Aims Naturally secreted nanovesicles, known as exosomes, play important roles in stem cell-mediated cardioprotection. We have previously demonstrated that atorvastatin (ATV) pretreatment improved the cardioprotective effects of mesenchymal stem cells (MSCs) in a rat model of acute myocardial infarction (AMI). The aim of this study was to investigate if exosomes derived from ATV-pretreated MSCs exhibit more potent cardioprotective function in a rat model of AMI and if so to explore the underlying mechanisms. Methods and results Exosomes were isolated from control MSCs (MSC-Exo) and ATV-pretreated MSCs (MSCATV-Exo) and were then delivered to endothelial cells and cardiomyocytes in vitro under hypoxia and serum deprivation (H/SD) condition or in vivo in an acutely infarcted Sprague-Dawley rat heart. Regulatory genes and pathways activated by ATV pretreatment were explored using genomics approaches and functional studies. In vitro, MSCATV-Exo accelerated migration, tube-like structure formation, and increased survival of endothelial cells but not cardiomyocytes, whereas the exosomes derived from MSCATV-Exo-treated endothelial cells prevented cardiomyocytes from H/SD-induced apoptosis. In a rat AMI model, MSCATV-Exo resulted in improved recovery in cardiac function, further reduction in infarct size and reduced cardiomyocyte apoptosis compared to MSC-Exo. In addition, MSCATV-Exo promoted angiogenesis and inhibited the elevation of IL-6 and TNF-α in the peri-infarct region. Mechanistically, we identified lncRNA H19 as a mediator of the role of MSCATV-Exo in regulating expression of miR-675 and activation of proangiogenic factor VEGF and intercellular adhesion molecule-1. Consistently, the cardioprotective effects of MSCATV-Exo was abrogated when lncRNA H19 was depleted in the ATV-pretreated MSCs and was mimicked by overexpression of lncRNA H19. Conclusion Exosomes obtained from ATV-pretreated MSCs have significantly enhanced therapeutic efficacy for treatment of AMI possibly through promoting endothelial cell function. LncRNA H19 mediates, at least partially, the cardioprotective roles of MSCATV-Exo in promoting angiogenesis.

Background Bone marrow mesenchymal stem cells (MSCs) are among the most common cell types to be used and studied for cardiac regeneration. Low survival rate and difficult retention of delivered MSCs in infarcted heart remain as major challenges in the field. Co-delivery of stem cell-derived exosomes (Exo) is expected to improve the recruitment and survival of transplanted MSCs. Methods Exo was isolated from MSCs and delivered to an acute myocardial infarction (AMI) rat heart through intramyocardial injection with or without intravenous infusion of atrovastatin-pretreated MSCs on day 1, day 3, or day 7 after infarction. Echocardiography was performed to evaluate cardiac function. Histological analysis and ELISA test were performed to assess angiogenesis, SDF-1, and inflammatory factor expression in the infarct border zone. The anti-apoptosis effect of Exo on MSCs was evaluated using flow cytometry and Hoechst 33342 staining assay. Results We found that intramyocardial delivery of Exo followed by MSC transplantation (in brief, Exo+MSC treatment) into MI hearts further improved cardiac function, reduced infarct size, and increased neovascularization when compared to controls treated with Exo or MSCs alone. Of note, comparing the three co-transplanting groups, intramyocardially injecting Exo 30 min after AMI combined with MSCs transplantation at day 3 after AMI achieved the highest improvement in heart function. The observed enhanced heart function is likely due to an improved microenvironment via Exo injection, which is exemplified as reduced inflammatory responses and better MSC recruitment and retention. Furthermore, we demonstrated that pre-transplantation injection of Exo enhanced survival of MSCs and reduced their apoptosis both in vitro and in vivo. Conclusions Combinatorial delivery of exosomes and stem cells in a sequential manner effectively reduces scar size and restores heart function after AMI. This approach may represent as an alternative promising strategy for stem cell-based heart repair and therapy.

Stem cell therapy is a promising approach to the treatment of ischemic heart disease via replenishing cell loss after myocardial infarction. Both preclinical studies and clinical trials have indicated that cardiac function improved consistently, but very modestly after cell-based therapy. This mainly attributed to low cell survival rate, engraftment and functional integration, which became the major challenges to regenerative medicine. In recent years, several new cell types have been developed to regenerate cardiomyocytes and novel delivery approaches helped to increase local cell retention. New strategies, such as cell pretreatment, gene-based therapy, tissue engineering, extracellular vesicles application and immunologic regulation, have surged and brought about improved cell survival and functional integration leading to better therapeutic effects after cell transplantation. In this review, we summarize these new strategies targeting at challenges of cardiac regenerative medicine and discuss recent evidences that may hint their effectiveness in the future clinical settings.

Background and Aims: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine that has been widely used to treat cardiovascular and cerebrovascular diseases. However, no studies have explored whether TXL can protect human cardiomyocytes (HCMs) from ischemia/reperfusion (I/R) injury. Reperfusion Injury Salvage Kinase (RISK) pathway activation was previously demonstrated to protect the hearts against I/R injury and it is generally activated via Akt or (and) Erk 1/2, and their common downstream protein, ribosomal protein S6 kinase (p70s6k). In addition, prior studies proved that TXL treatment of cells promoted secretion of VEGF, which could be stimulated by the increased phosphorylation of one p70s6k subtype, p70s6k1. Consequently, we hypothesized TXL could protect HCMs from I/R injury by activating p70s6k1 and investigated the underlying mechanism.Methods and Results: HCMs were exposed to hypoxia (18 h) and reoxygenation (2 h) (H/R), with or without TXL pretreatment. H/R reduced mitochondrial membrane potential, increased bax/bcl-2 ratios and cytochrome C levels and induced HCM apoptosis. TXL preconditioning reversed these H/R-induced changes in a dose-dependent manner and was most effective at 400 μg/mL. The anti-apoptotic effect of TXL was abrogated by rapamycin, an inhibitor of p70s6k. However, inhibitors of Erk1/2 (U0126) or Akt (LY294002) failed to inhibit the protective effect of TXL. TXL increased p70s6k1 expression and, thus, enhanced its phosphorylation. Furthermore, transfection of cardiomyocytes with siRNA to p70s6k1 abolished the protective effects of TXL. Among the micro-RNAs (miR-145-5p, miR-128-3p and miR-497-5p) previously reported to target p70s6k1, TXL downregulated miR-128-3p in HCMs during H/R, but had no effects on miR-145-5p and miR-497-5p. An in vivo study confirmed the role of the p70s6k1 pathway in the infarct-sparing effect of TXL, demonstrating that TXL decreased miR-128-3p levels in the rat myocardium during I/R. Transfection of HCMs with a hsa-miR-128-3p mimic eliminated the protective effects of TXL.Conclusions: The miR-128-3p/p70s6k1 signaling pathway is involved in protection by TXL against HCM apoptosis during H/R. Overexpression of p70s6k1 is, therefore, a potential new strategy for alleviating myocardial reperfusion injury.

Rationale: The crosstalk between cardiac microvascular endothelial cells (CMECs) and cardiomyocytes (CMs) has emerged as a key component in the development of, and protection against, cardiac diseases. For example, activation of endothelial nitric oxide synthase (eNOS) in CMECs, by therapeutic strategies such as ischemic preconditioning, plays a critical role in the protection against myocardial ischemia/reperfusion (I/R) injury. However, much less is known about the signals produced by CMs that are able to regulate CMEC biology. Here we uncovered one such mechanism using Tongxinluo (TXL), a traditional Chinese medicine, that alleviates myocardial ischemia/reperfusion (I/R) injury by activating CMEC eNOS. The aim of our study is to identify the signals produced by CMs that can regulate CMEC biology during I/R. Methods: Ex vivo, in vivo, and in vitro settings of ischemia-reperfusion were used in our study, with the protective signaling pathways activated in CMECs identified using genetic inhibition (p70s6k1 siRNA, miR-145-5p mimics, etc.), chemical inhibitors (the eNOS inhibitor, L-NNA, and the small extracellular vesicles (sEVs) inhibitor, GW4869) and Western blot analyses. TritonX-100 at a dose of 0.125% was utilized to inactivate the eNOS activity in endothelium to investigate the role of CMEC-derived eNOS in TXL-induced cardioprotection. Results: We found that while CMEC-derived eNOS activity was required for the cardioprotection of TXL, activation of eNOS in CMECs by TXL did not occur directly. Instead, eNOS activation in CMECs required a crosstalk between CMs and CMECs through the uptake of CM-derived sEVs. We further demonstrate that TXL induced CM-sEVs contain increased levels of Long Intergenic Non-Protein Coding RNA, Regulator Of Reprogramming (Linc-ROR). Upon uptake into CMECs, linc-ROR downregulates its target miR-145-5p leading to activation of the eNOS pathway by facilitating the expression of p70s6k1 in these cells. The activation of CMEC-derived eNOS works to increase survival in both the CMECs and the CMs themselves. Conclusions: These data uncover a mechanism by which the crosstalk between CMs and CMECs leads to the increased survival of the heart after I/R injury and point to a new therapeutic target for the blunting of myocardial I/R injury.

Aims Interleukin (IL)-5 mediates the development of eosinophils (EOS) that are essential for tissue post-injury repair. It remains unknown whether IL-5 plays a role in heart repair after myocardial infarction (MI). This study aims to test whether IL-5-induced EOS population promotes the healing and repair process post-MI and to reveal the underlying mechanisms. Method and results MI was induced by permanent ligation of the left anterior descending coronary artery in wild-type C57BL/6 mice. Western blot and real-time polymerase chain reaction revealed elevated expression of IL-5 in the heart at 5 days post-MI. Immunohistostaining indicated that IL-5 was secreted mainly from macrophages and type 2 innate lymphoid cells in the setting of experimental MI. External supply of recombinant mouse IL-5 (20 min, 1 day, and 2 days after MI surgery) reduced the infarct size and increased ejection fraction and angiogenesis in the border zone. A significant expansion of EOS was detected in both the peripheral blood and infarcted myocardium after IL-5 administration. Pharmacological depletion of EOS by TRFK5 pretreatment muted the beneficial effects of IL-5 in MI mice. Mechanistic studies demonstrated that IL-5 increased the accumulation of CD206+ macrophages in infarcted myocardium at 7 days post-MI. In vitro co-culture experiments showed that EOS shifted bone marrow-derived macrophage polarization towards the CD206+ phenotypes. This activity of EOS was abolished by IL-4 neutralizing antibody, but not IL-10 or IL-13 neutralization. Western blot analyses demonstrated that EOS promoted the macrophage downstream signal transducer and activator of transcription 6 (STAT6) phosphorylation. Conclusion IL-5 facilitates the recovery of cardiac dysfunction post-MI by promoting EOS accumulation and subsequent CD206+ macrophage polarization via the IL-4/STAT6 axis. Translational perspective Accumulating evidence suggests that modulation of innate and adaptive immune responses is a promising therapeutic strategy for myocardial infarction. In this study, we demonstrate that IL-5 exerts cardioprotective effects on infarcted myocardium by promoting eosinophil accumulation and subsequent CD206+ macrophage polarization via the IL-4/STAT6 axis. Hence, regulation of cardiac IL-5 level or eosinophil count may become a therapeutic approach for post-myocardial infarction cardiac repair in humans.

Background: The protection of endothelial cells (ECs) against reperfusion injury has received little attention. In this study, we used Tandem Mass Tag (TMT) labeling proteomics to investigate the modulated proteins in an in vitro model of cardiac microvascular endothelial cells (CMECs) subjected to ischemia/reperfusion (I/R) injury and their alteration by traditional Chinese medicine Tongxinluo (TXL). Methods: Human CMECs were subjected to 2 h of hypoxia followed by 2 h of reoxygenation with different concentrations of TXL Protein expression profiles of CMECs were determined using tandem mass spectrometry. We evaluated several proteins with altered expression in I/R injury and summarized some reported proteins related to I/R injury. Results: TXL dose-dependently decreased CMEC apoptosis, and the optimal concentration was 800 µg/mL. I/R significantly altered proteins in CMECs, and 30 different proteins were detected between a normal group and a hypoxia and serum deprivation group. In I/R injury, TXL treatment up-regulated 6 types of proteins including acyl-coenzyme A synthetase ACSM2B mitochondrial (ACSM2B), cyclin-dependent kinase inhibitor 1B (CDKN1B), heme oxygenase 1 (HMOX1), transcription factor SOX-17 (SOX17), sequestosome-1 isoform 1 (SQSTM1), and TBC1 domain family member 10B (TBC1D10B). Also, TXL down-regulated 5 proteins including angiopoietin-2 isoform c precursor (ANGPT2), cytochrome c oxidase assembly factor 5 (COA5), connective tissue growth factor precursor (CTGF), cathepsin L1 isoform 2 (CTSL), and eukaryotic elongation factor 2 kinase (LOC101930123). These types of proteins mainly had vital functions, including cell proliferation, stress response, and regulation of metabolic process. Conclusions: The study presented differential proteins upon I/R injury through a proteomic analysis. TXL modulated the expression of proteins in CMECs and has a protective role in response to I/R.

Background/Aims: Poor viability of transplanted mesenchymal stem cells (MSCs) within the ischemic heart limits their therapeutic potential for cardiac repair. Globular adiponectin (gAPN) exerts anti-apoptotic effects on several types of stem cells. Herein, we investigated the effect of gAPN on the MSCs against apoptosis induced by hypoxia and serum deprivation (H/SD). Methods: MSCs exposed to H/SD conditions were treated with different concentrations of gAPN. To identify the main type of receptor, MSCs were transfected with siRNA targeting adiponectin receptor 1 or 2 (AdipoR1 or AdipoR2). To elucidate the downstream pathway, MSCs were pre-incubated with AMPK inhibitor Compound C. Apoptosis, caspase-3 activity and mitochondrial membrane potential were evaluated. Results: H/SD-induced MSCs apoptosis and caspase-3 activation were attenuated by gAPN in a concentration-dependent manner. gAPN increased Bcl-2 and decreased Bax expressions. The loss of mitochondrial membrane potential induced by H/SD was also abolished by gAPN. The protective effect of gAPN was significantly attenuated after the knockdown of AdipoR1 rather than AdipoR2. Moreover, Compound C partly suppressed the anti-apoptotic effect of gAPN. Conclusions: gAPN inhibits H/SD-induced apoptosis in MSCs via AdipoR1-mediated pathway, possibly linked to the activation of AMPK. gAPN may be a novel survival factor for MSCs in the ischemic engraftment environment.