the diabetic milieu [2, 3]. Although the exact sequence of events and the relative importance of the early changes are poorly understood, capillary BL thickening is a hallmark of early DR and may be causal in endothelial and pericyte dysfunction. BL thickening is the result of extracellular matrix (ECM) remodeling, resulting in increased deposition of BL components such as collagen type IV, laminin, and fibronectin.

In addition, tissue fibrosis plays a role in PDR. Uncontrolled retinal neovascularization is followed by fibrosis, scarring, tractional retinal detachment, and blindness. PDR patients with established neovascularization and imminent fibrosis find themselves in a situation associated with poor prognosis, despite aggressive laser treatment or surgical procedures. The major mediator of vascular leakage and angiogenesis in PDR is VEGF- A, which is overexpressed in ischemic retina [2, 3, 5, 6]. However, little is known about growth factors that are involved in the subsequent fibrotic phase in PDR.

Connective tissue growth factor (CTGF) is a candidate for contributing to the fibrotic responses observed in both PCDR and PDR. CTGF acts as a mitogen for fibroblasts and induces increased ECM production [7–11]. CTGF functions as a downstream mediator of transforming growth factor (TGF)-b signaling in certain cell types and seems essential for effectuation of the profibrotic actions of TGF-b, such as ECM production [12].

In long-standing diabetes, structural and functional ECM alterations, including BL thickening, lead to microvascular diabetic complications, such as DR, nephropathy, cardiomyopathy, peripheral vascular disease, cerebrovascular disorders, and atherosclerosis [13, 14]. CTGF is involved in these diabetic microvascular complications [15–17]. In diabetic nephropathy, CTGF is strongly overexpressed in the kidney glomerulus [16, 18], and its levels in urine and plasma correlate with progression of the disease [19, 20].

Similarly to its involvement in fibrosis in diabetic nephropathy [7, 9, 10, 21], CTGF may have a causal role in capillary BL thickening in PCDR and in fibrosis in PDR. This chapter discusses the roles of CTGF in the pathogenesis of DR in relation to ECM remodeling and wound healing mechanisms, and explores whether CTGF is be a novel therapeutic target in the clinical management of early as well as late stages of DR.

ECM REMODELING AND WOUND HEALING MECHANISMS IN DIABETIC RETINOPATHY

ECM Remodeling in PCDR

Normal constituents of the retinal capillary BL are collagen type IV, which is the predominant component, laminin, and fibronectin. The latter is mainly located at pericyteendothelial cell contacts [22, 23]. Changes in the BL in diabetes are considered to result from a disturbed balance between synthesis and degradation of these matrix components [4, 24, 25]. The thickening BL may consist of matrix proteins that are normally found in the ECM, or proteins that are not present in the BL under physiological conditions, or both [4, 22, 26–28].

ECM alterations including BL thickening are hallmarks in all target organs affected by diabetes [29] and are directly related to loss of function of these organs [30]. It is considered to represent a pathological response to prolonged hyperglycemia, which is the major factor associated with the onset of microvascular complications, as has been

Fig. 1. Vascular basal lamina (BL) thickening in diabetic retinal capillaries. Electron microscopy of rat retina shows that retinal capillary BL (arrows) is susceptible to thickening after 12 months diabetes (compare nondiabetic (A) with diabetic (B)). (Reprinted by permission from Macmillan Publishers Ltd: Eye [34]Copyright (2009)).

shown in prospective studies for both type I and type II diabetes (DCCT and UKPDS, respectively) [31, 32].

The early thickening of the retinal capillary BL in diabetes was recognized already 60 years ago [33]. In the following decades, numerous electron microscopic studies have demonstrated increased thickness of the BL in diabetic humans and animals (Fig. 1) [34–36]. The only clear structural retinal change after 1–3 years of diabetes

was capillary BL thickening in a canine model of diabetes, which eventually displays all early features of diabetic retinal microvascular damage – BL thickening, loss of pericytes, the formation of microaneurysms, and capillary closure [37]. In this model, widespread loss of pericytes was noted only after 4 years of diabetes. BL thickening is likely to be instrumental in progression of early DR [38]. In an experimental model of diabetes in galactose-fed rats, downregulation of synthesis of the BL component fibronectin with the use of antisense oligos not only downregulated retinal BL thickening at least partly but also reduced other more advanced features of PCDR, such as apoptosis of pericytes and endothelial cells, as well as the development of acellular capillaries. In another study, concomitant downregulation of fibronectin, laminin, and collagen type IV expression with the use of antisense oligos injected in eyes of rats with streptozotocin-induced diabetes reduced vascular leakage [23]. These findings indicate that BL thickening is a crucial step in the progression of DR.

Thickening of the BL, often incorrectly referred to as “basement membrane,” is characterized by accumulation of ECM components, as well as a qualitative change in ECM composition [30]. Structurally, ECM consists of a complex network of collagens, elastins, structural glycoproteins, and proteoglycan–hyaluronans, and differs quantitatively and qualitatively in the various tissues. ECM provides mechanical support to cells in tissues, is involved in differentiation of cells, and regulates interactions between (vascular) cells and the ECM itself [39]. Under physiological circumstances, ECM continuously undergoes remodeling by synthesis and degradation, with a balanced turnover. ECM remodeling is required for maintaining the normal structure and function of tissues [40]. Multiple growth factors are involved in the induction of ECM synthesis, such as TGF-b [41], CTGF [42], insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) [30]. ECM degradation and remodeling is regulated by proteases such as the matrix metalloproteinases (MMPs) [43] and serine proteases [44], as well as their respective inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) [45] and plasminogen activator inhibitor-I (PAI-1) [43].

Different metabolic mechanisms underlie the hyperglycemia-induced changes in expression of growth factors and ECM turnover [35, 46]. Hyperglycemia affects various biochemical pathways, among which increased formation of glucose-derived AGEs [24, 47]. In DR, AGE formation has a causal role in changes in growth factor expression and ECM turnover [25, 48]. AGE formation also increases synthesis of BL components, most likely via upregulation of TGF-b signaling and its downstream effectors, including CTGF [49].

Wound Healing Mechanisms in PDR

The early features of retinal microvascular diabetic damage such as BL thickening eventually lead to retinal vascular occlusion and ischemia. In response, new vessels develop from the preexisting retinal vasculature, which hallmarks the progression of clinical DR from nonproliferative to proliferative disease. VEGF is the major mediator of this hypoxia-driven neovascularization [2, 3, 5, 6] and may act in concert with other angiogenic factors, such as angiopoietin-2 (Ang-2) [50]. The initial step in retinal neovascularization is degradation of the BL and ECM components surrounding the vascular cells, followed by invasion, migration, and proliferation of endothelial cells, and finally the formation of