Hypoxia-induced Paracrine Regulation

Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF), an endothelial cell (EC)–specific mitogen, stimulates angiogenesis in vivo, particularly in ischemic regions. VEGF/VPF expression by cells of hypoxic tissues coincides with expression of its two receptors, KDR and flt-1 , by ECs in the same tissues. We investigated whether hypoxia or hypoxia-dependent conditions operate in coordinating this phenomenon. Human umbilical vein and microvascular ECs were exposed to direct hypoxia or to medium conditioned (CM) by myoblasts maintained in hypoxia for 4 d. Control ECs were maintained in normoxia or normoxia-CM. Binding of 125 I-VEGF to ECs was then evaluated. Hypoxic treatment of ECs had no effect on 125 I-VEGF binding. However, treatment of ECs with hypoxia-CM produced a threefold increase in 125 I-VEGF binding, with peak at 24 h ( P , 0.001, ANOVA). Scatchard analysis disclosed that increased binding was due to a 13-fold increase in KDR receptors/cell, with no change in KDR affinity ( K d 5 260 6 51 pM, normoxia-CM versus K d 5 281 6 94 pM, hypoxia-CM) and no change in EC number (35.6 6 5.9 3 10 3 ECs/cm 2 , normoxia-CM versus 33.5 6 5.5 3 10 3 ECs/cm 2 , hypoxia-CM). Similar results were obtained using CM from hypoxic smooth muscle cells. KDR upregulation was not prevented by addition to the hypoxia-CM of neutralizing antibodies against VEGF, tumor necrosis factora , transforming growth factor b 1 or basic fibroblast growth factor. Similarly, addition of VEGF or lactic acid to the normoxia-CM had no effect on VEGF binding. We conclude that mechanism(s) initiated by hypoxia can induce KDR receptor upregulation in ECs. Hypoxic cells, normal or neoplastic, not only can produce VEGF/VPF, but can also modulate its effects via paracrine induction of VEGF/VPF receptors in ECs. ( J. Clin. Invest. 1996. 97:469–476.)