Effects of microenvironmental extracellular pH and extracellular matrix proteins on angiostatin's activity and on intracellular pH.

Antiangiogenic agents target migratory and proliferative endothelial cells (EC) in the process of forming new vessels, resulting in growth inhibition or cell death. Here we have shown that the antiangiogenic activity of angiostatin on EC is enhanced in culture when the microenvironmental extracellular pH (pH(e)) is reduced to levels similar to that of many tumors. In a migration/scratch assay and during tube formation, angiostatin in combination with reduced pH(e) synergistically resulted in an increased EC death--an effect not seen with either stimulus individually. Lowering of pH(e) decreased intracellular pH (pH(i)), and a further lowering of pH(i) occurred when low pH(e) was combined with angiostatin. These data suggest that low pH(e) plays a role in the relative specificity and efficacy of angiostatin for tumor neovasculature and indicate roles for both pH(e) and pH(i) in the mechanism of angiostatin action. A receptor for angiostatin, the alpha-subunit of ATP synthase, was found on the surface of EC. We show that cell surface receptor distribution is increased on Matrigel, a basement-like matrix, as opposed to fibronectin or RGD peptide substrates, and redistributed to a more punctuate appearance at low pH(e). Furthermore, positive cell surface histochemical staining for alpha-ATP synthase was blocked by preincubation with angiostatin. These data indicate that substrate and pH(e) are critical parameters in the evaluation of this antiangiogenic substance, and probably for others as well.