PEDF derived from glial Müller cells: a possible regulator of retinal angiogenesis

Abstract

A precise balance between stimulators and inhibitors of angiogenesis, such as vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), respectively, is essential for angiogenic homeostasis in ocular tissues. Retinal hypoxia is accompanied by some pathological conditions that may promote intraocular neovascularization. Here we demonstrate that retinal glial (Müller) cells express and release pigment epithelium-derived factor (PEDF). Decreasing oxygen concentrations cause strong attenuation of PEDF release resulting in enhanced VEGF/PEDF ratios. Exposure of Müller cells to VEGF suppressed PEDF release in a dose-dependent manner. This may represent a novel mechanism of ocular angiogenic homeostasis sufficient in the control of PEDF levels during normoxia or mild hypoxia but supplemented by other (hitherto unknown) mechanisms in cases of strong hypoxia. In spite of the enhanced VEGF/PEDF ratios resulting from hypoxia, conditioned media of Müller cells failed to stimulate additional proliferation of retinal endothelial cells. These findings suggest that in the ischemic retina, Müller cells generate a permissive condition for angiogenesis by secreting more VEGF and less PEDF, but the onset of retinal endothelial cell proliferation requires another triggering signal that remains to be identified.

Introduction

Retinal neovascularization is a visually threatening complication of various ocular diseases including diabetic retinopathy, central retinal vein occlusion, neovascular glaucoma, and retinopathy of prematurity. Numerous proangiogenic molecules have been proposed to play a role in retinal neovascularization, including basic fibroblast growth factor [1], growth hormone [2], insulin-like growth factor-1 [3], and hepatocyte growth factor [4]. In particular, the cytokine, vascular endothelial growth factor A (here called VEGF), was reported as a common pathological factor in neovascularizing ischemic retinopathies [5].

Increased VEGF levels were detected in the retina and vitreous of patients with ischemic ocular neovascular disorders as well as in animal models of ischemia-induced retinopathy or retinal vein occlusion [6], [7], [8], [9]. VEGF, acting as chemotactic factor, regulator of vascular permeability, and endothelial cell mitogen, is upregulated in the retina by hypoxia [10], [11], [12], [13]. VEGF binds to two different receptors, designated VEGF receptor 1 (VEGFR-1 or fms-like tyrosine kinase-1/Flt-1) and VEGF receptor 2 (VEGFR-2 or kinase-insert domain-containing receptor/KDR). Endothelial cells also express the co-receptors, neuropilin-1 and neuropilin-2, which bind selectively to the 165-amino acid form of VEGF (VEGF165) [14]. Angiogenic homeostasis is tightly controlled by the relative balance of stimulators and inhibitors of angiogenesis [15]. To initiate angiogenesis, the balance between the positive and negative regulators is likely to be shifted such that mitogenic factors are enhanced or inhibitory factors are decreased. However, whereas enhanced VEGF levels were consistently correlated with a stimulation of neovascularization in the retina, the role of angiostatic molecules is less well studied. TGF-β has been proposed as an inhibitor of retinal neovascularization [16], [17], and platelet factor-4 [18], angiostatin [19], endostatin [20], thrombospondin-1 [21], and chondromodulin-I [22] were found to inhibit retinal angiogenesis.

Recently, pigment epithelium-derived factor (PEDF), a 50-kDa glycoprotein initially isolated from the conditioned media of retinal pigment epithelial (RPE) cells and recognized for its neurotrophic activity on cells derived from neural crest [23], [24], [25], [26], [27], [28], was shown to be a potent inhibitor of angiogenesis [29]. A possible role for PEDF in the regulation of ocular neovascularization was suggested, as the molecule was detected in the vitreous and the aqueous humor and as it was shown to be one of the most potent known anti-angiogenic proteins found in humans (for recent reviews, see [30], [31]). It is noteworthy that PEDF inhibits VEGF-induced proliferation and migration of microvascular endothelial cells [32] and, most importantly, adenoviral vector-aided PEDF gene transfer has been found to cause vessel regression in established neovascularization [33]. The generally held belief is that RPE cells are the main producers of PEDF, which is released toward the neural retina into the interphotoreceptor matrix, as a diffusible factor [34]. On the other hand, we and others have shown that soluble mediators, inhibiting endothelial cell proliferation, are present in RPE-free retinal organ cultures as well as in cultures of isolated Müller glial cells [17], [35]. There is accumulating evidence that PEDF counteracts the angiogenic potential of VEGF in the retina, supported by the observation that ischemia-induced retinal neovascularization and proliferative diabetic retinopathy in patients are associated with decreased PEDF levels [36], [37], [38], [39]. However, it remained largely unclear whether RPE cells are the only significant source of PEDF or whether this factor may also be released by other cells in the retina.

Prior investigations have suggested that soluble factors released from ischemic retinal cells are essentially involved in pathological proliferative retinopathy. Several neovascularization-relevant cytokines have been identified (in particular, VEGF) that are produced by neurons, astrocytes, and Müller cells [8], [9], [13], [17] or by RPE cells, retinal microvascular endothelial cells, and pericytes [11], [16]. However, in spite of the observations that a hypoxia-induced expression of VEGF by Müller cells contributes to the development of the retinal vasculature [13] and that the expression of VEGF mRNA and protein in Müller cells can be stimulated in vitro by exposure to hypoxia [8], [42], other angiogenesis-relevant factors may be released by Müller cells as well. Here we show that Müller cells produce PEDF. Our data further support the view that these cells may play an important role to adjust the balance between VEGF and PEDF in the retina under both normoxic and hypoxic conditions, ensuring the cooperation of the two factors in the control of angiogenesis.