The human amniotic fluid stem cell secretome triggers intracellular Ca2+ oscillations, NF-κB nuclear translocation and tube formation in human endothelial colony-forming cells

Abstract

Second trimester foetal human amniotic fluid-derived stem cells (hAFS) have been shown to possess remarkable cardioprotective paracrine potential in different preclinical models of myocardial injury and drug-induced cardiotoxicity. The hAFS secretome, namely the total soluble factors released by cells in their conditioned medium (hAFS-CM), can also strongly sustain in vivo angiogenesis in a murine model of acute myocardial infarction (MI) and stimulates human endothelial colony-forming cells (ECFCs), the only truly recognized endothelial progenitor, to form capillary-like structures in vitro. Preliminary work demonstrated that the hypoxic hAFS secretome (hAFS-CM^Hypo ) triggers intracellular Ca²⁺ oscillations in human ECFCs, but the underlying mechanisms and the downstream Ca²⁺ -dependent effectors remain elusive. Herein, we found that the secretome obtained by hAFS undergoing hypoxic preconditioning induced intracellular Ca²⁺ oscillations by promoting extracellular Ca²⁺ entry through Transient Receptor Potential Vanilloid 4 (TRPV4). TRPV4-mediated Ca²⁺ entry, in turn, promoted the concerted interplay between inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-induced endogenous Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). hAFS-CM^Hypo -induced intracellular Ca²⁺ oscillations resulted in the nuclear translocation of the Ca²⁺ -sensitive transcription factor p65 NF-κB. Finally, inhibition of either intracellular Ca²⁺ oscillations or NF-κB activity prevented hAFS-CM^Hypo -induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS-CM^Hypo induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic-related myocardial injury.

Keywords: Ca2+ signalling; InsP3Rs; NAADP; NF-κB; TRPV4; angiogenesis; endothelial colony-forming cells; human amniotic fluid stem cell secretome; paracrine therapy; tubulogenesis.