Arachidonic acid-evoked Ca2+ signals promote nitric oxide release and proliferation in human endothelial colony forming cells

Abstract

Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca concentration ([Ca]), that, in turn, promotes nitric oxide (NO) release. AA-evoked Ca signals are mainly mediated by Transient Receptor Potential Vanilloid 4 (TRPV4) channels. Circulating endothelial colony forming cells (ECFCs) represent the only established precursors of ECs. In the present study, we, therefore, sought to elucidate whether AA promotes human ECFC (hECFC) proliferation through an increase in [Ca] and the following activation of the endothelial NO synthase (eNOS). AA induced a dose-dependent [Ca] raise that was mimicked by its non-metabolizable analogue eicosatetraynoic acid. AA-evoked Ca signals required both intracellular Ca release and external Ca inflow. AA-induced Ca release was mediated by inositol-1,4,5-trisphosphate receptors from the endoplasmic reticulum and by two pore channel 1 from the acidic stores of the endolysosomal system. AA-evoked Ca entry was, in turn, mediated by TRPV4, while it did not involve store-operated Ca entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca] and by inhibiting eNOS activity with NG-nitro-l-arginine methyl ester (l-NAME). Finally, AA per se did not stimulate hECFC growth, but potentiated growth factors-induced hECFC proliferation in a Ca- and NO-dependent manner. Therefore, AA-evoked Ca signals emerge as an additional target to prevent cancer vascularisation, which may be sustained by ECFC recruitment.

Liposomes as a Putative Tool to Investigate NAADP Signaling in Vasculogenesis

“…Previous studies showed the intracellular levels of NAADP may be increased by using NAADP-encapsulating liposomes. This finding, along with the observation that the NAADP-gated TPC1 is expressed in ECFCs [Zuccolo et al, 2016b], confirmed that PL formulations were suitable to load these cells with NAADP, thereby activating its prominent downstream signaling pathway, that is, intracellular Ca 2þ mobilization. The formulations were prepared by dissolving NAADP within a KCl buffer.…”

“…This finding reinforces the notion that the surface charge has a crucial role in permitting liposomes to be internalized into the cell. The involvement of NAADP receptors, that in ECFCs are represented only by TPC1 [Zuccolo et al, 2016b], was probed with NED-19, a specific blocker of NAADP-induced Ca 2þ release [Morgan et al, 2011;Ronco et al, 2015]. 4B), while empty liposomes did not alter intracellular Ca 2þ levels (not shown).…”

IP3 receptor signaling and endothelial barrier function

“…Recent studies have also revealed the presence of intracellular two-pore channels (TPCs) in endothelial cells that belong to a family of the voltage-gated ion channels [192], [193], [194]. The family is presented by three distantly related proteins (TPC1–3) with TPC2 specifically localized to the lysosomal, and TPC1 to the endolysosomal systems [192], [195].…”

“…In endothelial cells, TPCs are mainly involved in regulation of cell proliferation [193], [194]. TPC2, which promotes Ca 2+ release from lysosomal stores, potentiates pro-angiogenic effect of Vascular Endothelial Growth Factor (VEGF) on endothelial cells, suggesting a specific role of TPC2 in VEGFR2-mediated angiogenesis [193].…”

“…TPC2, which promotes Ca 2+ release from lysosomal stores, potentiates pro-angiogenic effect of Vascular Endothelial Growth Factor (VEGF) on endothelial cells, suggesting a specific role of TPC2 in VEGFR2-mediated angiogenesis [193]. In contrast, TPC1 is involved in intracellular calcium release from endolysosomal store in response to arachidonic acid and mediates specific calcium signaling associated with proliferation of circulating endothelial colony forming cells [194]. …”