Haemin attenuates intermittent hypoxia‐induced cardiac injury via inhibiting mitochondrial fission

Obstructive sleep apnoea (OSA) characterized by intermittent hypoxia (IH) is closely associated with cardiovascular diseases. IH confers cardiac injury via accelerating cardiomyocyte apoptosis, whereas the underlying mechanism has remained largely enigmatic. This study aimed to explore the potential mechanisms involved in the IH‐induced cardiac damage performed with the IH‐exposed cell and animal models and to investigate the protective effects of haemin, a potent haeme oxygenase‐1 (HO‐1) activator, on the cardiac injury induced by IH. Neonatal rat cardiomyocyte (NRC) was treated with or without haemin before IH exposure. Eighteen male Sprague‐Dawley (SD) rats were randomized into three groups: control group, IH group (PBS, ip) and IH + haemin group (haemin, 4 mg/kg, ip). The cardiac function was determined by echocardiography. Mitochondrial fission was evaluated by Mitotracker staining. The mitochondrial dynamics‐related proteins (mitochondrial fusion protein, Mfn2; mitochondrial fission protein, Drp1) were determined by Western blot. The apoptosis of cardiomyocytes and heart sections was examined by TUNEL. IH regulated mitochondrial dynamics‐related proteins (decreased Mfn2 and increased Drp1 expressions, respectively), thereby leading to mitochondrial fragmentation and cell apoptosis in cardiomyocytes in vitro and in vivo, while haemin‐induced HO‐1 up‐regulation attenuated IH‐induced mitochondrial fragmentation and cell apoptosis. Moreover, IH resulted in left ventricular hypertrophy and impaired contractile function in vivo, while haemin ameliorated IH‐induced cardiac dysfunction. This study demonstrates that pharmacological activation of HO‐1 pathway protects against IH‐induced cardiac dysfunction and myocardial fibrosis through the inhibition of mitochondrial fission and cell apoptosis.     

Mitophagy promotes the stemness of bone marrow-derived mesenchymal stem cells

Previous studies demonstrated that mitochondrial fission arguments the stemness of bone marrow-derived mesenchymal stem cells (BMSCs). Because mitophagy is critical in removing damaged or surplus mitochondrial fragments and maintaining mitochondrial integrity, the present study was undertaken to test the hypothesis that mitophagy is involved in mitochondrial fission-enhanced stemness of BMSCs. Primary cultures of rat BMSCs were treated with tyrphostin A9 (TA9, a potent inducer of mitochondrial fission) to increase mitochondrial fission, which was accompanied by enhanced mitophagy as defined by increased co-staining of MitoTracker Green for mitochondria and LysoTracker Deep Red for lysosomes, as well as the increased co-localization of autophagy markers (LC3B, P62) and mitochondrial marker (Tom20). A mitochondrial uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was used to promote mitophagy, which was confirmed by an increased co-localization of mitochondrial and lysosome biomarkers. The argumentation of mitophagy was associated with enhanced stemness of BMSCs as defined by increased expression of stemness markers Oct4 and Sox2, and enhanced induction of BMSCs to adipocytes or osteocytes. Conversely, transfection of BMSCs with siRNA targeting mitophagy-essential genes Pink1/Prkn led to diminished stemness of the stem cells, as defined by depressed stemness markers. Importantly, concomitant promotion of mitochondrial fission and inhibition of mitophagy suppressed the stemness of BMSCs. These results thus demonstrate that mitophagy is critically involved in mitochondrial fission promotion of the stemness of BMSCs.     

Paracrine action of HO-1-modified mesenchymal stem cells mediates cardiac protection and functional improvement

The aim has been to determine whether the supernatants of mesenchymal stem cells (MSCs) transfected with adenovirus carrying human heme oxygenase-1 (hHO-1) gene protect cardiomyocytes from ischemic injury. We have found that hHO-1 infected MSCs (hHO-1-MSCs) increased expression of hHO-1 protein. Apoptosis of cultured hHO-1-MSCs exposed to hypoxia was suppressed. Several cytokines, including HGF, bFGF, TGF-beta, VEGF and IL-1beta, were produced by hHO-1-MSCs, some being significantly enhanced under hypoxia stimulation. Meanwhile, those cytokines reduced caspase-3 level and activity in cultured adult rat ventricular cardiomyocytes (ARVCs) exposed to hypoxia. Supernatants obtained from hHO-1-MSCs improved left ventricular function, limited myocardial infarct size, increased microvessel density, and inhibited apoptosis of cardiomyocytes in rat myocardial infarction. It can be concluded hHO-1-modified MSCs prevent myocardial cell injury via secretion of paracrine-acting mediators.     

Knockdown of Mtfp1 can minimize doxorubicin cardiotoxicity by inhibiting Dnm1l‐mediated mitochondrial fission

The long‐term usage of doxorubicin (DOX) is largely limited due to the development of severe cardiomyopathy. Many studies indicate that DOX‐induced cardiac injury is related to reactive oxygen species generation and ultimate activation of apoptosis. The role of novel mitochondrial fission protein 1 (Mtfp1) in DOX‐induced cardiotoxicity remains elusive. Here, we report the pro‐mitochondrial fission and pro‐apoptotic roles of Mtfp1 in DOX‐induced cardiotoxicity. DOX up‐regulates the Mtfp1 expression in HL‐1 cardiac myocytes. Knockdown of Mtfp1 prevents cardiac myocyte from undergoing mitochondrial fission, and subsequently reduces the DOX‐induced apoptosis by preventing dynamin 1‐like (Dnm1l) accumulation in mitochondria. In contrast, when Mtfp1 is overexpressed, a suboptimal dose of DOX can induce a significant percentage of cells to undergo mitochondrial fission and apoptosis. These data suggest that knocking down of Mtfp1 can minimize the cardiomyocytes loss in DOX‐induced cardiotoxicity. Thus, the regulation of Mtfp1 expression could be a novel therapeutic approach in chemotherapy‐induced cardiotoxicity.     

Mineralocorticoid receptor deficiency improves the therapeutic effects of mesenchymal stem cells for myocardial infarction via enhanced cell survival

The poor survival of stem cells seriously limits their therapeutic efficacy for myocardial infarction (MI). Mineralocorticoid receptor (MR) activation plays an important role in the pathogenesis of multiple cardiovascular diseases. Here, we examined whether MR silencing in bone marrow derived mesenchymal stem cells (MSCs) could improve MSCs’ survival and enhance their cardioprotective effects in MI. MSCs from male Sprague‐Dawley rats were transfected with adenoviral small interfering RNA to silence MR (siRNA‐MR). MR silencing decreased hypoxia‐induced MSCs’ apoptosis, as demonstrated by Annexin V/7‐AAD staining. The mechanisms contributing to the beneficial effects of MR depletion were associated with inhibiting intracellular reactive oxygen species production and increased Bcl‐2/Bax ratio. In vivo study, 1 × 10⁶ of MSCs with or without siRNA‐MR were injected into rat hearts immediately after MI. Depletion of MR could improve the MSCs’ survival significantly in infarcted myocardium, associated with more cardiac function improvement and smaller infarct size. Capillary density were also significantly higher in siRNA group with increased expression of vascular endothelial growth factor. Our study demonstrated that silencing MR promoted MSCs’ survival and repair efficacy in ischaemic hearts. MR might be a potential target for enhancing the efficacy of cell therapy in ischaemic heart disease.     

Priming mesenchymal stem cells boosts stem cell therapy to treat myocardial infarction

Cardiovascular diseases are the number one cause of death globally and are projected to remain the single leading cause of death. Treatment options abounds, although efficacy is limited. Recent studies attribute discrete and ephemeral benefits to adult stem cell therapies, indicating the urge to improve stem cell based–therapy. In this study, we show that priming mesenchymal stem cells (MSC) towards cardiomyogenic lineage enhances their beneficial effects in vivo as treatment option for acute phase myocardial infarction. MSC were primed using cardiomyogenic media for 4 days, after which peak expression of key cardiomyogenic genes are reached and protein expression of Cx‐43 and sarcomeric α‐actinin are observed. MSC and primed MSC (pMSC) were characterized in vitro and used to treat infarcted rats immediately after left anterior descending (LAD) occlusion. Echocardiography analysis indicated that MSC‐treated myocardium presented discrete improvement in function, but it also showed that pMSC treatment lead to superior beneficial results, compared with undifferentiated MSC. Seven days after cell injection, MSC and pMSC could still be detected in the myocardium. Connexin‐43 expression was quantified through immunoblotting, and was superior in pMSC, indicating that this could be a possible explanation for the superior performance of pMSC therapy.     

miR‐155‐5p inhibition rejuvenates aged mesenchymal stem cells and enhances cardioprotection following infarction

Aging impairs the functions of human mesenchymal stem cells (MSCs), thereby severely reducing their beneficial effects on myocardial infarction (MI). MicroRNAs (miRNAs) play crucial roles in regulating the senescence of MSCs; however, the underlying mechanisms remain unclear. Here, we investigated the significance of miR‐155‐5p in regulating MSC senescence and whether inhibition of miR‐155‐5p could rejuvenate aged MSCs (AMSCs) to enhance their therapeutic efficacy for MI. Young MSCs (YMSCs) and AMSCs were isolated from young and aged donors, respectively. The cellular senescence of MSCs was evaluated by senescence‐associated β‐galactosidase (SA‐β‐gal) staining. Compared with YMSCs, AMSCs exhibited increased cellular senescence as evidenced by increased SA‐β‐gal activity and decreased proliferative capacity and paracrine effects. The expression of miR‐155‐5p was much higher in both serum and MSCs from aged donors than young donors. Upregulation of miR‐155‐5p in YMSCs led to increased cellular senescence, whereas downregulation of miR‐155‐5p decreased AMSC senescence. Mechanistically, miR‐155‐5p inhibited mitochondrial fission and increased mitochondrial fusion in MSCs via the AMPK signaling pathway, thereby resulting in cellular senescence by repressing the expression of Cab39. These effects were partially reversed by treatment with AMPK activator or mitofusin2‐specific siRNA (Mfn2‐siRNA). By enhancing angiogenesis and promoting cell survival, transplantation of anti‐miR‐155‐5p‐AMSCs led to improved cardiac function in an aged mouse model of MI compared with transplantation of AMSCs. In summary, our study shows that miR‐155‐5p mediates MSC senescence by regulating the Cab39/AMPK signaling pathway and miR‐155‐5p is a novel target to rejuvenate AMSCs and enhance their cardioprotective effects.     

Red wine antioxidant resveratrol‐modified cardiac stem cells regenerate infarcted myocardium

To study the efficiency of maintaining the reduced tissue environment via pre-treatment with natural antioxidant resveratrol in stem cell therapy, we pre-treated male Sprague-Dawley rats with resveratrol (2.5 mg/kg/day gavaged for 2 weeks). After occlusion of the left anterior descending coronary artery (LAD), adult cardiac stem cells stably expressing EGFP were injected into the border zone of the myocardium. One week after the LAD occlusion, the cardiac reduced environment was confirmed in resveratrol-treated rat hearts by the enhanced expression of nuclear factor-E2-related factor-2 (Nrf2) and redox effector factor-1 (Ref-1). In concert, cardiac functional parameters (left ventricular ejection fraction and fractional shortening) were significantly improved. The improvement of cardiac function was accompanied by the enhanced stem cell survival and proliferation as demonstrated by the expression of cell proliferation marker Ki67 and differentiation of stem cells towards the regeneration of the myocardium as demonstrated by the enhanced expression of EGFP 28 days after LAD occlusion in the resveratrol-treated hearts. Our results demonstrate that resveratrol maintained a reduced tissue environment by overexpressing Nrf2 and Ref-1 in rats resulting in an enhancement of the cardiac regeneration of the adult cardiac stem cells as demonstrated by increased cell survival and differentiation leading to cardiac function.     

Transplantation of mesenchymal stem cells overexpressing IL10 attenuates cardiac impairments in rats with myocardial infarction

Mesenchymal stem cell (MSC) has been well known to exert therapeutic potential for patients with myocardial infarction (MI). In addition, interleukin‐10 (IL10) could attenuate MI through suppressing inflammation. Thus, the combination of MSC implantation with IL10 delivery may extend health benefits to ameliorate cardiac injury after MI. Here we established overexpression of IL10 in bone marrow‐derived MSC through adenoviral transduction. Cell viability, apoptosis, and IL10 secretion under ischemic challenge in vitro were examined. In addition, MSC was transplanted into the injured hearts in a rat model of MI. Four weeks after the MI induction, MI, cardiac functions, apoptotic cells, and inflammation cytokines were assessed. In response to in vitro oxygen‐glucose deprivation (OGD), IL10 overexpression in MSC (Ad.IL10‐MSC) enhanced cell viability, decreased apoptosis, and increased IL10 secretion. Consistently, the implantation of Ad.IL10‐MSCs into MI animals resulted in more reductions in myocardial infarct size, cardiac impairment, and cell apoptosis, compared to the individual treatments of either MSC or IL10 administration. Moreover, the attenuation of both systemic and local inflammations was most prominent for Ad.IL10‐MSC treatment. IL10 overexpression and MSC may exert a synergistic anti‐inflammatory effect to alleviate cardiac injury after MI.     

Bone marrow mesenchymal stem cells for post-myocardial infarction cardiac repair: microRNAs as novel regulators

Transplantation of bone marrow-derived mesenchymal stem cells (MSCs) is safe and may improve cardiac function and structural remodelling in patients following myocardial infarction (MI). Cardiovascular cell differentiation and paracrine effects to promote endogenous cardiac regeneration, neovascularization, anti-inflammation, anti-apoptosis, anti-remodelling and cardiac contractility, may contribute to MSC-based cardiac repair following MI. However, current evidence indicates that the efficacy of MSC transplantation was unsatisfactory, due to the poor viability and massive death of the engrafted MSCs in the infarcted myocardium. MicroRNAs are short endogenous, conserved, non-coding RNAs and important regulators involved in numerous facets of cardiac pathophysiologic processes. There is an obvious involvement of microRNAs in almost every facet of putative repair mechanisms of MSC-based therapy in MI, such as stem cell differentiation, neovascularization, apoptosis, cardiac remodelling, cardiac contractility and arrhythmias, and others. It is proposed that therapeutic modulation of individual cardiovascular microRNA of MSCs, either mimicking or antagonizing microRNA actions, will hopefully enhance MSC therapeutic efficacy. In addition, MSCs may be manipulated to enhance functional microRNA expression or to inhibit aberrant microRNA levels in a paracrine manner. We hypothesize that microRNAs may be used as novel regulators in MSC-based therapy in MI and MSC transplantation by microRNA regulation may represent promising therapeutic strategy for MI patients in the future.     

Mesenchymal stem cell‐loaded cardiac patch promotes epicardial activation and repair of the infarcted myocardium

Cardiac patch is considered a promising strategy for enhancing stem cell therapy of myocardial infarction (MI). However, the underlying mechanisms for cardiac patch repairing infarcted myocardium remain unclear. In this study, we investigated the mechanisms of PCL/gelatin patch loaded with MSCs on activating endogenous cardiac repair. PCL/gelatin patch was fabricated by electrospun. The patch enhanced the survival of the seeded MSCs and their HIF‐1α, Tβ4, VEGF and SDF‐1 expression and decreased CXCL14 expression in hypoxic and serum‐deprived conditions. In murine MI models, the survival and distribution of the engrafted MSCs and the activation of the epicardium were examined, respectively. At 4 weeks after transplantation of the cell patch, the cardiac functions were significantly improved. The engrafted MSCs migrated across the epicardium and into the myocardium. Tendency of HIF‐1α, Tβ4, VEGF, SDF‐1 and CXCL14 expression in the infarcted myocardium was similar with expression in vitro. The epicardium was activated and epicardial‐derived cells (EPDCs) migrated into deep tissue. The EPDCs differentiated into endothelial cells and smooth muscle cells, and some of EPDCs showed to have differentiated into cardiomyocytes. Density of blood and lymphatic capillaries increased significantly. More c‐kit⁺ cells were recruited into the infarcted myocardium after transplantation of the cell patch. The results suggest that epicardial transplantation of the cell patch promotes repair of the infarcted myocardium and improves cardiac functions by enhancing the survival of the transplanted cells, accelerating locality paracrine, and then activating the epicardium and recruiting endogenous c‐kit⁺ cells. Epicardial transplantation of the cell patch may be applied as a novel effective MI therapy.     

Cell membrane damage is involved in the impaired survival of bone marrow stem cells by oxidized low‐density lipoprotein

Cell therapy with bone marrow stem cells (BMSCs) remains a viable option for tissue repair and regeneration. A major challenge for cell therapy is the limited cell survival after implantation. This study was to investigate the effect of oxidized low‐density lipoprotein (ox‐LDL, naturally present in human blood) on BMSC injury and the effect of MG53, a tissue repair protein, for the improvement of stem cell survival. Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox‐LDL, which caused significant cell death as reflected by the increased LDH release to the media. Exposure of MAPCs to ox‐LDL led to entry of fluorescent dye FM1‐43 measured under confocal microscope, suggesting damage to the plasma membrane. Ox‐LDL also generated reactive oxygen species (ROS) as measured with electron paramagnetic resonance spectroscopy. While antioxidant N‐acetylcysteine completely blocked ROS production from ox‐LDL, it failed to prevent ox‐LDL‐induced cell death. When MAPCs were treated with the recombinant human MG53 protein (rhMG53) ox‐LDL induced LDH release and FM1‐43 dye entry were significantly reduced. In the presence of rhMG53, the MAPCs showed enhanced cell survival and proliferation. Our data suggest that membrane damage induced by ox‐LDL contributed to the impaired survival of MAPCs. rhMG53 treatment protected MAPCs against membrane damage and enhanced their survival which might represent a novel means for improving efficacy for stem cell‐based therapy for treatment of diseases, especially in setting of hyperlipidemia.     

Exosomes derived from pro‐inflammatory bone marrow‐derived mesenchymal stem cells reduce inflammation and myocardial injury via mediating macrophage polarization

Exosomes are served as substitutes for stem cell therapy, playing important roles in mediating heart repair during myocardial infarction injury. Evidence have indicated that lipopolysaccharide (LPS) pre‐conditioning bone marrow‐derived mesenchymal stem cells (BMSCs) and their secreted exosomes promote macrophage polarization and tissue repair in several inflammation diseases; however, it has not been fully elucidated in myocardial infarction (MI). This study aimed to investigate whether LPS‐primed BMSC‐derived exosomes could mediate inflammation and myocardial injury via macrophage polarization after MI. Here, we found that exosomes derived from BMSCs, in both Exo and L‐Exo groups, increased M2 macrophage polarization and decreased M1 macrophage polarization under LPS stimulation, which strongly depressed LPS‐dependent NF‐κB signalling pathway and partly activated the AKT1/AKT2 signalling pathway. Compared with Exo, L‐Exo had superior therapeutic effects on polarizing M2 macrophage in vitro and attenuated the post‐infarction inflammation and cardiomyocyte apoptosis by mediating macrophage polarization in mice MI model. Consequently, we have confidence in the perspective that low concentration of LPS pre‐conditioning BMSC‐derived exosomes may develop into a promising cell‐free treatment strategy for clinical treatment of MI.     

Preconditioning strategies for improving the survival rate and paracrine ability of mesenchymal stem cells in acute kidney injury

Acute kidney injury (AKI) is a common, severe emergency case in clinics, with high incidence, significant mortality and increased costs. Despite development in the understanding of its pathophysiology, the therapeutic choices are still confined to dialysis and renal transplantation. Considering their antiapoptotic, immunomodulatory, antioxidative and pro‐angiogenic effects, mesenchymal stem cells (MSCs) may be a promising candidate for AKI management. Based on these findings, some clinical trials have been performed, but the results are contradictory (NCT00733876, NCT01602328). The low engraftment, poor survival rate, impaired paracrine ability and delayed administration of MSCs are the four main reasons for the limited clinical efficacy. Investigators have developed a series of preconditioning strategies to improve MSC survival rates and paracrine ability. In this review, by summarizing these encouraging studies, we intend to provide a comprehensive understanding of various preconditioning strategies on AKI therapy and improve the prognosis of AKI patients by regenerative medicine.     

Local activation of cardiac stem cells for post‐myocardial infarction cardiac repair

The prognosis of patients with myocardial infarction (MI) and resultant chronic heart failure remains extremely poor despite continuous advancements in optimal medical therapy and interventional procedures. Animal experiments and clinical trials using adult stem cell therapy following MI have shown a global improvement of myocardial function. The emergence of stem cell transplantation approaches has recently represented promising alternatives to stimulate myocardial regeneration. Regarding their tissue‐specific properties, cardiac stem cells (CSCs) residing within the heart have advantages over other stem cell types to be the best cell source for cell transplantation. However, time‐consuming and costly procedures to expanse cells prior to cell transplantation and the reliability of cell culture and expansion may both be major obstacles in the clinical application of CSC‐based transplantation therapy after MI. The recognition that the adult heart possesses endogenous CSCs that can regenerate cardiomyocytes and vascular cells has raised the unique therapeutic strategy to reconstitute dead myocardium via activating these cells post‐MI. Several strategies, such as growth factors, mircoRNAs and drugs, may be implemented to potentiate endogenous CSCs to repair infarcted heart without cell transplantation. Most molecular and cellular mechanism involved in the process of CSC‐based endogenous regeneration after MI is far from understanding. This article reviews current knowledge opening up the possibilities of cardiac repair through CSCs activation in situ in the setting of MI.     

Repair of senescent myocardium by mesenchymal stem cells is dependent on the age of donor mice

Myocardial infarction is one of the leading causes of mortality in aged people. Whether age of donors of mesenchymal stem cells (MSCs) affects its ability to repair the senescent heart tissue is unknown. In the present study, MSCs from young (2 months) and aged (18 months) green fluorescent protein expressing C57BL/6 mice were characterized with p16^INK4a and β‐gal associated senescence. Myocardial infarction was produced in 18‐month‐old wild‐type C57BL/6 mice transplanted with MSCs from young and aged animals in the border of the infarct region. Expression of p16^INK4a in MSCs from aged animals was significantly higher (21.5%± 1.2, P < 0.05) as compared to those from young animals (9.2%± 2.8). A decline in the tube‐forming ability on Matrigel was also observed in aged MSCs as well as down‐regulation of insulin‐like growth factor‐1, fibroblast growth factor (FGF‐2), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) compared to young cells. Mice transplanted with young MSCs exhibited significant improvement in their left ventricle (LV) systolic and diastolic function as demonstrated by dp/dt_max, dp/dt_min, P_max. Reduction in the LV fibrotic area was concomitant with neovascularization as demonstrated by CD31 and smooth muscle actin (SMA) expression. Real‐time RT‐PCR analysis for VEGF, stromal cell derived factor (SDF‐1α) and GATA binding factor 4 (GATA‐4) genes further confirmed the effect of age on MSC differentiation towards cardiac lineages and enhanced angiogenesis. These studies lead to the conclusion that repair potential of MSCs is dependent on the age of donors and the repair of senescent infarcted myocardium requires young healthy MSCs.