Supplementary Material for: R-Ras Deficiency in Pericytes Causes Frequent Microphthalmia and Perturbs Retinal Vascular Development

<b><i>Purpose:</i></b> The retinal vasculature is heavily invested by pericytes. Small GTPase R-Ras is highly expressed in endothelial cells and pericytes, suggesting importance of this Ras homolog for the regulation of the blood vessel wall. We investigated the specific contribution of pericyte-expressed R-Ras to the development of the retinal vasculature. <b><i>Methods:</i></b> The effect of R-Ras deficiency in pericytes was analyzed in pericyte-targeted conditional <i>Rras</i> knockout mice at birth and during the capillary plexus formation in the neonatal retina. <b><i>Results:</i></b> The offspring of these mice frequently exhibited unilateral microphthalmia. Analyses of the developing retinal vasculature in the eyes without microphthalmia revealed excessive endothelial cell proliferation, sprouting, and branching of the capillary plexus in these animals. These vessels were structurally defective with diminished pericyte coverage and basement membrane formation. Furthermore, these vessels showed reduced VE-cadherin staining and significantly elevated plasma leakage indicating the breakdown of the blood-retinal barrier. This defect was associated with considerable macrophage infiltration in the retina. <b><i>Conclusions:</i></b> The normal retinal vascular development is dependent on R-Ras expression in pericytes, and the absence of it leads to unattenuated angiogenesis and significantly weakens the blood-retinal barrier. Our findings underscore the importance of R-Ras for pericyte function during the normal eye development.