The Acid Sphingomyelinase Inhibitor Amitriptyline Ameliorates TNF-α-Induced Endothelial Dysfunction.

PURPOSE: Inflammation associated endothelial cell (EC) dysfunction is key to atherosclerotic disease. Recent studies have demonstrated a protective role of amitriptyline in cardiomyocytes induced by hypoxia/reoxygenation. However, the mechanism by which amitriptyline regulates the inflammatory reaction in ECs remains unknown. Thus, the aim of this study was to investigate whether amitriptyline protects against inflammation in TNF-α-treated ECs. METHODS: HUVECs were incubated with amitriptyline (2.5 µM) or TNF-α (20 ng/ml) for 24 h. EdU, tube formation, transwell, DHE fluorescence staining, and monocyte adhesion assays were performed to investigate endothelial function. Thoracic aortas were isolated from mice, and vascular tone was measured with a wire myograph system. The levels of ICAM-1, VCAM-1, MCP-1, phosphorylated MAPK and NF-κB were detected using western blotting. RESULTS: Amitriptyline increased the phosphorylation of nitric oxide synthase (eNOS) and the release of NO. Amitriptyline significantly inhibited TNF-α-induced increases in ASMase activity and the release of ceramide and downregulated TNF-α-induced expression of proinflammatory proteins, including ICAM-1, VCAM-1, and MCP-1 in ECs, as well as the secretion of sICAM-1 and sVCAM-1. TNF-α treatment obviously increased monocyte adhesion and ROS production and impaired HUVEC proliferation, migration and tube formation, while amitriptyline rescued proliferation, migration, and tube formation and decreased monocyte adhesion and ROS production. Additionally, we demonstrated that amitriptyline suppressed TNF-α-induced MAPK phosphorylation as well as the activity of NF-κB in HUVECs. The results showed that the relaxation response of aortic rings to acetylcholine in the WT-TNF-α group was much lower than that in the WT group, and the sensitivity of aortic rings to acetylcholine in the WT-TNF-α group and WT-AMI-TNF-α group was significantly higher than that in the WT-TNF-α group. CONCLUSION: These results suggest that amitriptyline reduces endothelial inflammation, consequently improving vascular endothelial function. Thus, the identification of amitriptyline as a potential strategy to improve endothelial function is important for preventing vascular diseases.

MEF2A alters the proliferation, inflammation-related gene expression profiles and its silencing induces cellular senescence in human coronary endothelial cells.

BACKGROUND: Myocyte enhancer factor 2A (MEF2A) plays an important role in cell proliferation, differentiation and survival. Functional deletion or mutation in MEF2A predisposes individuals to cardiovascular disease mainly caused by vascular endothelial dysfunction. However, the effect of the inhibition of MEF2A expression on human coronary artery endothelial cells (HCAECs) is unclear. In this study, expression of MEF2A was inhibited by specific small interference RNA (siRNA), and changes in mRNA profiles in response to MEF2A knockdown were analyzed using an Agilent human mRNA array. RESULTS: Silencing of MEF2A in HCAECs accelerated cell senescence and suppressed cell proliferation. Microarray analysis identified 962 differentially expressed genes (DEGs) between the MEF2A knockdown group and the negative control group. Annotation clustering analysis showed that the DEGs were preferentially enriched in gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to proliferation, development, survival, and inflammation. Furthermore, 61 of the 578 downregulated DEGs have at least one potential MEF2A binding site in the proximal promoter and were mostly enriched in the GO terms "reproduction" and "cardiovascular." The protein-protein interaction network analyzed for the downregulated DEGs and the DEGs in the GO terms "cardiovascular" and "aging" revealed that PIK3CG, IL1B, IL8, and PRKCB were included in hot nodes, and the regulation of the longevity-associated gene PIK3CG by MEF2A has been verified at the protein level, suggesting that PIK3CG might play a key role in MEF2A knockdown induced HCAEC senescence. CONCLUSIONS: MEF2A knockdown accelerates HCAEC senescence, and the underlying molecular mechanism may be involved in down-regulation of the genes related with cell proliferation, development, inflammation and survival, in which PIK3CG may play a key role.

Calycosin inhibits oxidative stress-induced cardiomyocyte apoptosis via activating estrogen receptor-α/ß.

Oxidative stress-induced myocardial apoptosis is a key step in the pathogenesis of ischemic heart disease. Calycosin is a phytoestrogen extracted from Radix astragali. In this study, we examined the effects and mechanisms of calycosin on oxidative stress-induced myocardial apoptosis. Molecular docking showed that calycosin can couple into binding site of ERα and ß. Pretreatment with calycosin increased the expression levels of ERα and ß. In H9C2 cells, H2O2 reduced cell viability and induced apoptosis, however, calycosin diminished the effects of H2O2 in a dose-dependent manner. Pretreatment with ICI 182,780, an estrogen receptor inhibitor, negated the protective effect of calycosin against H2O2-induced apoptosis. In addition, Akt phosphorylation was upregulated by calycosin mono treatment and downregulated by co-treatment with calycosin and ICI 182,780. These data demonstrated that calycosin exhibits anti-apoptotic effects by activating ERα/ß and enhancing Akt phosphorylation in cardiomyocytes.

Genotype Frequencies of CYP2C19, P2Y12 and GPIIIa Polymorphisms in Coronary Heart Disease Patients of Han Ethnicity, and Their Impact on Clopidogrel Responsiveness.

To investigate the genotype frequencies of cytochrome P450, family2, subfamily C, polypeptide19 (CYP2C19); P2Y12 receptor; and glycoprotein IIIa polymorphisms in patients with coronary heart disease and their impact on clopidogrel responsiveness and major adverse cardiac events (MACEs).A total of 146 coronary heart disease patients of Han ethnicity, on a clopidogrel regimen, were enrolled. Polymerase chain reaction and DNA sequencing were used to detect the genotype and allelic frequencies of CYP2C19 ((*)2,(*)3,(*)17), P2Y12 (C34T, G52T, T744C) and GPIIIa (T1565C) polymorphisms. Clinical and laboratory data were compared between the high on-treatment platelet reactivity (HTPR) versus normal groups.HTPR was identified in 35 (24%) patients. CYP2C19(*)2 (G681A) polymorphism was found to be significantly associated with HTPR (P < 0.05). A allele frequencies were significantly higher in the HTPR group versus the normal group (P < 0.05). On logistic regression analysis, CYP2C19(*)2 (G681A) polymorphism was found to be an independent risk factor associated with HTPR. No link could be established between genetic polymorphisms and recurrence of MACEs, or between HTPR and recurrence of MACEs.The genetic polymorphisms in CYP2C19(*)2 were closely associated with HTPR. The frequency of the A allele of CYP2C19(*)2 was significantly associated with HTPR, with A allele carriers being more likely to develop HTPR.

C-reactive protein stimulates RAGE expression in human coronary artery endothelial cells in vitro via ROS generation and ERK/NF-κB activation.

AIM: The receptor for advanced glycation end-products (RAGE) plays an important role in development of atherosclerosis, and C-reactive protein (CRP) has been found to stimulate its expression in endothelial cells. In this study we investigated how CRP regulated the expression of RAGE in human coronary artery endothelial cells (HCAECs). METHODS: HCAECs were treated in vitro with CRP (50 µg/mL) in combination with a variety of inhibitors. ROS generation was determined by immunocytochemistry and flow cytometry. The RAGE expression and phosphorylation of relevant signaling proteins were measured using Western blot analyses. RESULTS: CRP stimulated the expression of RAGE in the cells, accompanied by markedly increased ROS generation, phosphorylation of ERK1/2 and NF-κB p65, as well as translocation of NF-κB p65 to the nuclei. CRP also stimulated phosphorylation of JNK and p38 MAPK. Pretreatment of the cells with the ROS scavenger N-acetyl-L-cysteine, ERK inhibitor PD98059 or NF-κB inhibitor PDTC blocked CRP-stimulated RAGE expression, but pretreatment with the NADPH oxidase inhibitor DPI, JNK inhibitor SP600125 or p38 MAPK inhibitor SB203580 did not significantly alter CRP-stimulated RAGE expression. CONCLUSION: CRP stimulates RAGE expression in HCAECs in vitro via ROS generation and activation of the ERK/NF-κB signaling pathway.

Decreased plasma miR-140-3p is associated with coronary artery disease.

Background: Although many circulating miRNAs (c-miRNAs) are associated with coronary artery disease (CAD), they are far from being the biomarker for CAD diagnosis or risk prediction. Therefore, novel c-miRNAs discovery and validation are still required, especially evaluating their prediction capacity. Objectives: Identify novel CAD-related c-miRNAs and evaluate its risk prediction capacity for CAD. Methods: miRNAs associated with CAD were preliminarily investigated in three paired samples representing pre-CAD stage and CAD stage of three female individuals using the Applied Biosystems miRNA TaqMan® Low-Density Array (TLDA). Then, the candidate miRNAs were further verified in an independent case-control study including 129 CAD patients and 76 controls, and their potential practical value in prediction for CAD was evaluated using a machine learning (ML) algorithm. The accuracy of classification and prediction was assessed with the area under the receiver operating characteristic curve (AUC). Results: TLDA analysis shows that miR-140-3p decreased significantly in CAD-stage (FC = -3.01, P = 0.007). Further study shows that miR-140-3p was significantly lower in CAD group [1.26 (0.68, 2.01)] than in control group [2.07 (1.19, 3.21)] (P < 0.001) and independently associated with CAD (P < 0.001). The addition of miR-140-3p to the variables including smoking history, HDL-c, and APOA1 improved the accuracy of classification by logistic regression and of prediction for CAD by ML models. The ML models built with miR-140-3p and HDL-c, respectively, had a similar prediction accuracy. The feature importance of miR-140-3p and HDL-c in the ML models was also similar. Decision curve analysis showed that miR-140-3p and HDL-c had almost identical net benefits. Conclusion: Reduced levels of miR-140-3p is linked to CAD, and it is possible to use the plasma level of miR-140-3p as a means of evaluating the risk of CAD.

MicroRNA-26 was decreased in rat cardiac hypertrophy model and may be a promising therapeutic target.

MicroRNA (miR)-26 was found to be downregulated in cardiac diseases. In this study, the critical role of miR-26 in myocardial hypertrophy in both in vivo and in vitro was investigated. Sixteen male Wistar rats that underwent sham or transverse abdominal aortic constriction (TAAC) surgery were divided into control or TAAC group. Cardiomyocytes were isolated from neonatal Sprague-Dawley rats. Our study demonstrated that miR-26a/b was downregulated in both TAAC rat model and cardiomyocytes. The results of luciferase assays also suggested that glycogen synthase kinase 3ß (GSK3ß) may be a direct target of miR-26. The overexpression of miR-26 attenuated GSK3ß expression and inhibited myocardial hypertrophy. The downregulation of miR-26 reversed these effects. Furthermore, silence of GSK3ß gene phenocopied the anti-hypertrophy effects of miR-26, whereas overexpression of this protein attenuated the effects of miR-26. Taken together, these data suggest that miR-26 regulates pathological structural changes in the rat heart, which may be associated with suppression of the GSK3ß signaling pathway, and implicate the potential application of miR-26 in diagnosis and therapy of cardiac hypertrophy.

Myocyte Enhancer Factor 2A Plays a Central Role in the Regulatory Networks of Cellular Physiopathology.

Cell regulatory networks are the determinants of cellular homeostasis. Any alteration to these networks results in the disturbance of cellular homeostasis and induces cells towards different fates. Myocyte enhancer factor 2A (MEF2A) is one of four members of the MEF2 family of transcription factors (MEF2A-D). MEF2A is highly expressed in all tissues and is involved in many cell regulatory networks including growth, differentiation, survival and death. It is also necessary for heart development, myogenesis, neuronal development and differentiation. In addition, many other important functions of MEF2A have been reported. Recent studies have shown that MEF2A can regulate different, and sometimes even mutually exclusive cellular events. How MEF2A regulates opposing cellular life processes is an interesting topic and worthy of further exploration. Here, we reviewed almost all MEF2A research papers published in English and summarized them into three main sections: 1) the association of genetic variants in MEF2A with cardiovascular disease, 2) the physiopathological functions of MEF2A, and 3) the regulation of MEF2A activity and its regulatory targets. In summary, multiple regulatory patterns for MEF2A activity and a variety of co-factors cause its transcriptional activity to switch to different target genes, thereby regulating opposing cell life processes. The association of MEF2A with numerous signaling molecules establishes a central role for MEF2A in the regulatory network of cellular physiopathology.

Apoe-knockout induces strong vascular oxidative stress and significant changes in the gene expression profile related to the pathways implicated in redox, inflammation, and endothelial function.

Apolipoprotein E (APOE) was recognized as a key regulator of lipid metabolism, which prompted the Apoe-knockout (Apoe-/-) mouse to be the most widely used atherosclerotic model. However, with more and more important physiological roles of APOE being revealed, it is necessary to reacquaint its comprehensive function in the aorta. In this study, we aimed to reveal how Apoe-knockout impacts the gene pathways and phenotypes in the aorta of mice. We performed transcriptome sequencing to acquire the gene expression profile (GEP) for C57BL/6J and Apoe-/- mouse aorta, and used enrichment analysis to reveal the signal pathways enriched for differentially expressed genes (DEGs). In addition, we used immunofluorescence and ELISA to detect the phenotypic differences of vascular tissues and plasma in the two-group mice. Apoe-knockout resulted in significant changes in the expression of 538 genes, among which about 75% were up-regulated and 134 genes were altered more than twice. In addition to the lipid metabolism pathways, DEGs were also mainly enriched in the pathways implicated in endothelial cell proliferation, migration of epithelial cells, immune regulatory, and redox. GSEA shows that the up-regulated genes are mainly enriched in 'immune regulation pathways' and 'signal regulation' pathways, while the down-regulated genes are enriched in lipid metabolism pathways, 'regulation_of_nitric_oxide_synthase_activity' and the pathways involved in redox homeostasis, including 'monooxygenase regulation', 'peroxisomes' and 'oxygen binding'. A significant increase of reactive oxygen species and a remarkable reduction of GSH/GSSG ratio were respectively observed in the vascular tissues and plasma of Apoe-/- mice. In addition, endothelin-1 significantly increased in the vascular tissue and the plasma of Apoe-/- mice. Taken together, our results suggest that besides functioning in lipid metabolism, APOE may be an important signal regulator that mediates the expression of the genes related to the pathways involved in redox, inflammation, and endothelial function. Apoe-knockout-induced strong vascular oxidative stress is also the key factor contributing to atherosclerosis.

A Machine Learning Model Based on Genetic and Traditional Cardiovascular Risk Factors to Predict Premature Coronary Artery Disease.

BACKGROUND: Premature coronary artery disease (PCAD) has a poor prognosis and a high mortality and disability rate. Accurate prediction of the risk of PCAD is very important for the prevention and early diagnosis of this disease. Machine learning (ML) has been proven a reliable method used for disease diagnosis and for building risk prediction models based on complex factors. The aim of the present study was to develop an accurate prediction model of PCAD risk that allows early intervention. METHODS: We performed retrospective analysis of single nucleotide polymorphisms (SNPs) and traditional cardiovascular risk factors (TCRFs) for 131 PCAD patients and 187 controls. The data was used to construct classifiers for the prediction of PCAD risk with the machine learning (ML) algorithms LogisticRegression (LRC), RandomForestClassifier (RFC) and GradientBoostingClassifier (GBC) in scikit-learn. Three quarters of the participants were randomly grouped into a training dataset and the rest into a test dataset. The performance of classifiers was evaluated using area under the receiver operating characteristic curve (AUC), sensitivity and concordance index. R packages were used to construct nomograms. RESULTS: Three optimized feature combinations (FCs) were identified: RS-DT-FC1 (rs2259816, rs1378577, rs10757274, rs4961, smoking, hyperlipidemia, glucose, triglycerides), RS-DT-FC2 (rs1378577, rs10757274, smoking, diabetes, hyperlipidemia, glucose, triglycerides) and RS-DT-FC3 (rs1169313, rs5082, rs9340799, rs10757274, rs1152002, smoking, hyperlipidemia, high-density lipoprotein cholesterol). These were able to build the classifiers with an AUC >0.90 and sensitivity >0.90. The nomograms built with RS-DT-FC1, RS-DT-FC2 and RS-DT-FC3 had a concordance index of 0.94, 0.94 and 0.90, respectively, when validated with the test dataset, and 0.79, 0.82 and 0.79 when validated with the training dataset. Manual prediction of the test data with the three nomograms resulted in an AUC of 0.89, 0.92 and 0.83, respectively, and a sensitivity of 0.92, 0.96 and 0.86, respectively. CONCLUSIONS: The selection of suitable features determines the performance of ML models. RS-DT-FC2 may be a suitable FC for building a high-performance prediction model of PCAD with good sensitivity and accuracy. The nomograms allow practical scoring and interpretation of each predictor and may be useful for clinicians in determining the risk of PCAD.

MEF2A Is the Trigger of Resveratrol Exerting Protection on Vascular Endothelial Cell.

Both resveratrol and myocyte enhancer factor 2A (MEF2A) may protect vascular endothelial cell (VEC) through activating the expression of SIRT1. However, the relationship between resveratrol and MEF2A is unclear. We aimed to investigate the deeper mechanism of resveratrol in protecting vascular endothelial cells and whether MEF2A plays a key role in the protective function of resveratrol. Human umbilical vein endothelial cell (HUVEC) was used for in vitro study, and small interfere RNA was used for silencing MEF2A. Silencing MEF2A in the vascular endothelium (VE) of ApoE-/- mice was performed by tail injection with adeno associated virus expressing si-mef2a-shRNA. The results showed that treatment of HUVEC with resveratrol significantly up-regulated MEF2A, and prevented H2O2-induced but not siRNA-induced down-regulation of MEF2A. Under various experimental conditions, the expression of SIRT1 changed with the level of MEF2A. Resveratrol could rescue from cell apoptosis, reduction of cell proliferation and viability induced by H2O2, but could not prevent against that caused by silencing MEF2A with siRNA. Silencing MEF2A in VE of apoE-/- mice decreased the expression of SIRT1, increased the plasma LDL-c, and abrogated the function of resveratrol on reducing triglyceride. Impaired integrity of VE and aggravated atherosclerotic lesion were observed in MEF2A silenced mice through immunofluorescence and oil red O staining, respectively. In conclusion, resveratrol enhances MEF2A expression, and the upregulation of MEF2A is required for the endothelial protective benefits of resveratrol in vitro via activating SIRT1. Our work has also explored the in vivo relevance of this signaling pathway in experimental models of atherosclerosis and lipid dysregulation, setting the stage for more comprehensive phenotyping in vivo and further defining the molecular mechanisms.

Myocyte enhancer factor 2A delays vascular endothelial cell senescence by activating the PI3K/p-Akt/SIRT1 pathway.

Myocyte enhancer factor 2A (MEF2A) dysfunction is closely related to the occurrence of senile diseases such as cardiocerebrovascular diseases, but the underlying molecular mechanism is unclear. Here, we studied the effects of MEF2A on the senescent phenotype of vascular endothelial cells (VEC) and downstream signaling pathway, and the association between plasma MEF2A levels and coronary artery disease (CAD). Results showed that MEF2A silencing promoted cell senescence and down-regulated PI3K/p-AKT/Sirtuin 1 (SIRT1) expression. MEF2A overexpression delayed cell senescence and up-regulated PI3K/p-AKT/SIRT1. Hydrogen peroxide (H2O2) treatment induced cellular senescence and down-regulated the expression of MEF2A and PI3K/p-AKT/SIRT1. MEF2A overexpression inhibited cellular senescence and the down-regulation of PI3K/p-AKT/SIRT1 induced by H2O2. Further study revealed that MEF2A directly up-regulated the expression of PIK3CA and PIK3CG through MEF2 binding sites in the promoter region. Pearson correlation and logistic regression analysis showed that the plasma level of MEF2A was negatively correlated with CAD, and with age in the controls. These results suggested that MEF2A can directly up-regulate PI3K gene expression, and one of the molecular mechanisms of delaying effect of MEF2A on VEC cell senescence was SIRT1-expression activation through the PI3K/p-Akt pathway. Moreover, the plasma MEF2A levels may be a potential biomarker for CAD risk prediction.

The analysis of the entire HLA, partial non-HLA and HPV for Chinese women with cervical cancer.

Human leukocyte antigen (HLA) alleles have been associated with an increased or decreased risk of developing cervical cancer through infection with oncogenic forms of the human papillomavirus (HPV). The aim of this study was to analyze the interactions between HPV and HLA, particularly of the entire HLA and partial non-HLA regions, in invasive cervical cancers. The results showed the distribution of HPV genotypes among patients with cervical cancer in this group: the overall prevalence of HPV DNA was 91.3% (274/300). A total of 16 HPV genotypes, HPV16, 18, 58, 33, 81, 31, 68, 39, 67, 6, 11, 26, 52, 62, 66, and 70, were detected in patients with cervical cancer. Among the cervical cancer specimens, 68.7% were positive for HPV16. Genotyping was performed with 112 microsatellite markers spanning the entire HLA region and partial region of chromosome 6q15-16.3. D6S2766 and D6S2764, which are located near to the region containing the HLA-DPB genes, were negatively related with cervical cancer (OR for the D6S2766-195 allele carriers = 0.50), and positively related with cervical cancer (OR for the D6S2764-209 allele carriers = 2.44), respectively. Significant association was found for D6S1043 (allele 132) with HPV16-positive cervical cancer (OR = 4.52). There was an increased risk for carriers of alleles 112 and 132 at the D6S1043 locus, in particular for homozygotes. The present findings indicate that the antigenic presentation of HPV-derived peptides and non-HLA regions may be involved in the pathogenesis of HPV-associated cervical cancer.

Resveratrol inhibits angiotensin II­induced proliferation of A7r5 cells and decreases neointimal hyperplasia by inhibiting the CaMKII­HDAC4 signaling pathway.

Resveratrol has been reported to inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia following arterial injury; however, the underlying mechanisms remain unclear. The present study was designed to investigate the effects of resveratrol on angiotensin II (AngII)­induced proliferation of A7r5 cells and explore the molecular mechanisms responsible for the observed effects. Resveratrol inhibited cell proliferation and migration, and decreased the AngII­induced protein expression of α­smooth muscle actin (α­SMA), proliferating cell nuclear antigen (PCNA) and cyclin­dependent kinase 4 (CDK4). Resveratrol inhibited AngII­induced activation of intracellular Ca2+/calmodulin­dependent protein kinase II (CaMKII) and histone deacetylases 4 (HDAC4), as well as blocking AngII­induced cell cycle progression from the G0/G1 to S­phase. In vivo, 4­weeks of resveratrol treatment decreased the neointima area and the neointima/media area ratio in rats following carotid balloon injury. Resveratrol also inhibited the protein expression of total and phosphorylated CaMKII and HDAC4 in the injured arteries. In conclusion, the present study demonstrated that resveratrol attenuated AngII­induced cell proliferation and neointimal hyperplasia by inhibiting the CaMKII­HDAC4 signaling pathway. These findings suggest that resveratrol may potentially prevent arterial restenosis.

Human leukocyte antigen class II alleles and risk of cervical cancer in China.

Human leukocyte antigen (HLA) class II alleles have been associated with an increased or decreased risk of developing cervical cancer through infection with oncogenic forms of human papillomavirus (HPV). To verify whether HLA class II allelic polymorphism is related to cervical cancer in China, 133 cervical cancers and 98 healthy controls were analyzed for HLA typing. Our results showed that DPB1*1301 allele frequency was significantly higher in the cervical cancers compared with healthy controls (OR, 3.793; p = 0.002; Pc = 0.04). A significant relationship was found between DRB1*150101-DQB1*0602 haplotype (OR, 0.180; p < 0.0001; Pc < 0.003), DRB1*070101-DQB1*0201 haplotype (OR, 0.110; p = 0.001; Pc = 0.03), and decreased risk for cervical cancer. Similar tendencies were observed for DRB1*150101-DQB1*0602 haplotype with HPV16 positive cervical cancers (OR, 0.182; p = 0.001; Pc = 0.021), and for DRB1*070101-DQB1*0201 haplotype (OR, 0.144; p =0.003; Pc = 0.063). These results indicate that HLA-DPB1*1301 may confer susceptibility to cervical cancer, and the haplotypes DRB1*150101-DQB1*0602 and DRB1*070101-DQB1*0201 may contribute to the resistance to the development of cervical cancer among Chinese women. The study suggests that specific HLA class II alleles and haplotypes may influence the immune response to specific HPV-encoded epitopes and affect the risk of cervical cancer in a Chinese population from an area with a high incidence of this neoplasia.

Association between HLA-DQB1 alleles and susceptibility to coronary artery disease in Southern Han Chinese.

Accumulated evidence indicates that polymorphisms in human leukocyte antigens (HLA) are associated with susceptibility to coronary artery disease (CAD). However, whether HLA-DQB1 alleles are correlated with susceptibility to CAD is unclear. In this study, significantly lower frequencies of the allele groups (DQB1*03:01:01G and DQB1*05:03:01G) and the genotypes (DQB1*03:01:01G/DQB1*03:01:01G and DQB1*03:01:01G/DQB1*05:03:01G) were observed in the CAD group compared with that in the controls. However, notably higher frequencies of DQB1*04:01:01G and genotype DQB1*05:01:01G/DQB1*03:01:01G were observed in the CAD patients than in the controls. Further analysis in subgroups showed that DQB1*03:01:01G was present at a significantly lower frequency in both female and male CAD patients compared with the corresponding controls; however, DQB1*04:01:01G was overtly high only in male CAD patients. CAD patients with diabetes showed a negative association with DQB1*03:01:01G and DQB1*05:03:01G and a positive association with DQB1*04:01:01G, DQB1*03:02:01G and DQB1*03:03:02G. Results of logistic regression analysis indicated that DQB1*03:01:01G and DQB1*05:03:01G were significantly associated with reduced susceptibility to CAD, but DQB1*04:01:01G, DQB1*03:02:01G and DQB1*03:03:02G had no correlation with CAD. Together, these findings indicate that CAD in Southern Han Chinese is negatively associated with HLA-DQB1*03:01:01G and DQB1*05:03:01G, and males with HLA-DQB1*04:01:01G are likely to have high risk for CAD.

miR-34a modulates angiotensin II-induced myocardial hypertrophy by direct inhibition of ATG9A expression and autophagic activity.

Cardiac hypertrophy is characterized by thickening myocardium and decreasing in heart chamber volume in response to mechanical or pathological stress, but the underlying molecular mechanisms remain to be defined. This study investigated altered miRNA expression and autophagic activity in pathogenesis of cardiac hypertrophy. A rat model of myocardial hypertrophy was used and confirmed by heart morphology, induction of cardiomyocyte autophagy, altered expression of autophagy-related ATG9A, LC3 II/I and p62 proteins, and decrease in miR-34a expression. The in vitro data showed that in hypertrophic cardiomyocytes induced by Ang II, miR-34a expression was downregulated, whereas ATG9A expression was up-regulated. Moreover, miR-34a was able to bind to ATG9A 3'-UTR, but not to the mutated 3'-UTR and inhibited ATG9A protein expression and autophagic activity. The latter was evaluated by autophagy-related LC3 II/I and p62 levels, TEM, and flow cytometry in rat cardiomyocytes. In addition, ATG9A expression induced either by treatment of rat cardiomyocytes with Ang II or ATG9A cDNA transfection upregulated autophagic activity and cardiomyocyte hypertrophy in both morphology and expression of hypertrophy-related genes (i.e., ANP and ß-MHC), whereas knockdown of ATG9A expression downregulated autophagic activity and cardiomyocyte hypertrophy. However, miR-34a antagonized Ang II-stimulated myocardial hypertrophy, whereas inhibition of miR-34a expression aggravated Ang II-stimulated myocardial hypertrophy (such as cardiomyocyte hypertrophy-related ANP and ß-MHC expression and cardiomyocyte morphology). This study indicates that miR-34a plays a role in regulation of Ang II-induced cardiomyocyte hypertrophy by inhibition of ATG9A expression and autophagic activity.

MiR-30-regulated autophagy mediates angiotensin II-induced myocardial hypertrophy.

Dysregulated autophagy may lead to the development of disease. Role of autophagy and the diagnostic potential of microRNAs that regulate the autophagy in cardiac hypertrophy have not been evaluated. A rat model of cardiac hypertrophy was established using transverse abdominal aortic constriction (operation group). Cardiomyocyte autophagy was enhanced in rats from the operation group, compared with those in the sham operation group. Moreover, the operation group showed up-regulation of beclin-1 (an autophagy-related gene), and down-regulation of miR-30 in cardiac tissue. The effects of inhibition and over-expression of the beclin-1 gene on the expression of hypertrophy-related genes and on autophagy were assessed. Angiotensin II-induced myocardial hypertrophy was found to be mediated by over-expression of the beclin-1 gene. A dual luciferase reporter assay confirmed that beclin-1 was a target gene of miR-30a. miR-30a induced alterations in beclin-1 gene expression and autophagy in cardiomyocytes. Treatment of cardiomyocytes with miR-30a mimic attenuated the Angiotensin II-induced up-regulation of hypertrophy-related genes and decreased in the cardiomyocyte surface area. Conversely, treatment with miR-30a inhibitor enhanced the up-regulation of hypertrophy-related genes and increased the surface area of cardiomyocytes induced by Angiotensin II. In addition, circulating miR-30 was elevated in patients with left ventricular hypertrophy, and circulating miR-30 was positively associated with left ventricular wall thickness. Collectively, these above-mentioned results suggest that Angiotensin II induces down-regulation of miR-30 in cardiomyocytes, which in turn promotes myocardial hypertrophy through excessive autophagy. Circulating miR-30 may be an important marker for the diagnosis of left ventricular hypertrophy.

Inhibition of platelet activation and aggregation by furostanol saponins isolated from the bulbs of Allium macrostemon Bunge.

Three new furostanol saponins (FSs) were recently isolated from the dried bulbs of Allium macrostemon and were shown to have antiplatelet effects. This study investigated the inhibitory capabilities of these compounds on adenosine diphosphate (ADP)-induced human platelet activation. FS-1, when compared with the other 2, had a potent inhibitory effect on ADP-induced platelet aggregation and on the expression of P-selectin and integrin ß-3. FS-1 also inhibited Ca mobilization and significantly decreased phosphorylated AKT expression in ADP-activated platelets. The suppression by FS-1 of ADP-induced platelet activation and aggregation shown in this study indicate its potential for therapeutic applications.

HLA-DRB1*12:02:01 plays a protective role against coronary artery disease in women of southern Han Chinese descent.

The pathogenesis of coronary artery disease (CAD) is closely associated with inflammation, in which human leukocyte antigens (HLA), especially HLA-DR molecules, play important roles. However, whether various HLA-DRB1 alleles can contribute differing susceptibility to CAD has not been elucidated. In this study, we demonstrated a significantly lower frequency of HLA-DRB1*12:02:01 in the CAD group (9.82%) than in controls (18.22%) after age adjustment (odds ratio [OR] = 0.489, p = 0.0036). Logistic regression analysis indicated that carriers of HLA-DRB1*12:02:01 exhibited a lower risk of CAD events after adjustment for age, gender, and other confounders (p = 0.014, OR = 0.526 [95% confidence interval 0.314-0.878]). The female carriers of HLA-DRB1*12:02:01 exhibited a much lower risk of CAD events both before (OR = 0.424, p = 0.0387) and after age adjustment (OR = 0.302, p = 0.0058). In another female cohort, the frequency of HLA-DRB1*12:02:01 also differed between the female CAD group (8.23%) and the female controls (18.75%; OR = 0.389, p = 0.0116). Further analysis indicated that HLA-DRB1*12:02:01 was not frequent in participants with premature CAD or more diseased blood vessels. In summary, our data demonstrate that HLA-DRB1*12:02:01 plays a protective role against CAD in southern Han Chinese, especially in females. The mechanism behind the protective effect requires further exploration.