Insulin-Like Growth Factor 1

IGF-1, a polypeptide made up of 70 amino acids and with a molecular weight of 7.6 kDa, is a member of the IGF family of growth factors and related molecules. The IGF family is made up of ligands (IGF-I, IGF-II, and insulin), at least six well-characterized binding proteins (IGFBP-1 through -6), and cell surface receptors that mediate the actions of the ligands (IGF-I receptor, the insulin receptor, and the IGF-II mannose-6- phosphate receptor). IGF-1 stimulates growth, differentiation and metabolism in a variety of cell types by acting through the tyrosine kinase receptor IGF-1R, and plays an important role in both embryonic and postnatal growth. In addition, its systemic levels regulate growth hormone (GH) secretion through a negative feedback [17].

IGF-I is mainly synthesized by the liver and has been considered the main mediator of the growth-promoting and metabolic actions of GH. However, IGF-1 has also autocrine and paracrine effects, which are not related to GH levels. In fact, it has been shown in rats that IGF-1 expression is preserved in numerous tissues, including the retina, after hypophysectomy [17]. In addition, GH can itself produce direct effects mediated by GHR and not involving IGF-1. These dual observations suggest that GH has both direct and indirect (via IGF-1) effects on growth and emphasizes local, autocrine/paracrine action by IGF-1.

IGF-1 expression is increased by GH and insulin and decreased by malnutrition. Most IGF- 1 circulates bound to IGFBPs and less than 1% circulates as free form. Free IGF-1 represents the active form and, indeed, it is the main factor responsible for inhibiting the pituitary production of GH. The affinity of IGF-1 for each of the six IGFBPs is 5- to 50-fold greater than IGF-I affinity for IGF-IR. Therefore, the in vivo equilibrium favors the binding of IGF-I to the IGFBPs [17]. In serum, most IGF-1 circulates as a ~150-kDa complex (ternary complex) which consists of IGF-I (γ-subunit) bound to IGFBP-3 (β-subunit) and

acid-labile subunit (α-subunit). IGFBPs are mainly synthesized in the liver and they have major functions that are essential for coordinating and regulating the biological activities of IGF-1 [17].

Several in vitro studies have shown that IGF- 1 is expressed in microvascular endothelial cells, pericytes, Müller cells, and RPE cells. In addition, IGFR-1 expression has been found in cultures of HREC, Müller cells, and RPE cells. Furthermore, IGFBPs are also synthesized by retinal cells [2, 18]. These findings suggest that the IGF-1/IGF- 1R/IGFBPs complex participates in the physiological events that occur in the retina. Indeed, IGF-1 as well as IGF-1 analogues prolong the survival of neuroretinal cells in vitro, under conditions of hypoxia or serum starvation. In addition, IGF-I (10 ng/ml) also protects HREC from apoptosis induced by high glucose and serum starvation. Hellström et al. [19] examined retinal vessel morphology by digital image analysis of ocular fundus photographs in patients with genetic defects of the GH/IGF-I axis and low levels of IGF-I and demonstrated that these patients had significantly less retinal vascularization, thus providing evidence that IGF-1 is critical for normal vascularization of the human retina. In this regard, we have found that the contribution of free IGF-1 to total IGF-1 in vitreous fluid was 42% in nondiabetic controls. This percentage greatly exceeds that obtained in serum (<1%), thereby suggesting not only that a significant amount of free IGF-1 is produced intraocularly, but also that it plays a relevant role in retinal homeostasis [20].

Although extensive claims have been made for the stimulating or accelerating the role of serum IGF-1 in the development of DR, the results of clinical studies have been controversial, and this concept has not been supported by clinical intervention trials. More important than circulating IGF-1 is its intraocular production (fig. 4). As mentioned above, several retinal cell types express both IGF-1 and its receptor, and this expression is independent of GH levels. The proliferative effect of IGF-1 on retinal endothelial cells

has been reported in several in vitro studies, and the essential role of IGF-1 in regulating not only retinal neovascularization, but also VEGF action, has been established in RPE cells and in the ox- ygen-induced retinopathy (OIR) mouse model. In addition, it has been shown that IGF-1 injected intravitreally induces retinal neovascularization and/or blood-retinal barrier (BRB) breakdown in several experimental models. In this way, transgenic mice overexpressing IGF-1 in the retina developed most of the alterations seen in human diabetic eye disease and neovascularization was consistent with increased IGF-1 induction of VEGF expression in retinal glial cells [21]. However, it should be noted that in all these studies the concentrations of IGF-1 within the eye were supraphysiological and nondiabetic animal models were used. In fact, in another model of transgenic mice with subtle IGF-1 overexpression (2.5- fold elevation of IGF-1 mRNA and 29% increase in IGF-1 protein in the retina) a lack of spontaneous ocular neovascularization was observed [22]. In addition, several authors have failed to detect mitogenic effects when IGF-1 was added in physiological concentrations to cultured retinal

endothelial cells. Moreover, it has been demonstrated in rats that either hypoxia or diabetes produces a significant decrease in retinal IGF-1 mRNA levels, and also that both the immunoreactive protein and mRNA for IGF-1 are reduced in HREC of diabetic origin as compared to nondiabetic HREC cultures [2]. Gerhardinger et al. [23] have reported a three-fold decrease in IGF- 1 mRNA levels in retinas obtained post-mortem fromdiabetichumandonorswithincipientretinal microangiopathy compared with retinas of agematched nondiabetic donors. Finally, although elevated intravitreous levels of IGF-1 have been found in the vitreous fluid of diabetic patients with PDR [2], we have demonstrated that serum diffusion is the main factor accounting for this enhancement [20]. In fact, we observed a deficit of free IGF-1 in the vitreous fluid of PDR patients in comparison with nondiabetic control subjects, thus suggesting that in patients with PDR there is a lower production of free IGF-1 by the retina [20]. In addition, a relationship between intravitreous free IGF-1 and either PDR activity or intravitreous VEGF was not detected [24]. Taken together, these results suggest that although IGF-1

participates in the pathogenesis of DR it is not the main cause of ocular neovascularization and its precise role remains to be established.

The observation that circulating GH-IGF-1 production is reduced by somatostatin (SST) has been the basis for using SST analogues in both PDR and macular edema [25]. However, as mentioned above, circulating IGF-1 does not seem to be an important factor in the pathogenesis of DR. Supporting this idea, the results obtained in the multicentered clinical trial with a long-act- ing SST analogue (Sandostatin LAR®, Novartis Pharmaceuticals) given by intramuscular injections every 4 weeks in patients with severe nonPDR and non-high risk PDR have been inconclusive. In recent years, growing evidence has accumulated indicating that SS analogues have an angiostatic effect [2]. In addition, an SST deficit in the vitreous fluid and a lower expression in the retina have been found in diabetic patients [26, 27]. Furthermore, a significantly worsened neovascularization has been detected in a model of transgenic mice lacking the SST receptor type 2 (SSTR-2), whereas a chronic overexpression of SSTR-2 attenuated the hypoxia induced neovascularization by limiting VEGF increase [18]. Therefore, a treatment to target SST receptors can be theoretically effective in reducing neovascularization. However, given that SST analogues have inadequate penetration of the BRB following systemic administration and poor tissue distribution in the retina, novel formulations specifically addressing DR are needed. Alternatively, new SST analogues for intravitreous or intraretinal delivery could be envisaged as a new strategy in DR treatment.

Platelet-Derived Growth Factor

Platelet-derived growth factor (PDGF), a ≈35-kDa protein originally isolated from human platelets, is one of the most ubiquitous growth factors that stimulates cellular proliferation and directs

cellular movement. The PDGF family of growth factors is composed of four different polypeptide chains encoded by four different genes. The classical PDGF chains, PDGF-A and PDGF-B, are well characterized, while the novel PDGFs, PDGF-C and PDGF-D, are less well known. PDGF isoforms exert their effects on target cells by activating two structurally related protein tyrosine kinase receptors (α- and β-receptors) [28].

PDGF induces mitogenic, chemoattractant and survival cell responses on fibroblasts and endothelial and vascular smooth muscle cells. In addition, it appears to be an important factor in endothelial/pericyte interactions and in tissue regeneration [29]. PDGF has been implicated in PDR as well as in proliferative vitreoretinopathy (PVR). It has been shown that PDGF acts as a paracrine growth factor for RPE cells in culture stimulating their proliferation and chemotaxis and mediates contraction of the fibrovascular tissue that produces retinal detachment. Moreover, PDGF-B induces the expression of VEGF and en- dothelin-1 in cultured bovine retinal pericytes. Immunocytochemical studies have shown the presence of PDGF and its receptors in epiretinal membranes in DR. In addition, increased levels of PDGF have been described in vitreous fluid of patients with PVR and PDR [2].

Hypoxia and hyperglycemia increase PDGF production in cultured human vascular endothelial cells and bovine pericytes. PDGF-B is a potent survival factor for the retinal microvasculature in general and pericytes in particular [7]. Inhibition of PDGF in a OIR model promotes pericyte loss and increases VEGF and VEGFR-2 retinal expression. On the other hand, transgenic mice showing overexpression of PDGF-B develop a proliferation of endothelial cells, pericytes and glial cells leading to traction retinal detachment as seen in the end stages of DR. Therefore, it seems that PDGF acts as a survival factor and is necessary for normal retinal vascularization but its overexpression could be deleterious and, in consequence, it is a key mediator in the pathogenesis

of PDR. In recent years, several therapeutic approaches aimed at blocking PDGF action have been successfully used in experimental models.

Fibroblast Growth Factor

The fibroblast growth factor family contains over 20 structurally related heparin-binding proteins, and bFGF or FGF-2 is the prototype member. FGF-2 exists in four different molecular weight isoforms owing to differential splicing or initiation from alternative start codons. FGF is a potent angiogenic inducer, and its biological effects are mediated by binding to four distinct high-af- finity tyrosine kinase receptors (FGFR1-FGFR4) which in order to be fully activated operate in conjunction with low affinity HSPGs. In addition, the binding of FGF-2 to heparan sulphate serves as a storage of inactive FGF-2 and protects FGF-2 from degradation by heparanases [2].

In the retina, FGF-2 expression has been reported in the ganglion inner and outer nuclear layers, basement membranes of Müller cells, blood vessels and RPE cells. FGF receptors are also widely distributed in the neuroretina, and their expression is most abundant in photoreceptors. In this regard it should be noted that FGF- 2 neurotrophic effects protecting against photoreceptor damage and retinal degeneration have been extensively reported [2]. FGF-2 participates in retinal physiological vascularization. However, it seems that FGF-2 itself does not play an essential role in retinal neovascularization.

Hepatocyte Growth Factor

HGF, also named scatter factor, is a 90-kDa cytokine mainly synthesized by the liver. HGF regulates cell growth, cell motility, and the morphogenesis of various types of cells. Its name originates from its capacity to induce the mitogenesis in hepatocytes, but it is also a potent

angiogenic factor. HGF targets and signals epithelial and endothelial cells in a paracrine manner via its high-affinity c-Met surface receptor, a tyrosine kinase receptor [30].

In the retina, vascular endothelial cells, fibroblasts, glial cells and RPE cells have the ability to produce and release HGF. HGF can act as an antiapoptotic factor for endothelial cells, and prevents endothelial cell death which is induced by either serum deprivation, high glucose concentrations, hypoxia or glutathione depletion. Furthermore, intravitreous injection of recombinant human HGF is neuroprotective in a rat model of retinal ischemia-reperfusion injury [2]. Therefore, HGF could be considered as a survival growth factor physiologically synthesized by the retina.

Several authors have shown elevated levels of HGF in the vitreous fluid of PDR patients [2]. We have found 25-fold higher levels of HGF in the vitreous fluid than in the serum of diabetic patients with PDR, and no relationship between intravitreous and serum concentrations was detected [31]. These findings strongly suggest that intraocular synthesis is a significant contributing factor to the high intravitreous levels of HGF observed in patients with PDR. It has been recently reported in experimental models that HGF may play an important role in the initial stages of retinal angiogenesis as well as in increasing retinal vascular permeability. However, we have found that HGF levels per mg of intravitreal proteins were lower in diabetic patients than in nondiabetic subjects, and no relationship between intravitreous levels of HGF and either retinopathy activity or intravitreous VEGF was found [32]. In addition, it should be pointed out that, unlike VEGF, both high glucose concentration and hypoxia downregulate HGF expression in endothelial cells. Finally, we have not found any difference in the expression of HGF receptor (c- Met) on the epiretinal membranes from PDR patients in comparison with the idiopathic epiretinal membranes [33]. All this leads us to think that the role of HGF might be more important in