Overexpression of long non-coding RNA ANRIL promotes post-ischaemic angiogenesis and improves cardiac functions by targeting Akt.

Angiogenesis is critical for re-establishing the blood supply to the surviving myocardium after myocardial infarction (MI). Long non-coding RNA ANRIL (lncRNA-ANRIL) has been reported to regulate endothelial functions in cardiovascular diseases. This study was to determine the role of lncRNA-ANRIL in Akt regulation and cardiac functions after MI. Human umbilical vein endothelial cells (HUVECs) were exposed to oxygen-glucose deprivation (OGD) to mimic in vivo ischaemia. The MI model in mice was induced by ligating left anterior descending coronary artery. OGD remarkably decreased lncRNA-ANRIL expression level, reduced the phosphorylated levels of Akt and eNOS proteins, and inhibited NO release and cell viability, which were duplicated by shRNA-mediated gene knockdown of lncRNA-ANRIL. Conversely, all these effects induced by OGD were abolished by adenovirus-mediated overexpression of lncRNA-ANRIL in HUVECs. Further, OGD impaired cell migrations and tube formations in HUVECs, which were reversed by lncRNA-ANRIL overexpression or Akt up-regulation. RNA immunoprecipitation analysis indicated that the affinity of lncRNA-ANRIL to Akt protein was increased in OGD-treated cells. In animal studies, adenovirus-mediated lncRNA-ANRIL overexpression increased the phosphorylated levels of Akt and eNOS, promoted post-ischaemic angiogenesis and improved heart functions in mice with MI surgery. LncRNA-ANRIL regulates Akt phosphorylation to improve endothelial functions, which promotes angiogenesis and improves cardiac functions in mice following MI. In this perspective, targeting lncRNA-ANRIL/Akt may be considered to develop a drug to treat angiogenesis-related diseases.

Circulating miR-182-5p and miR-5187-5p as biomarkers for the diagnosis of unprotected left main coronary artery disease.

BACKGROUND: Patients with unprotected left main coronary artery disease (uLMCAD) have high mortality rate due to sudden heart failure and acute myocardial infarction, for which reliable diagnostic biomarkers to detect this disease at an early stage are in urgent need. Circulating microRNAs (miRNAs) have emerged as a class of novel biomarkers for cardiovascular diseases. The purpose of this study was to investigate utility of miRNAs as biomarkers for early detection of uLMCAD. METHODS: High-throughput sequencing (NGS) was initially employed to compare circulating miRNA expression profiles in uLMCAD patients to that in patients without coronary artery disease (CAD) to identify candidate miRNA biomarkers. We further validated the expression of candidate miRNAs by quantitative polymerase chain reaction (qPCR) in a larger cohort. Receiver operating characteristic (ROC) analysis with multivariate logistic regression was used to evaluate the diagnostic power of candidate miRNAs individually and combined. RESULTS: MiR-182-5p, miR-199a-5p and miR-5187-5p were found significantly differentially expressed through NGS (fold changes =1.35, 1.65, 0.5, P values =0.018, 0.046, 0.030, respectively, n=5 for both uLMCAD group and non-CAD control group). In a larger cohort (n=27 for uLMCAD patient and n=38 for non-CAD controls), qPCR confirmed that expression of miR-182-5p was up-regulated (2.57-fold, P=0.011) and expression of miR-5187-5p was down-regulated (0.47-fold, P=0.018) in the plasma of uLMCAD patients. ROC analysis with multivariate logistic regression show that miR-182 and miR-5187 have an AUC score of 0.97 and 0.94 respectively, indicating high diagnostic power as biomarkers for uLMCAD. Interestingly, correlation analysis suggests that the expression of two miRNAs were independent to each other. CONCLUSIONS: These results suggested that circulating miR-182-5p and miR-5187-5p were suitable diagnostic biomarkers for uLMCAD, both potentially providing diagnostic information for discriminating uLMCAD patients from non-CAD population prior to invasive diagnostic coronary angiography (CAG).

MicroRNA-155 inhibition attenuates endoplasmic reticulum stress-induced cardiomyocyte apoptosis following myocardial infarction via reducing macrophage inflammation.

Endoplasmic reticulum stress (ERS)-induced cardiomyocyte apoptosis plays an important role in the pathological process following myocardial infarction (MI). Macrophages that express microRNA-155 (miR-155) mediate cardiac inflammation, fibrosis, and hypertrophy. Therefore, we investigated if miR-155 regulates ERS-induced cardiomyocyte apoptosis after MI using a mouse model, lipopolysaccharide (LPS)-induced rat bone marrow derived macrophages (BMDMs)and hypoxia-induced neonatal rat cardiomyocytes (NRCMs). In vivo, miR-155 levelswere significantly higher in the MI group compared to the sham group. MI increasedmacrophage infiltration, nuclear factor-κB (NF-κB) activation, ERS induced-apoptosis, and SOCS1 expression, all of which were attenuated by the miR-155 antagomir, with the exception of SOCS1 expression. Additionally, post-MI cardiac dysfunction was significantly improved by miR-155 inhibition. In vitro, LPS upregulated miR-155 expression in BMDMs, and the miR-155 antagomir decreased LPS-induced macrophage inflammation and NF-κB pathway activation, but increased expression of SOCS1. Hypoxia increased NF-κB pathway activation, ERS marker expression, and apoptosis in NRCMs. Interestingly, conditioned medium from LPS-induced macrophages in combination with the miR-155 antagomir decreased, while the miR-155 agomir increased, the hypoxia-induced effects in NRCM's. The miR-155 agomir effects were reversed by inhibiting the NF-κB pathway in cardiomyocytes. Moreover, SOCS1 knockdown in LPS-induced macrophages promoted NF-κB pathway activation and ERS-induced cardiomyocyte apoptosis in the hypoxia-induced NRCMs, but the SOCS1-siRNA-induced effects were markedly decreased by miR-155 antagomir treatment. These data suggest that miR-155 inhibition attenuates ERS-induced cardiomyocyte apoptosis after MI via reducing macrophage inflammation through the SOCS1/NF-κB pathway.

Inhibition of microRNA-155 attenuates sympathetic neural remodeling following myocardial infarction via reducing M1 macrophage polarization and inflammatory responses in mice.

Inflammation plays an important role in sympathetic neural remodeling induced by myocardial infarction (MI). MiR-155 is a vital regulator of inflammatory responses, and macrophage-secreted miR-155 promotes cardiac fibrosis and hypertrophy. However, whether miR-155 influences MI-induced sympathetic neural remodeling is not clear. Therefore, we examined the role of miR-155 in MI-induced sympathetic neural remodeling and the related mechanisms in both an mouse model and in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs). Our data showed that miR-155 expression was significantly enhanced in the myocardial tissues of MI mice compared to sham mice. Also, MI up-regulated the electrophysiological parameters, M1 macrophage polarization, inflammatory responses, and suppressor of cytokine signaling 1 (SOCS1) expression, which coincided with the increased expression of sympathetic nerve remodeling markers(nerve growth factor, tyrosine hydroxylase and growth-associated protein 43). Except for SOCS1, these proteins were attenuated by miR-155 antagomir. In vitro, LPS-stimulation promoted miR-155 expression in BMDMs. Consistent with the in vivo findings, miR-155 antagomir diminished the LPS-induced M1 macrophage polarization, nuclear factor (NF)-κB activation, and the expression of pro-inflammatory factors and nerve growth factor; but it increased the expression of SOCS1. Inversely, miR-155 agomir significantly potentiated LPS-induced pathophysiological effects in BMDMs. MiR-155 agomir-induced effects were reversed by the NF-κB inhibitor. Mechanistically, treatment with siRNA against SOCS1 augmented the aforementioned LPS-mediated activities, which were antagonized by the addition of miR-155 antagomir. In conclusion, miR-155 inhibition downregulated NGF expression via decreasing M1 macrophage polarization and inflammatory responses dependent on the SOCS1/NF-κB pathway, subsequently diminishing MI-induced sympathetic neural remodeling and ventricular arrhythmias (VAs).

Endogenous reduction of miR-185 accelerates cardiac function recovery in mice following myocardial infarction via targeting of cathepsin K.

Angiogenesis is critical for re-establishing the blood supply to the surviving myocardium after myocardial infarction (MI) in patients with acute coronary syndrome (ACS). MicroRNAs are recognised as important epigenetic regulators of endothelial function. The aim of this study was to determine the roles of microRNAs in angiogenesis. Eighteen circulating microRNAs including miR-185-5p were differently expressed in plasma from patients with ACS by high-throughput RNA sequencing. The expressional levels of miR-185-5p were dramatically reduced in hearts isolated from mice following MI and cultured human umbilical vein endothelial cells (HUVECs) under hypoxia, as determined by fluorescence in situ hybridisation and quantitative RT-PCR. Evidence from computational prediction and luciferase reporter gene activity indicated that cathepsin K (CatK) mRNA is a target of miR-185-5p. In HUVECs, miR-185-5p mimics inhibited cell proliferations, migrations and tube formations under hypoxia, while miR-185-5p inhibitors performed the opposites. Further, the inhibitory effects of miR-185-5p up-regulation on cellular functions of HUVECs were abolished by CatK gene overexpression, and adenovirus-mediated CatK gene silencing ablated these enhancive effects in HUVECs under hypoxia. In vivo studies indicated that gain-function of miR-185-5p by agomir infusion down-regulated CatK gene expression, impaired angiogenesis and delayed the recovery of cardiac functions in mice following MI. These actions of miR-185-5p agonists were mirrored by in vivo knockdown of CatK in mice with MI. Endogenous reductions of miR-185-5p in endothelial cells induced by hypoxia increase CatK gene expression to promote angiogenesis and to accelerate the recovery of cardiac function in mice following MI.

A minimally invasive approach to induce myocardial infarction in mice without thoracotomy.

Acute myocardial infarction (MI) is a leading cause of morbidity and mortality in the world. Traditional method to induce MI by left coronary artery (LCA) ligation is typically performed by an invasive approach that requires ventilation and thoracotomy, causing serious injuries in animals undergoing this surgery. We attempted to develop a minimally invasive method (MIM) to induce MI in mice. Under the guide of ultrasound, LCA ligation was performed in mice without ventilation and chest-opening. Compared to sham mice, MIM induced MI in mice as determined by triphenyltetrazolium chloride staining and Masson staining. Mice with MIM surgery revealed the reductions of LVEF, LVFS, E/A and ascending aorta (AAO) blood flow, and the elevations of S-T segment and serum cTn-I levels at 24 post-operative hours. The effects of MI induced by MIM were comparable to the effects of MI produced by traditional method in mice. Importantly, MIM increased the survival rates and caused less inflammation after the surgery of LCA ligation, compared to the surgery of traditional method. Further, MIM induced angiogenesis and apoptosis in ischaemic hearts from mice at postoperative 28 days as similarly as traditional method did. Finally, the MIM model was able to develop into the myocardial ischaemia/reperfusion model by using a balloon catheter with minor modifications. The MI model is able to be efficiently induced by a minimally invasive approach in mice without ventilation and chest-opening. This new model is potentially to be used in studying ischaemia-related heart diseases.

Induction of microRNA-199 by Nitric Oxide in Endothelial Cells Is Required for Nitrovasodilator Resistance via Targeting of Prostaglandin I2 Synthase.

BACKGROUND: Nitrates are widely used to treat coronary artery disease, but their therapeutic value is compromised by nitrate tolerance, because of the dysfunction of prostaglandin I2 synthase (PTGIS). MicroRNAs repress target gene expression and are recognized as important epigenetic regulators of endothelial function. The aim of this study was to determine whether nitrates induce nitrovasodilator resistance via microRNA-dependent repression of PTGIS gene expression. METHODS: Nitrovasodilator resistance was induced by nitroglycerin (100 mg·kg-1·d-1, 3 days) infusion in Apoe-/- mice. The responses of aortic arteries to nitric oxide donors were assessed in an organ chamber. The expression levels of microRNA-199 (miR-199)a/b were assayed by quantitative reverse transcription polymerase chain reaction or fluorescent in situ hybridization. RESULTS: In cultured human umbilical vein endothelial cells, nitric oxide donors induced miR-199a/b endogenous expression and downregulated PTGIS gene expression, both of which were reversed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt or silence of serum response factor. Evidence from computational and luciferase reporter gene analyses indicates that the seed sequence of 976 to 982 in the 3'-untranslated region of PTGIS mRNA is a target of miR-199a/b. Gain functions of miR-199a/b resulting from chemical mimics or adenovirus-mediated overexpression increased PTGIS mRNA degradation in HEK293 cells and human umbilical vein endothelial cells. Furthermore, nitroglycerin-decreased PTGIS gene expression was prevented by miR-199a/b antagomirs or was mirrored by the enforced expression of miR-199a/b in human umbilical vein endothelial cells. In Apoe-/- mice, nitroglycerin induced the ectopic expression of miR-199a/b in the carotid arterial endothelium, decreased PTGIS gene expression, and instigated nitrovasodilator resistance, all of which were abrogated by miR-199a/b antagomirs or LNA-anti-miR-199. It is important that the effects of miR-199a/b inhibitions were abolished by adenovirus-mediated PTGIS deficiency. Moreover, the enforced expression of miR-199a/b in vivo repressed PTGIS gene expression and impaired the responses of aortic arteries to nitroglycerin/sodium nitroprusside/acetylcholine/cinaciguat/riociguat, whereas the exogenous expression of the PTGIS gene prevented nitrovasodilator resistance in Apoe-/- mice subjected to nitroglycerin infusion or miR-199a/b overexpression. Finally, indomethacin, iloprost, and SQ29548 improved vasorelaxation in nitroglycerin-infused Apoe-/- mice, whereas U51605 induced nitrovasodilator resistance. In humans, the increased expressions of miR-199a/b were closely associated with nitrate tolerance. CONCLUSIONS: Nitric oxide-induced ectopic expression of miR-199a/b in endothelial cells is required for nitrovasodilator resistance via the repression of PTGIS gene expression. Clinically, miR-199a/b is a novel target for the treatment of nitrate tolerance.

Association of CKIP-1 P21A polymorphism with risk of chronic heart failure in a Chinese population.

Pathological cardiac hypertrophy is an independent risk factor for chronic heart failure. Casein kinase-2 interacting protein-1 (CKIP-1) can inhibit pathological cardiac hypertrophy. Therefore, we investigated whether CKIP-1 nonsynonymous polymorphism rs2306235 (Pro21Ala) contributes to risk and prognosis of chronic heart failure in a Chinese population.A total of 923 adult patients with chronic heart failure and 1020 age- and gender-matched healthy controls were recruited. CKIP-1 rs2306235 polymorphism was genotyped using PCR-restriction fragment length polymorphism. Additional follow-up data for 140 chronic heart failure patients was evaluated. The rs2306235 G allele was associated with an increased risk of chronic heart failure (OR = 1.38, 95% CI = 1.09-1.75, p = 0.007), especially in patients with hypertension (OR = 1.45, 95% CI = 1.09-1.75, p = 0.006) and coronary heart disease (OR = 1.41, 95% CI = 1.09-1.83, p = 0.010) after adjustment for multiple cardiovascular risk factors. However, rs2306235 polymorphism was not associated with cardiovascular mortality in chronic heart failure (p = 0.875). CKIP-1 rs2306235 polymorphism may be a risk factor for chronic heart failure in a Chinese Han population.

HMGB1 induces endothelial progenitor cells apoptosis via RAGE-dependent PERK/eIF2α pathway.

Studies have demonstrated that the high-mobility group 1B protein (HMGB1) could regulate endothelial progenitor cell (EPC) homing, but the effect of HMGB1 on EPC apoptosis and associated mechanisms are still unclear. The aim of this study was to investigate the effects of HMGB1 on EPC apoptosis and the possible involvement of the endoplasmic reticulum (ER) stress pathway. EPC apoptosis was determined by flow cytometry. The expressions of PERK, eIF2α, and CHOP were detected by western blotting. Additionally, the effects of PERK shRNA on the biological behaviors of EPCs were assessed. Our results showed that incubation of EPCs with HMGB1 (0.1-1 µg/ml) for 12-48 h induced apoptosis as well as activated ER stress transducers, as assessed by up-regulating PERK protein expression and eIF2α phosphorylation in a dose or time-dependent manner. Moreover, HMGB1-mediated EPC apoptosis and CHOP expression were dramatically suppressed by PERK shRNA or a specific eIF2α inhibitor (salubrinal). Importantly, a blocking antibody specifically targeted against RAGE (anti-RAGE antibody) markedly inhibited HMGB1-induced EPC apoptosis and ER stress marker protein (PERK, eIF2α, and CHOP) expression levels. Our novel findings suggest that HMGB1 triggered EPC apoptosis in a manner of RAGE-mediated activation of the PERK/eIF2α pathway.

MiR-15b-5p Regulates Collateral Artery Formation by Targeting AKT3 (Protein Kinase B-3).

OBJECTIVE: To identify circulating microRNAs that are differentially expressed in severe coronary heart disease with well or poorly developed collateral arteries and to investigate their mechanisms of action in vivo and in vitro. APPROACH AND RESULTS: In our study, we identified a circulating microRNA, miR-15b-5p, with low expression that, nevertheless, characterized patients with sufficient coronary collateral artery function. Moreover, in murine hindlimb ischemia model, in situ hybridization identified that miR-15b-5p was specifically expressed in vascular endothelial cells of adductors in sham group and was remarkably downregulated after femoral artery ligation. Overexpressed miR-15b-5p significantly inhibited arteriogenesis and angiogenesis in mice. In vitro, both under basal and vascular endothelial growth factor stimulation, loss-of-function or gain-of-function studies suggested that miR-15b-5p significantly promoted or depressed the migration and proliferation of endothelial cells. We identified AKT3 (protein kinase B-3) as a direct target of miR-15b-5p. Interestingly, AKT3 deficiency by injection with Chol-AKT3-siRNA obviously suppressed arteriogenesis and the recovery of blood perfusion after femoral ligation in mice. CONCLUSIONS: These results indicate that circulating miR-15b-5p is a suitable biomarker for discriminating between patients with well-developed or poorly developed collaterals. Moreover, miR-15b-5p is a key regulator of arteriogenesis and angiogenesis, which may represent a potential therapeutic target for ischemic disease.

Basigin rs8259 Polymorphism Confers Decreased Risk of Chronic Heart Failure in a Chinese Population.

Left ventricular remodeling is an essential risk factor contributing to the pathogenesis of chronic heart failure (CHF). Basigin (BSG) promotes cardiovascular inflammation and myocardial remodeling processes by induction of extracellular matrix metalloproteinases and inflammatory cytokines. BSG rs8259 polymorphism was associated with BSG expression and risk of acute coronary syndrome. Therefore, we investigated whether rs8259 polymorphism contributes to risk and prognosis of CHF in Chinese patients. In total 922 adult patients with CHF and 1107 matched healthy controls were enrolled. BSG rs8259 polymorphism was genotyped using PCR-restriction fragment length polymorphism. Whole blood BSG mRNA expression data from Genotype-Tissue Expression project was accessed. Evaluation of follow-up data was performed in only 15.2% (140) of the patients with CHF. BSG rs8259 TT genotype was associated with a decreased risk of CHF (OR = 0.83, 95% CI = 0.72-0.96, p = 0.010), especially in patients with hypertension (OR = 0.80, 95% CI = 0.68-0.95, p = 0.011) and coronary heart disease (OR = 0.81, 95% CI = 0.69-0.96, p = 0.013) after adjustment for multiple cardiovascular risk factors. Rs8259 T allele was associated with decreased BSG mRNA in whole blood from 338 healthy normal donors (p = 1.31 × 10-6). However, rs8259 polymorphism failed to exhibit an association with cardiovascular mortality (p = 0.283). BSG rs8259 polymorphism may contribute to decreased risk of CHF in a Chinese Han population.

Plasma Omentin-1 Level as a Predictor of Good Coronary Collateral Circulation.

AIM: Coronary collateral circulation (CCC) is crucial during an acute ischemic attack. Evidences showed that omentin-1 exhibited remarkable antiatherogenic effects and ischemia-induced revascularization. The aim of this study was to investigate the relationship between plasma omentin-1 levels and CCC in patients with ≥90% angiography-proven coronary occlusion. METHODS: 142 patients with ≥90% luminal diameter stenosis in at least one major epicardial coronary artery were recruited. Among them, 79 patients with Rentrop 0-1 grade were classified into the poor CCC group and 63 patients with Rentrop 2-3 grade were included into the good CCC group. The association between plasma omentin-1 levels and CCC status was assessed. RESULTS: Plasma omentin-1 level was significantly higher in patients with good CCC than those with poor CCC (566.57±26.90 vs. 492.38±19.70 ng/mL, p=0.024). Besides, omentin-1 was positively correlated with total cholesterol (TC), high-density lipoprotein, and gensini score but inversely with hyperlipidemia and body mass index (all p values＜0.05). Multivariate regression analysis indicated that omentin-1 [odds ratio (OR)=1.002, 95% confidence interval (CI): 1.000-1.004, p=0.041)], TC, the number of the diseased vessels, a higher frequency of left circumflex artery and right coronary artery, chronic total occlusion, and gensini score remained as the independent predictors of good CCC. CONCLUSION: Higher plasma omentin-1 level was associated with better CCC development. Our findings suggest that omentin-1 may be an alternative marker for adequate CCC in patients with ≥90% coronary occlusion.

DDAH1-V3 transcript might act as miR-21 sponge to maintain balance of DDAH1-V1 in cultured HUVECs.

OBJECTIVE: To investigate whether microRNA (miRNA) miR-21 regulates dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression through binding 3'-UTR region directly in human umbilical venous endothelial cells (HUVECs) and to explore whether DDAH1-V2/V3 transcripts can function as microRNA sponge, thereby modulating DDAH1-V1 expression. METHODS: The DDAH1 3'-UTR containing miR-21 recognizing sequence was cloned into PmirGLO dual-luciferase miRNA target expression plasmid to construct PmirGLO-miR-21. The plasmid and miR-21 (at concentrations of 25, 50, 100 nM, respectively) or negative control (100 nM) were co-transfected into HUVECs, luciferase activity was detected at 24 h. HUVECs were incubated with 2 µg/ml Actinomycin D for the indicated time after miR-21 (25 nM) transfection, half-lives of DDAH1 mRNA were determined. HUVECs were transfected with PmirGLO-miR-21 alone or co-transfected with miR-21 for 24 h, DDAH1 transcripts mRNA, eNOS activity and DDAH1 protein expression were determined. RESULTS: MiR-21 decreased luciferase activity of PmirGLO-miR-21 in a dose-dependent manner (P < 0.05 for 25 nM miR-21, P < 0.01 for 50 nM and 100 nM miR-21), and miR-21 inhibitor increased reporter activity of PmirGLO-miR-21 and mRNA expression of all three DDAH1 transcript variants significantly (P < 0.05, respectively). The degree of increase in endogenous DDAH1 mRNA expression by miR-21 inhibitor was more obvious for DDAH1-V3. Overexpression of miR-21 decreased mRNA expression and mRNA half-life time of all DDAH1 transcripts significantly (P < 0.05), and DDAH1-V2 displayed significantly decreased half-life time than DDAH1-V1 and -V3 with or without miR-21 transfection (P < 0.05, respectively). MiR-21 (100 nM) decreased DDAH1 protein expression and eNOS activity significantly (P < 0.05), which was reversed by PmirGLO-miR-21 transfection (P < 0.05). Transfection of PmirGLO-miR-21 alone increased intracellular miR-21 expression by approximately 5.6-fold, but only showed a trend of increase in DDAH1 protein expression. CONCLUSION: Our results confirmed DDAH1 3'-UTR as a target for miR-21, and endogenous miR-21 showed increased inhibitory effect on DDAH1-V3 transcript. DDAH1 3'-UTR, especially for DDAH1-V3, may function as miR-21 sponge to regulate DDAH1 protein expression. Modulation of miR-21-DDAH1 interaction may provide a new approach for tackling cardiovascular diseases.

Considerable impacts of AGXT2 V140I polymorphism on chronic heart failure in the Chinese population.

BACKGROUND AND AIMS: Alanine-glyoxylate aminotransferase 2 (AGXT2) polymorphisms have been extensively studied to be associated with many cardiovascular diseases, with the exception of chronic heart failure (CHF). The aim of this study was to determine whether the AGXT2 rs37369 (V140I) polymorphism is associated with risk for and prognosis of CHF in Chinese patients. METHODS: 1000 CHF patients and 1200 healthy controls were recruited and polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) was used to determine the genotypes of rs37369 polymorphism. Tube formation assay and transwell migration assay were performed to assess the effects of asymmetric dimethylarginine (ADMA) and to explore the significance of rs37369 polymorphism in the pathogenesis of CHF. 140 CHF patients underwent a median follow-up of 38.7 months by telephone. RESULTS: The rs37369 GG genotype was significantly over-represented in CHF patients compared to controls (18.9% vs 14.7%, p = 0.009) and was significantly associated with increased risk of CHF (p = 0.030), especially in patients with hypertension (p = 0.021). Besides, the rs37369 GG genotype marginally increased the risk for CHF in smokers. ADMA stimulated migration and inhibited tube formation of cultured human umbilical vein endothelial cells (HUVECs). Overexpression of AGXT2 with pcAGXT2-rs37369-A or G plasmid reversed ADMA-induced HUVECs migration and tube formation. AGXT2 rs37369-A showed increased ADMA degradation activity and marginally prolonged the lifetime of CHF patients. CONCLUSIONS: ADMA might accelerate the progression of CHF possibly by inhibiting angiogenesis and promoting migration of HUVECs. AGXT2 rs37369 polymorphism is associated with increased risk for CHF, which may due to distinct disparities of alleles in ADMA degradation.

Association of the AGXT2 V140I polymorphism with risk for coronary heart disease in a Chinese population.

AIM: Asymmetric dimethylarginine (ADMA) is a nitric oxide synthase (NOS) inhibitor that decreases NO production and promotes the development of cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) plays an important role in ADMA metabolism. This study was designed to explore the association of the AGXT2 V140I (rs37369 G＞A) polymorphism with risk for coronary heart disease (CHD) in a Chinese population. METHODS: A case-control study including 1103 controls and 942 CHD patients was performed. The patients were genotyped for rs37369 using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Plasma ADMA concentration in healthy controls was measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: The rs37369 GG genotype was significantly overrepresented in CHD patients compared to the controls (18.5% versus 14.8%, p=0.025), and it was significantly associated with increased risk for CHD in smokers (OR=2.21, 95% CI: 1.24-3.92, p=0.007) and marginally increased CHD risk for individuals with diabetes mellitus (OR=1.92; 95% CI: 0.94-3.91, p=0.074). The association between rs37369 and CHD risk was further increased in smokers with diabetes mellitus (OR=3.32, 95% CI:1.14-9.67, p=0.028). Patients who smoked and were rs37369 GG homozygous showed significantly higher plasma ADMA levels than carriers of the rs37369 A allele (p=0.004). However, in non-smokers, patients homozygous for rs37369 GG showed significantly lower plasma ADMA concentrations than carriers of the rs37369 A allele (p=0.003). Furthermore, smokers homozygous for rs37369 GG showed significantly higher plasma ADMA concentrations than non-smokers with the same genotype (p=0.012). CONCLUSION: The AGXT2 rs37369 polymorphism is associated with increased risk for CHD in smokers and in diabetes mellitus patients. This increased risk may be due to increased plasma ADMA levels.

Association of platelet ITGA2B and ITGB3 polymorphisms with ex vivo antiplatelet effect of ticagrelor in healthy Chinese male subjects.

Ticagrelor (TIC) is the first reversible P2Y12 receptor antagonist that exhibits rapid antiplatelet effect by indirect inhibition of the GPIIb/IIIa complex. Polymorphisms in genes coding GPIIb/IIIa, namely ITGA2B and ITGB3, are associated with aspirin resistance and risk for thrombotic diseases. We assessed whether ITGA2B and ITGB3 polymorphisms can influence the ex vivo antiplatelet activity of ticagrelor in Chinese population. A total of 196 healthy Chinese male individuals were recruited. ADP-induced platelet aggregation was determined using optical aggregometry at baseline and after incubation of the platelet-rich plasma with 15 and 50 µM ticagrelor, respectively. Single nucleotide polymorphisms in ITGA2B (rs5911 G>T) and ITGB3 (rs4642 A>G and rs4634 G>A) were genotyped by sequencing. TIC at both concentrations of 15 and 50 µM decreased ADP-induced platelet aggregation significantly (P < 0.05, respectively). As compared to ITGA2B rs5911 GG homozygotes, individuals with the rs5911 TG genotype showed significantly increased inhibition of platelet aggregation (IPA) by both 15 and 50 µM ticagrelor incubation (P < 0.05, respectively). Neither rs4642 nor rs4634 polymorphism affected ticagrelor-induced IPA. We suggest that the ITGA2B rs5911 GG genotype is associated with decreased ex vivo antiplatelet activity of ticagrelor in healthy Chinese male subjects.

Correlations of DDAH1 transcript variants with human endothelial asymmetric dimethylarginine metabolizing activity.

BACKGROUND: Dimethylarginine dimethylaminohydrolases 1 (DDAH1) is the major enzyme responsible for inactivation of asymmetric dimethylarginine (ADMA). This study seeks to clarify the correlations between mRNA expression levels of DDAH1 transcript variants and the relationship with ADMA metabolizing activity in human. METHODS: The mRNA expression levels of DDAH1 transcript variants in primarily cultured human umbilical vein endothelial cells (HUVECs) and peripheral blood mononuclear cells (PBMCs) from healthy control subjects and patients suffering from both acute ischemic stroke (AIS) and acute myocardial infarction (AMI) were determined by real-time polymerase chain reaction. ADMA metabolizing activity of the cell lysates from HUVECs was determined by enzyme-linked immunosorbent assay. RESULTS: A novel DDAH1 transcript variant DDAH1-V3 was identified. DDAH1-V3 mRNA expression correlated significantly with that of both -V2 (R = 0.811; P = 0.000008) and -V1 (R = 0.454; P = 0.04) in HUVECs. In PBMCs from healthy subjects, significant correlation was observed only between DDAH1-V2 and -V3 (R = 0.571; P = 0.001; n = 36). Delta threshold cycle (DCT) values for both DDAH1-V2 and -V3 transcripts were increased significantly in PBMCs from AIS patients (P < 0.05, respectively). In PBMCs from patients suffering from both AIS and AMI, positive pairwise correlations between mRNA levels of DDAH1 transcripts were also observed as analyzed by partial correlation analysis (P < 0.05, respectively). However, only mRNA expression level of the DDAH1-V1 transcript correlated significantly with intracellular ADMA metabolizing activity in HUVECs (R = 0.805; P=0.002). CONCLUSIONS: This study demonstrated that although there are positive correlations between mRNA expression levels of DDAH1 transcript variants, only the DDAH1-V1 transcript is responsible for ADMA metabolism, and transcript specific primers are recommended to determine DDAH1 mRNA expression.

4-HNE increases intracellular ADMA levels in cultured HUVECs: evidence for miR-21-dependent mechanisms.

OBJECTIVE: To investigate whether 4-hydroxynonenal (4-HNE) regulates asymmetric dimethylarginine (ADMA) metabolism through pathway independent of direct adduct formation with ADMA metabolizing enzyme and the involvement of microRNA (miRNA) miR-21 in human umbilical venous endothelial cells (HUVECs). METHODS: Cultured HUVECs were treated with 4-HNE (at concentrations of 1, 5, and 10 µM, respectively) or 1‰ DMSO (vehicle control) for 24 h. MiR-21 inhibitor (final concentration of 100 nM) was transfected at 1 h before 4-HNE treatment. HUVECs were also transfected with miR-21 (at concentrations of 50 nM and 100 nM) and cultured for 12, 24, and 48 h, respectively. DDAH mRNA and miR-21 expression in the HUVECs were determined by semi-quantitative real time PCR. DDAH1 and DDAH2 protein expression were analyzed by Western blot. ADMA in the cell medium and cell lysates were analyzed by ELISA. ADMA metabolizing activity of the cell lysates was also determined. RESULTS: MiR-21 decreased DDAH1 and DDAH2 expression and ADMA metabolic activity significantly, while increased intracellular ADMA accumulation significantly in HUVECs. 10 µM 4-HNE treatment for 24 h increased the expression of miR-21 and intracellular ADMA concentration, decreased the expression of DDAH1/2 mRNA and protein, decreased ADMA metabolizing activity of the cell lysates significantly. MiR-21 inhibitor reversed the inhibitory effects of 4-HNE on DDAH1 expression completely, and partially reversed the changes in ADMA metabolizing activity and intracellular ADMA accumulation challenged by 10 µM 4-HNE. CONCLUSION: 4-HNE down-regulates DDAH1 expression and increases intracellular ADMA accumulation in HUVECs through a miR-21-dependent mechanism.