We examined the role of hypoxia and HIF factors in embryonic angiogenesis and correlated the degree of hypoxia with the level of HIF and VEGF expression and blood vessel formation. Quail eggs were incubated in normoxic and hypoxic (16% O(2)) conditions. Tissue hypoxia marker, pimonidazol hydrochloride, was applied in vivo for 1 hr and detected in sections with Hypoxyprobe-1 Ab. VEGF and HIF expression was detected by in situ hybridization. HIF-1alpha protein was detected in sections and by Western blot. Endothelial cells were visualized with QH-1 antibody. Hypoxic regions were detected even in normoxic control embryos, mainly in brain, neural tube, branchial arches, limb primordia, and mesonephros. The expression patterns of HIF-1alpha and HIF-1beta factors followed, in general, the Hypoxyprobe-1 marked regions. HIF-2alpha was predominantly expressed in endothelial cells. Diffuse VEGF expression was detected in hypoxic areas of neural tube, myocardium, digestive tube, and most prominently in mesonephros. Growing capillaries were directed to areas of VEGF positivity. Hypoxic regions in hypoxic embryos were larger and stained more intensely. VEGF and HIF-1 factors were proportionately elevated in Hypoxyprobe-1 marked regions without being expressed at new sites and were followed by increased angiogenesis. Our results demonstrate that normal embryonic vascular development involves the HIF-VEGF regulatory cascade. Experimentally increasing the level of hypoxia to a moderate level resulted in over-expression of HIF-1 factors and VEGF followed by an increase in the density of developing vessels. These data indicate that embryonic angiogenesis is responsive to environmental oxygen tension and, therefore, is not entirely genetically controlled.

Institute of Anatomy Charles University 1st Faculty of Medicine Prague Czech Republic

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