MicroRNA-200c/429 mediated regulation of Zeb1 augments N-Cadherin in mouse cardiac mesenchymal cells.

Cardiac mesenchymal cells (CMCs) are a promising cell type that showed therapeutic potential in heart failure models. The analysis of the underlying mechanisms by which the CMCs improve cardiac function is on track. This study aimed to investigate the expression of N-Cadherin, a transmembrane protein that enhances cell adhesion, and recently gained attention for differentiation and augmentation of stem cell function. The mouse CMCs were isolated and analyzed for the mesenchymal markers using flow cytometry. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were used to assess the expression of N-Cadherin along with its counteracting molecule E-Cadherin and their regulator Zeb1 in CMCs and dermal fibroblast. The expression level of miR-200c and miR-429 was analyzed using miRNA assays. Transient transfection of miR-200c followed by qRT-PCR, western blot analysis, and immunostaining was done in CMCs to analyze the expression of Zeb1, N-Cadherin, and E-Cadherin. Flow cytometry analysis showed that CMCs possess mesenchymal markers and absence for hematopoietic and immune cell markers. Increased expression of N-Cadherin and Zeb1 in CMCs was observed in CMCs at both RNA and protein levels compared to fibroblast. We found significant downregulation of miR-200c and miR-429 in CMCs. The ectopic expression of miR-200c in CMCs significantly downregulated Zeb1 and N-Cadherin expression. Our findings suggest that the significant downregulation of miR-200c/429 in CMCs maintains the expression of N-Cadherin, which may be important for its functional integrity.

Interplay between BMP2 and Notch signaling in endothelial-mesenchymal transition: implications for cardiac fibrosis.

Background: The endothelial-to-mesenchymal transition (EndoMT) is a crucial process in cardiovascular development and disorders. Cardiac fibrosis, characterized by excessive collagen deposition, occurs in heart failure, leading to the organ remodeling. Embryonic signaling pathways such as bone morphogenetic protein 2 (BMP2) and Notch are involved in its regulation. However, the interplay between these pathways in EndoMT remains unclear. Methods: This study investigates the downstream targets of Notch and BMP2 and their effect on EndoMT markers in cardiac mesenchymal cells (CMCs) and human umbilical vein endothelial cells (HUVECs). We transduced cell cultures with vectors carrying intracellular domain of NOTCH1 (NICD) and/or BMP2 and evaluated gene expression and activation of EndoMT markers. Results: The results suggest that the Notch and BMP2 signaling pathways have common downstream targets that regulate EndoMT. The activation of BMP2 and Notch is highly dependent on cell type, and co-cultivation of CMCs and HUVECs produced opposing cellular responses to target gene expression and α-smooth muscle actin (α-SMA) synthesis. Conclusions: The balance between Notch and BMP2 signaling determines the outcome of EndoMT and fibrosis in the heart. The study's findings highlight the need for further research to understand the interaction between Notch and BMP2 in the heart and develop new therapeutic strategies for treating cardiac fibrosis.

Mechanisms of Regenerative Potential Activation in Cardiac Mesenchymal Cells.

Recovery of the contractile function of the heart and the regeneration of the myocardium after ischemic injury are contemporary issues in regenerative medicine and cell biology. This study aimed to analyze early transcriptional events in cardiac tissue after infarction and to explore the cell population that can be isolated from myocardial tissue. We induced myocardial infarction in Wistar rats by permanent ligation of the left coronary artery and showed a change in the expression pattern of Notch-associated genes and Bmp2/Runx2 in post-MI tissues using RNA sequencing and RT-PCR. We obtained primary cardiac mesenchymal cell (CMC) cultures from postinfarction myocardium by enzymatic dissociation of tissues, which retained part of the activation stimulus and had a pronounced proliferative potential, assessed using a "xCELLigence" real-time system. Hypoxia in vitro also causes healthy CMCs to overexpress Notch-associated genes and Bmp2/Runx2. Exogenous activation of the Notch signaling pathway by lentiviral transduction of healthy CMCs resulted in a dose-dependent activation of the Runx2 transcription factor but did not affect the activity of the Bmp2 factor. Thus, the results of this study showed that acute hypoxic stress could cause short-term activation of the embryonic signaling pathways Notch and Bmp in CMCs, and this interaction is closely related to the processes of early myocardial remodeling after a heart attack. The ability to correctly modulate and control the corresponding signals in the heart can help increase the regenerative capacity of the myocardium before the formation of fibrotic conditions.

Physiological Oxygen Tension Enhances Competence and Functional Properties of Murine Cardiac Mesenchymal Cells.

Cardiac mesenchymal cells (CMCs), a newly-discovered and promising type of progenitor cells, are effective in improving cardiac function in rodents after myocardial infarction. Stem/progenitor cells are usually cultured at atmospheric O2 tension (21%); however, the physiologic O2 tension in the heart is ~5%, raising the concern that 21% O2 may cause toxicity due to oxidative stress. Thus, we compared mouse CMCs cultured at 21% or 5% O2 beginning at passage 2. At passage 5, CMCs underwent severe hypoxic stress (1% O2 for 24 h). Compared with CMCs cultured at 21% O2, culture at 5% O2 consistently improved cell morphology throughout 5 passages, markedly decreased cell size, increased cell number, shortened cell doubling time, and dramatically reduced lactate dehydrogenase release from CMCs into culture media after hypoxic stress. Furthermore, culture at 5% O2 increased telomerase activity and telomere length, implying that 21% O2 tension impairs telomerase activity, resulting in telomere shortening and decreased cell proliferation. Thus far, almost all preclinical and clinical studies of cell therapy for the heart disease have used atmospheric (21%) O2 to culture cells. Our data challenge this paradigm. Our results demonstrate that, compared with 21% O2, 5% O2 tension greatly enhances the competence and functional properties of CMCs. The increased proliferation rate at 5% O2 means that target numbers of CMCs can be achieved with much less time and cost. Furthermore, since this increased proliferation may continue in vivo after CMC transplantation, and since cells grown at 5% O2 are markedly resistant to severe hypoxic stress, and thus may be better able to survive after transplantation into scarred regions of the heart where O2 is very low, culture at 5% O2 may enhance the reparative properties of CMCs (and possibly other cell types). In conclusion, our data support a change in the methods used to culture CMCs and possibly other progenitor cells.

Cardiac Mesenchymal Cells Cultured at Physiologic Oxygen Tension Have Superior Therapeutic Efficacy in Heart Failure Caused by Myocardial Infarction.

Stem/progenitor cells are usually cultured at atmospheric O2 tension (21%); however, since physiologic O2 tension in the heart is ∼5%, using 21% O2 may cause oxidative stress and toxicity. Cardiac mesenchymal cells (CMCs), a newly discovered and promising type of progenitor cells, are effective in improving left ventricle (LV) function after myocardial infarction (MI). We have previously shown that, compared with 21% O2, culture at 5% O2 increases CMC proliferation, telomerase activity, telomere length, and resistance to severe hypoxia in vitro. However, it is unknown whether these beneficial effects of 5% O2 in vitro translate into greater therapeutic efficacy in vivo in the treatment of heart failure. Thus, murine CMCs were cultured at 21% or 5% O2. Mice with heart failure caused by a 60-min coronary occlusion followed by 30 days of reperfusion received vehicle, 21% or 5% O2 CMCs via echocardiography-guided intraventricular injection. After 35 days, the improvement in LV ejection fraction effected by 5% O2 CMCs was > 3 times greater than that afforded by 21% O2 CMCs (5.2 vs. 1.5 units, P < 0.01). Hemodynamic studies (Millar catheter) yielded similar results both for load-dependent (LV dP/dt) and load-independent (end-systolic elastance) indices. Thus, two independent approaches (echo and hemodynamics) demonstrated the therapeutic superiority of 5% O2 CMCs. Further, 5% O2 CMCs, but not 21% O2 CMCs, significantly decreased scar size, increased viable myocardium, reduced LV hypertrophy and dilatation, and limited myocardial fibrosis both in the risk and non-infarcted regions. Taken together, these results show, for the first time, that culturing CMCs at physiologic (5%) O2 tension provides superior therapeutic efficacy in promoting cardiac repair in vivo. This concept may enhance the therapeutic potential of CMCs. Further, culture at 5% O2 enables greater numbers of cells to be produced in a shorter time, thereby reducing costs and effort and limiting cell senescence. Thus, the present study has potentially vast implications for the field of cell therapy.

The Effect of Cardiogenic Factors on Cardiac Mesenchymal Cell Anti-Fibrogenic Paracrine Signaling and Therapeutic Performance.

Intrinsic cardiogenic factor expression, a proxy for cardiomyogenic lineage commitment, may be an important determinant of donor cell cardiac reparative capacity in cell therapy applications; however, whether and how this contributes to their salutary effects remain largely ambiguous. Methods: The current study examined the consequences of enhanced cardiogenic factor expression, via lentiviral delivery of GMT (GATA4, MEF2C, and TBX5), on cardiac mesenchymal cell (CMC) anti-fibrogenic paracrine signaling dynamics, in vitro, and cardiac reparative capacity, in vivo. Proteome cytokine array analyses and in vitro cardiac fibroblast activation assays were performed using conditioned medium derived from either GMT- or GFP control-transduced CMCs, to respectively assess cardiotrophic factor secretion and anti-fibrogenic paracrine signaling aptitude. Results: Relative to GFP controls, GMT CMCs exhibited enhanced secretion of cytokines implicated to function in pathways associated with matrix remodeling and collagen catabolism, and more ably impeded activated cardiac fibroblast Col1A1 synthesis in vitro. Following their delivery in a rat model of chronic ischemic cardiomyopathy, conventional echocardiography was unable to detect a therapeutic advantage with either CMC population; however, hemodynamic analyses identified a modest, yet calculable supplemental benefit in surrogate measures of global left ventricular contractility with GMT CMCs relative to GFP controls. This phenomenon was neither associated with a decrease in infarct size nor an increase in viable myocardium, but with only a marginal decrease in regional myocardial collagen deposition. Conclusion: Overall, these results suggest that CMC cardiomyogenic lineage commitment biases cardiac repair and, further, that enhanced anti-fibrogenic paracrine signaling potency may underlie, in part, their improved therapeutic utility.

Pro-Angiogenic Actions of CMC-Derived Extracellular Vesicles Rely on Selective Packaging of Angiopoietin 1 and 2, but Not FGF-2 and VEGF.

While the fundamental mechanism by which cardiac cell therapy mitigates ventricular dysfunction in the post ischemic heart remains poorly defined, donor cell paracrine signaling is presumed to be a chief contributor to the afforded benefits. Of the many bioactive molecules secreted by transplanted cells, extracellular vesicles (EVs) and their proteinaceous, nucleic acid, and lipid rich contents, comprise a heterogeneous assortment of prospective cardiotrophic factors-whose involvement in the activation of endogenous cardiac repair mechanism(s), including reducing fibrosis and promoting angiogenesis, have yet to be fully explained. In the current study we aimed to interrogate potential mechanisms by which cardiac mesenchymal stromal cell (CMC)-derived EVs contribute to the CMC pro-angiogenic paracrine signaling capacity in vitro. Vesicular transmission and biological activity of human CMC-derived EVs was evaluated in in vitro assays for human umbilical vein endothelial cell (HUVEC) function, including EV uptake, cell survival, migration, tube formation, and intracellular pathway activation. HUVECs incubated with EVs exhibited augmented cell migration, tube formation, and survival under peroxide exposure; findings which paralleled enhanced activation of the archetypal pro-survival/pro-angiogenic pathways, STAT3 and PI3K-AKT. Cytokine array analyses revealed preferential enrichment of a subset of prototypical angiogenic factors, Ang-1 and Ang-2, in CMC EVs. Interestingly, pharmacologic inhibition of Tie2 in HUVECs, the cognate receptors of angiopoietins, efficiently attenuated CMC-EV-induced HUVEC migration. Further, in additional assays a Tie2 kinase inhibitor exhibited specificity to inhibit Ang-1-, but not Ang-2-, induced HUVEC migration. Overall, these findings suggest that the pro-angiogenic activities of CMC EVs are principally mediated by Ang-1-Tie2 signaling.