HYPOXIA INDUCES VASCULAR ENDOTHELIAL GROWTH-FACTOR IN CULTURED HUMAN ENDOTHELIAL-CELLS

Smooth muscle cells, macrophages, glial cells, keratinocytes, and tran sformed cells have been established as synthesis sites for vascular en dothelial growth factor (VEGF). The modulating effects of VEGF are ess entially limited to endothelial cells (ECs), the only cell type consis tently shown to express VEGF receptors. VEGF has thus been considered to act exclusively via a paracrine pathway. We sought to determine whe ther the role of human ECs might, under selected conditions, extend be yond that of a target to involve contingency synthesis of VEGF. In bot h unstimulated human umbilical vein ECs (HUVECs) and human derma-deriv ed microvascular ECs (HUVECs), Northern analysis detected no VEGF tran scripts. Phorbol-12-myristate 13 acetate (10(-7) M) treatment, however , induced VEGF mRNA expression in both HUVECs and HMECs, peaking at 3 and 6 h, respectively, and returning to undetectable levels by 12 h. I n vitro exposure of HUVECs to a hypoxic environment (pO(2) = 35 mm of mercury) for 12, 24, and 48 h and exposure of HMECs for 6, 12, 24, and 48 h induced VEGF mRNA in a time-dependent fashion. Re-exposure to no rmoxia (pO(2) = 150 mm of mercury) for 24 h after 24 h of hypoxia retu rned VEGF mRNA transcripts to undetectable levels in HUVECs. Cobalt ch loride and nickel chloride treatment each induced VEGF mRNA in ECs. Cy clo-heximide treatment further augmented expression of VEGF mRNA induc ed by cobalt chloride, nickel chlo ride, and hypoxia in HUVECs. VEGF p rotein production in hypoxic HUVECs was demonstrated immunohistochemic ally. Conditioned media from hypoxic HUVECs caused a 2-fold increase i n the incorporation of tritiated thymidine. Finally, immune precipitat es of anti-KDR probed with anti-Tyr(P) antibodies demonstrated evidenc e of receptor autophosphorylation in hypoxic but not normoxic HUVECs. These findings thus establish the potential for an autocrine pathway t hat may augment and/or amplify the paracrine effects of VEGF in stimul ating angiogenesis.