Recent Advances in the Pathogenesis, Prevention and Management of Type 2 Diabetes and its Complications

RECENT ADVANCES IN THE PATHOGENESIS, PREVENTION AND MANAGEMENT OF TYPE 2 DIABETES AND ITS COMPLICATIONS Edited by Mark B...

Author: Mark B. Zimering | MD | PhD

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RECENT ADVANCES IN THE PATHOGENESIS, PREVENTION AND MANAGEMENT OF TYPE 2 DIABETES AND ITS COMPLICATIONS Edited by Mark B. Zimering

Recent Advances in the Pathogenesis, Prevention and Management of Type 2 Diabetes and its Complications Edited by Dr Mark B. Zimering

Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright © 2011 InTech All chapters are Open Access articles distributed under the Creative Commons Non Commercial Share Alike Attribution 3.0 license, which permits to copy, distribute, transmit, and adapt the work in any medium, so long as the original work is properly cited. After this work has been published by InTech, authors have the right to republish it, in whole or part, in any publication of which they are the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published articles. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. Publishing Process Manager Mirna Cvijic Technical Editor Teodora Smiljanic Cover Designer Jan Hyrat Image Copyright David Watkins, 2010. Used under license from Shutterstock.com First published August, 2011 Printed in Croatia A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from [email protected]

Recent Advances in the Pathogenesis, Prevention and Management of Type 2 Diabetes and its Complications, Edited by Mark B. Zimering p. cm. ISBN 978-953-307-597-6

free online editions of InTech Books and Journals can be found at www.intechopen.com

Contents Preface IX Part 1

Diabetic Neuropathy 1

Chapter 1

New Treatment Strategies in Diabetic Neuropathy 3 Anders Björkman, Niels Thomsen and Lars Dahlin

Chapter 2

Sonographic Imaging of the Peripheral Nerves in Patients with Type 2 Diabetes Mellitus 15 Tsuneo Watanabe, Shin-ichi Kawachi and Toshio Matsuoka

Chapter 3

Diabetic Neuropathy – Nerve Morphology in the Upper Extremity 33 Niels Thomsen, Anders Bjorkman and Lars B. Dahlin

Part 2

Diabetes and Cardiovascular Disease 43

Chapter 4

Residual Vascular Risk in T2DM: The Next Frontier 45 Michel P. Hermans, Sylvie A. Ahn and Michel F. Rousseau

Chapter 5

Type 2 Diabetes and Fibrinolysis Ján Staško, Peter Chudý, Daniela Kotuličová, Peter Galajda, Marián Mokáň and Peter Kubisz

Chapter 6

Diabetes and Aspirin Resistance 91 Subhashini Yaturu and Shaker Mousa

Chapter 7

Diabetic Cardiomyopathy 105 Dike Bevis Ojji

Chapter 8

AF and Diabetes Prognosis and Predictors 119 Carlo Pappone, Francesca Zuffada and Vincenzo Santinelli

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VI

Contents

Chapter 9

Chapter 10

Part 3

Detection of Silent Ischemia in Patients with Type 2 Diabetes 129 Babes Elena Emilia and Babes Victor Vlad Effects of Type 2 Diabetes on Arterial Endothelium 155 Arturo A. Arce-Esquivel, Aaron K. Bunker and M. Harold Laughlin Hypertension, Nephropathy and Diabetes

179

Chapter 11

Antihypertensive Treatment in Type 2 Diabetic Patients 181 Angelo Michele Carella

Chapter 12

Managing Hypertension in Patients with Diabetes Arthur L.M. Swislocki and David Siegel

Chapter 13

Treatments for Hypertension in Type 2 Diabetes-Non-Pharmacological and Pharmacological Measurements 233 Kazuko Masuo and Gavin W. Lambert

Chapter 14

Diabetic Nephropathy; Clinical Characteristics and Treatment Approaches 263 Derun Taner Ertugrul, Emre Tutal and Siren Sezer

Chapter 15

Anemia of Chronic Kidney Disease in Diabetic Patients: Pathophysiologic Insights and Implications of Recent Clinical Trials 273 Victoria Forte, Miriam Kim, George Steuber, Salma Asad and Samy I. McFarlane

Chapter 16

A Diachronic Study of Diabetic Nephropathy in Two Autochthonous Lines of Rats to Understand Diabetic Chronic Complications 283 Juan Carlos Picena, Silvana Marisa Montenegro, Alberto Enrique D´Ottavio, María Cristina Tarrés and Stella Maris Martínez

Part 4

207

Diverse Organ Involvement/Dysfunction in Diabetes

Chapter 17

Pathogenic Features of Insulin Resistance and Critical Organ Damage in the Liver, Muscle and Lung 303 Kei Nakajima, Toshitaka Muneyuki, Masafumi Siato and Masafumi Kakei

Chapter 18

Implications of Type II Diabetes Mellitus on Gastrointestinal Cancers 323 Diana H. Yu and M. Mazen Jamal

301

Contents

Chapter 19

Part 5

Prevention for Micro- and Macro-Vascular Complications in Diabetic Patients 337 Kyuzi Kamoi Aspects of the Treatment of Type 2 Diabetes

373

Chapter 20

Emerging Challenge of Type 2 Diabetes: Prospects of Medicinal Plants 375 Rokeya Begum, Mosihuzzaman M, Azad Khan AK, Nilufar Nahar and Ali Liaquat

Chapter 21

Nutritional Therapy in Diabetes: Mediterranean Diet 391 Pablo Pérez-Martínez, Antonio García-Ríos, Javier Delgado-Lista, Francisco Pérez-Jiménez and José López-Miranda

Chapter 22

Pharmacogenetics for T2DM and Anti-Diabetic Drugs 413 Qiong Huang and Zhao-qian Liu

VII

Preface Type 2 diabetes mellitus affects nearly 120 million persons worldwide- and according to the World Health Organization this number is expected to double by the year 2030. Owing to a rapidly increasing disease prevalence- the medical, social and economic burdens associated with the microvascular and macrovascular complications of type 2 diabetes are likely to increase dramatically in the coming decades. In this volume, leading contributors to the field review the pathogenesis, treatment and management of type 2 diabetes and its complications. They provide invaluable insight and share their discoveries about potentially important new techniques for the diagnosis, treatment and prevention of diabetic complications. Neuropathy is the most common microvascular complication in diabetes. In the Section on Diabetic Neuropathy, Bjorkman et al. provide evidence for how a novel technique known as ‘targeted plasticity’ may be useful for stimulating recovery of somatosensory function in diabetic neuropathy affecting the hand. Watanabe and co-workers describe the use of ultrasound of peripheral nerve as a potentially promising new modality for the evaluation of patients having distal polyneuropathy. Thomsen et al summarize the differences in nerve fiber morphology between large vs small fiber neuropathy. Type 2 diabetes is associated with a two-four- fold increased risk for cardiovascular disease. In the section Diabetes and Cardiovascular Disease, Hermans et al. review the evidence supporting atherogenic dyslipidemia as a potentially important component of residual vascular risk in diabetes. Stasko et al. treat the important topic of diabetes and fibrinolysis, including evidence for plasminogen activator inhibitor-1 as a predictor of cardiovascular risk in type 2 diabetes. Yaturu and Mousa discuss evidence for aspirin resistance in diabetes. Ojji reviews the pathogenesis of diabetic cardiomyopathy focusing on the unique contribution of diastolic dysfunction to heart failure in diabetes. Pappone et al summarize the results of a large prospective observational study in which type 2 diabetes was associated with an increased risk for progression from paroxysmal to persistent atrial fibrillation. Silent ischemia is highlighted in an excellent chapter by Emilia and Vlad Babes. Finally, Arce-Esquivel and colleagues review the beneficial effects of exercise on endothelial function. Hypertension frequently complicates type 2 diabetes contributing to a substantially increased risk for cardiovascular disease or progression of nephropathy. In three

X

Preface

excellent chapters written by Carella, Swislocki & Siegel, and Masuo & Lambert the latest advances in treatment of hypertension in type 2 diabetes are summarized. Ertugrul et al review treatment advances in diabetic nephropathy. MacFarlane and colleagues summarize the management of chronic anemia in diabetic nephropathy. Martinez et al describe a rat model which mimics certain key features of human diabetic nephropathy which may be useful for improving understanding and screening potential treatments to prevent this debilitating microvascular complication. Diabetes is a multi-systemic disease which can affect many different target organs. In a chapter by Nakajima et al, liver, muscle and lung involvement in diabetes are reviewed with a focus on the association between diabetes and restrictive lung disease. Diana Yu and Jamal Mohammad summarize evidence supporting an association between type 2 diabetes and an increased risk for certain types of cancer affecting the gastrointestinal tract. Kamoi summarizes evidence supporting the role for high blood pressure and techniques for improving the predictive value of high blood pressure as an important mediator of microvascular and macrovascular complications in diabetes. Finally, three excellent chapters by Begum et al, Perez-Martinez et al, and Huang & Liu informatively review aspects of the use of medicinal plants, nutritional therapy and the pharmacogenetics of anti-diabetic drugs in the treatment of type 2 diabetes.

Mark B. Zimering, MD, PhD Chief, Endocrinology Veterans Affairs New Jersey Healthcare System East Orange, New Jersey, USA Associate Professor of Medicine University of Medicine and Dentistry of New Jersey/ Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA

Part 1 Diabetic Neuropathy

1 New Treatment Strategies in Diabetic Neuropathy Anders Björkman, Niels Thomsen and Lars Dahlin

Department of Hand Surgery, Skåne University Hospital, Lund University Sweden 1. Introduction

Diabetes mellitus is associated with complications from a variety of tissues in the human body. Among them, diabetic neuropathy is a devastating complication leading to severe disability and even mortality. Neuropathy is common among type 1 and 2 diabetic patients and may yet be detected at the time of diagnosis among type 2 diabetic patients. The symptoms the patients experience vary from sensory disturbances with pain to muscle wasting. The results of autonomic neuropathy should also be considered as a severe problem in the patients, but will not be discussed in the present chapter. The possibilities to treat diabetic neuropathy are limited and a strict glycaemic control has mainly been advocated. The pathophysiology of diabetic neuropathy is complex and includes e.g. biochemical disturbances and vascular factors. Of the former, the polyol pathway has been the target for pharmacological attention. Based on the detailed knowledge of the polyol pathway, obtained from experimental models and human studies, that sorbitol accumulates in peripheral nerve trunks, pharmacological substances were developed with the purpose to decrease sorbitol levels. Such aldose reductase inhibitors have in different studies shown promising results (Bril et al., 2009; Hotta et al., 2008; Oates, 2008; Schemmel et al., 2010), but are still at a stage of development. Presently, various strategies are emerging on the management of diabetic neuropathy, where it is stressed that an appropriate diagnosis is crucial and that the condition is not untreatable (Perkins & Krolewski, 2005). In the present chapter, we present a different approach than treatment with pharmacological substances or focusing on the glycaemic level; namely, utilization of the capacity of the brain to adapt to alterations. Thus, our intention is to use the plasticity of the brain as a treatment strategy – i.e. targeted plasticity.

2. Peripheral neuropathy A peripheral neuropathy in diabetes may affect both the upper and lower extremities, where the latter location is more common and has gained more attention in research on diabetic neuropathy. Up to 50% of patients with diabetes in the United States may have a neuropathy in the lower extremity (Dyck et al., 1993). The prevalence of neuropathy in diabetes may vary from different studies depending on which diabetic population is examined, e.g. differences in Europe and Asia have been reported (Abbott et al., 2010; Rubino et al., 2007). In addition, various techniques, such as electrophysiology, examination of vibrotactile sense, skin and

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Recent Advances in the Pathogenesis, Prevention and Management of Type 2 Diabetes and its Complications

nerve biopsies and monofilament tests, have been used to detect neuropathy in diabetes. However, although the intense research over the years on various aspects of diabetic neuropathy the mechanisms, which include biochemical and vascular components, behind the different types of neuropathies in diabetes are not clarified. The pathophysiology of the neuropathy is multifaceted and not completely elucidated, although peripheral nerve dysfunction and established neuropathy in diabetes seem to be related to the degree of hyperglycaemia (Lehtinen et al., 1989; Malik et al., 1993). Recently, some studies on diabetic neuropathy with long term data on HbA1c levels available have not found any association between glycaemic level and larger fibre neuropathy (vibrotactile sense) (Dahlin et al., 2011). A specific issue in this context is if even impaired glucose tolerance can induce neuropathy, but so far large myelinated nerve fibre neuropathy is probably not associated with impaired glucose tolerance (Dahlin et al., 2008). However, reports indicate that small nerve fibre dysfunction is present in patients with impaired glucose intolerance (Dahlin et al., 2008). The latter condition and the question of possible presence of neuropathy is a complex issue and are beyond the focus of the present chapter. 2.1 Diabetic foot ulcers A particular problem in diabetes is the diabetic foot ulcer, which induces severe problems for the patients (Bengtsson et al., 2008) and causes tremendous costs for society (Prompers et al., 2008). It has long been known that diabetes may itself play an active part in the causation of perforating foot ulcers (Londahl et al., 2010). Foot ulcers are common in diabetic patients and associated with high morbidity and mortality. The prevalence of diabetic foot ulcer is 1.7 – 2.9% and the annual population based incidence among diabetic patients is 1.9 – 3.6%. Interestingly, the annual incidence rates of foot ulcers in patients with diabetic neuropathy vary from 5 to 7% (Abbott et al., 2010) and the recurrence rate is high. It is estimated that 70% of healed foot ulcers recur within five years (Apelqvist et al., 1993). It is generally accepted that the majority of amputations in diabetes are preceded by foot ulcers on the same leg with a lifetime risk of a foot ulcer estimated to reach 15-25%, where the majority of the ulcers are located to the toes. The main cause of diabetic foot ulcers are neuropathy and macro- and microvascular disease, but also other factors may increase the risk for an ulceration (Londahl et al., 2010). Patients with loss of sensation in the foot seem to have a sevenfold increased risk of developing foot ulcers as compared to diabetic patients without neuropathy (Young & Harris, 1994). In addition, a defect proprioception due to neuropathy may also cause impaired balance and postural instability contributing to the risk for foot ulceration. In clinical practice, sensory neuropathy is usually evaluated using monofilaments, 128 Hz tune fork or biothesiometer, where the latter is considered to be the most appropriate method (Edmonds, 2004). However, a multifrequency technique to examine vibrotactile sense has not previously been used to evaluate neuropathy in the foot, particularly as related to the risk for recurrence of foot ulcers. It may be an exiting approach in the future to refine detection of neuropathy. Interestingly, adjunct hyperbaric oxygen therapy, used in a multidisciplinary setting, can improve healing of chronic diabetic foot ulcers (Londahl et al., 2010); a therapy that may also beneficial in nerve regeneration after injury. 2.2 Carpal tunnel syndrome in diabetes Diabetic patients have an increased prevalence of one of the most common peripheral nerve compression lesions, i.e. carpal tunnel syndrome (CTS), which is compression of the median

New Treatment Strategies in Diabetic Neuropathy

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nerve at wrist level. It has a prevalence of 2-4% in the general population, while in diabetes it may be as high as 15%. Furthermore, if the subject has diabetic neuropathy in the lower extremity, the prevalence of CTS may approach 30% (Perkins et al., 2002). Interestingly, it has been shown that there seems to be an increased general susceptibility to peripheral nerve compression in diabetic rats (Dahlin et al., 2008), which can be related to disturbed axonal transport and a propensity to inhibit such transport in compression of diabetic nerves (Dahlin et al., 1987; Dahlin et al., 1986). Previously, it has been stated that surgical release of the median nerve in the carpal tunnel has no benefit for the patients and their symptoms from the CTS. Two previous studies showed diverse results (Mondelli et al., 2004; Ozkul et al., 2002), and proper conclusions can be difficult due to definition and selection of patients, extent of neuropathy and many other factors. Recently, we presented a prospective study where the outcome after surgical release of the carpal ligament was examined in diabetic patients with CTS and compared with ageand gender-matched healthy patients with CTS. The overall conclusion was that diabetic patients with CTS do benefit from surgical release of the carpal ligmament. This statement is relevant irrespective of the severity of the compression lesion or if signs of peripheral neuropathy are present (Thomsen et al., 2009). However, our data do not support a general view that any peripheral nerve trunk in diabetic patients should be surgically released.

3. Introduction to brain plasticity The brain has been seen as a rather static organ until about 20 years ago. It was widely believed by neuroscientists that no new neural connections could be formed in the adult brain (Kandel et al., 2000; Purves, 2004). It was assumed that once connections had been established in foetal life, or in early infancy, they hardly changed later in life. This stability of connections in the adult brain has often been used to explain why there is usually very little functional recovery after damage to the nervous system. On the other hand, memory and learning require that some changes are possible also in the adult brain (Kandel et al., 2000). It has often been assumed that these phenomena are based on small changes at the synaptic level and do not necessarily involve alterations in the basic circuit of the brain. The picture has changed radically in the last decades. One of the most interesting questions in neuroscience concerns the manner in which the nervous system can modify its organisation and ultimately its function throughout an individuals lifetime based on sensory input, experience, learning and injury (Donoghue, 1996; Kaas, 1991); a phenomenon that is often referred to as brain plasticity (Kandel et al., 2000; Purves, 2004). 3.1 Plasticity in the adult somatosensory pathways There is a complete somatotopic map of the entire body surface in the somatosensory cortex of primates (Kaas et al., 1983; Merzenich et al., 1983). Merzenich et al (Merzenich et al., 1984) showed that after amputation of the middle finger of adult primates, the area in the cortex corresponding to the amputated digit began, within two months, to respond to touch stimuli presented to the adjacent digits; i.e. this area is “taken over” by sensory input from adjacent digits. Merzenich et al (Merzenich et al., 1984) also showed that if a monkey “used” one finger excessively, for an hour and a half a day, then, after 3 months, the area of cortex corresponding to that finger “expanded” at the expense of adjacent fingers. Furthermore, if a monkey was forced to always use two fingers jointly by suturing two of its fingers together, it was found at seven weeks that single neurons in area 3b in the primary

6


somatosensory cortex had receptive fields that spanned the border separating the two digits. Interestingly, if more than one finger was amputated there was no “take over” beyond about 1 mm of cortex. Merzenich et al (Merzenich et al., 1987) concluded from this that the expansion is probably mediated by arborisation of thalamo-cortical axons that typically do not extend beyond 1 mm. The figure 1 mm has often been cited as the fixed upper limit of reorganization of sensory pathways in adult animals (Calford, 1991). Pons et al (Pons et al., 1991), however, suggested that this view might be incorrect. They found that after long-term (12 years) deafferentation of an upper limb, the cortical area originally corresponding to the hand in the primary somatosensory cortex was taken over by sensory input from the face. The cells in “the cortical hand area” now started to respond to stimuli applied to the lower face region. Since this patch of cortex is more than 1 cm wide, they concluded that sensory reorganisation could occur over at least this distance, i.e. an order of magnitude ten times greater than the original 1 mm limit. In addition to these long-term changes that are typically seen weeks or months after deprivation or stimulation, Calford and Tweelade (Calford, 1990) reported rapid, short term changes that are based, presumably, on the unmasking of pre-existing connections rather than on anatomical “sprouting”. They anaesthetized the middle finger of flying foxes and found that within 20 minutes the cortical neurons in the primary somatosensory cortex that originally responded to the middle finger could then be activated by touching the adjacent digits, indicating that the receptive fields had expanded to include adjacent digits. Calford and Tweedale (Calford, 1990) also showed that a small unilateral peripheral denervation in adult flying foxes lead to expansion of the cortical receptive field for neighboring skin areas as predicted from the work of Merzenich et al (Merzenich et al., 1984). Rapid plasticity changes are typically seen minutes after injury or an intervention, and are often based on decreased inhibition. Decreased inhibition would theoretically increase the receptive field size and enable more neurons to be activated by the stimulus. This is sometimes referred to as unmasking of synapses or neural structures. Surprisingly, the receptive field of the homotopic region in the other hemisphere mirrored the change. In other words, the second hemisphere learned what the first had done; it copied the revised sensory map. Maintaining symmetric sensory representation of the two sides in the cerebral cortex may be important for the control of symmetric bilateral motor activity. Experience dependent plasticity refers to the ability of the adult brain to adjust itself to changes in environmental conditions. It relates to the learning of special skills that requires special training and it often requires motivation and concentration on the task. Another example of brain plasticity is the so called cross-modal plasticity. This phenomenon implies that one sensory modality can substitute for another (Bavelier & Neville, 2002). The most well known example is in blind persons where an improved sensory function is noticed. It has also been shown that when a blind person reads Braille activation in the occipital lobe occurs implying that the somatosensory stimuli from reading activates the cortical area responsible for vision (Gizewski et al., 2003). Another example is persons in whom the lack of sensibility can be substituted with hearing. Through small microphones on the fingers the persons can, after a short training period, listen to what they feel (Lundborg et al., 1999). A crucial element in such cross-modal plasticity seems to be training, in order for a sensory modality to “take over” another sensory modality.


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3.2 Mechanisms of plasticity Several cellular mechanisms by which the adult brain can adjust to changes in the environment or in sensory input have been defined, including the following (Kandel et al., 2000; Purves, 2004). Decreased inhibition Many connections between the periphery and the cortex as well as intracortical connections are physiologically “silent” because of inhibitory influences (Wall, 1977). Sensory stimulation of a point on the skin activates neurons in the somatosensory system near the centre of the area of cortical representation and inhibits activity in neurons near the edges. In this way the receptive field appears smaller than its actual size. The inhibition is due to activation of inhibitory interneurons near the edges of the receptive field. Decreased inhibition would theoretically increase the receptive field size and enable more neurons to be activated by the stimulus; this is sometimes referred to as unmasking of synapses or neural structures. Gamma-aminobutyric acid (GABA) is the most important inhibitory neurotransmittor in the brain (Jones, 1993) and evidence is strong that reduction of GABAergic inhibition is crucial in mediating short term plasticity changes (Chen et al., 2002). Increase in synaptic strength The effectiveness of synaptic connections is continuously adopted in response to functional demands. Synaptic transmission becomes facilitated in a pathway that is frequently used, while those that lay dormant atrophy. In this way, repeated practice of a task leads to increased speed and accuracy of performance. Increased synaptic strength may be a mechanism for learning and also for recovery from brain injury. Repetitive stimulation results in increased excitability and facilitation of transmission in the synapses. These effects persist for some time after the initial stimulus and subsequently show gradual declines (long term potentiation, LTP). Calcium channels in the neuronal membrane appear to be crucial in this process. LTP is probably one of the major mechanism by which learning and memory consolidation takes place in the brain (Kandel et al., 2000). Axonal and dendritic sprouting The sprouting and elongation of new dendrites and axons is a common response to injury and cell loss at all levels in the nervous system. Sprouting can also be seen in response to increased functional demand, such as exposure to conditions requiring more complex motor activity (Kleim et al., 1996). Axons at the edges of a lesion send new axonal branches into the damaged area and re-innervate dendrites that have lost their synaptic input. This leads to new synaptic formation at the point of contact of axonal sprouts with these dendritic trees. This mechanism for recovery has been suggested in, for example, the reaction of the somatosensory cortex to loss of its input from the skin (Merzenich et al., 1984; Pons et al., 1991). 3.3 Targeted plasticity The primary somatosensory-and motorcortex is organized somatotopically, where different body parts project to different parts of the primary somatosensory-and motor cortex (Figure 1). The somatotopic map does not represent the body in its actual proportions. Instead, larger cortical areas are being assigned to sensitive parts or parts with complex motor demands, such as the hands and face. The cortical representation of different body parts alters constantly, depending on the pattern of afferent nerve impulses, injury and increased or decreased use.

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Fig. 1. Sensory information is sent from, in this case, the hand via the peripheral nerve to the dorsal root ganglia, spinal cord and thalamus to the primary somatosensory cortex. Motor information is sent from the primary motor cortex to the spinal cord and the effector muscles. Both the primary somatosensory and motor cortex are arranged somatotopically. Thus in each hemisphere there is a complete somatotopic map of the body both in the primary somatosensory and motor cortex. To utilize the central nervous systems’ (CNS) ability to change for therapeutic purposes, guided plasticity is an attractive concept with promising results (Duffau, 2006). The potential for cerebral plasticity is, for example, used in treatment of patients to strengthen or promote CNS functions that are lost or weakened. The plastic potential of the brain might be guided using neurosurgical methods, rehabilitation and different pharmacological drugs in order to improve lost or damaged functions (Duffau, 2006). The use of neurosurgical methods is very complicated, sometimes including complex surgical interventions, which limit the usefulness. The use of potent drugs affecting the central nervous system, such as amphetamine (Walker-Batson et al., 2001) and norepinephrine (Plewnia et al., 2004), in order to improve recovery of damaged function, have been described. However, few patients currently benefit from such treatments due to incomplete knowledge of optimal treatment regimes and side effects from the drugs.


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Our main objective when starting to develop a treatment regime for diabetic neuropathy was to look for a method more suitable for patient usage, giving a cortical deafferentation “large” enough to induce changes in peripheral function but not unnecessarily large. The method should also be safe with no side effects, pain free, and easy to us for both patient and therapist. Furthermore, it should be specific for sensory functions not affecting the motor function as this would affect the person’s ability to perform motor tasks. It is well known from animal and human experiments that temporary cutaneous anesthesia of one body part leads to cortical re-organization resulting in a corresponding silent area in the sensory cortex. This allows adjacent nearby body parts to rapidly expand at the expense of the silent cortical area this is likely mediated by unmasking of existing synapses. The forearm is located next to the hand in the somatotopic map and by anaesthetizing the forearm, the cortical hand area can rapidly expand over the forearm area resulted in improved sensory function of the hand in healthy controls (Bjorkman et al., 2009). Thus, more nerve cells can be available for the hand, resulting in improved hand function. In a randomized, controlled trial, sensory re-learning in combination with cutaneous forearm anesthesia, using an anesthetic cream, EMLA® containing 2.5% lidocain and 2.5% prilokain, improved sensory function of the hand compared with sensory re-learning and placebo in patients with ulnar or median nerve repair (Rosen et al., 2006). The participants received treatment twice a week for two consecutive weeks, and the effects lasted 4 weeks after the last EMLA® treatment. These results suggest that sensory recovery is enhanced by temporary anesthesia of adjacent body parts. The long lasting effect indicates that this treatment is clinically useful and relevant. Recently, the same principle of temporary cutaneous anesthesia as that used for the hand has been applied on the foot in uninjured subjects. In a randomized controlled trial, improvement in sensory function of the foot was observed after EMLA® treatment of the lower leg compared to placebo (Rosen et al., 2009). There is no specific treatment for neuropathy in diabetes except a strict control of the glycaemic level. However, recent data in healthy subjects and diabetic patients show that the sensory function in the foot and hand, measured by the monofilament test, can be improved by using the central nervous systems ability to change, i.e. brain plasticity. In a recent double blind randomized placebo controlled study male (n=26) or female (n=5) diabetic patients with type 1 (n=30) or type 2 (n=7) with a median duration of diabetes of 35 years, all with insulin treated diabetes, were either treated with EMLA® cream or placebo cream applied to the skin of the lower leg for 1.5 hours (n=18 and n=19, respectively). All the subjects in the EMLA® group with pre-treatment diminished protective sensibility at the first metatarsal head showed improved touch threshold below limits for protective sensibility after 1.5 and 24 hours, while no such changes were observed after the treatment with placebo cream (Fig 2). Furthermore, the touch thresholds improved at four other assessment sites (third metatarsal head, fifth metatarsal head, pulp of big toe and central of heal) together with increased vibration threshold at 125 Hz. However, the patients observed no subjective improvement, based on examination with a visual analogue scale, after treatment. This new strategy to improve the thresholds of touch creates new possibilities to treat disturbances in sensation of the diabetic foot. Hypothetically, the local anaesthetic cream results in a deafferentation of the lower leg in the primary somatosensory cortex, which allows the foot to expand. Thus, more nerve cells are available for the foot resulting in the observed improved sensory function.

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A challenge is to create a long lasting improvement of the sensory function. Studies using cutaneous anaesthesia in the upper extremity in patients with nerve injuries and neuropathy have shown that a lasing improvement of sensibility is possible using repeated sessions with cutaneous anaesthesia (Rosen et al., 2008; Rosen et al., 2006). In conclusion, treatment of diabetic neuropathy is complicated. However, new knowledge on the effect of a peripheral nerve injury and neuropathy on the central nervous system opens new perspectives to treat neuropathy by targeted plasticity. Cutaneous anaesthesia of the lower leg in diabetic patients is a good example of how targeted plasticity is used in order to improve foot sensibility in patients with diabetic neuropathy. The method is simple, safe, and cost-effective, although future studies are needed to work out the optimal treatment regime for a long lasting or permanent improvement in sensibility.

Fig. 2. Change of touch threshold in first metatarsal head in diabetic patients between pretreatment and after 1.5 h of EMLA® treatment compared with placebo (P < 0.001).

4. Acknowledgement The studies on diabetic neuropathy from our group was supported by the Swedish Research Council (Medicine), Crafoord’s Fund for Medical Research, Svenska Diabetesförbundet, Diabetesföreningen Malmö, Konsul Thure Carlsson Fund for Medical Research, Region


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Skåne, Stiftelsen Sigurd och Elsa Goljes Minne and Funds from the University Hospital Malmö, Sweden.

5. References Abbott, CA., Chaturvedi, N., Malik, RA., Salgami, E., Yates, AP., Pemberton, PW., & Boulton, AJ. (2010). Explanations for the lower rates of diabetic neuropathy in Indian Asians versus Europeans. Diabetes Care, Vol. 33, No. 6, pp 1325-1330 Apelqvist, J., Larsson, J., & Agardh, C.D. (1993). Long-term prognosis for diabetic patients with foot ulcers. Journal of Internal Medicine, Vol. 233, No. 6, pp 485-491 Bavelier, D., & Neville, HJ. (2002). Cross-modal plasticity: where and how? Nat Rev Neurosci, Vol. 3, No. 6, pp 443-452 Bengtsson, L., Jonsson, M., & Apelqvist, J., (2008). Wound-related pain is underestimated in patients with diabetic foot ulcers. Journal of Wound Care, Vol. 17, No. 10, pp 433-435 Bjorkman, A., Weibull, A., Rosen, B., Svensson, J., & Lundborg, G. (2009). Rapid cortical reorganisation and improved sensitivity of the hand following cutaneous anaesthesia of the forearm. European Journal of Neuroscience, Vol. 29, No. 4, pp 837844 Bril, V., Hirose, T., Tomioka, S., Buchanan, R., & Ranirestat Study Group. (2009). Ranirestat for the management of diabetic sensorimotor polyneuropathy. Diabetes Care, Vol. 32, No. 7, pp 1256-1260 Calford, M. (1991). Neurobiology. Curious cortical change. Nature, Vol. 352, No. 6338, pp 759-760 Calford, MB., & Tweedale, R. (1990). Interhemispheric transfer of plasticity in the cerebral cortex. Science, Vol. 249, pp 805-807 Chen, R., Cohen, LG., & Hallett, M. (2002). Nervous system reorganization following injury. Neuroscience, Vol. 111, No. 4, pp 761-773 Dahlin, LB., Archer, DR., & McLean, WG. (1987). Treatment with an aldose reductase inhibitor can reduce the susceptibility of fast axonal transport following nerve compression in the streptozotocin-diabetic rat. Diabetologia, Vol. 30, pp 414-418 Dahlin, LB., Granberg, V., Rolandsson, O., Rosen, I., & Dahlin, E. (2011). Disturbed vibrotactile sense in finger pulps in patients with type 1 diabetes – correlations with glycaemic level, clinical examination and electrophysiology. Diabetic Medicine, Vol. In press Dahlin, LB., Meiri, KF., McLean, WG., Rydevik, B., & Sjöstrand, J. (1986). Effects of nerve compression on fast axonal transport in streptozotocin-induced diabetes mellitus. Diabetologia, Vol. 29, pp 181-185 Dahlin, LB., Thrainsdottir, S., Cederlund, R., Thomsen, NO., Eriksson, KF., Rosen, I., Speidel, T., & Sundqvist, G. (2008). Vibrotactile sense in median and ulnar nerve innervated fingers of men with Type 2 diabetes, normal or impaired glucose tolerance. Diabetic Medicine, Vol. 25, No. 5, pp 543-549 Donoghue, JP., Hess, G., & Sanes, JN. Motor cortical substrates and mechanisms for learning. In: Bloedel JR, Ebner, T.J., Wise, S.P. (ed). Acquisition of Motor Behaviour in Vertebrates. Cambridge, MA: MIT; 1996. pp. 363-386. Duffau, H. (2006). Brain plasticity: from pathophysiological mechanisms to therapeutic applications. J Clin Neurosci, Vol. 13, No. 9, pp 885-897

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Dyck, PJ., Kratz, KM., Karnes, JL., Litchy, WJ., Klein, R., Pach, JM., Wilson, DM., O'Brien, PC., Melton, LJ., 3rd, & Service, FJ. (1993). The prevalence by staged severity of various types of diabetic neuropathy, retinopathy, and nephropathy in a population-based cohort: the Rochester Diabetic Neuropathy Study. Neurology, Vol. 43, No. 4, pp 817-824 Edmonds, ME, (2004). The diabetic foot, 2003. Diabetes/Metabolism Research and Reviews, Vol. 20 Suppl 1, pp S9-S12 Gizewski, ER., Gasser, T., de Greiff A., Boehm, A., & Forsting, M. (2003). Cross-modal plasticity for sensory and motor activation patterns in blind subjects. Neuroimage, Vol. 19, No. 3, pp 968-975 Hotta, N., Kawamori, R., Atsumi, Y., Baba, M., Kishikawa, H., Nakamura, J., Oikawa, S., Yamada, N., Yasuda, H., & Shigeta, Y.; ADCT Study Group. (2008). Stratified analyses for selecting appropriate target patients with diabetic peripheral neuropathy for long-term treatment with an aldose reductase inhibitor, epalrestat. Diabetic Medicine, Vol. 25, No. 7, pp 818-825 Jones, EG. (1993). GABAergic neurons and their role in cortical plasticity in primates. Cerebral Cortex, Vol. 3, No. 5, pp 361-372 Kaas, J. (1991). Plasticity of sensory and motor maps in adult mammals. Ann Rev neurosci, Vol. 14, pp 137-168. Kaas, JH., Merzenich, MM., Killackey, HP. (1983). The reorganization of somatosensory cortex following peripheral nerve damage in adult and developing mammals. Annual Review of Neuroscience, Vol. 6, pp 325-356 Kandel, ER., Schwartz, JH., & Jessel, TM. Principles of neural science. McGraw-Hill; 2000. Kleim JA, Lussnig E, Schwarz ER et al., (1996). Synaptogenesis and Fos expression in the motor cortex of the adult rat after motor skill learning. Journal of Neuroscience, Vol. 16, No. 14, pp 4529-4535 Lehtinen, JM., Uusitupa, M., Siitonen, O. & Pyorala, K. (1989). Prevalence of neuropathy in newly diagnosed NIDDM and nondiabetic control subjects. Diabetes, Vol. 38, No. 10, pp 1307-1313 Londahl, M., Katzman, P., Hammarlund, C., Nilsson, A., & Landin-Olsson, M. (2010). Relationship between ulcer healing after hyperbaric oxygen therapy and transcutaneous oximetry, toe blood pressure and ankle-brachial index in patients with diabetes and chronic foot ulcers. Diabetologia, Vol. 54, No. 1, pp 65-68 Lundborg, G., Rosén, B., & Lindberg, S. (1999). Hearing as substitution for sensation - a new principle for artificial sensibility. J Hand Surg, Vol. 24A, pp 219-224 Malik, RA., Tesfaye, S., Thompson, SD., Veves, A., Sharma, AK., Boulton, AJ., & Ward, JD. (1993). Endoneurial localisation of microvascular damage in human diabetic neuropathy. Diabetologia, Vol. 36, No. 5, pp 454-459 Merzenich, MM., Kaas, JH., Wall, RJ., Nelson, M., Sur, D., & Felleman, D. (1983). Topographic reorganization of somatosensory cortical areas 3B and 1 in adult monkeys following restricted deafferentation. Neuroscience, Vol. 8, pp 33-55 Merzenich, MM., Nelson, RJ., KaasJH., Stryker, MP., Jenkins, WM., Zook, JM., Cynader, MS., & Schoppmann, A. (1987). Variability in hand surface representations in areas 3 b and 1 in adult own and squirrel monkeys. Journal of Comparative Neurology, Vol. 258, pp 281-297


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Merzenich, MM., Nelson, RJ., Stryker, MS., Cynader, MS., Schoppmann, A., & Zook, JM. (1984). Somatosensory cortical map changes following digit amputation in adult monkeys. Journal of Comparative Neurology, Vol. 224, pp 591-605 Mondelli, M., Padua, L., Reale, F., Signorini, AM., & Romano, C. (2004). Outcome of surgical release among diabetics with carpal tunnel syndrome. Archives of Physical Medicine and Rehabilitation, Vol. 85, No. 1, pp 7-13 Oates, PJ. (2008). Aldose reductase, still a compelling target for diabetic neuropathy. Curr Drug Targets, Vol. 9, No. 1, pp 14-36,1873-5592 (Electronic) 1389-4501 (Linking). Ozkul, Y., Sabuncu, T., Kocabey, Y., & Nazligul, Y. (2002). Outcomes of carpal tunnel release in diabetic and non-diabetic patients. Acta Neurologica Scandinavica, Vol. 106, No. 3, pp 168-172 Perkins, BA., & Krolewski, AS. (2005). Early nephropathy in type 1 diabetes: a new perspective on who will and who will not progress. Curr Diab Rep, Vol. 5, No. 6, pp 455-463, Perkins, BA., Olaleye, D., & Brilm, V. (2002). Carpal tunnel syndrome in patients with diabetic polyneuropathy. Diabetes Care, Vol. 25, No. 3, pp 565-569 Plewnia, C., Hoppe, J., Cohen, LG., & Gerloff, C. (2004). Improved motor skill acquisition after selective stimulation of central norepinephrine. Neurology, Vol. 62, No. 11, pp 2124-2126 Pons, T., Preston, E., & Garraghty K, (1991). Massive cortical reorganization after sensory deafferentation in adult macaques. Science, Vol. 252, pp 1857-1860 Prompers, L., Huijberts, M., Schaper, N., Apelqvist, J., Bakker, K., Edmonds, M., Holstein, P., Jude, E., Jirkovska, A., Mauricio, D., Piaggesi, A., Reike, H., Spraul, M., Van Acker, K., Van Baal, S., Van Merode, F., Uccioli, L., Urbancic, V., & Ragnarson Tennvall, G. (2008). Resource utilisation and costs associated with the treatment of diabetic foot ulcers. Prospective data from the Eurodiale Study. Diabetologia, Vol. 51, No. 10, pp 1826-1834 Purves, D., Augustine, GJ., Fitzpatrick, D., Hall, WC., La Mantia AS., McNamara JO., & Williams SM. Neuroscience (Sinauer Associates Inc. Sunderland, MA, USA, third edition). 2004. Rosen, B., Bjorkman A, & Lundborg G, (2008). Improved hand function in a dental hygienist with neuropathy induced by vibration and compression: The effect of cutaneous anaesthetic treatment of the forearm. Scandinavian Journal of Plastic and Reconstructive Surgery and Hand Surgery, Vol. 42, No. 1, pp 51-53 Rosen, B., Bjorkman, A., & Lundborg, G. (2006). Improved sensory relearning after nerve repair induced by selective temporary anaesthesia - a new concept in hand rehabilitation. Journal of Hand Surgery British Volume, Vol. 31, No. 2, pp 126-132 Rosen, B., Bjorkman, A., Weibull, A., Svensson, J., & Lundborg, G. (2009). Improved sensibility of the foot after temporary cutaneous anesthesia of the lower leg. Neuroreport, Vol. 20, No. 1, pp 37-41 Rubino, A., Rousculp, MD., Davis, K., Wang, J., Bastyr, EJ., & Tesfaye, S. (2007). Diagnosis of diabetic peripheral neuropathy among patients with type 1 and type 2 diabetes in France, Italy, Spain, and the United Kingdom. Prim Care Diabetes, Vol. 1, No. 3, pp 129-134

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Schemmel, KE., Padiyara, RS., & D'Souza, JJ. (2010). Aldose reductase inhibitors in the treatment of diabetic peripheral neuropathy: a review. Journal of Diabetes and Its Complications, Vol. 24, No. 5, pp 354-360,1873-460X (Electronic) 1056-8727 (Linking). Thomsen, NO., Cederlund, R., Rosen, I., Bjork, J., & Dahlin, LB. (2009). Clinical outcomes of surgical release among diabetic patients with carpal tunnel syndrome: prospective follow-up with matched controls. J Hand Surg Am, Vol. 34, No. 7, pp 1177-1187 Walker-Batson, D., Curtis, S., Natarajan, R., Ford, J., Dronkers, N., Salmeron, E., Lai, J., & Unwin, DH. (2001). A double-blind, placebo-controlled study of the use of amphetamine in the treatment of aphasia. Stroke, Vol. 32, No. 9, pp 2093-2098,15244628 (Electronic). Wall PD. (1977). The presence of ineffective synapses and the circumstances which unmask them. Philosophical Transactions of the Royal Society of London Series B: Biological Sciences, Vol. 278, No. 961, pp 361-372 Young, TK., & Harris, SB, (1994). Risk of clinical diabetes in a northern Native Canadian cohort. Arctic Medical Research, Vol. 53, No. 2, pp 64-70

2 Sonographic Imaging of the Peripheral Nerves in Patients with Type 2 Diabetes Mellitus Tsuneo Watanabe, Shin-ichi Kawachi1 and Toshio Matsuoka

Department of Sports Medicine and Sports Science, Gifu University Graduate School of Medicine 1Departments of Diabetes and Endocrinology, Gifu University Graduate School of Medicine Japan

1. Introduction The World Health Organization estimates that more than 220 million people worldwide have diabetes mellitus (DM). This figure is estimated to more than double by 2030 (Wild et al., 2004). In Japan, the number of diabetic patients has increased to 8 million, and it is assumed that 35% to 45% of diabetic patients have diabetic symmetric polyneuropathy (DPN). Advanced DPN causes serious complications, such as diabetic foot ulcers, gangrene, and Charcot joint, all of which worsen the quality of life of diabetic patients (Ogawa et al., 2006). Therefore, early detection of nerve dysfunction is important to provide appropriate care for patients with DPN (Chudzik et al., 2007). The diagnosis of diabetic neuropathy is based primarily on characteristic symptoms and is confirmed with nerve conduction studies (NCS), which are time-consuming, slightly invasive, and occasionally not well tolerated for repeated evaluations (Colak et al., 2007). In contrast, ultrasonographic examinations can be performed to assess peripheral nerves with less discomfort and have already been used for the evaluation of several disorders of the peripheral nervous system such as carpal tunnel syndrome (Wiesler et al., 2006a; Abe et al., 2004; Jayaraman et al., 2004; Duncan et al., 1999; Lee et al., 1999), cubital tunnel syndrome (Okamoto et al., 2000; Wiesler et al., 2006b), and traumatic nerve lesions (Cartwright., 2007; Peer et al., 2001). High-resolution diagnostic ultrasonography (US) has improved greatly, allowing for evaluation of minute peripheral nerves (Fornage., 1993; Solbiati et al., 1985). We previously showed that the cross-sectional area (CSA) of the median nerve in the carpal tunnel of patients with DPN is greater than that of controls and correlates with NCS (Watanabe et al., 2009). Furthermore, it appears that the percentage of the hypoechoic area of the peripheral nerves was significantly greater in patients with lower motor nerve conduction velocity (MCV) and DM than in controls or patients with higher MCV and DM (Watanabe et al., 2010). The purpose of this chapter is to review the current knowledge regarding the overview and diagnosis of the most common forms of neuropathy in type 2 DM. Furthermore, our current sonographic technique and preliminary studies are presented for some cases. In this chapter,

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we focus mainly on a simple and noninvasive approach to the evaluation of peripheral nerves in patients with type 2 DM. Although the exact mechanisms contributing to our study have not been clearly identified and are not the main focus of this chapter, they will be discussed in brief.

2. Clinical aspects of diabetic neuropathy Diabetic neuropathy is a neuropathic disorder that is associated with DM. This condition is thought to result from both diabetic microvascular injury involving small blood vessels that supply nerves and macrovascular conditions that can culminate in DM. More than 80% of patients with clinical diabetic neuropathy have a distal, symmetrical form of the disorder (Said., 2007). In general, the symptoms included numbness, burning feet, pins-and-needles sensations, and lightning pains. These symptoms start in the feet go on to affect more proximal parts of the lower limbs, and eventually affect the distal parts of the upper limbs. The unified clinical criteria and classification for DPN do not represent the international standard because the causes of peripheral nerve disorders associated with DM are complex and probably involve a variety of causative mechanisms. In this chapter, DPN is classified into 3 groups (hyperglycemic neuropathy, symmetric polyneuropathy, and focal and multifocal neuropathy) according to the classification of the Thomas et al (1997). This classfication is the easiest. The most common of these groups seen in the clinical setting is symmetric distal polyneuropathy.

3. Pathology of diabetic neuropathy Abnormalities reported in diabetic neuropathy include axonal degeneration in nerve fibers, primary demyelination resulting from Schwann cell dysfunction, secondary segmental demyelination related to impairment of axonal control of myelination, remyelination, proliferation of Schwann cells, atrophy of denervated bands of Schwann cells, onion-bulb formations, and hypertrophy of the basal lamina. Early morphological changes include minimal alteration of myelinated and unmyelinated fibers and axonal regeneration (Yagihashi et al., 2007; Said et al., 2007). The pathophysiology of DPN is multifactorial and involves genetic, environmental, behavioral, metabolic, neurotrophic, and vascular factors (Vink et al., 1999; Oates, 2002; Vincent, et al., 2002; Perkins, et al., 2003). The vascular concept of peripheral diabetic neuropathy implies that diabetes-induced endothelial dysfunction with a resultant decrease in nerve blood flow, vascular reactivity, and endoneurial hypoxia plays a key role in functional and morphological changes in the diabetic nerve (Cameron et al., 2001). Endothelial changes in the vasa nervorum have been attributed to multiple mechanisms, including increased aldose reductase activity, nonenzymatic glycation and glycoxidation, activation of protein kinase C, oxidative–nitrosative stress, and changes in arachidonic acid and prostaglandin metabolism (Cameron et al., 2001). The complex and interrelated effects of hyperglycemia include increased metabolic flux through the polyol pathway with consequent sorbitol and fructose accumulation and reduced sodium-potassium ATPase levels, altered fatty acid metabolism, alterations in the redox state, reduced myoinositol and sodium-potassium ATPase activity, accumulation of advanced glycated end products, accelerated neuronal apoptosis, immunological alterations, changes in blood flow, and

Sonographic Imaging of the Peripheral Nerves in Patients with Type 2 Diabetes Mellitus

17

increased oxidative stresses. The exact mechanisms of DPN are uncertain, but may involve activation of the polyol pathway due to hyperglycemia; the polyol pathway is considered to play a major role in diabetic neuropathy (Greene, et al., 1987). Excellent reviews of this information are available in previous publications.

4. NCS Diagnosis of DPN on clinical grounds alone is not accurate, and it is difficult to detect small alterations in neuropathies (Feldman et al, 1994; Perkins et al, 2001). Therefore, as a surrogate measure, NCS are widely used as an evaluation of DPN. NCS measure the ability of peripheral nerves to conduct electrical signals, and this ability is impaired when pathological changes are present in the myelin, nodes of Ranvier, and axons. Routine NCS include evaluation of the motor function of the median, ulnar, peroneal, and tibial nerves, and evaluation of the sensory function of the median, ulnar, radial, and sural nerves. Velocities are universally reported in meters per second, motor amplitudes in millivolts, and sensory amplitudes in microvolts. These measurements of upper- and lower-limb motor and sensory nerve functions show the presence, distribution, and severity of peripheral nerve disease (Albers, et al., 1995). The attribution of peripheral nerve dysfunction to either primary demyelination or primary axonal loss is usually based on nerve conduction velocity and action potential amplitude data. In general, demyelination is indicated by a decreased nerve conduction velocity, conduction block, or increased temporal dispersion, whereas axonal loss is indicated by a reduction in the amplitude or area of the sensory nerve action potential (SNAP) or compound muscle action potential (CMAP). However, there has been considerable disagreement in terms of the clinical and the electrophysiological criteria for the diagnosis of DPN. Recently, various calculated indices such as the residual latency, terminal latency index, and modified F-wave ratio were introduced as more sensitive electrophysiological tools than a conventional NCS in patients with diverse types of peripheral neuropathies (Attarian et al, 2001; Kaplan, et al, 1978; Radziwill et al, 2003).

5. Sonography 5.1 Sonographic features of normal peripheral nerves US is a widely utilized diagnostic tool for gynecological purposes and examinations of the heart and intra-abdominal and superficial organs. With the advancement of sonographic resolution, normal peripheral nerves also can be clearly demonstrated. US is a useful technique for the investigation of a number of musculoskeletal disorders. Although US has the well-known advantages of low cost, accessibility, portability, noninvasiveness, and multiplanar imaging, one of its most important diagnostic advantages over other techniques is considered to be its real-time imaging capability, allowing for dynamic evaluation of the musculoskeletal field (Khoury et al., 2007). US can be used to determine the location, extent, type of lesion as well as the presence of nerve swelling and inflammation. Major peripheral nerves in the extremities, such as the median, ulnar, radial, sciatic, and posterior tibial nerves can be seen using conventional US performed with 5- to 12-MHz probes (Stokvis et al., 2009). In controls, peripheral nerves are seen as hypoechoic neuronal fascicles surrounded by echogenic connective tissue (Silvestri

18


et al., 1995). The basic units of the peripheral nerve consist of a neural fiber embedded in the endoneurium. Because the endoneurium is too thin to reflect the sound beam, it is hypoechogenic on the US scan. The neural fascicle consists of several neural fibers and is embedded in a capsule called the perineurium. This capsule consists of connective tissue, vessels, and lymphatic ducts and is thick enough to reflect the sound beam, resulting in hyperechoic lines on the US scan. The trunk of the peripheral nerve consists of several neural fascicles and is embedded in a thicker membrane called the epineurium, which is seen as bold echogenic lines on the US. Therefore, a peripheral nerve is seen as several parallel hyperechoic lines and bold hypoechoic lines on longitudinal images and as a faveolate pattern on transverse images (Fig 1).

(a)

(b)

(c) Fig. 1. Sonographic imaging of the median nerve. Transverse sonogram of the median nerve in 5cm proximal to the wrist (a), and longitudinal sonogram of the median nerve (b). Multislice views using three-dimensional volumetric ultrasonography images (c). The transverse planes at continuous segment of the median nerves are visualized.


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5.2 Sonographic features of the peripheral nerves in patients with type 2 DM We previuosly reported that peripheral nerves in patients with low MCV and type 2 DM showed enlarged and hypoechoic patterns as compared with those of controls or patients with high MCV and type 2 DM. There are 2 sonographic methods of measuring nerve CSA: the indirect method (ellipsoid formula) and direct method (tracing). Recently, Alemán et al.(2008) reported that median nerve CSA measurements are reproducible by either the direct or indirect method when a standardized ultrasonographic examination protocol is applied. Sernik et al. (2008) also reported a high correlation (r = 0.99) between the areas calculated by the indirect and direct methods. In patients with DPN, peripheral nerves showed enlarged and hypoechoic patterns. Figure 2 shows sonographic images of several nerves in the controls and in patients with type 2 DM. The CSAs of the median, ulnar, and tibial nerves in patients with DM were significantly larger than those in controls. It is likely that these findings reflect the pathological changes, although the pathogenesis of nerve enlargement and increased percentage of the hypoechoic area in peripheral nerves is uncertain because our study did not include histological evidence. In a 1H-nuclear magnetic resonance study, Suzuki et al. (1994) reported that sorbitol and the sodium accumulation caused by an increase in sorbitol may be major contributors to the increase in intracellular hydration. It has further been hypothesized that peripheral nerves are swollen in individuals with DM because of increased water content related to an increase in the aldose reductase-mediated conversion of glucose to sorbitol. We hypothesize that the increase in the hypoechoic area of peripheral nerves in diabetic patients may be because of increased water content, which is also a cause of enlargement of peripheral nerves. 5.3 Assessment of the internal echo of the peripheral nerves The ultrasonographic images of the peripheral nerves were saved as JPEG files and transferred to a personal computer for analysis. The monochrome US image was quantized to 8 bits (i.e., 256 gray levels). The brightness of the pixels ranged from 0 (black) to 255 (white). Histogram analysis in US has been expected to offer an objective index for estimating the echo intensity, such as in the diagnosis of fatty liver or hepatitis (Lee et al., 2006; Osawa et al., 1996). The region of interest was set to cover the entire nerve, excluding its hyperechoic rim. We used the percentage of the hypoechoic area as the index after the effects of gain shift on echo intensity in the median nerve were confirmed (Fig. 3). The normal appearance of a peripheral nerve should be readily recognized. Peripheral nerves consist of multiple hypoechoic bands corresponding to neuronal fascicles, which are separated by hyperechoic lines that correspond to the epineurium. Thus, the value obtained by the discriminant analysis method of Otsu was used as a threshold level for the analysis of the percentage of the hypoechoic area because the echogenicity of peripheral nerves was obtained as a graded echo density from black to white. Otsu’s method (1979), which selects a global threshold value by maximizing the separability of the classes in gray levels, is one of the better tequniques for image segmentation. Mathematically, this can be expressed as: fi pi = N , pi ≧ 0,

L

pi i=1

= 1.

(1)

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Fig. 2. Comparison of the nerves in controls and in diabetic patients. Transverse image of the median nerve in control’ wrist (a), and in diabetic patient’s wrist (b). Transverse image of the ulnar nerve in control’ wrist (c), and in diabetic patient’s wrist (d). Transverse image of the tibial nerve in control’ ankle (e), and in diabetic patient’s ankle (f). Transverse sonogram of the sural nerve in control’ ankle (g), and in diabetic patient’s ankle (h).

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Fig. 3. The effects of the gain shift on the echo intensity in the median nerve. Open bars showed a change of mean, closed bars showed a change of SD, and solid line connecting solid circles showed a change of percentage according to the gain shift. Assume that the image can be represented in L gray levels (1, 2,…, L). If the number of pixels at level i is denoted by fi, then the total number of pixels equals N = f1 + f2 + …+fL. If an image can be divided into 2 classes (C1 and C2) by a threshold at level t, where class C1 consists of gray levels from 0 to t, and class C2 contains the other gray levels with t + 1 to L, then the cumulative probabilities (w1 and w2) and mean levels (μ1 andμ2) for classes C1 and C2, respectively, are given by ｔ

w1 = pi ,

(2)

i=1

L

pi ,

w2 =

(3)

i=t+1

and ｔ

μ1 = ipi/w1 ,

(4)

i=1

L

μ2 = ipi/w2 .

(5)

i=t+1 2

Otsu selects an optimal threshold t* that maximizes the between-class varianceσB in Eq. (6) based on the discriminant analysis, where μT is the mean intensity of the image. t* = and

arg

1≦t＜L

2

2

max{σB (t)|σB = w1(μ1 –μT)2 + w2(μ2 –μT)2}

(6)

22

Recent Advances in the Pathogenesis, Prevention and Management of Type 2 Diabetes and its Complications L

μT = ipi .

(7)

i=1

The percentage of the hypoechoic area was studied using computer analysis. Using ImageJ software, the amount of the hypoechoic area falling below the threshold echo intensity was calculated. In our computerized method, the flowchart of the echo intensity evaluation process is demonstrated in Figure 4.

Fig. 4. Illustration of the working flow of the evaluation of the echo intensity. Representative sonographic images and three-dimensional graphic using ImageJ software of diabetic patients and controls are shown in Fig 5.


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Fig. 5. Distributions of the echo intensity in the median nerve. Transverse sonogram of the median nerve in a control participant’s wrist (a). Transverse sonogram of the median nerve in a diabetic patient’s wrist (b).Three-dimensional graphic of the distribution of the echo intensity in the control’s wrist (c). Three-dimensional graphic of the distribution of the echo intensity in the diabetic patient’s wrist (d). Hypoechoic area was displayed “light blue”, and hyperechoic area was displayed “pink”.

6. Sonography and NCS in patients with type 2 DM 6.1 Relationship between sonography and NCS We studied 144 peripheral nerves (40 median, 40 ulnar, 40 tibial, and 24 sural nerves) of 40 subjects who underwent both US and NCS (unpublished data). Overall, 20 type 2 diabetes patients [10 men and 10 women; age range, 50-88 years (mean, 68.5 ± 10.7 years)] and 20 healthy volunteers [12 men and 8 women; age range, 26-59 years (mean, 40.0 ± 12.3 years)] were enrolled in this study. All participants whose wrists had symptoms of carpal tunnel syndrome were excluded from the study. This study was approved by the Institutional Review Board of Gifu University Hospital, and informed consent was obtained from all participants.

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Ultrasonographic examination was performed by a board-certified sonographer who was blinded to the knowledge of the electrodiagnostic results. A 6.0- to 14.0-MHz linear array probe was used (portable real-time apparatus: Aplio XG; Toshiba Medical Systems, Japan). All subjects were seated on the examination table with their arms on a pillow and fingers semi-extended during examination of the median or ulnar nerves, and in the prone position during examination of the tibial and sural nerves. The CSA of the median nerve was measured at the carpal tunnel (MA) and at 5 cm proximal to the wrist (MB). The CSA of the ulnar nerve was measured at 5 cm proximal to the wrist (UA). The CSA of the tibial nerve was measured at the posterior medial malleolus (TA). The CSA of the sural nerve was measured at the lower third of the crus (SA). US images were quantitatively analyzed using the ImageJ software (National Institutes of Health, USA). We evaluated the relationship between the US and NCS results. Routine NCS were performed using conventional procedures and standard electromyography (Neuropack MEB-2200; Nihon Kohden Corp., Japan). All examinations were performed in a room with an ambient temperature of 25°C. The skin surface temperature in all cases was 31°C to 34°C. Mann-Whitney U test was used to compare data between the 2 groups. Pearson’s correlation coefficients were used to investigate the correlation of CSA and the percentage of the hypoechoic area with several NCS parameters. Results are given as mean ± SD, and statistical significance was assessed at P < 0.05. The US and NCS results of diabetic patients and controls are shown in Table 1. The CSAs in the patients with DM group were 0.13 ± 0.05 cm2 in the MA, 0.09 ± 0.03 cm2 in the MB, 0.0.7 ± 0.02 cm2 in the UA, 0.18 ± 0.04 cm2 in the TA, and 0.05 ± 0.01 cm2 in the SA. The CSAs in the controls were 0.08 ± 0.02 cm2 in the MA, 0.07 ± 0.02 cm2 in the MB, 0.04 ± 0.01 cm2 in the UA, 0.10 ± 0.03 cm2 in the TA, and 0.04 ± 0.01 cm2 in the SA. The CSAs was a significant increase in the diabetic patients compared with that in the controls, with the exception of the sural nerve. The percentage of the hypoechoic area was significantly increased in the diabetic patients compared with that in the controls (P < 0.05). The MCV and sensory nerve conduction velocity of all nerves in the diabetic patients showed a significant decrease compared with those in the controls (P < 0.001). The CMAP and SNAP of all nerves in the diabetic patients showed a significant decrease compared with those in the controls, with exception of the CMAPs of both the median and tibial nerves (P < 0.001). On the other hand, distal latency (DL) was significantly lesser in the diabetic patients than in the controls, with the exception of the sensory ulnar and motor tibial nerves. The relationships between US findings and NCS parameters are shown in Figure 6. The motor DL period of the median nerve was divided into 3 groups: DL of 4.0 ms. The MCV of the median nerve was also divided into 3 groups: MCV of 55 m/s. The categorization of subjects into tertiles of DL yielded 3 separate groups. Compared with the first tertile, the CSAs of the median nerve increased significantly with each tertile. Moreover, after combining tertiles of DL and MCV, even more comprehensive CSA stratification was possible, with all-cause CSA ranging from 0.07 cm2 in subjects in the lowest tertile of both parameters to 0.17 cm2 in subjects in the highest tertile (Fig. 6a). The hypoechoic area of the median nerve also increased significantly with each tertile. The hypoechoic area of the median nerve stratification was 52.5% in subjects in the highest tertile of both parameters (Fig. 6b). These results correlated with the electrophysiological severity.


Parameters Sonographic measurements (CSA) MA (cm2) MB (cm2) UA (cm2) TA (cm2) SA (cm2) Sonographic measurements (echo intensity) MB (%) UA (%) TA (%) SA (%) Electrophysiologic measurements MCV (m/s) DL (ms) CMAP (mV) SCV (m/s) DL (ms) SNAP (uV) MCV (m/s) DL (ms) CMAP (mV) SCV (m/s) DL (ms) SNAP (uV) MCV (m/s) DL (ms) CMAP (mV) SCV (m/s) DL (ms) SNAP (uV)

Nerve

Controls

Patients with type 2 DM

Median Median Ulnar Tibial Sural

0.08 ± 0.02 0.07 ± 0.02 0.04 ± 0.01 0.10 ± 0.03 0.04 ± 0.01

0.13 ± 0.05*** 0.09 ± 0.03** 0.07 ± 0.02*** 0.18 ± 0.04*** 0.05 ± 0.01

Median Ulnar Tibial Sural

43.7 ± 5.1 43.6 ± 4.1 44.8 ± 4.5 40.4 ±7.1

50.0 ± 9.3* 49.6 ± 10.6* 51.4 ± 7.5* 48.8 ± 3.7*

Median Median Median Median Median Median Ulnar Ulnar Ulnar Ulnar Ulnar Ulnar Tibial Tibial Tibial Sural Sural Sural

59.9 ± 4.4 3.3 ± 0.5 11.2 ± 3.7 69.1 ± 8.6 2.7 ± 0.4 38.3 ±11.7 64.7± 6.1 2.5 ± 0.4 14.9 ± 3.8 72.0 ± 5.4 3.3 ± 0.7 20.4 ± 10.0 49.4 ± 2.4 3.9 ± 0.5 16.1 ± 5.3 55.7± 2.5 2.5 ± 0.1 10.3± 4.1

50.7 ± 5.7*** 4.5 ± 1.3*** 11.6 ± 4.4 57.3 ± 5.7*** 3.3 ± 0.4*** 12.5 ± 8.3*** 52.1± 6.7*** 2.9 ± 0.5* 9.4 ± 2.5*** 61.7 ± 10.5*** 3.9 ± 0.8 6.3 ± 2.9*** 40.3 ± 5.1*** 4.4 ± 0.9 13.1 ± 6.9 49.1± 5.3*** 2.9 ± 0.3*** 3.6 ± 2.5***

25

Table 1. US and NCS measurements of controls and patients with type 2 DM (unpublished data). Mann-Whitney U test: *P 0.05).

Table 3. Atherogenic dyslipidemia prevalence, lipid-lowering drug(s) therapy, and baseline vs. current lipids and lipoproteins values in unselected T2DM patients c. Microangiopathic residual vascular risk Diabetic eye disease includes diabetic retinopathy (DRP) and non-vessel comorbidities, such as cataract or glaucoma [96]. DRP is a microangiopathy affecting the retinal blood vessels resulting predominantly from poor metabolic control, which produces microvascular occlusion and leakage, leading to progressive retinal damage which may end in complete visual loss as a result of maculopathy or ischemic/proliferative retinopathy [96-100]. Few modifiable risk factors are identified with respect to DRP: poor metabolic (i.e. glycemic) control; hypertension, especially if poorly-controlled; LDL and non-LDL dyslipidemia; anemia and possibly, tobacco smoking. The recently published ACCORD Eye substudy highlighted the limits of conventional approaches based on near-normalization of two key modifiable variables (HbA1c and high blood pressure). In ACCORD, DRP outcomes were defined as a ≥3-steps DRP progression on the EDTRS scale, or development of DRP requiring laser therapy or vitrectomy [101-103]. While glycemic control intensification significantly reduced the rate of progression of DRP (by a relative 33% and an absolute 3.1%, respectively), it was associated with serious safety

56


concerns, as higher all-cause mortality was observed in the intensive glycemic control group [104]. This led to premature termination of the glycemic arm of the ACCORD trial. Another source of disappointment from the ACCORD trial for microvascular RVR reduction was the lack of efficacy of BP normalization to improve primary DRP outcomes [105]. These findings highlights the current pitfalls of current approaches to DRP management based on a “the lower the better” paradigm for the two major standard modifiable risk factors, i.e. HbA1c and BP [104]. As regards lipids, landmark statin trials in T2DM or in subgroups with T2DM failed to identify a beneficial effect of LDL- lowering on any type on diabetic microangiopathies. On the other hand, epidemiological evidence links AD with RVR for diabetic retinopathy and/or nephropathy in T2DM [4-6,100,104]. The REsiduAl risk, Lipids and Standard Therapies (REALIST) studies are two Residual Risk Reduction Initiative-initiated worldwide epidemiological retrospective case-control surveys designed to assess AD-related macroand microvascular residual risk. These are performed in non-diabetic and T2DM patients (REALIST MICRO) receiving current standards of care, with LDL-C levels at or near goal, treated or not with a statin. Pilot results from REALIST MICRO show a highly-significant and strong association between AD and microangiopathy incidence, even when LDL-C is controlled [107,108]. In the ACCORD Eye substudy, fenofibrate decreased RVR of DRP progression, assessed using the validated EDTRS scale, and irrespective of baseline lipids. The efficacy of fenofibrate was most obvious in patients with DRP at baseline, i.e. those in secondary DRP prevention [101-104]. Such a beneficial effect confirms the previously documented benefits of fenofibrate on DRP. This, in the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study, a previous other large randomised controlled trial, a decreased requirement for DRP-related laser therapy was observed in T2DM treated with fenofibrate (vs. placebo). Such decreased requirement was however neither a primary outcome endpoint nor a “hard endpoint” for DRP progression assessment [88,104,106]. Results from ACCORD Eye therefore markedly raise the level of clinical evidence for a beneficial effect of this PPAR-α agonist on DRP. The FIELD study also showed that fenofibrate decreased new-onset albuminuria and diabetes-related non-traumatic amputations in T2DM patients [109]. In a recent assessment of fenofibrate's renal effects (FIELD washout sub-study), decreased albuminuria and reduced estimated glomerular filtration rate (eGFR) loss over 5 years were observed in patients allocated to the fenofibrate arm [110]. These observations confirm that fenofibrate not only reduces albuminuria or delay its onset, but also diminishes the natural history of progressive eGFR impairment in T2DM, despite an early and reversible increase in plasma creatinine [104,110-114]. As regards other LLD, future trials will establish whether targeting low HDL-C with nicotinic acid, alone or on top of background statin, reduces RVR of macroangiopathy in high-risk T2DM patients [90-93,115]. Safe and effective combination of LLDs (bi- or tritherapies) targeting most aspects of dyslipidemia are likely to become standards of care in high-risk populations such as patients with T2DM [45,75,91,104,115-118].

8. Benchmarking Innovative approaches are needed to improve individual and overall target achievement with the aim of reducing RVR. Benchmarking targeting physicians is one of those newer approaches. The goal of the non-interventional randomised OPTIMISE trial was to

Residual Vascular Risk in T2DM: The Next Frontier

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investigate in 6 European countries the effect of physician’s benchmarking on quality of care, assessed according to the percentage of T2DM patients achieving pre-set targets for three key modifiable variables (HbA1c, LDL-C and SBP), as recommended by international guidelines. At baseline, the percentage of patients achieving targets was highly unsatisfactory: 51% (HbA1c); 27% (SBP); and 35% (LDL-C), with a mere 5% reaching all three targets at study entry. Physicians were randomly assigned to receive either benchmarked feedback or non-benchmarked feedback on their patients’ modifiable outcome indicators (HbA1c, fasting glycaemia, total cholesterol, HDL-C, LDL-C and TG). At study end, the percentage of patients achieving all targets almost doubled, suggesting that benchmarking may be an innovative approach to improve target attainment of modifiable variables affecting RVR in T2DM patients [21,119,120].

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Vergès B. Role for fibrate therapy in diabetes: evidence before FIELD. Curr Opin Lipidol 2005;16:648-51. Jun M, Foote C, Lv J, Neal B, Patel A, Nicholls SJ, Grobbee DE, Cass A, Chalmers J, Perkovic V. Effects of fibrates on cardiovascular outcomes: a systematic review and metaanalysis. Lancet 2010;375:1875-84. Taylor AJ, Villines TC, Stanek E, Devine PJ, Griffen L, Miller M, Weissman NJ, Turco M. Extended-release niacin or ezetimibe and carotid intima-media thickness. New Engl J Med 2009;361: 2113-22. Chapman MJ, Redfern JS, McGovern ME, Giral P. Niacin and fibrates in atherogenic dyslipidemia: pharmacotherapy to reduce cardiovascular risk. Pharmacol Ther 2010;126:314-45. AIM HIGH: Niacin plus statin to prevent vascular events. ClinicalTrials.gov identifier: NCT00120289. HPS2-THRIVE. Treatment of HDL to reduce the incidence of vascular events. ClinicalTrials.gov Identifier: NCT00461630. ACCORD Study Group, Ginsberg HN, Elam MB, Lovato LC, Crouse JR 3rd, Leiter LA, Linz P, Friedewald WT, Buse JB, Gerstein HC, Probstfield J, Grimm RH, Ismail-Beigi F, Bigger JT, Goff DC Jr, Cushman WC, Simons-Morton DG, Byington RP. Effects of combination lipid therapy in type 2 diabetes mellitus. N Engl J Med 2010;362:156374. Fruchart J-C, Sacks FM, Hermans MP. Implications of the ACCORD lipid study : perspective from the Residual Risk Reduction Initiative (R3i). Curr Med Res Opin 2010;26:17937. Hermans MP, Ahn SA, Rousseau MF. Statin therapy and cataract in Type 2 diabetes. Diabetes Metab 2011;37:139-43. UK Prospective Diabetes Study Group. Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes. UKPDS 38. BMJ 1998;317:703-13. Misra A, Kumar S, Kishore Vikram A, Kumar A. The role of lipids in the development of diabetic microvascular complications: implications for therapy. Am J Cardiovasc Drugs 2003;3:325-338. Dodson PM. Medical treatment for diabetic retinopathy: do the FIELD microvascular study results support a role for lipid lowering? Practical Diabetes International 2008;25:76-79. Dodson PM. Diabetes and the Eye. In: Diabetic Retinopathy, Dodson PM (ed). Oxford University Press, Oxford, pp 33-44, 2009. Chew EY, Ambrosius WT, Howard LT, Greven CM, Johnson S, Danis RP, Davis MD, Genuth S, Domanski M; ACCORD Study Group. Rationale, design, and methods of the Action to Control Cardiovascular Risk in Diabetes Eye Study (ACCORD-EYE). Am J Cardiol 2007;99(12A):103i-11i. ACCORD Study Group; ACCORD Eye Study Group, Chew EY, Ambrosius WT, Davis MD, Danis RP, Gangaputra S, Greven CM, Hubbard L, Esser BA, Lovato JF, Perdue LH, Goff DC Jr, Cushman WC, Ginsberg HN, Elam MB, Genuth S, Gerstein HC, Schubart U, Fine LJ. Effects of medical therapies on retinopathy progression in type 2 diabetes. N Engl J Med 2010;363;233-44.


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Matthews DR. Fenofibrate and statin therapy, compared with placebo and statin, slows the development of retinopathy in type 2 diabetes patients of 10 years duration: the ACCORD study. Evid Based Med 2011 Apr;16(2):45-6. Hermans MP. Prevention of microvascular diabetic complications by fenofibrate: lessons from FIELD and ACCORD. Diabetes Vasc Dis Res 2011 (in press). Cushman WC, Grimm RH, Cutler JA for the ACCORD study group. Rationale and Design for the Blood Pressure Intervention of the Action to Control Cardiovascular Risk in Diabetes (ACCORD) Trial. Am J Cardiol 2007;99(suppl):44i-55i. Hermans MP. Impact of fenofibrate on type 2 diabetes patients with features of the metabolic syndrome: subgroup analysis from FIELD. Curr Cardiol Rev 2010;6:1128. Carey VJ, Bishop L, Laranjo N, Harschfield BJ, Kwiat C, Sacks FM. Contribution of high plasma triglycerides and low high-density lipoprotein cholesterol to residual risk of coronary heart disease after establishment of low-density lipoprotein cholesterol control. Am J Cardiol 2010;106:757-63. Rajamani K, Colman PG, Li LP, Best JD, Voysey M, D'Emden MC, Laakso M, Baker JR, Keech AC; FIELD study investigators. Effect of fenofibrate on amputation events in people with type 2 diabetes mellitus (FIELD study): a prespecified analysis of a randomised controlled trial. Lancet 2009;373:1780-8. Millán Núñez-Cortés J, Montoya JP, Salas XP, Hernández Mijares A, Carey VJ, Hermans MP, Sacks FM, Fruchart JC. The REALIST (REsiduAl risk, LIpids and Standard Therapies) study: an analysis of residual risk attributable to lipid profile in acute coronary syndrome. Endocrinol Nutr 2011;58:38-47. Davis TM, Ting R, Best JD, Donoghoe MW, Drury PL, Sullivan DR, Jenkins AJ, O'Connell RL, Whiting MJ, Glasziou PP, Simes RJ, Kesäniemi YA, Gebski VJ, Scott RS, Keech AC; Fenofibrate Intervention and Event Lowering in Diabetes Study investigators. Effects of fenofibrate on renal function in patients with type 2 diabetes mellitus: the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) Study. Diabetologia 2011;54:280-90. Keech A, Simes RJ, Barter P, Best J, Scott R, Taskinen MR, Forder P, Pillai A, Davis T, Glasziou P, Drury P, Kesaniemi YA, Sullivan D, Hunt D, Colman P, d'Emden M, Whiting M, Ehnholm C, Laakso M. Effects of long-term fenofibrate therapy on cardiovascular events in 9795 people with type 2 diabetes mellitus (the FIELD study): randomised controlled trial. Lancet 2005;366:1849-61. Scott R, O’Brien R, Fulcher G, Pardy C, D'Emden M, Tse D, Taskinen MR, Ehnholm C, Keech A; Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) Study Investigators. The effects of fenofibrate treatment on cardiovascular disease risk in 9795 people with type 2 diabetes and various components of the metabolic syndrome: the FIELD study. Diabetes Care 2009;32:493-8. Effect of fenofibrate on progression of coronary-artery disease in type 2 diabetes: the Diabetes Atherosclerosis Intervention Study, a randomised study. Lancet 2001;357:905-10. Ansquer JC, Foucher C, Rattier S, Taskinen MR, Steiner G; DAIS Investigators. Fenofibrate reduces progression to microalbuminuria over 3 years in a placebo-controlled study in type 2 diabetes: results from the Diabetes Atherosclerosis Intervention Study (DAIS). Am J Kidney Dis 2005;45:485-93.

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Polonsky TS, Davidson MH. Reducing the residual risk of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor therapy with combination therapy. Am J Cardiol 2008;101(8A):27B-35B. Rosenson RS. Management of non-high-density lipoprotein abnormalities. Atherosclerosis 2009;207:328-35. Rosenson RS, Davidson MH, Pourfarzib R. Underappreciated opportunities for low-density lipoprotein management in patients with cardiometabolic residual risk. Atherosclerosis 2010;213/1-7. Sacks FM, Carey VJ, Fruchart JC. Combination lipid therapy in type 2 diabetes. N Engl J Med 2010;363:692-4. Hermans MP, Muls E, Nobels F, Krzentowski G, Claes N, Debacker N, Matthys A. Evaluating benchmarking to optimize management of type 2 diabetes patients: the Belgian data from the OPTIMISE study. Acta Cardiol 2011;66:124-5. Brotons C, Elisaf M, Hermans MP, Michel G, Muls E, Matthys A. Evaluating benchmarking to optimize management of type 2 diabetic patients: HbA1c control in the European OPTIMISE study. Diabetes 2011 (in press).

5 Type 2 Diabetes and Fibrinolysis Ján Staško, Peter Chudý, Daniela Kotuličová, Peter Galajda, Marián Mokáň and Peter Kubisz

Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin Slovakia 1. Introduction Type 2 diabetes (DM2) is associated with complex hemostasis disorders characterised with enhanced coagulation, platelet dysfunction and hypofibrinolysis. These pathological conditions predispose type 2 diabetics towards increased morbidity and mortality due to thrombotic complications. In fact, DM2 is considered the most frequent acquired thrombophilic state. Under physiological conditions, fibrinolytic system is responsible for the lysis of fibrin clot, thus maintaining the blood fluidity. It was first described by Astrup in 1956. The most important molecule in fibrinolytic system is plasmin, which cleaves fibrin clot. It is released in its inactive form called plasminogen from the liver to the blood stream and gets activated by tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), clotting factor XII (FXII) and kallikrein. Plasmin deactivation occurs via several molecules. The best known and most important is α2-plasmin inhibitor (α-2PI). Another one, which was described in early nineteen nineties, is called thrombin-activatable fibrinolysis inhibitor (TAFI). Plasminogen activation and thus plasmin formation is inhibited by plasminogen activator inhibitor type 1 (PAI-1), PAI-2 and PAI-3. Fibrinolysis disorder due to disequilibrium between plasminogen activators and its inhibitors is the most characteristic feature of prothrombotic state associated with diabetes. The fibrinolytic activity in DM2 is influenced by numerous factors, the most crucial ones are glycaemic control, insulinaemia, blood lipid profile, blood pressure, genetic factors and different sorts of medication (Brummel-Ziedins et al., 2009, Alzahrani & Ajjan, 2010a).

2. Fibrinolysis 2.1 Plasminogen and its activation Under physiological conditions, fibrinolytic system is responsible for the lysis of fibrin clot, thus maintaining the blood fluidity. The central protein in whole fibrinolytic system is plasmin, a serin protease, which cleaves fibrin clot. Plasminogen, the single-chain proenzyme of plasmin, is a protein of 791 amino acids, m.w. 92 000 synthesized primarily in the liver (Raum et al., 1980). The lysine-binding domains of plasminogen mediate its specific interactions with fibrin, cell surface receptors and other proteins, including its circulating inhibitor α2-plasmin inhibitor (α2-PI), which is also known as α2-antiplasmin (Hajjar et al., 1986; Plow et al., 1991). Activation of plasminogen, by cleavage of a single Arg-Val peptide bond at position 560–561 (Holvoet et al., 1985), gives rise to the active serine protease, plasmin.

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Plasminogen can be converted to plasmin by both t-PA and u-PA. t-PA is a molecule of m.w. 72 000 which consists of 527 amino acids (Pennica et al., 1983). Cleavage of the Arg275Ile276 peptide bond by plasmin converts t-PA to a disulphide-linked, two-chain form (Pennica et al., 1983). While single-chain t-PA is less active than two-chain t-PA in the fluid phase, both forms demonstrate equivalent activity when fibrin bound. The t-PA is synthesized and secreted primarily by endothelial cells. u-PA is the second endogenous plasminogen activator. Single-chain u-PA or prourokinase, is a glycoprotein of m.w. 54 000, consisting of 411 amino acids. Cleavage of the Lys158-Ile159 peptide bond by plasmin or kallikrein converts single-chain u-PA to a disulphide-linked two-chain derivative. The u-PA has much lower affinity for fibrin than t-PA, and is an effective plasminogen activator in both the presence and the absence of fibrin (Gurewich et al., 1984). Fibrin, the major plasmin substrate, regulates its own degradation by binding both plasminogen and t-PA on its surface, thereby localizing and enhancing plasmin generation. The affinity between t-PA and plasminogen is low in the absence of fibrin, but increases significantly in its presence. Once formed, plasmin cleaves fibrin, generating soluble degradation products, and exposing carboxy-terminal lysine (Lys) residues. ‘Kringles’ 2 of t-PA, and 1 and 4 of plasminogen contain lysine-binding sites, which mediate further binding to fibrin, leading to enhanced plasmin generation and fibrin removal (Plow et al., 1991). This binding can be blocked by the recently discovered thrombin-activatable fibrinolysis inhibitor (TAFI). When activated by thrombin-thrombomodulin complex, TAFI removes carboxy-terminal Lys residues, thereby attenuating plasmin generation, stabilizing fibrin thrombi, and establishing a regulatory connection between coagulation and fibrinolysis. Fibrin dissolution is also regulated by inhibitors of plasminogen activation, such as PAI-1, and by inhibitors of plasmin itself, such as α2-PI. In addition, plasmin bound to fibrin is protected from α2-PI, due to occupancy of its lysine-binding sites. TAFI, on the other hand, decreases this protection by deleting plasmin-binding Lys residues on fibrin. Moreover, diverse cell types promote plasmin generation through their expression of cell surface receptors (Cesarman-Maus & Hajjar, 2005). Endothelial cells, monocytes, macrophages, neutrophils and some tumour cells, all bind plasminogen, as well as t-PA and/or u-PA. Their receptors localize cell surface fibrinolytic activity, serve as cofactors in acute or ongoing plasmin generation, and provide specialized environments that are protected from circulating inhibitors. Under certain conditions, proteases that are traditionally classified within the intrinsic arm of the coagulation cascade have been shown to activate plasminogen directly. These include kallikrein, factor XIa and factor XIIa, but normally account for no more than 15% of total plasmin-generating activity in plasma (CesarmanMaus & Hajjar, 2005). In addition, the membrane type 1 matrix metalloproteinase (MT1MMP) appears to exert fibrinolytic activity in the absence of plasminogen. Factor VIIactivating protease has also been reported to serve as an in vitro activator of single-chain PLG activators, but its role in vivo remains to be determined (Römisch et al., 1999). Plasminogen together with its activators t-PA and u-PA, urokinase receptor (uPAR), plasminogen activator inhibitors PAI-1, PAI-2, PAI-3 (which is identical to the protein C inhibitor) and protease nexin forms the plasminogen activator system (PAS). (BrummelZiedins et al., 2009; Schmitt et al., 1997). 2.2 Inhibition of fibrinolysis The major plasmin inhibitor is α2-PI. It is a single-chain glycoprotein of m.w. 70 000. α2-PI is also a constituent of platelet  granules (Plow & Collen, 1981). Thus, plasmin released into

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flowing blood or in the vicinity of a platelet-rich thrombus, is immediately neutralized by α2-PI. α2-PI is cleaved at its Arg364-Met365 peptide bond, and then forms a lysine-binding site-dependent α2-PI–plasmin complex, which is cleared in the liver (Plow et al., 1991). The PAI-1 is the most important and most rapidly acting physiological inhibitor of both tPA and uPA. This single chain glycoprotein of m.w. 52 000 is released by endothelial cells, monocytes, macrophages, hepatocytes, adipocytes and platelets (Ny et al, 1986). Release of PAI-1 is stimulated by many cytokines, growth factors and lipoproteins common to the global inflammatory response. After the release, PAI-1 quickly converts to an inactive form and circulates in plasma. Activation occurs by conformational change through binging to negatively charged phospholipids. It builds up a 1:1 complex binding with t-PA, thus inactivating it. This process is catalyzed by fibrin, which binds PAI-1 and enhances the t-PA plasma clearance. The complex dissociates from fibrin and is removed from circulation by liver (Cesarman-Maus & Hajjar, 2005). PAI-2, the less important inhibitor of plasmin activation, is a 393 amino acid member of the serpin family. It was originally purified from human placenta. Functionally, PAI-2 inhibits both two-chain t-PA and two-chain u-PA with comparable efficiency, but it is less effective towards single-chain t-PA, and does not inhibit prourokinase. Significant levels of PAI-2 are found in human plasma only during pregnancy (Ye et al, 1987). 2.3 Plasminogen activator inhibitor type 1 2.3.1 Regulation of plasminogen activator inhibitor type 1 levels PAI-1 is synthesized after stimulation. Its gene is localized on chromosome 7. In the circulation PAI-1 is rapidly converted (half-life 5 min.) to a latent inactive form which represents the major form of PAI-1 in plasma. Vitronectin stabilizes the active form of PAI-1 in plasma and prolonges its half-life 2 to 4-fold. The vitronectin levels are increased in relation to the cytokine dependent synthesis in the liver and hence the PAI-1 active form circulates in plasma during inflammation at high concentration. PAI-1 is removed from the circulation in the liver, in particular through binding at low density lipoprotein receptorrelated protein (LRP), with the half-life of 10 min. (Lijnen, 2004; Loskutoff & Mimuro, 1991; Schleef & Loskutoff, 1988; Wiman, 1995). According to multicompartmental and multifactorial model, the regulation of the PAI-1 plasma levels is complex and depends on multiple sites of PAI-1 synthesis and various stimuli, including hormones involved in the lipid and glucose metabolism. PAI-1 is synthesized in vascular (endothelium, vascular smooth muscles, platelets storage pool) and extravascular, i.e. metabolic (adipocytes, hepatocytes) compartments. These two PAI-1 compartments are regulated in a different way with various effect of insulin on PAI-1 synthesis. Factors regulating the PAI-1 production include stimulating effects of insulin, proinsulin, very low density lipoproteins (VLDL), inflammatory cytokines and oxidative stress (Galajda et al., 1997b, 1998a, 1998e, 1999; Galajda & Mokáň, 2001). 2.3.1.1 PAI-1 metabolic compartment PAI-1 metabolic compartment is formed by visceral adipocytes, stromal fat cells (in particular macrophages) and hepatocytes. These cells regulate the postprandial changes of fibrinolysis in splanchnic system. The principle site of PAI-1 production in mice is adipose tissue. The cultures of visceral adipocytes are more important source of PAI-1 in vitro than hepatocytes. Experimental studies of PAI-1 in vivo found that PAI-1 production in obese mice is 7-fold increased in adipocytes whilst it is only 2-fold higher in hepatocytes compared to lean mice. In

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human adipose tissue, PAI-1 production is localized mainly in the stromal fat cells and macrophages. Adipose tissue can be the principal source of PAI-1. Visceral adipose tissue produces 5-fold higher amount of PAI-1 compared to subcutaneous adipose tissue and levels of PAI-1 in obese subjects are related rather to the amount of visceral than subcutaneous fat. Thus PAI-1 levels are better correlated with indices of the central obesity (waist-to-hip ratio WHR). Insulin stimulates PAI-1 production in adipose tissue with synergistic effect of VLDL and free fat acids (FFAs). Therefore the adipose tissue is the most important source of PAI-1 related to insulin. Moreover, experimental studies in mice proved that the insulin application increases the expression of PAI-1 mRNA much more in adipose tissue than in the liver. Locally formed corticoids, inflammatory cytokines (TNF) and growth factors (TGF-β) can also stimulate the PAI-1 production in visceral adipose tissue. Under certain circumstances, hepatocytes can be an important source of PAI-1, especially in fatty liver disease. Insulin, proinsulin (PI) including des-31,32-split form and insulin like growth factor (IGF-1) stimulate production of PAI-1 in the liver. The most potent form among them is des-31,32-split PI, whilst PI and insulin stimulate the production of PAI-1 in liver with lower effect. The PAI-1 production is also stimulated by hyperlipidemia. VLDL act relatively rapidly and in synergy with insulin (Alessi et al., 1988, 1997, 2006; Hamsten & Eriksson, 1994; Juhan-Vague et al., 1991; Juhan-Vague & Alessi, 1997; Lijnen, 2004; Lijnen et al., 2005; Loskutoff & Mimuro, 1991; Loskutoff & Samad, 1998; Schneider & Sobel, 1996; Samad & Loskutoff, 1997). 2.3.1.2 PAI-1 vascular compartment PAI-1 vascular compartment includes endothelium, subendothelial pool (vascular smooth muscles, fibroblasts, macrophages) and a rapid release pool (platelets). PAI-1 is released to circulation mainly from the endothelium. Vascular smooth muscles can participate in regulation of PAI-1 levels under certain conditions. The platelets are the only cells in human body which store PAI-1. The total amount of PAI-1 is 4-fold higher in platelets compared to plasma, but they do not significantly impact on the PAI-1 plasma levels. The platelet PAI-1 plays a role in the local accumulation and action of the inhibitor. In our DM2 patients, hyperinsulinemic (HI) non-diabetics and healthy controls the PAI-1 levels did not correlate with platelet factor 4 (PF4) - the marker of platelet activation. This finding supports the hypothesis about more significant effect of the endothelium on PAI-1 plasma levels than the release of PAI-1 from platelet  granules (Galajda et al., 1997a; Galajda & Mokáň, 2001). Regulation of PAI-1 production in the endothelium has some specifics and differs from its regulation in metabolic compartment. The inflammatory cytokines as tumor necrosis factor (TNF), interleukin 1 (IL-1), transforming growth factor β (TGF-β), thrombin and angiotensin II and metabolic factors as proinsulin, hyperglycaemia, oxidative stress and lipids including VLDL and FFAs represent important stimulators of PAI-1 synthesis in the endothelium. On the contrary insulin inhibits synthesis of PAI-1 in the endothelium induced by TGF-β, probably due to activated cGMP and cAMP systems. Insulin along with leptin also stimulates the PAI-1 production in vascular smooth muscles (Eriksson et al., 1998; Hamsten & Eriksson, 1994; Kawai et al., 1996; Kim et al., 1997; Li et al., 1997; Peiretti et al., 1997; Vaughan, 1997, 2005; Yamauchi et al., 1997). 2.3.2 The control of PAI-1 levels in circulation PAI-1 levels depend on the PAI-1 expression and release from various tissues which are induced by the stimulatory effects of different factors. Adipose tissue, liver and vascular endothelium are considered to be the most important sources of PAI-1 production.


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2.3.2.1 Genetic factors Up to 100-fold individual variability of PAI-1 levels has been described in the general population. Genetic factors take part in this variability in only as much as 3-5%. Increased PAI-1 levels are associated with several known PAI-1 gene polymorphisms. One of them is the PAI-1 promotor region gene polymorphism -844 G/A (Xho I) which represents a potential binding site for Ets transcription factor. Another one is the polymorphism -675 4G/5G which affects the binding of nuclear proteins involved in the regulation of PAI-1 gene transcription. The PAI-1 5G allele leads to binding of a repressor protein thereby decreasing gene transcription. Consequently, higher plasma PAI-1 concentrations have been found among homozygotes for the 4G- allele compared to heterozygotes and homozygotes for the 5G-allele (Burzotta et al, 2003; Dawson et al., 1993; Eriksson et al, 1995). Majority of the europoid population are heterozygous carriers of the 4G/5G allele with a frequency of approximately 50%. Total frequency of 4G allele associated with a higher PAI-1 production is 52-54% in europoid, 30-46% in mongoloid and 24% in negroid population. Another polymorphism Hind III in the 3´-end of PAI-1 gene is associated with different regulation of PAI-1 by metabolic factors. Allele 2/2 is linked with more potent stimulatory effect of VLDL on PAI-1 production in the endothelial cell culture while a less frequent allele 1/1 (present in 20% of general population), is characterized by the stimulatory effect of insulin on PAI-1 production in endothelial cells in vitro. Recently identified G12078A dimorphism in 3´-end of PAI-1 can influence the PAI-1 levels by modifying of post-transcription mechanisms (Green, 1996; Lijnen, 2004; Mansfield et al., 1997; McCormack et al., 1996). 2.3.2.2 Insulin, proinsulin and insulin resistance The grade of insulin resistance (IR) is responsible for 49% interindividual variability of the PAI-1 levels in europoid men and 29% variability in women. It is a principle factor associated with PAI-1 levels but it includes various stimulatory mechanisms. Insulin is considered to be one of the main physiologic regulators of PAI-1 levels, it is responsible for a linkage of PLG system with human metabolic activity. The effect of insulin is different in various compartments of the PAI-1 production. Adipose tissue is the most important source of circulating PAI-1, its in vivo production is insulin dependent. Insulin stimulates the PAI-1 synthesis in synergy with VLDL and FFAs. Insulin also enhances the PAI-1 production in the liver. The effect of insulin on PAI-1 production in the endothelium is rather more complex. Insulin impacts neither PAI-1 basal production in the culture of human endothelial cells in vitro nor PAI-1 production in the vessels of mice splanchnic system after insulin application in vivo. On the contrary, insulin can inhibit PAI-1 production in the culture of endothelial cells induced by TGFβ in vitro. The inhibitory effect of insulin on the PAI-1 endothelial production can be mediated by cGMP and cAMP. Insulin increases the activity of cGMP by stimulation of NO production, it also increases the amount of cAMP by subsequent cGMP dependent inhibition of specific phosphodiesterase (PDE3). The decrease of PAI-1 during postprandial HI can be explained by inhibitory effect of insulin on the PAI-1 production in endothelium in vivo. Similarly, we confirmed the significantly decreased PAI1 levels which were independent from other metabolic parameters in our DM2 patients with endothelial dysfunction on long-term insulin treatment compared to those treated with oral antidiabetics (Table 1 and 2) (Galajda & Mokáň, 2001). These findings verify the inhibitory effect of insulin on PAI-1 production in the endothelial cells. On the other hand some studies found locally increased PAI-1 levels in human antebrachial vessels which were

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induced by the insulin application. This finding can be explained either by the release of PAI-1 from the vascular smooth muscles, as insulin stimulates PAI-1 release from the muscles in vitro or by heterogeneity of insulin effect on different types of endothelium. Another explanation can be found in the interindividual differences in human endothelial response to insulin as it was proved in Hind III polymorphism of PAI-1 gene. In this polymorphism a less frequent allele 1/1 is associated with stimulatory effect of insulin on PAI-1 production in the culture of human umbilical vein endothelial cells (HUVEC). Proinsulin (PI) including its incomplete split forms (des-31,32; des-61,62) stimulates the PAI-1 synthesis in liver and endothelial cells. The des-31,32 split PI form has the most potent stimulatory effect on PAI-1 production in hepatocytes whereas PI stimulates PAI-1 production with the same effect as insulin. Insulin-like growth factor 1 (IGF-1) stimulates, similarly to insulin, the PAI-1 production in the liver but not in the endothelium. C-peptide, which possesses certain hormonal activity of insulin, does not influence the PAI-1 production (Alessi et al., 1988, 1997, 2007; Alessi & Juhan-Vague, 2006; Grant et al., 1990; Gray et al., 1997; Juhan-Vague et al., 1991; Lijnen 2004; Nagi et al., 1990; Nordt et al., 1994; Panahloo et al., 1996; Pandolfi et al., 1996; Yamauchi, et al., 1997). 2.3.2.3 Free fat acids and lipoproteins with very low density VLDL are important molecules stimulating PAI-1 synthesis. From among them, the bigger VLDL1 stimulate PAI-1 production more than the smaller VLDL2. Unsaturated FFAs are proper stimulators of PAI-1 released from the VLDL, which intracellularly activate specific VLDL/FA - inducible transcription factor. This factor binds at -672 to -657 site of the PAI-1 gene promotor region marked as VLDL/FA-RE. This site has 67% homology with binding sites of so called peroxisome proliferator-activated receptors (PPARs) and is situated next to -675G promotor which is responsible for the binding of PAI-1 transcription repressor (factor B). Binding of VLDL/FA - inducible transcription factor at VLDL/FA-RE decreases the binding of transcription repressor (factor B) resulting in the increased PAI-1 production. VLDL increase synthesis of PAI-1 in the endothelium by this mechanism whilst in hepatocytes VLDL stimulate the PAI-1 synthesis synergistically with insulin by stabilization of mRNA. The monounsaturated, -3 and -6 polyunsaturated FFAs have a stimulatory effect on PAI-1 production in hepatocytes and endothelium in vitro while saturated FFAs do not influence PAI-1 production. Regulation of PAI-1 by PPARs has different mechanisms. Binding of PPARγ to corresponding bond sites (PPRE) on PAI-1 gene promotor can be associated with the increased basal mRNA expression for PAI-1 but the main effect is inhibition of PAI-1 production induced by inflammatory cytokines TNF and IL-1. Also the application of glitazones for patients with DM2 is associated with decreased PAI-1 independently on metabolic control and in correlation with decreased insulin and FFAs. Activation of PPARα by fibrates do not influence basal production of PAI-1 in the endothelium in vitro, but significantly decreases PAI-1 production mediated by TGFβ. The fibrates decrease PAI-1 production stimulated by insulin in hepatocytes in vitro. (Alessi & Juhan-Vague, 2006; Alessi et al., 2007; Asplund-Carlson et al., 1993; Eriksson et al., 1998; Li et al., 1997; Nilsson et al., 1998; Schneider & Sobel, 1996; Stiko-Rahm et al., 1990). 2.3.2.4 Inflammatory cytokines, growth factors, leptin Inflammatory cytokines TNF, IL-1, IL-6 stimulate very effectively PAI-1 synthesis. TNF induces PAI-1 production in endothelium and adipose tissue more than in hepatocytes. IL-1


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is an important stimulator of PAI-1 production in endothelium. Inflammatory cytokines have an increased effect on the 4G/5G promotor region PAI-1 gene polymorphism. The 5G allele has a bond site for inhibitory transcription factor and therefore the 4G insertion causes dramatic response to IL-1 with the increased PAI-1 levels. IL-6 also increases PAI-1 production in hepatocytes. Leptin, hormon of adipocytes, stimulates PAI-1 production in vascular smooth muscles. Adiponectin inhibits PAI-1 production in adipose tissue. TGFβ stimulates the endothelial PAI-1 production while growth factors related to the receptor protein tyrosine kinase (PTK) like insulin and IGF-1, do not act on PAI-1 synthesis in the endothelium but stimulate PAI-1 production in hepatocytes. This regulation is connected with indirect inhibitory effect of PAI-1 on the endothelial proliferation that needs proteolytic phenotype mediated by plasmin. Mentioned growth factors related to the receptor PTK induce endothelial proliferation. On the contrary, TGFß inhibits their growth effect, directly blocks the endothelial proliferation and increases PAI-1 endothelial production with an indirect antiproliferative effect. TGFß increases PAI-1 production in adipocytes and its effect is several times higher compared to insulin (Alessi & JuhanVague, 2006; Kawai et al., 1996; Loskutoff & Samad, 1998; Peiretti et al., 1997; Samad & Loskutoff, 1997; Vaughan, 2005). 2.4 Tissue plasminogen activator t-PA is a serine protease which cleaves plasminogen to plasmin. t-PA has a structure of glycoprotein with two polypeptide chains (m.w.70 kDa) and disulphide-linked bond. Fibrin acts as a main cofactor of t-PA activity in the activation of fibrinolysis. tPA is synthesized particulary in the endothelium. After its synthesis by gene localized at 8th chromosome the t-PA is either stored in reserved compartment of the endothelium which is different from Weibel-Palade bodies or released to the circulation. t-PA is bound in circulation on the surface proteoglycan layer of endothelium or minority of t-PA circulates in plasma bound in complexes with its inhibitor PAI-1. Release of t-PA from the endothelium differs according to the types of vessels and takes part on heterogeneity of the fibrinolytic potential of vessels. The most of t-PA is localized on the surface of veins, especially in the upper part of body (release of t-PA in the veins of arms is 10 times higher than in the veins of legs), less of t-PA is produced in microvascular circulation and in aorta, and at least is localized in the endothelium of arteries. The highest release of t-PA was found in splanchnic system and coronary arteries. Liver is the site of t-PA uptake with a half-life of 5 min. (Hinsgbergh, 1988; Jern et al., 1997b). 2.4.1 The effects on t-PA levels The t-PA antigen and activity levels depend on various factors. 2.4.1.1 Release of t-PA from stories in granules The t-PA is stored in reserved granules of the endothelium which are different from WeibelPalade bodies for vWF and small vesicules for protein S. Rapid degranulation and the t-PA release to plasma appears after the Cai-dependent stimulus. Factors activating receptors of phosphoinositol system by G-protein signaling pathway such as thrombin, acetylcholine, bradykinin, histamin, platelet activating factor (PAF) and endothelin stimulate the t-PA release by this way. Hormones like catecholamines activating cAMP system via β-receptors and vasopressin via V2-receptor also increase the t-PA release to plasma. Physical or mental

74


stress is associated with the activation of fibrinolysis and increased t-PA but without changes in PAI-1 levels. The t-PA released by stress is dependent on the intensity and duration of the stressor action. Strong or long-acting stressor against decreases the release of t-PA and increases PAI-1 production (Chandler et al., 1995; Kooistra et al., 1994; TegerNilsson et al., 1991). 2.4.1.2 Formation of complexes with PAI-1 The most of circulating t-PA antigen is coupled in complex with PAI-1 and only 5% of total t-PA circulates in free form. Measurement of the total t-PA by enzyme immunosorbent assay (EIA) does not distinguish the amount of free and bound t-PA. Close correlation between tPA and PAI-1 can be explained especially by the increased level of t-PA/PAI-1 complexes because the PAI-1 level is fundamental for t-PA level. But only a small amount of circulating t-PA is in active form and therefore the t-PA activity does not correlate with t-PA antigen (Loo et al., 1995; Wiman, 1991). 2.4.1.3 Endothelial damage t-PA is bound on the surface proteoglycan layer of endothelium and can be released to plasma after the endothelial damage. The increased t-PA levels correlate with vWF a TM in patients with coronary or peripheral atherosclerosis supporting the role of t-PA as a marker of endothelial damage. The t-PA levels are also an independent predictive marker for the risk of acute coronary events. So, the increased t-PA levels reflect severity of the endothelial dysfunction and are associated with a deterioration of fibrinolytic activity in plasma (Blann et al., 1995; Galajda et al., 1998d; Galajda & Mokáň, 2001). 2.5 Thrombin activatable fibrinolysis inhibitor Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase and member of the family called metallocarboxypeptidases. Activation of TAFI occurs by trypsin, plasmin, thrombin and meizothrombin. The catalytic efficiency of thrombin to activate TAFI is increased about three orders of magnitude in the presence of thrombomodulin. Thus, the thrombin-thrombomodulin complex is most likely the physiologic activator of TAFI. TAFI removes C-terminal lysine residues from partially degraded fibrin, thus inhibiting further plasminogen activation. It could be stated that TAFI acts as an important link between coagulation and fibrinolysis (Bajzar et al., 1996; Staško et al., 2004).

3. Fibrinolysis in diabetes mellitus type 2 Fibrinolysis disorder due to excessive PAI-1 production is the most characteristic feature of the prothrombotic state associated with DM2. Of all the various haemostatic measures, PAI1 has been most consistently associated with insulin resistance (IR) and is generally accepted as being an important part of the risk cluster. 3.1 PAI-1 in patients with diabetes mellitus type 2 PAI-1 levels are strongly correlated with components of the IR syndrome such as body mass index (BMI), blood pressure, plasma triglycerides (TG) and insulin in healthy subjects with IR, DM2 patients, and patients with known coronary heart disease (CHD). Elevated PAI-1 levels were an independent risk factor for the development of DM2 in healthy subjects in the Insulin Resistance Atherosclerosis Study (IRAS), suggesting that


75

they may be a very early risk marker for the development of the metabolic syndrome and DM2. This view was supported by subsequent serial analysis of PAI-1 in the IRAS in which baseline and follow-up PAI-1 levels were higher in subjects who subsequently converted to DM2. This link is strengthened by interventional studies, in which PAI-1 levels fell following improved insulin sensitivity (weight loss, exercise, metformin therapy). Festa et al. suggested that a fall in PAI-1 may in itself be associated with a reduction in conversion to DM2. However, the observation that elevated PAI-1 precedes the development of DM2 and is, in the presence of IR, independent of glycaemia (Natali et al., 2006) provides compelling evidence that abnormalities in fibrinolysis occur very early in the course of these disorders. (Juhan-Vague et al., 1987, 1989, 1993; Mansfield et al., 1997; Grant, 2007; Sakkinen et al., 2000; Vague et al., 1986; Landin et al., 1990a, 1990b; Hamsten et al., 1987; Sundell et al., 1989; Asplund-Carlson et al., 1993; Meigs et al., 2000; Festa et al., 1999, 2002, 2006; Natali et al., 2006). Our work also proved PAI-1 to be increased in early stages of diabetes (Kubisz et al., 2010). 3.1.1 PAI-1 as a predictive factor of cardiovascular risk The role of PAI-1 as a predictive factor of cardiovascular risk is different compared to parameters of subclinical inflammation (CRP, fibrinogen) and endothelial dysfunction (von Willebrand factor - vWF) that are the independent cardiovascular risk factors but their predictive role for the risk of DM2 development is dependent on presence of obesity. PAI-1 was supposed as a risk factor for development of coronary heart disease (CHD) due to the results of Northwick Park Heart Study that found an independent relation of decreased fibrinolytic activity to the risk of future CHD. However, metaanalysis of the studies showed that PAI-1 levels have not independent effect as a predictor of the CHD risk. Paradoxically the increased levels of t-PA can be predictive for development of CHD. The IRAS study confirmed that increased PAI-1 levels have strong effect as a predictor of DM2 risk that is independent from obesity (Alessi & Juhan-Vague, 2006; Festa et al., 2002; Lowe et al., 2004; Lowe, 2005; Meade et al. 1993). 3.1.2 Heterogeneity of the insulin effects on PAI-1 levels in diabetes mellitus type 2 It was considered in previous model that the overproduction of PAI in IR states is a result of direct stimulatory effect of the portal hyperinsulinaemia (HI). This was confirmed by a stimulatory effect of insulin in the culture of hepatocytes in vitro, by the increased PAI-1 levels during oral glucose tolerance test (OGTT) and clinically proved by the correlation of PAI-1 levels to insulinaemia. The endothelial production of PAI-1 was considered as independent on insulin. This model was modified by a discovery that the principal source of PAI-1 which is dependent on insulin is adipose tissue but there are some controversies. The effect of insulin is different in adipose tissue, liver and vascular system, dependent on duration and character of concurrent factors and influenced by clearence of PAI-1 in liver and endothelial cells (Jokl et al., 1994; Juhan-Vague et al., 1991; Potter van Loon et al., 1993; Galajda & Mokáň, 2001). 3.1.2.1 Dependence on compartment Insulin stimulates production of PAI-1 in visceral adipose tissue and liver. On the contrary insulin in the endothelial cells inhibits production of PAI-1 induced by cytokines. However, insulin has a stimulatory effect on PAI-1 synthesis in the subendothelial compartment,

76


which is probably responsible for a local release of PAI-1 in the vascular stream of forearm muscles during infusion of insulin. The increased PAI-1 levels in subjects with IR can be a result of either the stimulatory effect of HI on adipose tissue mass in obesity or the missing inhibitory effect of insulin on endothelium in IR (Jern et al., 1997a; Juhan-Vague et al., 1991; Negri et al., 1997; Yamauchi et al., 1997). 3.1.2.2 Dependence on duration of insulin action Decreased PAI-1 levels were found after a short-term insulin infusion or HI clamp. The same PAI-1 decrease was showed also in the control group with an administration of physiological solution without HI induction. These results confirmed that short-term application of insulin does not influence the PAI-1 levels and that PAI-1 decrease is a result of the PAI-1 diurnal rhytm variability. The PAI-1 levels after a long-term application of insulin in DM2 subjects were found decreased independently on other metabolic parameters. This effect can be caused by the stimulatory action of proinsulin during insulin treatment. We proved the inhibitory effect of insulin especially in DM2 subjects with endothelial dysfunction. This result supports, in accordance with an evidence of the inhibitory effect of PAI-1 on endothelium in vitro, the possibility of a direct inhibitory action of insulin during its long-term application (Galajda et al., 1998a, 1998b; Galajda & Mokáň, 2001; Nordt et al., 1993; Panahloo & Yudkin, 1996). 3.1.2.3 Dependence on concurrent factors Short-term infusion of insulin, lipids or glucose does not influence PAI-1 levels but their mixed application leads to the increased PAI-1 levels. Synergistical effect of insulin and lipids on PAI-1 levels was confirmed also in the culture of hepatocytes. Insulin supports the stimulatory effect of glucose on PAI-1 synthesis in the endothelial cells but it inhibits the PAI-1 production in the endothelium mediated by cytokines (Calles-Escandon et al., 1998; Panahloo & Yudkin, 1996; Schneider & Sobel, 1996; Yamauchi et al., 1997). 3.1.2.4 Dependence on PAI-1 clearence. PAI-1 synthesized by a visceral adipose tissue under the stimulation by insulin forms complexes with the increased t-PA produced in splanchnic system. Liver is a site of the rapid and effective uptake of these complexes. Rapid uptake of PAI-1/t-PA complexes can be responsible for the postprandial PAI-1 decrease. Intravenous application of glucose leads to the rapid PAI-1 decrease accelerated by sulodexid. This result supports the endothelial uptake of PAI-1. The similar finding was confirmed in subjects with diabetes mellitus type 1 (DM1) and it supposes the idea that postprandial PAI-1 levels variability can be independent on insulin (Ceriello et al., 1993; Jern et al., 1997b). 3.1.3 The effect of insulin treatment on PAI-1 levels in subjects with DM2 Insulin treatment in DM2 is associated with a decrease of PAI-1 levels. This is opposite to the confirmed stimulatory effect of insulin on PAI-1 levels in adipose tissue and liver. Although insulin has not the effect on basal PAI-1 production in endothelial culture in vitro, it inhibits the PAI-1 production induced by cytokines. This effect can exist also in vivo. In our clinical study we examined two groups of subjects with DM2 treated by sulphonylurea derivates (DM2-SU) and those treated 2-3 months by insulin (DM2-INS) (table 1, table 2).

77


DM2 - SU (n=17)

DM2 - INS (n=11)

p

PAI-1 (ng/ml)

48,1 (14-108)

27,9 (2-53)

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