Vascular endothelial growth factor (VEGF) and inducible nitricoxide synthase (iNOS) have been implicated in ischemia-induced retinal neovascularization. Retinal ischemia has been shown to induce VEGF and iNOS expression. It has been postulated that one of the crucial consequences of iNOS expression in the ischemic retina is the inhibition of angiogenesis. Furthermore, iNOS was shown to be overexpressed in Mü ller cells from patients with diabetic retinopathy. YC-1, a small molecule inhibitor of hypoxia-inducible factor (HIF)-1␣, has been shown to inhibit iNOS expression in various tissue models. Our aim was to assess the pleiotropic effects of YC-1 in an oxygen-induced retinopathy (OIR) mouse model and evaluate its therapeutic potential in HIF-1-and iNOS-mediated retinal pathologies. Dual-injections of YC-1 into the neovascular retinas decreased the total retinopathy score, inhibited vaso-obliteration and pathologic tuft formation, and concomitantly promoted physiological retinal revascularization, compared with dimethyl sulfoxide (DMSO)-treated group. Furthermore, YC-1-treated retinas exhibited a marked increase in immunoreactivities for CD31 and von Willebrand factor and displayed significant inhibition in HIF-1␣ protein expression. Furthermore, YC-1 down-regulated VEGF, erythropoietin, endothelin-1, matrix metalloproteinase-9, and iNOS message and protein levels. When hypoxic Mü ller and neuoroglial cells were treated with YC-1, iNOS mRNA and protein levels were reduced in a dosedependent fashion. We demonstrate that YC-1 inhibits pathological retinal neovascularization by exhibiting antineovascular activities, which impaired ischemia-induced expression of HIF-1 and its downstream angiogenic molecules. Furthermore, YC-1 enhanced physiological revascularization of the retinal vascular plexuses via the inhibition of iNOS mRNA and protein expressions. The pleiotropic effects of YC-1 allude to its possible use as a promising therapeutic iNOS inhibitor candidate for the treatment of retinal neovascularization.Retinal neovascularization (NV) is the major cause of severe vision loss and irreversible blindness in developed countries, affecting people of all ages (Lee et al., 1998). These clinical and pathologic manifestations occur in diabetic retinopathy, retinopathy of prematurity (ROP), age-related macular degeneration and retinal vein occlusion. The early stages of retinopathy result from retinal ischemia as a result of nonperfusion of the retina or a decrease in oxygen tension (Barinapa, 1995). In the late stages, the ischemia-induced pathologic growth of new blood vessels can cause catastrophic loss of vision.

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The zinc (Zn++) transporter ZnT8 plays a crucial role in zinc homeostasis. It’s been reported that an acute decrease in ZnT8 levels impairs β cell function and Zn++ homeostasis, which contribute to the pathophysiology of diabetes mellitus (DM). Although ZnT8 expression has been detected in the retinal pigment epithelium (RPE), its expression profile in the retina has yet to be determined. Furthermore, the link between diabetes and ischemic retinopathy is well documented; nevertheless, the molecular mechanism(s) of such link has yet to be defined. Our aims were to; investigate the expression profile of ZnT8 in the retina; address the influence of ischemia on such expression; and evaluate the influence of YC-1; (3-(50-hydroxymethyl-20-furyl)-1-benzyl indazole), a hypoxia inducible factor-1 (HIF-1) inhibitor, on the status of ZnT8 expression. We used real-time RT-PCR, immunohistochemistry, and Western blot in the mouse model of oxygen-induced retinopathy (OIR) and Müller cells to evaluate the effects of ischemia/hypoxia and YC-1 on ZnT8 expression. Our data indicate that ZnT8 was strongly expressed in the outer nuclear layer (ONL), outer plexiform layer (OPL), ganglion cell layer (GCL), and nerve fiber layer (NFL), whereas the photoreceptor layer (PRL), inner nuclear layer (INL) and inner plexiform layer (IPL) showed moderate ZnT8 immunoreactivity. Furthermore, we demonstrate that retinal ischemic insult induces a significant downregulation of ZnT8 at the message and protein levels, YC-1 rescues the injured retina by restoring the ZnT8 to its basal homeostatic levels in the neovascular retinas. Our data indicate that ischemic retinopathy maybe mediated by aberrant Zn++ homeostasis caused by ZnT8 downregulation, whereas YC-1 plays a neuroprotective role against ischemic insult. Therefore, targeting ZnT8 provides a therapeutic strategy to combat neovascular eye diseases.