Expression and Activation Status of VEGF Signaling Pathway During Choroidal Development and in the Adult.

Abstract

Abstract: : Purpose: Although a significant body of evidence indicates a role for retinal pigment epithelium (RPE) in the development and maintenance of the choroidal vasculature, the mechanims that underlie this relationship are unknown. In addition to an important function in the normal retina, the interactions between RPE and the choroidal vasculature are central to a number of retinal pathologies, including age–related macular diseases such as geographic atrophy. The aim of this study is to determine the function of RPE–secreted vascular endothelial growth factor (VEGF–A) in the formation, differentiation and survival of the underlying choriocapillaris. Methods: A transgenic mouse in which LacZ has been "knocked into" the 3’ portion of the VEGF gene was used to examine VEGF expression in the RPE. mRNA expression of VEGF family members and receptors in the RPE–choroid complex was assessed by RT–PCR. VEGF receptor activation was determined by immunoprecipitation followed by phosphotyrosine blotting. Results: Cryosections of mouse retinas co–stained for beta–galactosidase activity and the endothelial markers CD31 and VEGFR2 revealed strong expression of VEGF in the RPE layer throughout development and in the adult. The major VEGF isoforms expressed in the adult RPE corresponded to VEGF120 and VEGF164 whereas no VEGF188 was detected. RT–PCR analysis showed expression of VEGF–A receptors (VEGFR1 and VEGFR2) and co–receptors (neuropilin–1 and neuropilin–2) in the RPE–choroid complex. Immunoprecipitation of VEGFR2 followed by phospho–tyrosine blotting revealed that VEGFR2 is activated in the fetal choroid as well as in the endothelial cells of adult mouse and bovine choroid. Conclusion: Our data demonstrate that the VEGF–A signaling pathway is expressed and activated during choroidal development and in adult choroid, suggesting that RPE–secreted VEGF is not only involved in choroidal vessel formation, but also in maintenance of the choriocapillaris.

Keywords: choroid • retinal pigment epithelium • vascular cells