Growth Factors in Proliferative Diabetic Retinopathy

Many growth factors are implicated in the pathogenesis of proliferative diabetic retinopathy. Alteration of growth factors and their receptors in diabetes has been shown in both experimental and clinical studies. Sustained hyperglycemia resulting from long-standing diabetes leads to several biochemical abnormalities that consequently result in retinal hypoxia. Retinal oxygenation state regulates various growth factors that promote angiogenesis in order to meet the oxygen demands of the tissue. However, unregulated expression of these growth factors and induction of complex cascades leading to augmentation of other proangiogenic factors, which may not be regulated by tissue oxygenation, leads to uncontrolled retinal neovascularization and blindness in diabetic patients.


INTRODUCTION
Diabetic retinopathy is a vascular complication of both type I and type II diabetes. Nearly all people with type I and more than half with type II diabetes develop complications involving the retina (Fong et al., 2003). Clinically, diabetic retinopathy can be classified as background retinopathy (BDR), preproliferative retinopathy (PPDR), and proliferative retinopathy (PDR) (Alder et al., 1997;Davis, 1992;Hudson, 1996;Neely et al., 1998). BDR is the earliest stage, which is character-ized by capillary basement membrane thickening, pericyte loss, microaneurysms, increased permeability, exudate deposits, and retinal microinfarcts. Preproliferative retinopathy, on the other hand, is an advanced stage of retinopathy that subsequently leads to the proliferative stage. Progression to the proliferative stage results in neovascularization and accompanying hemorrhages.
Retinal changes in diabetes are thought to be initiated by sustained hyperglycemia leading to biochemical anomalies and alterations of various vasoactive factors and growth factors (Brownlee, 2001;Engerman et al., 1985;Diabetes Control and Complications Trial Research Group, 1993;King and Brownlee, 1996;Sheetz and King, 2002). Biochemical changes that are believed to alter the structural and functional properties of the retina include nonenzymatic glycation (Bierhaus et al., 1998;Brownlee et al., 1988;Kern and Engerman, 2001;Vlassara, 2001;Vlassara, 1997;Takagi et al., 1995), augmented polyol pathway and pseudohypoxia due to redox imbalance (Costantino et al., 1999;Engerman et al., 1993;Greene et al., 1987;Ido et al., 1997;Pugliese et al., 1991;Williamson et al., 1993), oxidative stress (Brownlee, 2001;Pugliese et al., 1991;Sheetz and King, 2002), and activation of protein kinase C (PKC) (Derubertis and Craven, 1994;Ishii et al., 1996;Ishizuka et al., 1989;Johannes et al., 1994;Koya and King, 1998;Lynch et al., 1990;Okumura et al., 1991;Williams et al., 1997;Xia et al., 1994). In addition, there is also alteration in the expression of several growth factors, vasoactive factors, and their respective receptors. Early in the disease course, these biochemical anomalies lead to alteration of retinal blood flow, in part because of impaired nitric oxide activity and up-regulated endothelin expression (Brownlee, 2001;Sheetz and King, 2002). With progression of the disease, vascular cells lose intercellular tight junctions and become susceptible to degeneration, the 287 288 Z. ALI KHAN AND S. CHAKRABARTI aftermath being increased permeability. Elaboration of growth factors such as vascular endothelial growth factor (VEGF) and endothelins, stimulated by hyperglycemia and cellular degeneration, lead to further increased permeability and increased extracellular matrix (ECM) protein deposition. A schematic outline of these events and their possible interactions are presented in Figure 1.
Angiogenesis is a complex but normal process under various physiological conditions. However, when unregulated, it may be a principle mechanism of tissue damage in specific pathological conditions, as is the case in PDR. Angiogenesis may be the most devastating outcome of growth factor alterations in PDR. Retinal neovascularization is believed to be the consequence of hypoxia due to retinal capillary closure. This idea is supported by studies that have shown concurrent occurrence of hypoxia and neovascularization (Patz, 1982;Shimizu et al., 1981). This hypoxia-induced state is believed to cause functional alterations in various cell types, including glial cells, endothelial cells, retinal pigment epithelial cells, vascular smooth muscle cells, fibroblasts, and inflammatory cells. However, the primary target of glucose-induced dysfunction is endothelial cells (Chakir and Plante, 1996;De Vriese et al., 2001;Hink et al., 2001;King et al., 1994) and they are therefore most susceptible to proliferating signals.
Numerous growth factors have been implicated in the pathogenesis of PDR. Growth factor alterations are believed to be important in both early and late stages of diabetic retinopathy. Early events in the disease course, dictated in part by growth factor alterations, include basement membrane thickening, a hallmark of chronic diabetic complications. Pericyte loss and endothelial cell damage are two factors that may further aggravate a growth factor-mediated proliferative response, resulting in increased ECM deposition and basement membrane thickening (Williamson and Kilo, 1983). In addition, various growth factors are involved in later stages of diabetic retinopathy, leading to proliferation and migration of endothelial cells and subsequent neovascularization (Casey and Li, 1997;D'Amore, 1994;Grant et al., 1987;Gross et al., 1983;Sholley et al., 1984). Several of these growth factors are also believed to participate in the balance of ECM protein expression and degradation, a phenomenon integral to angiogenesis (Hink et al., 2001;Ishii et al., 1996;Patz, 1982;Shimizu et al., 1981;Williams et al., 1997). Proteinases called plasminogen activators (PAs) carry out the conversion of plasminogen to plasmin, which plays an important role in the proteolysis of the ECM proteins. Other proteases, matrix metalloproteinases (MMPs), also degrade various ECM proteins. Naturally occurring inhibitors of PAs and MMPs, such as plasminogen activator inhibitors (PAIs) and tissue inhibitor of matrix metalloproteinases (TIMPs) are, in addition to PAs and MMPs, believed to be regulated by various growth factors (Casey and Li, 1997;D'Amore, 1994;Grant et al., 1987;Gross et al., 1983;Sholley et al., 1984).
The involvement of growth factors in PDR is supported by studies that have shown increased detection in vitreous samples and whole retinal tissues. Although conflicting reports have been published over the years, it is clear that alteration in growth factor expression is an important event that may in part be responsible for the development and progression of diabetic retinopathy to the proliferative stage. We will discuss the pathophysiological role of some specific growth factors that are believed to be involved in PDR.

INSULIN-LIKE GROWTH FACTOR-I (IGF-I)
The IGFs, IGF-I and IGF-II, share considerable sequence homology with insulin (D'Amore, 1994). These growth factors are produced by a number of cell types, although liver seems to be the major contributor. These growth factors act by binding to cell surface receptors, IGF-IR and IGF-IIR (LeRoith and Roberts, 1993). IGF-IR binds IGF-I with higher affinity than IGF-II and insulin. IGF-IIR, on the other hand, preferentially binds IGF-II. Several circulating proteins, insulinlike growth factor binding proteins (IGFBPs), are responsible for the bioavailability and half-life of the IGFs (LeRoith and Roberts, 1993). The first indication as to the role of IGF-I in diabetic retinopathy came from a study in which hypophysectomy lead to a reduction in the severity of the diabetic eye condition (Poulsen, 1953). A possible role of growth hormone was promptly proposed following this observation as growth hormone mediates its effects via IGF-I. Consequently, the role of IGF-I in diabetic complications, including diabetic retinopathy has been extensively studied. There are contradictory reports as to the correlation between IGF-I and the clinical stage of diabetic retinopathy (Agardh et al., 1992;Boulton et al., 1997;Danis and Bingaman, 1997;Dills et al., 1991;Lee et al., 1994;Lowe et al., 1995;Meyer-Schwickerath et al., 1993;Pfeiffer et al., 1997;Wang et al., 1995). It should be noted that autocrine and paracrine tissue production of IGF-I might contribute to the inconsistency in correlation studies. In addition, when IGF-I levels are corrected for age, a significant increase in IGF-I levels in diabetic subjects is evident (Boulton et al., 1997). IGF-I is also increased in vitreous samples from patients with PDR (Merimee et al., 1983). In support of a possible connection between IGF-I and diabetic retinopathy, is the observation that administration of recombinant IGF-I in to the vitreous cavity of animals results in diabetic retinopathy-like pathologic changes (Thrailkill et al., 1999).

FIGURE 1
Schematic illustration of the pathogenesis of proliferative diabetic retinopathy. Chronic hyperglycemia leads to various biochemical abnormalities that ultimately lead to retinal ischemia and susceptibility to unregulated angiogenesis. Many growth factors are up-regulated along with their respective receptors. An imbalance between proangiogenic and antiangiogenic factors leads to new blood vessel formation in the retina. Due to the fragile nature of the newly formed blood vessels and inadequate cell-cell junctions, the vessels are prone to leakage and hemorrhage. The course of diabetic retinopathy ultimately leads to retinal and vitreous hemorrhage and retinal detachment. endothelial cells to proliferate and migrate (Grant et al., 1987). Furthermore, implantation of IGF-I pellet in corneas of rabbits causes neovascularization . However, it is to be noted that the dose used in this study was significantly higher than that found in retinal and vitreous samples from patients with PDR. Interestingly, IGF-I is capable of increasing expression of VEGF in vivo and in vitro (Punglia et al., 1997). IGF-I can also increase transforming growth factor-β bioavailability via increased PA activity (Grant et al., 1990). This suggests that IGF-I might be important in directing angiogenesis by itself and also by regulating various other growth factors.

PLATELET-DERIVED GROWTH FACTOR (PDGF)
PDGFs are produced by a number of cells including platelets, endothelial cells, vascular smooth muscle cells, fibroblasts, and macrophages (Heldin and Westermark, 1999;Raines, 1993). There are three forms of this growth factor, which are produced by homo-and heterodimerization of two polypeptides, PDGF A and PDGF B. Two receptors for PDGF have been cloned (Heldin and Westermark, 1999;Raines, 1993). PDGF AA polypeptide binds to α receptors whereas PDGF BB polypeptide and PDGF heterodimer (PDGF AB) binds to both α and β receptors. Similar to other growth factor receptors, PDGF receptors also posseses intrinsic tyrosine kinase activity. Vascular endothelial cells express both PDGF A and B polypeptides along with β receptors (Bar et al., 1989;Dicorleto and Brown-Pope, 1983;Raines et al., 1990;Smits et al., 1989). Generally, β receptors are involved in cell growth, which is the case with endothelial cells. Activation of β receptors on endothelial cells leads to transduction of strong mitogenic signals (Battegay et al., 1994;Kuwabara et al., 1995;Raines et al., 1990).
Both in vivo and in vitro studies indicate that PDGF possesses an angiogenic property (Marx et al., 1994;Risau et al., 1992;Sato et al., 1993). PDGF levels have been shown to be upregulated in vitreous samples from patients with PDR (Cassidy et al., 1998;Endo et al., 2000;Freyberger et al., 2000). In addition, PDGF has been shown to augment VEGF expression in response to hypoxia . Whether PDGF augments or aggravates the effects of angiogenic factors in the context of PDR, other than VEGF, remains to be determined. However, in a recent study, it was demonstrated that PDGF BB causes increased expression of endothelin-1 in bovine retinal pigment epithelial cells and endothelial cells, which was inhibited by a general PKC inhibitor (Yokota et al., 2003). PDGF is also believed to be important in the early pathogenetic changes in the retina. It has been demonstrated that PDGF ablation in endothelial cells produces morphological changes, such as microaneurysms and acellular capillaries (Enge et al., 2002), which are reminiscent of early vascular changes in diabetic retinopathy.

BASIC FIBROBLAST GROWTH FACTOR (bFGF)
FGF is a heparin-binding peptide (Esch et al., 1985;Gospodarowicz et al., 1986;Sporn and Roberts, 1988). FGF is produced by a variety of cells, including fibroblasts, macrophages, and endothelial cells (Enge et al., 2002;Esch et al., 1985;Gospodarwicz et al., 1986;Schweigerer et al., 1987Schweigerer et al., , 1988Sporn and Roberts, 1988;Sternfeld et al., 1989). Two isoforms of FGFs have been cloned, acidic FGF (aFGF) and basic FGF (bFGF). These two isoforms share 53% sequence homology and possess mitogenic properties toward fibroblasts and endothelial cells (De Juan et al., 1990;Schweigerer et al., 1987). bFGF is found in nearly all tissues, whereas, aFGF is mainly present in neural tissues. Generally, bFGF is a protein localized to the ECM, bound to heparin, which protects it from degradation (Bashkin et al., 1989;Prestrelski et al., 1992;Saksela et al., 1988;Sommer and Rifkin, 1989). A lack of secretory signal on bFGF suggests that it might be introduced to the ECM by tissue damage or an exocytotic process (Gajdusek and Carbon, 1989;Muthukrishnan et al., 1991). Expression of FGF and its receptor in the retina prompted researchers to study the role of FGF in PDR in great detail (Baird et al., 1985;Hanneken et al., 1991;Khaliq et al., 1995).
In vivo angiogenesis assays have shown that bFGF is a very potent angiogenic factor (Glaser et al., 1980a(Glaser et al., , 1980bWong et al., 2001). It is also increased in vitreous samples from patients with PDR (Sivalingam et al., 1990). Exogenous administration of bFGF has been shown to induce endothelial proliferation and VEGF expression . Both of these effects suggest important roles in retinal neovascularization. However, there still remains quite a controversy as to the exact in vivo role played by these factors. In vivo and in vitro studies have demonstrated increased bFGF expression in various systems. However, bFGF immunostaining has only been localized to mature nonproliferating blood vessel cells (Hanneken et al., 1991). In addition, hyperglycemia has been shown to cause glycosylation of bFGF (Giardino et al., 1994), which reduces its mitogenic activity. It is plausible that bFGF is involved in increased expression of other secreted angiogenic factors, such as VEGF, which promote proliferation of endothelial cells.

TRANSFORMING GROWTH FACTOR-β (TGF-β)
TGF-β is a very important cytokine in fibrotic diseases of the liver, kidney, and lungs. TGF-β is a member of a large family of regulatory proteins that include activin, inhibin, and bone morphogenic protein (Barnard et al., 1990;Ruscetti et al., 1998). Mammals express three TGF-β isoforms (β 1 , β 2 , and β 3 ), which are encoded by separate genes. TGF-β shows widespread tissue distribution and biological actions. In diabetic retinopathy, the role of TGF-β seems to be complex. TGF-β has been shown to be up-regulated by high glucose levels in human retinal endothelial cells (Pascal et al., 1999). In addition, TGF-β 2 levels were found to be increased in vitreous samples from patients with PDR (Hirase et al., 1998). Although, TGF-β exihibits antiproliferative activity in vitro, it is implicated in a positive regulation of angiogenesis in vivo (Battegay, 1995;Beck and D'Amore, 1997;Bussolino et al., 1997;Folkman and Klagsburn, 1987;Merwin et al., 1991;Muller et al., 1987;Roberts et al., 1986;Yang and Moses, 1990). This discrepancy is the least understood aspect of TGF-β action. Several theories have been proposed to reconcile these contradictory reports. The most attractive being the chemoattractant theory. According to this postulate, TGF-β acts as a chemoattractant for various cell types, including fibroblasts and monocytes, both of which are capable of producing angiogenic factors such as VEGF, PDGF, and tumor necrosis factor (TNF)-α (Phillips et al., 1993;Sakamoto et al., 2000). Furthermore, some in vitro evidence exists that implicates expression of TNF-α in TGF-β-mediated angiogenesis (Iruela-Arispe and Sage, 1993; Vinals and Pouyssegur, 2001). This theory suggests an indirect role of TGF-β in angiogenesis mediated either by bystander cells or by the induction of angiogenic factors. Further support for its involvement in PDR is the observation that TGF-β modulates VEGF expression in endothelial cells (Pertovaara et al., 1994). Endothelial cell permeability is another aspect that is in part regulated by TGF-β (Behzadian et al., 2001). It has been demonstrated that TGF-β regulates retinal endothelial permeability by modulating proteases which degrade ECM proteins.
Interestingly, various other growth factors that are altered in chronic diabetic complications (VEGF, IGF-I, bFGF) increase the activity of PAs, which convert plasminogen to plasmin. Plasmin is involved in producing a conformational change in the latent TGF-β-binding protein, leading to release of TGF-β in bioactive form. Increased TGF-β, in turn, can modulate ECM protein synthesis and degradation, a balance that is altered in diabetic retinopathy.

VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF)
VEGFs are a family of growth factors with five isoforms that are produced from a single gene by alternative splicing (Robinson and Stringer, 2001). These growth factors are also produced by a variety of cells, including macrophages, vascular smooth muscle cells, vascular endothelial cells, and retinal pigment epithelial cells. These growth factors act on VEGF receptors (flt-1 and flk-1), which are mainly localized on vascular endothelial cells and probably to some extent on vascular smooth muscle cells (De Vries et al., 1992;Hewett and Murray, 1996;Terman et al., 1992). VEGF has been shown to be mitogenic for endothelial cells and its expression is increased in various animal models prior to neovascularization Favard et al., 1996;Gerhardinger et al., 1998;Grunwald, 1998;Lu and Adamis, 2002;Miller et al., 1997;Noma et al., 2002;Ohno-Matsui et al., 2002;Pe'er et al., 1996;Tanaka et al., 1997). In addition, it is up-regulated in hypoxic conditions and is also modulated by various other growth factors as mentioned earlier (Donahue et al., 1996;Marsh et al., 2000;Oh et al., 1999). It has been shown to be up-regulated in vitreous samples of patients with active PDR (Aiello et al., 1994;Endo et al., 2001;Funatsu et al., 2001;Mitamura et al., 2002;Ogata et al., 2002;Shinoda et al., 1999;Simo et al., 2002). This suggests that VEGF might be the perfect candidate to carry out signals for retinal neovascularization. A PKCdependent pathway has been thought to mediate VEGF activation (Zhou et al., 2002). On the other hand, the mechanism by which VEGF carries out its action also seems to involve PKC activation. VEGF binding to the receptor results in increased phosphatidylinositol 3-kinase (PI 3 kinase) activity and phospholipase-Cγ (PLCγ ) activation Gliki et al., 2002;Mathews et al., 1997;Seymour et al., 1996;Teicher et al., 2002;Wellner et al., 1999;Zhou et al., 2002). The subsequent increase in diacylglycerol (DAG) results in activation of PKC isoforms α and β. It has been shown that inhibition of PKC prevents VEGF-mediated increased permeability and proliferation of endothelial cells Gliki et al., 2002;Teicher et al., 2002;Wellner et al., 1999). Furthermore, downstream effector molecules of VEGF receptor-mediated signaling also include guanine 5 -triphosphate (GTP)ase-activating protein, in addition to PKC and PI 3 kinase (Qi and Claesson-Welsh, 2001;Suzuma et al., 2000;Takahashi and Shibuya, 1997).
In animal models, chimeric VEGF receptor proteins, which sequester VEGF and prevent it from binding to the receptors on cell surfaces, can suppress neovascularization in almost all animals studied (Aiello et al., 1995;Adamis et al., 1996;Ozaki et al., 2000). Similar results have been obtained in experiments utilizing antisense oligonucleotides against VEGF, which lead to in decreased VEGF production (Robinson et al., 1996). The use of antisense oligonucleotides resulted in suppression of neovascularization in almost 75% of animals studied. These findings certainly provide evidence of an integral role of VEGF in retinal neovascularization.

ENDOTHELINS (ETs)
Endothelins are potent vasoactive peptides that regulate vascular tone by their action on vascular endothelial and smooth muscle cells (Levin, 1995;Rubanyi and Polokof, 1994). The ET family is comprised of three isoforms, ET-1, ET-2, and ET-3 (Inoue et al., 1989;Levin, 1995;Rubanyi and Polokof, 1994). These peptides are produced by a number of tissues. The major source of ET-1, the most potent vasoconstrictor, is vascular endothelium (Yanagisawa et al., 1988). Regulation of ETs is achieved in a transcription-dependent manner (Levin, 1995;Rubanyi and Polokof, 1994). These vasoactive peptides interact with specific cell surface receptors, ET A , ET B , and ET C (Sakurai et al., 1992;Sumner et al., 1992). Only ET A and ET B receptor types are present in mammals. These receptors are coupled to PLC through G proteins. ET A receptors are localized primarily on vascular smooth muscle cells and are involved in vasoconstriction (Sakurai et al., 1992;Sumner et al., 1992). Activation of ET A receptors results in calcium influx via PLCmediated DAG and inositol trisphosphate production. ET B receptors are involved in generation of nitric oxide by endothelial cells and thus regulate vasodilation.
In addition to a number of inducing factors, tissue hypoxia and ischemia cause up-regulation of ETs (Blauw et al., 1995;Karmazyn, 1996). Alteration of ETs has been demonstrated in both type I and type II diabetes (Bertello et al., 1994;De Mattia et al., 1998;Donatelli et al., 1994;Haak et al., 1992;Kamoi et al., 1994;Kawamura et al., 1992;Laurenti et al., 1997;Letizia et al., 1997;Morise et al., 1995;Takahashi et al., 1990). Although, a number of studies can be cited that provide contradictory reports of plasma ET levels in diabetic patients, it should be noted that these peptides act in both an autocrine and paracrine fashion. Therefore plasma levels may not provide an adequate assessment of their biological activity in regards to the retina (Levin, 1995;Rubanyi and Polokof, 1994). In a recent study, vitreous ET-1 levels were found to be significantly elevated in patients with PDR as compared to patients with nondiabetes associated ocular conditions (Oku et al., 2001).
ETs exhibit mitogenic property on vascular endothelial cells and may be involved in diabetes-induced retinal neovascularization (Bek and McMillen, 2000;Brennan and Zaki, 2000;Chollet et al., 1993;Morbidelli et al., 1995;Salani et al., 2000). The mitogenic property was demonstrated in the early 1990s by DNA synthesis assays. Administration of ET-1 was shown to induce DNA synthesis in brain capillary endothelial cells (Vigne et al., 1990). In addition, it has been demonstrated that selective ET B receptor antagonist can prevent endothelial cell proliferation and migration, two fundamental steps in the process of angiogenesis. In further support of the role of ETs in the development and progression of PDR is the study from our laboratory that has demonstrated that ETs are involved in hyperglycemia-induced increased permeability (Chen et al., 2000). The downstream effector molecule regulating ET-mediated mitogenic effects and increased permeability seems to be PKC activation (Chen et al., 2000;Stanimirovic et al., 1994). This suggests a possible role of these vasoactive factors in mediating hyperglycemia-induced retinal structural and functional changes associated with PDR.

PIGMENT EPITHELIUM-DERIVED FACTOR (PEDF)
Recent advances in diabetic retinopathy research have reframed our thinking in regards to the role of growth factors promoting angiogenesis. Initially, retinal neovascularization was thought to be the result of augmented growth factor expression, which promotes new blood vessel growth. It is now being accepted that pathologic angiogenesis in the eye is the result of not only increased vessel growth-stimulating factors but also decreased antiangiogenic factors. PEDF is such an antiangiogenic factor, which is believed to carry out its activity by inhibiting proliferation of endothelial cells (Dawson et al., 1999;Hutchings et al., 2002). PEDF has been shown to be present in the vitreous humor with high antiangiogenic activity, keeping this ocular portion avascular (Alberdi et al., 1999;Singh et al., 1998;Wu et al., 1995). Removal of PEDF from vitreous results in invasion of numerous blood vessels. In addition, induction of corneal wounds does not normally induce angiogenesis but does develop neovascular structure when facilitated with antibodies against PEDF (Dawson et al., 1999). The mechanism by which PEDF carries out its antiangiogenic activity is still not clear. However, a receptor has been isolated from retinoblastoma cells and also retinal neural cells (Alberdi et al., 1999;Becerra, 1997). Binding of PEDF to endothelial cells with high affinity also suggests the presence of a receptor, possibly similar to the one isolated from neural cells, on endothelial cells.
The role played by PEDF has not been fully elucidated in the context of retinal neovascularization. However, it has been recently shown that there is a negative correlation between PEDF levels and the degree of ischemia-induced neovasularization in rats (Gao et al., 2001). Low levels of PEDF have also been associated with angiogenic ocular diseases in humans (Ogata et al., 2001;Spranger et al., 2001). Furthermore, the antiangiogenic activity that is believed to be suppressed in retinal neovascularization can be recovered by administration of recombinant PEDF as determined by prevention of retinopathy (Duh et al., 2002;Mori et al., 2001;Stellmach et al., 2001). These observations suggest that an imbalance in angiogenic and antiangiogenic factors, produced by suppressed PEDF and augmented proangiogenic factors such as VEGF, promote new blood vessel formation in the retina in PDR.

OTHER FACTORS
Apart from the growth factors that we have described so far, a number of potent factors, which by their angiogenic or other activities, are involved in diabetic retinopathy. We will briefly describe our current knowledge in regards to the involvement of such factors in PDR.

Nerve Growth Factor (NGF)
NGF belongs to the neurotrophin family of polypeptides that play important roles in survival of neurons in both central and peripheral nervous system (Greene and Shooter, 1980). Recent studies have indicated that NGF and NGF-mediated signaling might be important in other biological functions, including wound healing and inflammation (Lawman et al., 1985;Matsuda et al., 1998). NGF has also been demonstrated to promote proliferation of microvascular endothelial cells (Raychaudhuri et al., 2001). This suggests that NGF alteration might play an important role in the progression of diabetic retinopathy. Although no difference has been observed in NGF and NGF receptor immunoreactivity between normal and diabetic retina in spontaneously diabetic BB rats (Chakrabarti et al., 1990), the idea cannot be excluded that some alteration of NGF takes place in the retina of diabetic subjects. A more recent study has demonstrated increased serum levels of NGF in patients with type I diabetes compared to age-matched control subjects and type II patients (Azar et al., 1999). In addition, several other growth factors that are altered in long-standing diabetes, such as b-FGF and epidermal growth factor (EGF), can induce the expression of NGF by fibroblasts and possibly other cells (Hattori et al., 1993).

Hepatocyte Growth Factor (HGF)
HGF was initially isolated from serum of hepatectomized rats (Nakamura et al., 1984). This growth factor is produced by many mesenchymal cells including fibroblasts and endothelial cells. HGF has been shown to promote endothelial growth and migration and the formation of blood vessels in vivo (Bussolino et al., 1992;Cai et al., 2000;. Only recently, have alterations in the vitreous levels of HGF been demonstrated in patients with diabetes-induced retinopathy (Nishimura et al., 1999(Nishimura et al., , 2000. The levels of HGF in vitreous samples parallel the severity of PDR as determined by vitreous hemorrhage, fibrovascular proliferation, and tractional retinal detachment. In addition, the levels of HGF found in this study were in the range that has previously been shown to induce growth and proliferation of cultured endothelial cells (Nakamura et al., 1996). Although HGF is not as aggresively studied as other factors, its contributions to the development of PDR should not be ruled out.

Epidermal Growth Factor (EGF)
EGF is a highly mitogenic polypeptide produced by a variety of cell types (Carpenter, 1985;Lui and Grandis, 2002). A number of studies have concluded that EGF is mitogenic for corneal endothelial cells and is involved in corneal neovascularization (Nezu et al., 1992;Woost et al., 1992). However, the involvement of EGF and its possible role in regulating retinal angiogenesis is less characterized compared to this function by other growth factors. One recent study has shown increased EGF and EGF receptor immunoreactivity in retinal samples from subjects with PDR (Patel et al., 1994). Although EGF possesses limited angiogenic activity as compared to FGF, it is highly mitogenic for human retinal pigment epithelial cells and may act synergistically with FGF (Leschey et al., 1990). This suggests that EGF signaling might be involved in preretinal membrane formation. This finding, along with localization of EGF in preretinal membranes of patients with PDR (Fredj-Reygrobellet et al., 1991;Patel et al., 1994), points to the involvement of EGF in the progression of diabetic retinopathy.

Tumor Necrosis Factor-α (TNF-α)
TNF-α is a proinflammatory cytokine primarily produced by phagocytes (Pober and Cotran, 1990). The principle target of TNF-α is vascular endothelium (Madge and Pober, 2001;Pober, 1998). The mechanistic basis of TNF-α-mediated endothelial activation is not completely understood. However, accumulating evidence suggests that TNF-α may mediate alteration of vasoregulation and leukocyte adhesion, leading to endothelial dysfunction. In addition, it is well documented that TNF-α increases endothelial permeability (Friedl et al., 2002;Mark and Miller, 1999), an activity important in cell invasion and migration during angiogenesis. Vitreous and serum from diabetic subjects with PDR do show an elevated level of TNF-α (Doganay et al., 2002;Spranger et al., 1995;Yuuki et al., 2001). In addition, in vitro studies have demonstrated a potential role of TNF-α in TGF-β-induced angiogenesis (Iruela-Arispe and Sage, 1993; Vinals and Pouyssegur, 2001). The stage of diabetic retinopathy at which TNF-α might play a role is still obscure. However, mere elevation of this proinflammatory cytokine in PDR suggests an association that might be worth pursuing.

CONCLUDING REMARKS
PDR is a very complex condition that results from altered expression of various growth factors and vasoactive factors. In the retina, hyperglycemia causes several metabolic defects, including oxidative stress, nonenzymatic glycation, and formation of advanced glycation end (AGE) products, sorbitol/myo-inositolmediated changes, and redox potential alteration. These events set the background for pericyte loss, basement membrane thickening, microaneurysms, acellular capillaries, nonperfusion, ischemia and, therefore, susceptibility towards mitogenic stimuli. The increase in angiogenic factors or inducers of these factors results in a propagating complex that interplays between different pathways, resulting in nonregulated angiogenesis. In addition, hyperinsulinemia due to insulin resistance may also influence growth factor alteration. It is to be noted that an obligatory role of a particular growth factor in the development and progression of diabetic retinopathy is controversial. Our current knowledge suggests that a very heterogenous pattern of growth factor expression, with cross-talks between different pathways resulting in ocular neovascularization. Derubertis, F. R., and Craven, P. A. (1994) Activation of protein kinase C in glomerular cells in diabetes. Mechanisms and potential links to the pathogenesis of diabetic glomerulopathy. Diabetes, 43, 1-8. Diabetes Control and Complications Trial Research Group. (1993) The Diabetes, 26, 760-769. Engerman, R. L., Kern, T. S., and Garment, M. B. (1993) Capillary basement membrane in retina, kidney, and muscle of diabetic dogs and galactosemic dogs and its response to 5 years aldose reductase inhibition. J. Diabetes Complications, 7, 241-245. Esch, F., Baird, A., Ling, N., Ueno, N., Hill, F., Denoroy, L., Klepper, R., Gospodarowicz, D., Bohlen, P., and Guillemin, R. (1985) Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc. Natl. Acad. Sci. U. S. A., 82, 6507-6511. Favard, C., Ortega, N., Bayard, F., and Plouet, J. (1996) Vascular endothelial growth factor and retinal neovascularisation: A new