Lack of R-Ras Leads to Increased Vascular Permeability in Ischemic Retinopathy

Abstract

PurposeThe role of R-Ras in retinal angiogenesis and vascular permeability was evaluated in an oxygen-induced retinopathy (OIR) model using R-Ras knockout (KO) mice and in human diabetic neovascular membranes.MethodsMice deficient for R-Ras and their wild-type (WT) littermates were subjected to 75% oxygen from postnatal day 7 (P7) to P12 and then returned to room air. At P17 retinal vascularization was examined from whole mounts, and retinal vascular permeability was studied using Miles assay. Real-time RT-PCR, Western blotting, and immunohistochemistry were used to assess the expression of R-Ras in retina during development or in the OIR model. The degree of pericyte coverage and vascular endothelial (VE)-cadherin expression on WT and R-Ras KO retinal blood vessels was quantified using confocal microscopy. The correlation of R-Ras with vascular endothelial growth factor receptor 2 (VEGFR2) and human serum albumin on human proliferative diabetic retinopathy membranes was assessed using immunohistochemistry.ResultsIn retina, R-Ras expression was mostly restricted to the vasculature. Retinal vessels in the R-Ras KO mice were significantly more permeable than WT controls in the OIR model. A significant reduction in the direct physical contact between pericytes and blood vessel endothelium as well as reduced VE-cadherin immunostaining was found in R-Ras–deficient mice. In human proliferative diabetic retinopathy neovascular membranes, R-Ras expression negatively correlated with increased vascular leakage and expression of VEGFR2, a marker of blood vessel immaturity.ConclusionsOur results suggest that R-Ras has a role in controlling retinal vessel maturation and stabilization in ischemic retinopathy and provides a potential target for pharmacologic manipulation to treat diabetic retinopathy.

Prolonged activation of cAMP signaling leads to endothelial barrier disruptionviatranscriptional repression ofRRAS

“…One of the important functions of R-Ras is to enhance the blood vessel wall integrity and reduce blood leakage by stabilizing VE-cadherin at the endothelial adherens junction (2, 10). In the current study, we showed compelling evidence that cAMP is a negative regulator of R-Ras expression.…”

“…A strong down-regulation of R-Ras is associated with leaky blood vessels in human malignant tumors and diabetic retinopathy (2, 10). In this study, we demonstrated that the activation of cAMP signaling results in CREB3-dependent down-regulation of RRAS in both macrovascular and microvascular ECs.…”

“…R-Ras also promotes an intimate interaction between pericytes and ECs, and it stabilizes VE-cadherin clustering by limiting Ser 665 phosphorylation, which collectively enhance the integrity of blood vessel wall (2, 9). The Rras gene deletion in mice results in increased blood leakage in various pathologic angiogenesis models (2, 10). The up-regulation of R-Ras signaling, however, reduces vessel permeability (2).…”

“…The up-regulation of R-Ras signaling, however, reduces vessel permeability (2). In a recent study of oxygen-induced retinopathy, R-Ras–deficient mice exhibited significantly increased blood leakage in the retina (10). This result is consistent with clinical observations of vessel leakiness in diabetic retinopathy, as R-Ras expression is found strongly suppressed in the retinal vessels of diabetic patients (10).…”

“…In a recent study of oxygen-induced retinopathy, R-Ras–deficient mice exhibited significantly increased blood leakage in the retina (10). This result is consistent with clinical observations of vessel leakiness in diabetic retinopathy, as R-Ras expression is found strongly suppressed in the retinal vessels of diabetic patients (10). We have previously cloned the promoter and 5′ upstream sequence of the human RRAS gene from ECs and partially characterized the cis elements that are important for the transcriptional regulation (11).…”