Veronika Yu. Sysoeva

Adipose Stromal Cells Stimulate Angiogenesis via Promoting Progenitor Cell Differentiation, Secretion of Angiogenic Factors, and Enhancing Vessel Maturation

Adipose-derived stromal cells (ASCs) are suggested to be potent candidates for cell therapy of ischemic conditions due to their ability to stimulate blood vessel growth. ASCs produce many angiogenic and anti-apoptotic growth factors, and their secretion is significantly enhanced by hypoxia. Utilizing a Matrigel implant model, we showed that hypoxia-treated ASCs stimulated angiogenesis as well as maturation of the newly formed blood vessels in vivo. To elucidate mechanisms of ASC angiogenic action, we used a co-culture model of ASCs with cells isolated from early postnatal hearts (cardiomyocyte fraction, CMF). CMF contained mature cardiomyocytes, endothelial cells, and progenitor cells. On the second day of culture CMF cells formed spontaneously beating colonies with CD31+ capillary-like structures outgrowing from those cell aggregates. However, these vessel-like structures were not stable, and disassembled within next 5 days. Co-culturing of CMF with ASCs resulted in the formation of stable and branched CD31+ vessel-like structures. Using immunomagnetic depletion of CMF from vascular cells as well as incubation of CMF with mitomycin C-treated ASCs, we showed that in co-culture ASCs enhance blood vessel growth not only by production of paracrine-acting factors but also by promoting the endothelial differentiation of cardiac progenitor cells. All these mechanisms of actions could be beneficial for the stimulation of angiogenesis in ischemic tissues by ASCs administration.

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

BackgroundModified 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 methodsHuman 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.ResultsWe 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.ConclusionsTransplantation 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.

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 (β-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.

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 β2-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.

Local angiotensin II promotes adipogenic differentiation of human adipose tissue mesenchymal stem cells through type 2 angiotensin receptor

Obesity is often associated with high systemic and local activity of renin-angiotensin system (RAS). Mesenchymal stem cells of adipose tissue are the main source of adipocytes. The aim of this study was to clarify how local RAS could control adipose differentiation of human adipose tissue derived mesenchymal stem cells (ADSCs). We examined the distribution of angiotensin receptor expressing cells in human adipose tissue and found that type 1 and type 2 receptors are co-expressed in its stromal compartment, which is known to contain mesenchymal stem cells. To study the expression of receptors specifically in ADSCs we have isolated them from adipose tissue. Up to 99% of cultured ADSCs expressed angiotensin II (AngII) receptor type 1 (AT1). Using the analysis of Ca2+ mobilization in single cells we found that only 5.2±2.7% of ADSCs specifically respond to serial Ang II applications via AT1 receptor and expressed this receptor constantly. This AT1const ADSCs subpopulation exhibited increased adipose competency, which was triggered by endogenous AngII. Inhibitory and expression analyses showed that AT1const ADSCs highly co-express AngII type 2 receptor (AT2), which was responsible for increased adipose competency of this ADSC subpopulation.

Activation of β-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 Ca2+ 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 β-adrenergic receptors and adenylate cyclase activation. The data for the first time clarify mechanisms of regulation of MSCs sensitivity to noradrenaline.

UROKINASE AND UROKINASE RECEPTOR PARTICIPATE IN REGULATION OF NEURONAL MIGRATION, AXON GROWTH AND BRANCHING

PURPOSE Recent findings indicate the significant contribution of urokinase and urokinase receptor (uPA and uPAR) in the processes of nerve regeneration, however, their role in axonal growth and branching is unclear. Using a 3D model of mouse Dorsal Root Ganglia (DRG) explants, differentiated into neurons Neuro 2a cells and transgenic mice lacking the urokinase gene, we studied the involvement of the uPA/uPAR system in the neural cell migration, neurite outgrowth, elongation and branching. RESULTS uPA and uPAR are expressed in the growth cones of axons. Using an ex vivo model of DRG explants in Matrigel we have found that uPA inhibition attenuates neural cell migration and axonal growth, pointing to an important role of urokinase in these processes. Apparently, uPA mediates its effects through its specific receptor uPAR: anti-uPAR antibody, which blocks the uPA binding to uPAR, stimulates axon branching and attenuates neural cell migration from DRG explants. Simultaneous inhibition of uPA and uPAR almost completely prevents the axonal outgrowth from explants into the Matrigels. Experiments in vitro using Neuro 2a cells differentiated into neurons demonstrate that administration of exogenous uPA increases the neurite growth rate (elongation), most likely via the interaction of uPA with uPAR. Blocking of uPAR stimulates neurite formation and enhances branching of preexisting neurites. The results obtained on DRG explants from transgenic mice lacking uPA gene support the assumption that uPA stimulates neurite growth via uPA/uPAR interaction and uPAR role in axons branching and neural cell migration. CONCLUSIONS The uPA/uPAR system plays an essential role in neural cell migration, axonal growth and branching.

T-Cadherin Expression in Melanoma Cells Stimulates Stromal Cell Recruitment and Invasion by Regulating the Expression of Chemokines, Integrins and Adhesion Molecules

T-cadherin is a glycosyl-phosphatidylinositol (GPI) anchored member of the cadherin superfamily involved in the guidance of migrating cells. We have previously shown that in vivo T-cadherin overexpression leads to increased melanoma primary tumor growth due to the recruitment of mesenchymal stromal cells as well as the enhanced metastasis. Since tumor progression is highly dependent upon cell migration and invasion, the aim of the present study was to elucidate the mechanisms of T-cadherin participation in these processes. Herein we show that T-cadherin expression results in the increased invasive potential due to the upregulated expression of pro-oncogenic integrins, chemokines, adhesion molecules and extracellular matrix components. The detected increase in chemokine expression could be responsible for the stromal cell recruitment. At the same time our previous data demonstrated that T-cadherin expression inhibited neoangiogenesis in the primary tumors. We demonstrate that T-cadherin overexpression leads to the increase in the expression of anti-angiogenic molecules and reduction in pro-angiogenic factors. Thus, T-cadherin plays a dual role in melanoma growth and progression: T-cadherin expression results in anti-angiogenic effects in melanoma, however, this also stimulates transcription of genes responsible for migration and invasion of melanoma cells.

Calcium-gated K+ channels of the KCa1.1- and KCa3.1-type couple intracellular Ca2+ signals to membrane hyperpolarization in mesenchymal stromal cells from the human adipose tissue

Electrogenesis in mesenchymal stromal cells (MSCs) remains poorly understood. Little is known about ion channels active in resting MSCs and activated upon MSC stimulation, particularly, by agonists mobilizing Ca2+ in the MSC cytoplasm. A variety of Ca2+-gated ion channels may couple Ca2+ signals to polarization of the plasma membrane. Here, we studied MSCs from the human adipose tissue and found that in cells responsive to ATP and adenosine with Ca2+ transients or exhibiting spontaneous Ca2+ oscillations, Ca2+ bursts were associated with hyperpolarization mediated by Ca2+-gated K+ channels. The expression analysis revealed transcripts for KCNMA1 and KCNN4 genes encoding for Ca2+-activated K+ channels of large (KCa1.1) and intermediate (KCa3.1) conductance, respectively. Moreover, transcripts for the Ca2+-gated cation channel TRPM4 and anion channels Ano1, Ano2, and bestrophin-1, bestrophin-3, and bestrophin-4 were revealed. In all assayed MSCs, a rise in cytosolic Ca2+ stimulated K+ currents that were inhibited with iberiotoxin. This suggested that KCa1.1 channels are invariably expressed in MSCs. In ATP- and adenosine-responsive cells, iberiotoxin and TRAM-34 diminished electrical responses, implicating both KCa1.1 and KCa3.1 channels in coupling agonist-dependent Ca2+ signals to membrane voltage. Functional tests pointed at the existence of two separate MSC subpopulations exhibiting Ca2+-gated anion currents that were mediated by Ano2-like and bestrophin-like anion channels, respectively. Evidence for detectable activity of Ano1 and TRPM4 was not obtained. Thus, KCa1.1 channels are likely to represent the dominant type of Ca2+-activated K+ channels in MSCs, which can serve in concert with KCa3.1 channels as effectors downstream of G-protein-coupled receptor (GPCR)-mediated Ca2+ signaling.

Data supporting that adipose-derived mesenchymal stem/stromal cells express angiotensin II receptors in situ and in vitro

This article contains results of analyses of angiotensin II receptors expression in human adipose tissue and stem/stromal cells isolated from adipose tissue. We also provide here data regarding the effect of angiotensin II on intracellular calcium mobilization in adipose tissue derived stem/stromal cells (ADSCs). Discussion of the data can be found in (Sysoeva et al., 2017) [1].