Victoria Florea

S-nitrosoglutathione reductase (GSNOR) enhances vasculogenesis by mesenchymal stem cells

Although nitric oxide (NO) signaling promotes differentiation and maturation of endothelial progenitor cells, its role in the differentiation of mesenchymal stem cells (MSCs) into endothelial cells remains controversial. We tested the role of NO signaling in MSCs derived from WT mice and mice homozygous for a deletion of S-nitrosoglutathione reductase (GSNOR−/−), a denitrosylase that regulates S-nitrosylation. GSNOR−/− MSCs exhibited markedly diminished capacity for vasculogenesis in an in vitro Matrigel tube–forming assay and in vivo relative to WT MSCs. This decrease was associated with down-regulation of the PDGF receptorα (PDGFRα) in GSNOR−/− MSCs, a receptor essential for VEGF-A action in MSCs. Pharmacologic inhibition of NO synthase with L-NG-nitroarginine methyl ester (L-NAME) and stimulation of growth hormone–releasing hormone receptor (GHRHR) with GHRH agonists augmented VEGF-A production and normalized tube formation in GSNOR−/− MSCs, whereas NO donors or PDGFR antagonist reduced tube formation ∼50% by murine and human MSCs. The antagonist also blocked the rescue of tube formation in GSNOR−/− MSCs by L-NAME or the GHRH agonists JI-38, MR-409, and MR-356. Therefore, GSNOR−/− MSCs have a deficient capacity for endothelial differentiation due to downregulation of PDGFRα related to NO/GSNOR imbalance. These findings unravel important aspects of modulation of MSCs by VEGF-A activation of the PDGFR and illustrate a paradoxical inhibitory role of S-nitrosylation signaling in MSC vasculogenesis. Accordingly, disease states characterized by NO deficiency may trigger MSC-mediated vasculogenesis. These findings have important implications for therapeutic application of GHRH agonists to ischemic disorders.

Activation of growth hormone releasing hormone (GHRH) receptor stimulates cardiac reverse remodeling after myocardial infarction (MI)

Both cardiac myocytes and cardiac stem cells (CSCs) express the receptor of growth hormone releasing hormone (GHRH), activation of which improves injury responses after myocardial infarction (MI). Here we show that a GHRH-agonist (GHRH-A; JI-38) reverses ventricular remodeling and enhances functional recovery in the setting of chronic MI. This response is mediated entirely by activation of GHRH receptor (GHRHR), as demonstrated by the use of a highly selective GHRH antagonist (MIA-602). One month after MI, animals were randomly assigned to receive: placebo, GHRH-A (JI-38), rat recombinant GH, MIA-602, or a combination of GHRH-A and MIA-602, for a 4-wk period. We assessed cardiac performance and hemodynamics by using echocardiography and micromanometry derived pressure-volume loops. Morphometric measurements were carried out to determine MI size and capillary density, and the expression of GHRHR was assessed by immunofluorescence and quantitative RT-PCR. GHRH-A markedly improved cardiac function as shown by echocardiographic and hemodynamic parameters. MI size was substantially reduced, whereas myocyte and nonmyocyte mitosis was markedly increased by GHRH-A. These effects occurred without increases in circulating levels of growth hormone and insulin-like growth factor I and were, at least partially, nullified by GHRH antagonism, confirming a receptor-mediated mechanism. GHRH-A stimulated CSCs proliferation ex vivo, in a manner offset by MIA-602. Collectively, our findings reveal the importance of the GHRH signaling pathway within the heart. Therapy with GHRH-A although initiated 1 mo after MI substantially improved cardiac performance and reduced infarct size, suggesting a regenerative process. Therefore, activation of GHRHR provides a unique therapeutic approach to reverse remodeling after MI.

Agonists of growth hormone-releasing hormone stimulate self-renewal of cardiac stem cells and promote their survival

Significance Stem cell therapy is an emerging approach to the treatment of heart failure. Endogenous or transplanted stem cells have limited repair capacity due to damage by exposure to stress. Agonists of growth hormone-releasing hormone receptor (GHRH-R) have been previously shown to increase the number of endogenous cardiac stem cells (CSCs) after myocardial infarction; enhance vasculogenesis of mesenchymal stem cells; and improve growth, engraftment, and survival of transplanted pancreatic islets in experimental models. This study shows that CSCs isolated from different species express GHRH-R and agonists of GHRH-R stimulate their proliferation and survival. Our findings support the potential use of GHRH agonists for endogenous stem cell stimulation and preconditioning prior to transplantation to improve CSC mobilization, survival, and probably enhancement of their angiomyogenic potential in the infarcted myocardium. The beneficial effects of agonists of growth hormone-releasing hormone receptor (GHRH-R) in heart failure models are associated with an increase in the number of ckit+ cardiac stem cells (CSCs). The goal of the present study was to determine the presence of GHRH-R in CSCs, the effect of GHRH-R agonists on their proliferation and survival, and the mechanisms involved. We investigated the expression of GHRH-R in CSCs of different species and the effect of GHRH-R agonists on their cell proliferation and survival. GHRH-R is expressed in ckit+ CSCs isolated from mouse, rat, and pig. Treatment of porcine CSCs with the GHRH-R agonist JI-38 significantly increased the rate of cell division. Similar results were observed with other GHRH-R agonists, MR-356 and MR-409. JI-38 exerted a protective effect on survival of porcine CSCs under conditions of oxidative stress induced by exposure to hydrogen peroxide. Treatment with JI-38 before exposure to peroxide significantly reduced cell death. A similar effect was observed with MR-356. Addition of GHRH-R agonists to porcine CSCs induced activation of ERK and AKT pathways as determined by increased expression of phospho-ERK and phospho-AKT. Inhibitors of ERK and AKT pathways completely reversed the effect of GHRH-R agonists on CSC proliferation. Our findings extend the observations of the expression of GHRH-R by CSCs and demonstrate that GHRH-R agonists have a direct effect on proliferation and survival of CSCs. These results support the therapeutic use of GHRH-R agonists for stimulating endogenous mechanisms for myocardial repair or for preconditioning of stem cells before transplantation.

Dose Comparison Study of Allogeneic Mesenchymal Stem Cells in Patients With Ischemic Cardiomyopathy (The TRIDENT Study)

Rationale: Cell dose and concentration play crucial roles in phenotypic responses to cell-based therapy for heart failure. Objective: To compare the safety and efficacy of 2 doses of allogeneic bone marrow–derived human mesenchymal stem cells identically delivered in patients with ischemic cardiomyopathy. Methods and Results: Thirty patients with ischemic cardiomyopathy received in a blinded manner either 20 million (n=15) or 100 million (n=15) allogeneic human mesenchymal stem cells via transendocardial injection (0.5 cc per injection × 10 injections per patient). Patients were followed for 12 months for safety and efficacy end points. There were no treatment-emergent serious adverse events at 30 days or treatment-related serious adverse events at 12 months. The Major Adverse Cardiac Event rate was 20.0% (95% confidence interval [CI], 6.9% to 50.0%) in 20 million and 13.3% (95% CI, 3.5% to 43.6%) in 100 million (P=0.58). Worsening heart failure rehospitalization was 20.0% (95% CI, 6.9% to 50.0%) in 20 million and 7.1% (95% CI, 1.0% to 40.9%) in 100 million (P=0.27). Whereas scar size reduced to a similar degree in both groups: 20 million by −6.4 g (interquartile range, −13.5 to −3.4 g; P=0.001) and 100 million by −6.1 g (interquartile range, −8.1 to −4.6 g; P=0.0002), the ejection fraction improved only with 100 million by 3.7 U (interquartile range, 1.1 to 6.1; P=0.04). New York Heart Association class improved at 12 months in 35.7% (95% CI, 12.7% to 64.9%) in 20 million and 42.9% (95% CI, 17.7% to 71.1%) in 100 million. Importantly, proBNP (pro-brain natriuretic peptide) increased at 12 months in 20 million by 0.32 log pg/mL (95% CI, 0.02 to 0.62; P=0.039), but not in 100 million (−0.07 log pg/mL; 95% CI, −0.36 to 0.23; P=0.65; between group P=0.07). Conclusions: Although both cell doses reduced scar size, only the 100 million dose increased ejection fraction. This study highlights the crucial role of cell dose in the responses to cell therapy. Determining optimal dose and delivery is essential to advance the field, decipher mechanism(s) of action and enhance planning of pivotal Phase III trials. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02013674.

c-Myc is essential to prevent endothelial pro-inflammatory senescent phenotype.

The proto-oncogene c-Myc is vital for vascular development and promotes tumor angiogenesis, but the mechanisms by which it controls blood vessel growth remain unclear. In the present work we investigated the effects of c-Myc knockdown in endothelial cell functions essential for angiogenesis to define its role in the vasculature. We provide the first evidence that reduction in c-Myc expression in endothelial cells leads to a pro-inflammatory senescent phenotype, features typically observed during vascular aging and pathologies associated with endothelial dysfunction. c-Myc knockdown in human umbilical vein endothelial cells using lentivirus expressing specific anti-c-Myc shRNA reduced proliferation and tube formation. These functional defects were associated with morphological changes, increase in senescence-associated-β-galactosidase activity, upregulation of cell cycle inhibitors and accumulation of c-Myc-deficient cells in G1-phase, indicating that c-Myc knockdown in endothelial cells induces senescence. Gene expression analysis of c-Myc-deficient endothelial cells showed that senescent phenotype was accompanied by significant upregulation of growth factors, adhesion molecules, extracellular-matrix components and remodeling proteins, and a cluster of pro-inflammatory mediators, which include Angptl4, Cxcl12, Mdk, Tgfb2 and Tnfsf15. At the peak of expression of these cytokines, transcription factors known to be involved in growth control (E2f1, Id1 and Myb) were downregulated, while those involved in inflammatory responses (RelB, Stat1, Stat2 and Stat4) were upregulated. Our results demonstrate a novel role for c-Myc in the prevention of vascular pro-inflammatory phenotype, supporting an important physiological function as a central regulator of inflammation and endothelial dysfunction.

Allogeneic Mesenchymal Stem Cells Ameliorate Aging Frailty: A Phase II Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Abstract Background Aging frailty, characterized by decreased physical and immunological functioning, is associated with stem cell depletion. Human allogeneic mesenchymal stem cells (allo-hMSCs) exert immunomodulatory effects and promote tissue repair. Methods This is a randomized, double-blinded, dose-finding study of intravenous allo-hMSCs (100 or 200-million [M]) vs placebo delivered to patients (n = 30, mean age 75.5 ± 7.3) with frailty. The primary endpoint was incidence of treatment-emergent serious adverse events (TE-SAEs) at 1-month postinfusion. Secondary endpoints included physical performance, patient-reported outcomes, and immune markers of frailty measured at 6 months postinfusion. Results No therapy-related TE-SAEs occurred at 1 month. Physical performance improved preferentially in the 100M-group; immunologic improvement occurred in both the 100M- and 200M-groups. The 6-minute walk test, short physical performance exam, and forced expiratory volume in 1 second improved in the 100M-group (p = .01), not in the 200M- or placebo groups. The female sexual quality of life questionnaire improved in the 100M-group (p = .03). Serum TNF-α levels decreased in the 100M-group (p = .03). B cell intracellular TNF-α improved in both the 100M- (p < .0001) and 200M-groups (p = .002) as well as between groups compared to placebo (p = .003 and p = .039, respectively). Early and late activated T-cells were also reduced by MSC therapy. Conclusion Intravenous allo-hMSCs were safe in individuals with aging frailty. Treated groups had remarkable improvements in physical performance measures and inflammatory biomarkers, both of which characterize the frailty syndrome. Given the excellent safety and efficacy profiles demonstrated in this study, larger clinical trials are warranted to establish the efficacy of hMSCs in this multisystem disorder. Clinical Trial Registration www.clinicaltrials.gov: CRATUS (#NCT02065245).

Cell Therapy Augments Myocardial Perfusion and Improves Quality of Life in Patients With Refractory Angina

Refractory angina pectoris is a chronic disabling condition affecting ≈850 000 patients in the United States.1 It is characterized by frequent angina attacks unresponsive to maximal medical therapy and obstructive coronary artery disease not amenable to coronary revascularization.2 Although major progress has been made in medical therapy and cardiovascular interventions,1 up to 43% of patients continue to experience symptoms and 33% have positive exercise tests after angioplasty.3 It is now well recognized that these patients have concomitant microvascular disease, with reduced coronary and systemic flow reserve at a microvascular level and impaired endothelium-mediated vasorelaxation, that is, endothelial dysfunction.4 Currently, the treatment of these patients remains a major clinical challenge. Article, see p 984 To address this large unmet therapeutic need, research has focused on biological strategies for refractory angina. A key effort is the use of cell therapy, which has the potential to restore the microcirculation and improve myocardial tissue perfusion by stimulating neoangiogenesis.5 In this regard, accumulating evidence supports the idea that cell-based therapy can improve clinical outcomes, including frequency of angina episodes, myocardial infarction (MI) rate, and exercise tolerance, in patients with refractory angina5,6 and, thus, should be subject to further trials to evaluate this treatment option for this patient population. In this issue of Circulation Research , Khan et al present a comprehensive meta-analysis of cell-based therapy for refractory angina. Importantly, their analysis addresses the heterogeneity of the included trials, the problem of missing data, and limitations of the study.5 Six single- and double-blinded, randomized clinical trials were included in this meta-analysis, comprising a patient population that had class III-IV Canadian Cardiovascular Society angina, were refractory to medical therapy, and were not coronary revascularization candidates (Table). The study included 192 patients who received cell therapy plus standard …

Hypoxic Stress Decreases c-Myc Protein Stability in Cardiac Progenitor Cells Inducing Quiescence and Compromising Their Proliferative and Vasculogenic Potential

Cardiac progenitor cells (CPCs) have been shown to promote cardiac regeneration and improve heart function. However, evidence suggests that their regenerative capacity may be limited in conditions of severe hypoxia. Elucidating the mechanisms involved in CPC protection against hypoxic stress is essential to maximize their cardioprotective and therapeutic potential. We investigated the effects of hypoxic stress on CPCs and found significant reduction in proliferation and impairment of vasculogenesis, which were associated with induction of quiescence, as indicated by accumulation of cells in the G0-phase of the cell cycle and growth recovery when cells were returned to normoxia. Induction of quiescence was associated with a decrease in the expression of c-Myc through mechanisms involving protein degradation and upregulation of p21. Inhibition of c-Myc mimicked the effects of severe hypoxia on CPC proliferation, also triggering quiescence. Surprisingly, these effects did not involve changes in p21 expression, indicating that other hypoxia-activated factors may induce p21 in CPCs. Our results suggest that hypoxic stress compromises CPC function by inducing quiescence in part through downregulation of c-Myc. In addition, we found that c-Myc is required to preserve CPC growth, suggesting that modulation of pathways downstream of it may re-activate CPC regenerative potential under ischemic conditions.

New insights into cell-based therapy for heart failure from the CHART-1 study

Cardiopoietic cells (C3BS-CQR-1) are bone marrow mesenchymal stem cells (MSCs) that have been pre-treated with a cocktail of factors: transforming growth factor-β1, fibroblast growth factor-2, insulin-like growth factor-1, activin-A, retinoic acid, α-thrombin, bone morphogenetic protein 4, and interleukin-6. The preconditioning of MSCs with these factors guides them towards a cardiogenic oriented cell type. These cells were tested in a large, multicentre, phase I, randomized clinical trial: Cardiopoietic stem Cell therapy in heart failURE (C-CURE).2 The results of this trial showed no treatment-associated adverse events or systemic toxicity. Moreover, superior improvements in cardiac function and physical performance measures were reported in the MSC-preconditioned group compared to standard of care alone.2 The promising results from C-CURE led to the largest phase II/III, cardiac stem cell trial to date: the Congestive Heart Failure Cardiopoietic Regenerative Therapy (CHART-1) study, which evaluated the safety and efficacy of autologous cardiopoietic cell therapy in patients with ischaemic heart failure.3

Insights Into Signaling in Cell-Based Therapy for Heart Disease

Over the past several decades, stem cell therapy for heart disease has been translated from the bench to the bedside and in clinical trials improves cardiac structure and function in both ischemic and nonischemic cardiac disease. Although the regenerative effects of stem cells in cardiac disease are mediated by both paracrine and cell-to-cell contact mechanisms, many of the downstream signaling pathways remain to be fully elucidated. This review outlines what is currently known about the main signaling pathways involved in mesenchymal stem cell and cardiac stem cell survival, proliferation, and migration and mechanisms of action to repair the damaged heart.