Our data suggest that erythropoietin is a potent ischemia-induced angiogenic factor that acts independently of VEGF during retinal angiogenesis in proliferative diabetic retinopathy.

Background-Inadequate angiogenic response to ischemia in the myocardium of diabetic patients could result in poor collateral formation. Yet, excessive neovascularization in the retina causes proliferative diabetic retinopathy. Since vascular endothelial growth factor (VEGF) is the major angiogenic factor expressed in response to hypoxia, we have characterized expression of VEGF and its receptors in retina, renal glomeruli, aorta, and myocardium in insulin-resistant and diabetic states. Methods and Results-The expression of mRNA and protein for VEGF and its receptors, VEGF-R1 and VEGF-R2, in the myocardium was decreased significantly by 40% to 70% in both diabetic and insulin-resistant nondiabetic rats. Twofold reductions in VEGF and VEGF-R2 were observed in ventricles from diabetic patients compared with nondiabetic donors. In contrast, expression of VEGF and its receptors were increased 2-fold in retina and glomeruli from diabetic or insulin-resistant rats. Insulin treatment of diabetic rats normalized changes in both cardiac and microvascular tissues. Insulin increased VEGF mRNA expression in cultured rat neonatal cardiac myocytes. Conclusions-The results documented for the first time that differential regulation of VEGF and its receptors exist between microvascular and cardiac tissues, which can be regulated by insulin. These results provide a potential explanation for concomitant capillary leakage and neovascularization in the retina and inadequate collateral formation in the myocardium of insulin-resistant and diabetic patients.

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Diabetic macular edema, resulting from increased microvascular permeability, is the most prevalent cause of vision loss in diabetes. The mechanisms underlying this complication remain poorly understood. In the current study, diabetic vascular permeability (blood-retinal barrier breakdown) is demonstrated to result from a leukocyte-mediated Fas-FasL-dependent apoptosis of the retinal vasculature. Following the onset of streptozotocin-induced diabetes, FasL expression was increased in rat neutrophils (P<0.005) and was accompanied by a simultaneous increase in Fas expression in the retinal vasculature. Static adhesion assays demonstrated that neutrophils from diabetic, but not control, rats induced endothelial cell apoptosis in vitro (P<0.005). The latter was inhibited via an antibody-based FasL blockade (P<0.005). In vivo, the inhibition of FasL potently reduced retinal vascular endothelial cell injury, apoptosis, and blood-retinal barrier breakdown (P<0.0001) but did not diminish leukocyte adhesion to the diabetic retinal vasculature. Taken together, these data are the first to identify leukocyte-mediated Fas-FasL-dependent retinal endothelial cell apoptosis as a major cause of blood-retinal barrier breakdown in early diabetes. These data imply that the targeting of the Fas-FasL pathway may prove beneficial in the treatment of diabetic retinopathy.

Retinal neovascularization is a major cause of blindness and requires the activities of several signaling pathways and multiple cytokines. Activation of protein kinase C (PKC) enhances the angiogenic process and is involved in the signaling of vascular endothelial growth factor (VEGF). We have demonstrated a dramatic increase in the angiogenic response to oxygen-induced retinal ischemia in transgenic mice overexpressing PKC␤2 isoform and a significant decrease in retinal neovascularization in PKC␤ isoform null mice. The mitogenic action of VEGF, a potent hypoxiainduced angiogenic factor, was increased by 2-fold in retinal endothelial cells by the overexpression of PKC␤1 or ␤2 isoforms and inhibited significantly by the overexpression of a dominantnegative PKC␤2 isoform but not by the expression of PKC ␣, ␦, and isoforms. Association of PKC␤2 isoform with retinoblastoma protein was discovered in retinal endothelial cells, and PKC␤2 isoform increased retinoblastoma phosphorylation under basal and VEGF-stimulated conditions. The potential functional consequences of PKC␤-induced retinoblastoma phosphorylation could include enhanced E2 promoter binding factor transcriptional activity and increased VEGF-induced endothelial cell proliferation.

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The effects of insulin on vascular endothelial growth factor (VEGF) expression in cultured vascular cells and in angiogenesis were characterized. Insulin increased VEGF mRNA levels in mouse aortic smooth muscle cells from 10 ؊9 to 10 ؊7 M with an initial peak of 3.7-fold increases at 1 h and a second peak of 2.8-fold after 12 h.