Modification of vascular endothelial growth factor interactions and activity by extracellularpH

Angiogenesis is the process by which endothelial cells sprout from pre-existing vessels to form new vessels. Angiogenesis is essential for tissue repair and tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells. Heparan sulfate proteoglycans (HSPG) modulate VEGF activity and are upregulated at sites of active angiogenesis. Hypoxia induces angiogenesis and has been found to upregulate VEGF expression. A characteristic of hypoxic tissues is decreased extracellular pH. Wound beds and certain tumors have been shown to have an extracellular pH ∼6. While the effects of hypoxia on cellular activity have been studied, less attention has been given to the role of pH in modulating signals outside the cell. Therefore, the consequences of extracellular pH changes on VEGF interactions and activity were investigated. Interestingly, both heparin binding and non-heparin binding isoforms of VEGF, VEGF₁₆₅ and VEGF₁₂₁ respectively, showed increased binding to HSPG on endothelial cells and in the extracellular matrix (ECM) at acidic pH. In addition, VEGF₁₆₅ and VEGF₁₂₁ binding to fibronectin increased at acidic pH and this binding was enhanced in the presence of heparin, confirming that VEGF₁₂₁ is able to interact with heparan sulfate. Activation of extracellular regulated kinases 1/2 (Erk1/2) by VEGF at various pHs was measured. VEGF was unable to activate Erk1/2 at acidic pH. However, VEGF that was bound to endothelial cells at acidic pH could stimulate Erk1/2 activation when the pH was increased. Therefore, the data suggests that VEGF may be stored in the ECM at acidic pH such that it cannot activate Erk1/2 in endothelial cells. In fact, once VEGF was bound to fibronectin at acidic pH, the rate of dissociation from this complex increased when the pH was returned to pH 7.5. It was further demonstrated that VEGF released from fibronectin retained the ability to stimulate Erk1/2 activation in endothelial cells. It is proposed that within hypoxic tissues, a pH gradient is established, serving to create reservoirs of stored VEGF. As the pH is increased by the recruitment of new vasculature, VEGF is released from these storage sites thus activating endothelial cells and further propagating angiogenesis.