Expression of serum inflammatory cytokines and oxidative stress markers and their correlation with coronary artery calcium score in patients with coronary heart disease.

Introduction: The present study was conducted to explore the expression of serum inflammatory cytokines and oxidative stress markers in patients with coronary heart disease (CHD), with an attempt to analyze their relationship with the coronary artery calcium score (CACS) by coronary computed tomography angiography (CCTA). Material and methods: It total 81 patients with coronary heart disease and 81 healthy adults were included as the observation group and the control group, respectively. The levels of serum interleukin (IL)-6 and IL-12 of the two groups were detected by ELISA, and serum superoxide dismutase (SOD) was detected by the hydroxylamine oxidation method. Micro-RNA-497-5p (miR-497-5p) was screened out as a possible new CHD biomarker and its serum level was measured by real-time fluorescence quantitative PCR. The CACS of patients in the observation group was calculated by the Agatston method to analyze the correlation between the abovementioned indexes and CACS. Results: With increase in the number of CHD lesions, the levels of IL-6, IL-12 and miR-497-5p rose gradually while the level of SOD decreased gradually. In the observation group, IL-6, IL-12 and miR-497-5p were positively correlated with CACS while SOD was negatively correlated with CACS. Conclusions: Abnormal expression levels of serum IL-6, IL-12, SOD and miR-497-5p may be able to reveal the severity of the disease, and the combination with CACS is of potential value in terms of evaluating the condition of patients harboring coronary heart disease.

Myeloid-specific knockout of SHP2 regulates PI3K/PLCγ signaling pathway to protect against early myocardial infarction injury.

OBJECTIVES: To study the effects of myeloid-specific knockout of SHP2 on early myocardial infarction and explore its molecular mechanism. METHODS: The model of myocardial infarction was established by using SHP2 in myeloid-specific knockout mice, and the effect of SHP2MAC-KO on myocardial function was detected by echocardiography. The effects of SHP2 on myocardial infarct size in myeloid-specific knockout mice was examined by TTC assay and Masson staining. Then, the detection of apoptosis was performed using TUNEL staining and inflammatory cell infiltration was observed using immunohistochemical staining. Moreover, macrophages in mouse hearts were selected by Flow Cytometry and treated with PI3K inhibitors respectively. Western blotting was then used to detect protein expression of p-SHP2 and PI3K/PLCγ signaling pathway. The phagocytic ability of cells was detected by endocytosis test, and the expression of inflammatory cytokines was detected by ELISA. RESULTS: Specific knockout of SHP2 in mice with myocardial infarction can improve the cardiac function, decrease infarct size, and reduce apoptosis as well as inflammatory cell infiltration. It also can mediate the PI3K/PLCγ signaling pathway in macrophages, which in turn enhances the endocytosis of macrophages and reduces the expression of inflammatory cytokines in macrophages. CONCLUSIONS: Myeloid-specific knockout of SHP2 regulates PI3K/PLCγ signaling pathway to protect against early myocardial infarction injury.

miR-432 exerts a protective effect against myocardial ischemia/reperfusion injury by activating the ß-catenin/HIF-1α pathway and augmenting NRF2-mediated anti-oxidative stress.

OBJECTIVE: MicroRNAs (miRNAs) have been shown to play an important role in myocardial ischemia/reperfusion (MI/R) injury. This study aimed to determine the role of miR-432 in MI/R injury. METHODS: We established a MI/R injury model by ligation/untying of the left anterior descending coronary artery, and used viral infection to regulate gene expression, such as that of miR-432 in vitro and in vivo, and used RT-qPCR to detect the expression of the gene at mRNA level. Finally, western blotting and immunochemistry analyses were used to determine the protein level. RESULTS: The results of this study show that miR-432 is upregulated in the heart following MI/R injury and that miR-432 overexpression showed a significant decrease, while miR-432 knockdown showed a significant increase in the ratio of the infarct area (IA) to the area at risk (AAR) and levels of serum creating phosphokinase (CPK). Moreover, miR-432 augmented the activation of the ß-catenin pathway and decreased the rate of apoptosis in the mice heart at 24 hours after MI/R injury by targeting RBM5. At the same time, miR-432 overexpression enhanced HIF-1α activation, while ß-catenin deletion attenuated HIF-1α activation induced by miR-432 overexpression. Importantly, ß-catenin and HIF-1α knockdown significantly increased the rate of apoptosis and the ratio of IA to AAR and levels of serum CPK induced by miR-432 overexpression at 24 hours after MI/R injury. miR-432 overexpression strongly decreased levels of SOD and GSH-PX activity, and increased levels of MDA activity and the expression of the gp91phox protein in the mice hearts at 24 hours after MI/R injury, while miR-432 knockdown exerted an opposite effect. miR-432 was also found to have increased NRF2 protein levels by targeting KEAP1 protein expression. NRF2 knockdown reversed the downregulation of the levels of gp91phox protein and MDA, while it also reversed the upregulation of the levels of SOD and GSH-PX induced by miR-432 overexpression in the heart of the mice at 24 hours after MI/R injury. CONCLUSION: miR-432 protects against MI/R injury by activating the ß-catenin/HIF-1α pathway and augmenting NRF2-mediated anti-oxidative stress.

DNA methyltransferase 1 (DNMT1) suppresses mitophagy and aggravates heart failure via the microRNA-152-3p/ETS1/RhoH axis.

DNA methyltransferase 1 (DNMT1) shows close link with heart disease. This study aimed to define the role DNMT1 plays in heart failure and determine the underlying mechanism. Expression of microRNA (miR)-152-3p, DNMT1, E26 transformation specific-1 (ETS1) and ras homolog gene family member H (RhoH) was determined by RT-qPCR and/or western blot analysis. The interaction between miR-152-3p and ETS1 was predicted and verified. Methylation of the miR-152-3p promoter region was assessed using methylation-specific PCR. H9c2 cells were chosen for in vitro assays to examine the regulatory role of DNMT1 in autophagy and mitophagy with respect to miR-152-3p/ETS1/RhoH. Doxorubicin (DOX)-induced rat models of heart failure were employed for in vivo validation. DNMT1 expression was upregulated in the heart tissues of DOX-induced rats, where it showed an inverse correlation with miR-152-3p expression. Moreover, DNMT1 was shown to enhance methylation of the miR-152-3p promoter region and suppress its expression, leading to inhibition of mitophagy in H9c2 cells. In addition, DNMT1 enhanced expression of ETS1, which further elevated RhoH expression. Moreover, ETS1-elevated RhoH reduced cell viability and promoted autophagy and mitophagy in H9c2 cells upon treatment with DOX. Next, in vivo results demonstrated that depletion of DNMT1 protected rats from heart failure in a miR-152-3p/ETS1/RhoH-dependent manner. Overall, these findings indicate that DNMT1 may inhibit expression of miR-152-3p by promoting the methylation of miR-152-3p and enhancing the expression of ETS1, thereby inducing RHOH transcriptional activation and inhibiting mitochondrial autophagy, ultimately promoting the development of heart failure.

Nesfatin-1 inhibits myocardial ischaemia/reperfusion injury through activating Akt/ERK pathway-dependent attenuation of endoplasmic reticulum stress.

Nesfatin-1 (encoded by NUCB2) is a cardiac peptide possessing protective activities against myocardial ischaemia/reperfusion (MI/R) injury. However, the regulation of NUCB2/nesfatin-1 and the molecular mechanisms underlying its roles in MI/R injury are not clear. Here, by investigating a mouse MI/R injury model developed with transient myocardial ischaemia followed by reperfusion, we found that the levels of NUCB2 transcript and nesfatin-1 amount in the heart were both decreased, suggesting a transcriptional repression of NUCB2/nesfatin-1 in response to MI/R injury. Moreover, cardiac nesfatin-1 restoration reduced infarct size, troponin T (cTnT) level and myocardial apoptosis, supporting its cardioprotection against MI/R injury in vivo. Mechanistically, the Akt/ERK pathway was activated, and in contrast, endoplasmic reticulum (ER) stress was attenuated by nesfatin-1 following MI/R injury. In an in vitro system, similar results were obtained in nesfatin-1-treated H9c2 cardiomyocytes with hypoxia/reoxygenation (H/R) injury. More importantly, the treatment of wortmannin, an inhibitor of Akt/ERK pathway, abrogated nesfatin-1 effects on attenuating ER stress and H/R injury in H9c2 cells. Furthermore, nesfatin-1-mediated protection against H/R injury also vanished in the presence of tunicamycin (TM), an ER stress inducer. Lastly, Akt/ERK inhibition reversed nesfatin-1 effects on mouse ER stress and MI/R injury in vivo. Taken together, these findings demonstrate that NUCB2/nesfatin-1 inhibits MI/R injury through attenuating ER stress, which relies on Akt/ERK pathway activation. Hence, our study provides a molecular basis for understanding how NUCB2/nesfatin-1 reduces MI/R injury.

Platelet Microparticles: A Tool to Predict Infarction Area in Rats.

Background: Whether there is a quantitative correlation between platelet microparticles (PMPs)/calpain and infarction area is still unclear. Whether present antiplatelet agents can improve myocardial infarction by influencing PMPs need to be revealed. The object of our study was to answer those questions. Methods: Male Wistar rats were used for all studies. All rats were randomly divided into five groups: sham-operated group, myocardial infarction group (blank control group), aspirin intervention group, aspirin combined with clopidogrel intervention group, and aspirin combined with ticagrelor intervention group. Venous blood and hearts were collected at day 7 following MI. ELISA was applied to detect PMPs level. Infarction size was determined by TTC staining method. The comparisons of multiple means were tested with analysis of variance. And the two-two comparisons among the means were done by Student-Newman-Keuls and LSD method. Results: PMPs level and infarction area did not differ between aspirin combined with clopidogrel intervention group and aspirin combined with ticagrelor intervention group. However, significant differences were detected between any two other groups. PMPs were decreased more in dual antiplatelet intervention group. Pearson correlation analysis showed a strong correlation between PMPs and infarction area (r = 0.90) as well as calpain 10 and infarction area (r = 0.84). We created a regression model: y = 4.61 + 0.28*x (y: infarction area, x: PMPs) to assess myocardial infarction area by PMPs level. Conclusions: Antiplatelet agents may decrease infarction areas by modifying PMPs. There was a strong correlation between PMPs and infarction area. Therefore, PMPs could be used as a tool to assess infarction area.

Beneficial effects of combined administration of Clopidogrel and Aspirin on the levels of proinflammatory cytokines, cardiac function, and prognosis in ST-segment elevation myocardial infarction: A comparative study.

OBJECTIVE: Both Aspirin and Clopidogrel are considered as effective drugs in decreasing ischemic events, which potentially contribute to a promising application regarding the cardiovascular events. In the present study, we evaluated the efficacy of the combination of both Clopidogrel and Aspirin to determine the influence among inflammatory factors, cardiac function, and treatment outcome of patients suffering from ST-segment elevation myocardial infarction (STEMI) in the Hebei province of China. METHODS: To compare the efficacy of this combination therapy with a single Aspirin treatment, we experimented in 68 patients with the administration of both Clopidogrel and Aspirin as well as another 68 patients administered only with Aspirin. An enzyme-linked immunosorbent assay was used to measure the expression of inflammatory factors, thereby evaluating the effect on inflammation. In addition, a series of indexes related to cardiac function and renal function were monitored by use of a color Doppler ultrasound and an automatic biochemical analyzer, respectively. Myocardial injury-related indicators were detected. A multivariate logistic regression analysis was performed so we could identify potential risk factors. In addition, both postoperative hemorrhages and cardiac events were observed to evaluate the treatment outcome of patients with STEMI. RESULTS: Initially, the treatment outcome revealed a better efficacy in patients treated with the combination of both Clopidogrel and Aspirin, with the patients also showing more obviously alleviated myocardial injury, better cardiac and renal functions with lower serum levels of inflammatory factors. The lower incidence of postinfarction angina, recurrent myocardial infarction, stroke, and death also provide evidence that patients showed a better outcome after treatment with both Clopidogrel and Aspirin. CONCLUSION: Taken together, the combination therapy of Clopidogrel and Aspirin provided a better improvement on both the cardiac function and outcome of STEMI patients in the Hebei province of China, with reduced inflammation as well.

Protective effects of scoparone against ischemia­reperfusion­induced myocardial injury.

The present study aimed to investigate the protective effects and molecular mechanisms of scoparone on ischemia­reperfusion (I/R) injury in primary cultured cardiac myocytes and rats. An in vivo rat model of I/R injury and an in vitro primary cultured cardiac myocyte model of oxygen­glucose deprivation/reoxygenation were used to investigate the protective effects of scoparone. Cell viability, lactate dehydrogenase (LDH) release, superoxide dismutase (SOD), creatine kinase (CK) and malondialdehyde (MDA) levels, and reactive oxygen species (ROS) production were subsequently measured. In addition, cell apoptosis was assessed by terminal deoxynucleotidyl­transferase­mediated dUTP nick end labeling staining, and myocardial infarct area (IA) was determined by triphenyl tetrazolium chloride staining. Furthermore, the protein expression levels of B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax), cytochrome c (Cyt C) and caspase­3 were assessed by western blotting. The results demonstrated that treatment with scoparone markedly increased cell viability, SOD levels and Bcl­2 protein expression, and decreased LDH release, MDA production, CK levels, ROS concentration, cell apoptotic rate, myocardial IA, and Bax, caspase­3 and Cyt C protein expression. These findings indicated that scoparone may have a protective effect against I/R injury, thus suggesting that scoparone may be a considered a potential drug for the treatment of I/R injury via the inhibition of oxidative stress and cell apoptosis.

Aging is protective against pressure overload cardiomyopathy via adaptive extracellular matrix remodeling.

When challenged by hemodynamic stress, aging hearts respond differently to young hearts. Preclinical models of heart disease should take into account the effects of age. However, in the transverse aortic constriction (TAC) model of pressure-overload cardiomyopathy, the larger aorta of aging mice has not previously been taken into account. First, we studied the aortic size in mice, and found that the aortic cross-sectional area (CSA) is 28% larger in aging mice than in young adult mice (P=0.001). We then performed TAC to make the same proportional reduction in CSA in young and aging mice. This produced the same pressure gradient across the constriction and the same rise in B-type natriuretic peptide expression. Young mice showed acute deterioration in systolic function assessed by pressure-volume loops, progressive LV remodeling on echocardiography, and a 50% mortality at 12 weeks post-TAC. In contrast, aging mice showed no acute deterioration in systolic function, much less ventricular remodeling and were protected from death. Aging mice also showed significantly increased levels of matrix metalloproteinase-3 (MMP-3; 3.2 fold increase, P<0.001) and MMP-12 (1.5-fold increase, P<0.001), which were not seen in young mice. Expression of tissue inhibitor of MMP-1 (TIMP-1) increased 8.6-fold in aging hearts vs 4.3-fold in young hearts (P<0.01). In conclusion, following size-appropriate TAC, aging mice exhibit less LV remodeling and lower mortality than young adult mice. This is associated with induction of protective ECM changes.

PGC-1α ameliorates AngiotensinII-induced eNOS dysfunction in human aortic endothelial cells.

Increasing evidences support that PGC-1α participates in regulating endothelial homeostasis, in part by mediating endothelial nitric oxide (NO) synthase (eNOS) activity and NO production. However, the molecular mechanisms by which PGC-1α regulates eNOS activity are not completely understood. In the present study, we investigated the effects of PGC-1α on eNOS dysfunction and further explore the underlying mechanisms. The results showed that PGC-1α expression was downregulated after AngiotensinII (AngII) treatment and paralleled with the decreased NO generation in human aortic endothelial cells. Overexpression of PGC-1α with adenovirus or pharmacological agonist ameliorated AngII-induced the decrease of NO generation, evidenced by the restoration of cGMP and nitrite concentration. Rather than affecting eNOS expression and uncoupling, PGC-1α inhibited AngII-induced decrease of eNOS serine 1177 phosphorylation through activation of PI3K/Akt signaling. In addition, PGC-1α overexpression suppressed AngII-induced the increase of PP2A-A/eNOS interaction and PP2A phosphatase activity, with a concomitant decrease in PP2A phosphorylation, leading to eNOS serine 1177 phosphorylation. However, pharmacological inhibition of PI3K/Akt signaling blunted the observed effect of PGC-1α on PP2A activity. Taken together, our findings suggest that PGC-1α overexpression improves AngII-induced eNOS dysfunction and that improved eNOS dysfunction is associated with activated PI3K/Akt pathway, impaired PP2A activity and reduced PP2A-A/eNOS association. These date indicate that forced PGC-1α expression may be a novel therapeutic approach for endothelial dysfunction.