The occlusion of capillary vessels results in low
oxygen tension in adjacent tissues which triggers a signaling cascade that culminates in neovascularization. Using bovine
retinal capillary endothelial cells (BRCEC), we investigated the effects of short-term
hypoxia on
DNA synthesis,
phosphotyrosine induction, changes in the expression of
basic fibroblast growth factor receptor (bFGFR),
protein kinase C (PKC alpha),
heat shock protein 70 (HSP70), and SH2-containing
protein (SHC). The effect of
protein tyrosine kinase (PTK) and
phosphatase inhibitors on
hypoxia-induced
phosphotyrosine was also studied. Capillary endothelial cells cultured in standard normoxic (pO2 = 20%) conditions were quiesced in low serum containing medium and then exposed to low
oxygen tension or
hypoxia (pO2 = 3%) in humidified, 5% CO2, 37 degrees C, tissue culture chambers, on a time-course of up to 24 h.
DNA synthesis was potentiated by
hypoxia in a time-dependent manner. This response positively correlated with the cumulative induction of
phosphotyrosine and the downregulation of bFGFR (M(r) approximately 85 kDa).
Protein tyrosine kinase inhibitors,
herbimycin-A, and
methyl 2,5-dihydroxycinnamate, unlike
genistein, markedly blocked
hypoxia-induced
phosphotyrosine. Prolonged exposure of cells to
phosphatase inhibitor,
sodium orthovanadate, also blocked
hypoxia-induced
phosphotyrosine. The expression of HSP70, PKC alpha, and SHC were not markedly altered by
hypoxia. Taken together, these data suggest that short-term
hypoxia activates endothelial cell proliferation in part via
tyrosine phosphorylation of cellular
proteins and changes in the expression of the
FGF receptor. Thus, endothelial cell mitogenesis and neovascularization associated with low
oxygen tension may be controlled by abrogating signaling pathways mediated by
protein tyrosine kinase and
phosphatases.