COVID-19 vaccination affects short-term anti-coagulation levels in warfarin treatment.

Vaccines against SARS-CoV-2 have been recommended across the world, yet no study has investigated whether COVID-19 vaccination influences short-term warfarin anti-coagulation levels. Patients on stable warfarin treatment who received anti-SARS-CoV-2 vaccination were prospectively enrolled and followed up for three months. INR values less than 10 days before vaccination (baseline), 3-5 days (short-term) and 6-14 days (medium-term) after vaccination were recorded as INR0, INR1, and INR2, respectively. The variations of INR values within individuals were compared, and the linear mixed effect model was used to evaluate the variations of INR values at different time points. Logistic regression analysis was performed to determine covariates related to INR variations after COVID-19 vaccination. Vaccination safety was also monitored. There was a significant difference in INR values between INR0 and INR1 (2.15 vs. 2.26, p = 0.003), yet no marked difference was found between INR0 and INR2. The linear mixed effect model also demonstrated that INR variation was significant in short-term but not in medium-term or long-term period after vaccination. Logistic regression analysis showed that no investigated covariates, including age, vaccine dose, genetic polymorphisms of VKORC1 and CYP2C9 etc., were associated with short-term INR variations. Two patients (2.11%) reported gingival hemorrhage in the short-term due to increased INR values. The overall safety of COVID-19 vaccines for patients on warfarin was satisfying. COVID-19 vaccines may significantly influence warfarin anticoagulation levels 3-5 days after vaccination. We recommend patients on warfarin to perform at least one INR monitoring within the first week after COVID-19 vaccination.

Low expression of miR­532­3p contributes to cerebral ischemia/reperfusion oxidative stress injury by directly targeting NOX2.

NADPH oxidase 2 (NOX2) is a major subtype of NOX and is responsible for the generation of reactive oxygen species (ROS) in brain tissues. MicroRNAs (miRNAs/miRs) are important epigenetic regulators of NOX2. The present study aimed to identify the role of NOX2 miRNA­targets in ischemic stroke (IS). A rat cerebral ischemia/reperfusion (CI/R) injury model and a SH­SY5Y cell hypoxia/reoxygenation (H/R) model were used to simulate IS. Gene expression levels, ROS production and apoptosis in tissue or cells were determined, and bioinformatic analysis was conducted for target prediction of miRNA. In vitro experiments, including function­gain and luciferase activity assays, were also performed to assess the roles of miRNAs. The results indicated that NOX2 was significantly increased in brain tissues subjected to I/R and in SH­SY5Y cells subjected to H/R, while the expression of miR­532­3p (putative target of NOX2) was significantly decreased in brain tissues and plasma. Overexpression of miR­532­3p significantly suppressed NOX2 expression and ROS generation in SH­SY5Y cells subjected to H/R, as well as reduced the relative luciferase activity of cells transfected with a reporter gene plasmid. Collectively, these data indicated that miR­532­3p may be a target of NOX2 and a biomarker for CI/R injury. Thus, the present study may provide a novel target for drug development and IS therapy.

AGXT2 rs37369 polymorphism predicts the renal function in patients with chronic heart failure.

Patients with chronic heart failure (CHF) are often accompanied with varying degrees of renal diseases. The purpose of this study was to identify rs37369 polymorphism of AGXT2 specific to the renal function of CHF patients. A total of 1012 southern Chinese participants, including 487 CHF patients without history of renal diseases and 525 healthy volunteers, were recruited for this study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genotypes of AGXT2 rs37369 polymorphism. Levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected to indicate the renal function of the participants. BUN level was significantly higher in CHF patients without history of renal diseases compared with healthy volunteers (p=0.000). And the similar result was also obtained for SCr (p=0.000). Besides, our results indicated that the level of BUN correlated significantly with SCr in both the CHF patients without renal diseases (r=0.4533, p<0.0001) and volunteers (r=0.2489, p<0.0001). Furthermore, we found that the AGXT2 rs37369 polymorphism could significantly affect the level of BUN in CHF patients without history of renal diseases (p=0.036, AA+AG vs GG). Patients with rs37369 GG genotype showed a significantly reduced level of BUN compared to those with the AA genotype (p=0.024), and the significant difference was still observed in the smokers of CHF patients without renal diseases (p=0.023). In conclusion, we found that CHF might induce the impairment of kidney and cause deterioration of renal function. AGXT2 rs37369 polymorphism might affect the renal function of CHF patients free from renal diseases, especially in patients with cigarette smoking.

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.

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.