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.