CHANGES ASSOCIATED WITH TYROSINE PHOSPHORYLATION DURING SHORT-TERM HYPOXIA IN RETINAL MICROVASCULAR ENDOTHELIAL-CELLS IN-VITRO

The occlusion of capillary vessels results in low oxygen tension in ad jacent tissues which triggers a signaling cascade that culminates in n eovascularization. Using bovine retinal capillary endothelial cells (B RCEC), we investigated the effects of short-term hypoxia on DNA synthe sis, phosphotyrosine induction, changes in the expression of basic fib roblast growth factor receptor (bFGFR), protein kinase C (PKC alpha), heat shock protein 70 (HSP70), and SH2-containing protein (SHC). The e ffect of protein tyrosine kinase (PTK) and phosphatase inhibitors on h ypoxia-induced phosphotyrosine was also studied. Capillary endothelial cells cultured in standard normoxic (pO(2) = 20%) conditions were qui esced in low serum containing medium and then exposed to low oxygen te nsion or hypoxia (pO(2) = 3%) in humidified, 5% CO2, 37 degrees C, tis sue culture chambers, on a time-course of up to 24 h. DNA synthesis wa s potentiated by hypoxia in a time-dependent manner. This response pos itively correlated with the cumulative induction of phosphotyrosine an d the downregulation of bFGFR (M(r) similar to 85 kDa). Protein tyrosi ne kinase inhibitors, herbimycin-A, and methyl 2,5-dihydroxycinnamate, unlike genistein, markedly blocked hypoxia-induced phosphotyrosine. P rolonged exposure of cells to phosphatase inhibitor, sodium orthovanad ate, also blocked hypoxia-induced phosphotyrosine. The expression of H SP70, PKC alpha, and SHC were not markedly altered by hypoxia. Taken t ogether, these data suggest that short-term hypoxia activates endothel ial cell proliferation in part via tyrosine phosphorylation of cellula r proteins and changes in the expression of the FGF receptor. Thus, en dothelial cell mitogenesis and neovascularization associated with low oxygen tension may be controlled by abrogating signaling pathways medi ated by protein tyrosine kinase and phosphatases. (C) 1995 Wiley-Liss, Inc.