HYPOXIA AND VASCULAR ENDOTHELIAL GROWTH-FACTOR STIMULATE ANGIOGENIC INTEGRIN EXPRESSION IN BOVINE RETINAL MICROVASCULAR ENDOTHELIAL-CELLS

Purpose. Integrins alpha v beta 3 and alpha v beta 5 are cell-to-matri x adhesion molecules that have been reported to mediate vascular cell proliferation and migration, The authors investigated the regulation o f expression of these angiogenic integrins by hypoxia and vascular end othelial growth factor (VEGF) in retinal microvascular endothelial cel ls in culture. METHODS. Cultured bovine retinal capillary endothelial cells were exposed to human recombinant VEGF under normoxic (95% air, 5% CO2) conditions to assess the effects of VEGF. Hypoxia studies were performed under lower oxygen concentration (0.5%-1.5% O-2) induced by nitrogen replace ment in constant 5% CO2 conditions. Integrin family mRNA and protein expression were assessed by northern blot analysis an d immunoprecipitation. RESULTS. VEGF (25 ng/ml) increased integrin alp ha v, beta 3, and beta 5 mRNA after 24 hours 6.1 +/- 0.8-fold (P < 0.0 01), 5.9 +/- 1.1-fold (P < 0.001), and 1.9 +/- 0.2-fold (P < 0.01), re spectively. Similarly,, hypoxia stimulated gene expression of integrin alpha v and beta 3 after 24 hours by 5.1 +/- 1.7-fold (P < 0.01) and 3.0 +/- 0.5-fold (P < 0.01), respectively, and integrin beta 5 after 9 hours 1.4 +/- 0.2-fold (P < 0.05). This hypoxia-induced, integrin alp ha v mRNA elevation was inhibited significantly by anti-VEGF neutraliz ing antibody. Also, a conditioned medium from confluent endothelial ce lls maintained under hypoxic conditions for 24 hours produced a 7.1 +/ - 1.1-fold increase (P < 0.001) in integrin alpha v mRNA expression af ter 24 hours, which was reversed by anti-VEGF neutralizing antibody. I nduction of integrin alpha v by VEGF and hypoxia was confirmed in the protein level. CONCLUSIONS. These data suggest that hypoxia stimulates expression of vascular integrins alpha v beta 3 and alpha v beta 5 in retinal microvascular endothelial cells partially through autocrine-p aracrine action of VEGF induced by the hypoxic slate.