Peripheral 5-HT/HTR6 axis is responsible for obesity-associated hypertension.

Hypertension is one of the major life-threatening complications of obesity. Recently adipose multipotent mesenchymal stromal cells (MSCs) were implicated to the pathogenesis of obesity-associated hypertension. These cells amplify noradrenaline-induced vascular cell contraction via cAMP-mediated signaling pathway. In this study we tested the ability of several cAMP-mediated hormones to affect the adrenergic sensitivity of MSCs and their associated contractility. Despite that adipose MSCs express a plethora of receptors capable of cAMP signaling activation, only 5-HT was able to elevate α1A-adrenoceptor-induced Ca2+ signaling in MSCs. Furthermore, 5-HT markedly enhanced noradrenaline-induced MSCs contractility. Using HTR isoform-specific antagonists followed by CRISPRi-mediated knockdown, we identified that the observed 5-HT effect on MSCs was mediated by the HTR6 isoform. This receptor was previously associated exclusively with 5-HT central nervous system activity. Discovered effect of HTR6 on MSCs contractility points to it as a potential therapeutic target for the prevention and treatment of obesity-associated hypertension.

Functional Heterogeneity of Protein Kinase A Activation in Multipotent Stromal Cells.

Multipotent stromal cells (MSC) demonstrate remarkable functional heterogeneity; however, its molecular mechanisms remain largely obscure. In this study, we explored MSC response to hormones, which activate Gs-protein / cyclic AMP (cAMP) / protein kinase A (PKA) dependent signaling, at the single cell level using genetically encoded biosensor PKA-Spark. For the first time, we demonstrated that about half of cultured MSCs are not able to activate the cAMP/PKA pathway, possibly due to the limited availability of adenylyl cyclases. Using this approach, we showed that MSC subpopulations responding to various hormones largely overlapped, and the share of responding cells did not exceed 40%. Using clonal analysis, we showed that signaling heterogeneity of MSC could be formed de novo within 2 weeks.

Arachidonic acid hyperpolarizes mesenchymal stromal cells from the human adipose tissue by stimulating TREK1 K+ channels.

The current knowledge of electrogenesis in mesenchymal stromal cells (MSCs) remains scarce. Earlier, we demonstrated that in MSCs from the human adipose tissue, transduction of certain agonists involved the phosphoinositide cascade. Its pivotal effector PLC generates DAG that can regulate ion channels directly or via its derivatives, including arachidonic acid (AA). Here we showed that AA strongly hyperpolarized MSCs by stimulating instantly activating, outwardly rectifying TEA-insensitive K+ channels. Among AA-regulated K+ channels, K2P channels from the TREK subfamily appeared to be an appropriate target. The expression of K2P channels in MSCs was verified by RT-PCR, which revealed TWIK-1, TREK-1, and TASK-5 transcripts. The TREK-1 inhibitor spadin antagonized the electrogenic action of AA, which was simulated by the channel activator BL 1249. This functional evidence suggested that TREK-1 channels mediated AA-dependent hyperpolarization of MSCs. Being mostly silent at rest, TREK-1 negligibly contributed to the "background" K+ current. The dramatic stimulation of TREK-1 channels by AA indicates their involvement in AA-dependent signaling in MSCs.

Flow cytometry analysis of adrenoceptors expression in human adipose-derived mesenchymal stem/stromal cells.

Mesenchymal stem/stromal cells (MSCs) were identified in most tissues of an adult organism. MSCs mediate physiological renewal, as well as regulation of tissue homeostasis, reparation and regeneration. Functions of MSCs are regulated by endocrine and neuronal signals, and noradrenaline is one of the most important MSC regulators. We provided flow cytometry analysis of expression of adrenergic receptors on the surface of human MSCs isolated from ten different donors. We have found that the expression profile of adrenergic receptors in MSCs vary significantly between donors. We also showed that alpha1A-adrenoceptor expression is upregulated under the action of noradrenaline. We share our flow cytometry raw data, as well as processing of these data on a flow cytometry repository for freely downloading.

Coupling of P2Y receptors to Ca2+ mobilization in mesenchymal stromal cells from the human adipose tissue.

The purinergic transduction was examined in mesenchymal stromal cells (MSCs) from the human adipose tissue, and several nucleotides, including ATP, UTP, and ADP, were found to mobilize cytosolic Ca2+. Transcripts for multiple purinoreceptors were detected in MSC preparations, including A1, A2A, A2B, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y13, P2Y14, P2X2, P2X4, and P2X7. Cellular responses to nucleotides were insignificantly sensitive to bath Ca2+, pointing at a minor contribution of Ca2+ entry, and were suppressed by U73122 and 2-APB, implicating the phosphoinositide cascade in coupling P2Y receptors to Ca2+ release. While individual cells were sensitive to several P2Y agonists, responsiveness to a given nucleotide varied from cell to cell, suggesting that particular MSCs could employ different sets of purinoreceptors. Caged Ca2+ stimulated Ca2+-induced Ca2+ release (CICR) that was mediated largely by IP3 receptors, and resultant Ca2+ transients were similar to nucleotide responses by magnitude and kinetics. A variety of findings hinted at CICR to be a universal mechanism that finalizes Ca2+ signaling initiated by agonists in MSCs. Individual MSCs responded to nucleotides in an all-or-nothing manner. Presumably just CICR provided invariant Ca2+ responses observed in MSCs at different nucleotide concentrations. The effects of isoform specific agonists and antagonists suggested that both P2Y1 and P2Y13 were obligatory for ADP responses, while P2Y4 and P2Y11 served as primary UTP and ATP receptors, respectively. Extracellular NAD+ stimulated Ca2+ signaling in each ATP-responsive MSC by involving P2Y11. The overall data indicate that extracellular nucleotides and NAD+ can serve as autocrine/paracrine factors regulating MSC functions.

Activation of ß-adrenergic receptors is required for elevated α1A-adrenoreceptors expression and signaling in mesenchymal stromal cells.

Sympathetic neurons are important components of mesenchymal stem cells (MSCs) niche and noradrenaline regulates biological activities of these cells. Here we examined the mechanisms of regulation of MSCs responsiveness to noradrenaline. Using flow cytometry, we demonstrated that α1A adrenergic receptors isoform was the most abundant in adipose tissue-derived MSCs. Using calcium imaging in single cells, we demonstrated that only 6.9 ± 0.8% of MSCs responded to noradrenaline by intracellular calcium release. Noradrenaline increases MSCs sensitivity to catecholamines in a transitory mode. Within 6 hrs after incubation with noradrenaline the proportion of cells responding by Ca(2+) release to the fresh noradrenaline addition has doubled but declined to the baseline after 24 hrs. Increased sensitivity was due to the elevated quantities of α1A-adrenergic receptors on MSCs. Such elevation depended on the stimulation of ß-adrenergic receptors and adenylate cyclase activation. The data for the first time clarify mechanisms of regulation of MSCs sensitivity to noradrenaline.

Functional expression of adrenoreceptors in mesenchymal stromal cells derived from the human adipose tissue.

Cultured mesenchymal stromal cells (MSCs) from different sources represent a heterogeneous population of proliferating non-differentiated cells that contains multipotent stem cells capable of originating a variety of mesenchymal cell lineages. Despite tremendous progress in MSC biology spurred by their therapeutic potential, current knowledge on receptor and signaling systems of MSCs is mediocre. Here we isolated MSCs from the human adipose tissue and assayed their responsivity to GPCR agonists with Ca(2+) imaging. As a whole, a MSC population exhibited functional heterogeneity. Although a variety of first messengers was capable of stimulating Ca(2+) signaling in MSCs, only a relatively small group of cells was specifically responsive to the particular GPCR agonist, including noradrenaline. RT-PCR and immunocytochemistry revealed expression of α1B-, α2A-, and ß2-adrenoreceptors in MSCs. Their sensitivity to subtype-specific adrenergic agonists/antagonists and certain inhibitors of Ca(2+) signaling indicated that largely the α2A-isoform coupled to PLC endowed MSCs with sensitivity to noradrenaline. The all-or-nothing dose-dependence was characteristic of responsivity of robust adrenergic MSCs. Noradrenaline never elicited small or intermediate responses but initiated large and quite similar Ca(2+) transients at all concentrations above the threshold. The inhibitory analysis and Ca(2+) uncaging implicated Ca(2+)-induced Ca(2+) release (CICR) in shaping Ca(2+) signals elicited by noradrenaline. Evidence favored IP3 receptors as predominantly responsible for CICR. Based on the overall findings, we inferred that adrenergic transduction in MSCs includes two fundamentally different stages: noradrenaline initially triggers a local and relatively small Ca(2+) signal, which next stimulates CICR, thereby being converted into a global Ca(2+) signal.

Novel mechanism regulating endothelial permeability via T-cadherin-dependent VE-cadherin phosphorylation and clathrin-mediated endocytosis.

T-cadherin is a unique member of the cadherin superfamily of adhesion molecules. In contrast to "classical" cadherins, T-cadherin lacks transmembrane and cytoplasmic domains and is anchored to the cell membrane via a glycosilphosphoinositol moiety. T-cadherin is predominantly expressed in cardiovascular system. Clinical and biochemical studies evidence that expression of T-cadherin increases in post-angioplasty restenosis and atherosclerotic lesions-conditions associated with endothelial dysfunction and pathological expression of adhesion molecules. Here, we provide data suggesting a new signaling mechanism by which T-cadherin regulates endothelial permeability. T-cadherin overexpression leads to VE-cadherin phosphorylation on Y731 (ß-catenin-binding site), VE-cadherin clathrin-dependent endocytosis and its degradation in lysosomes. Moreover, T-cadherin overexpression results in activation of Rho GTPases signaling and actin stress fiber formation. Thus, T-cadherin up-regulation is involved in degradation of a key endothelial adhesion molecule, VE-cadherin, resulting in the disruption of endothelial barrier function. Our results point to the role of T-cadherin in regulation of endothelial permeability and its possible engagement in endothelial dysfunction.

Transplantation of modified human adipose derived stromal cells expressing VEGF165 results in more efficient angiogenic response in ischemic skeletal muscle.

BACKGROUND: Modified cell-based angiogenic therapy has become a promising novel strategy for ischemic heart and limb diseases. Most studies focused on myoblast, endothelial cell progenitors or bone marrow mesenchymal stromal cells transplantation. Yet adipose-derived stromal cells (in contrast to bone marrow) are abundantly available and can be easily harvested during surgery or liposuction. Due to high paracrine activity and availability ADSCs appear to be a preferable cell type for cardiovascular therapy. Still neither genetic modification of human ADSC nor in vivo therapeutic potential of modified ADSC have been thoroughly studied. Presented work is sought to evaluate angiogenic efficacy of modified ADSCs transplantation to ischemic tissue. MATERIALS AND METHODS: Human ADSCs were transduced using recombinant adeno-associated virus (rAAV) serotype 2 encoding human VEGF165. The influence of genetic modification on functional properties of ADSCs and their angiogenic potential in animal models were studied. RESULTS: We obtained AAV-modified ADSC with substantially increased secretion of VEGF (VEGF-ADSCs). Transduced ADSCs retained their adipogenic and osteogenic differentiation capacities and adhesion properties. The level of angiopoetin-1 mRNA was significantly increased in VEGF-ADSC compared to unmodified cells yet expression of FGF-2, HGF and urokinase did not change. Using matrigel implant model in mice it was shown that VEGF-ADSC substantially stimulated implant vascularization with paralleling increase of capillaries and arterioles. In murine hind limb ischemia test we found significant reperfusion and revascularization after intramuscular transplantation of VEGF-ADSC compared to controls with no evidence of angioma formation. CONCLUSIONS: Transplantation of AAV-VEGF- gene modified hADSC resulted in stronger therapeutic effects in the ischemic skeletal muscle and may be a promising clinical treatment for therapeutic angiogenesis.