HSF-1 enhances cardioprotective potential of stem cells via exosome biogenesis and their miRNA cargo enrichment.

Stem cell therapy provides a hope to no option heart disease patient group. Stem cells work via different mechanisms of which paracrine mechanism is reported to justify most of the effects. Therefore, identifying the control arms for paracrine cocktail production is necessary to tailor stem cell functions in disease contextual manner. In this study, we describe a novel paracrine cocktail regulatory axis, in stem cells, to enhance their cardioprotective abilities. We identified that HSF1 knockout resulted in reduced cardiac regenerative abilities of mesenchymal stem cells (MSCs) while its overexpression had opposite effects. Altered exosome biognesis and their miRNA cargo enrichment were found to be underlying these altered regenerative abilities. Decreased production of exosomes by MSCs accompanied their loss of HSF1 and vice versa. Moreover, the exosomes derived from HSF1 depleted MSCs showed significantly reduced candidate miRNA expression (miR-145, miR-146, 199-3p, 199b and miR-590) compared to those obtained from HSF1 overexpressing MSCs. We further discovered that HSF1 mediates miRNAs' enrichment into exosomes via Y binding protein 1 (YBX1) and showed, by loss and gain of function strategies, that miRNAs' enrichment in mesenchymal stem cell derived exosomes is deregulated with altered YBX1 expression. It was finally demonstrated that absence of YBX1 in MSCs, with normal HSF1 expression, resulted in significant accumulation of candidate miRNAs into the cells. Together, our data shows that HSF1 plays a critical role in determining the regenerative potential of stem cells. HSF1 does that by affecting exosome biogenesis and miRNA cargo sorting via regulation of YBX1 gene expression.

Augmentation of Histone Deacetylase 6 Activity Impairs Mitochondrial Respiratory Complex I in Ischemic/Reperfused Diabetic Hearts.

BACKGROUND: Diabetes augments activity of histone deacetylase 6 (HDAC6) and generation of tumor necrosis factor α (TNFα) and impairs the physiological function of mitochondrial complex I (mCI) which oxidizes reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide to sustain the tricarboxylic acid cycle and ß-oxidation. Here we examined how HDAC6 regulates TNFα production, mCI activity, mitochondrial morphology and NADH levels, and cardiac function in ischemic/reperfused diabetic hearts. METHODS: HDAC6 knockout, streptozotocin-induced type 1 diabetic, and obese type 2 diabetic db/db mice underwent myocardial ischemia/reperfusion injury in vivo or ex vivo in a Langendorff-perfused system. H9c2 cardiomyocytes with and without HDAC6 knockdown were subjected to hypoxia/reoxygenation injury in the presence of high glucose. We compared the activities of HDAC6 and mCI, TNFα and mitochondrial NADH levels, mitochondrial morphology, myocardial infarct size, and cardiac function between groups. RESULTS: Myocardial ischemia/reperfusion injury and diabetes synergistically augmented myocardial HDCA6 activity, myocardial TNFα levels, and mitochondrial fission and inhibited mCI activity. Interestingly, neutralization of TNFα with an anti-TNFα monoclonal antibody augmented myocardial mCI activity. Importantly, genetic disruption or inhibition of HDAC6 with tubastatin A decreased TNFα levels, mitochondrial fission, and myocardial mitochondrial NADH levels in ischemic/reperfused diabetic mice, concomitant with augmented mCI activity, decreased infarct size, and ameliorated cardiac dysfunction. In H9c2 cardiomyocytes cultured in high glucose, hypoxia/reoxygenation augmented HDAC6 activity and TNFα levels and decreased mCI activity. These negative effects were blocked by HDAC6 knockdown. CONCLUSIONS: Augmenting HDAC6 activity inhibits mCI activity by increasing TNFα levels in ischemic/reperfused diabetic hearts. The HDAC6 inhibitor, tubastatin A, has high therapeutic potential for acute myocardial infarction in diabetes.

Hypoxia-inducible factor 1-alpha enhances the secretome to rejuvenate adult cardiosphere-derived cells.

OBJECTIVE: After cardiac injury, endogenous repair mechanisms are ineffective. However, cell-based therapies provide a promising clinical intervention based on their ability to restore and remodel injured myocardium due to their paracrine factors. Recent clinical trials have demonstrated that adult cardiosphere-derived cell therapy is safe for the treatment of ischemic heart failure, although with limited regenerative potential. The limited efficiency of cardiosphere-derived cells after myocardial infarction is due to the inferior quality of their secretome. This study sought to augment the therapeutic potential of cardiosphere-derived cells by modulating hypoxia-inducible factor-1α, a regulator of paracrine factors. METHODS: Cardiosphere-derived cells were isolated and expanded from the right atrial appendage biopsies of patients undergoing cardiac surgery. To study the effect of hypoxia-inducible factor-1α on the secretome, cardiosphere-derived cells were transduced with hypoxia-inducible factor-1α-overexpressing lentivirus, and various cardioprotective factors within the secretome were quantified using enzyme-linked immunosorbent assays. Comparative analysis of the regenerative potential of cardiosphere-derived cells was performed in a rat myocardial infarction model. RESULTS: Mechanistically, overexpression of hypoxia-inducible factor-1α in adult cardiosphere-derived cells led to the enrichment of the secretome with vascular endothelial growth factor A, angiopoietin 1, stromal cell-derived factor 1α, and basic fibroblast growth factor. Intramyocardial administration of cardiosphere-derived cells transduced with hypoxia-inducible factor-1α after myocardial infarction significantly improved left ventricular ejection fraction, fractional shortening, left ventricular end-systolic volume, and cardiac output. Functional improvement of the rat heart correlated with improved adaptive remodeling of the infarcted myocardium by enhanced angiogenesis and decreased myocardial fibrosis. We also showed that hypoxia-inducible factor-1α expression in cardiosphere-derived cells was adversely affected by aging. CONCLUSIONS: Hypoxia-inducible factor-1α improves the functional potency of cardiosphere-derived cells to preserve myocardial function after myocardial infarction by enriching the cardiosphere-derived cells' secretome with cardioprotective factors. This strategy may be useful for improving the efficacy of allogeneic cell-based therapies in future clinical trials.

Transplanted allogeneic cardiac progenitor cells secrete GDF-15 and stimulate an active immune remodeling process in the ischemic myocardium.

BACKGROUND: Despite promising results in clinical studies, the mechanism for the beneficial effects of allogenic cell-based therapies remains unclear. Macrophages are not only critical mediators of inflammation but also critical players in cardiac remodeling. We hypothesized that transplanted allogenic rat cardiac progenitor cells (rCPCs) augment T-regulatory cells which ultimately promote proliferation of M2 like macrophages by an as-yet undefined mechanism. METHODS AND RESULTS: To test this hypothesis, we used crossover rat strains for exploring the mechanism of myocardial repair by allogenic CPCs. Human CPCs (hCPCs) were isolated from adult patients undergoing coronary artery bypass grafting, and rat CPCs (rCPCs) were isolated from male Wistar-Kyoto (WKY) rat hearts. Allogenic rCPCs suppressed the proliferation of T-cells observed in mixed lymphocyte reactions in vitro. Transplanted syngeneic or allogeneic rCPCs significantly increased cardiac function in a rat myocardial infarct (MI) model, whereas xenogeneic CPCs did not. Allogeneic rCPCs stimulated immunomodulatory responses by specifically increasing T-regulatory cells and M2 polarization, while maintaining their cardiac recovery potential and safety profile. Mechanistically, we confirmed the inactivation of NF-kB in Treg cells and increased M2 macrophages in the myocardium after MI by transplanted CPCs derived GDF15 and it's uptake by CD48 receptor on immune cells. CONCLUSION: Collectively, these findings strongly support the active immunomodulatory properties and robust therapeutic potential of allogenic CPCs in post-MI cardiac dysfunction.

Safety and efficacy of transcoronary transfer of human neonatal stem cells to ischemic myocardium using a novel cell-delivery system (CIRCULATE catheter) in swine model of acute myocardial infarction.

Introduction: Stem cell-based therapies have shown promise in adults with ischemic cardiomyopathy and children with congenital heart diseases, especially those without available therapeutic options. Human neonatal mesenchymal stem cells (nMSCs) have greater regenerative potential than adult stem cells. Aim: To describe our experience with a novel catheter system for transcoronary delivery of cell-based therapies (CIRCULATE catheter) in the intra-coronary delivery of nMSCs in a swine acute myocardial infarct model. Material and methods: A newly developed catheter system (CIRCULATE catheter) with several unique features, including an expandable intra-coronary reservoir with spirally placed side holes of varying diameter, was used. nMSCs together with their secretome were used for the treatment. Pigs underwent myocardial infarction by inflating a 2.5 mm angioplasty balloon in the left anterior descending artery for 60 min. After reperfusion, stem cell therapy or placebo was administered via the novel catheter. TTE was performed at baseline, 1 h after the procedure, and before the euthanasia. Troponin blood concertation was evaluated at baseline, and after 48 h. The heart was harvested, sliced, and stained with triphenyl tetrazolium chloride (TTC). Infarct size to area-at-risk ratio was calculated. Troponin was assessed at baseline and after 48 h. Results: Thirty-nine pigs were operated with the mortality rate of 5.13% (exclusively malignant arrhythmia). Infarct size to area-at-risk ratio was significantly lower in the treatment group. Treated animals had higher ejection fraction than controls. Conclusions: Intra-coronary delivery of neonatal mesenchymal stem cells reduces the infarct size and restores myocardial function in a swine model. The novel catheter system (CIRCULATE catheter) tested in this study was safe and effective in transcoronary cell delivery of human neonatal mesenchymal stem cells.

Stem Cell Therapy in Single-Ventricle Physiology: Recent Progress and Future Directions.

Current surgical and medical treatment options for single ventricle physiology conditions remain palliative. On the long term, despite treatment, the systemic ventricle has a significant risk of developing failure. There are unmet needs to develop novel treatment modalities to help ameliorate the ventricular dysfunction. Advances in the field of stem cell therapy have been promising for the treatment of heart failure. Numerous stem cell populations have been identified. Preclinical studies in small and large animal models provide evidence for effectiveness of this treatment modality and reveal several mechanisms of action by which stem cells exert their effect. Many clinical trials have been designed to further investigate the therapeutic potential that stem cell therapy may hold for pediatric populations with single ventricle physiology. In this review, we discuss the stem cell types used in these populations, some preclinical studies, and the clinical trials of stem cell therapy in single ventricle patients.

Circulating exosomes derived from transplanted progenitor cells aid the functional recovery of ischemic myocardium.

The stem cell field is hindered by its inability to noninvasively monitor transplanted cells within the target organ in a repeatable, time-sensitive, and condition-specific manner. We hypothesized that quantifying and characterizing transplanted cell-derived exosomes in the recipient plasma would enable reliable, noninvasive surveillance of the conditional activity of the transplanted cells. To test this hypothesis, we used a human-into-rat xenogeneic myocardial infarction model comparing two well-studied progenitor cell types: cardiosphere-derived cells (CDCs) and c-kit+ cardiac progenitor cells (CPCs), both derived from the right atrial appendage of adults undergoing cardiopulmonary bypass. CPCs outperformed the CDCs in cell-based and in vivo regenerative assays. To noninvasively monitor the activity of transplanted CDCs or CPCs in vivo, we purified progenitor cell-specific exosomes from recipient total plasma exosomes. Seven days after transplantation, the concentration of plasma CPC-specific exosomes increased about twofold compared to CDC-specific exosomes. Computational pathway analysis failed to link CPC or CDC cellular messenger RNA (mRNA) with observed myocardial recovery, although recovery was linked to the microRNA (miRNA) cargo of CPC exosomes purified from recipient plasma. We further identified mechanistic pathways governing specific outcomes related to myocardial recovery associated with transplanted CPCs. Collectively, these findings demonstrate the potential of circulating progenitor cell-specific exosomes as a liquid biopsy that provides a noninvasive window into the conditional state of the transplanted cells. These data implicate the surveillance potential of cell-specific exosomes for allogeneic cell therapies.

Stem Cell Therapy for Hypoplastic Left Heart Syndrome: Mechanism, Clinical Application, and Future Directions.

Hypoplastic left heart syndrome is a type of congenital heart disease characterized by underdevelopment of the left ventricle, outflow tract, and aorta. The condition is fatal if aggressive palliative operations are not undertaken, but even after the complete 3-staged surgical palliation, there is significant morbidity because of progressive and ultimately intractable right ventricular failure. For this reason, there is interest in developing novel therapies for the management of right ventricular dysfunction in patients with hypoplastic left heart syndrome. Stem cell therapy may represent one such innovative approach. The field has identified numerous stem cell populations from different tissues (cardiac or bone marrow or umbilical cord blood), different age groups (adult versus neonate-derived), and different donors (autologous versus allogeneic), with preclinical and clinical experience demonstrating the potential utility of each cell type. Preclinical trials in small and large animal models have elucidated several mechanisms by which stem cells affect the injured myocardium. Our current understanding of stem cell activity is undergoing a shift from a paradigm based on cellular engraftment and differentiation to one recognizing a primarily paracrine effect. Recent studies have comprehensively evaluated the individual components of the stem cells' secretomes, shedding new light on the intracellular and extracellular pathways at the center of their therapeutic effects. This research has laid the groundwork for clinical application, and there are now several trials of stem cell therapies in pediatric populations that will provide important insights into the value of this therapeutic strategy in the management of hypoplastic left heart syndrome and other forms of congenital heart disease. This article reviews the many stem cell types applied to congenital heart disease, their preclinical investigation and the mechanisms by which they might affect right ventricular dysfunction in patients with hypoplastic left heart syndrome, and finally, the completed and ongoing clinical trials of stem cell therapy in patients with congenital heart disease.

Regenerative medicine therapy for single ventricle congenital heart disease.

One of the most complex forms of congenital heart disease (CHD) involving single ventricle physiology is hypoplastic left heart syndrome (HLHS), characterized by underdevelopment of the left ventricle (LV), mitral and aortic valves, and narrowing of the ascending aorta. The underdeveloped LV is incapable of providing long-term systemic flow, and if left untreated, the condition is fatal. Current treatment for this condition consists of three consecutive staged palliative operations: the first is conducted within the first few weeks of birth, the second between 4 to 6 months, and the third and final surgery within the first 4 years. At the conclusion of the third surgery, systemic perfusion is provided by the right ventricle (RV), and deoxygenated blood flows passively to the pulmonary vasculature. Despite these palliative interventions, the RV, which is ill suited to provide long-term systemic perfusion, is prone to eventual failure. In the absence of satisfying curative treatments, stem cell therapy may represent one innovative approach to the management of RV dysfunction in HLHS patients. Several stem cell populations from different tissues (cardiac and non-cardiac), different age groups (adult- vs. neonate-derived), and different donors (autologous vs. allogeneic), are under active investigation. Preclinical trials in small and large animal models have elucidated several mechanisms by which these stem cells affect the injured myocardium, and are driving the shift from a paradigm based upon cellular engraftment and differentiation to one based primarily on paracrine effects. Recent studies have comprehensively evaluated the individual components of the stem cells' secretomes, shedding new light on the intracellular and extracellular pathways at the center of their therapeutic effects. This research has laid the groundwork for clinical application, and there are now several trials of stem cell therapies in pediatric populations that will provide important insights into the value of this therapeutic strategy in the management of HLHS and other forms of CHD. This article reviews the many stem cell types applied to CHD, their preclinical investigation and the mechanisms by which they might affect RV dysfunction in HLHS patients, and finally, the completed and ongoing clinical trials of stem cell therapy in patients with CHD.

Effects of small platform catheter-based left ventricular assist device support on regional myocardial signal transduction.

OBJECTIVES: Left ventricular (LV) assist device (LVAD) support reduces pathological loading. However, load-induced adaptive responses may be suppressed. Pathological loading dysregulates cardiac G protein-coupled receptor (GPCR) signaling. Signaling through G proteins is deleterious, whereas beta (ß)-arrestin-mediated signaling is cardioprotective. We examined the effects of pathological LV loading/LV dysfunction and treatment via LVAD, on ß-arrestin-mediated signaling, and genetic networks downstream of load. METHODS: An ovine myocardial infarction (MI) model was used. Sheep underwent sham thoracotomy (n = 3), mid-left anterior descending coronary artery ligation to produce MI (n = 3), or MI with placement of a small-platform catheter-based LVAD (n = 3). LVAD support was continued for 2 weeks. Animals were maintained for a total of 12 weeks. Myocardial specimens were harvested and analyzed. RESULTS: MI induced ß-arrestin activation. Increased interactions between epidermal growth factor receptor and ß-arrestins were observed. LVAD support inhibited these responses to MI (P < .05). LVAD support inhibited the activation of cardioprotective signaling effectors Akt (P < .05), and, to a lesser extent, extracellular regulated kinase 1/2 (P not significant); however, MI resulted in regional activation of load-induced GPCR signaling via G proteins, as assessed by the induction of atrial natriuretic peptide mRNA expression in the MI-adjacent zone relative to the MI-remote zone (P < .05). MI-adjacent zone atrial natriuretic peptide expression was renormalized with LVAD support. CONCLUSIONS: LVAD support inhibited cardioprotective ß-arrestin-mediated signaling. However, net benefits of normalization of load-induced GPCR signaling were observed in the MI-adjacent zone. These findings may have implications for the optimal extent and duration of unloading, and for the development of adjunctive medical therapies.