Therapeutic Efficacy For Myocardial Infarction Research Articles

Downregulation of ALKBH5 rejuvenates aged human mesenchymal stem cells and enhances their therapeutic efficacy in myocardial infarction.

Despite promising results in myocardial infarction (MI), mesenchymal stem cell (MSC)-based therapy is limited by cell senescence. N6-methyladenosine (m6A) messenger RNA methylation has been reported to be closely associated with cell senescence. Nonetheless, its role in the regulation of MSC senescence remains unclear. We examined the role of ALKB homolog 5 (ALKBH5) in regulating MSC senescence and determined whether ALKBH5 downregulation could rejuvenate aged MSCs (AMSCs) to improve their therapeutic efficacy for MI. RNA methylation was determined by m6A dot blotting assay. MSC senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining. A mouse model of acute MI was established by ligation of the left anterior decedent coronary artery (LAD). Compared with young MSCs (YMSCs), m6A level was significantly reduced but ALKBH5 was greatly increased in AMSCs. Overexpression of ALKBH5 reduced m6A modification and accelerated YMSC senescence. Conversely, ALKBH5 knockdown increased m6A modifications and alleviated AMSC senescence. Mechanistically, ALKBH5 regulated the m6A modification and stability of CDKN1C mRNA, which further upregulated CDKN1C expression, leading to MSC senescence. CDKN1C overexpression ameliorated the inhibition of cellular senescence of ALKBH5 siRNA-treated AMSCs. More importantly, compared with AMSCs, shALKBH5-AMSCs transplantation provided a superior cardioprotective effect against MI in mice by improving MSC survival and angiogenesis. We determined that ALKBH5 accelerated MSC senescence through m6A modification-dependent stabilization of the CDKN1C transcript, providing a potential target for MSC rejuvenation. ALKBH5 knockdown rejuvenated AMSCs and enhanced cardiac function when transplanted into the mouse heart following infarction.

Buyang Huanwu Decoction suppresses cardiac inflammation and fibrosis in mice after myocardial infarction through inhibition of the TLR4 signalling pathway

Ethnopharmacological relevanceIt has been reported that cardiac inflammation and fibrosis participate in the development of heart failure (HF) following myocardial infarction (MI). Anti-inflammatory and anti-fibrotic treatments exhibit therapeutic efficacy in MI. Buyang Huanwu Decoction (BYHWD) has cardioprotective properties. However, whether BYHWD regulates cardiac inflammation and fibrosis in HF after MI, and the underlying mechanisms, are still unknown. Aim of the studyThis study aimed to explore the effects and potential mechanisms of BYHWD on cardiac inflammation and fibrosis after MI. Materials and methodsAn MI model was constructed through ligation of the left anterior descending coronary artery (LAD) in mice. The cardioprotective effects of BYHWD were determined by echocardiography, Masson trichrome staining, wheat germ agglutinin (WGA) staining and haematoxylin and eosin (HE) staining. The effects of BYHWD on inflammation and fibrosis, and on the TLR4 signalling pathway, were explored through immunohistochemistry (IHC), Western blot (WB), enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) in vivo. Next, the effects of BYHWD on primary cardiac fibroblasts (CFs) inflammation and collagen synthesis, and on the TLR4 signalling pathway, were detected using WB, immunofluorescence (IF) and qRT-PCR in vitro. In addition, the suppression and overexpression of TLR4 in CFs were further explored. ResultsBYHWD dose-dependently reduced cardiac inflammation, fibrosis and ventricular dysfunction. The expression levels of collagen Ⅰ/Ⅲ, IL-1β and IL-18, as well as critical proteins in the TLR4 signalling pathway and the NLRP3 inflammasome, were suppressed by BYHWD in the in vivo experiment. BYHWD inhibited CFs inflammation and collagen synthesis, as well as critical proteins in the TLR4 signalling pathway and the NLRP3 inflammasome, in the in vitro experiment. TLR4 suppression mitigated these inhibitory effects of BYHWD while overexpression of TLR4 markedly reversed these inhibitory effects of BYHWD. ConclusionBYHWD exerts anti-inflammatory and anti-fibrotic effects in mice after MI, and suppresses CFs inflammation and collagen synthesis through suppression of the TLR4 signalling pathway.

Integrin α4 Positive Subpopulation in Adipose Derived Stem Cells Effectively Reduces Infarct Size through Enhanced Engraftment into Myocardial Infarction.

The efficacy of adipose-derived stem cells (ASCs) on myocardial infarction is limited due to poor survival and engraftment. Integrin-mediated cell adhesion is a prerequisite for its survival and homing. ASCs expressed insufficient integrin α4, limiting their homing capacity. This study aims to characterize integrin α4＋ ASC subpopulation and investigate their therapeutic efficacy in myocardial infarction. We used fluorescence-activated cell sorting to harvest integrin α4＋ ASCs subpopulation, which were characterized in vitro and transplanted into myocardial infarction model. Positron emission tomography imaging were performed to measure infarction size. Cardiac cine magnetic resonance imaging was used to evaluate heart contractile function. Compared with the unfractionated ASCs, integrin α4＋ ASCs subpopulation secreted a higher level of angiogenic growth factors, migrated more rapidly, and exhibited a stronger anti-apoptotic capacity. Vascular cell adhesion molecule-1 was obviously up-regulated at 3 days after myocardial infarction, which interacted with integrin α4 receptor on the surface of ASCs to enhance the survival and adhesion. Thus, we implanted unfractionated ASCs or integrin α4＋ ASCs subpopulation into the 3-day infarcted myocardium. Integrin α4＋ ASCs subpopulation exhibited more robust engraftment into the infarcted myocardium. Integrin α4＋ ASCs subpopulation more effectively decreased infarct size and strengthen cardiac function recovery than did the unfractionated ASCs. Integrin α4＋ ASCs subpopulation is superior to unfractionated ASCs in ameliorating ischemic myocardial damage in animal model. Mechanistically, their more robust engraftment into the infarct area, higher migratory capacity and their increased release of paracrine factors contribute to enhanced tissue repair.

Supramolecular Assemblies of Glycopeptides Enhance Gap Junction Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes via Inducing Spheroids Formation to Optimize Cardiac Repair.

Stem cell-based therapies have demonstrated significant potential for use in heart regeneration. An effective paradigm for heart repair in rodent and large animal models is the transplantation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Despite this, the functional and phenotypical immaturity of two-dimensional (2D)-cultured hiPSC-CMs, particularly their low electrical integration, poses a caveat for clinical translation. In this study, a supramolecular assembly of a glycopeptide containing a cell adhesion motif-RGD and saccharide-glucose (Bio-Gluc-RGD) was designed to enable the three-dimensional (3D) spheroid formation of hiPSC-CMs, promoting cell-cell and cell-matrix interactions that occur during spontaneous morphogenesis. HiPSC-CMs in spheroids were prone to be phenotypically mature and developed robust gap junctions via activation of the integrin/ILK/p-AKT/Gata4 pathway. Monodispersed hiPSC-CMs encapsulated in the Bio-Gluc-RGD hydrogel were more likely to form aggregates and, therefore, survive in the infarcted myocardium of mice, accompanied by more robust gap junction formation in the transplanted cells, and hiPSC-CMs delivered with the hydrogels also displayed angiogenic effect and anti-apoptosis capacity in the peri-infarct area, enhancing their overall therapeutic efficacy in myocardial infarction. Collectively, our findings illustrate a novel concept for modulating hiPSC-CM maturation by spheroid induction, which has the potential for post-MI heart regeneration. This article is protected by copyright. All rights reserved.

An Intrapericardial Injectable Hydrogel Patch for Mechanical-Electrical Coupling with Infarcted Myocardium.

Although hydrogel-based patches have shown promising therapeutic efficacy in myocardial infarction (MI), synergistic mechanical, electrical, and biological cues are required to restore cardiac electrical conduction and diastolic-systolic function. Here, an injectable mechanical-electrical coupling hydrogel patch (MEHP) is developed via dynamic covalent/noncovalent cross-linking, appropriate for cell encapsulation and minimally invasive implantation into the pericardial cavity. Pericardial fixation and hydrogel self-adhesiveness properties enable the MEHP to highly compliant interfacial coupling with cyclically deformed myocardium. The self-adaptive MEHP inhibits ventricular dilation while assisting cardiac pulsatile function. The MEHP with the electrical conductivity and sensitivity to match myocardial tissue improves electrical connectivity between healthy and infarcted areas and increases electrical conduction velocity and synchronization. Overall, the MEHP combined with cell therapy effectively prevents ventricular fibrosis and remodeling, promotes neovascularization, and restores electrical propagation and synchronized pulsation, facilitating the clinical translation of cardiac tissue engineering.

Apelin-13 Pretreatment Promotes the Cardioprotective Effect of Mesenchymal Stem Cells against Myocardial Infarction by Improving Their Survival.

Although mesenchymal stem cell- (MSC-) based therapy has shown promising results for myocardial infarction (MI), low cell survival heavily limits its beneficial effects. Apelin plays an essential regulatory role in cell proliferation. This study was aimed at determining whether Apelin-13 pretreatment could improve the survival of MSCs in the ischemic heart and enhance their cardioprotective efficacy against MI. MSCs were pretreated with or without Apelin-13 for 24 hours and then exposed to serum deprivation and hypoxia (SD/H) for 48 hours. The mitochondrial morphology of MSCs was assessed by MitoTracker staining. The apoptosis of MSCs was determined by TUNEL staining. The level of mitochondrial reactive oxygen species (ROS) of MSCs was detected by Mito-Sox staining. MSCs and Apelin-13-pretreated MSCs were transplanted into the peri-infarct region in a mouse MI model. Apelin-13 pretreatment protected MSCs against SD/H-induced mitochondrial fragmentation and apoptosis. Apelin-13 pretreatment reduced ROS generation induced by SD/H in MSCs. Furthermore, Apelin-13 pretreatment enhanced the angiogenesis of MSCs under SD/H conditions. Mechanistically, Apelin-13 pretreatment inhibited SD/H-induced MSC apoptosis by downregulating mitochondrial fission via activation of the ERK pathway, and these effects were partially abrogated by ERK inhibitor U0126. Apelin-13 pretreatment promoted the survival of MSCs in the ischemic heart. Moreover, transplantation with Apelin-13-pretreated MSCs improved heart function and increased angiogenesis accompanied by decreased fibrosis compared with MSC transplantation at 28 days following MI. These findings reveal that pretreatment with Apelin-13 improves MSCs survival and enhances their therapeutic efficacy for MI. Our study provides a novel approach to improve MSC-based therapy for cardiovascular disease.

Abstract 12070: Extracellular Vesicles, Derived Early After Myocardial Infarction, Inhibits Staurosporin Induced Cardiac Cell Death

Introduction: Adverse ventricular remodeling may occur after myocardial infarction (MI) and is associated with an increased risk of heart failure and death. Extracellular vesicles (EVs) derived exogenously from pluripotent stem cells exert cardioprotective effects after injury and being tested for therapeutic efficacy for MI. However role of endogenous EVs from MI patients in this regard is not yet addressed. Hypothesis: We aim to characterize EVs derived from MI patients and explore their cardioprotective potential on human primary cardiomyocytes (HCM) upon chemically induced cell death. Methods: The EVs from plasma of MI patients with preserved or reduced left ventricular ejection fraction (LVEF) and healthy controls (HC) were purified and characterized by flow cytometry and transmission electron microscopy (TEM). HCM and human cardiac microvascular endothelial cells (hCMVECs) were used to study functional effects of EVs upon staurosporin (STS), etoposide and rapamycin induced cell death. Results: Higher concentrations of platelet-, leukocyte-, endothelial- and erythrocyte-derived EVs were found in MI patients compared to HC. TEM analysis demonstrated the presence of heterogenous vesicles of different sizes (50-1000-nm) across all tested samples. Human cardiac cells- hCMVECs and HCM when pre-incubated with both preserved and reduced LVEF MI EVs showed significant attenuation to STS-induced caspase 3 activity compared to HC EVs or mock treatment. Lactate dehydrogenase (LDH) release, as a marker for membrane integrity, upon etoposide stimulation was not affected in hCMVECs and partly increased in HCM, whereas pre-incubation with HC EVs or MI EVs did not affect eotoposide treatment outcome by these cells. While rapamycin induced cleavage of LC3-I to LC3-II in hCMVECs, the presence of EVs from either HC or MI patients did not influence autophagic response. Whereas, baseline LC3-II expression in HCM was observed to be too high to be perturbed by the presence of rapamycin. Conclusions: Diverse nature of MI EVs in terms of cells of origin can be further elaborated to consolidate their biomarker potential. Cardioprotective ability of endogenously presented heterogenous mixture of MI EVs opens new avenues to consider in preclinical EV/MI research.

Apelin Rejuvenates Aged Human Mesenchymal Stem Cells by Regulating Autophagy and Improves Cardiac Protection After Infarction.

The protective effects of mesenchymal stem cell (MSC)-based therapy for myocardial infarction (MI) are largely hampered as they age. Apelin is an endogenous ligand of its receptor APJ and plays an essential role in regulating multiple biological activities including MSC proliferation and survival. In this study, we investigated whether Apelin regulates MSC senescence and whether its overexpression could rejuvenate aged MSCs (AMSCs) to improve cardiac protection following infarction in mice. MSC senescence was evaluated by senescence-associated β-galactosidase assays. Apelin level was examined by western blotting. Autophagy was determined by transmission electron microscopy. The cardioprotective effect of AMSCs with Apelin overexpression (Apelin-AMSCs) was assessed in a mouse MI model. Apelin expression was dramatically reduced in AMSCs. Interestingly, knockdown of Apelin induced young MSCs (YMSC) senescence, whereas overexpression rescued AMSC senescence. Apelin overexpression also increased AMSC angiogenic capacity. Mechanistically, Apelin overexpression upregulated the autophagy level of AMSCs by activating AMP-activated protein kinase (AMPK) signaling, thereby rejuvenating AMSCs. Compared with AMSCs, transplantation of Apelin-AMSCs achieved better therapeutic efficacy for MI by enhancing cell survival and angiogenesis. In conclusion, our results reveal that Apelin activates AMPK to rejuvenate AMSCs by increasing autophagy and promotes cardioprotection following infarction in mice. This study identified a novel target to rejuvenate AMSCs and enhance their therapeutic efficacy.

Co-delivery of curcumin and miRNA-144-3p using heart-targeted extracellular vesicles enhances the therapeutic efficacy for myocardial infarction

Curcumin exerts therapeutic effects in heart disease, but has limited bioavailability. Extracellular vesicles (EVs) have gained attention as nanovehicles; however, the poor targeting ability of systemically administered EVs still remains a crucial issue. Herein, we generated heart-targeted EVs (CTP-EVs) by functionalizing EVs surface with cardiac targeting peptide (CTP) using genetic modification of EVs-secreting cells, and further loaded curcumin into CTP-EVs (CTP-EVs-Cur). Consequently, CTP-EVs were able to specifically deliver curcumin to the heart. In addition, curcumin-loaded CTP-EVs possess improved bioavailability, and are fully functional with a high cardioprotective efficiency. Moreover, we loaded miR-144-3p in CTP-EVs-Cur following validation of miR-144-3p as a major contributor in curcumin-mediated therapeutic effects. The simultaneous packing of curcumin and miR-144-3p in CTP-EVs not only retains the active heart-targeting ability but also achieves enhanced cardioprotective effects both in vitro and in vivo, indicating the possibility of combining and sustaining their therapeutic potential by simultaneously loading in CTP-EVs. Therefore, CTP-EVs could be a potential and effective strategy for the delivery of therapeutic molecules, thereby providing a promising nanomedicine for MI therapy.

FNDC5/irisin improves the therapeutic efficacy of bone marrow-derived mesenchymal stem cells for myocardial infarction

BackgroundThe beneficial functions of bone marrow mesenchymal stem cells (BM-MSCs) decline with decreased cell survival, limiting their therapeutic efficacy for myocardial infarction (MI). Irisin, a novel myokine which is cleaved from its precursor fibronectin type III domain-containing protein 5 (FNDC5), is believed to be involved in a cardioprotective effect, but little was known on injured BM-MSCs and MI repair yet. Here, we investigated whether FNDC5 or irisin could improve the low viability of transplanted BM-MSCs and increase their therapeutic efficacy after MI.MethodsBM-MSCs, isolated from dual-reporter firefly luciferase and enhanced green fluorescent protein positive (Fluc+–eGFP+) transgenic mice, were exposed to normoxic condition and hypoxic stress for 12 h, 24 h, and 48 h, respectively. In addition, BM-MSCs were treated with irisin (20 nmol/L) and overexpression of FNDC5 (FNDC5-OV) in serum deprivation (H/SD) injury. Furthermore, BM-MSCs were engrafted into infarcted hearts with or without FNDC5-OV.ResultsHypoxic stress contributed to increased apoptosis, decreased cell viability, and paracrine effects of BM-MSCs while irisin or FNDC5-OV alleviated these injuries. Longitudinal in vivo bioluminescence imaging and immunofluorescence results illustrated that BM-MSCs with overexpression of FNDC5 treatment (FNDC5-MSCs) improved the survival of transplanted BM-MSCs, which ameliorated the increased apoptosis and decreased angiogenesis of BM-MSCs in vivo. Interestingly, FNDC5-OV elevated the secretion of exosomes in BM-MSCs. Furthermore, FNDC5-MSC therapy significantly reduced fibrosis and alleviated injured heart function.ConclusionsThe present study indicated that irisin or FNDC5 improved BM-MSC engraftment and paracrine effects in infarcted hearts, which might provide a potential therapeutic target for MI.

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.

Macrophage migration inhibitory factor rejuvenates aged human mesenchymal stem cells and improves myocardial repair.

The beneficial functions of mesenchymal stem cells (MSCs) decline with age, limiting their therapeutic efficacy for myocardial infarction (MI). Macrophage migration inhibitory factor (MIF) promotes cell proliferation and survival. We investigated whether MIF overexpression could rejuvenate aged MSCs and increase their therapeutic efficacy in MI. Young and aged MSCs were isolated from the bone marrow of young and aged donors. Young MSCs, aged MSCs, and MIF-overexpressing aged MSCs were transplanted into the peri-infarct region in a rat MI model. Aged MSCs exhibited a lower proliferative capacity, lower MIF level, greater cell size, greater senescence-associated-β-galactosidase activity, and weaker paracrine effects than young MSCs. Knocking down MIF in young MSCs induced cellular senescence, whereas overexpressing MIF in aged MSCs reduced cellular senescence. MIF rejuvenated aged MSCs by activating autophagy, an effect largely reversed by the autophagy inhibitor 3-methyladenine. MIF-overexpressing aged MSCs induced angiogenesis and prevented cardiomyocyte apoptosis to a greater extent than aged MSCs, and had improved heart function and cell survival more effectively than aged MSCs four weeks after MI. Thus, MIF rejuvenated aged MSCs by activating autophagy and enhanced their therapeutic efficacy in MI, suggesting a novel MSC-based therapeutic strategy for cardiovascular diseases in the aged population.

The cytoptrotection of small intestinal submucosa-derived gel in HL-1 cells during hypoxia/reoxygenation-induced injury.

Small intestinal submucosa (SIS)-derived gel injected into infarcted myocardium has been shown to promote repair and regeneration after myocardial infarction (MI); however, the specific impact of SIS gel on cardiomyocytes remained unknown. The aim of this study was to characterise SIS gel function in hypoxia-reoxygenation (H/R)-induced cardiomyocyte damage and its potential mechanism. HL-1 cardiomyocytes seeded on SIS matrix-coated plates, SIS gel, and uncoated plates were subjected to H/R, cell viability, apoptosis, expression of caspase-3, Bcl-2, and Bax were investigated. SIS gel and SIS matrix as coating substrates markedly improved cell viability, preventing cell apoptosis compared with uncoated plates, with SIS gel yielding the best cytoprotective effects. SIS gel down-regulated expression of pro-inflammatory cytokines (TNF-α, CCL2, and IL-6) by inhibiting the JNK-mitogen-activated protein kinase (MAPK)/NF-κB pathways. Furthermore, SIS gel protected cardiomyocytes from apoptosis by activating protein kinase B (AKT) and extracellular-signal-regulated kinase (ERK) pathways, and markedly up-regulated antiapoptotic Bcl-2 expression but inhibited that of proapoptotic Bax and c-caspase 3. Together, these findings show that SIS gel could decrease H/R-induced cell apoptosis through a mechanism potentially related to its ability to regulate expression of inflammatory cytokines and antiapoptosis signalling pathways to prevent cell apoptosis. Our findings thereby shed light on the mechanism related to SIS gel therapeutic efficacy for MI.

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.

GW29-e1697 Activation of Farnesoid X Receptor enhances the survival of engrafted adipose-derived mesenchymal stem cells in mouse post-MI heart

The poor survival of engrafted stem cells in the ischemic environment limits their therapeutic efficacy for myocardial infarction (MI) treatment. Previously, we demonstrated that GW4064, an farnesoid X receptor (FXR) agonist, ameliorated post-MI cardiac dysfunction and remodeling. The current study

Blocking Nox2 improves mesenchymal stem cells therapy in myocardial infarction via antagonizing oxidant and promoting survival.

An increase in reactive oxygen species (ROS) plays a key role in aging and apoptosis in mesenchymal stem cells derived from bone marrow (BMSCs). NADPH oxidase Nox2 serves as an important source of intracellular ROS formation. This study is designed to determine if blocking Nox2 enhances anti-apoptotic and anti-aging ability of BMSCs to oxidant stress, and thus improves therapeutic efficacy in myocardial infarction (MI). Nox2 inhibitor (Acetovanillone) and Nox2 siRNA were used to block Nox2 in BMSCs, and the cell viability, apoptosis, senescence and survival of BMSCs were determined by CCK-8, Edu staining, TUNEL staining, β-galactosidase (β-gal) assay and DAPI labeling. Here we found that both Nox2 inhibitor and Nox2 knockdown remarkably countered the decrease of viability, and the increase of aging and apoptosis of BMSCs by H2 O2 . Whereas, Nox2 overexpression exacerbated the viability reduction, senescence and apoptosis of BMSCs. The ROS accumulation in BMSCs was also suppressed by Nox2 blocking. Further study uncovered that Nox2 inhibitor caused the downregulation of p-p53, p21, p-FoxO1 and Bax, and the upregulation of anti-apoptotic protein Bcl-2. In vivo, Nox2 knockdown in grafted BMSCs led to the improvement of EF and FS in infarcted myocardium than BMSCs without Nox2 knockdown. Consistently, more retention and survival of BMSCs were found after Nox2 knockdown. Taken together, Nox2 inhibition enhances anti-aging and anti-apoptotic ability of BMSCs, and thus promotes survival and retention of BMSCs, which provides a new strategy for improving BMSCs-based therapy.

Pre-Treatment with Melatonin Enhances Therapeutic Efficacy of Cardiac Progenitor Cells for Myocardial Infarction

Background/Aims: Melatonin possesses many biological activities such as antioxidant and anti-aging. Cardiac progenitor cells (CPCs) have emerged as a promising therapeutic strategy for myocardial infarction (MI). However, the low survival of transplanted CPCs in infarcted myocardium limits the successful use in treating MI. In the present study, we aimed to investigate if melatonin protects against oxidative stress-induced CPCs damage and enhances its therapeutic efficacy for MI. Methods: TUNEL assay and EdU assay were used to detect the effects of melatonin and miR-98 on H₂O₂-induced apoptosis and proliferation. MI model was used to evaluate the potential cardioprotective effects of melatonin and miR-98. Results: Melatonin attenuated H₂O₂-induced the proliferation reduction and apoptosis of c-kit⁺ CPCs in vitro, and CPCs which pretreated with melatonin significantly improved the functions of post-infarct hearts compared with CPCs alone in vivo. Melatonin was capable to inhibit the increase of miR-98 level by H₂O₂ in CPCs. The proliferation reduction and apoptosis of CPCs induced by H₂O₂ was aggravated by miR-98. In vivo, transplantation of CPCs with miR-98 silencing caused the more significant improvement of cardiac functions in MI than CPCs. MiR-98 targets at the signal transducer and activator of the transcription 3 (STAT3), and thus aggravated H₂O₂-induced the reduction of Bcl-2 protein. Conclusions: Pre-treatment with melatonin protects c-kit+ CPCs against oxidative stress-induced damage via downregulation of miR-98 and thereby increasing STAT3, representing a potentially new strategy to improve CPC-based therapy for MI.

Paracrine regulation in mesenchymal stem cells: the role of Rap1.

Since the first report more than a decade ago that bone marrow (BM) stem cells transplantation could promote heart regeneration following myocardial infarction (MI),1 different types of stem cells have been examined. Among those investigated to date, mesenchymal stem cells (MSCs) have attracted huge interest owing to some of their unique properties that include easy isolation, high expandability and low immunogenicity. Nonetheless despite the promising results of animal studies and clinical trials, MSC-based therapy for MI still faces some fundamental hurdles.2 One of the major challenges is the poor cell engraftment and low cell-survival rate following transplantation caused by the hostile environment of the injured heart and change to the immunologic milieu of transplanted MSCs. In addition, the mechanisms of stem cell-mediated tissue repair are not fully understood. Low cell retention and poor myocardial differentiation cannot explain the remarkable recovery of heart function following MSCs treatment. Therefore, the paracrine effects of MSCs are currently thought to have a predominant role. Although many cytokines released from MSCs contribute to heart function recovery,3 some are useless and even harmful.4 Thus, there is an urgent need to optimize MSCs before their transplantation to improve cell survival and augment their paracrine effects. Recently, Rap1, a telomeric repeat binding factor 2 interacting protein 1, has been identified as an important modulator of the nuclear factor kappa-B (NF-κB) pathway.5, 6 This pathway has been reported to regulate MSCs secretion profiling and survival.7, 8 On the basis of these findings, modulation of the NF-κB pathway to mediate MSCs therapy is feasible and important. Nonetheless total deletion of NF-κB is lethal to cells.9 Identification of an important regulator that can mediate activity of the NF-κB pathway and subsequently regulate MSC therapeutic efficacy for MI is vital. The roles of Rap1 in regulation of MSCs and the underlying mechanisms have not been classified, thus to understand how Rap1 regulates the paracrine effects and cell survival of MSC-mediated heart repair following infarction by regulation of the NF-κB pathway, therefore, is important.10

Rap1-mediated nuclear factor-kappaB (NF-κB) activity regulates the paracrine capacity of mesenchymal stem cells in heart repair following infarction.

Paracrine effect is the major mechanism that underlies mesenchymal stem cells (MSC)-based therapy. This study aimed to examine how Rap1, telomeric repeat-binding factor 2-interacting protein 1 (Terf2IP), which is a novel modulator involved in the nuclear factor-kappaB (NF-κB) pathway, regulates the paracrine effects of MSC-mediated heart repair following infarction. NF-κB activity of stromal cells was increased by Rap1 as measured by pNF-κB-luciferase reporter activity, and this was abolished by IkB-dominant-negative protein. Knockdown of Rap1 with shRap1 resulted in diminished translocation of p65-NF-κB from the cytoplasm to nuclei in response to tumor necrosis factor-α (TNF-α) stimulation. Compared with BM-MSCs, Rap1−/−-BM-MSCs displayed a significantly reduced ratio of phosphorylated NF-κB to NF-κB-p65 and of Bax to Bcl-2, and increased resistance to hypoxia-induced apoptosis by the terminal deoxynucleotidal transferase-mediated dUTP nick end labeling (TUNEL) assay. In contrast, re-expression of Rap1 in Rap1−/−-BM-MSCs resulted in loss of resistance to apoptosis in the presence of hypoxia. Moreover, absence of Rap1 in BM-MSCs led to downregulation of NF-κB activity accompanied by reduced pro-inflammatory paracrine cytokines TNF-α, IL (interleukin)-6 and monocyte chemotactic protein-1 in Rap1−/−-BM-MSCs compared with BM-MSCs. The apoptosis of neonatal cardiomyocytes (NCMCs) induced by hypoxia was significantly reduced when cocultured with Rap1−/−-BM-MSC hypoxic-conditioned medium (CdM). The increased cardioprotective effects of Rap1−/−-BM-MSCs were reduced when Rap1−/−-BM-MSCs were reconstituted with Rap1 re-expression. Furthermore, in vivo study showed that transplantation of Rap1−/−-BM-MSCs significantly improved heart function, decreased infarct size, prevented cardiomyocyte apoptosis and inhibited inflammation compared with controls and BM-MSCs (P<0.01). This study reveals that Rap1 has a critical role in the regulation of MSC paracrine actions. Compared with BM-MSCs, Rap1−/−-BM-MSCs decreased NF-κB sensitivity to stress-induced pro-inflammatory cytokine production and reduced apoptosis. Selective inhibition of Rap1 in BM-MSCs may be a novel strategy to enhance MSC-based therapeutic efficacy in myocardial infarction.

Pretreatment of mesenchymal stem cells with angiotensin II enhances paracrine effects, angiogenesis, gap junction formation and therapeutic efficacy for myocardial infarction

BackgroundPretreatment of mesenchymal stem cells (MSCs) with growth factors is reported to be an effective route for improving cell-based therapy of myocardial infarction (MI). Angiotensin II (Ang II) triggers vascular endothelial growth factor (VEGF) synthesis in MSCs. This study aimed to investigate the effects and mechanisms of Ang II pretreatment in enhancing the therapeutic efficacy of MSCs in MI. MethodsMSCs and endothelial cells (ECs) were isolated from Sprague-Dawley rats. After pretreated with or without 100nM of Ang II for 24h, the MSCs were directly injected into the border zones of the ischemic heart. Cardiac function, fibrosis, infarct size, VEGF expression, angiogenesis, and cell differentiation in the infarcted myocardium were determined after 30days. The cell apoptosis of MSCs post hypoxia was assessed using flow cytometry. The angiogenic activity of MSCs was analyzed using tube formation assay. The gap junction protein connexin-43 (Cx43) expression was detected. ResultsCompared with the MSC group, pretreatment of MSCs with Ang II resulted in better cardiac function, less cardiac fibrosis, smaller infarct size, and higher expression of VEGF and Von Willebrand Factor in ischemic myocardium, but no promotion of cardiomyocyte-like differentiation of MSCs. Ang II pretreatment enhanced the survival of MSCs and the H9c2 cells surrounding MSCs, and augmented the tube formation of ECs and MSCs. Ang II pretreatment up-regulated the Cx43 expression. ConclusionsThe pretreatment of MSCs with Ang II improved the outcome of MSC-based therapy for MI via the mechanisms of enhancing the paracrine production of VEGF, angiogenesis, and gap junction formation.