Extracellular vesicles from hypoxia-preconditioned mesenchymal stem cells alleviates myocardial injury by targeting thioredoxin-interacting protein-mediated hypoxia-inducible factor-1α pathway.

BACKGROUND: Extracellular vesicles (EVs) derived from hypoxia-preconditioned (HP) mesenchymal stem cells (MSCs) have better cardioprotective effects against myocardial infarction (MI) in the early stage than EVs isolated from normoxic (NC)-MSCs. However, the cardioprotective mechanisms of HP-EVs are not fully understood. AIM: To explore the cardioprotective mechanism of EVs derived from HP MSCs. METHODS: We evaluated the cardioprotective effects of HP-EVs or NC-EVs from mouse adipose-derived MSCs (ADSCs) following hypoxia in vitro or MI in vivo, in order to improve the survival of cardiomyocytes (CMs) and restore cardiac function. The degree of CM apoptosis in each group was assessed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling and Annexin V/PI assays. MicroRNA (miRNA) sequencing was used to investigate the functional RNA diversity between HP-EVs and NC-EVs from mouse ADSCs. The molecular mechanism of EVs in mediating thioredoxin-interacting protein (TXNIP) was verified by the dual-luciferase reporter assay. Co-immunoprecipitation, western blotting, and immunofluorescence were performed to determine if TXNIP is involved in hypoxia-inducible factor-1 alpha (HIF-1α) ubiquitination and degradation via the chromosomal region maintenance-1 (CRM-1)-dependent nuclear transport pathway. RESULTS: HP-EVs derived from MSCs reduced both infarct size (necrosis area) and apoptotic degree to a greater extent than NC-EVs from CMs subjected to hypoxia in vitro and mice with MI in vivo. Sequencing of EV-associated miRNAs showed the upregulation of 10 miRNAs predicted to bind TXNIP, an oxidative stress-associated protein. We showed miRNA224-5p, the most upregulated miRNA in HP-EVs, directly combined the 3' untranslated region of TXNIP and demonstrated its critical protective role against hypoxia-mediated CM injury. Our results demonstrated that MI triggered TXNIP-mediated HIF-1α ubiquitination and degradation in the CRM-1-mediated nuclear transport pathway in CMs, which led to aggravated injury and hypoxia tolerance in CMs in the early stage of MI. CONCLUSION: The anti-apoptotic effects of HP-EVs in alleviating MI and the hypoxic conditions of CMs until reperfusion therapy may partly result from EV miR-224-5p targeting TXNIP.

Circular RNA Fbxl5 Regulates Cardiomyocyte Apoptosis During Ischemia Reperfusion Injury via Sponging microRNA-146a.

Objective: Cardiomyocyte apoptosis critically contributes to ischemia reperfusion injury (IRI), which lacks effective therapeutic strategies. Circular RNAs (circRNAs) serve as novel diagnostic and therapeutic targets in various cardiovascular diseases. CircRNA Fbxl5 is one of the abundantly expressed circRNAs in the heart and its role in myocardial IRI remains elusive. Materials and Methods: Wild-type (WT) mice and neonatal mice ventricular myocytes (NMVMs) were used and subjected to myocardial IRI and anoxia reoxygenation (AR), respectively. Molecular and histological analyses and echocardiography were used to determine the extent of apoptosis, infarct size, and cardiac function. Results: We found that circRNA Fbxl5 was significantly upregulated in the myocardium, as well as in NMVMs subjected to AR. Knockdown of circRNA Fbxl5 ameliorated cardiomyocyte apoptosis, thereby decreasing infarct size and preserving cardiac function. Additionally, in vitro knockdown of circRNA Fbxl5 in NMVMs subjected to AR recapitulated the in vivo findings. Mechanistically, we identified that circRNA Fbxl5 directly sponged and suppressed the endogenous microRNA-146a (miR-146a), thereby weakening its inhibitory effect on MED1, which could further promote the apoptosis of cardiomyocytes. Conclusion: Our findings revealed a novel and critical role for circRNA Fbxl5 in regulating cardiomyocyte apoptosis, and added additional insight into circRNAs mediated during myocardial IRI. The underlying miR-146a-MED1 signaling serves as an important cascade in regulating the apoptosis of cardiomyocytes.

VSTM1 regulates monocyte/macrophage function via the NF-κB signaling pathway.

OBJECTIVE: V-set and transmembrane domain-containing protein 1 (VSTM1) is negatively correlated with inflammation. However, its effect on atherosclerosis (AS) remains largely unexplored. In this study, we aimed to assess the effect of VSTM1 on the biological function of human peripheral blood mononuclear cells /macrophages stimulated by oxidized low-density lipoprotein (ox-LDL). METHODS: U937 cells were divided into three groups as follows: control group, pLenti-VSTM1 shRNA group (VSTM1 depletion), and pLenti-VSTM1 group (VSTM1 overexpression). Cellular migration, chemotaxis, apoptosis, and secretion of inflammatory factors of monocytes/macrophages stimulated by ox-LDL were studied. RESULTS: Overexpression of VSTM1 decreased the proliferation of U937 cells and induced cellular apoptosis. Depletion of VSTM1 enhanced the invasiveness and chemotaxis, increased the inflammatory response, and reduced the incidence of cell necrosis and apoptosis. Nuclear factor κ of B cells (NF-κB) was activated in VSTM1-depleted U937 cells. CONCLUSION: VSTM1 might play an important role in the activation of monocytes/macrophages and participate in the pathogenesis of AS via regulating NF-κB activity.

MicroRNA-221 inhibits the transition of endothelial progenitor cells to mesenchymal cells via the PTEN/FoxO3a signaling pathway.

BACKGROUND: Coronary heart disease is one of the most common cardiovascular diseases worldwide and is often associated with vascular endothelial injury. Endothelial-mesenchymal transition (EndMT) is an important process in vascular endothelial injury. OBJECTIVES: This study investigated the function of miR-221 in the EndMT process of endothelial progenitor cells (EPCs). MATERIAL AND METHODS: Transforming growth factor beta (TGF-ß1) was used to induce EndMT in EPCs, and SM22α expression was detected using immunocytochemistry. Western blot was used to detect alpha smooth muscle actin (αSMA) expression, and miR-221 function was evaluated using inhibitors or mimics of the miR-221 sequences that were transfected into EPCs. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of miR-221 and western blot was used to detect the expression of αSMA, myocardin, phosphatase and tensin homolog (PTEN), p-FoxO3a, and FoxO3a in EPCs. Finally, the expression of the miR-221 target genes was determined using RT-PCR. RESULTS: The expression of SM22α and αSMA increased in EPCs treated with TGF-ß1, while the expression of miR-221 was decreased in EPCs on the 5th day, when compared with the control. The expression of SM22α increased after inhibiting miR-221 in EPCs treated with TGF-ß1 and this was reversed by the overexpression of miR-221. The expression of αSMA and myocardin was significantly increased after inhibiting miR-221 in EPCs treated with TGF-ß1 and decreased in EPCs overexpressing miR-221. Conversely, PTEN was increased in TGF-ß1-treated EPCs and decreased following the overexpression of miR-221. The decrease in phosphorylated-FoxO3a expression in EPCs was accompanied by an increase in αSMA expression, which was reversed in the presence of miR-221 mimics. This effect was nearly abolished following the addition of PTEN cDNA. CONCLUSIONS: The overexpression of miR-221 inhibits EndMT in EPCs, possibly by interacting with PTEN to regulate FoxO3a signaling, to facilitate the repair of the endothelium by EPCs.

Nicotine exacerbates atherosclerosis through a macrophage-mediated endothelial injury pathway.

Evidence suggests that nicotine intake promotes atherosclerosis. We enrolled 100 patients with coronary heart disease (CHD) and found that plaque burden, TXNIP expression, and inflammatory chemokine levels were higher in smokers than non-smokers. Additionally, patients with higher TXNIP expression in peripheral blood mononuclear cells (PBMCs) had a higher Gensini Scores and higher plasma IL-1ß and IL-18 levels. Treating bone marrow-derived macrophages (BMDMs) with nicotine in vitro led to enhanced lipid phagocytosis, chemotaxis, and increased production of reactive oxygen species (ROS), which activated TXNIP/NLRP3 inflammasome signaling and promoted pyroptosis, as evidenced by caspase-1 cleavage and increased production of IL-1ß, IL-18, and gasdermin D. Nicotine intake by ApoE(-/-) mice fed a high-fat diet recapitulated those phenotypes. The effects of nicotine on pyroptotic signaling were reversed by N-acetyl-cysteine, a ROS scavenger. Silencing TXNIP in vivo reversed the effects of nicotine on macrophage invasion and vascular injury. Nicotine also induced pyroptotic macrophages that contributed to the apoptotic death of endothelial cells. These findings suggest that nicotine accelerates atherosclerosis in part by promoting macrophage pyroptosis and endothelial damage. Therefore, targeting the TXNIP/NLRP3-mediated pyroptotic pathway in macrophages may ameliorate nicotine-induced endothelial damage.

Extracellular vesicles from anoxia preconditioned mesenchymal stem cells alleviate myocardial ischemia/reperfusion injury.

Extracellular vesicles (EVs) produced by anoxia-preconditioned mesenchymal stem cells (MSCs) may afford greater cardioprotection against myocardial ischemia-reperfusion injury (MIRI) than EVs derived from normoxic MSCs. Here, we isolated EVs from mouse adipose-derived MSCs (ADSCs) subjected to anoxia preconditioning or normoxia and evaluated their ability to promote survival of mouse cardiomyocytes following MIRI in vivo and anoxia/reoxygenation (AR) in vitro. Injection of anoxia-preconditioned ADSC EVs (Int-EVs) reduced both infarct size and cardiomyocyte apoptosis to a greater extent than normoxic ADSC EVs (NC-EVs) in mice subjected to MIRI. Sequencing EV-associated miRNAs revealed differential upregulation of ten miRNAs predicted to bind thioredoxin-interacting protein (TXNIP), an inflammasome- and pyroptosis-related protein. We confirmed direct binding of miRNA224-5p, the most upregulated miRNA in Int-EVs, to TXNIP and asserted through western blotting and apoptosis assays a critical protective role for this miRNA against AR-induced cardiomyocyte death. Our results suggest that ischemia-reperfusion triggers TXNIP-induced inflammasome activation in cardiomyocytes, which leads to apoptosis rather than pyroptosis due to low basal levels of the pyroptosis executioner protein gasdermin D in these cells. The antiapoptotic effect of EV-associated miRNA224-5p would in turn result from TXNIP downregulation, which prevents caspase-1-mediated degradation of GATA4 and sustains the expression of Bcl-2.

MicroRNA-21 Mediates a Positive Feedback on Angiotensin II-Induced Myofibroblast Transformation.

OBJECTIVE: Post myocardial infarction (MI) fibrosis has been identified as an important factor in the progression of heart failure. Previous studies have revealed that microRNA-21 (miR-21) plays an important role in the pathogenesis of fibrosis. The purpose of this study was to explore the role of miR-21 in post-MI cardiac fibrosis. MATERIAL AND METHODS: MI was established in wild-type (WT) and miR-21 knockout (KO) mice. Primary mice cardiac fibroblasts (CFs) were isolated from WT and miR-21 KO mice and were treated with angiotensin II (Ang II) or Sprouty1 (Spry1) siRNA. Histological analysis and echocardiography were used to determine the extent of fibrosis and cardiac function. RESULTS: Compared with WT mice, miR-21 KO mice displayed smaller fibrotic areas and decreased expression of fibrotic markers and inflammatory cytokines. In parallel, Ang II-induced myofibroblasts transformation was partially inhibited upon miR-21 KO in primary CFs. Mechanistically, we found that the expression of Spry1, a previously reported target of miR-21, was markedly increased in miR-21 KO mice post MI, further inhibiting ERK1/2 activation. In vitro studies showed that Ang II activated ERK1/2/TGF-ß/Smad2/3 pathway. Phosphorylated Smad2/3 further enhanced the expression of α-SMA and FAP and may promote the maturation of miR-21, thereby downregulating Spry1. Additionally, these effects of miR-21 KO on fibrosis were reversed by siRNA-mediated knockdown of Spry1. CONCLUSION: Our findings suggest that miR-21 promotes post-MI fibrosis by targeting Spry1. Furthermore, it mediates a positive feedback on Ang II, thereby inducing the ERK/TGF-ß/Smad pathway. Therefore, targeting the miR-21-Spry1 axis may be a promising therapeutic option for ameliorating post-MI cardiac fibrosis.

NT-proBNP test with improved accuracy for the diagnosis of chronic heart failure.

The circulating concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) has been shown to be a diagnostic tool for the detection of heart failure. Several factors influence NT-proBNP levels including age, sex, and body mass index (BMI). Therefore, the diagnostic sensitivity of NT-proBNP level for heart failure is relatively higher, but its specificity is low. This study aims to improve the diagnostic accuracy rate of this test by including multiple variables in the diagnostic test.The suspected chronic heart failure outpatients were divided into heart failure with reduced ejection fraction, heart failure with mid-range ejection fraction, heart failure with preserved ejection fraction, and normal heart function groups. Area under the receiver-operating characteristic (ROC) curve, cut-off value, and logistic regression analysis were used to select the model variables, sensitivity and specificity.In all, 436 subjects enrolled into this study were divided in 2 groups: model establishment (n = 300) and model validation (n = 136). In the model establishment group, the area under the curve (AUC) and cut-off value of NT-proBNP was 0.926 and 257.4 pg/mL, respectively. When age, glomerular filtration rate, BMI, atrial fibrillation, and sex were entered into the diagnosis model, AUC, sensitivity, and specificity further increased to 0.955 (95% confidence interval [CI] 0.934, 0.976), 94.2% (from 93.0%), and 86.7% (from 74.2%). The ROC curve of corrected NT-proBNP diagnostic formula for heart failure was also significantly higher (P = .037).The corrected NT-proBNP diagnostic formula was found to improve the diagnostic accuracy of chronic heart failure.