Impact of hemodialysis on efficacies of the antiplatelet agents in coronary artery disease patients complicated with end-stage renal disease.

It is controversial whether hemodialysis affects the efficacy of the antiplatelet agents. We aimed to investigate the impact of hemodialysis on efficacies of the antiplatelet agents in coronary artery disease (CAD) patients complicated with end-stage renal disease (ESRD). 86 CAD patients complicated with ESRD requiring hemodialysis were consecutively enrolled. After 5-day treatment with aspirin and clopidogrel or ticagrelor, the platelet aggregations induced by arachidonic acid (PLAA) or adenosine diphosphate (PLADP), and the P2Y12 reaction unit (PRU) were measured before and after hemodialysis. The propensity matching score method was adopted to generate a control group with normal renal function from 2439 CAD patients. In patients taking aspirin, the PLAA remained unchanged after hemodialysis. In patients taking clopidogrel, the PLADP (37.26 ± 17.04 vs. 31.77 ± 16.09, p = 0.029) and corresponding clopidogrel resistance (CR) rate (23 [48.9%] vs. 14 [29.8%], p = 0.022) significantly decreased after hemodialysis, though PRU remained unchanged. Subgroup analysis indicated that PLADP significantly decreased while using polysulfone membrane (36.8 ± 17.9 vs. 31.1 ± 14.5, p = 0.024). In patients taking ticagrelor, PLADP, and PRU remained unchanged after hemodialysis. ESRD patients had higher incidences of aspirin resistance (AR) and CR compared to those with normal renal function (AR: 16.1% vs. 0%, p = 0.001; CR: 48.4% vs. 24.8%, p = 0.024). Hemodialysis does not have negative effect on the efficacies of aspirin, clopidogrel and ticagrelor in ESRD patients with CAD. ESRD patients have higher incidences of AR and CR compared with those with normal renal function.Trial registration ClinicalTrials.gov Identifier: NCT03330223, first registered January 4, 2018.

Exploration and bioinformatic prediction for profile of mRNA bound to circular RNA BTBD7_hsa_circ_0000563 in coronary artery disease.

BACKGROUND: As a novel circRNA, BTBD7_hsa_circ_0000563 has not been fully investigated in coronary artery disease (CAD). Our aim is to reveal the possible functional role and regulatory pathway of BTBD7_hsa_circ_0000563 in CAD via exploring genes combined with BTBD7_hsa_circ_0000563. METHODS: A total of 45 peripheral blood mononuclear cell (PBMC) samples of CAD patients were enrolled. The ChIRP-RNAseq assay was performed to directly explore genes bound to BTBD7_hsa_circ_0000563. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted to reveal possible functions of these genes. The interaction network was constructed by the STRING database and the Cytoscape software. The Cytoscape software were used again to identify clusters and hub genes of genes bound to BTBD7_hsa_circ_0000563. The target miRNAs of hub genes were predicted via online databases. RESULTS: In this study, a total of 221 mRNAs directly bound to BTBD7_hsa_circ_0000563 were identified in PBMCs of CAD patients via ChIRP-RNAseq. The functional enrichment analysis revealed that these mRNAs may participate in translation and necroptosis. Moreover, the interaction network showed that there may be a close relationship between these mRNAs. Eight clusters can be further subdivided from the interaction network. RPS3 and RPSA were identified as hub genes and hsa-miR-493-5p was predicted to be the target miRNA of RPS3. CONCLUSIONS: BTBD7_hsa_circ_0000563 and mRNAs directly bound to it may influence the initiation and progression of CAD, among which RPS3 and RPSA may be hub genes. These findings may provide innovative ideas for further research on CAD.

Circular RNA ZBTB46 depletion alleviates the progression of Atherosclerosis by regulating the ubiquitination and degradation of hnRNPA2B1 via the AKT/mTOR pathway.

BACKGROUND: CircZBTB46 has been identified as being associated with the risk of coronary artery disease (CAD) and has the potential to be a diagnostic biomarker for CAD. However, the specific function and detailed mechanism of circZBTB46 in CAD are still unknown. METHODS: The expression levels and properties of circRNAs were examined using qRT‒PCR, RNA FISH, and subcellular localization analysis. ApoE-/- mice fed a high-fat diet were used to establish an atherosclerosis model. HE, Masson, and Oil Red O staining were used to analyze the morphological features of the plaque. CCK-8, Transwell, and wound healing assays, and flow cytometric analysis were used to evaluate cell proliferation, migration, and apoptosis. RNA pull-down, silver staining, mass spectrometry analysis, and RNA-binding protein immunoprecipitation (RIP) were performed to identify the interacting proteins of circZBTB46. RESULTS: CircZBTB46 is highly conserved and is significantly upregulated in atherosclerotic lesions. Functional studies revealed that knockdown of circZBTB46 significantly decreased the atherosclerotic plaque area, attenuating the progression of atherosclerosis. In addition, silencing circZBTB46 inhibited cell proliferation and migration and induced apoptosis. Mechanistically, circZBTB46 physically interacted with hnRNPA2B1 and suppressed its degradation, thereby regulating cell functions and the formation of aortic atherosclerotic plaques. Additionally, circZBTB46 was identified as a functional mediator of PTEN-dependent regulation of the AKT/mTOR signaling pathway and thus affected cell proliferation and migration and induced apoptosis. CONCLUSION: Our study provides the first direct evidence that circZBTB46 functions as an important regulatory molecule for CAD progression by interacting with hnRNPA2B1 and regulating the PTEN/AKT/mTOR pathway.

Correlation between the Serum Platelet-Derived Growth Factor, Angiopoietin-1, and Severity of Coronary Heart Disease.

BACKGROUND: The expression of the platelet-derived growth factor (PDGF), angiopoietin-1 (Ang-1) in patients with coronary artery disease of different studies was inconsistent. This study was to investigate the expression of the PDGF and Ang-1 in peripheral blood and coronary artery in patients with acute coronary syndrome (ACS) and the relationship between the expression of the PDGF and Ang-1 and the severity of coronary artery disease. METHODS: A total of 81 patients with acute coronary syndrome undergoing coronary angiography were enrolled from September 2012 to December 2013. Patients with ACS included 61 patients with acute myocardial infarction (AMI group) and 20 patients with unstable angina pectoris (UAP group). The 29 patients who were hospitalized for chest pain undergoing coronary angiography without stenosis and with TIMI level 3 blood flow were selected as the control group. During coronary arteriography (CAG) or percutaneous coronary intervention (PCI), blood in the peripheral artery and in the local coronary artery was collected from all the patients. Serum PDGF and Ang-1 levels were measured by ELISA. We calculated the Gensini score of each patient with CHD according to the result of CAG. Patients with ACS were followed up, and the major adverse cardiovascular and cerebrovascular adverse events were recorded. RESULTS: In peripheral blood, the concentration of the PDGF was significantly elevated in the ACS group than that of the control group. The level of the PDGF in the AMI group was significantly higher than that in the UAP group. In coronary artery blood, the level of the PDGF in the ACS group was significantly higher than that of the UAP group. There was no significant difference in the concentration of Ang-1 in peripheral blood between patients with coronary heart disease and the control group. The concentration of Ang-1 in the coronary artery was significantly lower than that in peripheral blood. The coronary Ang-1 concentrations in the ACS group were significantly higher than those in the UAP group. The concentrations of the PDGF and Ang-1 in peripheral and coronary artery blood were positively correlated with the severity of coronary lesions. Patients with MACCE had higher PDGF and Ang-1 levels in the coronary sinus. CONCLUSION: The serum PDGF concentration in patients with acute coronary syndrome was significantly increased, especially in the local coronary artery. The serum Ang-1 in the coronary artery was significantly increased in patients with acute myocardial infarction and was related to the degree of coronary artery stenosis. Coronary sinus PDGF and Ang-1 levels can reflect the severity of lesions in patients with acute coronary syndrome.

Safety and Efficacy of Adenovirus Carrying Hepatocyte Growth Factor Gene by Percutaneous Endocardial Injection for Treating Post-infarct Heart Failure: A Phase IIa Clinical Trial.

OBJECTIVE: Our previous phase I clinical trial has confirmed the safety of Adenovirus carrying Hepatocyte Growth Factor gene (Ad-HGF) by intracoronary administration for treating severe coronary artery disease. This study was performed to evaluate the safety and efficacy of Ad-HGF by percutaneous endocardial injection for treating post-infarct heart failure. METHODS: A total of 30 patients (15 in the experimental group and 15 in the control group) with postinfarct heart failure who were not indicated to revascularization and had received the optimal standardized medication therapy were included in the study. Percutaneous endocardial Ad-HGF gene transfer was injected with a catheter-based intramyocardial delivery system in the experimental group. Safety parameters were measured and compared between baseline and follow-ups in the experimental group. The Mean Difference (MD) of efficacy parameters from baseline to 6-month follow-up was measured in both groups and compared with each other. RESULTS: No one suffered from serious adverse events in the experimental group during the 6-month follow-up. The experimental group revealed significant lower left ventricular end-diastolic dimension (LVDd) (68.5 vs. 65.8 MD: -2.69±1.08, P=0.03) and higher LVEF of both echocardiograph (35.2 vs. 39.3, MD: 4.05±0.86, P=0.0005) and single photon emission computed tomography (27.7 vs. 30.6, MD: 2.9±0.8, P=0.003) in the 6-month follow-up than that in the baseline, but the control group did not (P>0.05). Compared to the control group, the experimental group showed significant improvement ranges of lower LVDd (2.6 vs. -2.69, MD: -5.3±1.4, P=0.0009) and higher echocardiographic LVEF (-2 vs. 4.05, MD: 6.1±1.6, P=0.0008) from baseline to 6-month follow-up. CONCLUSION: Percutaneous endocardial administration of Ad-HGF is safe and potentially efficient in improving LVEF and lowering LVDd of patients with post-infarct heart failure.

Beneficial effects of muscone on cardiac remodeling in a mouse model of myocardial infarction.

Musk has been traditionally used in East Asia to alleviate the symptoms of angina pectoris. However, it remains unclear as to whether muscone, the main active ingredient of musk, has any beneficial effects on persistent myocardial ischemia in vivo. The aim of the present study was to investigate whether muscone can improve cardiac function and attenuate myocardial remodeling following myocardial infarction (MI) in mice. Mice were subjected to permanent ligation of the left anterior descending coronary artery to induce MI, and then randomly treated with muscone (2 mg/kg/day) or the vehicle (normal saline) for 3 weeks. Sham-operated mice were used as controls and were also administered the vehicle (normal saline). Treatment with muscone significantly improved cardiac function and exercise tolerance, as evidenced by the decrease in the left ventricular end-systolic diameter, left ventricular end-diastolic diameter, as well as an increase in the left ventricular ejection fraction, left ventricular fractional shortening and time to exhaustion during swimming. Pathological and morphological assessments indicated that treatment with muscone alleviated myocardial fibrosis, collagen deposition and improved the heart weight/body weight ratio. Muscone inhibited the inflammatory response by reducing the expression of transforming growth factor (TGF)­ß1, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and nuclear factor (NF)-κB. Treatment with muscone also reduced myocardial apoptosis by enhancing Bcl-2 and suppressing Bax expression. Muscone also induced the phosphorylation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS). Our results demonstrate that muscone ameliorates cardiac remodeling and dysfunction induced by MI by exerting anti-fibrotic, anti-inflammatory and anti-apoptotic effects in the ischemic myocardium.

Diagnostic and prognostic value of minor elevated cardiac troponin levels for percutaneous coronary intervention-related myocardial injury: a prospective, single-center and double-blind study.

Cardiac troponin-I (cTnI) and -T (cTnT) are sensitive and specific markers of myocardial injury. However, the role of increased cTnI and cTnT in percutaneous coronary intervention (PCI)-related myocardial injury remains controversial. In this prospective, single-center and double-blind study, we aimed to determine the diagnostic and prognostic value of cTnI as well as cTnT (cTns) in PCI-related myocardial injury in a Chinese population. A total of 1,008 patients with stable angina pectoris and non-ST-segment elevation acute coronary syndrome were recruited. The levels of cTnI and cTnT were examined before and after PCI. All patients were followed up for 26±9 months to observe the incidence of major adverse cardiac events (MACEs). Our results showed that post-PCI cTnI and/or cTnT levels were increased to more than the 99(th) percentile upper reference limit (URL) in 133 (13.2%) patients, among which 22 (2.2%) were more than 5 × 99(th) percentile URL. By univariate analysis, an elevation in cTns after PCI was not an independent predictor of increased MACEs, HR 1.35 (P  =  0.33, 95%CI: 0.74-2.46). In conclusion, our data demonstrate that the incidence of PCI-related myocardial injury is not common in a Chinese population and minor elevated cTns levels may not be a sensitive prognostic marker for MACEs.

Cardiac-specific expression of the hepatocyte growth factor (HGF) under the control of a TnIc promoter confers a heart protective effect after myocardial infarction (MI).

OBJECTIVE: Uncontrolled therapeutic gene expression and neovascularization in non-specific tissues has lowered the safety of gene therapy. The aim of the study was to identify a cardiac-specific promoter to control target gene expression in heart tissue in vitro and in vivo. METHODS: Adenovirus vectors containing the firefly luciferase or hepatocyte growth factor (HGF) genes under control of the Troponin I (TnIc) or Cytomegalievirus (CMV) promoters were transfected into cell lines or injected into the left ventricular wall of Sprague Dawley (SD) rats via thoracotomy. Myocardial infarction (MI) was induced immediately before direct injection. In vivo luciferase expression was assessed using a bioluminescence imaging system. Heart function was monitored via echocardiograph intermittently for eight weeks after injection. RESULTS: The constitutively active CMV promoter yielded luciferase expression throughout the body while luciferase expression driven by the TnIc promoter was largely restricted to the hypoxic heart. The CMV promoter was more efficient, yielding 100-1000 fold more light output than the TnIc promoter. Four weeks after injection, we observed a significant decline in the ejection fraction (EF) in saline and Ad-Null groups but a 17% increase in the Ad-CMV-HGF group. No change in EF was observed in the Ad-TnIc-HGF group. CONCLUSIONS: The adenovirus vector combined with the TnIc promoter largely restricts gene-targeted therapy in the hypoxic heart and prevents heart failure after myocardial infarction.

Beneficial effects of schisandrin B on the cardiac function in mice model of myocardial infarction.

The fruit of Schisandra chinensis has been used in the traditional Chinese medicine for thousands of years. Accumulating evidence suggests that Schisandrin B (Sch B) has cardioprotection effect on myocardial ischemia in vitro. However, it is unclear whether Sch B has beneficial effects on continuous myocardial ischemia in vivo. The aim of the present study was to investigate whether Sch B could improve cardiac function and attenuate myocardial remodeling after myocardial infarction (MI) in mice. Mice model of MI was established by permanent ligation of the left anterior descending (LAD) coronary artery. Then the MI mice were randomly treated with Sch B or vehicle alone. After treatment for 3 weeks, Sch B could increase survival rate, improve heart function and decrease infarct size compared with vehicle. Moreover, Sch B could down-regulate some inflammatory cytokines, activate eNOS pathway, inhibit cell apoptosis, and enhance cell proliferation. Further in vitro study on H9c2 cells showed similar effects of Sch B on prevention of hypoxia-induced inflammation and cell apoptosis. Taken together, our results demonstrate that Sch B can reduce inflammation, inhibit apoptosis, and improve cardiac function after ischemic injury. It represents a potential novel therapeutic approach for treatment of ischemic heart disease.

A correlation between acute kidney injury and myonecrosis after scheduled percutaneous coronary intervention.

Slight elevations in cardiac troponin I and T are frequently observed after percutaneous coronary intervention (PCI). Contrast-induced acute kidney injury (CI-AKI) is a complex syndrome induced by exposure to intravascular contrast media (CM). Currently, the relationships between the CM, pre-existing kidney insufficiency, CI-AKI, and myonecrosis after elective PCI are unclear. To investigate the relationship between CI-AKI and post-procedural myonecrosis (PMN) after PCI, we analyzed 327 non-ST-segment elevation acute coronary syndrome subjects undertaking elective PCI. The levels of cardiac troponins (cTns), cTnI and cTnT, at baseline and on at least one occasion 18-24 h after PCI were measured. We also recorded serum levels of creatinine (SCr) and the urine albumin:creatinine ratio (ACR) before coronary angiography, and 24-48 h and 48-72 h after contrast administration. A post-procedure increase in cTns was detected in 16.21% (53/327) of subjects with cTns levels >99th to 5×99th percentile upper reference limit (URL). Twenty-seven patients (8.26%) developed CI-AKI. CI-AKI occurred more often in subjects with PMN than in those without PMN (20.8% versus 5.8%, respectively, P=0.001). Multiple logistic regression analysis revealed that pre-existing microalbuminuria (MA) was an important independent predictor of PMN (OR: 3.31; 95% CI: 1.26-8.65, P=0.01). However, there was no correlation between the incidence of CI-AKI and PMN (OR: 2.38; 95% CI: 0.88-6.46, P=0.09). We conclude that pre-existing MA was not only an important independent predictor of CI-AKI but also of PMN.

Catheter-based intramyocardial delivery (NavX) of adenovirus achieves safe and accurate gene transfer in pigs.

BACKGROUND: Hepatocyte growth factor (HGF) is one of the major angiogenic factors being studied for the treatment of ischemic heart diseases. Our previous study demonstrated adenovirus-HGF was effective in myocardial ischemia models. The first clinical safety study showed a positive effect in patients with severe and diffused triple coronary disease. METHODS: 12 Pigs were randomized (1:1) to receive HGF, which was administered as five injections into the infarcted myocardium, or saline (control group). The injections were guided by EnSite NavX left ventricular electroanatomical mapping. RESULTS: The catheter-based injections caused no pericardial effusion, malignant arrhythmia or death. During mapping and injection, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, serum creatinine and creatine kinase-MB levels have no significant increase as compared to those before and after the injection in HGF group(P>0.05). HGF group has high HGF expression with Western blot, less myocardial infarct sizes by electroanatomical mapping (HGF group versus after saline group, 5.28 ± 0.55 cm(2) versus 9.06 ± 1.06 cm(2), P<0.01), better cardiac function with Gated-Single Photon Emission Computed Tomography compared with those in saline group. Histological, strongly increased lectin-positive microvessels and microvessel density were found in the myocardial ischemic regions in HGF group. CONCLUSION: Intramyocardial injection guided by NavX system provides a method of feasible and safe percutaneous gene transfer to myocardial infarct regions.

Beneficial effects of ginsenoside-Rg1 on ischemia-induced angiogenesis in diabetic mice.

Neovascularization and the formation of collateral vessels are often impaired in diabetes mellitus (DM) population compared with non-diabetics. Alterations in vascular endothelial growth factor (VEGF) signaling and endothelial nitric oxide synthase (eNOS) dysfunction have been confirmed to play a crucial role in impaired neovascularization in diabetic mice. Accumulating data have suggested that Rg1, a main component of Panax ginseng, has the ability to promote tubulogenesis of human umbilical vein endothelial cells (HUVECs) in vitro, and that the mechanism involves increased expression level of VEGF as well as increased eNOS activation. Thus, we speculated that Rg1 might also have therapeutic effects on the impairment of neovascularization in diabetic individuals. The aim of the present study was to investigate whether Rg1 could improve angiogenesis in ischemic hindlimb of diabetic mice in vivo. Our data demonstrated that Rg1 treatment resulted in improved angiogenesis in the diabetic ischemic hindlimb, and the potential mechanism might involve increased eNOS activation, upregulated VEGF expression, and inhibited apoptosis. Our results suggest that Rg1 may be used as a novel and useful adjunctive drug for the therapy of peripheral arterial disease in DM.

Effect of the calcium sensing receptor on rat bone marrow-derived mesenchymal stem cell proliferation through the ERK1/2 pathway.

Migration and proliferation of bone marrowderived mesenchymal stem cells (BMSCs) is critical to treatment of ischemic injury. The calcium sensing receptor (CaSR) has an important role in maintaining systemic calcium homeostasis, which is related to cell proliferation, apoptosis and paracrine signaling. We hypothesize that CaSR may enhance BMSC proliferation. Rat BMSCs were incubated with various calcium concentrations for 48 h in vitro to activate CaSR. To investigate potential mechanisms responsible for growth enhancement by calcium, the rat BMSC cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS), and induction of apoptosis confirmed by cytofluorimetric analysis using propidium iodide and Annexin V-FITC double staining. Since the mitogen-activated protein kinase (MAPK) signaling pathway was one of the most significantly affected by CaSR, MAPK activation was measured by western blotting. Calcium exposure significantly enhanced rat BMSCs proliferation, as well as the proportion of the population in S phase, in a dose-dependent manner, effects which were abolished by NPS2390 (a CaSR antagonist) and U0126 (a MEK1/2 inhibitor). These results demonstrate that CaSR is involved in rat BMSC proliferation, as seen by an increased proliferation index, decreased apoptosis, and ERK1/2 activation, and provide important insight into the cellular and molecular mechanisms by which CaSR affects cell proliferation. A CaSR agonist may prove useful to enhance BMSC survival during transplantation.

[Activation of calcium-sensing receptors is associated with apoptosis in cardiomyocytes under simulated ischemia/reperfusion].

OBJECTIVE: To examine the association of activation of calcium-sensing receptors (CaSR) with apoptosis in cardiomyocytes under simulated ischemia/reperfusion. METHODS: Ventricular cardiomyocytes of neonatal rats were incubated in ischemia-mimetic solution for 2 h, then re-incubated in normal culture medium for 24 h to establish a model of simulated ischemia/reperfusion (I/R). Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay). The expression of CaSR mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of Caspase -3 and Bcl-2 was detected by Western blotting. RESULT: The simulated I/R enhanced the expression of CaSR and cardiomyocyte apoptosis. GdCl(3), a specific activator of CaSR, further increased the expression of CaSR and cardiomyocyte apoptosis, along with upregulation of Caspase-3 and downregulation of Bcl-2. CONCLUSION: CaSR is associated with I/R injury and apoptosis in neonatal rat ventricular cardiomyocytes via suppressing Bcl-2 and promoting Caspase -3 expression.

Toxic effects of a high dose of non-ionic iodinated contrast media on renal glomerular and aortic endothelial cells in aged rats in vivo.

Iodinated contrast media (CM) can induce apoptosis and necrosis of renal tubular cells. The injuries of endothelial cells induced by CM on the systemic condition have not been fully understood. To assess the toxic effects of non-ionic CM on the glomerular and aortic endothelial cells, iopromide and iodixanol, two kinds of representative non-ionic CM, were used for the in vivo study. Sixty aged rats were respectively received the agents or normal sodium intravascularly. No obvious apoptosis and morphological change was detected in the glomerular and aortic endothelial cells apart from renal tubules after CM administration. However, expressions of the nitric oxide synthase (eNOS) in glomerular endothelium were decreased at 12h after CM injection. Furthermore, plasma creatinine and endothelin-1 were increased and plasma nitric oxide (NO) was decreased significantly after CM administration. However, we failed to observe the significant increase of plasma von Willebrand Factor. These results suggest that non-ionic iodinated CM do not induce apoptosis and necrosis of glomerular and aortic endothelial cells in vivo. Decreased eNOS expression and increased plasma endothelin-1 may be involved in non-ionic iodinated CM-induced endothelial dysfunction and kidney injury.

Coexpression of VEGF and angiopoietin-1 promotes angiogenesis and cardiomyocyte proliferation reduces apoptosis in porcine myocardial infarction (MI) heart.

VEGF and angiopoietin-1 (Ang1) are two major angiogenic factors being investigated for the treatment of myocardial infarction (MI). Targeting VEGF and Ang1 expression in the ischemic myocardium can increase their local therapeutic effects and reduce possible adverse effects. Adeno-associated viral vectors (AAVs) expressing cardiac-specific and hypoxia-inducible VEGF [AAV-myosin light chain-2v (MLC)VEGF] and Ang1 (AAV-MLCAng1) were coinjected (VEGF/Ang1 group) into six different sites of the porcine myocardium at the peri-infarct zone immediately after ligating the left descending coronary artery. An identical dose of AAV-Cytomegalovirus (CMV)LacZ or saline was injected into control animals. AAV genomes were detected in the liver in addition to the heart. RT-PCR, Western blotting, and ELISA analyses showed that VEGF and Ang1 were predominantly expressed in the myocardium in the infarct core and border of the infarct heart. Gated single-photon emission computed tomography analyses showed that the VEGF/Ang1 group had better cardiac function and myocardial perfusion at 8 wk than at 2 wk after vector injection. Compared with the saline and LacZ controls, the VEGF/Ang1 group expressed higher phosphorylated Akt and Bcl-xL, less Caspase-3 and Bad, and had higher vascular density, more proliferating cardiomyocytes, and less apoptotic cells in the infarct and peri-infarct zones. Thus, cardiac-specific and hypoxia-induced coexpression of VEGF and Ang1 improves the perfusion and function of porcine MI heart through the induction of angiogenesis and cardiomyocyte proliferation, activation of prosurvival pathways, and reduction of cell apoptosis.

Inhibitory effect of hepatocyte growth factor on cardiomyocytes apoptosis is partly related to reduced calcium sensing receptor expression during a model of simulated ischemia/reperfusion.

Calcium-sensing receptors (CaSR) are G-protein coupled receptors which maintain systemic calcium haemeostasis, participate in hormone secretion, activation of iron channel, cell apoptosis, proliferation and differentiation. Previous studies have show CaSR induce apoptosis in isolated rat adult heart and in normal rat neonatal cardiomyocytes by G-protein-PLC-IP3 signaling transinduction. A few of studies had demonstrated that CaSR induce apoptosis in cultured neonatal rat cardiomyocytes during ischemia/reperfusion. Hepatocyte growth factor (HGF), as a mesenchymally derived heterodimeric glycoprotein, play vital role in mitogenesis, angiogenesis, cellular motility and growth and anti-apoptosis after postinfarction heart failure via activation of transmembrane tyrosine kinase cell surface receptor c-Met. However, little knowledge exists about whether anti-apoptotic role of HGF in preventing cardiomyocytes injury induced by ischemia/reperfusion is associated with downregulation of CaSR expression. We incubated primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2 h, then reincubated them in normal culture medium for 24 h to establish a model of simulated ischemia/reperfusion (I/R). Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The expression of CaSR mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). In addition, we analyzed the expression of Caspase-3, Bcl-2 and Phosphoinositide 3-kinase (PI3K) by Western blotting. The simulated I/R enhances the expression of CaSR and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSR, further increase the expression of CaSR and Cardiomyocyte apoptosis, along with upregulation of Caspase-3, downregulation of Bcl-2 and inhibiting PI3K phosphorylation. Combination of GdCl3 with LY294002 (a selective PI3K inhibitor) increased Cardiomyocytes apoptosis but did not increased CaSR expression. Treatment of HGF decreased I/R- and GdCl3-induced apoptosis by suppressing Caspase-3 and promoting Bcl-2 and PI3K phosphorylation expression in accordance with downregulation of CaSR expression. HGF exerts protective role in I/R-induced apoptosis at least in part by inhibiting CaSR expression along with promoting Bcl-2, suppressing Caspase-3 expression and stimulating PI3K phosphorylation signaling pathway.

[Hepatocyte growth factor attenuates ischemia/reperfusion induced cardiomyocyte apoptosis via downregulating calcium sensing receptor expression].

OBJECTIVE: To examine whether the anti-apoptotic effect of hepatocyte growth factor (HGF) in cardiomyocytes underwent ischemia/reperfusion (I/R) is associated with downregulation of calcium sensing receptor (CaSR) mRNA expression. METHODS: Neonatal rat cardiomyocytes were isolated and randomly divided into 7 groups: control, I/R, GdCl(3), GdCl(3) + NiCl(2) + CdCl(2), GdCl(3) + LY294002, GdCl(3) + HGF, GdCl(3) + HGF + LY294002.I/R was established by incubating primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2 h, then reincubated in normal culture medium for 24 h. Cardiomyocyte apoptosis was detected by TUNEL. The expression of CaSR mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of Caspase-3, Bcl-2 and Phosphoinositide-3 Kinase (PI3K) was analyzed by Western blot. RESULTS: I/R enhanced the expression of CaSR mRNA (I/R: 2.62 ± 0.41, control: 1.00 ± 0.31, P < 0.01) and cardiomyocyte apoptosis [I/R: (15.32 ± 2.54)%, control: (2.90 ± 1.45)%, P < 0.01]. GdCl(3) further increased the expression of CaSR mRNA (GdCl(3): 4.46 ± 0.62, I/R: 2.62 ± 0.41, P < 0.01) and cardiomyocyte apoptosis [GdCl(3): (25.36 ± 2.60)%, I/R: (15.32 ± 2.54)%, P < 0.01], along with upregulation of Caspase-3 (GdCl(3): 1.93 ± 0.28, I/R: 1.50 ± 0.21, P < 0.01), downregulation of Bcl-2 (GdCl(3): 0.82 ± 0.18, I/R: 1.71 ± 0.30, P < 0.01) and PI3K phosphorylation inhibition (I/R: 0.87 ± 0.08, GdCl(3): 0.61 ± 0.07, P < 0.01). Combination of GdCl(3) with LY294002 further enhanced cardiomyocytes apoptosis [GdCl(3) + LY294002: (32.6 ± 3.42)%, GdCl(3): (25.36 ± 2.60)%, P < 0.01] but did not affect CaSR mRNA expression (GdCl(3) + LY294002: 4.27 ± 0.56, GdCl(3): 4.46 ± 0.62, P > 0.05). HGF decreased I/R- and GdCl(3)-induced apoptosis [GdCl(3) + HGF: (11.8 ± 1.89)%, GdCl(3): (25.36 ± 2.60)%, P < 0.05] by suppressing Caspase-3 (GdCl(3) + HGF: 1.12 ± 0.23, (GdCl(3): 1.93 ± 0.28, P < 0.05; GdCl(3) + HGF + LY294002: 1.87 ± 0.31, GdCl(3) + LY294002: 3.86 ± 0.47, P < 0.05) and promoting Bcl-2 (GdCl(3) + HGF: 2.56 ± 0.54, GdCl(3): 0.82 ± 0.18, P < 0.05; GdCl(3) + HGF + LY294002: 1.68 ± 0.28, GdCl(3) + LY294002: 0.68 ± 0.13, P < 0.05) and PI3K phosphorylation expression (GdCl(3) + HGF: 2.87 ± 0.21, GdCl(3): 0.61 ± 0.07, P < 0.05; GdCl(3) + HGF + LY294002: 2.01 ± 0.14, GdCl(3) + LY294002: 0.44 ± 0.10, P < 0.05) in accordance with downregulation of CaSR mRNA expression (GdCl(3) + HGF: 1.46 ± 0.37, GdCl(3): 4.46 ± 0.62, P < 0.01). CONCLUSION: HGF exerts protective role in I/R-induced apoptosis at least in part by inhibiting CaSR mRNA expression along with promoting Bcl-2, suppressing Caspase-3 expression and stimulating PI3K phosphorylation signaling pathway.

HGF percutaneous endocardial injection induces cardiomyocyte proliferation and rescues cardiac function in pigs.

OBJECTIVE: To investigate the effect of cardiomyocyte proliferation induced by human hepatocyte growth factor (HGF) in pigs with chronic myocardial infarction (CMI). METHODS: A steerable, deflectable 7F catheter incorporating a 27-guage needle was advanced percutaneously to the left ventricular myocardium of 18 pigs with CMI. Pigs were randomized (1:1:1) to receive adenoviral vector HGF (total dose, 1×10(10) genome copies), which was administered as five injections into the infarcted myocardium (total, 1.0 mL), or saline, or Ad-null (control groups). Injections were guided by Ensite NavX left ventricular electroanatomical mapping. HGF and cyclin proteins were detected by western blot and immunoprecipitation analysis. Histological and immunohistochemical analysis determined proliferating cardiomyocytes. Myocardial perfusion and cardiac function were estimated by Gated-Single Photon Emission Computed Tomography (G-SPECT). RESULTS: Western blot analyses showed that HGF were predominantly expressed in the infarct core and border in the myocardium of the infarcted heart. G-SPECT analysis indicated that the HGF group had better cardiac function and myocardial perfusion four weeks after the injection of Ad-HGF than before the injection of Ad-HGF. After treatment there were more proliferating cardiomyocytes in the HGF group compared to either of the control groups. Furthermore, the HGF group myocardial samples expressed higher levels of p-Akt, cyclin A, cyclin E, cyclin D1, cdk2, cdk4 than those in the control groups. CONCLUSION: The over-expression of HGF activates pro-survival pathways, induces cardiomyocyte proliferation, and improves the perfusion and function of the porcine CMI heart.

Activation of calcium-sensing receptors is associated with apoptosis in a model of simulated cardiomyocytes ischemia/reperfusion.

OBJECTIVE: Calcium-sensing receptors (CaSRs) are G-protein coupled receptors which maintain systemic calcium homeostasis and participate in hormone secretion, activation of ion channels, cell apoptosis, proliferation, and differentiation. Previous studies have shown that CaSRs induce apoptosis in isolated adult rat heart and in normal neonatal rat cardiomyocytes by G-protein-PLC-IP3 signaling transduction. However, little knowledge is presently available concerning the role of CaSRs in the apoptosis induced by ischemia and reperfusion in neonatal cardiomyocytes. METHODS: Primary neonatal rat ventricular cardiomyocytes were incubated in ischemiamimetic solution for 2 h, and then re-incubated in normal culture medium for 24 h to establish a model of simulated ischemia/reperfusion (I/R). Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The expression of CaSRs mRNA was detected by real-time reverse transcription polymerase chain reaction (RT-PCR). In addition, the expressions of caspase-3 and Bcl-2 were analyzed by western blot. RESULTS: The simulated I/R enhanced the expression of CaSRs and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSRs, further increased the expression of CaSRs and cardiomyocyte apoptosis, along with up-regulation of caspase-3 and down-regulation of Bcl-2. CONCLUSION: CaSRs are associated with I/R injury and apoptosis in neonatal rat ventricular cardiomyocytes via suppressing Bcl-2 and promoting caspase-3 expression.