Balance between Pigment Epithelium-Derived Factor and Vascular Endothelial Growth Factor in Diabetic Retinopathy

Abstract

Formation and maintenance of the normal vasculature is dependent on interactions between several agonists and inhibitors of angiogenesis. There is strong evidence that two key endogenous molecules with opposing effects on angiogenesis are critical in maintaining the structural and functional integrity of blood vessels in the body. These are the well-characterized proteins, vascular endothelial derived growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Overexpression of VEGF, a potent endothelial cell mitogen, is potentiated in response to hypoxia, hyperglycemia, and chronic inflammation to generate pathological angiogenesis. At nonphysiological levels, VEGF transmits increased proangiogenic signals by binding to its receptors on endothelial cells, which, in turn, activates discrete molecular pathways that perturb endothelial cell proliferation, adhesion, migration, tight junction formation, and vascular permeability. PEDF, on the other hand blocks endothelial cell proliferation and migration and in so doing checks the excessive actions of VEGF on blood vessel growth. Both of these molecules are found in a state of equilibrium in the eye, which is essential to maintain healthy retinal vasculature. Disruption in the function or synthesis of these factors by environmental stresses can contribute to vessel abnormalities. In diabetic retinopathy, formation of microvascular lesions is a hallmark event in the development and progression

of the disease. Disturbances in the levels of VEGF and PEDF have been noted in this condition in experimental and clinical findings. This chapter highlights recent developments that have widened our understanding of how modulations in expression levels of these opposing angiogenesis factors may exacerbate diabetic retinopathy, the need for better surveillance that would identify early disturbances in their synthesis and secretion in the diabetic retina, and the importance of developing treatments to restore physiological levels of both molecules in the prevention of diabetic retinopathy.

Copyright © 2010 S. Karger AG, Basel

Vascular endothelial growth factor (VEGF), a dimeric glycoprotein of approximately 40 kDa, is a potent endothelial cell mitogen that stimulates proliferation, migration, and tube formation, important processes in the development of new blood vessels. VEGF is essential for angiogenesis during normal embryological development. In mammals, the VEGF family consists of seven members: VEGF-A typically referred to as VEGF, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and VEGF-F. VEGF itself has many variants which are generated from a single gene by alternative splicing [1, 2]. In humans, these include the

relatively abundant VEGF121, VEGF165, VEGF189, and VEGF206, and several less abundant forms.

The VEGF isoforms are specifically mitogenic for vascular endothelial cells, and are implicated in inducing permeability at blood-tissue barriers [1, 2]. The abnormal expression of VEGF has become a focal point of current research on the pathogenesis of diabetic retinopathy, as well as other retinal and choroidal vascular diseases. Because unchecked production and activity of VEGF can have severe pathological consequences, antiangiogenic factors are quintessential in maintaining physiological levels of this protein or blocking its mitogenic actions on endothelial cells.

One such factor secreted by almost all tissues in the body is pigment epithelium-derived factor (PEDF), a 50-kDa polypeptide first identified in the conditioned-medium of human retinal pigment epithelial cells [3, 4]. This protein was described in initial studies as a potent differentiation factor that induced the formation of neuron-like structures in primitive cultured retinoblastoma cells [4]. Sequence analysis of the human PEDF gene showed that it is a member of the serine protease inhibitor (serpin) gene family, but to date a target substrate has not been identified to validate protease inhibitory activity [5]. However, another important function for PEDF emerged with the studies of Dawson et al. [6] who demonstrated that PEDF can block the migration of endothelial cells in vitro in a dose-dependent manner and was more effective than angiostatin, thrombospondin-1 and endostatin, other potent antagonists of blood vessel growth. The efficacy of PEDF in reducing angiogenesis was also clearly demonstrated in a murine model of ischemia-in- duced retinopathy [7]. One mechanism proposed for the antiangiogenic actions of PEDF on blood vessel formation is that it counteracts the mitogenic activity of VEGF by inducing apoptosis of activated endothelial cells during neovascularization [7]. These findings placed PEDF among the most potent natural inhibitors of angiogenesis.

Since then, many laboratories have focused on determining whether there is a physiological balance in levels of VEGF and PEDF that is critical in coordinating vessel formation.

VEGF and PEDF in the Eye

The expression level of VEGF in the eye is a focal point of research in the pathogenesis of diabetic retinopathy since increased vascularization and vessel permeability are hallmark features of this blinding disease. In normal developmental processes, VEGF expression decreases substantially after birth, but some cells still constitutively secrete picomolar amounts. It is estimated that cells of the neural retina can secrete ~20 pg per milligram of protein, while cells of the choroid and retinal pigment epithelium secrete ~ 50 pg per milligram of protein in the adult [8]. In vitro experiments have shown that VEGF can induce fenestrations in capillary endothelial cells derived from bovine adrenal cortex [9]. Increased endothelial fenestrations are most likely the predominant mechanism for vascular permeability in diabetic retinopathy.

One factor that is a major stimulus for VEGF expression and subsequent retinal neovascularization is hypoxia [10–13]. Reduced retinal blood flow that is associated with hypoxia may be present even before early signs of retinopathy, such as the loss of capillary pericytes and endothelial cells, are detected. These early pathological changes in the diabetic retina are likely to be triggered by an abnormal increase in the synthesis and secretion of VEGF by specific retinal cells that are responding to hypoxic conditions in the eye [14–16]. Immunocytochemical evidence to support this has been reported in studies which show an increase in the levels of the VEGF protein on nonvascular cells in the eyes of patients with diabetes even in the absence of retinopathy. Thus, diabetic retinopathy may actually have its earliest beginnings as a disease of retinal neurons

and glia with later involvement of the retinal vasculature [15, 16].

PEDF is also normally synthesized in the eye [17] and other tissues throughout the body. Not surprisingly, this antiangiogenic protein is secreted by cells of the cornea, lens, ciliary body, retina, choroid, and the retinal pigment epithelium, all of which are important structures in the eye that could lose function by the abnormal growth of blood vessels [17].

It is hypothesized that a critical balance in the production and secretion of VEGF and PEDF must be maintained to control the normal struc- ture-functionrelationship of the retinal and choroidal vasculature and the neural architecture of the retina.

There are numerous experimental studies that support a reciprocal relationship between PEDF and VEGF in the eye. For example, systemic (intraperitoneal) administration of the PEDF protein inhibits VEGF-induced retinal neovascularization in hyperoxygenated neonatalmice, an accepted model of human retinopathy of prematurity [7], and levels of VEGF are known to be increased while those of PEDF are decreased in proliferative diabetic retinopathy (PDR) [18–22]. Both experimental retinal and choroidal neovascularization in the mouse can be inhibited after an intravitreal injection of a rep- lication-deficient adenovirus containing the PEDF gene [23]. This would suggest that gene therapy with this agent in humans might be feasible.

PEDF/VEGF in the Diabetic Retinopathy

Abnormal production or secretion of VEGF and PEDF in retinal tissues may be an underlying cause of some retinal diseases. The vitreous from eyes with PDR contains high levels of VEGF [24– 26], which is produced by ischemic retinal cells [27, 28]. In addition, there is upregulation of the VEGF/VEGF receptor system in human diabetic retinas and those from STZ-induced diabetic retinopathy in rats [29, 30].

PEDF, on the other hand, has reduced expression levels in the vitreous of individuals with PDR 6, suggesting that loss of PEDF is involved in the pathogenesis of this condition [18–22]. Evidence to support this was shown in studies by Spranger et al. [19] who reported that PEDF concentrations in the vitreous of patients with PDR or with extensive nondiabetic retinal neovascularization caused by retinal-vein occlusion were significantly lower than those of control patients. We also observed a similar trend in PEDF expression in the vitreous of patients with diabetic retinopathy [20]. Correlations in the expression levels of these molecules were also noted in animal models with the degree of ischemia-induced retinal neovascularization in rats [18]. Retinas with neovascularization had a 5-fold increase in VEGF and a 2-fold decrease in PEDF compared to age-matched controls [18]. A decrease in the level of PEDF and an increase in the level of VEGF have been reported in the vitreous of human eyes with PDR [19–22]. In these studies, the vitreal concentration of PEDF was significantly lower at 1.11 ± 0.14 μg/ml (mean

± SE) in eyes with diabetic retinopathy than in eyes with macular hole at 1.71 ± 0.22 μg/ml (p = 0.021, fig. 1a), while the VEGF level was 1,799 ± 478 pg/ml in eyes with diabetic retinopathy and not detectable in eyes with macular hole (fig. 1b). Similarly, PEDF level in eyes with PDR (0.94 ± 0.12 μg/ml) was reduced in nonproliferative diabetic retinopathy (NPDR; 2.25 ± 0.32 μg/ml) and the level in active diabetic retinopathy (0.85 ± 0.14 μg/ ml) was lower than that in inactive diabetic retinopathy as well (1.59 ± 0.24 μg/ml; p = 0.01; fig. 1a). A comparative study showed that the VEGF level was as high as 2,025 ± 533 pg/ml in eyes with PDR compared to 215 ± 201 pg/ml in eyes with NPDR, and the concentration in active diabetic retinopathy (2,543 ± 673 pg/ml) was significantly higher than that in inactive diabetic retinopathy (395 ± 188 pg/ml; p = 0.0098; fig. 1b) [21]. These findings strongly indicate that inverse changes in the intraocular levels of PEDF and VEGF correlate with the degree of retinal neovascularization.

Fig. 1. a PEDF levels in the vitreous samples from the eyes with diabetic retinopathy. b VEGF levels in the vitreous samples from the eyes with diabetic retinopathy (* p < 0.05). Reprinted with permission from [21]. MH = idiopathic macular hole; DR = diabetic retinopathy.

Not only was this relationship noted in the vitreous of patients, but also in retinal cells where a decrease in the expression of PEDF was accompanied by an increase in the expression of VEGF in patients with PDR. Matsuoka et al. [31] observed strong expression of VEGF in the endothelial cells of newly formed vessels in the fibrovascular membranes in eyes with PDR and comparatively weak expression of PEDF in these cells. However, the level of PEDF was prominent in the extracellular matrix and fibrous tissue surrounding the new vessels (fig. 2).

The observations that PEDF blocks VEGFinduced retinal vascular hyperpermeability [32] and downregulates the expression of VEGF [33],

and that exogenously administered PEDF inhibits retinal angiogenesis [7, 23] suggest that the decrease in the level of PEDF is mechanistically involved in VEGF overexpression. It is proposed that changes in vitreous constituents including growth factors and cytokines, may exacerbate the diabetic retinopathy condition [21, 26, 34, 35], but changes in the levels of such factors may also be initiating events in the developing pathophysiology at an early stage since VEGF is shown to be increased in vitreous and in nonvascular cells of diabetic eyes without overt retinopathy [15, 16, 36]. Other ocular compartments such as the aqueous humor have similar changes in the levels of VEGF and PEDF in patients with

diabetes including those with no or mild retinopathy [37].

Modulations in the levels of these two angiogenic molecules are evident as well in diabetic macular edema (DME), a major cause of visual

loss in patientswith diabetes. When their levels in the vitreous were examined by Funatsu et al. [38], they found that VEGF was significantly higher in patients with DME compared to nondiabetic or diabetic individuals without retinopathy (p <

of nonperfused areas in the retina of SDT rats are different than in typical PDR in humans. Another interesting feature of this model and one that does not mimic the human condition is that both PEDF and VEGF expressions are upregulated in the SDT rat retinas (fig. 5) [40] as compared to the inverse relationship between these two molecules seen in human eyes with diabetic retinopathy

and ischemia-induced retinal neovascularization [6, 7,18–22, 33]. While the SDT model is useful in studying some aspects of diabetic retinopathy, the differences noted between the human and rat condition, especially in the expression of these two key angiogenic molecules, is a concern when studying the role of these factors in controlling retinal vasculature growth and permeability.

P10w P20w P40w~

SDT rat

Control

One explanation for the SDT retinopathy phenotype is the higher expression levels of PEDF in these animals. Recent studies show that hypoxiatreated Brown Norway rats have lower levels of PEDF, more nonperfused areas and increased retinal neovascularization when compared to hypox- ia-treated SD rats, which had higher levels of PEDF, fewer nonperfused areas, and less neovascularization [41]. Thus, the levels of PEDF in the retina may alter retinal susceptibility to neovascularization and the progression of diabetic retinopathy.

Some studies have also shown that PEDF can inhibit advanced glycation end-product (AGE)- induced death of pericytes [42] and monocyte chemoattractant protein-1 production in microvascular endothelial cells [43], suggesting that the increased PEDF levels in the SDT rat retina are likely to contribute to reduced AGE functions as well. Studies in support of this concept showed

that PEDF or pyridoxal phosphate, an AGE inhibitor, decreased retinal levels of 8-OHdG, an oxidative stress marker, and subsequently suppressed ICAM-1 gene expression and retinal leukostasis in diabetic rats [44]. Furthermore, PEDF was effective in blocking the increased expression of ICAM-1 as well as retinal leukostasis after intravenous administration of AGE to normal rats [44]. An additional affect on leukostasis was observed by Matsuoka et al. [45] who demonstrated that exposure of human umbilical vein endothelial cells (HUVECs) to VEGF increased the number of adhering monocytes, and that PEDF was efficacious in reducing VEGF-induced leukostasis in a dose-dependent manner (fig. 6). Therefore, PEDF may be a useful strategy to prevent retinal leukostasis induced by VEGF, diabetes, or AGE.

The effects of PEDF on blood vessel growth were also clearly demonstrated in a murine model

of ischemia-induced retinopathy, where administrated PEDF was shown to inhibit the aberrant growth of blood vessels by causing apoptosis of VEGF-activated endothelial cells [7]. Moreover, PEDF-deficient mice exhibit an increased rate of retinal vascular expansion and are more sensitive to hyperoxia-mediated vessel obliteration [46]. These findings strongly suggest that the increased expression of PEDF could attenuate the progression of diabetic retinopathy by inducing apoptosis of activated endothelial cells, suppressing VEGF functions, and attenuating the deleterious effects of AGE.

One mechanism through which PEDF may counteractVEGFactionsisthroughtheregulation of Poly (ADP-ribose) polymerase (PARP). PARP inhibitors are known to decrease angiogenesis by blocking VEGF-induced proliferation, migration, and tube formation of HUVECs, and PARP inhibition has been shown to be associated with the increased expression of PEDF in HUVECs [47].

A second mechanism for PEDF’s antiangiogenic actions is that it decreases the expression of VEGF. Studies suggested that PEDF is an endogenous negative regulator of VEGF in retinal capillary endothelial cells (RCECs) and in the retina of rats with oxygen-induced retinopathy [48]. Additional confirmation of this effect by PEDF was seen in Müller cells, where the silencing of the PEDF gene in these cells by siRNA resulted in a significant upregulation of VEGF expression.

The effect of PEDF on VEGF expression appears to be at the transcriptional level since PEDF inhibits hypoxia-induced increase in VEGF promoter activity, HIF-1 nuclear translocation and mito- gen-activated protein kinase phosphorylation.

A third mechanism of PEDF’s effect on VEGFs action may be mediated through the VEGF receptor. There are studies showing that PEDF can effectively inhibit VEGF binding to RCECs, and in vitro receptor-binding assays demonstrate that PEDF competes with VEGF for binding to VEGF receptor 2. On the other hand, VEGF has reciprocal effects on PEDF since it can decrease PEDF gene expression in RCECs, suggesting a VEGF receptor-mediated process for the expression of both genes. These results suggest that there is a reciprocal regulation between VEGF and PEDF that is important in angiogenic control [48].

Plasma PEDF Levels – Diabetes and

Nephropathy

PEDF is synthesized by a wide range of human tissues including the lung, brain, kidney, and especially by the liver [49], which may explain the high levels of PEDF in the blood. It has been reported that PEDF is present in the plasma of normal individuals at a concentration of approximately 5 μg/ml, indicating that this is one of the most abundantly circulating proteins in humans

PEDF (μg/ml)

a

10

*

9

8

7

6

5

4

3

Control NDR M-NPDR S-NPDR PDR

tion of circulating levels of PEDF in patients with diabetes or the importance of PEDF in the plasma of normal individuals.

AlthoughthePEDFlevelsintheeyesofpatients with diabetic retinopathy have been reported to be low [19–22], the plasma level of this polypeptide in patients with diabetes mellitus was found to be elevated [51–53] especially in those with PDR [51]. Quantitative analysis of blood samples from 112 patients with type 2 diabetes and 33 healthy volunteers indicated that the plasma PEDF level in the diabetic patients (6.68 ± 0.54 μg/ ml; mean ± SEM) was significantly higher than that in controls (4.38 ± 0.59 μg/ml, p = 0.03). The level of plasma PEDF was found to be 5.84 ± 1.72 μg/ml in individuals with no apparent diabetic retinopathy, 6.05 ± 1.02 μg/ml in those with mild to moderate NPDR, 5.95 ± 0.80 μg/ml in patients with severe NPDR, and 7.79 ± 0.98 μg/ml in the

especially high in patients with PDR compared to that of controls (p = 0.005; fig. 7) [51].

In addition, PEDF levels in the blood and clinical systemic status of diabetic patients, e.g. gender, age, insulin treatment, the levels of HbA1c, blood urea nitrogen (BUN), and triglycerides, when analyzed showed that plasma PEDF levels increased with aging in controls but not in the diabetic group and that gender (p = 0.03), BUN (p = 0.005), and triglycerides (p = 0.04) were all significant and independent determinants of plasma PEDF levels in diabetic patients (table 1) [51]. Among the diabetic patients studied, PEDF level in men was higher than that in women, but the reason for this difference is still unknown, although it was suggested that the hormonal environment may affect gender-related PEDF levels. The relationship between blood PEDF levels and the systemic status of diabetic patients is still

vague and further studies are warranted to understand the role of plasma PEDF in diabetes.

Diabetic nephropathy is also a serious vascular complication of diabetic mellitus [54]. It has been reported that there are high levels of PEDF in the serum of patients with end-stage renal disease [51, 55], that the levels of this protein in the kidney were reduced, but that serum levels were lower in a rat model of diabetic nephropathy [56].

Matsuyama et al. [57] evaluated the relationship between diabetic retinopathy, levels of PEDF, and renal function. They showed that the levels of BUN and creatinine increased significantly as the stage of diabetic retinopathy advanced and that plasma PEDF levels correlated with the levels of BUN and creatinine (r = 0.54, p < 0.0001; r = 0.57, p < 0.0001, respectively; fig. 8). Both retinopathy and nephropathy are common microvascular complications associated with diabetes and both are associated with increased plasma PEDF as these conditions progress. Thus, increased levels of PEDF in the blood may indicate microvascular damage in diabetic

patients and may be a predictor of the progression of both retinopathy and nephropathy.

In adipose tissue, the synthesis of PEDF is decreased during the differentiation of the cells to mature adipocytes [58]. This expression pattern is in contrast to that of adiponectin, and an association between PEDF plasma levels, obesity and insulin resistance was proposed [59]. Yamagishi et al. [60] reported that PEDF levels were higher in proportion to the number of components of the metabolic syndrome. They suggested that serum PEDF concentrations may increase as a mechanism to counteract coronary risk factors in metabolic syndrome. Together with the results of previous studies, PEDF is most likely associated with the metabolism of patients with diabetes mellitus and may be elevated to counteract vascular cell damage caused by chronic, low-grade inflammation [61, 62].

Although the PEDF receptor has not been cloned, a lipase-linked cell membrane protein (PLA2)thatinteractswithPEDFhasbeenreported

Fig. 8. Relationship between PEDF and renal function. a Correlation between PEDF levels and BUN. r = 0.54, p < 0.0001. b Correlation between PEDF levels and creatinine. r = 0.57, p < 0.0001. Reprinted with permission from [57].

in the retinal pigment epithelium by Notari et al. [63]. The derived polypeptide has putative transmembrane, intracellular and extracellular regions, and a phospholipase domain. This binding partner of PEDF [TTS-2.2/independent phospholipase A(2) (PLA(2)) zeta and mouse desnutrin/ATGL] has been described in adipose cells as a member of the new calcium-independent PLA(2)/nutrin/ patatin-like phospholipase domain-containing 2 (PNPLA2) family that possesses triglyceride lipase and acylglycerol transacylase activities. PLA2 is a regulator of several processes including inflammation, oxidative stress, release of fatty acids, insulin production, angiogenesis, and obesity.

This protein has specific and high binding affinity for PEDF, and has potent phospholipase A(2) activity that liberates fatty acids. Thus, it is likely that some of the effects of PEDF in diabetes may be mediated through PLA2 activity.

Recently, the relationship between plasma PEDF levels and anthropometric and metabolic variables in type 2 diabetic patients were examined [64]. The percentage change in serum levels of PEDF during a 1-year observational period showed a positive correlation with the patient’s BMI. In addition, the mRNA levels of PEDF in primary cultures of adipocytes, especially omental adipocytes, derived from these individuals