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‐CMHypo) 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‐CMHypo‐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‐CMHypo‐induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS‐CMHypo induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic‐related myocardial injury.

Nicotinic Acid Adenine Dinucleotide Phosphate Induces Intracellular Ca2+ Signalling and Stimulates Proliferation in Human Cardiac Mesenchymal Stromal Cells

“…Likewise, NAADP-AM, a membrane-permeable analogue of NAADP, could induce either a transient elevation in [Ca 2+ ] i or a burst of intracellular Ca 2+ oscillations. This latter observation is in accord with the evidence that: 1) intracellular delivery of NAADP may induce oscillatory Ca 2+ signals in human Jurkat T-lymphocytes (Berg et al, 2000), cytotoxic T lymphocytes (Davis et al, 2012), and human pancreatic β-cells (Johnson and Misler, 2002); 2) NAADP contributes to agonist-induced repetitive Ca 2+ spikes in several types of endothelial cells (Zuccolo et al, 2019;Berra-Romani et al, 2020;Balducci et al, 2021), and that 3) NAADP induces intracellular Ca 2+ oscillations in mouse cardiomyocytes during reperfusion injury (Davidson et al, 2015). Early work conducted on echinoderms first suggested that NAADP was able to elicit repetitive Ca 2+ oscillations by promoting a Ca 2+ -dependent crosstalk between two different Ca 2+ pools (Churchill and Galione, 2001), which were later shown to be located in acidic vesicles and ER (Churchill et al, 2002;Moccia et al, 2006).…”

Reactive Oxygen Species and Endothelial Ca2+ Signaling: Brothers in Arms or Partners in Crime?

“…Arachidonic acid is a conditionally essential polyunsaturated fatty acid that, in endothelial cells, plays a crucial role in regulating NO release and angiogenesis [ 43 , 96 , 97 ]. Arachidonic acid is cleaved by glycerophospholipids on the plasma membrane or the nuclear envelope by phospholipase A2 (PLA2), PLC, and phospholipase D (PLD) ( Figure 1 ) [ 98 ] and may be metabolized into an impressive array of bioactive eicosanoids, e.g., prostanoids, thromboxane, leukotrienes, and epoxyeicosatrienoic acids (EETs) ( Figure 1 ), by three distinct families of enzymes, respectively: COXs, LOXs, and CYP ω-hydroxylases and epoxygenases [ 98 , 99 ].…”

“…Thus, SOCE regulates most of endothelial functions, ranging from NO release and vWF secretion to the control of endothelial permeability and proliferation [ 9 , 26 , 166 , 167 , 168 ]. Similarly, SOCE is crucial to ensure proper intracellular Ca 2+ signaling in circulating ECFCs recruited to ischemic tissues to participate in vascular regrowth [ 97 , 111 , 169 ]. The molecular makeup of endothelial SOCE may change depending on the vascular bed, but briefly addressing this controversial issue is necessary to understand how redox signaling regulates agonist-evoked extracellular Ca 2+ entry in the endothelial lineage.…”

“…TRPV4 is a another polymodal channel that presents a P Ca /P Na ranging between 6 and 10 and, therefore, controls crucial Ca 2+ -dependent vascular functions, e.g., angiogenesis, permeability, NO release, and EDH [ 60 , 96 , 217 , 218 ]. In addition, TRPV4 is expressed and mediates proangiogenic Ca 2+ signals in circulating ECFCs [ 97 , 122 ]. TRPV4 is gated by a multitude of cues, including a moderate increase in temperature (>27 °C), pulsatile stretch, laminar shear stress, hypotonic cell swelling, arachidonic acid, EETs, and anandamide [ 217 , 218 ].…”

Small Extracellular Vesicles from Human Amniotic Fluid Samples as Promising Theranostics

“…Indeed, EVs concentrated from the human amniotic fluid progenitor secretome have been attracting increasing interest lately; the EV yield obtained from human hAF-MSC has been proven to be higher than human bone marrow-MSC [ 46 ]. Several independent studies have reported that the hAF-MSC and hAFSC secretome exert pro-survival, anti-apoptotic effects [ 47 ], while quenching inflammation [ 48 ], stimulating local angiogenesis [ 49 , 50 ], and supporting cardiac protection following myocardial infarction or cardiotoxicity [ 49 , 51 , 52 , 53 ]. In this scenario, hAFSC-EVs have been widely profiled to assess whether they can recapitulate such pro-regenerative potential with promising results in preclinical models of skeletal and cardiac muscle injury [ 19 , 47 , 49 , 54 ], lung [ 55 ] and neurodegenerative disease [ 56 , 57 ], osteoarthritis [ 58 ], osteoporosis [ 59 ] and cutaneous injury [ 60 ].…”