李清
中国医学科学院阜外医院 冠心病
The secreted factors from cardiac microvascular endothelial cells (CMECs) regulate the physiological activity of adjacent tissues and could be modulated by myocardial ischemia/reperfusion injury (MIRI). How this paracrine function of CMECs is regulated by MIRI and resveratrol remains to be elucidated. CMECs pretreated with/ without resveratrol were subjected to hypoxia/reoxygenation (H/R). Apoptosis was measured by flowcytometry. Protein antibody arrays were performed to find the alteration of cytokine secreted by CMECs. The Gene Ontology analysis was applied to interpret the function of modulated factors. We revealed resveratrol inhibited apoptosis of CMECs dose-dependently after H/R and reached its peak effect at the concentration of 100 μM. 29 factors were significantly changed by H/R, and resveratrol at 100 μM changed 98 types of factors compared with the H/R group. Among these factors, eight were increased by H/R and then were decreased by resveratrol. Eleven were attenuated by H/R and further decreased by resveratrol. Insulin-like growth factor binding protein-1 was upregulated by H/R and it was further increased by resveratrol. The altered factors were involved in cell proliferation, cell growth, cell motility, chemotaxis, angiogenesis and vasculogenesis. The study suggests that resveratrol inhibits the apoptosis and modulates the paracrine function of CMECs under ischemia/reperfusion condition.
Die Pharmazie 2022
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.
Cardiovascular research 2020
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.
Theranostics 2020
The SDF-1/CXCR4 signaling plays a critical role in the trafficking of mesenchymal stem cells (MSCs) to the sites of tissue damage. Our recent study demonstrated that atorvastatin (ATV) treatment improved the survival of MSCs, and ATV pretreated MSCs (ATV-MSCs) exhibited enhanced engraftment to injured myocardium. In this study, we investigated whether combined treatment with ATV and ATV-MSCs enhances cardiac repair and regeneration by activating SDF-1/CXCR4 signaling in a rat model of acute myocardial infarction. Rats were randomized into eight groups: the Sham, AMI control and 6 other groups that were subjected to AMI followed by treatment with MSCs, ATV, ATV+MSCs, ATV-MSCs, ATV+ATV-MSCs, ATV+ATV-MSCs+AMD3100 (SDF-1/CXCR4 antagonist), respectively. ATV+ATV-MSCs significantly potentiated targeted recruitment of MSCs to peri-infarct myocardium and resulted in further improvements in cardiac function and reduction in scar size compared with MSCs treatment alone at 4-week after AMI. More importantly, the cardioprotective effects conferred by ATV+ATV-MSCs were almost completely abolished by AMD3100 treatment. Together, our study demonstrated that ATV+ATV-MSCs significantly enhanced the targeted recruitment and survival of transplanted MSCs, and resulted in subsequent cardiac function improvement by augmenting SDF-1/CXCR4 signaling.
American journal of translational research 2019
Our previous studies showed that the combination of atorvastatin (ATV) and single injection of ATV-pretreated mesenchymal stem cells (MSCs) (ATV -MSCs) at 1 week post-acute myocardial infarction (AMI) promoted MSC recruitment and survival. This study aimed to investigate whether the combinatorial therapy of intensive ATV with multiple injections of ATV -MSCs has greater efficacy at different stages to better define the optimal strategy for MSC therapy in AMI. In order to determine the optimal time window for MSC treatment, we first assessed stromal cell-derived factor-1 (SDF-1) dynamic expression and inflammation. Next, we compared MSC recruitment and differentiation, cardiac function, infarct size, and angiogenesis among animal groups with single, dual, and triple injections of ATV -MSCs at early (Early1, Early2, Early3), mid-term (Mid1, Mid2, Mid3), and late (Late1, Late2, Late3) stages. Compared with AMI control, intensive ATV significantly augmented SDF-1 expression 1.5∼2.6-fold in peri-infarcted region with inhibited inflammation. ATV -MSCs implantation with ATV administration further enhanced MSC recruitment rate by 3.9%∼24.0%, improved left ventricular ejection fraction (LVEF) by 2.0%∼16.2%, and reduced infarct size in all groups 6 weeks post-AMI with most prominent improvement in mid groups and still effective in late groups. Mechanistically, ATV -MSCs remarkably suppressed inflammation and apoptosis while increasing angiogenesis. Furthermore, triple injections of ATV -MSCs were much more effective than single administration during early and mid-term stages of AMI with the best effects in Mid3 group. We conclude that the optimal strategy is multiple injections of ATV -MSCs combined with intensive ATV administration at mid-term stage of AMI. The translational potential of this strategy is clinically promising. Stem Cells Translational Medicine 2019;8:1068-1083.
Stem cells translational medicine 2019
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 research & therapy 2019
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.
Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 2017
What is the central question of this study? In a rat model of acute myocardial infarction (AMI), we investigated the effect of Tongxinluo (TXL) treatment. Does TXL activate autophagy and attenuate apoptosis of cardiomyocytes through the AMPK pathway to facilitate survival of cardiomyocytes and improve cardiac function? What is the main finding and its importance? Major findings are as follows: (i) TXL treatment preserved cardiac function and reduced ventricular remodelling, infarct size and inflammation in rat hearts after AMI; (ii) TXL treatment dramatically increased autophagy and inhibited apoptosis in myocardium; and (iii) the AMPK signalling pathway played a crucial role in mediating the beneficial effects of TXL. Tongxinluo (TXL) has been demonstrated to have a protective role during ischaemia-reperfusion after acute myocardial infarction, but the long-term effects and underlying mechanisms are still unknown. The aim of this study was to investigate whether TXL could have an effect on apoptosis or autophagy of cardiomyocytes through the AMP-activated protein kinase (AMPK) pathway. Male Sprague-Dawley rats (n = 75) were randomly divided to sham, control, TXL (4 mg kg-1 day-1 orally), compound C (i.p. injection of 10 mg kg-1 day-1 ) and TXL + compound C groups. The extent of fibrosis, infarct size and angiogenesis were determined by pathological and histological studies. Four weeks after acute myocardial infarction, TXL treatment significantly increased ejection fraction, promoted angiogenesis in the peri-infarct region and substantially decreased fibrosis and the size of the infarcted area (P < 0.05). Treatment with TXL also increased AMPK/mTOR phosphorylation, upregulated expression of the autophagic protein LC3 and downregulated expression of the apoptotic protein Bax in the infarcted myocardium (P < 0.05). Addition of the AMPK inhibitor, compound C, counteracted these beneficial effects significantly (P < 0.05). The cardioprotective benefits of TXL against myocardial infarction are related to the inhibition of apoptosis and promotion of autophagy in rat hearts after acute myocardial infarction. This effect may occur through the AMPK signalling pathway.
Experimental physiology 2017
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.
Frontiers in pharmacology 2017
Cardiac microvascular endothelial cells (CMECs) extensively secrete cytokines during myocardial ischemia/reperfusion injury (MIRI). Tongxinluo (TXL) has been demonstrated to preserve the function of the endothelium and myocardium against MIRI. This study was designed to identify alterations in the paracrine function of CMECs under hypoxia/reoxygenation (H/R) conditions and assess its modulation by TXL. CMECs were exposed to different concentrations of TXL for 30 min and then subjected to hypoxia and reoxygenation for 12 and 2 h, respectively. Apoptosis was measured to determine the optimal TXL concentration. Protein antibody arrays were used to assess changes in cytokines secreted into conditioned medium by CMECs. A Gene Ontology (GO) analysis was applied to interpret the functional implications of changes in cytokines. TXL inhibited CMEC apoptosis in a concentration-dependent manner after H/R, reaching peak efficacy at a concentration of 800 μg/ml. H/R significantly altered 33 cytokines, and TXL (800 μg/ml) changed the levels of 121 different cytokines compared with the H/R group. Among these cytokines, 10 that were increased by H/R were decreased by TXL, five that were decreased by H/R were increased by TXL, and eight that were attenuated by H/R were further decreased by TXL. Insulin-like growth factor binding protein-1 was up-regulated by H/R and was further increased by TXL. Significantly altered factors were found to be involved in cell proliferation, growth and differentiation, as well as chemotaxis and transport. TXL inhibited the apoptosis of CMECs and modulated their paracrine function in MIRI.
American journal of translational research 2016
