Adenosine triphosphate is a critical determinant for VEGFR signal during hypoxia

Hypoxia induces angiogenesis through the VEGF signaling pathway; however, signal propagation of VEGF in hypoxia is not fully understood. In this study, we examined alterations in VEGF signaling during hypoxia conditions and its determinant in endothelial cells. To analyze VEGF signaling during hypoxia, human umbilical vein endothelial cells (HUVECs) were exposed to 3 h of hypoxia (1% O₂) followed by 3 h of reoxygenation or 12 h of hypoxia. Hypoxia induced expression of VEGF mRNA, but it was not associated with an increase in tube formation by HUVECs. During 3 h of hypoxia, VEGF-induced phosphorylation of VEGF receptor-2 (VEGFR-2) and downstream molecules were significantly inhibited without a change in VEGFR-2 expression, but it was completely restored after reoxygenation. VEGF-mediated VEGFR-2 phosphorylation is associated with a reduction in cellular ATP in hypoxia conditions (65.93 ± 8.32% of normoxia, means ± SE, P < 0.01). Interestingly, attenuation of VEGFR-2 phosphorylation was restored by addition of ATP to prepared membranes from cells that underwent 3 h of hypoxia. In contrast to 3 h of hypoxia, exposure of cells to 12 h of hypoxia decreased VEGFR-2 expression and VEGF-mediated VEGFR-2 phosphorylation. The magnitude of VEGFR-2 phosphorylation was not fully restored by addition of ATP to prepared membranes from cells exposed to 12 h of hypoxia. These data indicate that ATP is an important determinant of VEGF signaling in hypoxia and suggest that the activation process of VEGFR-2 was modified by sustained hypoxia. These observations contribute to our understanding of signal alterations in VEGF in endothelial cells during hypoxia.