Identification and Characterization of Vascular Endothelial Growth Factor Receptor (Flt) in Bovine Retinal Pericytes

Abstract

Vascular endothelial growth factor (VEGF) plays an important role in the hypoxia-stimulated neovascularization of ischemic retinal diseases such as proliferative diabetic retinopathy. VEGF exerts its effect through two known high-affinity tyrosine kinase receptors, named kinase insert domain–containing receptor (KDR) and the fms-like tyrosine kinase (Flt). VEGF receptors are located primarily on endothelial cells, although receptors on a few other nonocular cell types also have been described. In the present study, we demonstrate the expression of Fit, but not KDR, in bovine retinal pericytes (BRPCs). Although KDR is expressed predominantly in retinal endothelial cells, Northern blot analysis demonstrated substantial expression of the Fit gene in BRPCs without detection of KDR despite using polyadenylated RNA. Hypoxia increased Fit gene expression in BRPCs (2.7-fold, P < 0.01). ¹²⁵I-labeled VEGF binding analysis on BRPCs demonstrated two apparent high-affinity receptor subtypes (K_d = 14 and 215 pmol/1), with 2.9 × 10⁴ and 1.4 × 10⁵ receptors/cell, respectively. ¹²⁵I-VEGF affinity cross-linking demonstrated VEGF-specific binding complexes at 150, 172, 187, and 200 kDa under reducing conditions. Western blot analysis using an anti-phosphotyrosine antibody demonstrated VEGF-induced tyrosine phosphorylation of several proteins. VEGF stimulation had little effect on initial BRPCs growth rates but significantly increased BRPCs number after 7 days. These results suggest that two classes of high-affinity VEGF receptors are present on BRPCs, at least one of which is analogous to Fit and is capable of intracellular protein phosphorylation. Thus, VEGF might regulate the function of both retinal endothelial cells and retinal pericytes to induce pathological angiogenesis and vascular remodeling during proliferative diabetic retinopathy and other ischemic retinal diseases.