Proceedings of The Physiological Society

University of Edinburgh (2011) Proc Physiol Soc 25, PC35

Poster Communications

VEGF-induced Ca2+ oscillations in umbilical cord blood-derived endothelial colony forming cells

S. Dragoni1, E. Bonetti2, U. Laforenza1, F. Lodola1, C. Bottino1, V. Rosti2, F. Tanzi1, F. Moccia1

1. Physiology, University of Pavia, Pavia, Italy. 2. Unit of Clinical Epidemiology, IRCCS Policlinico S. Matteo Foundation, Pavia, Italy.

Objective Endothelial colony forming cells (ECFCs) are endothelial progenitor cells capable of acquiring a mature endothelial phenotype. ECFCs are mobilized from bone-marrow to promote vascularization and represent a suitable tool for cell-based therapy (CBT). We have shown that VEGF stimulates ECFC proliferation and tubulogenesis by causing an intracellular Ca2+ oscillations. VEGF-induced Ca2+ spikes are driven by the interplay between inositol-1,4,5-trisphosphate (InsP3)-dependent Ca2+ release and store-operated Ca2+ entry (SOCE). Similar to peripheral ECFCs (PB-ECFCs), the therapeutic potential of umbilical cord blood-derived ECFCs (UCB-ECFCs) has recently been shown. Interestingly VEGF-induced proliferation and expansion of UCB-ECFCs are faster compared to their peripheral counterpart. Unlike PB-ECFCs, UCB-ECFCs express canonical transient receptor potential channel-3 (TRPC3), that mediated diacylglycerol (DAG)-dependent Ca2+ entry and promotes angiogenesis in endothelial cells. This study aimed at investigating whether the higher proliferative potential of UCB-ECFCs was associated to any difference in the molecular underpinnings of their Ca2+ response to VEGF. Results VEGF induced intracellular Ca2+ oscillations in UCB-ECFCs which did not occur in the absence of external Ca2+. Therefore, Ca2+ entry triggers the Ca2+ response to VEGF in these cells. To assess whether TRPC3 contributes to the onset of VEGF-elicited Ca2+ spikes, we exploited the membrane permeable DAG analogue, OAG. Similar to VEGF, OAG elicited a Ca2+ transient in presence, but not in absence, of extracellular Ca2+. This response was abolished by the TRPC3 blockers, flufenamic acid (FFA) and Pyr3. Ca2+ oscillations were inhibited by U73122, which prevent both InsP3 and DAG production. Consistently both FFA and Pyr3 abrogated VEGF-induced Ca2+ signals. Moreover, depletion of the InsP3-sensitive Ca2+ pools with cyclopiazonic acid (CPA), switched the oscillatory response to VEGF into a monotonic Ca2+ increase. Finally BTP-2, a selective inhibitor of SOCE, shortened the Ca2+ signal. Interfering with the Ca2+ response to VEGF by utilizing FFA, BAPTA, an intracellular Ca2+ buffer, and BTP-2 prevented UCB-ECFC proliferation and tubulogenesis. Conclusion These data indicates that VEGF induces Ca2+ oscillations in UCB-ECFCs which depend on TRPC3 recruitment by PLCγ-mediated production of DAG. The following Ca2+ entry elicits the oscillatory signal by promoting the interplay between InsP3Rs and SOCE: the repetitive Ca2+ spikes, in turn, drive cell proliferation and tubulogenesis. This signaling pathway is different to that observed in PB-ECFCs owing to the involvement of TRPC3. Future studies will have to outline whether TRPC3 overexpression in PB-ECFCs augments their proliferative rate in vitro and their regenerative potential in vivo.

Where applicable, experiments conform with Society ethical requirements