Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells

The retinal pigmented epithelium (RPE) is a monolayer of polarized cells lo cated between retinal photoreceptors and blood Vessels of the choroid. The basal surface of RPE cells rests on Bruch's membrane, a complex extracellul ar matrix structure which becomes abnormal in several disease processes, in cluding age-related macular degeneration (AMD). Ruptures or abnormalities i n Bruch's membrane are frequently accompanied by choroidal neovascularizati on. Disturbed interaction of RPE cells with their extracellular matrix (ECM ) could play a role in this process. The present study was undertaken to ex amine the complex interactions between hypoxia, integrin, and ECM in the re gulation of RPE functions. Antibody blocking experiments demonstrated that RPE cell adhesion to vitronectin is mediated primarily through alpha v beta 5 and adhesion to fibronectin occurs through alpha 5 beta 1. RPE adhesion to immobilized laminin demonstrated highest level of non-RGD-mediated adhes ion as compared to that with collagen IV or the RGD matrices such as vitron ectin (alpha v beta 5), fibronectin (alpha 5 beta 1), or thrombospondin (al pha 5 beta 1 + alpha v beta 5). Addition of soluble vitronectin, or fibrino gen to RPE cell cultures resulted in a small to moderate increase in VEGF a nd FGF2 in the media, while each of these growth factors was dramatically i ncreased after addition of thrombospondin 1 (TSP1). In contrast, soluble fi bronectin resulted in differential upregulation of VEGF but not FGF2. Simil arly, immobilized TSP1 resulted in differential greater upregulation in VEG F but not FGF2 release from RPE as compared to other ECMs under either norm oxic or hypoxic conditions. Additionally, Hypoxia resulted in a time-depend ent increase in VEGF, but not FGF2 release in the media. RPE cells grown on TSP1-coated plates showed increased VEGF and FGF2 in their media compared to cells grown on plates coated with type IV collagen, laminin, vitronectin , or fibronectin. The TSP1-induced increase in secretion of growth factors was partially blocked by anti-alpha 5 beta 1, anti-alpha v beta 3, and anti -alpha v beta 5 antibodies indicating that it may be mediated in part by TS P1 binding to those integrins. These data suggest that alterations in oxyge n levels (hypoxia/ischemia) and ECM of RPE cells, a prominent feature of AM D, can cause increased secretion of angiogenic growth factors that might co ntribute to the development of choroidal neovascularization. These data als o suggest the potential modulatory role of VEGF release from RPE by ECM and alpha v beta 5 and alpha 5 beta 1 integrins. I. Cell. Biochem. 74:13 5-143 , 1999. (C) 1999 Wiley-Liss, Inc.