Nicorandil-Pretreated Mesenchymal Stem Cell-Derived Exosomes Facilitate Cardiac Repair After Myocardial Infarction via Promoting Macrophage M2 Polarization by Targeting miR-125a-5p/TRAF6/IRF5 Signaling Pathway.

Background: Exosomes derived from bone marrow mesenchymal stem cells (MSC-exo) have been considered as a promising cell-free therapeutic strategy for ischemic heart disease. Cardioprotective drug pretreatment could be an effective approach to improve the efficacy of MSC-exo. Nicorandil has long been used in clinical practice for cardioprotection. This study aimed to investigate whether the effects of exosomes derived from nicorandil pretreated MSC (MSCNIC-exo) could be enhanced in facilitating cardiac repair after acute myocardial infarction (AMI). Methods: MSCNIC-exo and MSC-exo were collected and injected into the border zone of infarcted hearts 30 minutes after coronary ligation in rats. Macrophage polarization was detected 3 days post-infarction, cardiac function as well as histological pathology were measured on the 28th day after AMI. Macrophages were separated from the bone marrow of rats for in vitro model. Exosomal miRNA sequencing was conducted to identify differentially expressed miRNAs between MSCNIC-exo and MSC-exo. MiRNA mimics and inhibitors were transfected to MSCs or macrophages to explore the specific mechanism. Results: Compared to MSC-exo, MSCNIC-exo showed superior therapeutic effects on cardiac functional and structural recovery after AMI and markedly elevated the ratio of CD68+ CD206+/ CD68+cells in infarcted hearts 3 days post-infarction. The notable ability of MSCNIC-exo to promote macrophage M2 polarization was also confirmed in vitro. Exosomal miRNA sequencing and both in vivo and in vitro experiments identified and verified that miR-125a-5p was an effector of the roles of MSCNIC-exo in vivo and in vitro. Furthermore, we found miR-125a-5p promoted macrophage M2 polarization by inhibiting TRAF6/IRF5 signaling pathway. Conclusion: This study suggested that MSCNIC-exo could markedly facilitate cardiac repair post-infarction by promoting macrophage M2 polarization by upregulating miR-125a-5p targeting TRAF6/IRF5 signaling pathway, which has great potential for clinical translation.

Causal associations between atrial fibrillation and breast cancer: A bidirectional Mendelian randomization analysis.

BACKGROUND: Previous observational studies indicated that atrial fibrillation may increase the risk of breast cancer. Following a breast cancer diagnosis, the chance of developing atrial fibrillation may increase as well. However, it is uncertain whether the link is causal or just due to confounding factors. OBJECTIVE: Using bidirectional Mendelian randomization (MR) analysis, we sought to assess the bidirectional causal relationship between atrial fibrillation and breast cancer from a genetic level. METHODS: Large genome-wide association studies yielded summary-level data for atrial fibrillation and breast cancer. The preliminary estimate was inverse variance weighted (IVW) under a random model. MR-Egger, weighted median, simple mode, weighted mode, and multivariable MR (adjusting body mass index, smoking, and alcohol drinking) were performed as sensitivity analyses. RESULTS: Genetically predicted atrial fibrillation presented no statistically significant association with overall breast cancer (odds ratio [OR] = 1.00; 95% confidence interval [CI]: 0.97-1.04; p = 0.79), estrogen receptor (ER) + (OR = 1.00; 95% CI: 0.96-1.03; p = 0.89) or ER- subtypes (OR = 1.00; 95% CI: 0.97-1.04; p = 0.89). Similarly, genetically predicted overall breast cancer (OR = 1.01; 95% CI: 0.98-1.04; p = 0.37), ER+ (OR = 1.02; 95% CI: 0.99-1.05; p = 0.16) or ER- (OR = 0.98; 95% CI: 0.93-1.02; p = 0.32) subtypes had no causal effect on atrial fibrillation. Sensitivity analyses yielded similar results. Individual single nucleotide polymorphism had little effect on the total estimate. We did not observe any evidence of horizontal pleiotropy. CONCLUSIONS: Our bidirectional MR studies revealed that there may be no causal links between atrial fibrillation and breast cancer.

Development of heart failure with preserved ejection fraction is independent of eosinophils in a preclinical model.

The increasing burden of heart failure with preserved ejection fraction (HFpEF) has become a global health problem. HFpEF is characterized by systematic inflammation, cardiac metabolic remodeling, and fibrosis. Eosinophils act as an essential but generally overlooked subgroup of white blood cells, which participate in cardiac fibrosis, as reported in several recent studies. Herein, we explored the role of eosinophils in a "two-hit" preclinical HFpEF model. The peripheral eosinophil counts were comparable between the normal chow and HFpEF mice. Deficiency of eosinophils failed to alter the phenotype of HFpEF. Conclusively, the development of HFpEF is independent of eosinophils in terms of the functional, biochemical, and histological results.

Levosimendan Reverses Cardiac Malfunction and Cardiomyocyte Ferroptosis During Heart Failure with Preserved Ejection Fraction via Connexin 43 Signaling Activation.

PURPOSE: In recent decades, the occurrence of heart failure with preserved ejection fraction (HFpEF) has outweighed that of heart failure with reduced ejection fraction by degrees, but few drugs have been demonstrated to improve long-term clinical outcomes in patients with HFpEF. Levosimendan, a calcium-sensitizing cardiotonic agent, improves decompensated heart failure clinically. However, the anti-HFpEF activities of levosimendan and underlying molecular mechanisms are unclear. METHODS: In this study, a double-hit HFpEF C57BL/6N mouse model was established, and levosimendan (3 mg/kg/week) was administered to HFpEF mice aged 13 to 17 weeks. Different biological experimental techniques were used to verify the protective effects of levosimendan against HFpEF. RESULTS: After four weeks of drug treatment, left ventricular diastolic dysfunction, cardiac hypertrophy, pulmonary congestion, and exercise exhaustion were significantly alleviated. Junction proteins in the endothelial barrier and between cardiomyocytes were also improved by levosimendan. Among the gap junction channel proteins, connexin 43, which was especially highly expressed in cardiomyocytes, mediated mitochondrial protection. Furthermore, levosimendan reversed mitochondrial malfunction in HFpEF mice, as evidenced by increased mitofilin and decreased ROS, superoxide anion, NOX4, and cytochrome C levels. Interestingly, after levosimendan administration, myocardial tissue from HFpEF mice showed restricted ferroptosis, indicated by an increased GSH/GSSG ratio; upregulated GPX4, xCT, and FSP-1 expression; and reduced intracellular ferrous ion, MDA, and 4-HNE levels. CONCLUSION: Regular long-term levosimendan administration can benefit cardiac function in a mouse model of HFpEF with metabolic syndromes (namely, obesity and hypertension) by activating connexin 43-mediated mitochondrial protection and sequential ferroptosis inhibition in cardiomyocytes.

Atorvastatin-pretreated mesenchymal stem cell-derived extracellular vesicles promote cardiac repair after myocardial infarction via shifting macrophage polarization by targeting microRNA-139-3p/Stat1 pathway.

BACKGROUND: Extracellular vesicles (EVs) derived from bone marrow mesenchymal stem cells (MSCs) pretreated with atorvastatin (ATV) (MSCATV-EV) have a superior cardiac repair effect on acute myocardial infarction (AMI). The mechanisms, however, have not been fully elucidated. This study aims to explore whether inflammation alleviation of infarct region via macrophage polarization plays a key role in the efficacy of MSCATV-EV. METHODS: MSCATV-EV or MSC-EV were intramyocardially injected 30 min after coronary ligation in AMI rats. Macrophage infiltration and polarization (day 3), cardiac function (days 0, 3, 7, 28), and infarct size (day 28) were measured. EV small RNA sequencing and bioinformatics analysis were conducted for differentially expressed miRNAs between MSCATV-EV and MSC-EV. Macrophages were isolated from rat bone marrow for molecular mechanism analysis. miRNA mimics or inhibitors were transfected into EVs or macrophages to analyze its effects on macrophage polarization and cardiac repair in vitro and in vivo. RESULTS: MSCATV-EV significantly reduced the amount of CD68+ total macrophages and increased CD206+ M2 macrophages of infarct zone on day 3 after AMI compared with MSC-EV group (P < 0.01-0.0001). On day 28, MSCATV-EV much more significantly improved the cardiac function than MSC-EV with the infarct size markedly reduced (P < 0.05-0.0001). In vitro, MSCATV-EV also significantly reduced the protein and mRNA expressions of M1 markers but increased those of M2 markers in lipopolysaccharide-treated macrophages (P < 0.05-0.0001). EV miR-139-3p was identified as a potential cardiac repair factor mediating macrophage polarization. Knockdown of miR-139-3p in MSCATV-EV significantly attenuated while overexpression of it in MSC-EV enhanced the effect on promoting M2 polarization by suppressing downstream signal transducer and activator of transcription 1 (Stat1). Furthermore, MSCATV-EV loaded with miR-139-3p inhibitors decreased while MSC-EV loaded with miR-139-3p mimics increased the expressions of M2 markers and cardioprotective efficacy. CONCLUSIONS: We uncovered a novel mechanism that MSCATV-EV remarkably facilitate cardiac repair in AMI by promoting macrophage polarization via miR-139-3p/Stat1 pathway, which has the great potential for clinical translation.

Mendelian Randomization Study Does Not Support a Bidirectional Link between Atherosclerosis and Venous Thromboembolism.

AIM: Some observational studies suggested that atherosclerosis increased the risk of venous thromboembolism (VTE), and vice versa. However, the results were conflicting, and the causal relationship is yet to be established. Therefore, we applied Mendelian randomization (MR) analyses to assess the bidirectional causality between coronary heart disease (CHD) and VTE, deep venous thrombosis (DVT), and pulmonary embolism (PE). METHODS: A total of 184,305 individuals with CHD were included from the CARDIoGRAMplusC4D Consortium. Information on VTE, DVT, and PE were obtained from the FinnGen biobank. Genetic instruments for CHD and VTE were constructed using 37 and 12 single-nucleotide polymorphisms, respectively. Inverse-variance weighted meta-analysis under a random-effect model was used as the preliminary estimate. Five complementary MR methods were also used, including weighted median, MR-Egger, multivariable MR (adjusted for the body mass index), simple mode, and weighted mode methods. RESULTS: The genetically instrumented VTE (odds ratio [OR]: 1.05; 95% confidence interval [CI]: 1.00-1.11; P=0.06), DVT (OR: 1.03; 95% CI: 0.99-1.08; P=0.19), or PE (OR: 1.07; 95% CI: 0.98-1.16; P=0.11) showed no causal relationships with CHD. There was also no clear evidence showing the causal effects of CHD on VTE (OR: 1.00; 95% CI: 0.82-1.22; P=0.98), DVT (OR: 1.00; 95% CI: 0.79-1.27; P=0.97), or PE (OR: 0.98; 95% CI: 0.82-1.18; P=0.87). No pleiotropic bias was found in the MR analyses. As heterogeneity was significant, a random model was used to minimize the effect of heterogeneity. CONCLUSIONS: No causal associations existed between CHD and VTE. Arterial and venous thromboses may represent separate entities.

Identification of Key Non-coding RNAs and Transcription Factors in Calcific Aortic Valve Disease.

Background: Calcific aortic valve disease (CAVD) is one of the most frequently occurring valvular heart diseases among the aging population. Currently, there is no known pharmacological treatment available to delay or reverse CAVD progression. The regulation of gene expression could contribute to the initiation, progression, and treatment of CAVD. Non-coding RNAs (ncRNAs) and transcription factors play essential regulatory roles in gene expression in CAVD; thus, further research is urgently needed. Materials and Methods: The gene-expression profiles of GSE51472 and GSE12644 were obtained from the Gene Expression Omnibus database, and differentially expressed genes (DEGs) were identified in each dataset. A protein-protein-interaction (PPI) network of DEGs was then constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins database, and functional modules were analyzed with ClusterOne plugin in Cytoscape. Furthermore, Gene Ontology-functional annotation and Kyoto Encyclopedia of Genes and Genomes-pathway analysis were conducted for each functional module. Most crucially, ncRNAs and transcription factors acting on each functional module were separately identified using the RNAInter and TRRUST databases. The expression of predicted transcription factors and key genes was validated using GSE51472 and GSE12644. Furthermore, quantitative real-time PCR (qRT-PCR) experiments were performed to validate the differential expression of most promising candidates in human CAVD and control samples. Results: Among 552 DEGs, 383 were upregulated and 169 were downregulated. In the PPI network, 15 functional modules involving 182 genes and proteins were identified. After hypergeometric testing, 45 ncRNAs and 33 transcription factors were obtained. Among the predicted transcription factors, CIITA, HIF1A, JUN, POU2F2, and STAT6 were differentially expressed in both the training and validation sets. In addition, we found that key genes, namely, CD2, CD86, CXCL8, FCGR3B, GZMB, ITGB2, LY86, MMP9, PPBP, and TYROBP were also differentially expressed in both the training and validation sets. Among the most promising candidates, differential expressions of ETS1, JUN, NFKB1, RELA, SP1, STAT1, ANCR, and LOC101927497 were identified via qRT-PCR experiments. Conclusion: In this study, we identified functional modules with ncRNAs and transcription factors involved in CAVD pathogenesis. The current results suggest candidate molecules for further research on CAVD.

Tongxinluo-pretreated mesenchymal stem cells facilitate cardiac repair via exosomal transfer of miR-146a-5p targeting IRAK1/NF-κB p65 pathway.

BACKGROUND: Bone marrow cells (BMCs), especially mesenchymal stem cells (MSCs), have shown attractive application prospects in acute myocardial infarction (AMI). However, the weak efficacy becomes their main limitation in clinical translation. Based on the anti-inflammation and anti-apoptosis effects of a Chinese medicine-Tongxinluo (TXL), we aimed to explore the effects of TXL-pretreated MSCs (MSCsTXL) in enhancing cardiac repair and further investigated the underlying mechanism. METHODS: MSCsTXL or MSCs and the derived exosomes (MSCsTXL-exo or MSCs-exo) were collected and injected into the infarct zone of rat hearts. In vivo, the anti-apoptotic and anti-inflammation effects, and cardiac functional and histological recovery were evaluated. In vitro, the apoptosis was evaluated by western blotting and flow cytometry. miRNA sequencing was utilized to identify the significant differentially expressed miRNAs between MSCsTXL-exo and MSCs-exo, and the miRNA mimics and inhibitors were applied to explore the specific mechanism. RESULTS: Compared to MSCs, MSCsTXL enhanced cardiac repair with reduced cardiomyocytes apoptosis and inflammation at the early stage of AMI and significantly improved left ventricular ejection fraction (LVEF) with reduced infarct size in an exosome-dependent way. Similarly, MSCsTXL-exo exerted superior therapeutic effects in anti-apoptosis and anti-inflammation, as well as improving LVEF and reducing infarct size compared to MSCs-exo. Further exosomal miRNA analysis demonstrated that miR-146a-5p was the candidate effector of the superior effects of MSCsTXL-exo. Besides, miR-146a-5p targeted and decreased IRAK1, which inhibited the nuclear translocation of NF-κB p65 thus protecting H9C2 cells from hypoxia injury. CONCLUSIONS: This study suggested that MSCsTXL markedly facilitated cardiac repair via a new mechanism of the exosomal transfer of miR-146a-5p targeting IRAK1/NF-κB p65 pathway, which has great potential for clinical translation.

Sequential transplantation of exosomes and mesenchymal stem cells pretreated with a combination of hypoxia and Tongxinluo efficiently facilitates cardiac repair.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (MSCs), which possess immunomodulatory characteristic, are promising candidates for the treatment of acute myocardial infarction (AMI). However, the low retention and survival rate of MSCs in the ischemic heart limit their therapeutic efficacy. Strategies either modifying MSCs or alleviating the inflammatory environment, which facilitates the recruitment and survival of the engrafted MSCs, may solve the problem. Thus, we aimed to explore the therapeutic efficacy of sequential transplantation of exosomes and combinatorial pretreated MSCs in the treatment of AMI. METHODS: Exosomes derived from MSCs were delivered to infarcted hearts through intramyocardial injection followed by the intravenous infusion of differentially pretreated MSCs on Day 3 post-AMI. Enzyme linked immunosorbent assay (ELISA) was performed to evaluate the inflammation level as well as the SDF-1 levels in the infarcted border zone of the heart. Echocardiography and histological analysis were performed to assess cardiac function, infarct size, collagen area and angiogenesis. RESULTS: Sequential transplantation of exosomes and the combinatorial pretreated MSCs significantly facilitated cardiac repair compared to AMI rats treated with exosomes alone. Notably, compared to the other three methods of cotransplantation, combinatorial pretreatment with hypoxia and Tongxinluo (TXL) markedly enhanced the CXCR4 level of MSCs and promoted recruitment, which resulted in better cardiac function, smaller infarct size and enhanced angiogenesis. We further demonstrated that exosomes effectively reduced apoptosis in MSCs in vitro. CONCLUSION: Sequential delivery of exosomes and pretreated MSCs facilitated cardiac repair post-AMI, and combined pretreatment with hypoxia and TXL better enhanced the cardioprotective effects. This method provides new insight into the clinical translation of stem cell-based therapy for AMI.

Nanoparticles: Promising Tools for the Treatment and Prevention of Myocardial Infarction.

Despite several recent advances, current therapy and prevention strategies for myocardial infarction are far from satisfactory, owing to limitations in their applicability and treatment effects. Nanoparticles (NPs) enable the targeted and stable delivery of therapeutic compounds, enhance tissue engineering processes, and regulate the behaviour of transplants such as stem cells. Thus, NPs may be more effective than other mechanisms, and may minimize potential adverse effects. This review provides evidence for the view that function-oriented systems are more practical than traditional material-based systems; it also summarizes the latest advances in NP-based strategies for the treatment and prevention of myocardial infarction.

The pivotal roles of exosomes derived from endogenous immune cells and exogenous stem cells in myocardial repair after acute myocardial infarction.

Acute myocardial infarction (AMI) is one of the leading causes of mortality around the world, and the inflammatory response plays a pivotal role in the progress of myocardial necrosis and ventricular remodeling, dysfunction and heart failure after AMI. Therapies aimed at modulating immune response after AMI on a molecular and cellular basis are urgently needed. Exosomes are a type of extracellular vesicles which contain a large amount of biologically active substances, like lipids, nucleic acids, proteins and so on. Emerging evidence suggests key roles of exosomes in immune regulation post AMI. A variety of immune cells participate in the immunomodulation after AMI, working together to clean up necrotic tissue and repair damaged myocardium. Stem cell therapy for myocardial infarction has long been a research hotspot during the last two decades and exosomes secreted by stem cells are important active substances and have similar therapeutic effects of immunomodulation, anti-apoptosis, anti-fibrotic and angiogenesis to those of stem cells themselves. Therefore, in this review, we focus on the characteristics and roles of exosomes produced by both of endogenous immune cells and exogenous stem cells in myocardial repair through immunomodulation after AMI.