Angiogenesis, the growth of new blood vessels, is regulated by a number of factors, including hypoxia and vascular endothelial growth factor (VEGF). Although the effects of hypoxia have been studied intensely, less attention has been given to other extracellular parameters such as pH. Thus, the present study investigates the consequences of acidic pH on VEGF binding and activity in endothelial cell cultures. We found that the binding of VEGF 165 and VEGF 121 to endothelial cells increased as the extracellular pH was decreased from 7.5 to 5.5. Binding of VEGF 165 and VEGF 121 to endothelial extracellular matrix was also increased at acidic pH. These effects were, in part, a reflection of increased heparin binding, because VEGF 165 and VEGF 121 showed increased retention on heparin-Sepharose at pH 5.5 compared with pH 7.5. Consistent with these findings, soluble heparin competed for VEGF binding to endothelial cells under acidic conditions. However, at neutral pH (7.5) low concentrations of heparin (0.1-1.0 g/ml) potentiated VEGF binding. Extracellular pH also regulated VEGF activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/ 2). VEGF 165 and VEGF 121 activation of Erk1/2 at pH 7.5 peaked after 5 min, whereas at pH 6.5 the peak was shifted to 10 min. At pH 5.5, neither VEGF isoform was able to activate Erk1/2, suggesting that the increased VEGF bound to the cells at low pH was sequestered in a stored state. Therefore, extracellular pH might play an important role in regulating VEGF interactions with cells and the extracellular matrix, which can modulate VEGF activity.

Hypoxia is one of the major signals that induces angiogenesis. Hypoxic conditions lead to reduced extracellular pH. Vascular endothelial growth factor (VEGF) binding to endothelial cells and the extracellular matrix (ECM) increases at acidic pH (7.0 -5.5). These interactions are dependent on heparan sulfate proteoglycans, but do not depend on the presence of VEGF receptors. Here we report that VEGF 165 and VEGF 121 binding to fibronectin also increased at acidic pH, and that these interactions are further enhanced by the addition of heparin. These results reveal that the accepted nonheparin-binding isoform of VEGF (VEGF 121 ) is converted into a heparin-binding growth factor under acidic conditions. Interestingly, we did not observe increased binding of VEGF to collagen type I at acidic pH in the presence or absence of heparin, indicating that this effect is not a general property of all heparin-binding ECM proteins. The high level of VEGF binding at acidic pH was also rapidly reversed as demonstrated by increased rates of VEGF dissociation from fibronectin and fibronectin-heparin matrices as the pH was raised. The VEGF released from fibronectin retained its ability to stimulate the activation of extracellular-regulated kinase 1/2 in endothelial cells. These results suggest that VEGF may be stored in the extracellular matrix via interactions with fibronectin and heparan sulfate in tissues that are in need of vascularization so that it can aid in directing the dynamic process of growth and migration of new blood vessels.