Non-Canonical VEGF Receptor Signaling Regulates Retinal Neovascularization

Abstract

Purpose: : VEGF binds to its cognate receptors VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1) to regulate endothelial function and survival. Although classic canonical kinase signaling pathways can determine angiogenic outcome it is now realized that the intracellular trafficking and localization of VEGFRs make a critical contribution to cellular function. The purpose of this study was to investigate the role of the subcellular translocation of VEGFRs to the nucleus and adheren junctions (AJ) and to assess if the ratio of VEGFR1:VEGFR2 at these intracellular locations determines retinal neovascularization.

Methods: : Retinal microvascular endothelial cells (RMECs) exposed to VEGF, PEDF or VEGF+PEDF were subjected to subcellular fractionation followed by Western blot analysis with antibodies against the C-terminal domain of VEGFR1 and VEGFR2. Immunocytochemistry was used to characterize the subcellular translocation of VEGFRs. The reciprocal immunoprecipitation/Western blot approach was used to detect: 1) the association of VEGFRs with candidate transcription factors and 2) the association of VEGFRs with AJ proteins (e.g. VE-cadherin and β-catenin). To confirm the nuclear localization of VEGFRs in vivo, C57BL mice received an intravitreal injection of VEGF, PEDF or VEGF+PEDF and nuclear expression of VEGFR1 and VEGFR2 in retinal endothelial cells was determined by immunogold cytochemistry.

Results: : VEGF induced nuclear translocation of both VEGFRs in RMECs and decreased VEGFR1:VEGFR2 ratio compared to control. However, addition of PEDF blocked the VEGF-induced effect and maintained a high VEGFR1:VEGFR2 ratio. Full length VEGFRs were differentially associated with three transcript factors implicated in angiogenesis; CREB1, NFATc2 and ETS1. In vivo studies confirmed our in vitro results and showed intravitreal VEGF significantly increased VEGFR2 levels within the nucleus while VEGFR1 was reduced. This was blocked if VEGF was co-injected with PEDF. We also observed a strong and dynamic association between VEGFRs and AJ proteins. Under basal conditions, VE-cadherin and β-catenin were strongly associated with VEGFR1 but after VEGF treatment VEGFR1 association significantly decreased resulting in a reduced VEGFR1:VEGFR2 ratio which eventually resulted in dissociation of the VE-cadherin/β-catenin complex and increased permeability.

Conclusions: : A dynamic subcellular translocation of VEGFRs occurs in microvascular endothelial cells that is dependent on the balance of growth factors in the local microenvironment. The ratio of VEGFR1 to VEGFR2 is a critical determinant in retinal angiogenesis and vascular permeability.

Keywords: vascular endothelial growth factor • retinal neovascularization • receptors