persists, necrosis of the Müller cells occurs, leading to cystoid cavities in the retina also visible on OCT. The cavities often start in the external plexiform layer (fig. 3b). When cystoid maculopathy progresses, the walls of the pseudocysts disappear forming larger confluent cystoid cavities. Finally, cystoid maculopathy can involve the full thickness of the retina with atrophy of the retinal tissues, showing hyporeflective cavities on OCT (fig. 5). However, diabetic macular edema can also be caused by serous fluid that accumulates under the neurosensory retina leading to a serous detachment of the macula which usually does not show on biomicroscopy and fluorescein angiography [9]. It can be detected by OCT showing a hyporeflective area under the macula elevating the neurosensory retina. Serous foveolar

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a

b Fig. 2. a Nonproliferative diabetic retinopathy with numerous cotton wool spots and clinically significant diabetic macular edema with hard exudates. Arrow indicates scanline. b Diffuse diabetic macular edema with hyporeflective serous detachment of the neurosensory retina (arrows), high reflective hard exudates (arrowhead) in the deeper retinal layers shadowing the posterior layers, and hyperreflective cotton wool spot (asterisk).

retinal detachment is reported in up to 15% of diabetic patients [6]. The visual acuity significantly correlates with central foveal thickness measured by OCT [10]. Patients with fovea-involving macular edema show an overnight increase in retinal thickness of about 20 ␮m accompanied by a reduction in visual acuity


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a

b Fig. 3. a Cystoid diabetic macular edema with hard exudates. Arrow indicates scanline. b OCT shows cystoid diabetic macular edema with hyporeflective cystoid cavities (arrows) and high reflective hard exudates (arrowhead) in the deeper retinal layers shadowing the posterior layers.

being directly related to the nocturnal change in blood pressure, indicating a deficient regulation of retinal capillary filling pressure that promotes edema [11]. Yang et al. [7] found a significant correlation between OCT and fluorescein angiography in clinically significant macular edema. They suggested to categorize clinically significant macular edema into four types: type 1, thickening of the fovea with homogenous optical reflectivity throughout the whole layer of the retina; type 2, thickening of the fovea with markedly decreased optical reflectivity in the outer retinal layer; type 3, thickening of the fovea with subfoveal fluid accumulation and distinct outer border of detached retina,

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a

b

c

d Fig. 4. a Macular edema in diabetic retinopathy. b OCT showing diffuse macular edema with hyporeflective retina, loss of foveal depression and some cystoid cavities. c Early frame of the fluorescein angiography shows microaneurysms, hemorrhages and ischemic maculopathy with enlarged foveal avascular zone. d Late frame shows ischemic and cystoid maculopathy.

a

b Fig. 5. a Cystoid macular edema in diabetic retinopathy. b OCT shows large and small hyporeflective cystoid cavities.


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including type 3A, without vitreofoveal traction, and type 3B, with vitreofoveal traction. They found that the prevalence of OCT type 1 was higher in diabetic macular edema with focal leakage type and in the diffuse type than in the diffuse cystoid leakage type of fluorescein angiography. The prevalence of OCT types 2 and 3A was higher in the diffuse cystoid leakage type than in the focal type or diffuse leakage type. OCT type 1 and the focal leakage type of fluorescein angiography showed the least foveal thickness and the best visual acuity [7]. Types 2 and 3A increased with the existence of retinal vascular hyperpermeability. The external limiting membrane is not impermeable to fluid and albumin. With the disruption of the blood-retinal barrier, the excessive fluid might reach the subretinal space in large amounts, which cannot be removed by retinal pigment epithelium and may result in subvofeal detachment. Foveal detachment may lead to cystoid foveal changes. The proportion of diffuse leakage and diffuse cystoid leakage type of macular edema increases with proliferative diabetic retinopathy in comparison with nonproliferative diabetic retinopathy. This suggests that the large extent of ischemia in the eyes with proliferative diabetic retinopathy releases endogenous growth factors like vascular endothelial growth factor, which results in the breakdown of the blood-retinal barrier, and causes diffuse leakage from damaged capillaries [4]. Significant differences in retinal thickness between patients with diabetic retinopathy without clinically significant macular edema and controls can be detected by OCT, most likely in the superior nasal quadrant [12]. An excellent agreement between OCT and contact lens examination for the absence or presence of foveal edema is found when OCT thickness is normal (ⱕ200 ␮m) or moderately to severely increased (⬎300 ␮m). However, agreement is poor when foveal thickness is mildly increased on OCT (201–300 ␮m) [13]. This suggests that OCT is more sensitive to the detection of mild foveal thickening than slit-lamp biomicroscopy. Lattanzio et al. [5] found that macular thickness was greater in diabetics than in controls and tended to increase with diabetic retinopathy and macular edema severity. OCT is also a sensitive technique for quantifying treatment effects like reduction in macular thickness after laser photocoagulation. The change in macular profile and the internal retinal structure after laser photocoagulation of surgical treatment are well visible with OCT [14]. Diabetic macular edema can be accurately and prospectively measured with OCT [15]. In a multivariate logistic regression model, foveal thickness is a strong and independent predictor of clinically significant macular edema [16] suggesting that foveal thickness ⬎180 ␮m measured by OCT may be useful for the early detection of macular thickening and may be an indicator for a closer follow-up of the patients with diabetes mellitus.

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a

b

c

d Fig. 6. a Retinal thickness of the macular thickness mode of a patient with diabetes mellitus. b Retinal thickness of the fast macular mode of the same patient showing a thickened macula in the parafoveal area (normal thickness, gray area). c Retinal thickness of the macular thickness mode of a patient with cystoid macular edema showing markedly thickened macula and loss of foveal depression. d Retinal thickness of the fast macular mode of the same patient.

OCT allows to quantify retinal thickness in diabetic retinopathy with excellent reproducibility and is able to detect sight-threatening macular edema with great reliability [17]. Retinal thickness can be obtained either by the macular thickness mode or by the fast macular mode, which provides an agematched normal subject database (fig. 6). For fast-scan retinal thickness, measurements are taken at 128 points in each scan, for a total of 78 transverse points, 6 of which intersect at the fovea. Thus, the measurements are more precise at the center than at the periphery of the map. For macular thickness map scan, the retinal thickness measurements are taken at 512 points in each scan by default, but this number can be adjusted to 256 or 128 per scan line.


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b

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Cotton wool spots are ischemic infarctions of the nerve fiber layer. On ophthalmoscopy, they are white and located superficially. On OCT, they appear as hyperreflective, nodular or elongated lesions in the nerve fiber layer, which can cast a shadow on the posterior layers (fig. 2). Hemorrhages can be located pre-, intra- or subretinally. On ophthalmoscopy, they are flame shaped when located in the nerve fiber layer, and they are rounded or irregular when located in the deep retinal layers. They are hyperreflective on OCT and can produce a shadow cone on the posterior layer, especially if they are located preretinally (fig. 8a–e).

OCT Findings in Proliferative Diabetic Retinopathy, Vitreoretinal Traction and ERMs

Proliferative diabetic retinopathy is characterized by either neovascularization on the disc or elsewhere [19]. Preretinal neovascularization can be detected by OCT when a certain amount of fibroglial tissue is present, showing medium reflective preretinal structures shadowing the posterior layers (fig. 8a, c, d). Vitreous hemorrhage, when it is not too severe, shows preretinal high reflective structures shadowing the retinal layers. If vitreous hemorrhage is more severe, no good reflection can be obtained from retinal structures. On clinical grounds, it is often difficult to detect vitreomacular or vitreoretinal traction, when caused by partial vitreous detachment. The posterior hyaloid surface is visible as mid-reflective band inserted in the retina creating traction and resulting in retinal edema. In vitreomacular traction, a thin slightly hyperreflective band is visible adhering to the retina, sometimes at several points to the retinal surface, which is often elevated (fig. 9a, b). OCT can also detect traction-induced retinal detachment, which can occur in proliferative diabetic vitreoretinopathy. OCT accurately reveals the fibrovascular tissue, the points of traction and the detached retina. In patients with diabetic retinopathy, secondary ERMs can develop because of epimacular proliferating fibrocellular tissue, which grows across the inner retinal surface causing a cellophane maculopathy or a macular pucker (fig. 9a, b). It can cause a macular distortion. In the beginning, the ERMs often show a global retinal adherence. ERMs with tractional forces show focal adherence. The ERM is characterized by a slightly hyperreflective band on the inner surface of the retina. The ERMs can also detach from the retinal surface. ERMs can lead to pseudohole formation, loss of foveal depression or cystoid maculopathy. OCT is helpful in monitoring postoperative follow-up after pars plana vitrectomy and membrane peeling (fig. 9c).

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c

b

d a

e Fig. 8. a Proliferative diabetic retinopathy with neovascularization on the disc and elsewhere, vitreous hemorrhage and clinically significant macular edema. b OCT shows macular edema with hyporeflective subretinal edema and hyporeflective cystoid cavities. c Medium reflective preretinal neovascular tuft at the superior vascular arcade shadowing the posterior layers. d Medium reflective epipapillar neovascularization shadowing the posterior layers. e High reflective vitreous hemorrhage completely shadowing the posterior layers.

Conclusion

OCT can provide major contributions to the understanding of diabetic macular edema and diabetic retinopathy. It can help monitor treatment results objectively. This is important because new treatment concepts for diabetic retinopathy are under investigation and might be approved within the near future. New OCT developments are high-speed, high-resolution devices and three-dimensional data.


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a

b

c Fig. 9. a Cystoid diabetic macular edema with epiretinal macular membrane. b OCT shows vitreomacular traction (arrowhead), epiretinal membrane (arrow), hyporeflective cystoid cavities (asterisks), and serous macular detachment before surgery. c OCT of the same patient after pars plana vitrectomy and membrane peeling.

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References 1 2 3

4

5

6 7

8 9

10

11 12

13 14 15

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17 18 19

Brancato R, Lumbroso B: Guide to Optical Coherence Tomography Interpretation. Rome, Innovation-News Communication, 2004. Panozzo G, Parolini B, Gusson E, Mercanti A, Pinackatt S, Bertoldo G, Pignatto S: Diabetic macular edema: an OCT-based classification. Semin Ophthalmol 2004;19:13–20. Massin P, Erignay A, Haouchine B, Mehidi AB, Paques M, Gaudric A: Retinal thickness in healthy and diabetic subjects measured using optical coherence tomography mapping software. Eur J Ophthalmol 2002;12:102–108. Kang SW, Park CY, Ham DI: The correlation between fluorescein angiographic and optical coherence tomographic features in clinically significant diabetic macular edema. Am J Ophthalmol 2004;137:313–322. Lattanzio R, Brancato R, Pierro L, Bandello F, Iaccheri B, Fiore T, Maestranzi G: Macular thickness measured by optical coherence tomography (OCT) in diabetic patients. Eur J Ophthalmol 2002;12:482–487. Bresnick GH: Diabetic macular edema: a review. Ophthalmology 1986;93:989–992. Yang CS, Cheng CY, Lee FL, Hsu WM, Liu JH: Quantitative assessment of retinal thickness in diabetic patients with and without clinically significant macular edema using optical coherence tomography. Acta Ophthalmol Scand 2001;79:266–270. Otani T, Kishi S, Mauyama Y: Patterns of diabetic macular edema with optical coherence tomography. Am J Ophthalmol 1999;127:688–693. Özdek SC, Erdinc MA, Gürelik G, Aydin B: Optical coherence tomography assessment of diabetic macular edema: comparison with fluorescein angiographic and clinical findings. Ophthalmologica 2005;219:86–92. Hee MR, Puliafito CA, Duker JS, Reichel E, Coker JG, Wilkins JR, Schuma JS, Swanson EA, Fujimoto JG: Topography of diabetic macular edema with optical coherence tomography. Opthalmology 1998;105:360–370. Larsen M, Wang M, Sander B: Overnight thickness variation in diabetic macular edema. Invest Ophthalmol Vis Sci 2005;47:2313–2316. Schaudig UH, Glaefke C, Scholz F, Richard G: Optical coherence tomography for retinal thickness measurement in diabetic patients without clinically significant macular edema. Ophthalmic Surg Lasers 2000;31:182–186. Brown JC, Solomon SD, Bressler SB, Schachat AP, Di Bernardo C, Bressler N: Detection of diabetic foveal edema. Arch Ophthalmol 2004;122:330–335. Panozzo G, Gusson E, Parolini B, Mercanti A: Role of OCT in the diagnosis and follow up of diabetic macular edema. Semin Ophthalmol 2003;18:74–81. Strom C, Sander B, Laresen N, Larsen M, Lund-Andersen H: Diabetic macular edema assessed with optical coherence tomography and stereo fundus photography. Invest Ophthalmol Vis Sci 2002;43:241–245. Sanchez-Tocino H, Alvarez-Vidal A, Maldonado MJ, Moreno-Montanes J, Garcia-Layana A: Retinal thickness study with optical coherence tomography in patients with diabetes. Invest Ophthamol Vis Sci 2002;43:1588–1594. Goebel W, Kretzchmar-Gross T: Retinal thickness in diabetic retinopathy. A study using optical coherence tomography (OCT). Retina 2002;22:759–767. Browning DJ, Mc Owen MD, Bowen RM, O Marah TL: Comparison of the clinical diagnosis of diabetic macular edema with diagnosis by optical coherence tomography. Ophthalmology 2004;111: 712–715. Lang GE: Diabetische Retinopathie – Stadieneinteilung und Laserbehandlung. Klin Monatsbl Augenheilkd 2005;222:R1–R18. Prof. Dr. Gabriele E. Lang Universitätsklinikum Ulm, Augenklinik Prittwitzstrasse 43 DE–89075 Ulm (Germany) Tel. ⫹49 731 500 59001, Fax ⫹49 731 500 59002, E-Mail [email protected]


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Laser Treatment of Diabetic Retinopathy Gabriele E. Lang Augenklinik, Universitätsklinikum Ulm, Ulm, Germany

Abstract Laser treatment of diabetic retinopathy is still the gold standard of treatment for focal and diffuse diabetic macular edema and proliferative diabetic retinopathy. When properly treated, the 5-year risk of blindness is reduced by 90% in patients with proliferative diabetic retinopathy and the risk of visual loss from macular edema is reduced by 50%. However, only about 35–50% of patients with diabetes mellitus receive regular eye examinations, which are important for timely diagnosis and proper treatment. The necessary goals are better patient education to improve the control of diabetes and better screening programs to reduce the risk of blindness from diabetic retinopathy. Copyright © 2007 S. Karger AG, Basel

In 1959, photocoagulation for the treatment of diabetic retinopathy was first reported by Meyer-Schwickerath, who used a xenon arc photocoagulator. In the 1960s, laser treatment of diabetic retinopathy was introduced. Diabetic retinopathy is becoming a more prevalent cause of visual problems in the future. The number of diabetics is increasing in industrialized and also in developing countries, reaching a prevalence of up to 7%. Early detection of diabetic retinopathy and adequate treatment are crucial. At present, the gold standards of treatment are still laser photocoagulation and vitrectomy. Pathophysiology and Classification of Diabetic Retinopathy

The classification of diabetic retinopathy is based on intra- and preretinal microvascular changes. Diabetic retinopathy is broadly classified into nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). NPDR is characterized by retinal microvascular changes that are limited to the retina, whereas PDR shows growth of new vessels from the retinal surface into the vitreous space.

Fig. 1. Fluorescein angiography of mild NPDR with hyperfluorescent dots representing microaneurysms.

Retinal capillary microaneurysms are the first definite sign and a hallmark of diabetic retinopathy. They are most common on the posterior pole. Sometimes, they can only be diagnosed and differentiated from punctate hemorrhages with fluorescein angiography (fig. 1). Microaneurysms exhibit bright hyperfluorescent dots in the early frames, whereas hemorrhages block fluorescence. Microaneurysms are 15–60 m in size. Histologically, microaneurysms are hypercellular outpouchings of the capillary wall, subsequent also to a loss of intramural pericytes. Typical for early changes of diabetic retinopathy is the thickening of the basement membrane of retinal capillaries. Hemorrhages can also occur, but they disappear within 3 months so that they are not considered as diabetic retinopathy changes without accompanying microaneurysms [1]. Microaneurysms alone have no clinical significance concerning risk of vision loss or progression of diabetic retinopathy. However, they are associated with an increased risk of cardiovascular complications. Altered vascular permeability results in macular edema and deposits of hard exudates. Vascular permeability of the retinal capillaries can already occur in the early stages of diabetic retinopathy caused by increased expression of vascular endothelial growth factor (VEGF). This leads to extravasation of fluid and plasma constituents. Lipoproteins accumulate most often in the macular area. When the macular edema involves the center, this leads to visual loss. Macular edema can be detected with dilated pupil and slit-lamp biomicroscopy, stereoscopic fundus photography and optical coherence tomography. Fluorescein angiography can identify leaking microaneurysms and leakage from

Laser Treatment of Diabetic Retinopathy

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a

b Fig. 2. a PDR with neovascularization on the disc and elsewhere and vitreous hemorrhage. b Fluorescein angiography of the same patient showing dye leakage from preretinal neovascularizations, ischemic maculopathy and blockage of fluorescence caused by vitreous hemorrhage.

retinal capillaries. Fluorescein leakage alone does not always indicate the presence of macular edema, and in patients with significant macular edema, sometimes only mild leakage is found. Hard exudates, that often accompany macular edema, make it much easier to diagnose diabetic macular edema. They are lipid deposits located in the outer plexiform or Henle layer of the retina. Clinically hard exudates are yellow-white, well-defined, intraretinal deposits. On optical coherence tomography (OCT), they are visible as hyperreflective nodular lesions. The deposit of the hard exudates is associated with the damage of the inner blood-retinal barrier, i.e. the breakdown of the endothelial tight junctions in the capillaries and microaneurysms. The extent of the lipid deposits in the retina is associated with the degree of serum lipid elevation [2]. One serious consequence of diabetic retinopathy is the closure of retinal capillaries leading to areas of nonperfused retina. Findings associated with areas of nonperfusion and thus retinal ischemia are large intraretinal hemorrhages, intraretinal microvascular anomalies (IRMA) and venous beading (VB). The ischemia triggers the production of growth factors like VEGF and insulin-like growth factor 1. Increasing areas of nonperfusion result in PDR with preretinal neovascularization and vitreous hemorrhage (fig. 2). The NPDR is categorized into five levels of severity: very mild, mild, moderate, severe, very severe (table 1). The extent of intraretinal hemorrhages and microaneurysms, IRMA and VB in the 4 midperipheral quadrants (fig. 3, fields 4–7) are the factors that predict the level of NPDR (table 1). One problem of classification is the difficulty in recognizing IRMA and VB on clinical examination. They can be more easily diagnosed on fluorescein angiography.

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Table 1. Classification of severity of diabetic retinopathy Disease severity level

Definition

DR absent

microaneursyms and other characteristics absent

Very mild NPDR

no lesions other than microaneurysms

Mild NPDR

microaneurysms plus venous loops, IRMA or VB Q, retinal hemorrhages, HE, SE

Moderate NPDR

IRMA D/4–5 H/MA M/2–3 VB D/1

Severe NPDR

H/MA S/4–5 VB D/2–3 IRMA M/1

Very severe NPDR

two or more of the features described in severe NPDR

Mild PDR

NVE 1/2 DA in 1 field

Moderate PDR

NVE M/1 (M 1/2 DA in 1 field) NVD 1/4 to 1/3 DA NVE 1/2 DA and VH and PRH

High-risk PDR

NVD 1/4 to 1/3 DA on or within 1 DD of the disc VH or PRH M/1 (M 1 DA) NVE M/1 and VH or PRH

1–7 number of fields (fig. 3); D definitely present; DA disc area; DD disc diameter; DR diabetic retinopathy; HE hard exudates; H/MA hemorrhages and microaneurysms; M moderate; NVD neovascularisation of the disc; NVE neovascularization elsewhere; PRH preretinal hemorrhage; Q questionable; S severe; SE soft exudates; VH vitreous hemorrhage.

A total of 50.2% of eyes with severe NPDR will develop proliferative retinopathy within 1 year, and 14.6% will develop proliferative retinopathy with high-risk characteristics. In eyes with very severe NPDR (severe hemorrhages in 4 quadrants, VB in 2 quadrants, and IRMA in 1 quadrant), the risk of developing high-risk PDR is 45% [3]. PDR is categorized into three levels: mild, moderate and high risk (table 1). Macular edema can be associated with any severity level of diabetic retinopathy. It is the most common cause of visual loss in NPDR. The Early Treatment of Diabetic Retinopathy Study (ETDRS) investigators classified the severity by three characteristics concerning the relation to the center of the macula. The macular edema is defined as clinically significant macular edema if any of the three features described in table 2 is present.


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4

3

x2

5

1

7

Fig. 3. Seven standard fields of the ETDRS classification, with x representing the fovea [3].

Table 2. Classification of diabetic clinically significant macular edema Thickening of the retina at or within 500 m of the center of the macula Hard exudates at or within 500 m of the center of the macula associated with thickening of the adjacent retina A zone or zones of thickening of 1 disc area or larger, any part of which is within 1 disc diameter of the center of the macula

A more simplified international classification of diabetic retinopathy and macular edema was proposed by the Global Diabetic Retinopathy Project Group (tables 3, 4) in order to improve communication between ophthalmologists and primary care physicians [4]. This classification simplifies the ETDRS classification of diabetic retinopathy and diabetic macular edema for clinical use improving ophthalmological screening of diabetic patients and guiding the definition for laser treatment. Epidemiology

The Centers of Disease Control estimate that 18.2 million Americans have diabetes mellitus, of whom 90% have type 2 diabetes [5]. Diagnosed diabetes mellitus is most prevalent in the middle-aged and elderly populations.

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Table 3. Diabetic retinopathy disease severity scale [4] Disease severity level

Findings observable on dilated ophthalmoscopy

No retinopathy Mild NPDR Moderate NPDR Severe NPDR

no abnormalities miroaneurysms only more than just microaneurysms, but less than severe NPDR more than 20 intraretinal hemorrhages in each of 4 quadrants, definite VB in 2 quadrants, prominent IRMA in 1 quadrant and no signs of PDR neovascularization, vitreous/preretinal hemorrhage

PDR

Table 4. Diabetic macular edema disease severity scale [4] Proposed disease severity level

Findings on dilated ophthalmoscopy

Diabetic macular edema apparently absent

no apparent retinal thickening or hard exudates in posterior pole some apparent retinal thickening or hard exudates in posterior pole

Diabetic macular edema apparently present If diabetic macular edema is present, it can be categorized as follows: Mild diabetic macular edema Moderate diabetic macular edema Severe diabetic macular edema

some retinal thickening or hard exudates in posterior pole but distant from the center of the macula retinal thickening or hard exudates approaching the center of the macula but not involving the center retinal thickening or hard exudates involving the center of the macula

About 4.1 million adults over the age of 40 years have diabetic retinopathy [6]. In type 1 diabetic patients, ocular involvement occurs as early as 3–5 years after onset of diabetes mellitus. When the diagnosis of type 2 diabetes is made, up to 15% already have diabetic retinopathy. Occasionally, diabetic retinopathy is the initial sign of type 2 diabetes. The prevalence of any diabetic retinopathy is about 98% after 20 years and about 50% for PDR after 15 years of type 1 diabetes mellitus [7]. About 15% of type 1 diabetic patients have diabetic macular edema after 15 years. In type 2 diabetic patients, 50–80% have diabetic retinopathy after 20 years and 10–30% have PDR. A clinically significant macular edema is found in 25% of type 2 diabetic patients after 15 years [8]. However, the incidence, especially of PDR, seems to be decreasing in recent years due to better glycemic control.


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Risk Factors

In several observational studies, poorer glycemic control is associated with increased severity of diabetic retinopathy. When the Diabetes Control and Complications Trial [9] results were stratified by hemoglobin A1c (HbA1c) levels, there was a 40% reduction in the risk of retinopathy progression for every 10% decrease in HbA1c [2]. Therefore, the recommended levels of HbA1c are below 7%. In the UK Prospective Diabetes Study, a comparison of more intensive blood pressure control versus less intensive blood pressure control in type 2 diabetics demonstrated that better blood pressure control was associated with a decreased risk of retinopathy progression [10]. There might also be a benefit on the progression of diabetic retinopathy of angiotensin-converting enzyme inhibition and blood pressure reduction, even in normotensive persons [11]. The UK Prospective Diabetes Study compared -blockers and angiotensin-converting enzyme inhibitors in tight blood pressure control and found that benefits were present in both treatment groups. Good blood pressure control is considered below 130/80 mm Hg [10]. The Wisconsin Epidemiological Study of Diabetic Retinopathy and ETDRS found elevated serum levels of cholesterol being associated with increased severity of hard exudates [1]. The severity of retinal hard exudates is associated with decreased visual acuity and is a significant risk factor for moderate visual loss [12]. The strongest risk factor for the development of subretinal fibrosis in patients with diabetic macular edema was the presence of severe hard exudates [13]. The Diabetes Control and Complications Trial [14] found that the severity of retinopathy was associated with increasing triglycerides and inversely associated with high-density lipoprotein cholesterol. Elevated triglycerides and lowdensity lipoprotein cholesterol are associated with PDR [15]. These data suggest that lowering elevated serum lipids might reduce the risk of visual loss. Pregnancy may accelerate the progression of diabetic retinopathy [16], even though this seems to be rare. Pregnant diabetic patients with mild or moderate NPDR should be examined every 3 months, and those with severe NPDR every 1–3 months.

Examination of Diabetic Patients

Clinical examination should include best corrected visual acuity and intraocular pressure to rule out glaucoma. Slit-lamp examination is necessary to detect iris neovascularization. Fundus examination should be performed with ophthalmoscopy, a fundus contact lens, or with a 78- or 90-diopter fundus lens at the slit lamp with dilated

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Table 5. Ophthalmological examination in patients with diabetes mellitus Method

Reason for examination

Best corrected visual acuity Motility Pupillary function Intraocular pressure Slit-lamp examination Gonioscopy Dilated fundus examination Fundus photography Fluorescein angiography

to rule out visual loss to rule out ocular muscle paresis to rule out pupillary dysfunction increased risk of glaucoma to rule out iris neovascularization to rule out angle neovascularization staging of diabetic retinopathy and macular edema staging of diabetic retinopathy and macular edema staging of diabetic retinopathy diagnosis of ischemic maculopathy qualitative and quantitative diagnosis of macular edema monitor treatment

Optical coherence tomography

Table 6. Follow-up and laser treatment of diabetic retinopathy Stage of retinopathy

Follow-up (months)

Laser

Fluorescein angiography

No DR or very mild NPDR Mild and moderate NPDR NPDR without CSME NPDR with CSME NPDR with CME Severe and very severe NPDR Mild and moderate PDR PDR with high risk

12 6–12 4–6 3–4 2–4 3–4 2–3 3

no no no yes yes yes yes yes

no no occasionally yes yes occasionally yes occasionally

CME Cystoid macular edema; CSME clinically significant macular edema; DR diabetic retinopathy.

pupils to assess the severity of diabetic retinopathy. Pharmacological mydriasis improves image quality, allows better identification of maculopathy and the grading of diabetic retinopathy stage [17]. The risk of angle-closure glaucoma, that might be caused by mydriasis, is extremely rare, especially if the anterior chamber depth is examined by slit-lamp biomicroscopy prior to pupil dilation [18]. Gonioscopy can rule out iris neovascularization and angle closure (table 5). If indicated, fluorescein angiography and optical coherence tomography should be performed (tables 5, 6). If dense vitreous hemorrhage blocks the view on the fundus, retinal detachment should be ruled out by ultrasonography.


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This clinical evaluation in diabetic patients is essential to make the correct treatment decisions. Follow-up of the patients depends on the stage of retinopathy and macular involvement (table 6).

Treatment Recommendations

In patients with mild and moderate NPDR, the risk of progression to proliferative retinopathy is very low. Therefore, scatter photocoagulation is not recommended in eyes with mild or moderate NPDR, provided that careful followup can be maintained. In this group, the 5-year rate of severe visual loss is 1–3%. Good glycemic and blood pressure control and treatment of dyslipidemia, if present, are recommended [19].

Panretinal Laser Treatment of Diabetic Retinopathy

The laser treatment recommendations for diabetic retinopathy are based on the results of two randomized clinical trials of laser photocoagulation, the Diabetic Retinopathy Study (DRS) [20] and the ETDRS [21]. The results of the DRS showed a 50% reduction in severe visual loss in eyes with severe NPDR or PDR and visual acuity of 20/100 or better that had received photocoagulation compared with eyes that were not treated. The overall risk of severe visual loss with PDR at the 2-year follow-up examination was 6% in the treated eyes compared with 16% in the control group. With the DRS high-risk characteristics, the risk increased to 11% in the treated eyes compared with 26% in the control group. The reports of the DRS identified retinopathy characteristics with a high risk of severe visual loss which are neovascularization on the disc or any neovascularization accompanied by vitreous hemorrhage [1]. Panretinal laser treatment considerably improves the visual prognosis, especially in PDR. Wei et al. [22] found a complete resolution of neovascularization in 67% of eyes, and a partial resolution in 33% which needed additional photocoagulation. Postoperative preretinal and vitreous hemorrhage occurred in 9%. Qian et al. [23] report that panretinal argon laser photocoagulation is effective in 85% of eyes with NPDR and in 77% with PDR. Visual acuity improved in 23% and was unchanged in 61%. Rema et al. [24] studied the outcome of patients with type 2 diabetes and PDR after panretinal laser treatment. Seventy-three percent of patients with visual acuity of 6/9 or better maintained their vision. Of patients with visual acuity 6/12–6/36, 59% maintained the same vision and 20% improved their vision at 1-year follow-up. Of patients with visual acuity 6/60 or less, 70%

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a

b Fig. 4. a PDR with vitreous hemorrhage before laser treatment. b The same eye after panretinal laser treatment.

maintained their vision and 30% improved. On multiple regression analysis, diastolic blood pressure, duration of diabetes, fasting blood glucose and nephropathy were associated with decreased vision after panretinal laser treatment. Scatter laser photocoagulation should be considered in severe and very severe NPDR, especially in patients with poor compliance, proliferative disease in the fellow eye, pending cataract surgery, poor glycemic control, high blood pressure, advanced renal disease and extensive capillary closure [25]. In eyes with severe and very severe NPDR and clinically significant macular edema, the macular edema is treated first and panretinal scatter photocoagulation should be delayed until the macular edema has improved. In mild and moderate PDR, scatter laser photocoagulation should be performed because it reduces the risk of severe visual loss, especially in type 2 diabetes. The clinically significant macular edema and cystoid macular edema should be treated first, before performing panretinal laser treatment. In PDR with high-risk characteristics, extensive scatter laser photocoagulation should be performed immediately because of the high risk of visual loss (fig. 4). In the standard full-scatter panretinal photocoagulation, 1,200–1,600 burns of 500-m spot size on the retina are applied to the retina (table 7; fig. 4). The burns are placed from the vascular arcades to the equator, nasally 500 m apart from the optic disc and temporally 2 disc diameters temporal to the macular center (3,000 m). Treatment has to be extended to within the vascular arcades, if retinal neovascularization is located within this area with a spot size of 200 m when treating within 1,500 m from the center of the foveal avascular zone. Treatment should not be extended to closer than 500 m from the macular center. If a grid laser treatment has been performed for clinically significant macular edema, the panretinal burns should be placed temporally adjacent to the macular burns, if there are retinal edema or areas of nonperfused


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Table 7. Treatment of diabetic retinopathy Diabetic retinopathy

CSME

CME

Severe NPDR

High risk PDR

Mild and moderate PDR

Focal ME Diffuse ME Grid LP Consider panretinal LP Panretinal LP

Focal LP

Grid LP

With VH

Panretinal LP or vitrectomy

CME Cystoid macular edema; CSME clinically significant macular edema; LP laser photocoagulation; ME macular edema; VH vitreous hemorrhage.

retina that carry the risk of later development of neovascularization. The burns should have moderate intensity with a spacing of about 1 burn width apart. In patients with high-risk PDR and iris neovascularization, a number of 2,000 burns is recommended. Panretinal photocoagulation should be completed in two or more sessions within 3–6 weeks. One session should not extend 500–600 burns to avoid side effects that are described below. Further division of panretinal treatment sessions can be considered when clinically significant macular edema is present, because this reduces the risk of visual loss. The order of sessions in which the retina is treated is optional. If there is a risk of vitreous hemorrhage, the inferior quadrants should be treated first. Disc neovascularization and elevated neovascularization elsewhere are treated with scatter photocoagulation in an attempt to get a regression of the new vessels. Flat neovascularization elsewhere is directly treated with confluent laser burns with 200–500 m in size on the retina to close the new vessels. Panretinal scatter photocoagulation is very effective in iris neovascularization (at least 2,000 laser burns), especially if treatment is given prior to the development of neovascular glaucoma. In neovascular glaucoma, panretinal laser treatment should be combined with a cyclodestructive procedure. Preferably, the scatter treatment should be applied first, because after the cyclodestructive procedure, fibrin and cells in the anterior chamber might hinder laser treatment.

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Table 8. Panretinal laser treatment Laser spot size Exposure Intensity Number of burns Placement

Number of sessions Lesions treated directly Follow-up treatment

500 m at retina 0.1–0.2 s mild to moderate 1,200–1,600, in iris neovascularization 2,000 1 burn apart, 2 disc diameters from the fovea out to the equator, retreatment and iris neovascularization to the periphery 2–5 NVE with overlapping burns persistent or recurrent neovascularization

NVE Neovascularization elsewhere.

Panretinal photocoagulation significantly reduces the risk of severe visual loss, but in some patients, vision may worsen in spite of laser treatment. If in patients with disk neovascularization the regression of the neovascular vessels is slow, the risk of vitreous hemorrhage persists. The patients must return for follow-up visits at 3-month intervals for a successful result, since additional laser is needed in at least 30% of patients, because of the insufficient regression of neovascularization in at least one third of the patients. Additional fill-in laser treatment between prior laser scars and photocoagulation of the peripheral retina are required, if the new vessels do not regress sufficiently. The Goldmann three-mirror laser lens gives an upright image and can be used for retinal posterior pole and periphery treatment. For panretinal treatment, wide-angle lenses are usually employed. They provide a wider view and have an invert image. They differ in image magnification, field of view and laser spot magnification factor (table 8). The wider the field, the smaller the image magnification. The panretinal laser treatment can usually be performed under topical anesthesia. If a patient is experiencing more severe pain, the pace of the applications can be lowered, and the ciliary nerves at 3 and 9 o’clock should be avoided or subconjunctival anesthesia can be used. Laser lenses suitable for panretinal laser treatment are wide field lenses with a magnification of 1.4 for the area up to the equator and lenses with a magnification of 1.8 for the retinal periphery. The size of the laser burns in the retina depends on the laser spot size, the magnification of the lens, the power and duration of the applied laser burn, the transparency of the media (cataract requires more energy, pseudophakia less energy), and the pigmentation of the eye. The power of the laser burn is the minimum necessary to obtain a burn of medium, gray-white intensity.


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However, in a prospective randomized controlled clinical trial, Bandello et al. [26] found that light panretinal photocoagulation with a very light effect of the burn on the retina on biomicroscopy had the same efficacy in comparison with classic treatment with white burns in eyes with high-risk PDR. Light coagulation was associated with fewer complications and allowed reduction in number of treatment sessions. The regression of neovascularization was the same in both groups, the total mean session number was 7.4 for the light and 9.9 for the classic treatment group. Therefore, light effect of burns can also be considered for panretinial laser treatment. Zaninetti et al. [27] emphasize that eyes requiring vitrectomy because of vitreous hemorrhage or retinal detachment in PDR after panretinal laser photocoagulation are often a result of incomplete photocoagulation. Therefore, sufficient panretinal laser treatment is mandatory. Strong positive predictors of post-panretinal photocoagulation visual acuity outcome are pretreatment visual acuity and low age. Diabetes type and diabetes duration have no influence on visual outcome [28]. The visual prognosis is inversely related to the number of treatment sessions required. Exploration of symptoms revealed that the most frequently reported symptom due to diabetic retinopathy is blurred vision (55% of patients). First-time laser-treated patients report fewer symptoms than multi-treated patients. The patients’ expectations were basically met; however, the treatment had less of an impact than they had hoped for. Patients would have laser treatment again if they needed [29].

Laser Treatment of Diabetic Macular Edema

Diabetic macular edema may be present at any level of NPDR and PDR. It is caused by either focal or diffuse leakage. As the severity of diabetic retinopathy increases, the proportion of eyes with macular edema increases, ranging from 38% in eyes with moderate to severe NPDR to 71% in eyes with PDR [30]. The diagnosis of macular edema requires stereoscopic examination of the macula at the slit lamp with a fundus lens with dilated pupil. If the diagnosis of diabetic macular edema is made, a fluorescein angiography should be performed to rule out additional ischemic maculopathy. With the OCT, the macular edema can be quantified and posterior hyaloid traction can be detected. Secondary epiretinal membranes can also be associated with diabetic macular edema. Diabetic macular edema is classified into clinically significant and clinically insignificant according to the ETDRS (table 2) or, by the more simplified scale for diabetic macular edema, into mild, moderate and severe diabetic macular edema (table 4).

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a

b Fig. 5. a Clinically significant macular edema with focal area of retinal thickening with hard exudates. b The same eye after focal laser treatment.

In patients with diabetic macular edema, blood glucose and blood pressure control, serum lipids and proteinuria should be checked and treated adequately. Diabetic macular edema has to be differentiated from ischemic maculopathy, which is caused by capillary dropout in the center of the macula leading to an enlargement of the foveal acvascular zone. Isolated ischemic maculopathy is not treated with laser. However, if ischemic maculopathy is associated with clinically significant macular edema, laser treatment is indicated. Laser treatment reduced the risk of vision loss due to diabetic macular edema by 50–70%. About 17% of laser-treated eyes will experience a three-line improvement in visual acuity in 5 years [3]. Ohkoshi [31] found that visual acuity after grid laser photocoagulation in diffuse diabetic macular edema improved more than 0.2 levels in 41% of eyes, as well as in 60% of eyes in which preoperative visual acuity had been less than 0.5. Average visual acuity reached a plateau within 3 months after surgery. After argon laser treatment, Wei et al. [22] found an improvement in visual acuity by at least one line on the visual acuity chart in 35%, no change in 55%, and deterioration in 19% of eyes. Retinal edema and fluorescein leakage were reduced in 89% of eyes, with the rest requiring additional treatment. In focal diabetic macular edema, only the area of retinal thickening is treated (fig. 5). In circinate rings of hard exudates, the leaking microaneurysms that need to be treated are usually located in the center of the ring. Successfully treated leaking microaneurysms change the color to either white or dark red. Spots of 50 m have a higher risk of Bruch’s membrane perforation and should be avoided. Initial treatment must not be applied close to the center of the foveal avascular zone, because it results in central scotomas. Laser burns are not applied within 500 m of the center of the fovea [21]. For the patient, the most disturbing


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Table 9. Laser lenses and spot magnification Lens

Magnification of spot in retina

Mainster Standard/Focal Grid Mainster Wide Field Mainster Ultrafield Volk Area Centralis Volk Trans Equator Volk Quadra Aspheric Volk Superquad Goldmann three-mirror lens

1.05 1.47 1.89 1.0 1.43 1.92 2.0 1.08

Table 10. Treatment of diabetic macular edema Laser spot size Exposure Intensity Area Placement Number of sessions Lesions treated directly Follow-up treatment

100–200 m at retina 0.1–0.2 s mild to moderate focal in focal edema, grid in diffuse edema 1 burn apart 1 microaneurysms, areas of retinal thickening every 3 months if macular edema persists

scars are located at 3 and 9 o’clock. If the clinically macular edema persists after 3–6 months, leaking microaneurysms up to 300 m from the center of the fovea can be treated. Often mild intensity burns are as effective as more intense burns in reaching an improvement or resolution of the macular edema and they also allow more retreatment sessions if necessary. In diabetic macular edema, no confluent burns should be used in the macular area. It is important to retreat the patients in 3-month intervals, in areas where the macular edema persists. Focal macular edema is treated only in the area of leaking microaneurysms and retinal thickening between 500 and 3,000 m from the center of the macula. Individual microaneurysms are treated with a spot size of 50 or preferably 100 m. Minimal power should be used to get a color change (whitening or darkening) of the microaneurysms. Grid laser treatment is recommended for diffuse macular edema and is still the gold standard for treatment of diffuse macular edema. Prompt photocoagulation is indicated in eyes with center involvement of the macula. Light to moderate intensity of 100- to 200-m burns are placed 1 burn apart, producing a grid of equally spaced burns (tables 9–10; fig. 6). The patients should be

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a

b Fig. 6. a Diffuse macular edema with hard exudates in the center of the macula after laser retreatment. b The same patient after complete resorption of the macular edema and hard exudates.

examined at 3-month intervals and considered for additional treatment if clinically significant macular edema persists. Bandello et al. [32] studied light versus classic laser treatment for clinically significant macular edema. In light laser treatment, the energy employed was the lowest capable to produce barely visible burns at the level of the retinal pigment epithelium. It was as effective in decreasing the foveal retinal thickness on OCT and visual improvement or loss. Vitreomacular traction is very difficult to detect on clinical grounds but is easily visible on OCT. If it is combined with clinically significant macular edema, we perform a grid laser treatment first, if possible. Sometimes, there is a spontaneous complete vitreous detachment and resolution of the vitreomacular traction after laser treatment. If vitreomacular traction persists, vitrectomy is performed. Often after vitrectomy, further macular laser treatment is necessary because of persistent clinically significant macular edema. Epiretinal membranes also have to be treated by vitrectomy and membrane peeling, combined with laser treatment. However, they tend to recur after some time. If macular edema is combined with ischemic maculopathy, laser treatment should be performed if dye leakage is found on fluorescein angiography or retinal thickening is present on biomicroscopy or OCT examination. However, laser burns should spare about 500 m of the still perfused capillaries on the boarder to the foveal avascular zone. If the laser burns are placed right at the edge of the avascular foveal area, immediate visual loss often occurs because the capillaries are further compromised, leading to more ischemia. Subthreshold diode micropulse photocoagulation was introduced for the treatment of clinically significant macular edema. Visual acuity was stable or


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improved in 85% of treated eyes, with a mean follow-up of 12.2 months. Macular edema decreased in 96% and resolved in 79% of treated eyes. No adverse laser events occurred. No laser lesions were detectable on ophthalmoscopy or angiography after treatment, and no scarring occurred during the follow-up period. Subthreshold diode micropulse laser photocoagulation minimizes chorioretinal damage in the management of clinically significant macular edema and demonstrates a beneficial effect on visual acuity and edema resolution [33]. In a small study, Patel et al. [34] found no visual benefit for standard pars plana vitrectomy and removal of the posterior hyaloid compared with macular grid photocoagulation alone in eyes that showed persistent clinically significant macular edema despite previous macular photocoagulation. VEGF inhibitors were beneficial in diabetic macular edema in a phase II study. A combination therapy of laser and VEGF inhibitors might result in a better outcome and needs to be further investigated.

Wavelengths

The most commonly used wavelengths are double-frequency Nd:YAG (532 nm) or argon green (514 nm). Krypton red and dye lasers are equally effective, but they may be more painful. Gupta et al. [35] examined the efficacy of various wavelengths in the treatment of clinically significant macular edema. Reduction or elimination of macular edema was found in 93.3% of argon-, 88.5% of krypton-, 92.9% of frequency-doubled Nd:YAG-, and 84.8% of diode laser-treated eyes. Although there was no statistically significant difference between the groups, frequency-doubled Nd:YAG-treated eyes appeared to have the advantage of requiring fewer retreatment sessions.

Rules to Bear in Mind

The therapeutic effect of the laser occurs through absorption of the laser energy in the retinal pigment epithelium. Laser burns should not be directed on vitreous hemorrhage or larger intraretinal hemorrhages because the hemoglobin would absorb the laser energy and the vitreous traction, or nerve fiber damage would result. Hard exudates should not be treated directly either, because they do not absorb the laser energy. Major retinal vessels should not be treated directly because of the risk of vessel rupture or closure; however, this is very rare. Areas with proliferative vitreoretinopathy should not be

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treated directly because of the risk of shrinkage of the membrane, which then causes retinal traction and detachment. Chorioretinal scars should be avoided because the risk of visual field loss and secondary choroidal neovascularization increases. In unsatisfactory laser treatment results, one should always consider undertreatment as reason for failure of improvement in macular edema or proliferative changes. Either the treated area was too small, the number of applied burns not sufficient or there was no adequate retreatment.

Side Effects

The most common side effects of panretinal laser treatment are pain during the treatment, moderate visual loss, restriction of the visual fields and nyctalopia. Visual field loss occurs in 5% of argon laser-treated eyes [36]. Permanent visual loss of two or more lines is experienced in 3% of treated eyes. Other side effects are glare, exudative retinal detachment, ciliochoroidal effusion, elevated intraocular pressure, angle-closure glaucoma and subretinal or epiretinal fibrosis. The risk of ciliochoroidal effusion depends on burn intensity, burn size and number and axial length representing the percentage of the retinal surface area. Some degree of cilioretinal effusion occurs in up to 59–90% of patients, resolving within 2 weeks. Those side effects are less common when scatter treatment is carried out in two or more sessions [37]. Macular edema may exacerbate by panretinal laser treatment. Visual loss can be reduced by treating the macular edema prior to initiating panretinal photocoagulation, avoiding intense panretinal photocoagulation burns and dividing panretinal photocoagulation in several treatment sessions. Rare side effects are damage of the cornea, iris or lens (especially in wide field laser lenses). Transient myopia, accomodative pareses, retinal or choroidal hemorrhages, and uveitis are rare. After panretinal laser treatment, a breakdown of the blood-aqueous barrier is found. More pigmented irides showed a greater breakdown than blue irides [38]. The most common side effects of macular laser treatment are scotomas in laser burns close to the boarder of the foveal avascular zone. Subretinal fibrosis most likely develops in eyes with severe hard exudate deposition. Secondary choroidal neovascularization can develop in laser scars because of the rupture of Bruch’s membrane. The larger laser burns have a lower risk of breaks in Bruch’s membrane and therefore a lower risk of secondary choriodal neovascularization in the area of laser scars. If the patient is not cooperative, a foveal burn might occur if the patient moves the eye during laser exposure. Inadvertent foveal burns can be avoided by the laser surgeon if the center of the macula and the patient’s fixation point


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are clearly identified before the laser treatment is started. Treatment of macular edema can be challenging if the fovea is obscured by edema, exudates or hemorrhages. It must also be taken into consideration that laser scars expand with time. Maeshima et al. [39] found that 90% of the laser scars gradually increase in size. The mean annual expansion rates were 12.7% in the posterior pole and 7% in the midperiphery. The annual expansion rate (16.5%) more than 4 years after treatment was higher than that (8.8%) within 4 years of treatment. Lasers of a longer wavelength contributed to larger areas of chorioretinal atrophy. Delivery of laser energy using small spot sizes, short durations and high power increases the risk of perforation of Bruch’s membrane and choroidal neovascularization. Blue wavelength should not be used because of the damage of photoreceptors.

References 1 2 3 4

5 6

7 8 9

10 11 12

13 14

Chew EY, Ferris FL 3rd: Nonproliferative diabetic retinopathy; in Ryan S (ed): Retina. St Louis, Mosby, 1994, chap 67, pp 125–129. Chew EY, Klein ML, Ferris FL 3rd, et al: Association of elevated serum lipid levels with retinal hard exudate in diabetic retinopathy. Arch Ophthalmol 1996;114:1079–1084. Early Treatment Diabetic Retinopathy Study Research Group: Early photocoagulation for diabetic retinopathy. ETDRS report number 9. Ophthalmology 1991;98:766–785. Wilkinson CP, Ferris FL, Klein RE, Lee PP, Agardh CD, Davis M, Dills D, Kampik A, Pararajasegaram R, Verdaguer JT: Proposed international clinical diabetic retinopathy and diabetic macular edema disease severity scales. Ophthalmology 2003;100:1677–1682. Centers for Disease Control. www.cdc.gov/diabetes/news/docs/dpp.htm. Harris M, Flegal KM, Cowie CC, et al: Prevalence of diabetes, impaired fasting glucose and impaired glucose tolerance in US adults. The Third National Health and Nutrition Examination Survey, 1988–1994. Diabetes Care 1998;21:518–524. Frank R: Etiologic mechanisms in diabetic retinopathy; in Ryan S (ed): Retina, ed 4. St Louis, Mosby, 1994, pp 1241–1270. Lang GE: Diabetische Retinopathie – Stadieneinteilung und Laserbehandlung. Klin Monatsbl Augenheilkd 2005;222:R1–R18. The Diabetes Control and Complications Trial Research Group: The effects of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977–986. UK Prospective Diabetes Study Group: Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. BMJ 1998;317:703–713. Chaturvedi N, Sjolie AK, Stephen JM, et al: Effect of lisinopril on progression of retinopathy in normotensive people with type 1 diabetes. Lancet 1998;351:28–31. Klein BEK, Moss SE, Klein R, et al: The Wisconsin Epidemiologic Study of Diabetic Retinopathy. 13. Relationship of serum cholesterol to retinopathy and hard exudates. Ophthalmology 1991;98: 1261–1265. Fong DS, Segal PP, Myers F, et al: Subretinal fibrosis in diabetic macular edema. ETDRS report number 23. Arch Ophthalmol 1997;115:873–877. Lyons TJ, Jenkins AJ, Zhen D, et al: Diabetic retinopathy and serum lipoprotein subclasses in the DCCT/EDIC cohort. Invest Ophthalmol Vis Sci 2004;45:910–918.

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15 16 17

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27 28 29

30 31 32 33 34

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36 37

Kostraba JN, Klein R, Dorman JS, et al: The epidemiology of diabetes complications study. 4. Correlates of diabetic background and proliferative retinopathy. Am J Epidemiol 1991;133:381–391. The Diabetes Control and Complications Trial Research Group: Effect of pregnancy on the microvascular complications. Diabetes Care 2000;23:1084–1091. Deb-Joardar N, Germain N, Thuret G, Manoli P, Garcin AF, Millot L, Gavet Y, Gain P: Screening for diabetic retinopathy by ophthalmologists and endocrinologists with pupillary dilation and a nonmydriatic digital camera. Am J Ophthalmol 2005;140:814–821. Moss S, Klein R, Klein B: Factors associated with having eye examinations in persons with diabetes. Arch Fam Med 1995;4:529–534. Verdaguer JT: Photocoagulation for diabetic retinopathy; in Boyd S, Agarwal A, Boyd BF (eds): Laser Surgery of the Eye. El Dorado, Highlights of Ophthalmology, 2005, pp 283–295. Diabetic Retinopathy Study Research Group: Indications for photocoagulation treatment of diabetic retinopathy. DRS report No 14. Int Ophthalmol Clin 1987;27:239–253. Early Treatment Diabetic Retinopathy Study Research Group: Treatment techniques and clinical guidelines for photocoagulation of diabetic macular edema. ETDRS report number 2. Ophthalmology 1987;94:761–774. Wei ZY, Hu SX, Tang N, Wu J, Wang J: Effect of argon laser photocoagulation on diabetic retinopathy. Ci Yi Jun Yi Da Xue Xue Bao 2004;24:1313–1315. Qian Z, Zhu L, Zhao C: Observation on clinical effects of panretinal coagulation for diabetic retinopathy. Yan Ke Xue Bao 2002;18:99–101. Rema M, Sujatha P, Pradeepa R: Visual outcomes of pan-retinal photocoagulation in diabetic retinopathy at one-year follow-up and associated risk factors. Indian J Ophthalmol 2005;53: 93–99. Ferris F: Early photocoagulation in patients with either type I or type II diabetes. Trans Am Ophthalmol Soc 1996;94:505–537. Bandello F, Brancato R, Menchini U, Virgili G, Lanzetta P, Ferrari E, Incorvaia C: Light panretinal photocoagulation (LPRP) versus classic panretinal photocoagulation (CPRP) in proliferative diabetic retinopathy. Semin Ophthalmol 2001;16:12–18. Zaninetti M, Petropoulos IK, Pournaras CJ: Proliferative diabetic retinopathy: vitreo-retinal complications are often related to insufficient retinal photocoagulation. J Fr Ophtalmol 2005;28: 381–384. Bek T, Erlandsen M: Visual prognosis after panretinal photocoagulation for proliferative diabetic retinopathy. Acta Ophthalmol Scand 2006;84:16–20. Scanlon PH, Martin ML, Bailey C, Johnson E, Hykin P, Keightley S: Reported symptoms and quality-of-life impacts in patients having laser treatment for sight threatening diabetic retinopathy. Diabet Med 2006;23:60–66. Brenick GH: Diabetic macular edema, a review. Ophthalmology 1986;93:989–997. Ohkoshi K: Visual prognosis and prognostic risk factors after photocoagulation for diffuse diabetic macular edema. Nippon Ganka Gakkai Zasshi 2005;109:210–217. Bandello F, Polito A, Del Borrello M, Zemella N, Isola M: ‘Light’ versus ‘classic’ laser treatment for clinically significant diabetic macular edema. Br J Ophthalmol 2005;89:864–870. Luttrull JK, Musch DC, Mainster MA: Subthreshold diode micropulse photocoagulation for the treatment of clinically significant diabetic macular oedema. Br J Ophthalmol 2005;89:74–80. Patel JI, Hykin PG, Schadt M, Luong V, Bunce C, Fitzke C, Fitzke F, Gregor ZJ: Diabetic macular oedema: pilot randomised trial of pars plana vitrectomy vs macular argon photocoagulation. Eye 2006;20:873–881. Gupta V, Gupta A, Kaur R, Narang S, Dogra MR: Efficacy of various laser wavelengths in the treatment of clinically significant macular edema in diabetics. Ophthalmic Surg Lasers 2001;32: 397–405. Stoltz RA, Brucker AJ: Lasers in diabetes; in Fankhauser F, Kwasniewska S (eds): Lasers in Ophthalmology. The Hague, Karger Publications, 2003, pp 229–240. Liang H, Huamonte F: Reduction of immediate complications after panretinal photocoagulation. Retina 1984;4:166–170.


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Moriarty AP, Spalton DJ, Shilling JS, Ffytche TJ, Bulsara M: Breakdown of the blood-aqueous barrier after argon laser panretinal photocoagulation for proliferative diabetic retinopathy. Ophthalmology 1996;103:833–838. Maeshima K, Utsugi-Sutoh N, Otrani T, Kishi S: Progressive enlargement of scattered photocoagulation scars in diabetic retinopathy. Retina 2004;24:507–511.

Prof. Dr. Gabriele E. Lang Universitätsklinikum Ulm, Augenklinik Prittwitzstrasse 43 DE–89075 Ulm (Germany) Tel. 49 731 500 59001, Fax 49 731 500 59002, E-Mail [email protected]

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Benefits and Limitations in Vitreoretinal Surgery for Proliferative Diabetic Retinopathy and Macular Edema Antonia M. Joussena, Sandra Joeresb a

Department of Ophthalmology, University of Duesseldorf, Duesseldorf, and Department of Vitreoretinal Surgery, Center of Ophthalmology, University of Cologne, Cologne, Germany b

Abstract Surgical therapy for diabetic retinopathy has been refined since the 1960s (Early Treatment Diabetic Retinopathy Study). While the Early Treatment Diabetic Retinopathy Study abstained from panretinal photocoagulation at the time of surgery, today, endophotocoagulation is the most important singular reason for vitrectomy, e.g., in vitreous hemorrhage. Despite improved techniques, the surgical prognosis is lagging behind patient expectations, especially in cases of advanced proliferative stages. The following review addresses current surgical options and indications of diabetic retinopathy/maculopathy. Copyright © 2007 S. Karger AG, Basel

The advent of pars plana vitrectomy by Robert Machemer [1995] considerably improved the prognosis of advanced stages of diabetic retinopathy. Original indications for pars plana vitrectomy in diabetic retinopathy include: (1) persistent vitreous hemorrhage, (2) tractive detachment of the macula, (3) combined tractional and rhegmatogenous retinal detachment, and (4) progressive fibrovascular proliferation despite panretinal photocoagulation [Ho et al., 1992]. Photocoagulation is the only lasting treatment so far. The rationale of photocoagulation is to remedy retinal ischemia and thereby eliminate growth factors that would otherwise cause new vessel formation and blood-ocular barrier breakdown. In certain eyes, vitrectomy is a prerequisite of retinal photocoagulation. Relative indications for vitrectomy comprise: • persistent retrohyaloidal hemorrhage leading to massive fibrosis at the vitreoretinal interface [Smiddy and Flynn, 1999];

Table 1. Indications for vitrectomy in diabetic retinopathy Ischemia and complications Active proliferative retinopathy and consequences Neovascularization of the anterior segment in connection with secondary glaucoma Media opacities Persistent vitreous opacities Persistent subhyaloidal fibrosis Neovascularization of the anterior segment in connection with vitreous opacities Vitreous hemorrhage (e.g., postoperatively) in combination with ‘ghost cell glaucoma’ Traction-related complications Progressive fibrovascular proliferation Tractive macular detachment Combined tractional rhegmatogenous detachment Macular edema with a ‘taut hyaloid’ Nonvascularized epiretinal membranes (e.g., postoperatively)

•

tractional retinal detachment outside the macula, which may remain stable without progression [Smiddy and Flynn, 1999]; exceptions are cases presenting with newly formed active neovascularization, recurrent vitreous bleeding, or progression towards the macula area; • neovascular glaucoma [Bartz-Schmidt et al., 1999; Joussen et al., 2003]. It is agreed that vitreoretinal traction on the macula is best addressed by vitrectomy and membrane peeling, although surgery is unable to heal altered original retinal vessels. However, it may lower the diffusional barrier between the retinal compartment and the vitreous cavity. Vitrectomy alone seems to help with the resolution of exudative macular edema [Lewis et al., 1992; Pendergast et al., 2000; Lewis, 2001; Yamamoto et al., 2001]. A more complete clearing of potential diffusion barriers is achieved by induction of a posterior vitreous detachment and by removal of the inner limiting membrane (ILM) from the posterior pole [Gandorfer et al., 2000; Radetzky et al., 2004; Rosenblatt et al., 2005]. In conclusion, indications for surgery in diabetic eyes can be attributed to three areas: ischemia, media opacities and tractional forces (table 1). This review aims to critically discuss indications and expected results of surgical approaches to diabetic retinopathy and maculopathy.

Surgical Techniques

The 3-port access is standard for vitrectomy in diabetes. Besides the growing miniaturization of intraocular surgery tools, major advances of the past

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years include intraoperative heavy liquids [Chang, 1987; Imamura et al., 2003] and wide-field lenses, which together allow for a more safe, effective and atraumatic intraocular manipulation [Bartz-Schmidt et al., 1996]. In proliferative diabetic retinopathy (PDR), posterior vitreous separation is best achieved or already exists in the midperiphery of the fundus, especially in eyes previously treated with panretinal photocoagulation. Posterior vitreous separation outside the posterior pole facilitates finding the cleavage plane between the retina and epiretinal membranes. This is important when addressing adhesions and fibrovascular proliferations at the vascular arcades. Several techniques have been proposed to remove diabetic tractional membranes: segmentation technique, delamination and en-block resection of the epiretinal membranes [Smiddy and Flynn, 1999]. Most surgeons prefer a combination of the above. A bimanual technique, e.g., using illuminated infusion lines or illuminated scissors and forceps, allows to lift up a membrane with one hand, indicating its connections to the retina, and with the other hand to dissect those connections in a save and atraumatic way. The bimanual technique is especially useful for difficult preparations, e.g., widely adherent peripherally located membranes. Wide-angle systems improve visualization of the retinal periphery. A combined line for laser and illumination facilitates laser application to the peripheral retina, especially in phakic eyes, where the other hand is free for scleral indentation. Silicone oil is also being looked at as a surgical tool. It does not mix with blood and has been addressed as ‘styptic’ [Kroll et al., 1989], because eventual postoperative rebleeding remains localized. Therefore, visual rehabilitation may be accelerated by a silicone tamponade in eyes with active neovascularization. Rapid regression of rubeosis iridis in silicone-filled eyes compared with gas or aqueous tamponades initiated the hypothesis of a ‘compartmental’ effect of silicone [Hoerauf et al., 1995; Bartz-Schmidt et al., 1999]. The diffusion of the retina-based growth factor, namely vascular endothelial growth factor, to the anterior segment of the eye is supposedly compromised. If the detachment of the posterior vitreous remains incomplete, Peyman et al. [2000] suggest intraoperative visualization of the vitreous patches using triamcinolone acetonide [Kimura et al., 2004]. This technique may benefit from an additional vasoprotective effect of the steroid; however, it has not gained broad acceptance until now. The intermediate goals of vitrectomy during the intraoperative time course have been reviewed by Smiddy and Flynn [1999]: (1) removal of media opacities, (2) release of anterior-posterior traction, (3) release of tangential traction, (4) segmentation and preparation of epiretinal membranes, (5) support for hemostasis, (6) appropriate tamponade of retinal holes, and (7) photocoagulation.

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a

b Fig. 1. Effects of posterior vitreous detachment on diabetic retinopathy. a Posterior vitreous detachment without complications in a healthy individual. b Posterior vitreous detachment in a diabetic patient. Strands between the hyaloid and retina (e.g., neovascularization or fibrovascular complexes) are torn with the consequence of subhyaloidal or intravitreal hemorrhage.

Nevertheless, with respect to long-term prognosis, panretinal photocoagulation is the most important aim in vitreous surgery for PDR and should be listed first.

Early Vitrectomy for PDR and Vitreous Hemorrhage

One of the most frequent reasons for vitrectomy is persistent vitreous and preretinal hemorrhage. Vitreous hemorrhage is most likely a consequence of ruptured neovascularization at the vitreoretinal interface secondary to a (usually partial) posterior vitreous detachment (fig. 1). Vitreous hemorrhage was a major risk factor for severe visual loss in the Early Treatment Diabetic Retinopathy Study (ETDRS) [Flynn et al., 1992; Fong et al., 1999]. The Diabetic Retinopathy Vitrectomy Study (DRVS) demonstrated the efficacy of vitrectomy in a randomized, prospective study. Six hundred and sixteen eyes with severe diabetic vitreous hemorrhage (visual acuity ⬍1/50) were randomized to either immediate vitrectomy or a postponed treatment after 1 year. At the 2-year follow-up visit, 25% of the patients receiving early vitrectomy demonstrated a visual acuity of 10/20 or better in comparison with only 15% of the late vitrectomy group (p ⬍ 0.01). Surprisingly, there was a more obvious benefit in patients with type 1 compared with type 2 diabetes [DRVS Research Group, 1985a, b]. Later investigations which included an intraoperative panretinal photocoagulation confirmed this trend [Chaudhry et al., 1995].

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A retrospective analysis of eyes with vitreous hemorrhage demonstrated a final visual acuity of 20/60 in one third of the patients treated [Helbig et al., 1998a]. Accordingly, early vitrectomy should be considered in eyes with vitreous hemorrhage, precluding laser application, not resolving within 4–8 weeks. The general aim of the treatment is an early adequate panretinal photocoagulation (including the outer retinal periphery). As described above, it is advantageous to perform the panretinal photocoagulation intraoperatively using an illuminated laser probe and scleral indentation to complete the treatment of the peripheral retina. When is vitrectomy urgent? In cases of dense hemorrhage, a preoperative ultrasound examination (B-scan) is advisable to assess the macula. If retinal detachment is about to extend the macula, or if the macula detached only recently, then vitrectomy should be performed in due course. Early vitrectomy is further advisable in eyes lacking previous panretinal photocoagulation. A severe progressive proliferation of the fellow eye is another reason to perform vitrectomy instantly [DRVS Research Group, 1990; Smiddy et al., 1995]. In these conditions, surgical treatment of vitreous hemorrhage in fellow eyes may help to prevent progression to a tractional retinal detachment. In any case of associated anterior segment neovascularization (either rubeosis iridis or manifest neovascular glaucoma), vitreous hemorrhage is an indication for early surgical intervention. Only instant vitrectomy and complete panretinal photocoagulation are able to inhibit progression of the neovascular process and to prevent occlusion of the chamber angle [Ho et al., 1992]. Subhyaloidal hemorrhage develops if neovascular bridges tear between the retina and the posterior hyaloid surface. Spontaneous reabsorption can be postponed in most cases; however, with long-standing detachment, the subhyaloidal hemorrhage can serve as a scaffold for fibrovascular proliferation between ILM and the posterior vitreous surface. In these cases, vitrectomy is advisable. Nd:YAG laser rupture of the posterior hyaloid to allow draining and eventually resolution of the blood to and in the vitreous cavity should not be attempted. Incalculable pressure waves may damage the macula.

Early Vitrectomy for PDR with Vitreomacular Traction

Early vitrectomy should be discussed in eyes with fibrovascular proliferation as well as in those demonstrating moderately advanced neovascularization. The DRVS included 370 eyes with advanced active PDR, without vitreous hemorrhage and without macular detachment, but with traction on the macula and a visual acuity of 10/200 or more, which were randomized to either early vitrectomy or conventional treatment (photocoagulation only). Within 4 years,


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visual acuity of 10/20 or better was achieved in 44% of eyes after early vitrectomy and in 28% of the eyes within the conventional treatment group (p ⬍ 0.05). The proportion of patients with an insufficient visual outcome was equally distributed among both groups. The more advanced the neovascularization, the more advantageous early vitrectomy [DRVS Research Group, 1988a, b; Ho et al., 1992]. Thorough examination of the fellow eye is advisable in these cases, even if visual acuity is not affected yet. The decision for early vitrectomy in eyes with active PDR but no macular traction, no hemorrhage and therefore good visual acuity is controversial. As indicated earlier, the DRVS did not include photocoagulation during vitrectomy. Today, we demand a photocoagulation as complete as possible prior to and during vitrectomy, since panretinal photocoagulation is the only means to inhibit neovascularization and since posterior vitreous separation is facilitated or induced. Therefore, completion of panretinal photocoagulation is the treatment of choice, even though vitrectomy techniques are less traumatic today compared with those at the time of the DRVS.

Vitrectomy in PDR Refractive to Panretinal Laser Coagulation

In rare instances, neovascularization does not regress despite a supposedly complete panretinal coagulation. • If there is any doubt about a sufficient photocoagulation, it is advisable to further intensify the treatment and increase the number of laser spots per area. • If it is certain that panretinal photocoagulation is complete, then vitrectomy plus silicone oil tamponade aims at regression/prevention of rubeosis and ciliary body neovascularization, and at remedy of preretinal neovascularization. Preretinal neovascularization requires the presence of a hyaloid (fig. 2) [Wong et al., 1989].

Pars Plana Vitrectomy for Tractional Detachment

Despite the fact that pars plana vitrectomy for vitreous hemorrhage without retinal detachment provides considerable visual improvement in the patient, the functional results in cases of complicated tractional detachment are disappointing although a good anatomical result is achieved. Helbig et al. [1996, 1998a, b, 2002] report an overall intraoperative reattachment rate of 86% with persistent reattachment of 82% within 6 months postoperatively. In contrast, in cases of complete tractional detachment, a complete reattachment was only achieved in 9 out of 16 cases (56%), which is in

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a

b Fig. 2. a Right eye of a 52-year-old type 2 diabetic patient. Fibrovascular proliferation at the optic disc despite panretinal photocoagulation. Because of recurrent hemorrhages and persistent proliferation, the patient was scheduled for pars plana vitrectomy in order to induce a posterior vitreous detachment and remove the scaffold for proliferation. b The same eye 14 years later. The proliferation has settled and there is focal macula edema.

Table 2. Success rates of surgical intervention (final visual outcome ⬎20/400) Study

Macula attached (e.g., vitreous hemorrhage)

Tractional macular detachment

Remarks

Helbig et al., 1996

94%

52%

Blankenship, 1972

65%

32%

85%, if macular detachment ⬍6 months and lack of rubeosis 42%, if visual deterioration 0–2 months; 20%, if visual deterioration ⬎13 months

Thompson, 1986 Krampitz-Glas, 1986

79% 71%

64% 38%

accordance with recent reports [La Heij et al., 2004]. In general, long-standing detachments including the macula signal a bad visual prognosis. Helbig et al. [1996] demonstrate a 13-fold higher risk of unfavorable outcome (visual acuity ⬍20/400) in cases with preexisting macular detachment. This confirms previous results (table 2) and supports the concept of ‘early vitrectomy’. The improved results of more recent reports are likely to be attributable to a better visualization of the periphery, intraoperative photocoagulation and vitreous tamponade. Besides macular detachment, risk factors for a reduced final outcome are preoperative rubeosis iridis and subsequent neovascular glaucoma [Oldendoerp


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and Spitznas, 1989]. Along the same lines, Helbig et al. [1996] describe preexisting secondary glaucoma as a major risk factor for an unfavorable outcome. In eyes with tractive macular detachment and a preoperative visual acuity of hand movement or less, only 87% of the cases reached a postoperative visual acuity of ⬎20/400. On the other hand, eyes with rubeosis iridis and complete retinal detachment with a duration of more than 6 months have a chance of only 2% for a final visual acuity of ⬎20/400. Further, ischemic alterations in the macular as well as vitreopapillary traction damaging the anterior optic nerve and resulting in ischemic optic neuropathy are considered at risk of a poor outcome [La Heij et al., 2004]. Similarly, eyes with a preoperative vitreous hemorrhage as well as those lacking preoperative photocoagulation maintain a limited prognosis [Rice et al., 1983b]. Should those eyes be treated at all? In our opinion, vitrectomy is recommended in eyes with active neovascularization and long-standing macular detachment to preserve the eye. Treatment should be performed according to the guidelines for antineovascular therapy (see below) and aims to prevent phthisis or secondary glaucoma. Different considerations apply to tractional retinal detachments outside the macula. In these cases, the visual prognosis is far better. A defined peripheral detachment without active proliferation may be observed without surgical intervention. The risk of a severe visual loss in macular-sparing tractional detachment is reported to be 14% per year [Charles and Flinn, 1979; DRVS Research Group, 1993]. Nevertheless, these cases require a close follow-up to prevent progression of the ischemic disease including rubeosis and related negative consequences.

Combined PDR and Proliferative Vitreoretinopathy Tractional Retinal Detachment

Iatrogenic retinal holes or retinal tears complicating tractional diabetic membranes herald a severe risk of additional proliferative vitreoretinopathy type of vitreoretinal traction. We then expect inferior star folds independent of the retinal vasculature in addition to preexisting tractional membranes associated with retinal vessels. The prognosis will be guarded despite silicone oil tamponade and an additional encircling band. The band is meant to release anterior-posterior traction at the vitreous base and to support silicone-retinal contact in the inferior circumference [D. Wong, pers. commun.; Wetterquist et al., 2004]. A retrospective analysis of 215 consecutive patients with PDR operated on in Cologne demonstrated a rate of 4.1% encircling bands. This is comparable with the study results (3%) of Helbig et al. [1996]. However, in our

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study, the rate of encircling band procedures rose to one third (31.1%), considering only patients with tractional detachment.

Vitreoretinal Surgery in Neovascular Glaucoma

Neovascular glaucoma is the most important reason for enucleation [Naumann, 1996]. We encounter this problem at two levels of priority: firstly, clear optical media, rubeosis, chamber angle more or less closed, and intraocular pressure normal or elevated. At this stage, panretinal photocoagulation can bring the intraocular pressure to normal or prevent a raise [Tasman et al., 1980] in at least 5%. Secondly, opaque optical media (vitreous hemorrhage, cataract, corneal edema) prevent transpupillary laser coagulation of the retina. Controlled coagulation of the retina can only be enforced by eventual removal of the vitreous and/or lens. Regression of rubeosis iridis has been reported following vitrectomy and endophotocoagulation [Helbig et al., 1998b]. ‘Blind’ cryocoagulation of the peripheral retina instead of vitrectomy and endolaser has been advocated. However, this approach bears the risk of overtreatment and stimulation of chorioretinal neovascularization [Kirchhof, 1994]. Simultaneously, ‘blind’ cryocoagulation also bears the risk of undertreatment, since the vitreous cavity may not clear up early enough, and vitreoretinal traction may progress before cryocoagulation is effective and the concentration of growth factors in the vitreous subsides. Silicone oil tamponade besides compartmentalization (regression of rubeosis, see above) prevents recurrent vitreous hemorrhages [Bartz-Schmidt et al., 1999; Psichias et al., 1999] and supports rehabilitation of the patient. At the beginning of vitrectomy, a near normal intraocular pressure should be achieved, e.g., by paracentesis, to avoid hemorrhagic choroidal detachment during surgery. All procedures with extensive disseminated photocoagulation bear a high risk of choroidal swelling and pressure rise, which can be aggravated by postoperative bleeding. Additional endophotocoagulation of the ciliary processes can temporarily reduce intraocular pressure and improve retinal perfusion. Endocyclophotocoagulation should cover 75% of the ciliary processes. This treatment is best performed using scleral indentation and illumination by blue-green laser spot (approximately 100 mW with long-duration pulses until there is a whitish color change and shrinkage of the ciliary processes). Lasting regulation of the intraocular pressure can be achieved even in eyes with refractive neovascular glaucoma by additional partial retinectomy. In a middle-aged person, normal flow conductivity is reinstalled by a 3-clock-hour retinectomy, extending


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anterior-posteriorly between the ora serrata and half the distance to the retinal vascular arcade [Kirchhof, 1994, 1999; Joussen et al., 2003]. Since the retinal barrier against the transition of water cannot be recovered, the effect of retinectomy lasts. Randomized studies investigating this approach to drainage procedures are currently under way. Lensectomy is considered to be a risk factor for the development of a postoperative rubeosis in eyes with proliferative retinopathy. This raises the question whether lens or intraocular lens (IOL) removal is advisable in eyes with neovascular glaucoma. The incidence of neovascularization of the iris after vitrectomy is reported to be 8–26% in phakic, but 31–55% in aphakic eyes [Oldendoerp and Spitznas, 1989; Helbig et al., 1998a, b]. Nevertheless, lensectomy in combination with vitrectomy offers the advantage of a more effective and complete anterior vitrectomy and peripheral photocoagulation, which in turn helps to reduce the risk of the development of iris neovascularization [Bartz-Schmidt et al., 1999]. We value the original lens more than an artificial implant as a barrier against the diffusion of growth factors from the retina to the iris and leave the lens as long as the view to the retina is sufficient.

Vitreoretinal Surgery of Diabetic Maculopathy

A relatively new indication for vitrectomy is persistent macular edema. Diabetic macular edema is a consequence of blood-retinal barrier breakdown. Photocoagulation is limited to patients with focal edema and selected cases with diffuse maculopathy. Up to now, for the ischemic form of diffuse macular edema there is no promising approach, as for subretinal fibrosis secondary to long-standing macular edema [Fong et al., 1997]. Since 1996, surgical intervention for macular edema has been more frequently reported. The observation that a posterior vitreous detachment is less frequently found in patients with diffuse diabetic macular edema led to the assumption that a posterior vitreous detachment could be therapeutically efficient [Nasrallah et al., 1989, 1998; Lewis, 2001]. Hikichi et al. [1997] report on resorption of macular edema in 55% of the patients after posterior vitreous detachment compared with 25% with an attached posterior vitreous. Since then, multiple authors have demonstrated that vitrectomy including removal of the posterior vitreous results in a reduction in macular edema and potential improvement in visual acuity [Lewis et al., 1992; Harbour et al., 1996; Gandorfer et al., 2000]. Pendergast et al. [2000] demonstrated improvement in 2 lines in 27 out of 55 eyes (49.1%). In a total of 52 out of 55 vitrectomized eyes (94.5%), improvement in macular edema was achieved. In 45 eyes (81.8%), a

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a

b Fig. 3. Demonstrates a 34-year-old type 1 diabetic patient, which presented with a best corrected visual acuity of 20/200 (a). While scheduled for surgery, the patient developed a spontaneous posterior vitreous detachment after photocoagulation, and visual acuity improved to 20/30 (b).

complete resolution of the edema was demonstrated after 4.5 months. As expected, the results with ischemic maculopathy are considerably worse. Figure 3 demonstrates a young type 1 diabetic patient, who presented to the clinic with a best corrected visual acuity of 20/200. Scheduled for surgery, she developed a spontaneous posterior vitreous detachment after photocoagulation, and visual acuity improved to 20/30. However, there are also reports conflicting with the surgical approach to macular edema. Yamamoto et al. [2001] measured retinal thickness using optical coherence tomography in a series of 30 patients and demonstrated that vitrectomy is able to reduce macular edema; however, this effect is independent of preexisting posterior vitreous detachment. This leads to the assumption that macular edema, at least in part, is kept by cytokines and growth factors.


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Thus, even though in eyes with persistent diffuse diabetic macular edema with a taut premacular posterior hyaloid face unresponsive to laser therapy, vitrectomy with removal of the posterior hyaloid appears to be beneficial [Pendergast et al., 2000]; a careful selection of eyes with favorable preoperative clinical characteristics may be necessary to improve surgical outcomes [Harbour et al., 1996; Micelli Ferrari et al., 1999; Ikeda et al., 2000]. Up to date, there are no randomized multicenter trials which determine the effectiveness of vitrectomy in diabetic macular edema. The peeling of the ILM has been shown to be beneficial in patients with a macular hole by releasing tangential tractional forces [Haritoglou et al., 2002]. The ILM, a pseudomembrane built by the endplates of Müller cells, is thought to act as a diffusion barrier between the retina and the vitreous. Preliminary studies have demonstrated a beneficial effect of surgical removal of the ILM for macular edema. Gandorfer et al. [2000] report pars plana vitrectomy with peeling of the ILM in 12 eyes with diffuse diabetic macular edema. In 10 eyes, the posterior hyaloid was attached and thickened. Six eyes had undergone macular photocoagulation previously, and 2 other eyes had been vitrectomized previously. Intraoperatively, the posterior hyaloid was found to be thickened and completely attached to the macula in 10 eyes. The 2 previously vitrectomized eyes showed a glistening reflex of the vitreoretinal interface but no premacular membrane. The posterior hyaloid and the ILM were removed from the macula. Postoperatively, retinal thickening resolved or decreased in all eyes. Visual acuity improved by at least 2 lines in 11 eyes. Best corrected postoperative visual acuity developed within 4–12 weeks. No recurrence or deterioration in macular edema or epiretinal membrane formation were observed during the entire period of review (mean 16 months, range 8–31 months). Vitrectomy including removal of the ILM leads to expedited resolution of diffuse diabetic macular edema and improvement in visual acuity without subsequent epiretinal membrane formation. Thus, complete release of tractional forces and inhibition of reproliferation of fibrous astrocytes seem to be prudent in the eyes of patients with diabetes and advanced vitreoretinal interface disease of the macula [Gandorfer et al., 2000]. However, the effect of ILM peeling is still controversially discussed. A retrospective series of 19 patients with different underlying diseases failed to demonstrate a significant difference in the visual outcome [Radetzky et al., 2004]. This retrospective investigation analyzed a series of 23 eyes from 23 patients with persistent macular edema after pars plana vitrectomy with indocyanine green-assisted peeling of the ILM, which suggested that ILM peeling is ineffective in central retinal vein occlusion and PDR. Improvements were apparent only in non-PDR. A potential beneficial effect of the surgical therapy should be weighed against the risk of surgical complications [Radetzky et al.,

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2004]. The lack of long-term improvement in this study is in accordance with the hypothesis that ILM peeling does not interfere with the mechanism of macular edema. Currently, a randomized multicenter study is ongoing (TIME, Triamcinolone versus ILM Peeling in Persistent Diabetic Macular Edema), which investigates the benefit of ILM peeling in patients with persistent diabetic macular edema ([email protected]).

Cataract Surgery

Cataract surgery is the most frequent and most successful surgery in ophthalmology. Improvement in visual acuity after cataract surgery is achieved despite severe non-PDR in 55% of the patients [Chew et al., 1999]. Nevertheless, the postoperative results in diabetic patients are inferior to patients without diabetes. The ETDRS reports a gain of 2 or more lines in 64.5% of eyes with early and 59.3% of eyes with delayed photocoagulation 1 year after cataract surgery. A visual acuity of 0.5 after cataract surgery was only achieved in 46% of eyes with delayed photocoagulation. In our experience, worsening or development of a macular edema is the main reason for visual deterioration after cataract surgery. Therefore, every eye with increased central retinal thickness, even if according to ETDRS no clinically significant macular edema is apparent, should be treated by focal photocoagulation, if an adequate view of the fundus is present. Vice versa, a potential postoperative worsening of macular edema is no argument against cataract surgery, but should be treated as indicated below. If during the postoperative course the edema progresses to a cystic form that is difficult to approach with photocoagulation, early treatment with intravitreal triamcinolone should be considered. According to our own experience and published reports [Jonas et al., 2005], steroid injection is able to efficiently reduce the postsurgical edema as well as diabetic macular edema. Prospective randomized clinical trials regarding triamcinolone treatment for diabetic macular edema are currently under investigation (for further information, [email protected]). In eyes of dense cataract, not only the patient’s visual acuity, but also the fundus view is obscured, and the necessary panretinal photocoagulation is not adequately possible. In some cases, the use of a crypton laser with a wavelength in the range of 600 nm is advantageous in the penetration of a nuclear cataract. In cases of proliferative retinopathy, a panretinal photocoagulation prior to cataract surgery is urgently advised. If this is rendered impossible, small incision surgery allows for photocoagulation within a short time after cataract extraction.


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Cataract surgery itself requires some peculiarities: in order to facilitate later panretinal photocoagulation or vitrectomy, a large capsulorrhexis is required as well as an IOL with a large optic. Acryl is the recommended lens material, as these IOL can be folded and implanted in small incision surgery. Furthermore, the risk of unfavorable interactions with silicone oil, which could become necessary in subsequent vitreous surgery, is reduced if acryl is used as lens material compared with silicone. Silicone oil tends to stick like glue on silicone lenses if close contact occurs. Without any doubt, phacoemulsification with implantation of a posterior chamber lens into the capsular bag remains the method of choice for the diabetic eye. Nevertheless, there are also selected indications for lens extraction with subsequent aphakia. As indicated previously, coagulation of the ischemic periphery is an essential part of an antiproliferative therapy. The outermost periphery is easiest approached in aphakic eyes, as is the removal of anterior hyaloid proliferation. Lensectomy or removal of the IOL should be considered in eyes with revision surgery and reduced visual prognosis (e.g., rubeosis and persistent tractional detachment of the macula) and, according to our experience, is not associated with a higher complication rate. Previous reports on stimulation of rubeosis following lensectomy in aphakic eyes did not use the possibility of a facilitated peripheral photocoagulation. Combination of cataract removal, vitrectomy and endophotocoagulation was only reported in small case series to be associated with a higher risk of neovascularization of the iris [Blankenship et al., 1989; Kokame et al., 1989]. The inhibition of ischemia and thus of developing or existing rubeosis by a radical peripheral vitrectomy and endophotocoagulation predominates, in our opinion, the stimulation of rubeosis by aphakia.

Conclusion

The project ‘Diabetes 2000’, which was propagated in the 1990s, aimed to reduce the rate of diabetes patients with visual loss through early diagnosis and treatment of complications [Patz and Smith, 1994]. Refined materials and instrumentation, and thus improvement in surgical techniques, allow preventing complications such as severe anterior hyaloidal fibrovascular proliferation and severe fibrinoid syndrome [Ho et al., 1992] which have been frequently seen in the early years of vitrectomy. Nevertheless, even today, patient expectations of visual rehabilitation cannot be satisfied, and a large discrepancy lies between these expectations and the physician’s hope to limit secondary consequences of rubeotic glaucoma with phthisis bulbi. The spectrum of indications for surgical interventions in diabetic retinopathy did not change during the past decade. Helbig et al. [1997, 1998a,

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b, 2002] report on 389 eyes which underwent surgery between 1990 and 1994. In this series, indications for vitrectomy included 39% vitreous hemorrhages, 13% tractional detachments of the macula, 12% tractional rhegmatogenous detachment, and 36% severe progressing proliferative retinopathy. A retrospective analysis of the patients at the department in Cologne from 2000 to 2004, which analyzed 361 patients, demonstrated 54% vitreous hemorrhages, 9% tractional detachment including the macula, 4% tractional detachment without the macula, 7% rubeotic secondary glaucoma, 2% macular edema, and 1% others. At present, the major discussion about the value of vitrectomy in the treatment of diabetic retinopathy refers to the optimal time point for intervention [Helbig et al., 1996]. According to the DRVS, early vitrectomy in cases of severe vitreous hemorrhage without tractional detachment improves the longterm prognosis in type 1 diabetics despite the immediate risk of the surgical procedure [DRVS Research Group, 1990]. Earlier in this review, risk factors for progression have been discussed extensively, e.g., lack of previous panretinal photocoagulation or tractional detachment of the fellow eye in young diabetics with insufficient adjustment of blood glucose levels. Without risk profile, in patients with vitreous hemorrhages, it is allowed to wait until the hemorrhage clears up (for a maximum of 3 months) and photocoagulation is possible. Among several exceptions, single eyes, which require quick visual rehabilitation, should be reminded of. However, the frequency of iatrogenic holes and the risk of reproliferation can be reduced with the improved surgical techniques. Helbig et al. [1998a, b] found postoperative detachments in 18% of all vitrectomies. Selecting patients with pure vitreous hemorrhage reduces the proportion to 5%. Certainly only a small part of the detachments are induced by iatrogenic retinal holes. Reproliferation is a major reason [Messmer et al., 1992]. Thus, more recent publications report on a lower redetachment rate [Helbig et al., 1997, 1998a, b; Joussen, unpubl.]. The indication for revision surgery in cases with combined rhegmatogenous tractional detachment and cases of ‘ghost cell glaucoma’ after vitreous hemorrhage should be generously handled. In general, combined cataract and vitreoretinal surgery is possible in diabetic patients. If possible, we would prefer two separate procedures. In any case, it is important to perform a sufficient panretinal photocoagulation for reduction in ischemia and the proliferative stimulus. Only few years back, silicone oil tamponade was controversially discussed, but is now an integral component of surgical treatment in diabetic retinopathy. Silicone oil was described to increase the risk of progression of maculopathy, damage to the optic disc, and reproliferation [Messmer et al., 1992; Helbig et al.,


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1997]. Whether the differences reported here have any clinical relevance still remains questionable. Silicone oil tamponade, performed in cases of severe angiopathy, is not necessarily causally linked to disease progression. Similarly, reproliferation under oil is more likely attributable to incomplete membrane removal. Thus, ‘perisilicone proliferation’ [Lewis et al., 1988] should rather be ‘proliferation following incomplete peeling’. In fact, severe postoperative bleeding can lead to formation of epiretinal membranes requiring revision surgery. We suggest to perform the revision surgery after an interval of 8–12 weeks following primary surgery to avoid repeated bleeding and to lower the proliferative vitreoretinopathy rate. In general, silicone oil removal should be attempted after 3 months to prevent formation of secondary glaucoma or cataract formation [Messmer et al., 1992; Karel and Kalvodova, 1994; Sima and Zoran, 1994].

Summary

• •

• •

Despite anatomically satisfying results, vitreoretinal surgery can only partially meet the patient’s expectations of visual rehabilitation. A complete panretinal photocoagulation and thus reduction in the ischemic proliferation is key to surgery in eyes with active neovascularization and its complications. Even in prognostic unfavorable situations with preexisting rubeosis or persistent tractional detachment involving the macula, surgical treatment is worthwhile to prevent phthisis or neovascular glaucoma. There are only small case series regarding the effectiveness of pars plana vitrectomy in diabetic macular edema. The results of large, randomized clinical investigations are being awaited. Besides surgical therapy, a long-term optimization of the blood glucose levels is inevitable [Diabetes Control and Complications Trial Research Group, 1993; Davidson, 1994; UK Prospective Diabetes Study Group, 1998]. References

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Patz A, Smith RE: The ETDRS and Diabetes 2000. Ophthalmology 1994;101:1061–1070. Pendergast SD, Hassan TS, Williams GA: Vitrectomy for diffuse diabetic macular edema associated with a taut premacular posterior hyaloid. Am J Ophthalmol 2000;130:178. Peyman GA, Cheema R, Conway MD, Fang T: Triamcinolone acetonide as an aid to visualization of the vitreous and the posterior hyaloid during pars plana vitrectomy. Retina 2000;20:554–555. Psichias A, Bartz-Schmidt KU, Thumann G, Krieglstein GK, Heimann K: Vitreoretinale Chirurgie in der Behandlung des neovaskulären Glaukoms. Klin Monatsbl Augenheilkd 1999;214:61–70. Radetzky S, Walter P, Koizumi K, Kirchhof B, Joussen AM: Visual outcome of patients with macular edema after pars plana vitrectomy and indocyanine green-assisted internal limiting membrane peeling. Graefes Arch Clin Ophthalmol 2004;242:273–278. Rosenblatt BJ, Shah GK, Sharma S, Bakal J: Pars plana vitrectomy with internal limiting membranectomy for refractory diabetic macular edema without a taut posterior hyaloid. Graefes Arch Clin Exp Ophthalmol 2005;243:20–25. Rice TA, Michels RG, Rice EF: Vitrectomy for diabetic rhegmatogenous retinal detachment. Am J Ophthalmol 1983a;95:34–44. Rice TA, Michels RG, Rice EF: Vitrectomy for diabetic traction retinal detachment involving the macula. Am J Ophthalmol 1983b;95:22–33. Sima P, Zoran T: Long-term results of vitreous surgery for proliferative diabetic retinopathy. Doc Ophthalmol 1994;87:223–232. Smiddy WE, Feuer W, Irvine WD, Flynn HW Jr, Blankenship GW: Vitrectomy for complications of proliferative diabetic retinopathy. Functional outcomes. Ophthalmology 1995;102:1688–1695. Smiddy WE, Flynn HW Jr: Vitrectomy in the management of diabetic retinopathy. Surv Ophthalmol 1999;43:491. Tasman W, Magargal LE, Augsburger JJ: Effects of argon laser photocoagulation on rubeosis iridis and angle neovascularization. Ophthalmology 1980;87:400–402. UK Prospective Diabetes Study Group: Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. BMJ 1998;317:703–713. Wetterqvist C, Wong D, Williams R, Stappler T, Herbert E, Freeburn S: Tamponade efficiency of perfluorohexyloctane and silicone oil solutions in a model eye chamber. Br J Ophthalmol 2004;88: 692–696. Wong HC, Sehmi KS, McLeod D: Abortive neovascular outgrowths discovered during vitrectomy for diabetic vitreous haemorrhage. Graefes Arch Clin Exp Ophthalmol 1989;227:237–240. Yamamoto T, Akabane N, Takeuchi S: Vitrectomy for diabetic macular edema: the role of posterior vitreous detachment and epimacular membrane. Am J Ophthalmol 2001;132:369–377.

Antonia M. Joussen Department of Ophthalmology Heinrich-Heine University Duesseldorf Moorenstraße 5 DE–40225 Duesseldorf Tel. ⫹49 0211 81 17321, Fax ⫹49 0211 81 16241, E-Mail [email protected]


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Diffuse Diabetic Macular Edema: Pathology and Implications for Surgery Arnd Gandorfer Vitreoretinal and Pathology Unit, Augenklinik der Ludwig-Maximilians-Universität, München, Germany

Abstract Diffuse diabetic macular edema represents a common problem in diabetic patients. It is characterized by widespread and poorly demarcated leakage in the macular area. The vitreomacular interface in eyes with diffuse diabetic macular edema is composed of (1) a layer of native vitreous collagen covering the internal limiting membrane, (2) fibroblasts and fibrous astrocytes embedded in native vitreous collagen, and (3) mostly multilayered cellular membranes situated on a layer of vitreous collagen in eyes with tangential vitreomacular traction. Given the poor response of diffuse diabetic macular edema to grid laser photocoagulation, vitreoretinal surgical techniques including removal of the vitreous cortex and the internal limiting membrane of the retina have been proposed. In this chapter, the pathology of diffuse diabetic macular edema and the implications for surgery are discussed. Copyright © 2007 S. Karger AG, Basel

Diabetic macular edema is the most common cause of decreased visual acuity in patients with diabetes mellitus, with an incidence between 13.9 and 25.4% over a 10-year period [1]. In patients suffering from type 1 diabetes, more than 40% will develop macular edema during their lifetime [2]. In clinical terms, there are two patterns of diabetic macular edema: focal and diffuse. Focal macular edema is characterized by well-defined areas of leakage. Diffuse diabetic macular edema is characterized by widespread and poorly demarcated leakage. Whereas focal macular edema can be treated effectively by focal laser photocoagulation [3], diffuse diabetic macular edema represent a more challenging clinical situation, not responding to grid laser photocoagulation in up to 24.6% [4].

Pathophysiology

Causes of diabetic macular edema include increased vasopermeability and damage to the retinal capillaries and the barrier provided by the retinal pigment epithelium [5]. Alternatively, or additionally, increased vasopermeability resulting in macular edema may be induced by vitreomacular traction [6]. In a subset of eyes with diabetic macular edema, the role of the vitreous and – in particular – the role of the posterior vitreous cortex have become increasingly recognized [7].

Role of the Vitreous and Vitreoschisis

Evidence of a vitreous origin of development and exacerbation of diabetic macular edema arises from several clinical studies. The prevalence of posterior vitreous detachment (PVD) in patients with diabetic macular edema is significantly lower than in diabetic patients without macular edema [8]. Spontaneous vitreomacular separation can cause resolution of diabetic macular edema [9]. In 1993, Kishi and Shimizu [10] reported the clinical manifestations of the premacular vitreous in proliferative diabetic retinopathy. In 94% of their 134 studied eyes with partial PVD, a posterior precortical vitreous pocket was observed in front of the macula. The posterior border of this pocket was formed by the premacular cortical vitreous which remained attached to the macula. Schwartz et al. [11] identified cortical vitreous remaining attached to the macula (‘vitreoschisis’) during surgery in 145 (81%) of 179 patients with proliferative diabetic retinopathy and traction retinal detachment. By using immunochemical staining, they were able to show that the wall of the vitreoschisis cavity was composed of type II collagen, thus providing direct evidence of the occurrence of splits in the posterior vitreous cortex [11]. Histopathologic studies in patients suffering from proliferative diabetic retinopathy disclosed the growth of newly formed blood vessels into the posterior vitreous cortex [12], thereby providing a possible explanation for the low incidence of progressive diabetic retinopathy in patients with complete PVD and the significantly higher risk of aggressive proliferation of new blood vessels in patients with partial PVD [13]. In a recent clinicopathological series of 61 eyes with diffuse diabetic macular edema, only 11% showed complete PVD, whereas 89% had a partially or completely attached vitreous [14]. There was a higher incidence of complete PVD confirmed intraoperatively in patients with nonproliferative disease (29%) compared with patients with proliferative diabetic retinopathy (8%).

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In summary, vitreoschisis and the presence of a thickened posterior cortical vitreous have been considered to play a key role in disease progression – not only in terms of neovascularization but also in terms of diabetic macular edema – and removal of the cortical vitreous has been suggested as one treatment option of diffuse diabetic macular edema [15].

Ultrastructural Findings of the Vitreous Cortex

We investigated the ultrastructure of the vitreomacular interface in a consecutive series of patients with diffuse diabetic macular edema [14]. Our approach was based on en bloc removal of the internal limiting membrane (ILM) together with all epimacular tissue. We found native vitreous collagen covering the ILM in almost all specimens (60/61). Even in the presence of clinically complete PVD and in eyes which had been vitrectomized previously, there were remnants of the cortical vitreous present at the vitreal side of the ILM. These findings emphasize that in diabetic eyes, PVD rarely occurs between the ILM and the vitreous cortex. Splitting of the vitreous cortex is a common finding, leaving a layer of cortical vitreous at the vitreoretinal interface. Our results also show that surgical induction of PVD by suction does not separate the vitreous cortex from the ILM but leaves a layer of collagen attached to the ILM. Native vitreous collagen was also the major ultrastructural component of a clinically prominent premacular cortical vitreous which had previously been called ‘thickened and taut premacular hyaloid’. In these eyes, fibroblasts, fibrous astrocytes and macrophages were embedded in collagen or were localized on a layer of vitreous collagen [14].

Vascular Endothelial Growth Factor and the Cortical Vitreous

It has been hypothesized previously that the presence of factors capable of altering vascular permeability may be a more physiological rather than mechanical cause of macular edema [16]. Vascular endothelial growth factor (VEGF) and its receptors as well as interleukin-6 have been localized to cells of vascular and avascular epiretinal membranes in patients with diabetic retinopathy [17, 18]. The presence of these and other factors altering vasopermeability, which are produced by cells within the cortical vitreous, may promote persistence of macular edema. Antonetti et al. [19] showed that increased levels of VEGF in the vitreous decrease levels of occludin, a membrane spanning tight junction protein, which could alter the structure of the retinal endothelial junction and may account for the increased vasopermeability in patients with diabetic

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macular edema. This finding may also explain the poor response of diffuse diabetic macular edema to grid laser photocoagulation.

Epiretinal Cellular Proliferation

In our series, one third of the studied eyes showed biomicroscopically visible fibrocellular tissue covering the macula [14]. In 9 eyes, a prominent premacular cortical vitreous was associated with obvious signs of vitreomacular traction such as retinal striae and vessel distortion. The ultrastructure of the vitreomacular interface in these eyes was characterized by a single-layered or multilayered cellular component localized on a layer of native vitreous collagen. In all eyes, the vitreous was found partially attached to the posterior pole at the beginning of surgery. In clinical and ultrastructural terms, these eyes showed resemblances to the characteristics of vitreomacular traction syndrome in nondiabetic patients, such as a firm partial attachment of the vitreous to the posterior pole and a prominent cellular component exerting tangential traction at the vitreomacular interface [20]. Another 9 eyes demonstrated a prominent cortical vitreous at the macula and biomicroscopically visible fibrocellular tissue [14]. Specimens of these eyes showed mostly multilayered cellular membranes situated on a layer of native vitreous collagen and additional cells embedded within the collagen layer (fig. 1). In ultrastructural terms, they were hardly distinguishable from specimens removed from eyes with macular pucker in nondiabetic patients [21, 22]. However, in clinical terms, the specimens of the present study were not associated with PVD. There were attachments of the vitreous to the posterior pole in almost all eyes, and only 1 eye showed no attachment as it had been vitrectomized previously. This is in contrast to epimacular membranes in nondiabetic patients which are associated with PVD in most cases [23, 24].

Current Concepts of Treatment

Grid laser photocoagulation has been shown to reduce leakage in eyes with diffuse diabetic macular edema. However, up to 25% of eyes do not respond to therapy. Moreover, despite reduction in leakage in cases of successful grid laser photocoagulation, the central macular area frequently remains thickened and cystic spaces develop. In 1992, Lewis et al. [25] reported that vitrectomy was beneficial in a series of 10 eyes with diabetic macular traction and edema associated with a thickened and taut premacular posterior hyaloid. Since this report, there has


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been an increasing interest in vitreous surgery as a potential treatment option for diabetic macular edema [15, 25]. Several groups confirmed that vitreoretinal separation seemed to be beneficial to patients with diabetic macular edema associated with vitreomacular traction [26–32]. However, even in patients without visible evidence of posterior hyaloidal traction or thickening, separation of the vitreous from the retina has been reported to result in resolution of macular edema [17, 33–36]. The ultrastructural findings of the vitreomacular interface in patients with diffuse diabetic macular edema described above illustrate that resolution of macular edema following removal of the cortical vitreous may not only be related to the relief of tractional forces but may also be caused by eliminating factors enhancing vasopermeability, such as VEGF, and possibly by a better transport and penetration of oxygen and nutrients through the vitreous cavity to the macula [37–39]. The surgical technique of epimacular tissue removal is still a matter of debate. As mechanisms of action of resolution of macular edema following surgery include release of traction forces and elimination of factors enhancing vasopermeability which are produced or which are concentrated within the premacular cortical vitreous, removal of the vitreous cortex alone should be sufficient. However, complete separation of the vitreous cortex from the ILM is not feasible by mechanical means [40, 41]. In theory and in practice, removal of the ILM leads to a better resolution of macular edema by removal of the vitreous cortex and all fibrocellular proliferation [14]. At present, it is not clear by which other mechanisms ILM peeling works. Beside traction relief, glial cell proliferation may also play a role in edema resorption as the ILM which forms the basement membrane of Müller cells is removed [42–44]. In the future, pharmacologic therapies will have a major impact on retinal diseases, including diabetic retinopathy and diffuse diabetic macular edema. Clinical assessment of these therapies in morphological and functional terms must demonstrate in which patients an intravitreal injection is sufficient and when advanced vitreoretinal surgical techniques are still required.

Fig. 1. Proliferative retinopathy and diffuse diabetic macular edema associated with vitreomacular traction from a 66-year-old female. a, b Preoperative fluorescein angiogram showing diffuse leakage of dye and marked retinal distortion (a 1:25 min; b 5:44 min). c Transmission electron micrograph of the multilayered membrane shows fibrocytes embedded in vitreous collagen (cross). ⫻4,800. d A macrophage embedded in vitreous collagen. Asterisk indicates the ILM. ⫻9,600. e, f Postoperative fluorescein angiogram demonstrating no leakage of dye (e 0:57 min; f 3:31 min).


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Klein R, Klein B, Moss S, Cruickshanks KC: The Wisconsin Epidemiologic Study of Diabetic Retinopathy. 15. The long-term incidence of macular edema. Ophthalmology 1995;102:7–16. Javitt JC, Canner JK, Sommer A: Cost effectiveness of current approaches to the control of retinopathy in type I diabetes. Ophthalmology 1989;96:255–264. Focal photocoagulation treatment of diabetic macular edema. Relationship of treatment effect to fluorescein angiographic and other retinal characteristics at baseline: ETDRS report No 19. Early Treatment Diabetic Retinopathy Study Research Group. Arch Ophthalmol 1995;113:1144–1155. Lee CM, Olk RJ: Modified grid laser photocoagulation for diffuse diabetic macular edema: longterm visual results. Ophthalmology 1991;98:1594–1602. Bresnick GH: Diabetic macular edema. A review. Ophthalmology 1986;93:989–997. Schepens CL, Avila MP, Jalkh AE, Trempe CL: Role of the vitreous in cystoid macular edema. Surv Ophthalmol 1984;28(suppl):499–504. Sebag J: Diabetic vitreopathy. Ophthalmology 1996;103:205–206. Nasrallah FP, Jalkh AE, Van Coppenolle F, Kado M, Trempe CL, McMeel JW, Schepens CL: The role of the vitreous in diabetic macular edema. Ophthalmology 1988;95:1335–1339. Hikichi T, Fujio N, Akiba J, Azuma Y, Takahashi M, Yoshida A: Association between the shortterm natural history of diabetic macular edema and the vitreomacular relationship in type II diabetes mellitus. Ophthalmology 1997;104:473–478. Kishi S, Shimizu K: Clinical manifestations of posterior precortical vitreous pocket in proliferative diabetic retinopathy. Ophthalmology 1993;100:225–229. Schwartz SD, Alexander R, Hiscott P, Gregor ZJ: Recognition of vitreoschisis in proliferative diabetic retinopathy. A useful landmark in vitrectomy for diabetic traction retinal detachment. Ophthalmology 1996;103:323–328. Faulborn J, Bowald S: Microproliferations in proliferative diabetic retinopathy and their relationship to the vitreous: corresponding light and electron microscopic studies. Graefes Arch Clin Exp Ophthalmol 1985;223:130–138. Sebag J: The Vitreous. Structure, Function, Pathobiology. New York, Springer, 1989. Gandorfer A, Rohleder M, Grosselfinger S, Haritoglou C, Ulbig M, Kampik A: Epiretinal pathology of diffuse diabetic macular edema associated with vitreomacular traction. Am J Ophthalmol 2005;139:638–652. Lewis H: The role of vitrectomy in the treatment of diabetic macular edema. Am J Ophthalmol 2001;131:123–125. Jumper JM, Embabi SN, Toth CA, McCuen BW 2nd, Hatchell DL: Electron immunocytochemical analysis of posterior hyaloid associated with diabetic macular edema. Retina 2000;20:63–68. Yamamoto T, Akabane N, Takeuchi S: Vitrectomy for diabetic macular edema: the role of posterior vitreous detachment and epimacular membrane. Am J Ophthalmol 2001;132:369–377. Chen YS, Hackett SF, Schoenfeld DL, Vinores MA, Vinores SA, Campochiaro PA: Localisation of vascular endothelial growth factor and its receptors to cells of vascular and avascular epiretinal membranes. Br J Ophthalmol 1997;81:919–926. Antonetti DA, Barber AJ, Khin S, Lieth E, Tarbell JM, Gardner TW, Group PSRR: Vascular permeability in experimental diabetes associated with reduced endothelial occludin content. Diabetes 1998;47:1953–1959. Gandorfer A, Rohleder M, Kampik A: Epiretinal pathology of vitreomacular traction syndrome. Br J Ophthalmol 2002;86:902–909. Kampik A, Green WR, Michels RG, Nase PK: Ultrastructural features of progressive idiopathic epiretinal membrane removed by vitreous surgery. Am J Ophthalmol 1980;90:797–809. Kampik A, Kenyon KR, Michels RG, Green WR, de la Cruz ZC: Epiretinal and vitreous membranes. Comparative study of 56 cases. Arch Ophthalmol 1981;99:1445–1454. Appiah AP, Hirose T, Kado M: A review of 324 cases of idiopathic premacular gliosis. Am J Ophthalmol 1988;106:533–535. Wise GN: Clinical features of idiopathic preretinal macular fibrosis. Schoenberg Lecture. Am J Ophthalmol 1975;79:349–357.

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Lewis H, Abrams GW, Blumenkranz MS, Campo RV: Vitrectomy for diabetic macular traction and edema associated with posterior hyaloidal traction. Ophthalmology 1992;99:753–759. Gandorfer A, Messmer EM, Ulbig MW, Kampik A: Resolution of diabetic macular edema after surgical removal of the posterior hyaloid and the inner limiting membrane. Retina 2000;20:126–133. Harbour JW, Smiddy WE, Flynn HW Jr, Rubsamen PE: Vitrectomy for diabetic macular edema associated with a thickened and taut posterior hyaloid membrane. Am J Ophthalmol 1996;121: 405–413. Yang CM: Surgical treatment for severe diabetic macular edema with massive hard exudates. Retina 2000;20:121–125. Kaiser PK, Riemann CD, Sears JE, Lewis H: Macular traction detachment and diabetic macular edema associated with posterior hyaloidal traction. Am J Ophthalmol 2001;131:44–49. Pendergast SD: Vitrectomy for diabetic macular edema associated with a taut premacular posterior hyaloid. Curr Opin Ophthalmol 1998;9:71–75. Pendergast SD, Hassan TS, Williams GA, Cox MS, Margherio RR, Ferrone PJ, Garretson BR, Trese MT: Vitrectomy for diffuse diabetic macular edema associated with a taut premacular posterior hyaloid. Am J Ophthalmol 2000;130:178–186. van Effenterre G, Guyot-Argenton C, Guiberteau B, Hany I, Lacotte JL: Macular edema caused by contraction of the posterior hyaloid in diabetic retinopathy. Surgical treatment of a series of 22 cases. J Fr Ophtalmol 1993;16:602–610. Ikeda T, Sato K, Katano T, Hayashi Y: Attached posterior hyaloid membrane and the pathogenesis of honeycombed cystoid macular edema in patients with diabetes. Am J Ophthalmol 1999;127:478–479. Ikeda T, Sato K, Katano T, Hayashi Y: Vitrectomy for cystoid macular oedema with attached posterior hyaloid membrane in patients with diabetes. Br J Ophthalmol 1999;83:12–14. Tachi N, Ogino N: Vitrectomy for diffuse macular edema in cases of diabetic retinopathy. Am J Ophthalmol 1996;122:258–260. La Heij EC, Hendrikse F, Kessel AG, Derhaag PJ: Vitrectomy results in diabetic macular oedema without evident vitreomacular traction. Graefes Arch Clin Exp Ophthalmol 2001;239:264–270. Stefansson E, Landers MB 3rd, Wolbarsht ML: Increased retinal oxygen supply following panretinal photocoagulation and vitrectomy and lensectomy. Trans Am Ophthalmol Soc 1981;79: 307–334. Stefansson E, Landers MB 3rd, Wolbarsht ML: Vitrectomy, lensectomy, and ocular oxygenation. Retina 1982;2:159–166. Stefansson E: The therapeutic effects of retinal laser treatment and vitrectomy. A theory based on oxygen and vascular physiology. Acta Ophthalmol Scand 2001;79:435–440. Russell SR, Hageman GS: Optic disc, foveal, and extrafoveal damage due to surgical separation of the vitreous. Arch Ophthalmol 2001;119:1653–1658. Gandorfer A, Ulbig M, Kampik A: Plasmin-assisted vitrectomy eliminates cortical vitreous remnants. Eye 2002;16:95–97. Gandorfer A, Rohleder M, Charteris DG, Sethi C, Kampik A, Luthert P: Staining and peeling of the internal limiting membrane in the cat eye. Curr Eye Res 2005;30:977–987. Nakamura T, Murata T, Hisatomi T, Enaida H, Sassa Y, Ueno A, Sakamoto T, Ishibashi T: Ultrastructure of the vitreoretinal interface following the removal of the internal limiting membrane using indocyanine green. Curr Eye Res 2003;27:395–399. Gandorfer A, Haritoglou C, Kampik A, Charteris D: Ultrastructure of the vitreoretinal interface following removal of the internal limiting membrane using indocyanine green. Curr Eye Res 2004;29:319–320.

PD Dr. Arnd Gandorfer Vitreoretinal and Pathology Unit, Augenklinik der Ludwig-Maximilians-Universität Mathildenstasse 8 DE–80336 München (Germany) Tel. ⫹49 089 5160 3800, Fax ⫹49 089 5160 4778 E-Mail [email protected]


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Intravitreal Triamcinolone Acetonide for Diabetic Retinopathy Jost B. Jonas Department of Ophthalmology, Faculty of Clinical Medicine Mannheim, Ruprecht Karls University of Heidelberg, Heidelberg, Germany

Abstract Intravitreal triamcinolone acetonide (IVTA) has been applied in exponentially increasing frequency for various intraocular neovascular and edematous diseases, including diabetic macular edema, proliferating diabetic retinopathy, neovascular glaucoma due to proliferative diabetic retinopathy, and chronic prephthisical ocular hypotony as complication of the surgical treatment of diabetic retinopathy. In diabetic macular edema, the edema may almost completely resolve, and visual acuity may increase as much as macular ischemia and the tissue destruction by the diabetic process may allow. For proliferative diabetic retinopathy and neovascular glaucoma, investigations have suggested an antiangiogenic effect of IVTA. Using a side effect of IVTA, i.e. steroid-induced elevation of intraocular pressure, IVTA may be applied for the therapy of chronic prephthisical ocular hypotony due to an insufficiency of the ciliary body as consequence of a surgical treatment of proliferative diabetic retinopathy. The complications of IVTA include secondary ocular hypertension in about 40% of the eyes, medically uncontrollable high intraocular pressure leading to antiglaucomatous surgery in about 1–2%, posterior subcapsular cataract and nuclear cataract leading to cataract surgery in about 15–20%, especially in elderly patients within 1 year after injection, postoperative infectious endophthalmitis with a rate of about 1:500 or 1:1,000, noninfectious endophthalmitis, and pseudo-endophthalmitis. IVTA can be combined with other intraocular surgeries including cataract surgery, particularly in eyes with iris neovascularization due to diabetic retinopathy. Cataract surgery performed some months after the injection does not show a markedly elevated rate of complications. If vision increases and eventually decreases after an IVTA injection, the injection can be repeated. The duration of the effect of a single IVTA is dosage dependent (about 6–9 months with 20 mg, and about 2–4 months with 4 mg). Copyright © 2007 S. Karger AG, Basel

Injecting triamcinolone acetonide intravitreally, Robert Machemer, Yasuo Tano, Gholam Peyman, Stephan Ryan and other researchers were the pioneers to consider and use the vitreous cavity as drug reservoir for treatment of intraocular

diseases such as proliferative vitreoretinopathy [1–4]. Later on, the list of intraocular edematous and neovascular disorders, which may potentially be treatable by intravitreal triamcinolone acetonide (IVTA), was extended to exudative age-related macular degeneration and other diseases including various forms of diabetic retinopathy [5]. Accordingly, recent studies have suggested that IVTA may be useful in temporarily increasing visual acuity in patients with diffuse diabetic macular edema [6–37]. Patients of study groups receiving IVTA compared with patients of control groups without intravitreal injections of triamcinolone acetonide showed a significant increase in visual acuity during follow-up. The most convincing evidence of the effect of IVTA as treatment for diabetic macular edema comes from a recent randomized trial by Sutter et al. [26]. They performed a prospective, double-masked, placebo-controlled, randomized clinical trial on 69 eyes of 43 patients, with 34 eyes randomized to receive intravitreal triamcinolone (4 mg) and 35 eyes randomized to receive a placebo injection. Eighteen of 33 eyes (55%) treated with triamcinolone gained 5 or more letters of bestcorrected visual acuity compared with 5 of 32 eyes (16%) treated with placebo (p ⫽ 0.002). Macular edema was reduced by 1 or more grades as determined by masked semiquantitative contact lens examination in 25 of 33 treated eyes versus 5 of 32 untreated eyes (p ⬍ 0.0001). A similar result was reported from a previous intraindividual inter-eye comparison of patients with bilateral diabetic macular edema who received an unilateral intravitreal injection of about 20 mg triamcinolone acetonide into the more severely affected eye [16]. In the injected eyes, compared with the contralateral nontreated eyes, visual acuity increased significantly (p ⬍ 0.001) by 3.0 ⫾ 2.6 Snellen lines. In the contralateral eyes, differences between baseline visual acuity and visual acuity measured at any of the reexaminations during follow-up were not significant (p ⬎ 0.10). Correspondingly, gain in visual acuity was significantly higher (p ⬍ 0.05) in the injected eyes for the measurements obtained up to 4 months after baseline. In the study group, from a peak in visual acuity at about 2–6 months after the injection, visual acuity decreased significantly (p ⫽ 0.001) towards the end of the follow-up, at which visual acuity was still higher, though not significantly (p ⫽ 0.18) higher, than at baseline. In the control group, visual acuity at the end of follow-up was lower, though not significantly lower (p ⫽ 0.26), than at baseline. The study confirmed another recent investigation with a similar study design carried out by Massin et al. [21]. Massin’s study included 15 patients with bilateral diabetic macular edema unresponsive to laser photocoagulation. Performing a unilateral injection of 4 mg triamcinolone acetonide, Massin et al. [21] found a significant reduction in macular thickness. Perhaps due to the smaller number of patients involved in their study, or due to the smaller dosage of triamcinolone acetonide injected intravitreally, the authors detected a slight,

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though not statistically significant, increase in visual acuity in the injected eyes compared with the contralateral eyes without intravitreal injection. Using a dosage of about 20 mg triamcinolone acetonide, the increase in visual acuity was most marked for the first 3–6 months after the injection and could be observed for a period of about 6–8 months [5, 15, 16, 34]. Using a dosage of 4 mg triamcinolone acetonide, the duration of a reduction in the macular thickness as measured by optical coherence tomography was less than 6 months [7, 19–21, 26]. At the end of the follow-up, visual acuity measurements returned to baseline values with no significant difference between baseline values and the measurements obtained at the end of the follow-up. Another clinical investigation evaluated which factors influence change in visual acuity after intravitreal injection of triamcinolone acetonide as treatment for diffuse diabetic macular edema [36]. Improvement in visual acuity after IVTA was significantly correlated with a lower degree of macular ischemia (p ⬍ 0.001), higher preoperative visual acuity (p ⫽ 0.002), and a higher degree of macular edema. Change in visual acuity after the intravitreal triamcinolone injection was statistically independent (p ⬎ 0.20) of age, gender and pseudophakia. The effect of IVTA with respect to an increase in visual acuity has also been found in other studies. In a retrospective, interventional, noncomparative case series study, Ciardella et al. [13] performed an intravitreal injection of 4 mg of triamcinolone acetonide in 30 eyes of 22 consecutive patients with diabetic macular edema refractory to laser treatment. Mean visual acuity improved from 0.17 ⫾ 0.12 at baseline to 0.34 ⫾ 0.18, 0.36 ⫾ 0.16 and 0.31 ⫾ 0.17 at the 1-, 3- and 6-month follow-up, respectively. Twelve eyes received 2, 7 eyes 3, and 2 eyes 4 IVTA injections. The mean interval between the first and second IVTA injection was 5.7 ⫾ 2.7 months, and between the second and third injection 5.7 ⫾ 3.3 months. Hard exudates were present in the macula at baseline in all eyes. Progressive reduction in the number and size of the hard exudates was noted after IVTA in all patients. Intraocular pressure was raised above 21 mm Hg in 12 (40%) of 30 eyes. The authors concluded that IVTA is a promising treatment for patients with diabetic macular edema refractory to laser treatment. Similar results were reported in studies performed by Micelli et al. [22], Karacorlu et al. [18], Gharbiya et al. [14] and Negi et al. [23]. Ozkiris et al. [24] investigated the efficacy of IVTA by pattern electroretinography. They found that during follow-up (mean 6.1 months), mean visual acuity and mean P50 amplitude of the pattern electroretinogram improved significantly. Bandello et al. [12] reported on the combination of IVTA with panretinal laser coagulation in a patient with bilateral florid proliferative diabetic retinopathy. The contralateral eye only received the panretinal laser coagulation. They found a greater reduction in retinal thickening and fluorescein leakage from retinal new vessels in the eye with the combined treatment than in the eye treated only by laser.

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In a similar manner, Zacks and Johnson [37] described that a combination treatment of panretinal laser coagulation and IVTA may provide benefit in patients with diffuse diabetic macular edema who require urgent laser treatment for proliferative diabetic retinopathy by preventing an exacerbation of macular edema. Karacorlu et al. [38] reported on the clinical outcome of a patient with proliferative diabetic retinopathy and diabetic macular edema which progressed despite grid laser photocoagulation in the macular region. After a single intravitreal injection of 4 mg triamcinolone acetonide, visual acuity increased, macular edema decreased, and the optic nerve head neovascularization markedly regressed. It has remained unclear so far whether and how intensively triamcinolone acetonide crystals injected into the vitreous body may influence the vitreoretinal interface. One may suspect that the crystals, due to their weight, can lead to a posterior vitreous detachment if the vitreous was not already detached prior to the injection. A posterior vitreous detachment may have as disadvantage a possibly increased risk of rhegmatogenous retinal detachment. However, so far, there have been no reports in the literature on a markedly elevated rate of retinal rhegmatogenous detachments as complication in the follow-up of patients who received an intravitreal injection of triamcinolone acetonide [39–42]. The advantage of a posterior vitreous detachment in patients with diabetic retinopathy may be a reduction in macular edema, as suggested by studies on pars plana vitrectomy in patients with diffuse diabetic macular edema, and a decreased risk of retinovitreal proliferations. Interestingly, triamcinolone acetonide has not been found in clinically significant concentrations in the serum shortly after intravitreal injections of about 20 mg triamcinolone acetonide [43]. This agrees with clinical observations that the metabolic control of patients with diabetes mellitus is not markedly influenced by the intraocular application of the steroid. As a possible alternative to the intravitreal application of triamcinolone acetonide, the posterior sub-Tenon injection has been reported. Bakri and Kaiser [44] included 63 eyes of 50 patients with persistent clinically significant diabetic macular edema involving the center of the fovea 3 or more months after one or more treatments of focal macular photocoagulation. All patients received a posterior sub-Tenon injection of 40 mg triamcinolone acetonide. Mean visual acuity significantly improved from 20/80 to 20/50 at 1 month, then stabilized to 20/65 at 3 months, 20/68 at 6 months, and 20/63 at 12 months. Complications were rare, with a transient significant rise in intraocular pressure at the 3-month evaluation and ptosis in 2 patients. Correspondingly, Cardillo et al. [31] recently reported on a study in which safety and efficacy of intravitreal versus posterior sub-Tenon capsule injection of triamcinolone acetonide for diffuse diabetic macular edema were compared. Including 12 patients (24 eyes) with


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bilateral diffuse diabetic macular edema, 1 eye of each patient was randomly assigned to receive a single 4-mg triamcinolone acetonide intravitreal injection and the fellow eye to receive a 40-mg triamcinolone acetonide posterior subTenon capsule injection. Both intravitreal and sub-Tenon capsule injections of triamcinolone acetonide resulted in significant but transient improvements in central macular thickness. The mean central macular thickness in eyes with intravitreal injection was significantly thinner than in the sub-Tenon capsuleinjected eyes at 1 month (p ⫽ 0.002) and 3 months (p ⫽ 0.005) after the intervention. Correspondingly, mean visual acuity was significantly better in the intravitreally injected eyes than in the sub-Tenon capsule-injected eyes at 3 months after injection (p ⫽ 0.004). The authors concluded that the short-term performance preferred the intravitreal (4 mg) to the sub-Tenon (40 mg) capsule route for triamcinolone acetonide administration. Besides, for diffuse diabetic macular edema, IVTA has been used in combination with pars plana vitrectomy for patients with proliferative diabetic retinopathy in an attempt to use the anti-inflammatory and antiangiogenic effects of triamcinolone acetonide. A pilot case series study including 29 patients suggested that intravitreal injection of crystalline cortisone with most of the vehicle removed may be well tolerated [45]. A following nonrandomized comparative investigation consisted of a study group of 32 eyes undergoing pars plana vitrectomy with IVTA and a control group of 32 eyes which were matched with the study group eyes for preoperative and intraoperative parameters and which underwent pars plana vitrectomy for proliferative diabetic retinopathy without intravitreal injection of triamcinolone acetonide [46]. The study group and the control group did not vary significantly in the rate of postoperative retinal detachment, re-pars plana vitrectomy, postoperative enucleation and phthisis bulbi, in best postoperative visual acuity, visual acuity at the end of the study, and gain in visual acuity. It was concluded that IVTA did not show a higher than usual rate of postoperative complications and that as a corollary, the adjunct use of IVTA combined with pars plana vitrectomy as treatment for proliferative diabetic retinopathy did not show a marked therapeutic benefit. Neovascular glaucoma, a typical end-stage complication of proliferative diabetic retinopathy, has recently been treated by IVTA, again using the antiangiogenic effect of triamcinolone acetonide [47, 48]. Fourteen eyes with neovascular glaucoma due to proliferative diabetic retinopathy or ischemic central retinal vein occlusion received an IVTA of about 20 mg acetonide as only procedure (n ⫽ 4 eyes) or in combination with additional procedures such as goniosynchiolysis (n ⫽ 1) and transscleral peripheral retinal cryocoagulation. Postoperatively, the degree of iris neovascularization decreased significantly (p ⫽ 0.02). Considering the 4 patients for whom the intraocular cortisone injection was the only procedure performed, mean intraocular pressure decreased

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from 26.5 ⫾ 12.1 to 21.75 ⫾ 11.3 mm Hg. This suggests that IVTA, mostly in combination with a retinal ablative procedure, may be an additional option in the treatment of neovascular glaucoma. Progressive ocular hypotony or prephthisical ocular hypotony can be a complication of ciliary destructive procedures as surgical treatment for neovascular glaucoma due to proliferative diabetic retinopathy. In an attempt to use a side effect of steroids as desired effect, triamcinolone acetonide was injected intravitreally into 3 eyes with longstanding prephthisical ocular hypotony [49, 50]. In all 3 patients, intraocular pressure and visual acuity increased after the injection, associated with a stabilization of the eyes. It suggests that in some eyes with long-standing prephthisical ocular hypotony, intravitreal injection of triamcinolone acetonide can be beneficial to increase intraocular pressure and stabilize the eye. Cataract is one of the most common ophthalmologic diseases in the elderly population. Therefore, it is common that cataract is present in eyes additionally showing other age-related disorders, such as diabetic retinopathy. Since these diseases may be treatable by intraocular injections of triamcinolone acetonide, and because intraocular triamcinolone acetonide by itself may further increase a preexisting lens opacification, it may be useful to combine an intravitreal injection of triamcinolone acetonide with cataract surgery. Taking into account that one has just recently started to clinically evaluate intravitreal injections of triamcinolone acetonide, and in view of the already known complications and side effects of intraocular triamcinolone acetonide, any additional procedure may further increase the frequency and enlarge the spectrum of complications of the new therapy. However, in a recent clinical investigation, frequencies of postoperative infectious endophthalmitis, wound leakage or other corneal wound healing problems, persisting corneal endothelial decompensation, rhegmatogenous retinal detachment, marked postoperative pain, or a clinically significant decentration of the intraocular lens did not vary between a study group of 60 eyes undergoing cataract surgery with implantation of a posterior chamber lens and an additional intravitreal injection of about 20 mg triamcinolone acetonide and a control group of 290 eyes consecutively receiving IVTA without additional intraocular cataract surgery [51]. It was concluded that for a mean follow-up of about 9 months, the frequency and the amount of complications of an intravitreal injection of triamcinolone acetonide, such as increased intraocular pressure, do not markedly differ whether the injection is combined with a standard cataract surgery or not. A similar conclusion was drawn in a study by Lam et al. [19] on 19 eyes of 15 consecutive diabetic patients with cataract and diabetic macular edema, in which phacoemulsification with concurrent intravitreal injection of 4 mg triamcinolone acetonide appeared to be a safe option for managing diabetics with cataract and diabetic macular edema.


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Since steroids applied in a high dosage may lead to several changes, such as alterations in collagenous structures and the immunologic status, intraocular surgery performed after an intravitreal application of triamcinolone acetonide may have an unusual spectrum of complications. Addressing this question, a case series study included 22 patients presenting with cataract which had progressed after a single or repeated intravitreal injection of about 20 mg of triamcinolone acetonide for the treatment of exudative age-related macular degeneration or diffuse diabetic macular edema [52]. During routine phacoemulsification surgery, an intraoperative dialysis of the lens zonules with vitreous prolapse occurred in 1 eye (4.5%). During the postoperative follow-up, an optically significant decentration of the intraocular lens or infectious endophthalmitis was not encountered in any patient. It was concluded that cataract surgery after single or repeated intravitreal injections of about 20 mg triamcinolone acetonide may not harbor a markedly elevated frequency or a markedly changed profile of complications of standard cataract surgery. In patients with dense cataract and iris neovascularization due to proliferative diabetic retinopathy, the lens opacification prevents a transpupillary laser coagulation of the retina. However, an intraocular intervention such as cataract surgery will lead to a marked postoperative inflammation if iris neovascularization is additionally present. In that clinical situation, cataract surgery has been combined with an intravitreal injection of triamcinolone acetonide [53]. In the postoperative period, visual acuity increased, and without additional retinal ablative treatments, iris neovascularization markedly regressed within the first 5 weeks after surgery. The study suggested that IVTA can be a useful adjunctive treatment tool in eyes with iris neovascularization undergoing cataract surgery, and that IVTA may have an antiangiogenic effect. The use of IVTA is associated with several complications. One of the two most common side effects of IVTA was the steroid-induced elevation of intraocular pressure [54–56]. A recent prospective clinical interventional comparative nonrandomized study included 260 consecutive patients (293 eyes) receiving an intravitreal injection of 20–25 mg triamcinolone acetonide as treatment for diffuse diabetic macular edema, exudative age-related macular degeneration, retinal vein occlusions, uveitis and cystoid macular edema [56]. Intraocular pressure readings higher than 21, 30, 35 and 40 mm Hg were measured in 94 (36.2%), 22 (8.5%), 11 (4.2%) and 4 (1.5%) patients, respectively. Triamcinolone-induced elevation of intraocular pressure could be treated by antiglaucomatous medication in all but 3 eyes (1.0%), for which filtering surgery became necessary. About 40% of the patients developed a secondary ocular hypertension, starting about 1 week after the injection in few eyes and occurring about 1–2 months after the intravitreal injection of 20–25 mg triamcinolone acetonide in most eyes, developing an ocular hypertension. Using this

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dosage, the increase in intraocular pressure lasted about 7–9 months after which the intraocular pressure measurements return to the normal range without any further antiglaucomatous medication taken. Younger age was a significant factor contributing to the triamcinolone acetonide-induced increase in intraocular pressure. Diagnosis of diabetes mellitus or presence of a clinically significant diffuse diabetic macular edema did not influence the reaction of intraocular pressure after the injection. It agrees with previous randomized clinical trials in which diabetes mellitus was not a major risk factor for glaucoma [56]. Therefore, from a clinical point of view, diagnosis of diabetes mellitus may not be contradictory against IVTA. This fits with another aforementioned study, in which patients after IVTA did not show any, or only traces of, triamcinolone acetonide in the serum [43]. Those patients who received a second injection of 20–25 mg triamcinolone acetonide showed a similar reaction of intraocular pressure to that after the first injection [56]. This result suggests that if after a first injection intraocular pressure remained in the normal range, intraocular pressure may also remain in the normal range after a second injection. In a similar manner, if intraocular pressure increased after the first injection, a similar rise in intraocular pressure can be expected after a second injection. So far, there are no reports on a permanent rise in intraocular pressure after an intravitreal injection of triamcinolone acetonide. Comparing studies using different dosages of triamcinolone acetonide for intravitreal injection suggests that the higher the dosage, the longer the duration of the steroid-induced ocular hypertension [7, 10, 16, 17, 20, 21, 26, 27, 34, 35, 58]. The figures of the frequency of secondary ocular hypertension may not be directly correlated with the dosage injected. In the study performed by Smithen et al. [58] with the intravitreal use of 4 mg triamcinolone acetonide, a pressure elevation defined as a pressure of 24 mm Hg or higher during the follow-up was found in 36 (40.4%) out of 89 patients at a mean of 101 ⫾ 83 days after the injection. Out of nonglaucomatous patients with a baseline intraocular pressure of 15 mm Hg or above, 60.0% experienced a pressure elevation, compared with only 22.7% of those with baseline pressures below 15 mm Hg. In glaucoma patients, 6 of 12 (50%) experienced a pressure elevation, and this elevation was not correlated with baseline pressure. Thirty-two patients (36.0%) received repeated injections, and there was no difference in the incidence of procedure elevation in patients receiving multiple injections versus those receiving a single injection. Pressure elevation was controlled with topical medications in all patients. Using a dosage of 8 mg triamcinolone acetonide, Ozkiris and Erkilic [59] detected a transient elevation of intraocular pressure above 21 mm Hg in 20.8% of eyes. The average intraocular pressure rose by 28.5, 38.2, 16.7 and 4.2% from baseline at 1, 3, 6 and 9 months, respectively.


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If further studies confirm the assumption that the frequency of secondary ocular hypertension after an intravitreal injection of triamcinolone acetonide does not markedly depend on the dosage used, one may assume that relatively low triamcinolone acetonide dosages are already so high that all steroid receptors are occupied. One has to take into account that the eye makes out about 0.01% of the body volume. Assuming an equal distribution of triamcinolone acetonide throughout the body, an intravitreal injection of 4 mg is equal to a intragluteal injection of 40 g, and an intravitreal injection of 25 mg triamcinolone acetonide is equal to a quarter of a kilogram injected intragluteally. Another complication of IVTA is the postinjection infectious endophthalmitis. In recent studies on patients receiving an intravitreal injection of triamcinolone acetonide, the frequency of postinjection infectious endophthalmitis ranged between 0/700 and 8/992 (0.87%) [60–64]. The risk of an infectious endophthalmitis may partially depend on the setting of the injection itself. The studies suggest that if the injection is performed under sterile conditions, the risk may be less. Histologically, eyes with IVTA and infectious endophthalmitis show a marked destruction of the whole globe and a morphallaxia-like morphology [65]. It may go along with the clinical observation that patients with infectious endophthalmitis after IVTA usually have almost no pain. With respect to susceptibility to infectious endophthalmitis, a recent experimental study showed that rabbit eyes with IVTA have a significantly higher rate of apparent intraocular infection than rabbits without IVTA [66]. Concerning the multiple use of triamcinolone acetonide-containing bottles, another investigation [67] showed that even after 24 h of exposure to the benzyl alcohol preservative, four of five challenge organisms demonstrated moderate growth in the bottle so that the use of multiple-dose containers of triamcinolone for intravitreal injections may be discouraged. A ‘sterile endophthalmitis’ has been described to occur after an intravitreal injection of triamcinolone acetonide [63, 64, 68]. It has been inconclusive so far whether the solvent agent of triamcinolone acetonide is the cause for the sterile intraocular inflammation after the injection, and whether the solvent agent should be removed. The disadvantage of removal of the solvent agent is that the dosage gets inaccurate [69, 70]. Bakri et al. [71] reported on the use of a commercially available preservative-free solution of triamcinolone acetonide, and Hernaez-Ortega and Soto-Pedre [72] described the use of density gradient centrifugation to remove the preservative. Postinjection pseudo-endophthalmitis is present if triamcinolone acetonide crystals are washed from the vitreous cavity into the anterior chamber and settle down in the inferior anterior chamber angle mimicking a hypopyon [73–76]. The diagnostic problem is the differentiation between a painless hypopyon caused by postinjection infectious endophthalmitis and a pseudo-hypopyon due

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to triamcinolone acetonide crystals. So far, there have been no reports showing a corneal endothelial damage or a damage to the trabecular meshwork by the crystals. A postinjection, steroid-induced cataract is one of the most frequent complications or side effects of IVTA. In a recent study on 144 phakic eyes which consecutively received an intravitreal injection of about 20 mg triamcinolone acetonide for diffuse diabetic macular edema, exudative age-related macular degeneration and branch retinal vein occlusion, cataract surgery was performed in 20 eyes (13.9%) 17.4 ⫾ 9.1 months (median 12.7 months, range 8.0–35.5) after the intravitreal injection [77]. Out of the 20 eyes undergoing cataract surgery, 19 eyes (95%) had received one intravitreal injection, and 1 eye (5%) had received two previous injections. It was concluded that in the elderly population of patients with exudative age-related macular degeneration, diffuse diabetic macular edema or branch retinal vein occlusion, intravitreal high-dosage injection of triamcinolone acetonide leads to clinically significant cataract with eventual cataract surgery in about 15–20% of eyes within about 1 year after the intravitreal injection. In an analysis of longitudinal data from a randomized, double-masked, placebo-controlled trial of intravitreal triamcinolone for age-related macular degeneration, Gillies et al. [78] compared 57 phakic eyes in the treatment group with 4 mg triamcinolone acetonide versus 54 phakic eyes in the control group. They found that progression of posterior subcapsular cataract by 2 or more grades in the treatment group was significantly higher among 16 intraocular pressure responders (51% after 2 years) than among 37 nonresponders (3%; p ⬍ 0.0001). There was no significant progression of posterior subcapsular cataract in the placebo group or the opposite eye of the treatment group. Progression of cortical cataracts was also significantly higher among responders than among nonresponders (15 vs. 3%; p ⫽ 0.015). The progression of nuclear cataracts (13 vs. 3%) was not significantly different between intraocular pressure responders and nonresponders (p ⫽ 0.3). The authors concluded that although steroid-related cataracts were unlikely to develop in eyes that do not experience an elevation of intraocular pressure after intravitreal triamcinolone, those eyes that do experience an elevation also have a very high risk of rapidly experiencing posterior subcapsular lens opacification. They postulated that the strong association suggests a similar mechanism responsible for the development of steroid-induced posterior subcapsular cataract and for the elevation of intraocular pressure. Direct toxic effects of triamcinolone acetonide on the retina and optic nerve have not been observed yet, independently of the dosage used. Correspondingly, a recent safety and efficacy study of an intravitreal fluocinolone acetonide-sustained delivery device as treatment for cystoid macular


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edema in patients with uveitis and other clinical and experimental studies have not shown a toxic effect of intraocular steroids [39, 41]. The same was found by Hida et al. [79]. However, a recent study performed by Yeung et al. [80] reported a possible cytotoxic effect of triamcinolone acetonide. Yeung et al. [80] cultured a retinal pigment epithelium cell line (ARPE19) and added corticosteroids (0.01–1 mg/ml) or vehicle (benzyl alcohol, 0.025%), diluted in culture medium. Subsequently, the culture medium containing corticosteroid or vehicle was refreshed daily. After 1, 3 and 5 days, the proliferated amount of cells with and without corticosteroid treatment was determined. They found that triamcinolone acetonide caused a significant reduction in cell numbers throughout the whole range of concentrations when cells were exposed for more than 1 day. Compared with dexamethasone and hydrocortisone, triamcinolone acetonide showed a higher relative toxicity. The vehicle alone had no effect. In a similar study, Yeung et al. [81] compared the cytotoxic effect of triamcinolone acetonide on human retinal pigment epithelium (cell line ARPE19) and human glial cells over a range of concentrations and durations of exposure. They found that triamcinolone acetonide caused a significant reduction in the retinal pigment epithelium cell line ARPE19 that had been exposed to the substance for more than 1 day. Significant reductions in the number of glial cells were observed as early as day 1. The glial cells appeared to be more susceptible to triamcinolone acetonide. The vehicle of triamcinolone acetonide had no effect. In conclusion, the intravitreal injection of triamcinolone acetonide may possibly open new avenues for the treatment of intraocular edematous and neovascular diseases [82]. However, as for any new therapy, one has to be very careful since long-term experience has not been available yet. There are many open issues still to be addressed. What may be the best dosage for which disease and for which clinical situation? Is the proliferation of retinal pigment epithelium cells in high concentrations of triamcinolone acetonide decreased and, paradoxically, increased in low concentrations [83]? What is the best mode of application of triamcinolone acetonide? Is the sub-Tenon application, the subconjunctival application or the retrobulbar application better than the intravitreal injection? Are there other complications than those already described in clinical studies or after accidental injection of triamcinolone acetonide into the vitreous cavity? Is it necessary to remove the solvent agent prior to the intraocular injection, and how should the solvent agent be removed? The most fascinating point is that the intravitreal injection of triamcinolone acetonide together with previous clinical experiences on the use of intravitreal antibiotics and virustatic drugs makes one infer that retinal diseases may become locally treatable diseases. Unbelievably high intraocular concentrations of drugs become achievable, and systemic side effects may mostly be avoided.

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Jonas JB, Hayler JK, Söfker A, Panda-Jonas S: Intravitreal injection of crystalline cortisone as adjunctive treatment of proliferative diabetic retinopathy. Am J Ophthalmol 2001;131:468–471. Jonas JB, Söfker A, Degenring RF: Intravitreal triamcinolone acetonide as additional tool in pars plana vitrectomy for proliferate diabetic retinopathy. Eur J Ophthalmol 2003;13:468–473. Jonas JB, Hayler JK, Söfker A, Panda-Jonas S: Regression of neovascular iris vessels by intravitreal injection of crystalline cortisone. J Glaucoma 2001;10:284–287. Jonas JB, Kreissig I, Degenring RF: Neovascular glaucoma treated by intravitreal triamcinolone acetonide. Acta Ophthalmol 2003;81:540–541. Jonas JB, Hayler JK, Panda-Jonas S: Intravitreal injection of crystalline cortisone as treatment of pre-phthisical ocular hypotony. Graefes Arch Clin Exp Ophthalmol 2001;239:464–465. Rodriguez ML, Juarez CP, Luna JD: Intraocular steroids as a treatment for blind painful red eyes. Eur J Ophthalmol 2003;13:292–297. Jonas JB, Kreissig I, Budde WM, Degenring RF: Cataract surgery combined with intravitreal injection of triamcinolone acetonide. Eur J Ophthalmol 2005;15:329–335. Jonas JB, Kreissig I, Degenring RF: Cataract surgery after intravitreal injection of triamcinolone acetonide. Eye 2004;18:361–364. Jonas JB, Söfker A: IVTA for cataract surgery with iris neovascularization. J Cataract Refract Surg 2002;28:2040–2041. Wingate RJ, Beaumont PE: Intravitreal triamcinolone and elevated intraocular pressure. Aust NZ J Ophthalmol 1999;27:431–432. Bakri SJ, Beer PM: The effect of intravitreal triamcinolone acetonide on intraocular pressure. Ophthalmic Surg Lasers Imaging 2003;34:386–390. Jonas JB, Degenring RF, Kreissig I, Akkoyun I, Kamppeter BA: Intraocular pressure elevation after intravitreal triamcinolone acetonide injection. Ophthalmology 2005;112:593–598. Spandau UHM, Derse M, Schmitz-Valckenberg P, Papoulis C, Jonas JB: Dosage-dependency of intravitreal triamcinolone acetonide as treatment for diabetic macular edema. Br J Ophthalmol 2005;89:999–1003. Smithen LM, Ober MD, Maranan L, Spaide RF: Intravitreal triamcinolone acetonide and intraocular pressure. Am J Ophthalmol 2004;138:740–743. Ozkiris A, Erkilic K: Complications of intravitreal injection of triamcinolone acetonide. Can J Ophthalmol 2005;40:63–68. Benz MS, Murray TG, Dubovy SR, Katz RS, Eifrig CW: Endophthalmitis caused by Mycobacterium chelonae abscessus after intravitreal injection of triamcinolone. Arch Ophthalmol 2003;121:271–273. Jonas JB, Kreissig I, Degenring RF: Endophthalmitis after intravitreal injection of triamcinolone acetonide. Arch Ophthalmol 2003;121:1663–1664. Moshfeghi DM, Kaiser PK, Scott IU, Sears JE, Benz M, Sinesterra JP, Kaiser RS, Bakri SJ, Maturi RK, Belmont J, Beer PM, Murray TG, Quiroz-Mercado H, Mieler WF: Acute endophthalmitis following IVTA injection. Am J Ophthalmol 2003;136:791–796. Nelson ML, Tennant MT, Sivalingam A, Regillo CD, Belmont JB, Martidis A: Infectious and presumed noninfectious endophthalmitis after IVTA injection. Retina 2003;23:686–691. Parke DW: Intravitreal triamcinolone and endophthalmitis. Am J Ophthalmol 2003;136:918–919. Jonas JB, Bleyl U: Morphallaxia-like ocular histology after IVTA. Br J Ophthalmol 2004;88: 839–840. Bucher RS, Hall E, Reed DM, Richards JE, Johnson MW, Zacks DN: Effect of IVTA on susceptibility to experimental bacterial endophthalmitis and subsequent response to treatment. Arch Ophthalmol 2005;123:649–653. Bucher RS, Johnson MW: Microbiologic studies of multiple-dose containers of triamcinolone acetonide and lidocaine hydrochloride. Retina 2005;25:269–271. Roth DB, Chieh J, Spirn MJ, Green SN, Yarian DL, Chaudhry NA: Noninfectious endophthalmitis associated with intravitreal triamcinolone injection. Arch Ophthalmol 2003;121:1279–1282. Rodriguez-Coleman H, Yuan P, Kim H, Gravlin L, Srivastava S, Csaky KG, Robinson MR: Intravitreal injection of triamcinolone for diffuse diabetic macular edema. Arch Ophthalmol 2004;122:1085–1086; author reply 1086–1088.


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Spandau UHM, Derse M, Schmitz-Valckenberg P, Papoulis C, Sagstetter BU, Stiefvater KH, Jonas JB: Measurement of triamcinolone acetonide concentration after filtering of solvent agent. Am J Ophthalmol 2005;139:712–713. Bakri SJ, Shah A, Falk NS, Beer PM: Intravitreal preservative-free triamcinolone acetonide for the treatment of macular oedema. Eye 2005;19:686–688. Hernaez-Ortega MC, Soto-Pedre E: A simple and rapid method for purification of triamcinolone acetonide suspension for intravitreal injection. Ophthalmic Surg Lasers Imaging 2004;35: 350–351. Jonas JB, Hayler JK, Panda-Jonas S: Intravitreal injection of crystalline cortisone as adjunctive treatment of proliferative vitreoretinopathy. Br J Ophthalmol 2000;84:1064–1067. Sutter FK, Gillies MC: Pseudo-endophthalmitis after intravitreal injection of triamcinolone. Br J Ophthalmol 2003;87:972–974. Moshfeghi AA, Scott IU, Flynn HW Jr, Puliafito CA: Pseudohypopyon after IVTA injection for cystoid macular edema. Am J Ophthalmol 2004;138:489–492. Chen SD, Lochhead J, McDonald B, Patel CK: Pseudohypopyon after intravitreal triamcinolone injection for the treatment of pseudophakic cystoid macular oedema. Br J Ophthalmol 2004;88: 843–844. Jonas JB, Degenring RF, Vossmerbaeumer U, Kamppeter BA: Frequency of cataract surgery after intravitreal injection of high-dosage triamcinolone acetonide. Eur J Ophthalmol 2005;15: 462–464. Gillies MC, Kuzniarz M, Craig J, Ball M, Luo W, Simpson JM: Intravitreal triamcinolone-induced elevated intraocular pressure is associated with the development of posterior subcapsular cataract. Ophthalmology 2005;112:139–143. Hida T, Chandler D, Arena JE, Machemer R: Experimental and clinical observations of the intraocular toxicity of commercial corticosteroid preparations. Am J Ophthalmol 1986;101: 190–195. Yeung CK, Chan KP, Chan CK, Pang CP, Lam DS: Cytotoxicity of triamcinolone on cultured human retinal pigment epithelial cells: comparison with dexamethasone and hydrocortisone. Jpn J Ophthalmol 2004;48:236–242. Yeung CK, Chan KP, Chiang SW, Pang CP, Lam DS: The toxic and stress responses of cultured human retinal pigment epithelium (ARPE19) and human glial cells (SVG) in the presence of triamcinolone. Invest Ophthalmol Vis Sci 2003;44:5293–5300. Aiello LP, Brucker AJ, Chang S, Cunningham ET Jr, D’Amico DJ, Flynn HW Jr, Grillone LR, Hutcherson S, Liebmann JM, O’Brien TP, Scott IU, Spaide RF, Ta C, Trese MT: Evolving guidelines for intravitreous injections. Retina 2004;24(suppl 5):S3–S19. Blumenkranz MS, Claflin A, Hajek AS: Selection of therapeutic agents for intraocular proliferative disease. Cell culture evaluation. Arch Ophthalmol 1984;102:598–604.

Dr. Jost B. Jonas Universitäts-Augenklinik Theodor-Kutzer-Ufer 1–3 DE–68167 Mannheim (Germany) Tel. ⫹49 621 383 2652, Fax ⫹49 621 383 3803 E-Mail [email protected]

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Use of Long-Acting Somatostatin Analogue Treatment in Diabetic Retinopathy Bernhard O. Boehm Division of Endocrinology and Diabetes, Department of Medicine I, Ulm University, Ulm, Germany

Abstract The diabetes epidemic continues unabated, leading to an increasing number of acute and chronic complications, including sight-threatening proliferative diabetic retinopathy. Currently, there is no accepted pharmaceutical therapy for diabetic retinopathy besides nearnormal glycemia, treatment of hypertension, and dyslipidemia. For an effective treatment of retinopathy, one would recommend a concept leading to the downregulation of endogenous angiogenic stimulators and the upregulation of endogenous angiogenic inhibitors, resulting in a restoration of the balance in angiogenic control. The naturally occurring growth hormone inhibitor, somatostatin, has been suggested as candidate for developing novel therapies. Somatostatin may exert its antiangiogenic effects, both through antagonism of the growth hormone axis and through direct antiproliferative and apoptotic effects on endothelial cells. Therefore, the use of long-acting somatostatin analogues will lead to an upregulation of antiangiogenic signaling. Use of long-acting somatostatin analogues in diabetic retinopathy would be an important extension of the initial concept that somatostatin is a regulator of growth hormone secretion only. Currently available analogues have already allowed to modulate the expression of diabetic retinopathy at various disease stages. Somatostatin analogues remain the only nondestructive therapeutic alternative to patients with proliferative diabetic retinopathy who have failed to respond to panretinal photocoagulation. Copyright © 2007 S. Karger AG, Basel

Angiogenesis is a fundamental process of growth and differentiation of new blood vessels [1]. It involves new vessel formation from preexisting vessels, whereas vasculogenesis involves new vessel growth from endothelial cell precursors or stem cells [2–4]. Angiogenesis results from multiple signals acting on endothelial cells. Many peptide growth factors and cytokines have been found that regulate this process.

Endothelial cells are surrounded by pericytes that regulate the function of the blood vessels. Regulation of the barrier function by endothelial cells is an intricate process, requiring coordination of a large number of complex signaling pathways. The breakdown of the blood-retinal barrier, resulting in leakage of plasma from small blood vessels in the macula, the central portion of the retina, is responsible for the major part of impaired visual function. Therefore, macular edema is a clinical correlate of a compromised barrier function [5, 6]. The development of pathological neovascularization is often associated with hypoxia/ischemia. Hypoxia and ischemia can be observed both in malignant tumors and in proliferative retinopathies, which includes diabetic retinopathy. Hypoxia is known to stimulate important angiogenic mediators including vascular endothelial growth factor (VEGF). This occurs through activation of hypoxia-inducible factor (HIF)-1 that increases VEGF expression [7, 8]. The concept that growth factors mediate retinal angiogenesis has been introduced in 1948 by Michaelson [9]. There is now ample evidence that the development of diabetic retinopathy is a multifactorial process in which growth factors, including growth hormone and insulin-like growth factor (IGF)-1, play an important role. The lack of inhibitory signals of growth has also been recently advocated [10–12]. The pathological neovascularization seen in patients with diabetes mellitus is the response to a rise in the local concentration of molecules that induce such angiogenesis, but it is also due to a fall in the levels of endogenous molecules inhibiting angiogenesis (fig. 1). One of the most potent endogenous regulators is pigment epithelium-derived factor (PEDF), which serves both as a survival factor for neuronal components of the eye as well as an essential inhibitor of the growth of ocular blood vessels. In the presence of a pathological/diabetic milieu, a reduced gene expression of PEDF resulting in a reduced antiangiogenic activity has been found [13, 14]. This suggests that an unbalanced expression of angiogenic mediators and antiangiogenic factors is involved in the development and progression of pathological neovascularization in the diabetic eye [15, 16]. PEDF may also act as an endogenous antiinflammatory factor in the eye. Therefore, decreased ocular PEDF levels may contribute to an ongoing low-grade inflammation and vascular leakage in diabetic retinopathy [17]. For an effective treatment of retinopathy in people with diabetes mellitus, one would recommend a concept leading to the downregulation of endogenous angiogenic stimulators and the upregulation of endogenous angiogenic inhibitors, resulting in a restoration of balance in angiogenic control [18, 19]. In this review, we will address the potential role of growth factor inhibitory substances, i.e. long-acting somatostatin analogues, in diabetic patients with proliferative retinopathy.

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Quiescent vasculature Endogenous angiogenic factors

Endogenous angiogenic inhibitors

Angiogenesis Pro angiogenic factors • VEGF ↑ • GH, IGF-1 ↑ • Erythropoietin ↑

Angiogenic inhibitors • PEDF ↓ • SMS ↓

Fig. 1. Unbalanced expression of angiogenic mediators and antiangiogenic factors in the diabetic eye. GH ⫽ Growth hormone; SMS ⫽ somatostatin.


Diabetic retinopathy is the most severe of the several ocular complications of chromic hyperglycemia [20]. Diabetic retinopathy affects both type 1 and type 2 diabetic patients. Because diabetes is so common, although advances in treatment have greatly reduced the risk of blindness, retinopathy still remains a significant clinical problem in daily practice [20–22].

Current Approaches to Prevention and Treatment of Diabetic Retinopathy

Without intervention, proliferative retinopathy will eventually develop in 60% of persons with diabetes, resulting in profound visual loss in almost half of them. Randomized, controlled clinical trials have shown that medical therapy providing glucose control at near-normal levels by use of intensive conventional therapy or continuous subcutaneous insulin infusion significantly retard development and progression of retinopathy in patients with type 1 diabetes [23]. Likewise, intensified treatment of type 2 diabetes mellitus will lower the risk of microvascular complications [24]. Blood glucose control and the control of lipids also delay progression [25–27]. There is no doubt that an intensified diabetes treatment is effective; however, in the Diabetes Control and Complications Trial, two rather unexpected observations were made, both of which are of considerable importance. First, the differences in progression between the group with ‘tight’ blood glucose control and the group with standard control did not appear until approximately 2.5 years after the

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initiation of these treatment regimens. Second, about 10% of the patients with preexisting retinopathy had a transient worsening after the institution of tight blood glucose control [28]. Early worsening of their retinopathy was found to be related to increased systemic levels of IGF-1 plus the upregulation of the mitogenic cytokine VEGF and its receptor leading to an unbalanced increase in angiogenic mediators [29, 30]. In diabetic rats, acute intensive insulin therapy markedly increases VEGF mRNA and protein levels in the retinae. In this setting, retinal nuclear extracts revealed increased HIF-1␣ levels leading to an increased HIF1␣-dependent binding to hypoxia-responsive elements in the VEGF promoter [31]. This suggest that a treatment of choice, i.e. intensified diabetes treatment, may also increase the likelihood of proliferative retinopathy in the short term. Laser therapy, introduced during the 1960s, is the mainstay in the treatment of proliferative diabetic retinopathy and diabetic macular edema. However, it always has to be remembered that laser treatment is a destructive treatment [32, 33].

Novel Concepts – the Growth Factor Hypothesis

Knowledge of the major factors responsible for modulating neovascularization has had significant implications for the development of novel, nondestructive, pharmacologic treatment modalities [34–36]. Proliferative retinopathies could be prevented by improved metabolic control or by pharmacologically blunting the biochemical consequences of hyperglycemia. The angiogenesis in proliferative diabetic retinopathy could also be treated via growth factor blockade by either upregulating endogenous angiogenic inhibitors or by pharmacological blocking. Targets could be VEGF and its corresponding receptor molecule, IGF-1, as well as the blockade of integrin molecules [35, 36].

Inhibitors of Growth Hormone Action

Inhibition of growth hormone action might be a potential pharmacological treatment for diabetic retinopathy. Interest in the field has emerged when the spontaneous resolution of proliferative diabetic retinopathy in a woman in whom acute panhypopituitarism had developed stimulated interest in pituitary ablation as a treatment for vision-threatening retinopathy [37–39]. Therefore, destruction of the pituitary by surgery or radiation has been used to treat proliferative retinopathy [40]. Long-term follow-up of patients who underwent pituitary ablation due to yttrium-90 implantation for treatment of proliferative diabetic retinopathy revealed either stabilization or improvement in visual acuity, including improvement in the grading of hard exudates, microaneurysms and hemorrhages [41–43].

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Growth hormone action is mediated through the IGFs. Preclinical studies suggested that IGF-1 is not itself a vasoproliferative factor, but rather a strong permissive agent suggesting that neovascularization cannot occur in its absence but must be accompanied by other proangiogenic molecules such as VEGF to stimulate new vessel growth. These studies provide the rationale for the use of blockers of IGF-1 secretion, either by destroying the pituitary or by more specific inhibition of IGF-1 production [44, 45].

Long-Acting Somatostatin Analogue Treatment in Diabetic Retinopathy

The peptide somatostatin was defined in the 1960s as a molecule that inhibits the release of growth hormone. The physiological actions of somatostatin are primarily inhibitory. It affects calcium and potassium ion channels, leads to tyrosine phosphatase activation, modulates secretion of neuroendocrine cells, and may also affect cell proliferation [46, 47]. Somatostatin regulates several organ systems, including the retina and vascular endothelial cells, acts as a classical hormone, a neurohormone, as a neurotransmitter, and exerts autocrine and paracrine functions. The biological effects are mediated by 5 membrane-bound specific receptors, SSTR1–SSTR5. All receptors are G-protein-coupled receptors with 7 transmembrane-spanning domains linked to adenylate cyclase. SSTR genes are widely expressed in normal human eye tissues, with genes for SSTR1 and SSTR2 being the most widely expressed [48, 49]. SSTR2 and SSTR3 are the most important receptor subtypes mediating growth hormone secretion and endothelial cell cycle arrest, retinal endothelial cell apoptosis. SSTR expression suggests that somatostatin and its analogues will have a target in various compartments of the eye [50–53]. A number of uncontrolled clinical studies have used somatostatin analogue treatment in the context of diabetic retinopathy [for a summary, see ref. 54, 55]. Various dosages of the somatostatin analogues (minimal dosage per day 150 ␮g, maximal dosage per day 500 ␮g of SMS 201-995; 1,500 ␮g/day of BMI23014) have been applied to patients with proliferative retinopathy [56–61] and cystoid macular edema [62]. The drugs were used for a variable length of time, ranging from 12 weeks up to a maximum of 12 months. Some studies reported effects on the suppression of growth hormone levels, stabilization of neovascularizations, resorption of hemorrhages, and reduction in the number of microaneurysms, respectively. In a case report, effective treatment of a macular edema was also found [62].


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Two well-controlled trials have studied the delay to laser therapy and improvements in patients with persistence of proliferations following laser treatment [63, 64]. Grant et al. [63] have studied patients with severe nonproliferative diabetic retinopathy or early non-high-risk proliferative retinopathy. At this stage of diabetic retinopathy, the likelihood for the need of panretinal photocoagulation is high. The somatostatin analogue octreotide was titrated in 11 patients to the maximally tolerated dose for a 15-month period. From 200 up to 5,000 ␮g/day octreotide was used. Only 1 of 22 eyes of octreotide-treated patients required panretinal photocoagulation, whereas 9 of 24 eyes in the control group had to be laser treated. The incidence of ocular disease progression was only 27% in patients treated with octreotide compared with 42% in patients with conventional management. This study provided the first clear evidence that octreotide treatment retarded progression of advanced retinopathy and delayed the time for laser photocoagulation. Boehm et al. [64] reported the use of octreotide in a cohort of diabetic patients with a very advanced stage of proliferative diabetic retinopathy, i.e. the presence of active proliferations after full scatter laser treatment. Three hundred micrograms per day of octreotide was used in 9 patients, and 9 patients with standard diabetes management served as controls. The dose of 300 ␮g/day is roughly equivalent to a 30-mg dose of the long-acting LAR formulation of octreotide. Ophthalmologists who defined end points of this intervention were masked throughout the study. The observation period was the longest ever reported in a trial using a somatostatin analogue for the treatment of diabetic complications. After 3 years of treatment, the incidence of vitreous hemorrhages was significantly lower in the octreotidetreated patients. Visual acuity was also preserved and significantly improved over time in the octreotide-treated group. Only in the group of patients with octreotide treatment, a regression of proliferations and fibrovascular changes, as defined by stereoscopic photography and fluorescein angiography, was found.

Mechanisms of Efficacy

The effects of octreotide on the progression of retinopathy may be explained by a least partial systemic suppression of a system overproduction of growth hormone and a partial correction of the associated imbalance of the IGF-1 system components. In addition, the recognition that SSTR subtypes are expressed at the retina provides evidence that a direct inhibition of locally produced growth factor molecules, including a direct inhibition of angiogenic response, and an antifibrotic action may also take place. The expression pattern of both somatostatin and its receptors on various cellular components of the human eye makes it highly likely that somatostatin has

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regulatory functions. The observed positive effects in cystic maculopathy and the positive case reports in patients with macular edema make it highly likely that somatostatin plays a direct role in fluid evasion or resorption. Most probably, the pigment epithelium cell is responsible for a favorable exchange of fluids. This suggests that somatostatin may exert direct effects beyond a blockade of the IGF-1 system, since SSTR subtypes are expressed at various eye components, including the retina. This may also explain why the growth hormone receptor blocker pegvisomant was found ineffective in a recently published pilot trial [65]. In a 3-month, open-label study, pegvisomant did not cause regression of the new retinal vessels in patients with non-high-risk proliferative diabetic retinopathy, although plasma levels of IGF-1 decreased significantly by 50% [65].

Side Effects of Long-Acting Analogue Treatment

The side effect profile of long-acting analogue treatment includes the gut and hypoglycemic side effects. This side effect profile has been suggested to strongly argue against a clinical role for the current somatostatin analogues in the treatment of diabetes mellitus [66]. Since somatostatin can inhibit a large variety of physiological functions, including counterregulatory hormone response in the case of hypoglycemia, all trials (including the ongoing trails with octreotide LAR) have carefully monitored the safety of somatostatin use. In the Ulm trial, no severe hypoglycemic events, as defined by help needed from third parties or requirement of hospital admissions, did occur during an observational period of almost 3 years. Two patients complained of abdominal discomfort and increased bowel movements, which could be alleviated by use of an oral pancreatic enzyme supplementation. No gall bladder stones or sludges were noted on the routine ultrasound examinations of the abdomen. However, overall likelihood of gallstone formation is increased with long-acting somatostatin analogue treatment. Long-acting somatostatin analogues can also decrease thyrotropin secretion. Therefore, follow-up procedures should include endocrine management with thyroid hormone replacement when appropriate [67]. Use of thyroid hormone supplementation in patients with diabetes mellitus will reduce the risk of hypoglycemia.

Perspective

Knowledge of the major factors responsible for modulating neovascularization has had significant implications for the development of novel, nondestructive, pharmacologic treatment modalities [67–69]. Substantial


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efforts are under way to develop new therapies that do not result in tissue destruction inherent to laser treatment, including two large, randomized, phase III trials evaluating the efficacy and tolerability/safety of long-acting octreotide (Sandostatin LAR) in preparation for release of full trial results in 2007. Two phase III trials Sandostatin LAR (CSMS 802 trial: placebo vs. Sandostatin LAR® 20 and 30 mg and CSMS 804 trial: placebo vs. Sandostatin LAR® 30 mg) have reported a significant reduction in retinal bleeding. In both trials a risk reduction of vitreous hemorrhage of approximately 60% for octreotide compared to placebo was seen. In the 804 trial octreotide a delayed the time to progression of retinopathy as defined by ETDRS severity scale. Future approaches might include the use of somatostatin analogues as a treatment option for reentry retinopathy and as an adjunct to an ongoing laser therapy, or even in vitreoretinal surgery [53, 54, 70–72]. Whether such a therapy may also prove effective for other retinal vascular proliferative diseases such as retinopathy of prematurity and age-related macular degeneration remains an open question that deserves attention, given our new understanding of the cellular and molecular mechanisms by which somatostatin may exert its antiangiogenic effects. The use of long-acting analogues of the naturally occurring peptide, somatostatin, has evolved as a novel promising therapeutic option for retinopathy over the last decade. Current clinical evidence supports its use in diabetic retinopathy, but further clinical evidence from larger treatment groups of longer trial duration is required. Improved analogues may also help to make the use of somatostatin analogues an option far beyond the treatment of diabetes retinopathy [53, 54, 72]. References 1 2 3

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Poulaki V, Qin W, Joussen AM, Hurlbut P, Wiegand SJ, Rudge J, Yancopoulos GD, Adamis AP: Acute intensive insulin therapy exacerbates diabetic blood-retinal barrier breakdown via hypoxiainducible factor-1␣ and VEGF. J Clin Invest 2002;109:805–815. Early Treatment Diabetic Retinopathy Study Research Group. Photocoagulation for diabetic macular edema: Early Treatment Diabetic Retinopathy Study report number 1. Arch Ophthalmol 1985;103:1796–1806. Early Treatment Diabetic Retinopathy Study Research Group. Early photocoagulation for diabetic retinopathy: ETDRS report number 9. Ophthalmology 1991;98(suppl):766–785. Aiello LP: Clinical implications of vascular growth factors in proliferative retinopathies. Curr Opin Ophthalmol 1997;8:19–31. Duh E, Aiello LP: Vascular endothelial growth factor and diabetes: the agonist versus antagonist paradox. Diabetes 1999;48:1899–1906. Ferrara N, Gerber HP, LeCouter J: The biology of VEGF and its receptors. Nat Med 2003;9:669–676. Poulsen JE: Recovery from retinopathy in a case of diabetes with Simmonds’ disease. Diabetes 1953;2:7–12. Poulsen JE: Diabetes and anterior pituitary insufficiency. Final course and postmortem study of a diabetic patient with Sheehan’s syndrome. Diabetes 1966;15:73–77. Pi-Sunyer FX, Cushman P Jr: Sheehan’s syndrome and diabetes mellitus: observations on the Houssay phenomenon in man. Am J Med Sci 1972;264:143–147. Lundbaek K, Malmros R, Andersen HC, et al: Hypophysectomy for diabetic angiopathy: a controlled clinical trial; in Goldberg MF, Fine SL (eds): Symposium on the Treatment of Diabetic Retinopathy. Washington, Government Printing Office (Public Health Service publication No 1890), 1968. Sharp PS, Fallon TJ, Brazier OJ, Sandler L, Joplin GF, Kohner EM: Long-term follow-up of patients who underwent yttrium-90 pituitary implantation for treatment of proliferative diabetic retinopathy. Diabetologia 1987;30:199–207. Hyer SL, Kohner EM: Aspects of growth hormone control in diabetes. Aust NZ J Ophthalmol 1990;18:33–39. Balodimos MC: Treatment of diabetic retinopathy: pituitary ablation and retinal photocoagulation. Med Clin North Am 1971;55:989–999. Smith LE, Shen W, Perruzzi C, et al: Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor. Nat Med 1999;5:1390–1395. Smith LEH, Kopchick JJ, Chen W, et al: Essential role of growth hormone in ischemia-induced retinal neovascularization. Science 1997;276:1706–1709. von Wichert G, Jehle PM, Hoeflich A, Koschnick S, Dralle H, Wolf E, Wiedenmann B, Boehm BO, Adler G, Seufferlein T: Insulin-like growth factor-I is an autocrine regulator of chromogranin. A secretion and growth in human neuroendocrine tumor cells. Cancer Res 2000;60: 4573–4581. von Wichert G, Haeussler U, Greten FR, Kliche S, Dralle H, Boehm BO, Adler G, Seufferlein T: Regulation of cyclin D1 expression by autocrine IGF-I in human BON neuroendocrine tumour cells. Oncogene 2005;24:1284–1289. Klisovic DD, O’Dorisio MS, Katz SE, Sall JW, Balster D, O’Dorisio TM, Craig E, Lubow M: Somatostatin receptor gene expression in human ocular tissues: RT-PCR and immunohistochemical study. Invest Ophthalmol Vis Sci 2001;42:2193–2201. Patel YC: Somatostatin and its receptor family. Front Neuroendocrinol 1999;20:157–198. Baldysiak-Figiel A, Jong-Hesse YD, Lang GK, Lang GE: Octreotide inhibits growth factorinduced and basal proliferation of lens epithelial cells in vitro. J Cataract Refract Surg 2005;31: 1059–1064. Baldysiak-Figiel A, Lang GK, Kampmeier J, Lang GE: Octreotide prevents growth factor-induced proliferation of bovine retinal endothelial cells under hypoxia. J Endocrinol 2004;180:417–424. Sall JW, Klisovic DD, O’Dorisio MS, Katz SE: Somatostatin inhibits IGF-1 mediated induction of VEGF in human retinal pigment epithelial cells. Exp Eye Res 2004;79:465–476. Grant MB, Caballero S: Somatostatin analogues as drug therapies for retinopathies. Drugs Today (Barc) 2002;38:783–791. Boehm BO, Lustig RH: Use of somatostatin receptor ligands in obesity and diabetic complications. Best Pract Res Clin Gastroenterol 2002;16:493–509.

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Lamberts SWJ, Van Der Lely AJ, De Herder WW, Hofland LJ: Drug therapy: octreotide. N Engl J Med 1996;334:246–254. Hyer SL, Sharp PS, Brooks RA, Burrin JM, Kohner EM: Continuous subcutaneous octreotide infusion markedly suppresses IGF-I levels whilst only partially suppressing GH secretion in diabetics with retinopathy. Acta Endocrinol (Copenh) 1989;120:187–194. Mallet B, Vialettes B, Haroche S, Escoffier P, Gastaut P, Taubert JP, Vague P: Stabilization of severe proliferative diabetic retinopathy by long-term treatment with SMS 201-995. Diabetes Metab 1992;18:438–444. Lee HK, Suh KI, Koh CS, Min HK, Lee JH, Chung H: Effect of SMS 201-995 in rapidly progressive diabetic retinopathy. Diabetes Care 1988;11:441–443. Kirkegaard C, Norgaard K, Snorgaard O, Bek T, Larsen M, Lund-Andersen H: Effect of one year continuous subcutaneous infusion of a somatostatin analogue, octreotide, on early retinopathy, metabolic control and thyroid function in type I (insulin-dependent) diabetes mellitus. Acta Endocrinol (Copenh) 1990;122:766–772. Shumak SL, Grossman LD, Chew E, Kozousek V, George SR, Singer W, Harris AG, Zinman B: Growth hormone suppression and nonproliferative diabetic retinopathy: a preliminary feasibility study. Clin Invest Med 1990;13:287–292. McCombe M, Lightman S, Eckland DJ, Hamilton AM, Lightman SL: Effect of a long-acting somatostatin analogue (BIM23014) on proliferative diabetic retinopathy: a pilot study. Eye 1991;5: 569–575. Kuijpers RW, Baarsma S, van Hagen PM: Treatment of cystoid macular edema with octreotide. N Engl J Med 1998;338:624–626. Grant MB, Mames RN, Fitzgerald C, Hazariwala KM, Cooper-DeHoff R, Caballero S, Estes KS: The efficacy of octreotide in the therapy of severe nonproliferative and early proliferative diabetic retinopathy: a randomized controlled study. Diabetes Care 2000;23:504–509. Boehm BO, Lang GK, Jehle PM, Feldmann B, Lang GE: Octreotide reduces risk for vitreous hemorrhages and loss of visual acuity in patients with high risk proliferative diabetic retinopathy. Horm Metab Res 2001;33:300–306. Growth Hormone Antagonist for Proliferative Diabetic Retinopathy Study Group. The effect of a growth hormone receptor antagonist drug on proliferative diabetic retinopathy. Ophthalmology 2001;108:2266–2272. Davies RR, Turner SJ, Alberti KG, Johnston DG: Somatostatin analogues in diabetes mellitus. Diabet Med 1989;6:103–111. Colao A, Merola B, Ferone D, Marzullo P, Cerbone G, Longobardi S, Di Somma C, Lombardi G: Acute and chronic effects of octreotide on thyroid axis in growth hormone-secreting and clinically non-functioning pituitary adenomas. Eur J Endocrinol 1995;133:189–194. Aiello LP: Clinical implications of vascular growth factors in proliferative retinopathies. Curr Opin Ophthalmol 1997;8:19–31. Duh E, Aiello LP: Vascular endothelial growth factor and diabetes: the agonist versus antagonist paradox. Diabetes 1999;48:1899–1906. Ferrara N, Gerber HP, LeCouter J: The biology of VEGF and its receptors. Nat Med 2003;9: 669–676. Porta M, Allione A: Current approaches and perspectives in the medical treatment of diabetic retinopathy. Pharmacol Ther 2004;103:167–177. Croxen R, Baarsma GS, Kuijpers RW, van Hagen PM: Somatostatin in diabetic retinopathy. Pediatr Endocrinol Rev 2004;1(suppl 3):518–524.

Bernhard O. Boehm, MD Division of Endocrinology and Diabetes, Ulm University Robert-Koch-Strasse 8 DE–89081 Ulm (Germany) Tel. ⫹49 731 500 44504, Fax ⫹49 731 500 44506, E-Mail [email protected]


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Vascular Endothelial Growth Factor and the Potential Therapeutic Use of Pegaptanib (Macugen®) in Diabetic Retinopathy Carla Staritaa, Manju Patelb, Barrett Katzc, Anthony P. Adamisc a

Pfizer Ltd., Sandwich, UK; bPfizer Inc., and c(OSI) Eyetech, New York, N.Y., USA

Abstract Both clinical and preclinical findings have implicated vascular endothelial growth factor (VEGF) in the pathophysiology of diabetic macular edema (DME). VEGF is both a potent enhancer of vascular permeability and a key inducer of angiogenesis. VEGF levels are elevated in the eyes of patients with DME, and in animal models of diabetes this elevation coincides with the breakdown of the blood-retinal barrier. Moreover, injection of VEGF (the VEGF165 isoform in particular) into healthy eyes of animals can induce diabetes-associated ocular pathologies.Pegaptanib, a novel RNA aptamer currently used in the treatment of agerelated macular degeneration, binds and inactivates VEGF165 and has been shown in animal models to reverse the blood-retinal barrier breakdown associated with diabetes. These findings formed the basis of a phase II trial involving 172 patients with DME, in which intravitreous pegaptanib (0.3 mg, 1 mg, 3 mg) or sham injections were administered every 6 weeks for 12 weeks, with the option of continuing for 18 more weeks or undergoing laser treatment. Compared to sham, patients receiving 0.3 mg displayed superior visual acuity (p ⫽ 0.04) as well as a reduction in retinal thickness of 68 micrometers compared to a slight increase under sham treatment (p ⫽ 0.021). These data support the use of pegaptanib in the treatment of DME. Copyright © 2007 S. Karger AG, Basel

Diabetic retinopathy, a retinal vascular disorder that occurs as a complication of diabetes mellitus, is one of the leading causes of blindness worldwide. It accounts for an estimated 15–17% of the 2.7 million individuals suffering from blindness in the European Union [1]. In the United States, an estimated 4.1 million individuals aged 40 and over are affected by diabetic retinopathy, with nearly 900,000 having vision-threatening disease [2]. Furthermore, the

prevalence of diabetic retinopathy is expected to rise as the number of people with diabetes increases due to the demographic effects of population growth, aging and urbanization and the growing prevalence of obesity and physical inactivity [3]. This will further add to the human and economic burden that diabetic retinopathy and its ensuing vision loss are already imposing on our society [2]. Severe visual loss in patients with diabetes occurs primarily as a consequence of retinal neovascularization and complications resulting from intraocular angiogenesis; moderate visual loss results primarily from diabetic macular edema (DME) related to altered permeability of the retinal vasculature. Proliferative diabetic retinopathy is more commonly reported in patients with type 1 diabetes, whereas DME is more commonly associated with type 2 diabetes [4]. While the pathogenesis of diabetic retinopathy is incompletely understood, evidence suggests that it is one of several ocular diseases characterized by neovascularization and increased vascular leakage ultimately driven by the effects of vascular endothelial growth factor (VEGF) [5–7]. Laser photocoagulation is the current standard of care for the treatment of sight-threatening diabetic retinopathy. Focal photocoagulation, primarily used for treating DME, applies small-sized burns to leaking microaneurysms, while scatter (panretinal) photocoagulation is employed for proliferative retinopathy and indirectly treats neovascularization by placing burns throughout the fundus [8]. While the use of laser photocoagulation has greatly reduced the risk of developing severe vision loss, this is accomplished by attendant destruction of retinal tissue that can lead to side effects, such as loss of peripheral vision, alterations in color perception, and perceptions of night blindness. Pars plana vitrectomy is another option for treating complications of severe proliferative retinopathy and/or hemorrhage [5, 8]. Although instrumentation and surgical techniques have improved during the past decade [9], pars plana vitrectomy is still associated with several complications [5, 9–11], and for this reason, visual acuity (VA) outcomes are still poor [9]. The mechanism by which scatter laser photocoagulation reduces proliferative retinopathy is not known. It has been proposed that light energy absorbed by melanin in the retinal pigment epithelium destroys highly metabolically active outer retinal cells, reducing retinal oxygen consumption and facilitating improved oxygen diffusion from the choriocapillaris through the laser scars [12]. Increased oxygen tension may lead to vasoconstriction, further reducing the edema. Therefore, laser photocoagulation is directed at reducing the retinal neovascularization or macular edema rather than reversing the underlying biological process of diabetic retinopathy. The risk of VA loss is reduced, and substantial recovery of reduced VA is relatively unusual [4, 5, 13].

Pegaptanib (Macugen®) in Diabetic Retinopathy

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Because of the limitations, potential side effects and complications of currently available treatments for diabetic retinopathy, research continues to be directed toward the development of novel, more effective and nondestructive therapeutic modalities. Over the past decade, a large body of work has established VEGF as a major regulator of both physiological and pathological vessel growth and vascular permeability, and that it plays a key role in ocular neovascular diseases, such as age-related macular degeneration and diabetic retinopathy [6]. Importantly, studies in animal models have demonstrated that the single isoform VEGF164 (the rodent counterpart of human VEGF165) is particularly important in the pathogenesis of diabetic retinopathy [14]. Macugen® (pegaptanib sodium), a pegylated synthetic ribonucleic acid (RNA) oligonucleotide, specifically inhibits the actions of VEGF165 [15]. The oligonucleotide portion of the molecule, called an aptamer, was designed to bind selectively to extracellular VEGF165 as compared with antisense oligonucleotides, which have an intracellular site of action. To increase the in vivo residence time of pegaptanib, a 40-kDa branched polyethylene glycol molecule has been conjugated to the oligonucleotide. Based on the efficacy demonstrated in two large, multicenter, randomized clinical trials (the VEGF Inhibition Study in Ocular Neovascularization, or VISION, trials) [16], Macugen has been approved for the treatment of neovascular age-related macular degeneration in the United States, Canada and Brazil and has received a recommendation for market authorization by the Committee for Human Medicinal Products of the European Union. The selective pharmacologic blockade of the 165 isoform of VEGF with pegaptanib also has potential applicability in the treatment of other diseases characterized by retinal revascularization and increased retinal vascular permeability. Indeed, a phase II clinical trial exploring its safety and efficacy in patients with DME has reported encouraging early results [17]. This chapter will first review the role of VEGF165 in the pathogenesis of ocular neovascular diseases, including diabetic retinopathy. A description of the development of pegaptanib sodium as an anti-VEGF agent will follow, together with a review and discussion of the recent phase II clinical trial evaluating its use in patients with DME [17]. The findings from this trial not only validate the hypothesis that VEGF165 plays an important role in the pathogenesis of diabetic retinopathy, but also offer the promise of a new and less destructive treatment option for DME.

VEGF in Ocular Neovascular Disease

VEGF Is a Pluripotent Growth Factor VEGF (also known as VEGF-A) is a member of the VEGF-plateletderived growth factor family, which also includes VEGF-B, VEGF-C, VEGF-D

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Table 1. Pro- and antiangiogenic factors [21] Proangiogenic factors

Antiangiogenic factors

Adrenomedullin Angiogenin Angiopoietin-1 Angiopoietin-related growth factor Brain-derived neurotrophic factor Corticotropin-releasing hormone Cyr16 Erythropoietin Fibroblast growth factors: acidic and basic Follistatin Granulocyte colony-stimulating factor Hepatocyte growth factor/scatter factor IL-3, IL-8 Midkine Nerve growth factor Neurokinin A Neuropeptide Y Pigment epithelium-derived growth factor Placental growth factor Platelet-derived endothelial cell growth factor Platelet-derived growth factor Pleiotrophin Progranulin Proliferin Secretoneurin Substance P Transforming growth factor-␣ Transforming growth factor-␤ Tumor necrosis factor-␣ VEGF

Angioarrestin Angiostatin (plasminogen fragment) Antiangiogenic antithrombin III Cartilage-derived inhibitor CD59 complement fragment Endostatin (collagen XVIII fragment) Fibronectin fragment Growth-related oncogene (Gro-␤) Heparinases Heparin hexasaccharide fragment Human chorionic gonadotropin IL-12 Interferon-␣, -␤, -␥ Interferon-inducible protein (IP-10) Kringle 5 (plasminogen fragment) Metalloproteinase inhibitors Pigment epithelium-derived growth factor Placental ribonuclease inhibitor Plasminogen activator inhibitor Platelet factor 4 Prolactin, 16-kDa fragment Proliferin-related protein Retinoids Tetrahydrocortisol-S Thrombospondin-1 Transforming growth factor-␤ 2-Methoxyestradiol Vasculostatin Vasostatin (calreticulin fragment)

and VEGF-E [for a general review of VEGF, see ref. 18]. It was isolated independently by 2 groups, first as a vascular permeability factor [19] and second as a potent endothelial cell mitogen [20]. Although investigations during the 1980s suggested numerous proangiogenic and antiangiogenic factors, in a list that has since continued to grow (table 1) [21], only VEGF convincingly showed all the characteristics of a necessary and sufficient inducer of angiogenesis [22]. Alternative splicing of the VEGF gene yields at least 6 distinct biologically active human isoforms, each comprised of a differing number of amino acids (e.g., 121, 145, 165, 183, 189 and 206). VEGF165, the predominant


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isoform, is a 45-kDa homodimeric glycoprotein existing both free in the cytoplasm and bound through a heparin-binding domain to the cell surface and extracellular matrix and is the isoform principally responsible for mediating the pathological effects of VEGF in ocular neovascular diseases [23, 24]. VEGF189 and VEGF208 also contain this heparin-binding domain and are strongly basic, and for the most part, they are sequestered in the extracellular matrix while VEGF121 is acidic, lacks the heparin-binding domain and is secreted [25]. VEGF is a ligand for 2 receptor tyrosine kinases, VEGFR-1 and VEGFR-2, mediating their activation of downstream signal transduction cascades [18]. While VEGFR-2 is believed to be the principal receptor for VEGF signaling in angiogenesis [18], VEGFR-1 also plays a key role in pathological ocular neovascularization through mediating monocyte chemotaxis to VEGF [24, 26]. Much research effort has been applied toward understanding the function of VEGF with the goal of inhibiting, if not reversing, pathological angiogenesis. However, it is becoming increasingly evident that VEGF is a pluripotent growth factor that is active not only in angiogenesis but also in a variety of physiological contexts. For example, there is recent evidence that VEGF serves as a neurotrophic role, lending hope that the administration of VEGF may have benefits in the treatment of neurodegenerative diseases and optic neuropathies [27, 28]. In the eye, VEGF121 appears to be sufficient to exert this neuroprotective action, which may serve to counteract the effects of retinal ischemia [29]. In addition, VEGF secretion by the retinal pigment epithelium has been implicated in trophic maintenance of the choriocapillaris [30], much like the trophic role it plays in other vascular beds [31]. VEGF also has been implicated in a variety of other vital and required physiological processes, including bone growth [32, 33], wound healing [34, 35], female reproductive cycling [32, 36], vasorelaxation [37], skeletal muscle regeneration [38], glomerulogenesis [39] and protection of hepatic cells [40]. Given this wide range of actions, antiangiogenic therapies that target VEGF need vigorous monitoring of safety, an issue of particular relevance for systemic administration [41]. In this context, there is already evidence that intravenous administration of a monoclonal antibody that binds all VEGF isoforms is associated with an increased incidence of hypertension, thromboembolism and hemorrhage [42– 47]. VEGF in Physiological and Pathological Angiogenesis VEGF has a variety of properties in physiological and pathological neovascularization (table 2) [14, 18, 19, 23, 24, 26, 48–55]. In addition to its role as a potent endothelial cell mitogen, VEGF serves as an endothelial cell survival factor [56] and as a chemoattractant for bone marrow-derived endothelial cells [48, 57, 58]. It also induces the synthesis of several enzymes whose actions affect angiogenesis, including the matrix metalloproteinases and plasminogen activator; together, these promote degradation of the extracellular matrix,


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Table 2. Key properties of VEGF in physiological and pathological angiogenesis [14, 18, 19, 23, 24, 26, 48–55] Endothelial cell mitogen Endothelial cell survival factor Chemoattractant for bone marrow-derived endothelial cells Potent enhancer of vascular permeability Expression induced by hypoxia Chemoattractant for monocyte lineage cells Proinflammatory cytokine, promoting leukocyte adhesion Inducer of synthesis of key enzymes Matrix metalloproteinases Plasminogen activator Endothelial nitric oxide synthase

permitting blood vessel extravasation [49–51]. VEGF also induces endothelial nitric oxide synthase, leading to the upregulation of nitric oxide, a stimulator of angiogenesis [52, 53]. In addition, VEGF acts as a chemoattractant for monocyte lineage cells, which are believed to contribute to pathological ocular neovascularization [23, 24], and to promote local adhesion of leukocytes [14, 54]. Historically, much of the impetus for the isolation of angiogenic factors stems from the hypothesis that antiangiogenic approaches could serve to starve malignant tumors [59]. VEGF was evaluated in this context in the early 1990s when it was found that tumor vascularization and growth could indeed be inhibited by injections of a monoclonal antibody to VEGF [60]. Subsequently, the role of VEGF in supporting tumor growth was intensively examined, with the first anti-VEGF agent developed for clinical use (bevacizumab, an anti-VEGF monoclonal antibody) as an anticancer therapeutic [61]. VEGF has been implicated in several other classes of disorders involving dysregulation of angiogenesis, including hematological malignancies, inflammation, brain edema and several pathological conditions of the female reproductive tract [18]. In the course of evaluating the properties of VEGF, 2 were recognized that are of particular relevance to the pathogenesis of diabetic retinopathy. First, synthesis of VEGF is upregulated by hypoxia, which provides a mechanistic basis for VEGF-mediated ocular neovascularization in response to ischemia [18]. Secondly, VEGF is the most potent known inducer of vascular permeability, 50,000 times more potent than histamine [55]. Several additional mechanisms contributing to VEGF-mediated increases in vascular permeability have been elucidated, including induction of fenestrations in the endothelium [62], dissolution of tight junctions [63] and induction of leukostasis and subsequent injury to the endothelium [54, 64]. These properties of VEGF support a direct


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involvement of VEGF in the macular edema that often accompanies diabetic retinopathy. VEGF in Ocular Neovascularization An extensive series of clinical and preclinical investigations has confirmed that VEGF plays a central role in promoting ocular neovascularization [7, 65–73]. Clinical studies have demonstrated elevated ocular levels of VEGF in patients with anterior segment neovascularization [7], retinal vein occlusion [7], neovascular glaucoma [74], retinopathy of prematurity [75], and DME [76 –78]. In other studies, increased expression of VEGF was detected within the macula of patients with age-related macular degeneration when compared with controls [79] and in choroidal neovascular membranes from patients with either age-related macular degeneration or diabetic retinopathy [80–82]. Preclinical studies examining VEGF in a variety of animal models of ocular neovascularization demonstrated that increased intraocular levels of VEGF can induce ocular neovascularization and that inactivation of VEGF in the eye can prevent the occurrence of ocular neovascularization [67–73, 83–90]. In one of the first of these preclinical studies, Miller et al. [83] reported that experimentally induced retinal vein occlusion in monkeys resulted in iris neovascularization and an associated increase in ocular VEGF levels. The severity of iris neovascularization was proportional to the concentration of VEGF [83]. In other studies, injection of VEGF into the vitreous of monkeys produced many of the features characteristic of diabetic retinopathy, including intraretinal and preretinal neovascularization, microaneurysm formation, intraretinal hemorrhage and edema, and areas of capillary nonperfusion with endothelial cell hyperplasia [72, 84]. Qualitatively similar data were obtained using molecular biological techniques. Injection of recombinant adenovirus vectors expressing VEGF into rodent eyes increased VEGF production in the retinal pigment epithelium, with resulting choroidal neovascularization [85, 86]. The severity and extent of vascular proliferation correlated with the amount of virus delivered [86]. Similarly, ocular neovascularization occurred in transgenic mice engineered to overexpress VEGF in the retinal pigment epithelium [73] or in photoreceptors [87]. In the latter study, neovascularization was sufficient in some instances to cause retinal detachment [87]. A variety of models have also been employed to demonstrate that blockade of VEGF and its receptors can inhibit the development of ocular neovascularization. Injection of anti-VEGF antibodies was shown to prevent the neovascularization of the monkey iris that normally followed laser occlusion of the retinal vein [68], and antibodies or their Fab fragments were also effective in preventing choroidal neovascularization in a photocoagulation-induced model in monkeys [70] and in a rat corneal wound model [67]. Other approaches for


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inactivating VEGF that have prevented ocular neovascularization included administration of soluble VEGFR chimeric proteins by injection [69, 88] or expression from an adenovirus vector [89], injection of pegaptanib [14], and injection of an anti-VEGF antisense oligonucleotide [90]. VEGF in Diabetic Retinopathy While the pathophysiology of diabetic retinopathy involves a complex interaction between many factors, current evidence supports a pivotal role of VEGF. Progression of diabetic retinopathy begins with alterations in the retinal vasculature characterized by the degeneration of retinal capillary pericytes, thickening of the basement membrane, and adhesion of leukocytes to the endothelium. These changes are accompanied by blockages of retinal capillaries, loss of endothelial cells, and the formation of acellular vessels, resulting in areas of local nonperfusion [4, 91]. The resultant hypoxia leads to local upregulation of factors such as VEGF [92]. Many retinal cell types express VEGF, including all classes of neurons, glia, endothelial cells, pericytes, and retinal pigment epithelium cells [30, 93, 94]. Hypoxia leads to dramatic increases in VEGF expression from these cells [30, 93, 95]. Several biochemical pathways are believed to be important in linking hyperglycemia to vascular injury in the retina, including the accumulation of polyols, advanced glycation end products and reactive oxygen intermediates; these compounds can produce vascular injury by affecting cellular metabolites and by induction of growth factors [4, 96]. Both advanced glycation end products [97] and reactive oxygen intermediates [98] can directly induce VEGF expression. While causative mechanisms remain to be fully elucidated, increased VEGF levels have been consistently observed in eyes with diabetic retinopathy [7, 65, 66, 99–101]. Early studies demonstrated that VEGF levels were higher in eyes with proliferative diabetic retinopathy than those with nonproliferative diabetic retinopathy; this finding has since been corroborated by other investigators [99–101]. Several other factors were subsequently shown to be elevated in conjunction with VEGF in diabetic retinopathy, including interleukin (IL)-6 [100], stromal-derived factor 1 [101] and angiopoietin II [102]. Similarly, elevated levels of VEGF, together with angiotensin II [76], IL-6 [77], stromal-derived factor 1 [101], and intercellular adhesion molecule (ICAM)-1 [78], have also been demonstrated in association with DME. In recent work, both VEGF and erythropoietin levels were found to be independent predictors of proliferative diabetic retinopathy [103]. To what extent these various factors act independently of VEGF production is unclear; both angiotensin II [104, 105] and stromal-derived factor 1 [106] induce VEGF production while ICAM-1 is upregulated in response to VEGF [24, 107].


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Normalized VEGF mRNA (arbitrary units, mean)

40

p⬍ 0.0001

30

20

VEGF120 VEGF164 VEGF188

10

0 Control (n ⫽ 5)

Diabetes (n⫽ 6)

Fig. 1. Retinal VEGF mRNA levels are increased in early diabetes. Adapted from Qaum et al. [108].

More recent evidence suggests that the proinflammatory effects of VEGF are important in the pathogenesis of ocular neovascular diseases such as diabetic retinopathy. Specifically, VEGF-mediated upregulation of ICAM-1, an adhesion receptor for leukocytes, may provide a link connecting some of the vascular changes seen in diabetic retinopathy to elevated VEGF levels. This conclusion is based on 2 lines of evidence. First, studies demonstrated that expression of both retinal VEGF mRNA [108] and ICAM-1 [109] was upregulated in rodent models of diabetic retinopathy. Second, studies in nondiabetic rats found that retinal ICAM-1 was upregulated in response to VEGF [24, 54]. Subsequently to its upregulation, ICAM-1 may then contribute to the vascular damage characteristic of diabetic retinopathy by promoting leukocyte entrapment (leukostasis) in capillaries, with accompanying local nonperfusion, vascular leakage and endothelial cell damage. Importantly, VEGF165 has been established as the predominant pathological isoform responsible for inflammation and vascular injury characteristic of diabetic retinopathy as well as the ocular neovascularization that follows ischemia [14, 23, 108]. In rats made diabetic by injection of streptozotocin, retinal VEGF levels were increased 3.2-fold after 1 week (fig. 1) [108]. The effects of this elevation may have been compounded by the enhanced pathogenicity of VEGF164; VEGF164 has been shown to be approximately twice as potent as VEGF120 (the rodent counterpart of human VEGF121) in mediating the upregulation of


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ICAM-1 and in producing leukocyte adhesion and blood-retinal barrier breakdown in a diabetic retinopathy model [14]. Moreover, leukocyte adhesion and breakdown of the blood-retinal barrier were significantly suppressed in both early and late diabetes by intravitreous injection of pegaptanib, which specifically targets VEGF165/164 [14]. Although suppression of blood-retinal barrier breakdown was somewhat diminished in established diabetes, these results suggest that pegaptanib has the potential to reverse many of the features of diabetic retinopathy. Ishida et al. [23] have provided further evidence of the inflammatory role of VEGF165 in hypoxia-related ocular neovascularization, which may also have relevance to diabetic retinopathy. Using a mouse model system approximating retinopathy of prematurity, VEGF164 was dramatically increased, compared with VEGF120, in retinas undergoing pathological neovascularization. Injection of a VEGFR-Fc fusion protein that provides a pan-isoform blockade inhibited both physiological and pathological neovascularization, while pegaptanib inhibited only the pathological form [23]. Since pegaptanib is specific to VEGF164/165, this finding suggested that VEGF164/165 is especially important in mediating pathologic neovascularization yet is dispensable for normal physiological vascularization in the eye. Studies in transgenic mice lacking VEGF164 yielded similar results in that VEGF164-deficient mice exhibited no negative effects with respect to physiological vascularization [23]. VEGF164 has also been found to be dispensable for VEGF-mediated neuroprotective effects in the rat eye, providing further evidence that the actions of VEGF164/165 can be inactivated without producing adverse ocular effects [29]. These data support a proinflammatory role of VEGF165 in pathological neovascularization and suggest that pegaptanib, by targeting only the VEGF165 isoform, could be both an effective and safe treatment for ocular neovascular diseases, findings that have been confirmed in clinical trials of pegaptanib in the treatment of age-related macular degeneration and DME [16].

Development of the Anti-VEGF Aptamer Pegaptanib

Pegaptanib is a nuclease-resistant, 28-nucleotide RNA aptamer that binds to VEGF165 with high affinity (with a dissociation constant of approximately 0.2 nM) while showing little affinity for VEGF121 [110]. It is highly stable in biological fluids due to chemical modifications designed to increase resistance to nucleases and has also been modified by addition of a 40-kDa polyethylene glycol moiety to the 5⬘ terminus to increase bioavailability by decreasing clearance from the vitreous [data on file, (OSI) Eyetech, Inc.; 110 –112]. Pegaptanib is a potent inhibitor of the interaction between VEGF165 and its cellular receptors,


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with the concentration for 50% inhibition of 125I-labeled VEGF (10 ng/ml) binding to cultured human endothelial cells ranging from 0.75 to 1.4 nM; total inhibition of VEGF binding occurs at 10 nM [15]. Perhaps the finding most relevant to diabetic retinopathy is that pegaptanib is able to inhibit VEGF-mediated endothelial cell mitogenic and vascular permeability effects. Specifically, cultured endothelial cell proliferative responses to VEGF165, but not to VEGF121, were inhibited when cells were pretreated with pegaptanib [15]. In addition, vessel leakage produced in response to VEGF (as demonstrated using an animal microvascular permeability assay) was inhibited by 83% when VEGF was preincubated with 0.1 ␮M pegaptanib [110]. These findings were further supported by studies demonstrating that intravitreous injection of pegaptanib significantly reduced ocular neovascularization using rodent models of corneal angiogenesis and retinopathy of prematurity [113]. Studies in rhesus monkeys demonstrated that the pharmacokinetic properties of pegaptanib were appropriate for a therapeutic agent, with elimination half-lives following intravenous or subcutaneous injections of 9.3 and 12.0 h, respectively [114]. Additional work in monkeys established that pegaptanib was removed from the eye following intravitreous injection through plasma clearance, with a half-life of approximately 94 h [111]. Pegaptanib was detectable in the eye for 28 days after a single 0.5-mg intravitreous injection and retained full biological activity [111]. Such preclinical studies led to early phase I/II clinical trials testing the safety of pegaptanib when administered by intravitreous injection to patients with age-related macular degeneration [113, 115]. The efficacy of pegaptanib in treating age-related macular degeneration was demonstrated in 2 concurrent phase III trials, the VISION trials, involving a total of 1,208 patients [16]. Pegaptanib (0.3, 1 or 3 mg) or sham injections were administered intravitreously every 6 weeks for a period of 48 weeks. In a combined analysis, 70% (206/ 294) of patients receiving 0.3 mg of pegaptanib (p ⬍ 0.001), 71% (213/300) receiving 1 mg (p ⬍ 0.001) and 65% (193/296) receiving 3 mg (p ⫽ 0.03) lost ⬍15 letters of VA on the study eye chart between baseline and week 54, compared with 55% (164/296) of patients receiving sham injections (primary efficacy endpoint). There was no evidence of a dose-response relationship. The 0.3-mg pegaptanib dose also showed significant benefits for additional secondary endpoints (table 3) [16], including mean change in VA (fig. 2), VA loss of 30 letters or more, and the likelihood of maintaining or gaining VA. Pegaptanib demonstrated clinical benefits irrespective of angiographic subtype, size of lesion, baseline VA, sex, age, race or iris pigmentation [16, 116]. Early detection and treatment with pegaptanib appeared to result in superior vision outcomes [117].


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Table 3. Maintenance, gain and severe loss of VA with pegaptanib and sham injection [16] Endpoints

Pegaptanib 0.3 mg (n ⫽ 294)

Sham (n ⫽ 296)

Loss of ⬍15 letters p value versus sham injection Maintenance or gain of ⱖ0 letters p value versus sham injection Gain of ⱖ5 letters p value versus sham injection Gain of ⱖ10 letters p value versus sham injection Gain of ⱖ15 letters p value versus sham injection Loss of ⱖ30 letters p value versus sham injection

206 (70) ⬍0.001 98 (33) 0.003 64 (22) 0.004 33 (11) 0.02 18 (6) 0.04 28 (10) ⬍0.001

164 (55) 67 (23) 36 (12) 17 (6) 6 (2) 65 (22)

Data are indicated as number of patients, with figures in parentheses as percentages. Where data were missing, the last-observation-carried-forward method was used. Loss of 30 or more letters was defined as severe loss of VA. p values were calculated with the use of the Cochran-Mantel-Haenszel test.

Mean change in vision (letters)

0

Pegaptanib sodium 0.3 mg (n⫽294) Sham (n ⫽296)

⫺2 ⫺4 ⫺6 ⫺8 ⫺10 ⫺12

p ⬍0.05 at all prespecified endpoints (weeks 6, 12 and 54)

⫺14 ⫺16 0

6

12

18

24

30

36

42

48

54

Time (weeks)

Fig. 2. Mean change in VA at week 54 in patients receiving 0.3 mg of pegaptanib or sham injection in the pivotal phase III VISION trials. With permission from Ng and Adamis [116].


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The VISION trials established that pegaptanib was well tolerated with a low risk of serious complications, such as endophthalmitis, retinal detachment or traumatic lens injury [16]. These complications were related to the intravitreous injection procedure itself (and therefore modifiable) rather than to the drug. An important safety finding is that there was no evidence that pegaptanib was associated with major systemic adverse events, such as hypertension, thromboembolism or serious hemorrhage, that have been reported with systemically administered, pan-isoform VEGF blockade [42, 44–47]. The positive findings of the VISION trials led to US Food and Drug Administration approval of pegaptanib for the treatment of neovascular age-related macular degeneration. This was a notable milestone in that pegaptanib represented the first aptamer therapeutic approved by a government regulatory agency and the first antiVEGF agent for treatment of an ocular neovascular disease.

Phase II Trial of Pegaptanib in Patients with DME

The accumulating body of evidence supporting an important role of VEGF165 in the pathogenesis of diabetic retinopathy (see above), coupled with the preclinical findings demonstrating that pegaptanib was able to suppress leukostasis and blood-retinal barrier breakdown in established diabetes [14], provided strong theoretical support for evaluating pegaptanib as a therapeutic agent for DME. Intravitreous pegaptanib was evaluated in a phase I study, which identified no safety issues that would preclude the use of pegaptanib in patients with DME [data on file, (OSI) Eyetech, Inc.]. A phase II trial was conducted to further explore the safety and efficacy of pegaptanib in patients with DME [17]. The design and important outcomes of the phase II trial are described below. Study Design The randomized, sham-controlled, double-masked, dose-finding phase II trial enrolled patients of 18 years and older with type 1 or type 2 diabetes [17]. To be eligible, study eyes had to have macular edema involving the center of the macula, as confirmed by optical coherence tomography (OCT), together with leakage from microaneurysms, retinal telangiectasis, or both, demonstrable on fluorescein angiography, and retinal thickening of at least half a disc area involving the central macula. An independent fundus photograph and angiogram reading center confirmed eligibility and appropriate retinal thickness classification (both for study entry and subsequent randomization and stratification) according to baseline fluorescein angiography and OCT assessments. Only patients for whom the investigator judged that photocoagulation could be safely


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withheld for 16 weeks could be enrolled. A best-corrected VA in the study eye from 20/50 to 20/320 and at least 20/100 in the fellow eye were required. Principal exclusion criteria included a history of panretinal, focal photocoagulation, other retinal treatments within the previous 6 months, or abnormalities that would interfere with measurements of VA and fundus photography. Patients with glycosylated hemoglobin levels ⱖ13%, with evidence of severe cardiac disease, clinically significant peripheral vascular disease, or uncontrolled hypertension were excluded. In all, 172 patients were randomized to 4 treatment arms (0.3, 1 and 3 mg pegaptanib or sham injections). Randomization was stratified by study site, size of the thickened retina area (ⱕ2.5 vs. ⬎2.5 disc areas) and baseline VA (letter score ⱖ58 vs. ⬍58). Injections were given at baseline, week 6 and week 12 for a minimum of 3 injections. Thereafter, additional injections were given every 6 weeks up to week 30 (for a maximum of 6 injections) at the discretion of the investigators. Final assessments were made at week 36 or 6 weeks after the last injection. Refraction, VA, an ophthalmologic examination and OCT were performed at baseline and at each visit. Color fundus photography was performed at baseline and every 6 weeks while fluorescein angiography was carried out at baseline and 6 weeks after the last injection. Overall, 169 patients received at least 1 injection, and more than 90% of patients in each treatment group completed the study; among the pegaptanib-treated patients, 49% received the maximum of 6 injections from baseline to week 30 (table 4) [17]. Results Visual Outcomes. Visual outcomes were evaluated in terms of a mean change in best-corrected VA (number of lines and letters gained or lost) and the proportion of patients maintaining baseline VA (0 lines lost) or gaining ⱖ5 (1 line), ⱖ10 (2 lines) or ⱖ15 letters (3 lines). At week 36, all pegaptanib groups demonstrated better VA relative to the sham group. Compared with baseline, 93, 98, 93 and 90% of patients in the 0.3-, 1-, 3-mg and sham groups, respectively, avoided losing 3 or more lines of VA. In the same treatment groups, gains of ⱖ0 letters were seen in 73, 72, 60 and 51% (0.3 mg vs. sham; p ⫽ 0.023), gains of ⱖ5 letters were seen in 59, 44, 31 and 34% (0.3 mg vs. sham; p ⫽ 0.010), gains of ⱖ10 letters were seen in 34, 30, 14 and 10% (0.3 mg vs. sham; p ⫽ 0.003), and gains of ⱖ15 letters were seen in 18, 14, 7 and 7% (0.3 mg vs. sham; p ⫽ 0.012). In these same respective groups, mean changes in VA also favored pegaptanib treatment, with changes of ⫹4.7, ⫹4.7, ⫹1.1 and –0.4 letters (p ⫽ 0.04, 0.05 and 0.55 for the 0.3-, 1- and 3-mg groups compared with the sham group) (table 5) [17]. Moreover, more patients in all the pegaptanib groups maintained or gained acuity, with a 22% absolute increase and a 43% relative increase in the 0.3-mg group compared with the sham group (fig. 3) [17].


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Table 4. Treatment summary (safety population, n ⫽ 169) Pegaptanib

Sham

0.3 mg (n ⫽ 44)

1 mg (n ⫽ 42)

3 mg (n ⫽ 42)

(n ⫽ 41)

Injections received, day 0 to week 30 Mean ⫾ SD Median Range

5.0 ⫾ 1.2 5.0 1–6

5.2 ⫾ 1.0 6.0 3–6

5.0 ⫾ 1.3 6.0 1–6

4.5⫾ 1.5 5.0 1–6

Number of patients receiving 6 injections 5 injections 4 injections 3 injections 2 injections 1 injection 0 injection

21 (48) 11 (25) 5 (11) 6 (14) 0 1 (2) 0

24 (57) 8 (19) 6 (14) 4 (10) 0 0 0

23 (55) 7 (17) 5 (12) 6 (14) 0 1 (2) 0

15 (37) 8 (20) 6 (15) 8 (20) 3 (7) 1 (2) 0

Figures in parentheses are percentages. With permission from the Macugen Diabetic Retinopathy Study Group [17].

Table 5. Changes from baseline to week 36 in VA (intention-to-treat population, n ⫽ 172) Pegaptanib

Mean change in VA from baseline, letters Week 0 Week 6 Week 12 Week 30 Week 36 p value versus sham at week 361

Sham

0.3 mg (n ⫽ 44)

1 mg (n ⫽ 44)

3 mg (n ⫽ 42)

⫹0.4 ⫹1.8 ⫹3.5 ⫹5.4 ⫹4.7 0.04

–0.0 ⫹2.9 ⫹4.3 ⫹4.1 ⫹4.7 0.05

⫹0.2 ⫹3.6 ⫹2.5 ⫹2.3 ⫹1.1 0.55

(n ⫽ 42) ⫹0.9 ⫹1.4 ⫹1.3 ⫹0.6 –0.4

For missing baseline data, day 0 data were used for the analysis. For missing data at subsequent time points, the last observation was carried forward. There were missing relevant data for 1 patient each in the 1-mg and sham groups. 1 The analysis of covariance model was adjusted for the baseline retinal thickening area and baseline vision; p values of pairwise comparisons were unadjusted for multiplicity. With permission from the Macugen Diabetic Retinopathy Study Group [17].


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100 90

Pegaptanib 0.3mg (n ⫽44)

Pegaptanib 1mg (n ⫽44)

Pegaptanib 3mg (n ⫽44)

Sham (n⫽42)

80

Patients (%)

70

* * *

60 50 40

** *

30 20 10 0 ⱖ0 lines

ⱖ1 line

ⱖ2 lines

ⱖ3 lines

Gained

Fig. 3. Percentage of patients treated with pegaptanib maintaining or gaining VA from baseline to week 36 (intention-to-treat population, n ⫽ 172). *p ⬍ 0.05; **p ⬍ 0.01. With permission from the Macugen Diabetic Retinopathy Study Group [17].

Retinal Thickness. Changes in central retinal thickness were evaluated as an anatomic proxy for the presence and extent of macular edema. Mean changes in retinal thickness from baseline to week 36 as determined at the center point were –68.0, –22.7 and –5.3 ␮m for the 0.3-, 1- and 3-mg groups, respectively, compared with ⫹3.7 ␮m for the sham group (0.3 mg vs. sham; p ⫽ 0.021). More patients in the pegaptanib-treated groups experienced an absolute decrease of ⱖ75, ⱖ100 and ⱖ200 ␮m compared with the sham group. Differences in the 0.3-mg group were especially marked, with 49% showing a decrease of ⱖ75 ␮m compared with 19% in the sham group (p ⫽ 0.008); in addition, 42% of the 0.3-mg pegaptanib group had an decrease of ⱖ100 ␮m compared with 16% in the sham arm (p ⫽ 0.02) (table 6) [17]. Baseline and week 36 color fundus photographs and OCT images from a representative patient are presented in figure 4 [17]. Need for Laser Photocoagulation. In the sham group, 48% of patients required additional intervention with photocoagulation therapy between weeks 12 and 36 while only 25, 30 and 40% of patients in the 0.3-, 1- and 3-mg groups needed such treatment (p ⫽ 0.042, 0.090 and 0.537, respectively, compared


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Table 6. Changes from baseline to week 36 in retinal thickness of the center point of the central subfield (intention-to-treat population, n ⫽ 172) Pegaptanib

Sham

0.3 mg (n ⫽ 44)

1 mg (n ⫽ 44)

3 mg (n ⫽ 42)

Retinal thickness, microns Mean at baseline Mean change at week 36 95% confidence interval p value versus sham1

476.0 –68.0 –118.9 to –9.88 0.02

451.7 –22.7 –76.9 to 33.8 0.44

424.7 –5.3 –63.0 to 49.5 0.81

423.2 ⫹3.7

ⱖ75 ␮m decrease from baseline At week 36 Odds ratio 95% confidence interval p value versus sham2

21 (49) 4.1 1.5–11.3 0.008

11 (28) 1.7 0.6–5.0 0.283

9 (25) 1.4 0.5–4.4 0.596

7 (19)


18 (42) 3.7 1.3–10.8 0.021

10 (26) 1.8 0.6–5.5 0.303

7 (19) 1.3 0.4–4.2 0.829

6 (16)


5 (12) 4.7 0.5–42.5 0.126

3 (8) 3.0 0.3–30.2 0.304

2 (6) 2.1 0.2–24.4 0.678

1 (3)

(n ⫽ 42)

Figures in parentheses are percentages. For missing baseline data, day 0 data were used for the analysis. For missing data at week 36, the last observation was carried forward. There are missing relevant data for 1 patient in the 0.3-mg, 5 patients in the 1-mg, 6 patients in the 3-mg and 5 patients in the sham groups. 1 Analysis of covariance model adjusted for the baseline retinal thickening area, baseline vision, and baseline retinal thickness; p value indicates the difference in least square means between each dose group and the sham group. 2 The Cochran-Mantel-Haenszel test was adjusted for the baseline retinal thickening area and baseline vision; p value indicates the difference in odds ratios between each dose group and the sham group. With permission from the Macugen Diabetic Retinopathy Study Group [17].

with sham). For the 0.3-mg group, this difference meant a relative decrease of 44% compared with the sham group (table 7) [17]. Retinal Neovascularization. Fundus photographs of all study patients were graded for severity of diabetic retinopathy by an independent reading center at baseline, week 36 and week 52 using the Early Treatment Diabetic


138

a

b

c

d Fig. 4. a, b Baseline color fundus photograph (a) and optical coherence tomography image (b) before treatment with intravitreous pegaptanib 0.3 mg show intraretinal hemorrhage, microaneurysm formation and exudates as well as a retinal thickness of 422 ␮M with cystic spaces evident at the center of the macula. VA at the time of study entry was 68 Early Treatment Diabetic Retinopathy Study chart letters (Snellen acuity, approximately 20/50). Laser photocoagulation was administered 6 months before enrollment. Adapted from the Macugen Diabetic Retinopathy Study Group [17]. c, d Fundus photograph at week 36 (c) and optical coherence tomography image (d) after 4 intravitreous injections (at day 0, weeks 6, 12 and 24) of pegaptanib 0.3 mg show partial resolution of retinal microaneurysms, hemorrhages and exudates, as well as a marked decrease in retinal thickness to 267 ␮M. VA at week 36 was 79 Early Treatment Diabetic Retinopathy Study chart letters (Snellen acuity, approximately 20/25). No focal laser photocoagulation treatments were administered after enrollment. Adapted from the Macugen Diabetic Retinopathy Study Group [17].

Retinopathy Study severity scale; fluorescein angiograms were also graded in a masked fashion for the presence of neovascularization. A review of all subjects identified 19 of 172 patients who had retinal neovascularization (⬍0.5 disc area in one or more fields) in the study eye at baseline [118]. A retrospective analysis of these patients was done to evaluate the effects of pegaptanib on retinal neovascularization. One of the 19 patients was excluded due to a protocol violation (scatter photocoagulation 13 days before randomization), and 2 were excluded due to the unavailability of follow-up photographs. Of the remaining cohort of


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Table 7. Patients receiving focal/grid laser at week 12 or later in the study eye (intention-to-treat population, n ⫽ 172) Pegaptanib

Sham

0.3 mg (n ⫽ 44)

1 mg (n ⫽ 44)

3 mg (n ⫽ 42)

(n ⫽ 42)

Focal photocoagulation Yes No

11 (25) 33 (75)

13 (30) 31 (70)

17 (40) 25 (60)

20 (48) 22 (52)

Comparison versus sham Odds ratio 95% confidence interval p value

0.37 0.15–0.91 0.042

0.46 0.19–1.12 0.090

0.75 0.32–1.77 0.537

Figures in parentheses are percentages. p values based on the Cochran-Mantel-Haenszel test were adjusted for the baseline retinal thickening area and baseline vision. With permission from the Macugen Diabetic Retinopathy Study Group [17].

16 patients included in this analysis, 8 had photocoagulation more than 6 months prior to the study and 1 had photocoagulation during the study. Four patients had retinal neovascularization in the fellow eye [118]. Thirteen of these patients received pegaptanib, while the remaining 3 patients received sham injections. Eight of 13 patients (61%) in the pegaptanib group, including the patient receiving photocoagulation during follow-up, had regression of neovascularization demonstrated by fundus photography, diminished or absent fluorescein leakage on fluorescein angiography, or both at 36 weeks. In contrast, none of the 3 patients in the sham group and none of the 4 fellow eyes showed regression of neovascularization. Moreover, 3 of the 8 patients (including the patient receiving photocoagulation) who had regression of neovascularization while receiving pegaptanib experienced a return of neovascularization between weeks 36 and 52, when pegaptanib had been discontinued (fig. 5) [118]. These findings provide further support that VEGF165 plays an important role in the pathogenesis of diabetic retinopathy and suggest that early treatment, before the most destructive sequelae caused by neovascularization have occurred, is likely to be beneficial. Safety. Pegaptanib was well tolerated, with the majority of adverse events being transient, injection procedure related and mild to moderate in severity. Few serious adverse events were noted, and none were attributed to the pegaptanib drug itself. There was one incident of endophthalmitis (culture negative)


140

a

b

c

d

e

f

g

h

i

j

k

l

Fig. 5. a–d Baseline visit: magnification of retinal neovascularization elsewhere (a), red-free photograph showing the location of the neovascularization along the inferotemporal arcade (b), fluorescein angiograms with areas of capillary nonperfusion in the early phase (c), and leakage from the neovascularization elsewhere in the late-phase frame (d). e–h Thirty-six weeks after 6 periodic pegaptanib injections (and 6 weeks since most recent injection): regression of neovascularization elsewhere on red-free photographs (e, f ), with less apparent microaneurysms in the early-phase frame ( g) and regression of leakage from neovascularization elsewhere in the late phase (h). i–l Fifty-two weeks after study entry and 22 weeks since the last pegaptanib injection: reappearance of neovascularization elsewhere on red-free photographs (i, j), with reappearance of leakage from neovascularization elsewhere in the early- (k) and late-phase (l) frames. With permission from Macugen Diabetic Retinopathy Study Group [118].

after intravitreous injection among 652 injections (0.15%) administered in the pegaptanib arms, and there was no evidence of cataract formation/progression, sustained intraocular pressure elevation, or serious systemic events associated with pegaptanib therapy. Importantly, there was no evidence that pegaptanib


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treatment was associated with systemic thromboembolic events or the cardiac, gastrointestinal or hemorrhagic complications noted with pan-VEGF blockade.

Conclusions

Current treatment options for diabetic retinopathy are primarily restricted to laser photocoagulation and pars plana vitrectomy, both of which are destructive and do not address the underlying pathological mechanisms involved in the development of diabetic retinopathy. Preclinical studies support the concept of blocking the actions of VEGF165 in the eye as a sound therapeutic approach to the treatment of diabetic retinopathy. These concepts have been validated by a recent phase II study that demonstrated the clinical benefits of the VEGF165blocking aptamer, pegaptanib, in the treatment of DME. Outcomes included improvements in VA and a reduction in retinal thickness. Moreover, a separate retrospective analysis in a subset of these subjects who had concomitant retinal neovascularization demonstrated regression of neovascularization in 61% of eyes treated with pegaptanib. Confirmation of these findings and the more subtle effects of VEGF165 blockade upon the severity of underlying retinopathy, as well as the effects upon capillary nonperfusion await the results of definitive phase III trials that are under way.

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growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation. Am J Pathol 1999;155:421–428. Baffert F, Thurston G, Rochon-Duck M, Le T, Brekken R, McDonald DM: Age-related changes in vascular endothelial growth factor dependency and angiopoietin-1-induced plasticity of adult blood vessels. Circ Res 2004;94:984–992. Ryan AM, Eppler DB, Hagler KE, Bruner RH, Thomford PJ, Hall RL, Shopp GM, O’Neill CA: Preclinical safety evaluation of rhuMAbVEGF, an antiangiogenic humanized monoclonal antibody. Toxicol Pathol 1999;27:78–86. Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N: VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 1999;5:623–628. Nissen NN, Polverini PJ, Koch AE, Volin MV, Gamelli RL, DiPietro LA: Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing. Am J Pathol 1998;152:1445–1452. Deodato B, Arsic N, Zentilin L, Galeano M, Santoro D, Torre V, Altavilla D, Valdembri D, Bussolino F, Squadrito F, Giacca M: Recombinant AAV vector encoding human VEGF165 enhances wound healing. Gene Ther 2002;9:777–785. Fraser HM, Wilson H, Rudge JS, Wiegand SJ: Single injections of vascular endothelial growth factor trap block ovulation in the macaque and produce a prolonged, dose-related suppression of ovarian function. J Clin Endocrinol Metab 2005;90:1114–1122. Liu MH, Jin H, Floten HS, Ren Z, Yim AP, He GW: Vascular endothelial growth factor-mediated, endothelium-dependent relaxation in human internal mammary artery. Ann Thorac Surg 2002;73: 819–824. Arsic N, Zacchigna S, Zentilin L, Ramirez-Correa G, Pattarini L, Salvi A, Sinagra G, Giacca M: Vascular endothelial growth factor stimulates skeletal muscle regeneration in vivo. Mol Ther 2004;10:844–854. Kitamoto Y, Tokunaga H, Tomita K: Vascular endothelial growth factor is an essential molecule for mouse kidney development: glomerulogenesis and nephrogenesis. J Clin Invest 1997;99: 2351–2357. LeCouter J, Moritz DR, Li B, Phillips GL, Liang XH, Gerber HP, Hillan KJ, Ferrara N: Angiogenesisindependent endothelial protection of liver: role of VEGFR-1. Science 2003;299:890–893. van Wijngaarden P, Coster DJ, Williams KA: Inhibitors of ocular neovascularization: promises and potential problems. JAMA 2005;293:1509–1513. Kabbinavar F, Hurwitz HI, Fehrenbacher L, Meropol NJ, Novotny WF, Lieberman G, Griffing S, Bergsland E: Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 2003;21:60–65. Kabbinavar FF, Schulz J, McCleod M, Patel T, Hamm JT, Hecht JR, Mass R, Perrou B, Nelson B, Novotny WF: Addition of bevacizumab to bolus fluorouracil and leucovorin in first-line metastatic colorectal cancer: results of a randomized phase II trial. J Clin Oncol 2005;23:3697–3705. Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R, Kabbinavar F: Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004;350: 2335–2342. Johnson DH, Fehrenbacher L, Novotny WF, Herbst RS, Nemunaitis JJ, Jablons DM, Langer CJ, DeVore RF 3rd, Gaudreault J, Damico LA, Holmgren E, Kabbinavar F: Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 2004;22:2184–2191. Miller KD, Chap LI, Holmes FA, Cobleigh MA, Marcom PK, Fehrenbacher L, Dickler M, Overmoyer BA, Reimann JD, Sing AP, Langmuir V, Rugo HS: Randomized phase III trial of capecitabine compared with bevacizumab plus capecitabine in patients with previously treated metastatic breast cancer. J Clin Oncol 2005;23:792–799. Skillings JR, Johnson DH, Miller K, Kabbinavar F, Bergsland E, Holmgren E, Holden SN, Hurwitz H, Scappaticci F: Arterial thromboembolic events (ATEs) in a pooled analysis of 5


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Carla Starita, MD, PhD Pfizer Global Research and Development, Building 508/1.75 IPC 613 Ramsgate Road, Sandwich CT13 9NJ Kent (UK) Tel. ⫹44 1304 642915, Fax ⫹44 1304 652540, E-Mail [email protected]


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Pharmacologic Vitreolysis Arnd Gandorfer Vitreoretinal and Pathology Unit, Augenklinik der Ludwig-Maximilians-Universität, München, Germany

Abstract At present, surgical separation of the vitreous from the retina (posterior vitreous detachment, PVD) is achieved by mechanical means only. However, with this technique, complete removal of the cortical vitreous from the internal limiting membrane of the retina is not feasible. As incomplete PVD and an attached vitreous cortex are associated with the progression of common retinal diseases including diabetic retinopathy and maculopathy, central retinal vein occlusion and proliferative vitreoretinopathy, induction of complete PVD is a major issue both in vitreoretinal surgery and in medical retina. This chapter focuses on current concepts of pharmacologic vitreolysis. Agents capable of altering the molecular organization of the vitreous are introduced and discussed in terms of PVD induction and liquefaction of the vitreous gel. Copyright © 2007 S. Karger AG, Basel

As the limits of conventional vitrectomy are being approached, vitreoretinal surgeons look forward to a new generation of pharmacological techniques [1]. Several enzymes have been suggested as adjunctive therapy to vitreoretinal surgery or its replacement, including chondroitinase, hyaluronidase, dispase and plasmin enzyme (table 1). The goal of enzymatic vitreolysis is to manipulate the vitreous collagen pharmacologically, achieving liquefaction (synchisis) both centrally as well as along the vitreoretinal interface to induce posterior vitreous detachment (PVD, syneresis) and to create a cleavage plane more safely and cleaner than can currently be achieved by mechanical means [2].

A.G. is founder of the Microplasmin Study Group and has a financial interest in pharmacologic vitreolysis.

Table 1. Enzyme candidates for pharmacologic vitreolysis Enzyme

Target

Effect

Chondroitinase

chondroitin sulfate at the vitreoretinal interface

PVD in animal models

Hyaluronidase

hyaluronan

liquefaction

Dispase

type IV collagen

PVD, inner retinal damage

Plasmin/microplasmin

laminin and fibronectin at the vitreoretinal interface; matrix metalloproteinase-2 activation

PVD and liquefaction

Chondroitinase

A 240-kDa chondroitin sulfate proteoglycan is associated with the vitreoretinal interface [3]. The greatest immunoreactivity of this proteoglycan has been observed in regions of firm vitreoretinal adhesion, such as the vitreous base and the papillary margin, suggesting a major role in vitreoretinal adhesion. The enzyme chondroitinase cleaves this proteoglycan and has been studied as an adjunct in vitrectomy in 2 human donor eyes and in 57 cynomolgus monkeys [3]. Intravitreal injection of the enzyme separated the vitreous from the retina without damaging the inner limiting membrane (ILM). Three monkeys have been followed for 14–16 months after surgery without any adverse effects [3]. Chondroitinase has also been utilized to detach epiretinal membranes in 4 monkeys, providing evidence that chondroitin sulfate proteoglycan participates in the adhesion of epiretinal membranes to the ILM [3]. Unfortunately, no clinical results have been reported yet.

Hyaluronidase

Hyaluronan represents one of the two major macromolecules of the vitreous [4–6] and is supposed to maintain the 3-dimensional structure of the vitreous gel by coating the collagen fibrils and by bridging them with interconnecting filaments [7]. Hyaluronidase (Vitrase®) cleaves hyaluronan and has been suggested to liquify the vitreous. A recently published phase III clinical trial shows that 55 IU of highly purified ovine hyaluronidase (Vitrase) helps to clear eyes with vitreous

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hemorrhage 1 month after intravitreal application [8]. In a companion article on the safety results, no serious safety issues were reported [9]. In particular, the incidence of retinal detachment was not statistically different between treated and control groups. However, no assessment was performed in terms of PVD induction, and experimental trials of hyaluronidase in rabbits failed to achieve PVD [10].

Dispase

Dispase, a neutral 41-kDa protease isolated from Bacillus polymyxa, selectively cleaves type IV collagen and fibronectin [11]. The enzyme facilitated PVD in enucleated porcine and human eyes, and in pig eyes in vivo [12, 13]. However, partial digestion of the ILM was observed in postmortem eyes, exposing the mosaic pattern of Müller cell endfeet [12]. In rabbit eyes in vivo and in human eyes 15 min before enucleation, intravitreal injection of dispase caused intraretinal hemorrhages and ILM disruption at bleeding sites [14]. In this series, there was no effect of dispase on PVD induction. As dispase acts on type IV collagen, forming the main structural protein of basement membranes including the ILM, changes of the inner retina following application of the enzyme are not surprising [12]. The enzyme has been shown to effectively induce proliferative vitreoretinopathy in rabbits in a dose-dependent fashion, known as the dispase model of proliferative vitreoretinopathy, and future studies need to investigate the safety of dispase before clinical studies can be considered [14, 15].

Plasmin

Plasmin is a nonspecific serine protease mediating the fibrinolytic process. It also acts on a variety of glycoproteins including laminin and fibronectin, both of which are present at the vitreoretinal interface [16–18]. In 1993, PVD could be achieved in rabbit eyes by intravitreal injection of the enzyme followed by vitrectomy [19]. In 1999, Hikichi et al. [20] confirmed complete PVD after injection of 1 unit plasmin and 0.5 ml SF6 gas in the rabbit model, without evidence of retinal toxicity. We investigated the effect of plasmin in porcine postmortem eyes and in human donor eyes. In porcine eyes, we observed a dose-dependent separation of the vitreous cortex from the ILM after intravitreal injection, without additional vitrectomy or gas injection [21]. In scanning electron microscopy, a bare ILM was achieved by 1 unit of porcine plasmin 60 min after injection, and with

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2 units of plasmin 30 and 60 min after injection, respectively. In control fellow eyes which were injected with balanced salt solution, the cortical vitreous remained attached to the retina [21]. In human donor eyes, 2 units of human plasmin from pooled plasma achieved complete PVD 30 min after injection, whereas the vitreoretinal surface of the fellow eyes was covered by collagen fibrils [22]. In both studies, transmission electron microscopy revealed a clean and perfectly preserved ILM in plasmin-treated eyes, and no evidence of inner retinal damage [21, 22]. Li et al. [23] confirmed these results and reported a reduced immunoreactivity of the vitreoretinal interface for laminin and fibronectin following application of plasmin. In an experimental setting simulating the application of plasmin as an adjunct to vitrectomy, we injected human donor eyes with 1 unit of plasmin, followed by vitrectomy 30 min thereafter [24]. All plasmin-treated eyes showed complete PVD, whereas the control eyes which were vitrectomized conventionally had various amounts of the cortical vitreous still present at the vitreoretinal interface [24]. Plasmin is not available for clinical application, and alternative strategies have been pursued to administer the enzyme in vitreoretinal surgery. Tissue plasminogen activator was injected into the vitreous in an attempt to generate plasmin by intravitreal activation of endogenous plasminogen. In an animal model in rabbit eyes, complete PVD was observed in all eyes treated with 25 ␮g tissue plasminogen activator [25]. Breakdown of the blood-retinal barrier was necessary to allow plasminogen to enter the vitreous, and this was induced by cryocoagulation [25]. In two clinical pilot studies, 25 ␮g tissue plasminogen activator was injected into the vitreous of patients with proliferative diabetic retinopathy 15 min before vitrectomy [26, 27]. However, the results of both studies were contradictory in terms of PVD induction and clinical benefit. Recently, Peyman’s group demonstrated PVD induction in rabbit eyes by an intravitreal administration of recombinant lysine plasminogen and recombinant urokinase [28]. Autologous plasminogen purified from the patient’s own plasma by affinity chromatography was converted to plasmin by streptokinase in vitro. Autologous plasmin enzyme, 0.4 units, was injected into the vitreous in patients with pediatric macular holes, diabetic retinopathy and stage 3 idiopathic macular holes, followed by vitrectomy after 15 min [29–31]. All autologous plasmin enzyme-treated eyes achieved spontaneous or easy removal of the posterior hyaloid including 1 eye that had vitreoschisis over areas of detached retina. Recombinant microplasmin (ThromboGenics Ltd., Dublin, Ireland), a truncated molecule containing the catalytic domain of human plasmin, has been administered successfully into the vitreous of human [32] and porcine postmortem

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Fig. 1. Complete vitreoretinal separation following an intravitreal injection of microplasmin into a human donor eye.

Fig. 2. Collagen remnants at the vitreoretinal interface in a control eye.


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eyes [M. de Smet, Monte Carlo, 2004], and in rabbit and cat eyes in vivo [32, 33]. In all experimental settings, complete PVD was achieved in a dose-dependent fashion (figs. 1, 2). No alteration in the inner retina was seen, and there was no change in antigenicity of neurons and glial cells.

Summary

There are three reasons to pursue enzymatic-assisted vitreoretinal surgery. First, some retinal diseases that are currently managed in an operation room with mechanical manipulation of the vitreoretinal interface could be managed more safely by pharmacologic technique or even in an office setting. Second, enzymatic-assisted vitrectomy may achieve better anatomic and thus functional results by creating a cleaner cleavage plane between the vitreous and the retina than can be currently achieved by approaching the retina by mechanical means [2]. This is of particular importance in eyes with incomplete removal of the cortical vitreous from the retina, and in eyes with vitreoschisis, such as diabetic eyes [34]. Third, as incomplete PVD has been shown to be associated with both development of aggressive fibrovascular proliferation and macular edema, pharmacologic induction of complete PVD could prevent progression of diabetic retinopathy if given before advanced stages of diabetic eye disease. Plasmin holds the promise of inducing complete PVD without morphologic alteration in the retina. Several independent studies confirmed a dosedependent and complete vitreoretinal separation, associated with perfect preservation of the ultrastructure of the ILM and the retina [19–22, 24, 32]. In addition, a dose-dependent liquefaction of the vitreous induced by microplasmin has been demonstrated by dynamic light scattering in dissected porcine vitreous and in intact pig eyes [Ansari, Monte Carlo, 2004], making plasmin and microplasmin the most promising agents for pharmacologic vitreolysis at the moment. At present, clinical studies are performed to assess the safety and efficacy of microplasmin and other pharmacologic enzymes when used as an adjunct to vitrectomy, or even as its replacement.

References 1 2 3

Bhisitkul RB: Anticipation for enzymatic vitreolysis. Br J Ophthalmol 2001;85:1–2. Trese M: Enzymatic-assisted vitrectomy. Eye 2002;16:365–368. Hageman GS, Russell SR: Chondroitinase-mediated disinsertion of the primate vitreous body. Invest Ophthalmol Vis Sci 1994;35:1260.

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Sebag J: Pharmacologic vitreolysis. Retina 1998;18:1–3. Bishop P: The biochemical structure of mammalian vitreous. Eye 1996;10:664–670. Sebag J: The Vitreous. Structure, Function, Pathobiology. New York, Springer, 1989. Asakura A: Histochemistry of hyaluronic acid of the bovine vitreous body by electronmicroscopy. Nippon Ganka Gakkai Zasshi 1985;89:179–191. Kuppermann BD, Thomas EL, de Smet MD, Grillone LR: Pooled efficacy results from two multinational randomized controlled clinical trials of a single intravitreous injection of highly purified ovine hyaluronidase (Vitrase) for the management of vitreous hemorrhage. Am J Ophthalmol 2005;140:573–584. Kuppermann BD, Thomas EL, de Smet MD, Grillone LR: Safety results of two phase III trials of an intravitreous injection of highly purified ovine hyaluronidase (Vitrase) for the management of vitreous hemorrhage. Am J Ophthalmol 2005;140:585–597. Hikichi T, Kado M, Yoshida A: Intravitreal injection of hyaluronidase cannot induce posterior vitreous detachment in the rabbit. Retina 2000;20:195–198. Stenn KS, Link R, Moelmann G: Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase. J Invest Dermatol 1989;93:287–290. Tezel TH, Del Priore LV, Kaplan HJ: Posterior vitreous detachment with dispase. Retina 1998;18:7–15. Oliveira LB, Tatebayashi M, Mahmoud TH, Blackmon SM, Wong F, McCuen BW 2nd: Dispase facilitates posterior vitreous detachment during vitrectomy in young pigs. Retina 2001;21: 324–331. Jorge R, Oyamaguchi EK, Cardillo JA, Gobbi A, Laicine EM, Haddad A: Intravitreal injection of dispase causes retinal hemorrhages in rabbit and human eyes. Curr Eye Res 2003;26: 107–112. Frenzel EM, Neely KA, Walsh AW, Cameron JD, Gregerson DS: A new model of proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci 1998;39:2157–2164. Kohno T, Sorgente N, Ishibashi T, Goodnight R, Ryan SJ: Immunofluorescent studies of fibronectin and laminin in the human eye. Invest Ophthalmol Vis Sci 1987;28:506–514. Kohno T, Sorgente N, Goodnight R, Ryan SJ: Alterations in the distribution of fibronectin and laminin in the diabetic human eye. Invest Ophthalmol Vis Sci 1987;28:515–521. Liotta LA, Goldfarb RH, Brundage R, Siegal GP, Terranova V, Garbisa S: Effect of plasminogen activator (urokinase), plasmin, and thrombin on glycoprotein and collagenous components of basement membrane. Cancer Res 1981;41:4629–4636. Verstraeten TC, Chapman C, Hartzer M, Winkler BS, Trese MT, Williams GA: Pharmacologic induction of posterior vitreous detachment in the rabbit. Arch Ophthalmol 1993;111:849–854. Hikichi T, Yanagiya N, Kado M, Akiba J, Yoshida A: Posterior vitreous detachment induced by injection of plasmin and sulfur hexafluoride in the rabbit vitreous. Retina 1999;19: 55–58. Gandorfer A, Putz E, Welge-Lussen U, Gruterich M, Ulbig M, Kampik A: Ultrastructure of the vitreoretinal interface following plasmin assisted vitrectomy. Br J Ophthalmol 2001;85: 6–10. Gandorfer A, Priglinger S, Schebitz K, Hoops J, Ulbig M, Ruckhofer J, Grabner G, Kampik A: Vitreoretinal morphology of plasmin-treated human eyes. Am J Ophthalmol 2002;133: 156–159. Li X, Shi X, Fan J: Posterior vitreous detachment with plasmin in the isolated human eye. Graefes Arch Clin Exp Ophthalmol 2002;240:56–62. Gandorfer A, Ulbig M, Kampik A: Plasmin-assisted vitrectomy eliminates cortical vitreous remnants. Eye 2002;16:95–97. Hesse L, Nebeling B, Schroeder B, Heller G, Kroll P: Induction of posterior vitreous detachment in rabbits by intravitreal injection of tissue plasminogen activator following cryopexy. Exp Eye Res 2000;70:31–39. Hesse L, Chofflet J, Kroll P: Tissue plasminogen activator as a biochemical adjuvant in vitrectomy for proliferative diabetic vitreoretinopathy. Ger J Ophthalmol 1995;4:323–327. Le Mer Y, Korobelnik JF, Morel C, Ullern M, Berrod JP: TPA-assisted vitrectomy for proliferative diabetic retinopathy: results of a double-masked, multicenter trial. Retina 1999;19:378–382.


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PD Dr. Arnd Gandorfer Vitreoretinal and Pathology Unit Augenklinik der Ludwig-Maximilians-Universität Mathildenstrasse 8 DE–80336 München (Germany) Tel. ⫹49 089 5160 3800, Fax ⫹49 089 5160 4778, E-Mail [email protected]

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Treatment of Diabetic Retinopathy with Protein Kinase C Subtype ␤ Inhibitor Gabriele E. Lang Augenklinik, Universitätsklinikum Ulm, Ulm, Germany

Abstract Despite better options of controlling diabetes mellitus and although the prognosis of diabetic retinopathy has markedly improved by laser treatment and vitreoretinal surgery, diabetic retinopathy is still the leading cause of blindness in working-age people in industrialized countries. Little has changed in the last decades concerning the prognosis of ocular complications in diabetes mellitus. Therefore, we need better tools for prevention and treatment of diabetic ocular complications due to diabetic retinopathy that go beyond reduction in glycemia, blood pressure and cholesterol levels. Newer therapeutic options are directed at the causative mechanisms of diabetic retinopathy. Experimental and clinical evidence suggests that pharmacological compounds like protein kinase C subtype ␤ (PKC-␤) inhibitors may be effective in the treatment of diabetic retinopathy. One important pathomechanism in the development of diabetic retinopathy is the activation of PKC induced by high glucose due to an increased diacylglycerol level. The selective PKC-␤ inhibitor ruboxistaurin mesylate enables a new therapeutical approach for the treatment of diabetic retinopathy and diabetic macular edema. Ongoing prospective clinical trials investigate if treatment with the specific PKC-␤ inhibitor ruboxistaurin mesylate can prevent the progression of diabetic retinopathy and diabetic macular edema. Copyright © 2007 S. Karger AG, Basel

The manifestation of diabetes mellitus is increasing rapidly in developed countries. It is estimated to affect over 18 million Americans, and diabetic retinopathy is the most common diabetic microvascular complication occurring in nearly all patients after 20 years duration. Visual loss results primarily from either proliferative diabetic retinopathy or macular edema. Proliferative diabetic retinopathy accounts for severe visual loss, whereas diabetic macular edema is the leading cause of moderate visual loss in diabetes mellitus. Laser photocoagulation, the mainstay of treatment of diabetic retinopathy for 4 decades, is considered to be effective in only 60–70% of cases.

One of the main mechanisms by which the body regulates the activity of tissue proteins is adding and removing phosphate groups – whether they are receptors, enzymes, signal proteins, or transcription factors. In these reversible processes, kinases add phosphate groups to tissue proteins at tyrosine residues or at serine and threonine residues, and phosphatases remove the phosphate groups [1]. However, the exact mechanisms by which diabetes mellitus causes diabetic retinopathy remain incompletely understood. Four mechanisms have been implicated in the development of glycemic injury in vascular tissue: nonenzymatic glycation forming advanced glycation end products, oxidative stress, aldose reductase activation, and diacylglycerol (DAG)-mediated activation of protein kinase C (PKC). Inhibition of the enzyme PKC represents an exciting therapeutic approach to managing diabetic retinopathy because PKC is involved in the activation of the vascular endothelial growth factor (VEGF) gene. Inhibition of the ␤-isoform of PKC inhibits VEGF in animal experiments. VEGF inhibition is especially exciting in ophthalmology because VEGF is also involved in other ocular disorders [2]. Presently, the PKC pathway is the focus of intense investigation in completed and ongoing diabetic retinopathy trials investigating its effect on progression of diabetic retinopathy stage and macular edema. Hyperglycemia-induced synthesis of DAG results in activation of PKC, which is considered to play a central role in the development of diabetes complications (fig. 1). PKC adds phosphate groups to a host of protein substrates within vascular tissues at serine and threonine residues and is considered one of the major serine/threonine-specific protein kinases. By adding phosphate groups, PKC modifies the receptor status of the phosphorylated substrate. PKC is a family of at least 13 enzymes, of which the ␤-isoform has been closely linked to the development of diabetic microvascular complications. The activation of PKC appears to mediate increased vascular permeability and neovascularization. PKC activation is important in the intracellular signaling of VEGF, which is hypothesized to play a major role in the development of diabetic macular edema and proliferative diabetic retinopathy.

Protein Kinase C

PKC-␤ inhibitors act via influencing the cellular signal transduction by inhibition of specific protein kinases. The balance of kinases and phosphatases is important for cellular processes like growth, differentiation and motility. PKC consists of a family of about 13 isoforms, which differ in structure, substrate requirements, location and function. The ␤-isoform has been most closely linked to the development of diabetic retinopathy [3].

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Hyperglycemia

DAG

AGE

⫹ PKC-␤ inhibitor

⫺

Glycation

⫹ PKC-␤

Capillary leakage

⫹ VEGF ⫹

Capillary occlusion

Neovascularization

Fig. 1. Pathomechanism and treatment of diabetic retinopathy with PKC-␤ inhibitor. Hyperglycemia results in the production of advanced glycation endproducts (AGE) and leads to increased DAG levels. This results in an activation of PKC-␤, leading to an overexpression of VEGF. Therefore, PKC-␤ activation results in capillary leakage and neovascularization. These effects can be inhibited with the PKC-␤ inhibitor RBX.

The protein kinases can be divided into 4 classes according to the acceptor amino acids: serine/threonine-, tyrosine-, histidine- und aspartate/glutamatespecific protein kinases. Serine/threonine-specific kinases, which are found in all tissues, are divided into 3 groups: a cAMP-dependent protein kinase A, a protein kinase B, and a calcium/phospholipid-activated PKC [4]. The PKC family was first isolated in 1977 as a proteolytic activated kinase in rat brain [5]. PKC is a single polypeptide with an N-terminal regulatory region and C-terminal catalytic regions (fig. 2). The conventional and novel isoforms are activated by DAG. The group of atypical PKC is not activated by DAG [6]. The PKC pathways are responsible for cell growth and cell death. They are regulated isoenzyme and cell specific [7]. PKC acts by catalyzing the transfer of a phosphate group from ATP to various substrate proteins. Several studies showed that the activation of PKC via hyperglycemia in diabetics is associated with increased DAG levels in vascular tissue. This was also proven for the retina. In recent studies, it was shown that PKC-␤ is involved in vascular dysfunctions which are induced by hyperglycemia [8]. The intracellular release of DAG is the primary step for activation of PKC.

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PS cPKC

nPKC

C1

C2

N

C3

C4 COOH

N

aPKC

naPKC

C1

COOH

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N

COOH

COOH

Fig. 2. Schematic illustration of the primary structure of the PKC isoenzymes. The PKC isoenzymes consist of a regulatory and a catalytic region: C1–C4. DAG, phosphatidylserine and phorbol esters bind to the C1 domain, calcium to the C2 region and ATP to the C3 region. aPKC ⫽ atypical PKC; cPKC ⫽ conventional PKC; nPKC ⫽ novel PKC; naPKC ⫽ newatypical; PKC; PS ⫽ pseudosubstrate.

PKC and Diabetic Retinopathy

The activation of PKC via hyperglycemia plays a central role in the development and progression of diabetic retinopathy [9]. Glucose gets into the cells and is further metabolized via glycolysis. This results in the synthesis of DAG. Increased DAG levels have been found in the retina of diabetics [10]. Hyperglycemia results in an increased DAG-PKC signal transduction in the retina [11]. Furthermore, independent of DAG synthesis, lipid acids play an important role in the modulation of PKC activation. However, the PKC isoenzymes in the various tissues are activated differently. PKC-␤ is the dominating isoenzyme in the retina. One reason for the privileged activation of PKC-␤ in diabetics is the high sensitivity against DAG [11]. It has been established that PKC-␤ is activated very early in diabetes, well before clinically apparent retinopathy. The activation of the DAG-PKC metabolic pathway leads to longacting structural and functional changes, which are associated with different complications. The vascular endothelial cells play a key role in the regulation of homeostasis, the vascular tonus, vessel permeability and thrombocyte activation. Endothelial dysfunction and cell activation lead to the development of microangiopathy. Biochemial or mechanic stimulation releases a number of substances in endothelial cells, such as, among others, angiotensin II, endothelin-1, transforming growth factor-␤, VEGF and prostaglandins. The PKC activation is an important biochemical step in the hyperglycemia-induced endothelial dysfunction. PKC for example inhibits the nitric oxide-mediated vasodilation [12]. This

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is important because the inhibition of PKC can normalize the retinal microvascular hemodynamics. The adhesion of monocytes on endothelial cells is increased in diabetes mellitus. The membrane-associated activity of PKC in monocytes is markedly elevated in diabetics and leads to an increased adhesion of monocytes on endothelial vessel walls [13]. The activity of PKC plays an important role in the regulation of receptor density on the cell surfaces for hormones, in the intracellular signal response, the ion channel activity, the intracellular pH and the phosphorylation of proteins [14]. The increased reactive contractility of smooth muscles, which is observed in diabetic patients, is caused by hyperglycemia-induced PKC activation. Changes in the intracellular calcium concentration are associated with the PKC activation and modulate growth factor-induced mitogenesis and contraction. Finally, apoptosis of smooth vascular muscle cells are dependent on PKC [15]. The loss of endothelial cell barrier function is an early pathophysiological phenomenon in diabetic retinopathy. The PKC-mediated phosphorylation of junctional proteins and dissolution of tight junctions, as well as the relaxation of cytoskeletal and adhesion proteins like caldesmon, vimentin, talin and vinulin are responsible for increased vascular permeability caused by increased glucose levels. VEGF is not only the primary mediator of disturbed vascular permeability, but also of neoangiogenesis. In eyes of diabetics, high VEGF levels were found. When glucose levels are increased, the VEGF gene expression is dependent on PKC. Diabetic macular edema and the majority of the neovascular response in the retina is mediated by VEGF. Activation of PKC-␤ is involved in mediating VEGF-induced intracellular signaling. The VEGF-induced disturbed bloodretinal barrier, endothelial cell proliferation and neoangiogenesis can be blocked by ␤-specific PKC inhibition, although the PKC-␤ inhibitor is not primarily a VEGF inhibitor [16]. In cellular and animal models, its antiproliferative effect is weaker than its antipermeability activity [16, 17]. Over time, different growth factors and cytokines are involved in the pathogenesis of diabetic retinopathy. The thickening of the capillary basement membrane and the increase in extracellular matrix are the predominant vascular changes in the early phase of diabetes mellitus. The basement membrane plays an important role concerning vascular permeability, cellular adhesion, proliferation, differentiation and gene expression. The production of collagen types IV and VI as well as fibronectin is enhanced in diabetics. PKC inhibitors can prevent these effects. Transforming growth factor-␤ and connective tissue growth factor play a key role in the thickening of the basement membrane and the synthesis of extracellular matrix. The expression of these growth factors can be blocked by PKC inhibition [18].


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Treatment of Diabetic Retinopathy with PKC-␤ Inhibitors

Recently, an isoenzyme-selective PKC inhibitor, ruboxistaurin mesylate (RBX; Eli Lilly), was developed [19], which is orally administered. RBX is the first of a new class of compounds and the most potent and selective PKC-␤ inhibitor being investigated. The treatment of diabetic rats with RBX showed an amelioration of the retinal blood flow in a dose-responsive manner in parallel with its inhibition of the retinal PKC activity [20]. Aiello et al. [16] demonstrated that intravitreal injection of VEGF rapidly activates PKC in the retina at concentrations observed clinically and increases retinal vasopermeability in vivo by more than 3-fold. Intravitreal or oral administration of a PKC-␤ inhibitor resulted in ⬎95% inhibition of VEGF-induced permeability. RBX reduces the VEGF-induced retinal blood-retinal barrier breakdown and neovascularization in animals. PKC inhibitors abolished both VEGF-induced PKC activation and endothelial cell proliferation. The mitogenic effect of VEGFs was inhibited by RBX in a concentration-dependent manner [17]. The PKC-␤ inhibitor is effective in preventing diabetes-induced retinal vascular leakage in animal models and in preventing retinal neovascularization. Danis et al. [21] found that the PKC-␤ inhibitor RBX effectively inhibited preretinal and optic nerve head neovascularization in a pig model of branch retinal vein occlusion. They found a significantly decreased degree of neovascularization in pig eyes with no apparent systemic toxicity. The ameliorative effect seems to be a result of disruption of a key element of the intracellular signal cascade by angiogenic growth factors, in particular VEGF. It has been shown that the PKC pathway lies downstream of the VEGF receptor ligand binding and is involved in mediating the proliferative response of endothelial cells to VEGF. RBX treatment can reduce the retinal vascular leakage in eyes that have diabetic macular edema and markedly elevated leakage at doses between 4 and 32 mg/day. These data suggest that RBX treatment may be most prominent in patients with severe macular edema [22]. In patients receiving 16 mg RBX twice daily, the diabetes-induced increase in retinal circulation time was ameliorated. Increasing the RBX dose was linearly associated with greater effect on retinal circulation time. Similar results were observed with retinal blood flow [23]. Treatment of diabetic macular edema patients for 3 months with multitargeted kinase inhibitor, which also acts as a nonspecific PKC inhibitor, led to reduction in retinal thickening as evaluated by optical coherence tomography. The systemic applicability of this nonselective compound was limited by gastrointestinal side effects and dose-related problems with tolerability, glycemic control and liver toxicity [24].

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In a mulitcenter, double-masked, randomized, placebo-controlled study, the safety and efficacy of the orally administered PKC-␤ isoform selective inhibitor RBX was evaluated in subjects with moderately severe to very severe nonproliferative diabetic retinopathy. Two hundred and fifty-two subjects received placebo or RBX (8, 16 or 32 mg/day) for 36–46 months. Patients had an Early Treatment Diabetic Retinopathy Study retinopathy severity level between 47B and 53E inclusive, an Early Treatment Diabetic Retinopathy Study visual acuity of 20/125 or better, and no history of scatter photocoagulation. Efficacy measurements included progression of diabetic retinopathy, moderate visual loss and sustained moderate visual loss. RBX was well tolerated without significant adverse events, but had no significant effect on the progression of diabetic retinopathy. Compared with placebo, 32 mg/day RBX was associated with a delayed occurrence of moderate visual loss (p ⫽ 0.038) and sustained moderate visual loss (p ⫽ 0.226). This was evident only in eyes with definite diabetic macular edema at baseline (p ⫽ 0.017). In multivariable Cox proportional hazard analysis, 32 mg/day RBX significantly reduced the risk of moderate visual loss compared with placebo (p ⫽ 0.012) [25]. In this clinical trial, RBX was well tolerated and reduced the risk of visual loss but did not prevent diabetic retinopathy progression. The beneficial effect of RBX on moderate visual loss might be the result of improved retinal cell viability resulting from PKC-␤ inhibition. Reduction in PKC-␤ activity might result in greater resistance of retinal vascular and neural cells to the pathologic stress of hyperglycemia and changes in hemodynamics like blood flow. Further multicenter trials investigate if RBX can reduce the progression of diabetic macular edema and diabetic retinopathy. One of the phase 3 clinical trials was finished recently, and it was announced by Eli Lilly and Company that RBX significantly reduced the occurrence of sustained vision loss in patients with moderately severe to very severe nonproliferative diabetic retinopathy, showing a beneficial effect on the functional outcome. RBX reduced sustained moderate vision loss by 40%. Twice as many RBX-treated eyes gained 15 or more letters of visual acuity from baseline to endpoint. The detailed results of the study are expected to be published this year. RBX would be the first oral medication for treatment of diabetic retinopathy.

Side Effects

When considering systemic therapy, the safety profile of a compound is of substantial importance. This is particularly true when inhibiting a key signaling enzyme such as PKC, where substantial toxicity might be expected. Treatment of diabetic macular edema patients with an inhibitor of multiple kinases and PKC isoforms resulted in liver enzyme elevations, nausea, vomiting and diarrhea [24].


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In contrast, RBX is selective for the ␤-isoform of PKC and highly selective for PKC as compared with other kinases [25]. Indeed, RBX is very well tolerated without significant adverse events over 52 months of treatment. Mild side effects are rare. Only nine adverse events occurred, with an incidence exceeding 1%, that were statistically different among the groups. No serious adverse events were reported more frequently in the RBX treatment groups. The frequency of nonserious adverse event occurrence of diarrhea, flatulence, nephropathy, proteinuria and coronary artery disease was highest among patients in the 16-mg/day treatment group; there did not appear to be a RBX dose-response effect. In addition, the small number of events makes it likely that any disparity in the 16-mg group was due to chance. Patients taking the highest RBX dose of 32 mg/day did not experience these same events more frequently than placebo patients. To date, over 1,400 patients have been exposed to RBX, and no clinically significant increase in adverse effects has been identified [25]. In a small study with 29 persons, Aiello et al. [23] found a statistically significant difference among treatment groups, showing that abdominal pain was more common in the placebo group compared with RBX-treated persons.

Conclusions

New treatment modalities for diabetic retinopathy are clearly needed. The next years will demonstrate exciting new therapies for diabetic microvascular complications, the orally active RBX (Eli Lilly) for PKC inhibition possibly being one of them. RBX provides an effective blockade of hyperglycemiainduced vascular injury and is safe for administration in humans. RBX treatment reduces visual loss in patients with moderately severe to very severe nonproliferative diabetic retinopathy.

References 1 2 3 4 5

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Donnelly R: Molecular mechanisms, therapeutic targets. Adv Stud Med 2004;3:1008–1013. Dodsen PM: New trends in the management of diabetic retinopathy. Adv Stud Med 2004;3:1002–1012. Hayashi A, Seki N, Hattori A, et al: PKCv, a new member of the protein kinase C family, composes a fourth subfamily with PKC␮. Biochem Biophys Acta 1999;1450:99–106. Idris I, Gray S, Donnelly R: Protein kinase C activation: isozyme-specific effects on metabolism and cardiovascular complications in diabetes. Diabetologica 2001;44:659–673. Inoue M, Kishimoto A, Takai Y, Nishizuka Y: Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. 2. Proenzyme and its activation by calciumdependent protease from rat brain. J Biol Chem 1977;252:7610–7616. Kishimoto A, Takai Y, Mori T, et al: Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. J Biol Chem 1980;255:2273–2276.

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Lang GE, Kampmeier J: Die Bedeutung der Proteinkinase C in der Pathophysiologie der diabetischen Retinopathie. Klin Monatsbl Augenheilkd 2002;219:769–776. Lang GE: Pharmacological treatment of diabetic retinopathy. Ophthalmologica, in press. Xia P, Inoguchi T, Kern TS, et al: Characterization of the mechanism for the chronic activation of diacylglycerol-protein kinase C pathway in diabetes and hypergalactosemia. Diabetes 1994;43: 1122–1129. Craven PA, Davidson CM, DeRubertis FR: Increase in diacylglycerol mass in isolated glomeruli by glucose from de novo synthesis of glycerolipids. Diabetes 1990;39:667–674. Nishizuka Y: The molecular heterogeneity of protein kinase C and its implication for cellular regulation. Nature 1988;334:661–665. Chakravarthy U, Hayes R, Stitt A, McAuley E, Archer DB: Constitutive nitric oxide synthase expression in retinal vascular endothelial cells is suppressed by high glucose and advanced glycation end products. Diabetes 1998;47:945–952. Kreuzer J, Denger S, Schmidts A, et al: Fibrinogen promotes monocyte adhesion via a protein kinase C dependent mechanism. J Mol Med 1996;74:161–165. Malhotra A, Reich D, Nakouzi A, Sanghi V, Greenen DL, Buttrick PM: Experimental diabetes is associated with functional activation of protein kinase C ␧ and phosphorylation of troponin I in the heart, which are prevented by angiotensin II receptor blockade. Circ Res 1997;81: 1027–1033. Li PF, Maasch C, Haller H, et al: Requirement for protein kinase C in reactive oxygen speciesinduced apoptosis of vascular smooth muscle cells. Circulation 1999;100:967–973. Aiello LP, Bursell SE, Clermont A, et al: Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective ␤-isoformselective inhibitor. Diabetes 1997;46:1473–1480. Xia P, Aiello LP, Ishii H, Jiang ZY, Park DJ, Robbinson GS, Takagi H, Newsome WP, Jirousek MR, King GL: Characterization of vascular endothelial growth factor’s effect on the activation of protein kinase C, its isoforms, and endothelial cell growth. J Clin Invest 1996;98:2018–2026. Fumo P, Kuncio GS, Ziyadeh FN: PKC and high glucose stimulate collagen ␣1 (IV) transcriptional activity in a reporter mesangial cell line. Am J Physiol 1994;267:632–638. Liao DF, Monia B, Dean N, et al: Protein kinase C-␨ mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells. J Biol Chem 1997;272:6146–6150. Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clemont A, Bursell SE, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL: Amelioration of vascular dysfunctions in diabetic rats by an oral PKC-␤ inhibitor. Science 1996;272:728–731. Danis RP, Bingaman DP, Jirousek M, Yang Y: Inhibition of intraocular neovascularization caused by retinal ischemia in pigs by PKC␤ inhibition with LY333531. Invest Ophthalmol Vis Sci 1998;39:171–179. Strom C, Sander B, Klemp K, Aiello LP, Lund-Andersen H, Larsen M: Effect of ruboxistaurin on blood-retinal barrier permeability in relation to severity of leakage in diabetic macular edema. Invest Ophthalmol Vis Sci 2005;46:3855–3858. Aiello LP, Clermont A, Arora V, Davis MD, Sheetz MJ, Bursell SE: Inhibition of PKC␤ by oral administration of ruboxistaurin is well tolerated and ameliorates diabetes-induced retinal hemodynamic abnormalities in patients. Invest Ophthalmol Vis Sci 2006;47:86–92. Campochiaro PA: Reduction of diabetic macular edema by oral administration of the kinase inhibitor PKC412. Invest Ophthalmol Vis Sci 2004;45:922–931. The PKC-DRS Study Group. The effect of ruboxistaurin on visual loss in patients with moderately severe to severe nonproliferative diabetic retinopathy. Diabetes 2005;54:2188–2197.

Prof. Dr. Gabriele E. Lang Universitätsklinikum Ulm, Augenklinik Prittwitzstrasse 43 DE–89075 Ulm (Germany) Tel. ⫹49 731 500 27551, Fax ⫹49 731 500 27549, E-Mail [email protected]


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Subject Index

Adherens junctions, see Intercellular junctions Angiogenesis, regulators 111, 112, 125 Angiopoietin II, elevation in diabetic retinopathy 129 Basement membrane, macular edema changes 6 Blood-retinal barrier, breakdown in diabetes 2, 112 Cataract surgery diabetics 81, 82, 84 intravitreal triamcinolone acetonide combination 101, 102 Chondroitinase, pharmacologic vitreolysis 150 Cotton wool spots, optical coherence tomography findings 44 Diabetic retinopathy diabetes types and vision loss mechanisms 14, 123 epidemiology 14, 52, 53, 113, 122 initial stages 13–15 intravitreal triamcinolone acetonide, see Intravitreal triamcinolone acetonide Macugen therapy, see Macugen macular edema, see Macular edema nonproliferative stage clinical management 24–26, 56 features 14–17, 48, 50, 51 optical coherence tomography, see Optical coherence tomography

pathophysiology 49–52, 158 phenotypes characterization 17–22 phenotype/genotype correlations 22–24 photocoagulation, see Laser treatment progression under glycemic control 21, 22, 113, 115 protein kinase C inhibitor therapy, see Ruboxistaurin mesylate risk assessment and factors 26–29, 54 severity scale 52, 53 somatostatin analog management, see Somatostatin vitrectomy, see Vitrectomy Dispase, pharmacologic vitreolysis 151 Endothelial cell macular edema damage and apoptosis 8 Endothelial precursor cell (EPC), recruitment in macular edema 7, 8 Extracellular matrix (ECM), macular edema changes 5, 6 Fas ligand, endothelial cell apoptosis in macular edema 8 Gap junctions, see Intercellular junctions Glaucoma, neovascular intravitreal triamcinolone acetonide 100, 101 vitreoretinal surgery 77, 78 Gonioscopy, diabetic patient evaluation 55

166

Hard exudates, optical coherence tomography findings 43, 50 Hyaluronidase, pharmacologic vitreolysis 150, 151 Hypoxia, angiogenesis stimulation 112 Insulin-like growth factor-1 (IGF-1) diabetic retinopathy role 112, 114 therapeutic targeting 114, 115 Intercellular junctions diabetic retinopathy alterations 4, 5 regulation 5 retinal composition 4 Interleukin-6 (IL-6), elevation in diabetic retinopathy 129 Internal limiting membrane (ILM) peeling in macular edema surgery 80, 81, 93 ultrastructure in diffuse macular edema 90 Interretinal microvascular anomalies, diagnosis 50, 51 Intracellular adhesion molecule-1 (ICAM-1), elevation in diabetic retinopathy 129, 130 Intravitreal triamcinolone acetonide (IVTA) cataract surgery combination 101, 102 complications 102–106 diffuse macular edema management 97–99 indications 96, 97 mechanism of action 99 neovascular glaucoma management 100, 101 posterior sub-Tenon injection 99, 100 prospects 106 vitrectomy combination 100 Laser treatment diabetic retinopathy Diabetic Retinopathy Study 56, 57 mechanism of action 123 nonproliferative diabetic retinopathy 57 outcomes 60, 157 proliferative diabetic retinopathy 57 technique 57–60

Subject Index

guidelines 64, 65 historical perspective 48 macular edema 60–64 patient evaluation 54–56 refractive cases and vitrectomy 74 side effects 65, 66 wavelength 64 Macugen clinical trials Phase II trial retinal neovascularization 138–140 retinal thickness 137, 138 safety 140–142 study design 134, 135 vision outcomes 135 VISION trials 134 mechanism of action 131, 132 pharmacokinetics 132 preclinical studies 132 prospects in diabetic retinopathy treatment 142 Macular edema blood-retinal barrier breakdown in diabetes 2 diffuse macular edema epiretinal cellular proliferation 91 intravitreal triamcinolone acetonide 97–99 pathophysiology 89 treatment 91, 93 vitreoschisis 89, 90 vitreous cortex ultrastructural findings 90 vitreous origins 89 endothelial cell damage and apoptosis 8 endothelial precursor cell recruitment 7, 8 epidemiology in diabetes 88 extracellular matrix changes 5, 6 focal versus diffuse disease 88 intercellular junctions diabetic retinopathy alterations 4, 5 regulation 5 retinal composition 4 leukocyte infiltration 6, 7 Macugen therapy, see Macugen

167

Macular edema (continued) optical coherence tomography findings 35–38, 40, 41, 43 photocoagulation, see Laser treatment severity scale 52, 53 treatment options 9 vascular endothelial growth factor diffuse macular edema role 90 neovascularization 3 vascular hyperpermeability induction 3 vitrectomy, see Vitrectomy vitreoretinal surgery 78–81 Matrix metalloproteinases (MMPs), macular edema role 5, 6 Neovascular glaucoma, see Glaucoma, neovascular Octreotide, see Somatostatin Ophthalmoscopy, diabetic patient evaluation 54, 55 Optical coherence tomography (OCT) applications 32 cotton wool spots 44 hard exudates 43 interpretation 34, 35 macular edema findings 35–38, 40, 41, 43 principles 31–34 proliferative diabetic retinopathy findings 44 retinal hemorrhage 44 technique 33, 34 Pegaptanib, see Macugen Pharmacologic vitreolysis chondroitinase 150 dispase 151 hyaluronidase 150, 151 plasmin 151–164 prospects 154 rationale 149, 153, 154 Photocoagulation, see Laser treatment Pigment endothelial-derived factor (PEDF), angiogenesis regulation 112 Plasmin, pharmacologic vitreolysis 151–164

Subject Index

Protein kinase C (PKC) classification 159 diabetic retinopathy pathophysiology 160, 161 ruboxistaurin mesylate treatment outcomes 162–164 side effects 163, 164 vascular endothelial growth factor response 162 therapeutic targeting 26, 161 functional overview 159 vascular endothelial growth factor activation 158, 161 Retinal detachment, optical coherence tomography findings in proliferative diabetic retinopathy 44 Retinal hemorrhage, optical coherence tomography findings 44 Retinal thickness Macugen response 137, 138 macular edema 37, 38, 40, 41 optical coherence tomography 33, 34, 41 Ruboxistaurin mesylate (RBX) diabetic retinopathy outcomes 162–164 side effects 163, 164 vascular endothelial growth factor response 162 Silicone oil tamponade, vitrectomy in diabetes 71, 77, 83 Somatostatin analogs in diabetic retinopathy management dosing 115, 116 mechanisms of action 116, 117 octreotide advanced disease management 116 effects on progression 116 prospects 118 side effects 117 growth hormone antagonism 115 receptors 115 Stromal-derived factor-1, elevation in diabetic retinopathy 129

168

Triamcinolone acetonide, see Intravitreal triamcinolone acetonide Vascular endothelial growth factor (VEGF) angiogenesis regulation 125–127 biological activity 126 diabetic retinopathy role 129–131 diffuse macular edema role 90 hypoxia stimulation 112, 114 inflammation role 129–131 intercellular junction regulation 5 intracellular adhesion molecule-1 induction 129, 130 isoforms 124–126 neovascularization induction in eye 3, 128, 129 receptors 126 ruboxistaurin mesylate response 162 therapeutic targeting early macular edema 9 Macugen, see Macugen rationale 124, 126 vascular hyperpermeability induction 3, 49 Venous beading, diagnosis 50, 51 Vitrectomy

Subject Index

cataract surgery in diabetics 81, 82, 84 early vitrectomy for proliferative diabetic retinopathy and vitreous hemorrhage Diabetic Retinopathy Vitrectomy Study 72–74, 83 Early Treatment Diabetic Retinopathy Study 72 emergencies 73 vitreomacular traction 73, 74 indications in diabetic retinopathy 69, 70, 83 intravitreal triamcinolone acetonide combination 100 macular edema and vitreoretinal surgery 78–81 neovascular glaucoma and vitreoretinal surgery 77, 78 pars plana vitrectomy for tractional detachment 74–76, 123 proliferative vitreoretinopathy tractional retinal detachment 76, 77 refractive photocoagulation cases 74 technique 70–72 Vitreolysis, see Pharmacologic vitreolysis

169

Diabetic Retinopathy

Diabetic Retinopathy

Diabetic Retinopathy: The Essentials