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-1β 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-1β, 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.
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 IIinduced 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-β/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-β/Smad pathway. Therefore, targeting the miR-21-Spry1 axis may be a promising therapeutic option for ameliorating post-MI cardiac fibrosis.
BackgroundMyocardial ischaemia reperfusion injury (MIRI) is a difficult problem in clinical practice, and it may involve various microRNAs. This study investigated the role that endogenous microRNA-146a plays in myocardial ischaemia reperfusion and explored the possible target genes.MethodsMIRI models were established in microRNA-146a deficient (KO) and wild type (WT) mice. MicroRNA-146a expression was evaluated in the myocardium of WT mice after reperfusion. The heart function, area of myocardium infarction and in situ apoptosis were compared between the KO and WT mice. Microarray was used to explore possible target genes of microRNA-146a, while qRT-PCR and dual luciferase reporter assays were used for verification. Western blotting was performed to detect the expression levels of the target gene and related signalling molecules. A rescue study was used for further testing.ResultsMicroRNA-146a was upregulated 1 h after reperfusion. MicroRNA-146a deficiency decreased heart function and increased myocardial infarction and apoptosis. Microarray detected 19 apoptosis genes upregulated in the KO mice compared with the WT mice. qRT-PCR and dual luciferase verified that Med1 was one target gene of microRNA-146a. TRAP220, encoded by Med1 in the KO mice, was upregulated, accompanied by an amplified ratio of Bax/Bcl2 and increased cleaved caspase-3. Inhibition of microRNA-146a in H9C2 cells caused increased TRAP220 expression and more apoptosis under the stimulus of hypoxia and re-oxygenation, while knockdown of the increased TRAP220 expression led to decreased cell apoptosis.ConclusionsMicroRNA-146a exerts a protective effect against MIRI, which might be partially mediated by the target gene Med1 and related to the apoptosis signalling 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.
Introduction The aim of this study was to screen the leading compounds of natural origin with anti-angiogenic potential and to investigate their anti-angiogenic mechanism preliminarily. Materials and Methods An initial screening of 240 compounds from the Natural Products Collection of MicroSource was performed using the transgenic zebrafish strain Tg [fli1a: enhanced green fluorescent protein (EGFP)] y1 . The zebrafish embryos at 24 h post-fertilization were exposed to the natural compounds for an additional 24 h; then, morphological changes in the intersegmental vessels (ISVs) were observed and quantified under a fluorescence microscope. The expression profiles of angiogenesis-related genes in the zebrafish embryos were detected using quantitative real-time PCR. Results Five compounds were identified with potential anti-angiogenic activity on the zebrafish embryogenesis. Among them, deoxysappanone B 7.4ʹ-dimethyl ether (Deox B 7,4) showed anti-angiogenic activity on the formation of ISVs in a dose-dependent manner. The inhibition of ISV formation reached up to 99.64% at 5 μM Deox B 7,4. The expression of delta-like ligand 4 ( dll4 ), hes-related family basic helix-loop-helix transcription factor with YRPW motif 2, ephrin B2, fibroblast growth factor receptor ( fgfr ) 3, cyclooxygenase-2, protein tyrosine phosphatase, receptor type B ( ptp-rb ), phosphoinositide-3-kinase regulatory subunit 2, slit guidance ligand ( slit ) 2, slit3 , roundabout guidance receptor ( robo ) 1, robo2 , and robo4 were down-regulated, while vascular endothelial growth factor receptor-2, fgfr 1, and matrix metallopeptidase 9 were up-regulated in the zebrafish embryos treated with Deox B 7,4. Conclusion Deox B 7,4 has a therapeutic potential for the treatment of angiogenesis-dependent diseases and may exert anti-angiogenic activities by suppressing the slit2/robo1/2, slit3/robo4, cox2/ptp-rb/pik3r2, and dll4/hey2/efnb2a signaling pathways as well as activation of vegfr-2/fgfr1/mmp9.
The frequency and poor prognosis of patients with metastatic colorectal cancer (mCRC) emphasizes the requirement for improved biomarkers for use in the treatment and prognosis of mCRC. In the present study, somatic variants in exonic regions of key cancer genes were identified in mCRC patients. Formalin-fixed, paraffin-embedded tissues obtained by biopsy of the metastases of mCRC patients were collected, and the DNA was extracted and sequenced using the Ion Torrent Personal Genome Machine. For the targeted amplification of known cancer genes, the Ion AmpliSeq™ Cancer Panel, which is designed to detect 739 Catalogue of Somatic Mutations in Cancer (COSMIC) mutations in 604 loci from 46 oncogenes and tumor suppressor genes using as little as 10 ng of input DNA, was used. The sequencing results were then analyzed using the Ampliseq™ Variant Caller plug-in within the Ion Torrent Suite software. In addition, Ingenuity Pathway software was used to perform a pathway analysis. The Cox regression analysis was also conducted to investigate the potential correlation between alteration numbers and clinical factors, including response rate, disease-free survival and overall survival. Among 10 specimens, 65 genetic alterations were identified in 24 genes following the exclusion of germline mutations using the SNP database, whereby 41% of the alterations were also present in the COSMIC database. No clinical factors were found to significantly correlate with the alteration numbers in the patients by statistical analysis. However, pathway analysis identified ‘colorectal cancer metastasis signaling’ as the most commonly mutated canonical pathway. This analysis further revealed mutated genes in the Wnt, phosphoinositide 3-kinase (PI3K)/AKT and transforming growth factor (TGF)-β/SMAD signaling pathways. Notably, 11 genes, including the expected APC, BRAF, KRAS, PIK3CA and TP53 genes, were mutated in at least two samples. Notably, 90% (9/10) of mCRC patients harbored at least one ‘druggable’ alteration (range, 1–6 alterations) that has been linked to a clinical treatment option or is currently being investigated in clinical trials of novel targeted therapies. These results indicated that DNA sequencing of key oncogenes and tumor suppressors enables the identification of ‘druggable’ alterations for individual colorectal cancer patients.
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