Hydrogen sulphide triggers VEGF-induced intracellular Ca signals in human endothelial cells but not in their immature progenitors

Hydrogen sulphide (H₂S) is a newly discovered gasotransmitter that regulates multiple steps in VEGF-induced angiogenesis. An increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is central to endothelial proliferation and may be triggered by both VEGF and H₂S. Albeit VEGFR-2 might serve as H₂S receptor, the mechanistic relationship between VEGF- and H₂S-induced Ca²⁺ signals in endothelial cells is unclear. The present study aimed at assessing whether and how NaHS, a widely employed H₂S donor, stimulates pro-angiogenic Ca²⁺ signals in Ea.hy926 cells, a suitable surrogate for mature endothelial cells, and human endothelial progenitor cells (EPCs). We found that NaHS induced a dose-dependent increase in [Ca²⁺]_i in Ea.hy926 cells. NaHS-induced Ca²⁺ signals in Ea.hy926 cells did not require extracellular Ca²⁺ entry, while they were inhibited upon pharmacological blockade of the phospholipase C/inositol-1,4,5-trisphosphate (InsP₃) signalling pathway. Moreover, the Ca²⁺ response to NaHS was prevented by genistein, but not by SU5416, which selectively inhibits VEGFR-2. However, VEGF-induced Ca²⁺ signals were suppressed by dl-propargylglycine (PAG), which blocks the H₂S-producing enzyme, cystathionine γ-lyase. Consistent with these data, VEGF-induced proliferation and migration were inhibited by PAG in Ea.hy926 cells, albeit NaHS alone did not influence these processes. Conversely, NaHS elevated [Ca²⁺]_i only in a modest fraction of circulating EPCs, whereas neither VEGF-induced Ca²⁺ oscillations nor VEGF-dependent proliferation were affected by PAG. Therefore, H₂S-evoked elevation in [Ca²⁺]_i is essential to trigger the pro-angiogenic Ca²⁺ response to VEGF in mature endothelial cells, but not in their immature progenitors.