Hypoxia–Induced Up–Regulation of a Pro–Angiogenic Gene Family in Choroidal Retinal Endothelial (Rf/6a) Cells

Abstract

Abstract: : Purpose: The up–regulation of pro–angiogenic gene expression is associated with the onset of new blood vessel growth, or neovascularization, in stressed retinal endothelial cells. In this study we used brief periods of hypoxia to monitor up–regulation in the expression of a pro–angiogenic gene family, whose members include cytosolic phospholipase A₂ (cPLA₂), the cell death receptor FAS, beta amyloid precursor protein (ßAPP), the nuclear factor for interleukin 6 (NF–IL6), interleukin–1ß (IL–1ß), cyclooxygenase–2 (COX–2), ß–chemokine exodus protein (CEX–1) and vascular endothelial growth factor (VEGF). Methods: Choroidal retinal endothelial (RF/6A) cells were subjected to hypoxia (90% N₂/5% CO₂/5%O₂) for 0, 1 and 3 hours at which times RNA, under stringent conditions, was isolated and labeled using an Enzo Bioarray High Yield RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA). Total cellular proteins were isolated using whole cell lysates and were quanitified using a mini–Bradford assay. Gene expression patterns for cPLA₂, FAS, ßAPP, NF–IL6, IL–1ß, COX–2, CEX–1 and VEGF were examined using DNA array assay and HG U95Av2 GeneChip (Affymetrix) analysis and Western immunoblot assay. The extent of induced neovascularization (vessel tube formation) in RF/6A cells was monitored using phase contrast microscopy. Results: In contrast to several non–angiogenic genetic markers (ß–actin, cyclophilin, glial fibrillary acidic protein), during hypoxia, all 8 pro–angiogenic genes examined (cPLA₂, FAS, ßAPP, NF–IL6, IL–1ß, COX–2, CEX–1, and VEGF) exhibited a robust up–regulation in gene expression, at both the level of RNA and protein. At the level of RNA abundance, the mean increases for this pro–angiogenic gene family ranged from 2.5– to 4.1–fold over age–matched normoxic controls. Conclusions: The expression of a small family of pro–angiogenic genes, including those encoding cPLA₂, FAS, ßAPP, NF–IL6, IL–1ß, COX–2, CEX–1, and VEGF is strongly activated during brief periods of hypoxia in RF/6A cells. Coincident with the up–regulation in the expression of this gene family is the onset of de novo neovascularization. As these genes appear to be coordinately induced, pharmacotherapies targeting the up–regulation of one gene may also reduce the expression of other gene family members, and function to repress the inception of neovascularization in hypoxia–stressed RF/6A cells.

Keywords: hypoxia • gene/expression • retinal neovascularization