Crocin inhibits KBTBD7 to prevent excessive inflammation and cardiac dysfunction following myocardial infarction.

Myocardial infarction (MI) refers to myocardial ischemic necrosis that is caused by coronary artery disease. Notably, crocin has protective effects on the heart. The present study aimed to i) investigate the protective effect of crocin, an active ingredient in Gardenia jasminoides Ellis and Crocus sativus L., on myocardial ischemia and ii) to verify the interaction between crocin and kelch repeat and BTB domain­containing protein 7 (KBTBD7), which is a novel member of the BTB­kelch protein family. In the present study, the left anterior descending coronary artery was ligated to establish a myocardial ischemia­reperfusion injury (MIRI) model in rats and the protective effect of crocin on rat myocardial tissue was observed. The levels of the inflammatory cytokines, interleukin (IL)­1ß, IL­6 and tumor necrosis factor α (TNFα), in the sham, MI model, MI + crocin (100 mg/kg) and MI + crocin (200 mg/kg) groups were compared in the rat myocardial tissue. The TUNEL assay was used to detect apoptosis of myocardial cells. In addition, RAW264.7 cells were stimulated with the inflammatory factors recombinant mouse high mobility group box 1 (rmHMGB1) and recombinant mouse heat shock protein 60 (rmHSP60). The inhibitory effect of crocin on inflammatory cytokine levels was observed using ELISA. Western blotting was used to detect the inhibitory effect of crocin on KBTBD7. The inhibitory effect of KBTBD7 knockdown on MAPK and nuclear factor (NF)­κB signaling pathways was also analyzed. The expression levels of IL­1ß, IL­6 and TNFα were significantly decreased in the crocin­treated groups compared with in the model group. Crocin significantly reduced the apoptosis of myocardial cells and significantly inhibited the release of inflammatory cytokines induced by rmHMGB1 and rmHSP60. KBTBD7 was determined to be a target of crocin. Knockdown of KBTBD7 significantly inhibited p38 and NF­κB signaling pathways. Furthermore, the results demonstrated that KBTBD7 knockdown significantly reduced the production of inflammatory cytokines induced by rmHMGB1 and rmHSP60. KBTBD7 knockdown also significantly reduced p38 and NF­κB signaling in the rmHMGB1­ and rmHSP60­treated groups. The present study demonstrated the potential protective effect of crocin on MIRI in rats. The underlying mechanism may be through direct inhibition of KBTBD7, thereby inhibiting excessive inflammatory responses and myocardial cell apoptosis following myocardial infarction.

FinKENet: A Novel Financial Knowledge Enhanced Network for Financial Question Matching.

Question matching is the fundamental task in retrieval-based dialogue systems which assesses the similarity between Query and Question. Unfortunately, existing methods focus on improving the accuracy of text similarity in the general domain, without adaptation to the financial domain. Financial question matching has two critical issues: (1) How to accurately model the contextual representation of a financial sentence? (2) How to accurately represent financial key phrases in an utterance? To address these issues, this paper proposes a novel Financial Knowledge Enhanced Network (FinKENet) that significantly injects financial knowledge into contextual text. Specifically, we propose a multi-level encoder to extract both sentence-level features and financial phrase-level features, which can more accurately represent sentences and financial phrases. Furthermore, we propose a financial co-attention adapter to combine sentence features and financial keyword features. Finally, we design a multi-level similarity decoder to calculate the similarity between queries and questions. In addition, a cross-entropy-based loss function is presented for model optimization. Experimental results demonstrate the effectiveness of the proposed method on the Ant Financial question matching dataset. In particular, the Recall score improves from 73.21% to 74.90% (1.69% absolute).

Effects of Chronic Remote Ischemic Conditioning on Atrial Fibrillation Burden in Patients with Permanent Pacemakers.

This study aimed to evaluate the effects of chronic remote ischemic conditioning (CRIC) on atrial fibrillation burden in patients with an implanted pacemaker. Sixty-six patients with permanent pacemakers were randomly divided into the CRIC group and control group after 4 weeks of screening. CRIC treatment was performed twice daily for 12 weeks. The remote ischemic conditioning protocol consisted of 4 × 5 minutes inflation/deflation of the blood pressure cuff applied in the upper arm to create intermittent arm ischemia. Sixty-one patients (31 patients in the CRIC group and 30 patients in the control group) completed the study. CRIC was well tolerated by patients after 12 weeks of treatment. The burden of atrial fibrillation (AF) in the CRIC group decreased significantly at 4 weeks compared with that at 0 weeks (14.7% ± 18.5% versus 17.0% ± 20.7%, P < 0.001), which further decreased at 12 weeks compared with that at 0 weeks (8.6% ± 10.2% versus 17.0% ± 20.7%, P < 0.001) and that at 4 weeks (8.6% ± 10.2% versus 14.7% ± 18.5%, P < 0.001), which was not observed in the control group. AF burden also reduced significantly after 12-week CRIC compared with that in the control group (8.6% ± 10.2% versus 17.6% ± 19.5%, P = 0.013). Repeated measurement ANOVA showed that the changes in AF burden were associated with CRIC instead of time (P < 0.01). In addition, there were trends that the longest duration of AF and cumulative numbers of atrial high-rate episodes (AHREs) reduced after 12-week CRIC. This study suggests that a 12-week course of CRIC treatment could reduce AF burden in patients with permanent pacemakers, supporting the widespread use of CRIC in the daily lives of these patients, which needs to be verified in the future.

Circ-GTF2I/miR-590-5p Axis Aggravates Myocardial Ischemia-Reperfusion Injury by Regulating Kelch Repeat and BTB Domain-Containing Protein 7.

Purpose: We investigated the effect of the circular RNA (circRNA) general transcription factor IIi (GTF2I) on myocardial ischemia (MI) deterioration and neonatal rat cardiomyocyte damage. Methods: The cell experiment was performed by using neonatal rat cardiomyocytes. Moreover, a hypoxia/reoxygenation treatment model was established. Cell Counting Kit-8 assay was conducted, and EdU cell proliferation was detected. Cell apoptosis was detected via flow cytometry and quantitative RT-PCR (RT-qPCR). Binding detection was performed through a double-luciferase reporter assay. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and lactate dehydrogenase (LDH) were detected via enzyme-linked immunosorbent assay (ELISA). Results: Compared with that in the sham and control groups, circ-GTF2I expression in MIRI and the hypoxia/reoxygenation treatment model was significantly upregulated in vivo and in vitro. The knockdown of circ-GTF2I relieved neonatal rat cardiomyocyte damage and MI. Further detection through the double-luciferase reporter assay confirmed that the binding site of circ-GTF2I to miR-590-5p and miR-590-5p was Kelch repeat and BTB domain-containing protein 7 (KBTBD7). ELISA and RT-qPCR results showed that circ-GTF2I induced the abnormal expressions of IL-6 TNF-α, LDH, Bax, Bcl-2, and Cyt-c in MIRI and the hypoxia/reoxygenation treatment models by regulating miR-590-5p and the heart development transcription factor KBTBD7. Conclusions: CircRNA circ-GTF2I aggravated MIRI and neonatal rat cardiomyocyte damage in vivo and in vitro by regulating miR-590-5p and the heart development transcription factor KBTBD7.

Antithrombotic Therapy With Ticagrelor in Atrial Fibrillation Subjects After Percutaneous Coronary Intervention.

Background: Warfarin, along with aspirin and clopidogrel, has long been recommended for patients with atrial fibrillation (AF) who are undergoing percutaneous coronary intervention with a drug-eluting stent (PCI-DES). However, this triple therapy has been known to increase the risk of bleeding complications. Meanwhile, there is no evidence from prospective trials on the use of ticagrelor in a dual therapy. We here aimed to compare the antiplatelet drug ticagrelor as a dual antithrombotic agent to aspirin and clopidogrel in bleeding events. Methods: In this multicenter, active-controlled, open-label, randomized trial, patients with AF taking warfarin who had undergone PCI-DES were randomly assigned to the ticagrelor therapy group (Dual group) or the clopidogrel plus aspirin therapy group (Triple group). The primary and secondary endpoints were overall bleeding events and major bleeding events, respectively, according to the Thrombolysis in Myocardial Infarction (TIMI) criteria at 6 months. Cardiovascular events [re-PCI, surgical bypass, myocardial infarction (MI), heart failure, rehospitalization due to angina pectoris, stent thrombosis and death due to cardiovascular causes] at 6 months were also recorded. Results: A total of 296 patients from 12 medical centers in China were randomized after PCI-DES to either the Dual therapy group (n = 148) or the Triple group (n = 146) for 6 months. The overall incidence of bleeding events at 6 months was 36.49% in the Dual therapy group and 35.62% in the Triple group [hazard ratio, 0.930; 95% confidence interval (CI), 0.635 to 1.361; P = 0.7088]. The incidence of the secondary endpoint over 6 months was 4.73% in the Dual therapy group and 1.37% in the Triple group (hazard ratio, 0.273; 95% CI, 0.057 to 1.315; P = 0.1056). Cardiovascular event occurrence was also comparable in both groups at 6 months (18.24 vs. 16.44%; hazard ratio, 0.845; 95% CI, 0.488 to 1.465; P = 0.5484). Conclusions: The incidence of total bleeding events in AF patients treated with ticagrelor was comparable to that in patients treated with clopidogrel plus aspirin at 6 month; Meanwhile, the incidence of cardiovascular events were also comparable between the groups. Clinical Trial Registration: MANJUSRI, ClinicalTrials.gov# NCT02206815, 2014, August 1st.

PTBP1 Targets ILK to Regulate the Hypoxia-Induced Phenotypic Transformation of Pulmonary Artery Smooth Muscle Cells.

PURPOSE: Pulmonary hypertension (PH) is a pathological process mainly characterized by the progressive increase in pulmonary vascular resistance. The degradation of pulmonary artery smooth muscle cells (PASMCs) from contractile/differentiated phenotype to synthetic/dedifferentiated phenotype is a key factor for hypoxic pulmonary hypertension. MATERIALS AND METHODS: In this study, qPCR was performed to evaluate the gene expression of mRNAs. Western blot, immunofluorescence and RNA pull down were used to detect gene expression levels. RESULTS: We found that the gene expression of polypyrimidine tract-binding protein1 (PTBP1) was increased significantly in a time-dependent manner in rats PA tissues and PASMCs after hypoxia. PTBP1 knockdown can inhibit the phenotypic transition of PASMCs. PTBP1 inhibits the phenotypic transition of PASMCs. In addition, PTBP1 inhibits the integrin-linked kinase (ILK) expression under hypoxic conditions, thereby down-regulating the expression of downstream proteins. It inhibits the phenotypic transition of PASMCs and alleviates pulmonary hypertension. CONCLUSION: In conclusion, PTBP1/ILK axis promotes the development of PH via inducing phenotypic transition of PASMCs. This may provide a novel therapy for PH.

MiR-15a Functions as a Diagnostic Biomarker for Coronary Artery Disease.

BACKGROUND: The present study aimed to explore the diagnostic value of miR-15a in coronary artery disease (CAD). METHODS: After recruiting all the participants, peripheral blood samples were obtained according to the instructions. miR-15a expression was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and IL-6, TNF-α, and hs-CRP expressions were detected using ELISA kits. RESULTS: The results elucidated a significantly decreased expression of miR-15a in peripheral blood samples from CAD patients compared to non-CAD controls (p < 0.01). Meanwhile, miR-15a expression was negatively correlated with LDL-C and Gensini score (p = 0.0059, 0.0243, respectively). Moreover, miR-15a expression was negatively correlated with inflammatory cytokines IL-6, TNF-α, and hs-CRP (p = 0.0009, 0.0178, 0.0005, respectively). The receiver operating curve (ROC) curve analysis demonstrated that miR-15a was a promising biomarker for early diagnosis of CAD with an area under the curve (AUC) of 0.9368. CONCLUSIONS: The present study elucidated a decreased miR-15a expression in CAD patients and showed negative correlations with LDL-C, Gensini score, and inflammatory cytokines. miR-15a may serve as a promising biomarker for early diagnosis of CAD.

miR-142 is a Sensitive Biomarker for the Diagnosis and Prognosis of Acute Myocardial Infarction.

BACKGROUND: Acute myocardial infarction (AMI) is myocardial necrosis caused by acute and persistent ischemia and hypoxia of coronary arteries. AMI is one of the most common diseases in European countries and over 1.5 million AMI patients die of it in the United States annually. A collection of studies proposed that certain micro-RNAs play crucial roles in the onset and development of AMI. METHODS: Ninety-four AMI patients and 83 non-AMI healthy controls were recruited from Zhongda Hospital, Southeast University between July 2015 and September 2017. Serum samples were collected at admission and the expression of miR-142 was detected using real-time quantitative polymerase chain reaction (RT-qPCR) assays. RESULTS: miR-142 expression was markedly elevated in serum samples of AMI patients compared with the 83 non-AMI healthy controls. miR-142 expression was positively correlated with creatine kinase-KB (CK-MB; r = 0.6731, p = 0.0021) and troponin (r = 0.7138, p = 0.0013). The area under the curve (AUC) of miR-142, CK-MB, and troponin for the diagnosis of AMI were 0.9185, 0.8172, and 0.8717, respectively. Overall survival analysis implied that high miR-142 expression may predict poor survival (log-rank test, p = 0.0146). CONCLUSIONS: miR-142 may be a diagnostic and prognostic indicator for AMI, and therefore, it may contribute to AMI clinicopathologic prediction.

Circulating miR-130 is a potential bio signature for early prognosis of acute myocardial infarction.

BACKGROUND: As distinctive leading reasons of death globally, acute myocardial infarction (AMI). Accounts for major death ratio, caused by coronary artery disease (CAD). Its diagnosis relies on the presenting clinical symptoms, electrocardiograms (ECGs), and levels of circulating biomarkers. Recent studies have implicated microRNAs (miRNAs) in the pathogenesis of many diseases, including AMI. The present study inquire into feature value of miR-130 in AMI patients. METHODS: levels of expression of miR-130 in patient plasma, considered through simultaneous quantitative polymerase chain reaction (qRT-PCR). The method used for determining Plasma cardiac troponin I (cTnI) & creatine kinase-MB(CK-MB) degree set on by enzyme-linked immunosorbent assay (ELISA). The diagnostic value of miR-130 was measured using a receiver operating characteristic (ROC) curve. RESULTS: Plasma miR-130, cTnI, and CK-MB levels exist remarkably inflated in the AMI classification in comparison with control category (P<0.05). MiR-130 expression peaked 6 hours after disease onset, earlier than cTnI and CK-MB. The level of expression of miR-130 6 hours after disease onset was positively correlated with cTnI and CK-MB levels 12 hours after onset. The optimal cut-off point for miR-130 in peripheral blood, sensitivity, and specificity were 1.58 ng/mL, 82.5% and 77.5%, respectively. The area under curve (AUC) was 0.922. CONCLUSIONS: These results indicate that circulating miR-130 holds great promise as an effective biomarker for diagnosing AMI earlier.

miR-17 regulates the proliferation and apoptosis of endothelial cells in coronary heart disease via targeting insulin-like-growth factor 1.

Coronary heart disease (CHD) is one of the main risks of death, which is mainly caused by coronary arteries arteriosclerosis. The present study aims to investigate the potential roles of miR-17 in CHD. In the present study, Human umbilical vascular endothelial cells (HUVECs) were treated with oxidized low density lipoprotein (ox-LDL). qRT-PCR and western blot were used to examine the mRNA and protein levels, respectively. CCK-8 and flow cytomtry were conducted to determine the proliferation and apoptosis of ox-LDL treated HUVECs. Moreover, luciferase assay was performed to confirm whether insulin-like Growth Factor-1 (IGF-1) was a target of miR-17. The results showed that miR-17 was upregulated in ox-LDL treated HUVECs, while IGF-1 was downregulated. The luciferase activity of ox-LDL treated HUVECs was decreased after the treatment of miR-17 mimics and IGF-1 3'UTR WT. Moreover, overexpressed miR-17 promoted the cell viability and inhibited the apoptosis of ox-LDL treated HUVECs, which was more potent after the treatment of IGF-1 siRNA. Furthermore, the expression of Bax and Caspase3 was decreased, and Bcl-2 was increased in ox-LDL treated HUVECs transfected with miR-17 mimics, which was further decreased after transfection with IGF-1 siRNA. Taken together, miR-17 may regulate the proliferation and apoptosis of ox-LDL treated HUVECs. miR-17 may be a promising biomarker for CHD.

Baicalin Suppresses the Proliferation and Migration of Ox-LDL-VSMCs in Atherosclerosis through Upregulating miR-126-5p.

Atherosclerosis (AS) is a chronic inflammatory disease threatening human health, and vascular smooth muscle cells (VSMCs) are involved in AS processes. Baicalin is a flavonoid compound, which has anti-atherosclerotic effect. The aim of our study was to explore the molecular mechanism of baicalin on AS. The expression of miR-126-5p was measured in peripheral blood of AS patients and healthy control. We found miR-126-5p expression was decreased in AS. Then, high-mobility group box 1 (HMGB1) was verified as a target of miR-126-5p and its expression was increased in AS. Similarly, miR-126-5p and HMGB1 expression was downregulated and upregulated in oxidized low-density lipoprotein treated VSMCs (ox-LDL-VSMCs), respectively. Furthermore, baicalin upregulated miR-126-5p and downregulated HMGB1 expression. Functionally, baicalin significantly inhibited ox-LDL-VSMCs proliferation and migration, and miR-126-5p targets HMGB1 to enhance the inhibition induced by baicalin. Taken together, baicalin is able to prevent AS, which suppressed the proliferation and migration of ox-LDL-VSMCs through upregulating miR-126-5p by targeting HMGB1. These findings suggested that baicalin is an effective drug to alleviate AS, and miR-126-5p is a novel therapeutic target for AS.

DNA methylation regulates α-smooth muscle actin expression during cardiac fibroblast differentiation.

Cardiac fibroblast (CF) differentiation to myofibroblasts expressing α-smooth muscle actin (α-SMA) plays a key role in cardiac fibrosis. Therefore, a study of the mechanism regulating α-SMA expression is a means to understanding the mechanism of fibroblast differentiation and cardiac fibrosis. Previous studies have shown that DNA methylation is associated with gene expression and is related to the development of tissue fibrosis. However, the mechanisms by which CF differentiation is regulated by DNA methylation remain unclear. Here, we explored the epigenetic regulation of α-SMA expression and its relevance in CF differentiation. In this study, we demonstrated that α-SMA was overexpressed and DNMT1 expression was downregulated in the infarct area after myocardial infarction. Treatment of CFs with transforming growth factor-ß1 (TGF-ß1 ) in vitro upregulated α-SMA expression via epigenetic modifications. TGF-ß1 also inhibited DNMT1 expression and activity during CF differentiation. In addition, α-SMA expression was regulated by DNMT1. Conversely, increasing DNMT1 expression levels rescued the TGF-ß1 -induced upregulation of α-SMA expression. Finally, TGF-ß1 regulated α-SMA expression by inhibiting the DNMT1-mediated DNA methylation of the α-SMA promoter. Taken together, our research showed that inhibition of the DNMT1-mediated DNA methylation of the α-SMA promoter plays an essential role in CF differentiation. In addition, DNMT1 may be a new target for the prevention and treatment of myocardial fibrosis.

TWEAK promotes endothelial progenitor cell vasculogenesis to alleviate acute myocardial infarction via the Fn14-NF-κB signaling pathway.

Acute myocardial infarction (AMI) remains one of the leading causes of mortality worldwide; however, endothelial progenitor cell (EPC) transplantation has been proposed as a promising treatment strategy for EPC. High levels of tumor necrosis factor-related weak inducer of apoptosis (TWEAK) have been reported in AMI, although its effect on EPCs has not been reported. In the present study, immunofluorescence and flow cytometry were performed to assess the effect of TWEAK in isolated mouse EPCs. Echocardiography was used to evaluate the cardiac function of murine hearts following EPC treatment in the AMI model, while collagen synthesis within the heart tissue was assessed using Masson's trichrome staining. A tube formation assay and Transwell migration assay were performed to investigate the effects of TWEAK on vessel formation and EPC migration in vitro. Angiogenesis and arteriogenesis were assessed in vivo using immunohistochemistry and western blotting was performed to determine the effect of TWEAK-mediated nuclear factor (NF)-κB pathway activation in EPCs. The results revealed that TWEAK promotes EPC migration, tube formation and viability in vitro. Furthermore, TWEAK treatment resulted in improved cardiac function, decreased heart collagen and vasculogenesis in mice with AMI, which was mediated by the TWEAK- fibroblast growth factor-inducible 14 (Fn14)-NF-κB signaling pathway, as determined using Fn14 small interfering (si)RNA and Bay 11-7082 (an NF-κB inhibitor). In summary, the results of the present study suggest that activation of the TWEAK-Fn14-NF-κB signaling pathway exerts a beneficial effect on EPCs for the treatment of AMI.

Sustained Release of IGF-1 by 3D Mesoporous Scaffolds Promoting Cardiac Stem Cell Migration and Proliferation.

BACKGROUND/AIMS: C-kit-positive cardiac stem cells (CSCs) may have potential as a treatment for cardiovascular disease. However, the low survival rates of c-kit-positive CSCs present a major challenge during the transplantation process. METHODS: The hierarchical structure of the 3D cell scaffold was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and N2 adsorption-desorption isotherms. Analyses of the proliferation and migration performances of the IGF-1 scaffold on c-kit-positive CSCs were conducted by experiments including QuantiT PicoGreen dsDNA and transwell assays. RESULTS: In this study, we synthesized for the first time a novel hierarchical macro-mesoporous silica material (denoted MS15-c) in a one-pot procedure for the release of insulin-like growth factor-1 (IGF-1) and a three-dimensional (3D) cell scaffold. Both macropores and mesopores were visible in MS15-c and enabled the sustained release of IGF-1, extending its half-life and enhancing CSC proliferation and migration. Proliferation and migration were detected by QuantiT PicoGreen dsDNA and transwell assays, respectively. Moreover, an in vivo experiment was conducted to detect heart function with the addition of MS15-c. The new strategy proposed in this paper may extend the bio-applications of 3D cell scaffolds, thus permitting the sustained release of growth factors and efficient promotion of cell proliferation. CONCLUSION: This work successfully demonstrated an effective strategy for the construction of MS15-c cell scaffolds with hierarchical macro-mesoporous structures. The macro-mesoporous structures gave cell scaffolds the ability to release a growth factor to facilitate cell growth, while the scaffold structure promoted cell proliferation.

Insulin-like growth factor-1-mediated regulation of miR-193a expression promotes the migration and proliferation of c-kit-positive mouse cardiac stem cells.

BACKGROUND: C-kit-positive cardiac stem cells (CSCs) have been shown to be a promising candidate treatment for myocardial infarction and heart failure. Insulin-like growth factor (IGF)-1 is an anabolic growth hormone that regulates cellular proliferation, differentiation, senescence, and death in various tissues. Although IGF-1 promotes the migration and proliferation of c-kit-positive mouse CSCs, the underlying mechanism remains unclear. METHODS: Cells were isolated from adult mouse hearts, and c-kit-positive CSCs were separated using magnetic beads. The cells were cultured with or without IGF-1, and c-kit expression was measured by Western blotting. IGF-1 induced CSC proliferation and migration, as measured through Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. The miR-193a expression was measured by quantitative real-time PCR (qPCR) assays. RESULTS: IGF-1 enhanced c-kit expression in c-kit-positive CSCs. The activities of the phosphoinositol 3-kinase (PI3K)/AKT signaling pathway and DNA methyltransferases (DNMTs) were enhanced, and their respective inhibitors LY294002 and 5-azacytidine (5-AZA) blunted c-kit expression. Based on the results of quantitative real-time PCR (qPCR) assays, the expression of miR-193a, which is embedded in a CpG island, was down-regulated in the IGF-1-stimulated group and negatively correlated with c-kit expression, whereas c-kit-positive CSCs infected with lentivirus carrying micro-RNA193a displayed reduced c-kit expression, migration and proliferation. CONCLUSIONS: IGF-1 upregulated c-kit expression in c-kit-positive CSCs resulting in enhanced CSC proliferation and migration by activating the PI3K/AKT/DNMT signaling pathway to epigenetically silence miR-193a, which negatively modifies the c-kit expression level.

Effects of Remote Ischaemic Conditioning on Heart Rate Variability and Cardiac Function in Patients With Mild Ischaemic Heart Failure.

BACKGROUND: Cardioprotective effects of remote ischaemic conditioning (RIC) in the setting of ischaemic heart disease have been shown recently. But the effects of RIC on heart rate variability (HRV) and cardiac function in patients with stable ischaemic heart failure (IHF) are still unknown. METHODS: Fifty patients with stable IHF were enrolled and randomly divided into RIC group and control group. Remote ischaemic conditioning treatment was performed twice a day for 6 weeks. A remote is chaemic conditioning protocol consisted of 4×5min inflation/deflation of the blood pressure cuff applied in the upper arm to create intermittent arm ischaemia. B-type natriuretic peptide (BNP), left ventricular ejection fraction (LVEF), 24-hour ambulatory electrocardiogram, and 6-minute walk distance (6MWD) were all assessed in two groups. RESULTS: Forty-seven patients completed the study. Remote ischaemic conditioning was well-tolerated by patients in the RIC group after 6 weeks treatment and LVEF showed a significant increase, from 39.2% to 43.4% (p<0.001), as well as decreased BNP, increased 6MWD and HRV, but this was not observed in the control group. In addition, the patients treated with RIC also showed improved NYHA class, LVEF, 6MWD, BNP level and HRV compared to control group. CONCLUSIONS: This study suggests that a 6-week course of RIC treatment could improve cardiac function and HRV in patients with mild and stable IHF, supporting widespread use of RIC in the daily lives of these patients.

Hypoxic Preconditioning Inhibits Hypoxia-induced Apoptosis of Cardiac Progenitor Cells via the PI3K/Akt-DNMT1-p53 Pathway.

Research has demonstrated that hypoxic preconditioning (HP) can enhance the survival and proliferation of cardiac progenitor cells (CPCs); however, the underlying mechanisms are not fully understood. Here, we report that HP of c-kit (+) CPCs inhibits p53 via the PI3K/Akt-DNMT1 pathway. First, CPCs were isolated from the hearts of C57BL/6 mice and further purified by magnetic-activated cell sorting. Next, these cells were cultured under either normoxia (H0) or HP for 6 hours (H6) followed by oxygen-serum deprivation for 24 hours (24h). Flow cytometric analysis and MTT assays revealed that hypoxia-preconditioned CPCs exhibited an increased survival rate. Western blot and quantitative real-time PCR assays showed that p53 was obviously inhibited, while DNMT1 and DNMT3ß were both significantly up-regulated by HP. Bisulphite sequencing analysis indicated that DNMT1 and DNMT3ß did not cause p53 promoter hypermethylation. A reporter gene assay and chromatin immunoprecipitation analysis further demonstrated that DNMT1 bound to the promoter locus of p53 in hypoxia-preconditioned CPCs. Together, these observations suggest that HP of CPCs could lead to p53 inhibition by up-regulating DNMT1 and DNMT3ß, which does not result in p53 promoter hypermethylation, and that DNMT1 might directly repress p53, at least in part, by binding to the p53 promoter locus.

Regulation of c-kit+ progenitor cells by stromal cell derived factor-1α in adult murine heart.

BACKGROUND: c-kit-positive cardiac progenitor cells (CPCs) have been proven suitable for stem cell therapy. CPCs marker c-kit and its ligand, the stem cell factor (SCF), are associated with the functions of proliferation and differentiation. In our previous study, we found that stromal cell-derived factor-1α (SDF-1α) could enhance the expression of c-kit. However, the mechanism is unknown. METHODS AND RESULTS: CPCs were isolated from adult mouse hearts, and c-kit-positive CPCs were purified by magnetic-activated c-kit cell sorting magnetic beads. The cells were cultured with SDF-1α, c-kit expression was measured by western blotting and qPCR, the proliferation and migration of cells were measured by CCK-8 and transwell assay, DNA methyltransferase (DNMT) mRNA were measured by qPCR, global DNMT activity was measured by DNMT activity assay kit, and DNA methylation was analysed using Sequenom's MassARRAY platform. Results showed that SDF-1α could enhance the expression of c-kit, which results in the promoting of c-kit-positive CPCs proliferation and migration. SDF-1α stimulation inhibited the expression of DNMT1, DNMT3ß, and global DNMT activity, which led to significant demethylation in c-kit-positive CPCs. CONCLUSIONS: SDF-1α signalling, via CXCR4 activation, up-regulated c-kit expression by inhibiting DNMT1 and DNMT3ß expression and global DNMT activity, and by subsequent demethylation of the c-kit gene.

Epigenetic regulation of cardiac progenitor cells marker c-kit by stromal cell derived factor-1α.

BACKGROUND: Cardiac progenitor cells (CPCs) have been proven suitable for stem cell therapy after myocardial infarction, especially c-kit(+)CPCs. CPCs marker c-kit and its ligand, the stem cell factor (SCF), are linked as c-kit/SCF axis, which is associated with the functions of proliferation and differentiation. In our previous study, we found that stromal cell-derived factor-1α (SDF-1α) could enhance the expression of c-kit. However, the mechanism is unknown. METHODS AND RESULTS: CPCs were isolated from adult mouse hearts, c-kit(+) and c-kit(-) CPCs were separated by magnetic beads. The cells were cultured with SDF-1α and CXCR4-selective antagonist AMD3100, and c-kit expression was measured by qPCR and Western blotting. Results showed that SDF-1α could enhance c-kit expression of c-kit(+)CPCs, made c-kit(-)CPCs expressing c-kit, and AMD3100 could inhibit the function of SDF-1α. After the intervention of SDF-1α and AMD3100, proliferation and migration of CPCs were measured by CCK-8 and transwell assay. Results showed that SDF-1α could enhance the proliferation and migration of both c-kit(+) and c-kit(-) CPCs, and AMD3100 could inhibit these functions. DNA methyltransferase (DNMT) mRNA were measured by qPCR, DNMT activity was measured using the DNMT activity assay kit, and DNA methylation was analyzed using Sequenom's MassARRAY platform, after the CPCs were cultured with SDF-1α. The results showed that SDF-1α stimulation inhibited the expression of DNMT1 and DNMT3ß, which are critical for the maintenance of regional DNA methylation. Global DNMT activity was also inhibited by SDF-1α. Lastly, SDF-1α treatment led to significant demethylation in both c-kit(+) and c-kit(-) CPCs. CONCLUSIONS: SDF-1α combined with CXCR4 could up-regulate c-kit expression of c-kit(+)CPCs and make c-kit(-)CPCs expressing c-kit, which result in the CPCs proliferation and migration ability improvement, through the inhibition of DNMT1 and DNMT3ß expression and global DNMT activity, as well as the subsequent demethylation of the c-kit gene.

Transforming growth factor ß1 induces the expression of collagen type I by DNA methylation in cardiac fibroblasts.

Transforming growth factor-beta (TGF-ß), a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-ß induces collagen type I alpha 1 (COL1A1) expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-ß-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-ß1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs) were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-ß1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-ß1 for 48 h. TGF-ß1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-ß1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-ß1-induced COL1A1 gene expression.