The Anti–Angiogenic Activity of Vegf–A Is Masked by the Silencing of Vegfr–1 By Sparc

Abstract

Purpose: : To determine roles and interactions of VEGF–A, VEGFR–1, VEGFR–2, and the matricellular protein SPARC in the laser–injury model of choroidal neovascularization (CNV).

Methods: : CNV lesion volume was measured 7 days after laser injury by confocal imaging of Isolectin B4 stained choroidal flat mounts. VEGF–A, PlGF–1, VEGF–E, CoCl2, SPARC, or neutralizing Abs against VEGF–A, VEGFR–1, VEGFR–2, or SPARC were injected into the vitreous 1 day before or 1 day after laser injury. RPE/choroid lysates were interrogated for SPARC expression and VEGFR–1 or VEGFR–2 phosphorylation levels. VEGFR phosphorylation was studied in HUVECs. Whole mouse eye lysates were subject to reciprocal immunoprecipitation and western blot for VEGF–A and SPARC. FTDOCK simulation was performed to model the VEGF–SPARC complex.

Results: : VEGF–A, VEGF–E and CoCl2 before injury increased CNV, that was sensitive to VEGFR–2 Ab and SPARC Ab. Conversely, VEGF–A, PlGF–1 and CoCl2 after injury decreased CNV, that was sensitive to VEGFR–1 Ab and SPARC. VEGF–A and CoCl2 after injury increased CNV in Vegfr–1 tyrosine kinase –/– but not in SPARC –/– mice (all Ps < 0.05). VEGF–A–induced VEGFR–2 phosphorylation in HUVECs that was reduced by SPARC Ab. Specific in vivo interaction between VEGF–A and SPARC was demonstrated by co–immunoprecipitation. Docking simulations revealed heteromeric interaction between two VEGF–A molecules and SPARC. Side chain interactions at the VEGF–SPARC binding interface involved residues mediating VEGF–A binding to VEGFR–1.

Conclusions: : SPARC regulates whether VEGF–A signals through VEGFR–1 or VEGFR–2, in turn determining whether VEGF–A promotes or suppresses CNV. These observations indicate that therapeutic design of VEGF–A inhibition should include consideration of the level and activity of SPARC.

Keywords: choroid: neovascularization • neovascularization