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The vascular endothelium is the first line of defense to prevent cardiovascular disease. Its optimal functioning and health are maintained by the interaction of the proteins—endothelial nitric oxide synthase (eNOS), sirtuin 1 (SIRT1), and endothelin 1 (ET1)—and the genes that encode them—NOS3, SIRT1, and EDN1, respectively. Aerobic exercise improves endothelial function by allegedly increasing endothelial shear stress (ESS). However, there are no current data exploring the acute effects of specific exercise-induced ESS intensities on these regulatory proteins and genes that are associated with endothelial function. The purpose of this study was to assess the acute changes in endothelial proteins and gene expression after exposure to low-, moderate-, and high-intensity exercise-induced ESS. Human umbilical vein endothelial cells (HUVECs) were exposed to resting ESS (18 dynes/cm2, 60 pulses per minute (PPM)), low ESS (35 dynes/cm2, 100 PPM), moderate ESS (50 dynes/cm2, 120 PPM), and high ESS (70 dynes/cm2, 150 PPM). Protein and gene expression were quantified by fluorescent Western blot and RTqPCR, respectively. All exercise conditions showed an increase in eNOS and SIRT1 expression and a decrease in NOS3 and SIRT1 gene expression when compared to resting conditions. In addition, there was no expression of ET1 and an increase in EDN1 gene expression when compared to resting conditions. These results show that (1) exercise-induced ESS increases the expressions of vascular protective proteins and (2) there is an inverse relationship between the proteins and their encoding genes immediately after exercise-induced ESS, suggesting that exercise has a previously unexplored translational role catalyzing mRNA to proteins.

Purpose: Genetic factors are important in terms of athletic performance. Recent studies to determine the relationship between the genes that lead to physiological responses have attracted attention. In this respect, this meta-analysis study was designed to examine the relationship between genetic polymorphism (BDKRB2 rs5810761, GNB3 rs5443, HIF1A rs11549565, MCT1 rs1049434, NOS3 rs2070744) and endurance athlete's status. Methods: The search included studies published from 2009 to 2022. To determine the relevant studies, Pubmed, Web of Science databases were systematically scanned. Only case-control studies were included in the meta-analysis. To determine the relevant studies, Pubmed, Web of Science databases were systematically scanned, and a total of 31 studies met the criteria for inclusion in the meta-analysis. Relevant data from the included studies were collected and analyzed using a random effects or fixed effects model. The effect size was calculated as the odds ratio or a risk ratio the corresponding 95% confidence intervals. Results: According to the results of the analysis, BDKRB2 rs5810761 + 9 allele, and NOS3 rs2070744 T allele were significantly more prevalent in endurance athletes (p < 0.05). Genotype distributions of BDKRB2 rs5810761, MCT1 rs1049434, and NOS3 rs2070744 showed significant differences in the dominant model (p < 0.05). However, no significant association was found between endurance athlete status and GNB3 rs5443 and HIF1A rs11549465 polymorphisms. Conclusion: These results show that some gene polymorphisms play an important role in endurance athlete status and suggest that having a specific genetic basis may also confer a physiological advantage for performance. [ABSTRACT FROM AUTHOR]

Cerebral ischemia is a severe neurological disability related to neuronal apoptosis and cellular stress response. Circular RNAs (circRNAs) are emerging regulators of cerebral ischemia. Herein, this study proposed to probe the action of circ_0000115 in cerebral ischemia injury. The mouse neuroblastoma cells N2a and HT22 underwent oxygen–glucose deprivation (OGD) were used as a model of in vitro cerebral ischemia. Levels of genes and proteins were detected by qRT-PCR and western blotting. Cell proliferation and apoptosis were determined by EdU assay and flow cytometry. Western blotting was used to detect the protein level of pro-inflammatory factors. The oxidative stress injury was evaluated by detecting reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) generation. Dual-luciferase reporter and RIP assays were used to confirm the target relationship between miR-1224-5p and circ_0000115 or nitric oxide synthase 3 (NOS3). OGD exposure decreased circ_0000115 and NOS3 expression, and increased miR-1224-5p in N2a and HT22 cells in a time-dependent manner. Circ_0000115 silencing attenuated OGD-induced apoptosis, oxidative stress and inflammation in N2a and HT22 cells. Mechanistically, circ_0000115 directly sponged miR-1224-5p, which targeted NOS3. Furthermore, rescue experiments showed that miR-1224-5p overexpression abolished the neuroprotective effect of circ_0000115 in N2a and HT22 cells under OGD treatment. Besides that, silencing of miR-1224-5p protected N2a and HT22 cells against OGD-evoked injury, which was counteracted by NOS3 knockdown. Circ_0000115 protects N2a and HT22 cells against OGD-evoked neuronal apoptosis, inflammation, and oxidative stress via the miR-1224-5p/NOS3 axis, providing an exciting view of the pathogenesis of cerebral ischemia. [ABSTRACT FROM AUTHOR]

Background G6PD (glucose‐6‐phosphate‐dehydrogenase) is a key enzyme in the glycolytic pathway and has been implicated in the pathogenesis of cancer and pulmonary hypertension‐associated vascular remodeling. Here, we investigated the role of an X‐linked G6pd mutation (N126D polymorphism), which is known to increase the risk of cardiovascular disease in individuals from sub‐Saharan Africa and many others with African ancestry, in the pathogenesis of pulmonary hypertension induced by a vascular endothelial cell growth factor receptor blocker used for treating cancer. Methods and Results CRISPR‐Cas9 genome editing was used to generate the G6pd variant (N126D; G6pdN126D) in rats. A single dose of the vascular endothelial cell growth factor receptor blocker sugen‐5416 (SU; 20 mg/kg in DMSO), which is currently in a Phase 2/3 clinical trial for cancer treatment, was subcutaneously injected into G6pdN126D rats and their wild‐type littermates. After 8 weeks of normoxic conditions, right ventricular pressure and hypertrophy, pulmonary artery remodeling, the metabolic profile, and cytokine expression were assessed. Right ventricular pressure and pulmonary arterial wall thickness were increased in G6PDN126D+SU/normoxic rats. Simultaneously, levels of oxidized glutathione, inositol triphosphate, and intracellular Ca2+ were increased in the lungs of G6PDN126D+SU/normoxic rats, whereas nitric oxide was decreased. Also increased in G6PDN126D+SU/normoxic rats were pulmonary levels of plasminogen activator inhibitor‐1, thrombin‐antithrombin complex, and expression of proinflammatory cytokines CCL3 (chemokine [C‐C motif] ligand), CCL5, and CCL7. Conclusions Our results suggest G6PDN126D increases inositol triphosphate‐Ca2+ signaling, inflammation, thrombosis, and hypertrophic pulmonary artery remodeling in SU‐treated rats. This suggests an increased risk of vascular endothelial cell growth factor receptor blocker‐induced pulmonary hypertension in those carrying this G6PD variant.

• Variation on CβS , MTHFR and ACE1 genes are associated with leiomyoma onset. • Genetic variation on NOS3 is associated with a lower leiomyoma uterus volume. • Epistatic interaction between NOS3 and ACE1 genes modulate leiomyoma onset. • Genetics of the systemic vascular system can play a role in the onset of leiomyoma. The link between the systemic vasculature system and tumor biology is here investigated by studying the contribution of CβS (844ins68), MTHFR (677C > T), NOS3 (4a/4b), CYBA (C242T), and ACE1 (I/D) genes to leiomyoma onset, uterus and leiomyoma volumes. DNA samples from 130 women with leiomyomas and 527 from healthy women were genotyped by PCR or PCR-RFLP. Qui-square (χ2) or Fisher's exact test were used to test associations. All the mentioned tests were performed in IBM® SPSS® Statistics Version 28. Statistical significance was defined as a p-value < 0.05. Results revealed that CβS (in the codominant and allelic models, p = 0.044 and, p = 0.015, OR = 1.791 [1.114–2.879], respectively), MTHFR (in the codominant, allelic and dominant models, p = 0.009, p = 0.002, OR = 0.585 [0.416–0.824] and p = 0.003, OR = 0.527 [0.346–0.802], respectively) and ACE1 (dominant model, p = 0.045, OR = 0.639 [0.411–0.992]) genes are associated with leiomyoma onset. NOS3 4a4a genotype is associated with a lower uterus volume (p = 0.004). This study also uncovers intriguing epistatic interactions among some genes that further accentuate their roles in disease modulation. Indeed, the epistatic interactions between the CC genotype (MTHFR) and (+/+) (CβS; p = 0.003), 4b4b (NOS3; p = 0.006 , OR = 2.050 [1.223–3.439]) or DD (ACE1 ; p < 0.001, OR = 2.362 [1.438–3.880]) were shown to be associated with the disease, while 4a presence (NOS3) in epistasis with I presence (ACE1), increased the effect protection having just the I allele presence (p = 0.029, OR = 0.446 [0.214–0.930]). We conclude that variation in genes related to the systemic vascular system can play a role in the onset and development of leiomyoma. [ABSTRACT FROM AUTHOR]

Objective: Axitinib is an oral multi-target tyrosine kinase inhibitor used for the treatment of renal cell carcinoma (RCC). Because of the severe adverse events (AEs) associated with axitinib, patients often need dose reductions or discontinue its use, highlighting the need for effective biomarkers to assess efficacy and/or AEs. The aim of this study was to investigate the relationship between single nucleotide polymorphisms (SNPs) in genes involved in the pharmacodynamic action of axitinib and clinical prognosis and AEs in metastatic RCC (mRCC) patients. Methods: This study included 80 mRCC patients treated with first-, second-, or third-line axitinib (5 mg orally twice daily). Clinical parameters and genetic polymorphisms were examined in 75 cases (53 males and 22 females). We assessed three SNPs in each of three candidate genes namely, angiotensinconverting enzyme (ACE), nitric oxide synthase 3 (NOS3), and angiotensin II receptor type 1 (AT1R), all of which are involved in axitinib effects on vascular endothelial function. Results: Axitinib-treated patients carrying the ACE deletion allele suffered more frequently from handfoot syndrome and a deterioration in kidney function (p = .045 and p = 0.005, respectively) whereas those carrying the NOS3 G allele suffered more frequently from proteinuria and multiple AEs (p = .025 and p = 0.036, respectively). Conclusions: Our study found that the ACE deletion allele and the NOS3 G allele are associated with increased AEs. [ABSTRACT FROM AUTHOR]

Myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic stem-cell diseases with excessive proliferation of one or more blood cell lines. In this study, we evaluated the effect of different oxygen concentrations on HIF-1α and NOS3 gene expression to determine the effect of the bone marrow microenvironment on JAK2 V617F positive Philadelphia chromosome negative (Ph−) MPNs. Peripheral blood mononuclear cells (MNC) of 12 patients with Ph− MPN were collected. The presence of JAK2 V617F allele status was determined with allele-specific nested PCR analysis. MPN CD34+ and CD34depleted populations were isolated from MNC by magnetic beads. Separate cell cultures of CD34+/depleted populations were managed at different oxygen concentrations including anoxia (∼0%), hypoxia (∼3%), and normoxia (∼20%) conditions for 24 ​h. HIF-1α and NOS3 gene expression changes were examined in each population related to JAK2 V617F status with real time RT-PCR. It was revealed that relative HIF-1α and NOS3 expressions were significantly increased in response to decreased oxygen concentration in all samples. Relative HIF-1α and NOS3 expressions were found to be higher especially in CD34+ and CD34depleted populations carrying JAK2 V617F mutations compared to MPN patients carrying wild-type JAK2. JAK2 V617F might have specific role in HIF-1α and NOS3 regulations with respect to low oxygen concentrations in Ph− MPN. Further evaluations might reveal the effect of JAK2 V617F on Ph− MPN pathogenesis in bone marrow microenvironment. [ABSTRACT FROM AUTHOR]

There is a hypothesis that a sufficient level of endothelial nitric oxide synthase is important for reliable protection against COVID-19. Theoretical ideas about the NOS3 gene demonstrated that it can have an effect on links of the complications pathogenesis in COVID-associated pneumonia. We determined the goal – to investigate the association of the NOS3 gene variants with the occurrence of the disease and its clinical course in patients of the intensive care unit. The study group included 117 patients with a diagnosis of severe "viral COVID-19 pneumonia". Determination of NOS3 gene variants was performed using the PCR method. The probability of differences in the quantitative results were determined using ANOVA or Kruskal-Wallis test (depend of normality of studied parameters). Our results indicate that the presence of the NOS3 gene 4a allele increase the risk of complicated COVID-19-associated pneumonia (χ2 = 18.84, p = 0.00001, OR = 3.53 (1.95–6.39)). It was showed, that carriers of the 4aa genotype had a significantly higher ratio of SpO 2 /FiO 2 on the first and second days after hospitalization (p = 0.017 and p = 0.03, respectively). Patients with the 4aa genotype also had the acid-base imbalances, as showed by indicators of base deficiency and standard bicarbonate, which were beyond the reference values. Potassium and sodium concentrations on the first and second day after hospitalization were also significantly lower in patients with 4aa genotype (p = 0.009 and p = 0.048, respectively), for whom, in the same time, the concentrations of C-reactive protein and total bilirubin were significantly higher (p = 0.002 and p = 0.033, respectively). Our results confirmed that the rs61722009 variant of the NOS3 gene is associated with an increased risk of severe СOVID-19-associated pneumonia and its adverse clinical course with potential progression of kidney and liver damage, and occurrence risk of systemic inflammatory response syndrome. These results require further research for the new metabolic strategy formation, in order to prevent the severe COVID-19 associated pneumonia and its complications. • The presence of the NOS3 gene 4a allele increase the risk of complicated COVID-19-associated pneumonia. • Patients with the 4aa genotype had the acid-base imbalances. • Patients with the 4aa genotype had higher levels of total bilirubin and С-reactive protein. [ABSTRACT FROM AUTHOR]

Relevance – it is promising to develop a system for the diagnosis and prevention of primary open-angle glaucoma (POAG), which would be based on the determination of constitutive propensity factors and would be able to predict the onset and progression of the disease. The aim of the study was to develop a model for predicting the rate of progression of primary open-angle glaucoma depending on gender and endothelial NO-synthase (NOS3) gene polymorphism. There were genotyped 153 patients with POAG aged 36-84 years. Genotypes rs1799983 and rs207074 were determined in the blood of patients by real-time polymerase chain reaction (amplifier Gene Amp® PCR System 7500; USA) using the TaqMan Mutation Detection Assays Life-Technology test system (USA). For mathematical processing of the obtained results, the Statistica 10 program (StatSoft, Inc., USA) was used. The rate of progression of POAG was higher in the presence of risk alleles in the haplotype of the NOS3 gene polymorphisms: T rs1799983 and C rs2070744 (haplotypes TT-CC, GT-CC and GT-CT), which, when distributed by gender, was more pronounced in women than in men of each of the possible haplotypes. The maximum difference was noted for carriers of the TT-SS haplotype, in whom the rate of progression of POAG in women exceeded that in men by 1.4 times (p

Allele typing of single-nucleotide polymorphisms (SNPs) may be used in predictive medicine and to determine targets for the most effective treatment strategies for various diseases. The purpose of the present work was to investigate the association between the SNPs of inflammatory genes, e.g., IL10 (C819T; rs1800871; C592A; rs1800872); IL4 (C589T; rs2243250); fibrosis-related factors - TGFβ1 (G915C; rs1800471); MMP1 (1607insG; rs1799750); apoptosis-regulators (TNFRSF11B G1181C; rs2073618); vasoconstricting factors (CRP C3872T; rs1205); CYP1A1 (A2454G; rs1048943), endothelial dysfunction (EDN1 G925T; rs5370); (NOS3 C786T; rs2070744) and development of coronary heart disorders, breast cancer, bronchial asthma (BA) and threatened miscarriage in early pregnancy among population of the Republic of Adygea.DNA samples of unrelated donors and patients (n = 74) with verified diagnoses of bronchial asthma (n = 13), coronary heart disease (n = 10), breast cancer (n = 10) and threatened miscarriage in the first trimester of pregnancy (n = 8) were isolated from peripheral blood leukocytes and typed by allele-specific polymerase chain reaction with electrophoretic detection of results using commercial tests-systems of NPF “Litech”, Moscow.The study in a group of Adygea residents has revealed the statistical significance for the “normal” Arg25-allelic variant of the TGFβ1 gene (p < 0.05; F = 0.038; OR = 3.231; 95% CI = 1.081-9.656) in the development of bronchial asthma. There were no significant differences in SNP rs1800471 of the TGFβ1 gene in the groups with cardiovascular, oncological diseases and gestational disorders (p > 0.05). The frequency distribution of allelic variants NOS3 C786T; TNFRSF11B G1181C; 1607insG of the MMP1 gene; G925T of the EDN1 gene, and CYP1A1 2454G in the examined patients with cardiovascular disease and breast cancer did not significantly differ from the control group (p > 0.05). The statistical significance for the frequency of allelic variants rs1799750 (MMP1 gene) in cases of threatened early miscarriage and in women with a physiological course of pregnancy (F = 0.096; p < 0.05%: OR = 6.0) was close to reliable, but with a confidence interval > 1.0 (95% CI = 0,98036,716), thus requiring further research.The obtained data could be sufficient in order to suggest predisposition for bronchial asthma, as well as to develop a set of preventive measures taking into account the individual characteristics of each patient.

Nitric oxide synthase 3 (NOS3) is a major vasoprotective enzyme that catalyzes the conversion of l‐arginine to nitric oxide (NO) in response to a significant number of signaling pathways. Here, we provide evidence that NOS3 interactions with MAP kinases have physiological relevance. Binding interactions of NOS3 with c‐Jun N‐terminal kinase (JNK1α1), p38α, and ERK2 were characterized using optical biosensing with full‐length NOS3 and NOS3 specific peptides and phosphopeptides. Like p38α and ERK2, JNK1α1 exhibited high‐affinity binding to full‐length NOS3 (KD 15 nm). Rate constants exhibited fast‐on, slow‐off binding (kon = 4106 m−1s−1; koff = 6.2 × 10‐5 s−1). Further analysis using synthetic NOS3 peptides revealed two MAP kinase binding sites unique to NOS3. p38α evinced similar affinity with both NOS3 binding sites. For ERK2 and JNK1α1, the affinity at the two sites differed. However, NOS3 peptides with a phosphate at either S114 or S633 did not meaningfully interact with the kinases. Immunoblotting revealed that each kinase phosphorylated NOS3 with a unique pattern. JNK1α1 predominantly phosphorylated NOS3 at S114, ERK2 at S600, and p38α phosphorylated both residues. In vitro production of NO was unchanged by phosphorylation at these sites. In human microvascular endothelial cells, endogenous interactions of all the MAP kinases with NOS3 were captured using proximity ligation assay in resting cells. Our results underscore the importance of MAP kinase interactions, identifying two unique NOS3 interaction sites with potential for modulation by MAP kinase phosphorylation (S114) and other signaling inputs, like protein kinase A (S633).

Hypertension is an independent risk factor for atherosclerosis. However, few models of hypertensive atherosclerosis have been established in medical research. In this study, we crossed the ApoE knockout (ApoE-KO; ApoE−/−) atherosclerotic mouse model with the NOS3 knockout (NOS3-KO; NOS3−/−) hypertensive mouse model to establish an ApoE/NOS3 double knockout (ApoE/NOS3-KO; ApoE/NOS3−/−) hypertensive atherosclerosis mouse model. We found that ApoE/NOS3−/− mice reproduced normally, had a blood pressure of 133.00 ± 3.85 mmHg, and developed hypertensive fundus retinopathy and hypertensive nephropathy. In addition, serum total cholesterol (TC) and low-density lipoprotein (LDL) levels in the blood were abnormally elevated, steatosis was observed in the liver cells, and atherosclerotic lesions were observed in the aortic vessels in ApoE/NOS3−/− adult mice. In conclusion, ApoE/NOS3−/− adult mice are a satisfactory model of hypertension and atherosclerosis and can be utilized for studies on cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Relevance – it is promising to develop a system for the diagnosis and prevention of primary open-angle glaucoma (POAG), which would be based on the determination of constitutive propensity factors and would be able to predict the onset and progression of the disease. The aim of the study was to develop a model for predicting the rate of progression of primary open-angle glaucoma depending on gender and endothelial NO-synthase (NOS3) gene polymorphism. There were genotyped 153 patients with POAG aged 36-84 years. Genotypes rs1799983 and rs207074 were determined in the blood of patients by real-time polymerase chain reaction (amplifier Gene Amp® PCR System 7500; USA) using the TaqMan Mutation Detection Assays Life-Technology test system (USA). For mathematical processing of the obtained results, the Statistica 10 program (StatSoft, Inc., USA) was used. The rate of progression of POAG was higher in the presence of risk alleles in the haplotype of the NOS3 gene polymorphisms: T rs1799983 and C rs2070744 (haplotypes TT-CC, GT-CC and GT-CT), which, when distributed by gender, was more pronounced in women than in men of each of the possible haplotypes. The maximum difference was noted for carriers of the TT-SS haplotype, in whom the rate of progression of POAG in women exceeded that in men by 1.4 times (p<0.001). A regression model was built with satisfactory prediction indicators (multiple correlation coefficient R=0.963; coefficient of determination R² =0.928; p<0.001). The probable “hereditary” age of patients in which one or another stage of POAG should be expected is calculated. Gender and haplotypes rs1799983 and rs2070744 of the NOS3 gene were shown to be associated with the onset and rate of progression of POAG, which was implemented in the prognostic model of the disease. Separately for men and women, carriers of different haplotypes, the rate of progression and the possible age of POAG development by stages were calculated. [ABSTRACT FROM AUTHOR]

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Background: It is estimated that 1-5% of couples suffer from recurrent pregnancy loss (RPL). Recent studies have shown the effects of gene polymorphisms in RPL. Objective: The aim of this study was to evaluate 3 gene polymorphisms including rs1048943 of CYP1A1, rs28371725 of CYP2D6, and rs7830 of NOS3 in idiopathic RPL to identify their association with RPL. Materials and Methods: Blood samples were collected from 136 women with at least 2 consecutive idiopathic miscarriages (case group) and 136 women with no history of miscarriage and at least one successful pregnancy (control group) from the Iranian Azeri population. This study was carried out between April 2018-April 2020. Amplification-refractory mutation system polymerase chain reaction was used for the rs7830, rs1048943 and rs28371725 polymorphisms in order to genotype each extracted genomic DNA sample. After that, Chi-square, Fisher’s exact test and logistic regression were used to investigate whether each of these polymorphisms is associated with RPL. Results: Among these polymorphisms, only rs1048943 of CYP1A1 showed a statistically significant association with RPL in the Iranian Azeri women studied. Conclusion: Our results suggest that CYP1A1 gene polymorphisms might be associated with a reduced risk of RPL. Further studies in other populations and in the same population with a larger sample size, as well as functional genomics analyses such as gene expression analyses or epigenetic studies are required to validate our results. [ABSTRACT FROM AUTHOR]

Background: G6PD (glucose-6-phosphate-dehydrogenase) is a key enzyme in the glycolytic pathway and has been implicated in the pathogenesis of cancer and pulmonary hypertension-associated vascular remodeling. Here, we investigated the role of an X-linked G6pd mutation (N126D polymorphism), which is known to increase the risk of cardiovascular disease in individuals from sub-Saharan Africa and many others with African ancestry, in the pathogenesis of pulmonary hypertension induced by a vascular endothelial cell growth factor receptor blocker used for treating cancer., Methods and Results: CRISPR-Cas9 genome editing was used to generate the G6pd variant (N126D; G6pd N126D ) in rats. A single dose of the vascular endothelial cell growth factor receptor blocker sugen-5416 (SU; 20 mg/kg in DMSO), which is currently in a Phase 2/3 clinical trial for cancer treatment, was subcutaneously injected into G6pd N126D rats and their wild-type littermates. After 8 weeks of normoxic conditions, right ventricular pressure and hypertrophy, pulmonary artery remodeling, the metabolic profile, and cytokine expression were assessed. Right ventricular pressure and pulmonary arterial wall thickness were increased in G6PD N126D +SU/normoxic rats. Simultaneously, levels of oxidized glutathione, inositol triphosphate, and intracellular Ca 2+ were increased in the lungs of G6PD N126D +SU/normoxic rats, whereas nitric oxide was decreased. Also increased in G6PD N126D +SU/normoxic rats were pulmonary levels of plasminogen activator inhibitor-1, thrombin-antithrombin complex, and expression of proinflammatory cytokines CCL3 (chemokine [C-C motif] ligand), CCL5, and CCL7., Conclusions: Our results suggest G6PD N126D increases inositol triphosphate-Ca 2+ signaling, inflammation, thrombosis, and hypertrophic pulmonary artery remodeling in SU-treated rats. This suggests an increased risk of vascular endothelial cell growth factor receptor blocker-induced pulmonary hypertension in those carrying this G6PD variant.

The vascular endothelium is the first line of defense to prevent cardiovascular disease. Its optimal functioning and health are maintained by the interaction of the proteins-endothelial nitric oxide synthase (eNOS), sirtuin 1 (SIRT1), and endothelin 1 (ET1)-and the genes that encode them- NOS3 , SIRT1 , and EDN1 , respectively. Aerobic exercise improves endothelial function by allegedly increasing endothelial shear stress (ESS). However, there are no current data exploring the acute effects of specific exercise-induced ESS intensities on these regulatory proteins and genes that are associated with endothelial function. The purpose of this study was to assess the acute changes in endothelial proteins and gene expression after exposure to low-, moderate-, and high-intensity exercise-induced ESS. Human umbilical vein endothelial cells (HUVECs) were exposed to resting ESS (18 dynes/cm 2 , 60 pulses per minute (PPM)), low ESS (35 dynes/cm 2 , 100 PPM), moderate ESS (50 dynes/cm 2 , 120 PPM), and high ESS (70 dynes/cm 2 , 150 PPM). Protein and gene expression were quantified by fluorescent Western blot and RTqPCR, respectively. All exercise conditions showed an increase in eNOS and SIRT1 expression and a decrease in NOS3 and SIRT1 gene expression when compared to resting conditions. In addition, there was no expression of ET1 and an increase in EDN1 gene expression when compared to resting conditions. These results show that (1) exercise-induced ESS increases the expressions of vascular protective proteins and (2) there is an inverse relationship between the proteins and their encoding genes immediately after exercise-induced ESS, suggesting that exercise has a previously unexplored translational role catalyzing mRNA to proteins.

Nitric oxide synthase 3 (NOS3) is a major vasoprotective enzyme that catalyzes the conversion of L-arginine to nitric oxide (NO) in response to a significant number of signaling pathways. Here, we provide evidence that NOS3 interactions with MAP kinases have physiological relevance. Binding interactions of NOS3 with c-Jun N-terminal kinase (JNK1α1), p38α, and ERK2 were characterized using optical biosensing with full-length NOS3 and NOS3 specific peptides and phosphopeptides. Like p38α and ERK2, JNK1a1 exhibited high-affinity binding to full-length NOS3 (KD 15 nM). Rate constants exhibited fast-on, slow-off binding (kon = 4106 M-1 s-1 ; koff = 6.2 9 10-5 s-1 ). Further analysis using synthetic NOS3 peptides revealed two MAP kinase binding sites unique to NOS3. p38α evinced similar affinity with both NOS3 binding sites. For ERK2 and JNK1a1, the affinity at the two sites differed. However, NOS3 peptides with a phosphate at either S114 or S633 did not meaningfully interact with the kinases. Immunoblotting revealed that each kinase phosphorylated NOS3 with a unique pattern. JNK1α1 predominantly phosphorylated NOS3 at S114, ERK2 at S600, and p38α phosphorylated both residues. In vitro production of NO was unchanged by phosphorylation at these sites. In human microvascular endothelial cells, endogenous interactions of all the MAP kinases with NOS3 were captured using proximity ligation assay in resting cells. Our results underscore the importance of MAP kinase interactions, identifying two unique NOS3 interaction sites with potential for modulation by MAP kinase phosphorylation (S114) and other signaling inputs, like protein kinase A (S633). [ABSTRACT FROM AUTHOR]

Nitric oxide synthases (NOS) are the major sources of nitric oxide (NO), a small bioactive molecule involved in the regulation of many cellular processes. One of the most prominent functions of NO is regulation of vasodilatation and thereby control of blood pressure. Most important for vascular tone is NOS3. Endothelial NOS3-generated NO diffuses into the vascular smooth muscle cells, activates the soluble guanylate cyclase resulting in enhanced cGMP concentrations and smooth muscle cell relaxation. However, more and more evidence exist that also NOS1 and NOS2 contribute to vascular function. We summarize the current knowledge about the regulation of NOS expression in the vasculature by transcriptional, post-transcriptional and post-translational mechanisms, in regard to inflammation and innate immune pathways.

Polymorphisms located within NOS3 gene have been investigated as susceptibility variants for diabetic nephropathy (DN) in type 2 diabetes mellitus (T2DM) in a large number of studies. However, these previous articles yielded inconsistent results and we aimed at elucidating the impact of NOS3 variants on DN risk in T2DM by conducting an updated systematic data synthesis. A total of 36 studies (12,807 participants) were selected for qualitative data synthesis, while 33 records with 11,649 subjects were included in the meta-analysis. The pooled analysis demonstrated the association of minor alleles of rs2070744 and rs1799983 with an increased susceptibility to DN (P < 0.001 and P = 0.015 for allelic model, respectively). For both of these variants, a significant effect of subgrouping according to ethnicity was found. Rs869109213 displayed an association with DN susceptibility, with pooled effect measures indicating a predisposing effect of the minor allele a (P rec = 0.002, OR rec = 1.960, 95%CI 1.288–2.983; P aa vs. bb = 0.001, OR aa vs. bb = 2.014, 95%CI 1.316–3.083). These findings support the effects of NOS3 variants on the risk of developing DN in T2DM. [Display omitted] • A total of 36 studies were selected for qualitative data synthesis. • 33 records with 11,649 subjects were included in the meta-analysis. • Minor alleles of rs2070744 and rs1799983 associate with diabetic nephropathy risk. • Allele a of rs869109213 increases susceptibility to diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Objective: Axitinib is an oral multi-target tyrosine kinase inhibitor used for the treatment of renal cell carcinoma (RCC). Because of the severe adverse events (AEs) associated with axitinib, patients often need dose reductions or discontinue its use, highlighting the need for effective biomarkers to assess efficacy and/or AEs. The aim of this study was to investigate the relationship between single nucleotide polymorphisms (SNPs) in genes involved in the pharmacodynamic action of axitinib and clinical prognosis and AEs in metastatic RCC (mRCC) patients., Methods: This study included 80 mRCC patients treated with first-, second-, or third-line axitinib (5 mg orally twice daily). Clinical parameters and genetic polymorphisms were examined in 75 cases (53 males and 22 females). We assessed three SNPs in each of three candidate genes namely, angiotensin-converting enzyme (ACE), nitric oxide synthase 3 (NOS3), and angiotensin II receptor type 1 (AT1R), all of which are involved in axitinib effects on vascular endothelial function., Results: Axitinib-treated patients carrying the ACE deletion allele suffered more frequently from hand-foot syndrome and a deterioration in kidney function (p  = .045 and p  =  0.005, respectively) whereas those carrying the NOS3 G allele suffered more frequently from proteinuria and multiple AEs (p  = .025 and p  =  0.036, respectively)., Conclusions: Our study found that the ACE deletion allele and the NOS3 G allele are associated with increased AEs.

Essential hypertension (EH) is a significant health issue around the globe. The indifferent therapy regimen suggests varied physiological functions due to the lifestyle and genetic presentations of an individual. The endothelial nitric oxide synthase (NOS3) gene is a crucial vascular system marker in EH that contributes significantly to the phenotype. Hence, the present study aimed to employ the candidate gene approach and investigate the association between NOS3 single nucleotide polymorphism (SNP) E298D (G894T/rs1799983) by applying several in silico tools and validation through human samples screening. We corroborated computational findings through a case-control study comprising 294 controls and 299 patients; the 894T allele emerged significantly as the risk allele (odds ratio=2.07; P=6.38E−05). The in silico analyses highlighted the significance of E298D on the native structure and function of NOS3. The dynamics simulation study revealed that the variant type 298D caused structural destabilization of the protein to alter its function. Plasma nitrite levels were reduced in patients (P=0.0002), and the same correlated with the 894T allele. Furthermore, correlations were apparent between clinical, genotype, and routine biochemical parameters. To conclude, the study demonstrated a perceptible association between the SNP E298D and NOS3 protein structure stability that appears to have a bearing on the enzyme's function with a deleterious role in EH. [ABSTRACT FROM AUTHOR]

Metabolic syndrome (MetS) belongs to the serious health complications expanding in cardiovascular diseases, obesity, insulin resistance, and hyperglycemia. In this study, hypertriacylglycerolemic rats fed a high-fat-fructose diet (HFFD) were used as an experimental model of MetS to explore the effect of tested compounds. Effects of a new prospective pyridoindole derivative coded SMe1EC2 and the natural polyphenol rutin were tested. Endothelial nitric oxide synthase (NOS3) and nuclear factor kappa B (NF-κB) expression were assessed in the left ventricle immunohistochemically and left ventricle activity was monitored in isolated perfused rat hearts. NOS3 activity in the left ventricle decreased markedly as a result of a HFFD. NOS3 expression was upregulated by both substances. NF-κB expression was increased in the MetS group in comparison to control rats and the expression further increased in the SMe1EC2 treatment. This compound significantly improved the coronary flow in comparison to the control group during reperfusion of the heart followed after ischemia. Further, it tended to increase left ventricular systolic pressure, heart product, rate of maximal contraction and relaxation, and coronary flow during baseline assessment. Moreover, the compound SMe1EC2 decreased the sensitivity of hearts to electrically induced ventricular fibrillation. Contrary to this rutin decreased coronary flow in reperfusion. Present results suggest that despite upregulation of NOS3 by both substances tested, pyridoindole SMe1EC2 rather than rutin could be suitable in treatment strategies of cardiovascular disorders in MetS-like conditions. [ABSTRACT FROM AUTHOR]

Purpose This study aimed to detect the association between nitric oxide synthase 3 (NOS3) gene polymorphisms (rs1799983 and rs3918181) and the susceptibility to osteonecrosis of the femoral head (ONFH).Methods Total 88 ONFH patients (55 non-traumatic ONFH and 33 traumatic ONFH) and 90 healthy controls were recruited in this case–control study. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) was adopted for genotyping NOS3 rs1799983 and rs3918181 polymorphisms. χ2 test was used to calculate differences in genotype and allele frequencies of NOS3 gene polymorphisms between the cases and controls. Relative risk of ONFH was represented using odds ratios (ORs) with corresponding 95% confidence intervals (CIs).Results The T allele of the polymorphism rs1799983 showed significantly different frequencies between ONFH patients and control groups (p = .046) and carrying this allele significantly decreased the disease risk (OR = 0.521, 95%CI = 0.272–0.997), especially for non-traumatic ONFH (OR = 0.408, 95%CI = 0.179–0.929, p = .029). But genotype frequencies of the polymorphism rs1799983 had no obvious difference between the compared two groups (p > .05 for all). There was no remarkable association between NOS3 rs3918181 polymorphism and NOFH risk (p > .05 for all).Conclusions NOS3 rs1799983 polymorphism is obviously associated with ONFH and its T allele may be a protective factor against ONFH occurrence in Chinese Han population.

Context. Different polymorphisms of the endothelial nitric oxide synthase gene (NOS3) have been related to diabetic kidney disease. Objective. To evaluate the association between advanced diabetic chronic kidney disease (ACKD) and the rs1799983 and rs2070744 poymorphisms of NOS3 in a population from the Gran Canaria island. Design. Cross-sectional case-control study. Subjects and methods. Polymorphisms were genotyped in 152 subjects with ACKD secondary to type 2 diabetes [estimated glomerular filtration rate (eGFR) <30 mL/min/1.73 m2], 110 subjects with type 2 diabetes for 20 or more years since diagnosis without ACKD (eGFR =45 mL/min/1.73m2 and albumin/creatinine ratio <300 mg/g and/or 24-h urinary albumin excretion <300 mg) and 292 healthy controls. Association between both polymorphisms and established coronary heart disease (CHD) was also analyzed in both groups with diabetes. Results. A greater proportion of homozygous individuals for the risk allele C of rs2070744 was found among subjects with ACKD. Association between ACKD and rs2070744 was observed in a recessive genetic model, both for comparison to subjects with diabetes but no ACKD [OR 2.17 (95% CI: 1.17-4.00), p=0.014] and for comparison to healthy controls [OR 1.61 (1.03-2.52), p=0.036]. The frequency of the C allele was significantly higher among subjects with CHD, but only in the group with ACKD. No associations were found for rs1799983. Conclusions. NOS3 rs2070744 is associated with ACKD in population with type 2 diabetes from Gran Canaria. A link between this genetic variant and CHD in Canarian subjects with type 2 diabetes could be restricted to cases with ACKD. [ABSTRACT FROM AUTHOR]

Periplocymarin is an effective component of Periplocae Cortex, which was wildly used as an ingredient in Traditional Chinese Medicine. Our group previously reported that periplocymarin exerted cardiotonic role via promoting calcium influx. However, its exact role in the pathogenesis of myocardial fibrosis has not been elucidated yet. The present study was aimed at determining the potential effect and underlying mechanism of periplocymarin in isoproterenol (ISO)-induced myocardial fibrosis. C57BL/6 mice were subcutaneously injected with ISO (5 mg/kg/day) or saline for 1 week. The early-to-atrial wave ratio (E/A ratio) measured by echocardiography revealed that ISO-induced heart stiffness was remarkably reversed by administration of periplocymarin (5 mg/kg/day). Masson trichrome staining exhibited that treatment of periplocymarin reduced the excessive deposition of extracellular matrix (ECM). Further investigations employing real-time PCR and western blot demonstrated that periplocymarin suppressed the expression of fibrosis related genes (Col1a1, Col3a1, Acta2 and Tgfb1) and proteins (Collagen I, Collagen III, α-SMA and TGF-β1) induced by ISO. Metabolomics analysis demonstrated that periplocymarin ameliorated the disorders triggered by ISO and many of the differential metabolic substances were involved in amino acid, glucose and lipid metabolism. Further analysis using network pharmacology revealed that three key genes, namely NOS2, NOS3 and Ptgs2, may be the potential targets of periplocymarin and responsible for the disorders. Validation using heart tissues showed that the mRNA expression of NOS3 was decreased while Ptgs2 was increased upon ISO treatment, which were reversed by periplocymarin. Moreover, the expression of COX-2 (Ptgs2 encoded protein) was consistent with the aspect of Ptgs2 mRNA, while eNOS (NOS3 encoded protein) expression was unchanged. In vitro studies exhibited that periplocymarin exerts anti-fibrotic function via regulating at least eNOS and COX-2 in cardiomyocyte. Taken together, periplocymarin protects against myocardial fibrosis induced by β-adrenergic activation, the potential mechanism was that periplocymarin targeted on, at least eNOS and COX-2, to improve the metabolic processes of cardiomyocyte and thus attenuated the myocardial fibrosis. Our study highlighted that periplocymarin is a potential therapeutic agent for the prevention of myocardial fibrosis.

The identification of thalidomide–Cereblon-induced SALL4 degradation has brought new understanding for thalidomide embryopathy (TE) differences across species. Some questions, however, regarding species variability, still remain. The aim of this study was to detect sequence divergences between species, affected or not by TE, and to evaluate the regulated gene co-expression in a murine model. Here, we performed a comparative analysis of proteins experimentally established as affected by thalidomide exposure, evaluating 14 species. The comparative analysis, regarding synteny, neighborhood, and protein conservation, was performed in 42 selected genes. Differential co-expression analysis was performed, using a publicly available assay, GSE61306, which evaluated mouse embryonic stem cells (mESC) exposed to thalidomide. The comparative analyses evidenced 20 genes in the upstream neighborhood of NOS3 , which are different between the species who develop, or not, the classic TE phenotype. Considering protein sequence alignments, RECQL4, SALL4, CDH5, KDR, and NOS2 proteins had the biggest number of variants reported in unaffected species. In co-expression analysis, Crbn was a gene identified as a driver of the co-expression of other genes implicated in genetic, non-teratogenic, limb reduction defects (LRD), such as Tbx5 , Esco2 , Recql4 , and Sall4 ; Crbn and Sall4 were shown to have a moderate co-expression correlation, which is affected after thalidomide exposure. Hence, even though the classic TE phenotype is not identified in mice, a deregulatory Crbn-induced mechanism is suggested in this animal. Functional studies are necessary, especially evaluating the genes responsible for LRD syndromes and their interaction with thalidomide–Cereblon. [ABSTRACT FROM AUTHOR]

Peritoneal dissemination threatens the survival of patients with gastric cancer (GC). Bufalin is an extract of traditional Chinese medicine, which has been proved to have anticancer effect. The target of bufalin in suppressing gastric cancer peritoneal dissemination (GCPD) and the underlying mechanism are still unclear. In this research, GC cell line MGC‐803 and high‐potential peritoneal dissemination cell line MKN‐45P were treated with bufalin or L‐NAME. Malignant biological behavior and protein level of GC cell lines were detected with MTT, wound healing, transwell, adhesion, and western blotting. Bioinformatics analysis and patient tissues were used to verify the role of endothelial nitric oxide synthase (NOS3) in GC. Mice model was used to assess the effect of bufalin and role of NOS3 in vivo. We found that bufalin inhibited the proliferation, invasion, and migration in GC cell lines. NOS3, which was an independent prognostic factor of GC patients, was predicted to be a potential target of bufalin. Further experiments proved that bufalin reduced the phosphorylation of NOS3, thereby inhibiting the mitogen‐activated protein kinase (MAPK) signaling pathway, and ultimately suppressed GCPD by inhibiting EMT process. In conclusion, NOS3 was a potential therapeutic target and prognostic biomarker of GC. Bufalin could suppress GCPD through NOS3‐MAPK signaling pathway, which provided more evidence support for intraperitoneal perfusion of bufalin to treat GCPD. [ABSTRACT FROM AUTHOR]

Nansi López-Valverde,1 Antonio López-Valverde,1 Rafael Gómez de Diego,2 Clara Cieza-Borrella,3 Juan M Ramírez,4 Rogelio González-Sarmiento31Dental Clinic, Department of Surgery, Biomedical Research Institute of Salamanca (IBSAL), Salamanca, Spain; 2Dental Clinic, Alfonso X El Sabio University, Madrid, Spain; 3Molecular Medicine Unit, Department of Medicine, Biomedical Research Institute of Salamanca (IBSAL), Salamanca, Spain; 4Department of Morphological Sciences, School of Medicine, University of Córdoba, Córdoba, SpainAims: In this study we wanted to figure out if there was a correlation between OPRM1 N40D, TRPV1 I316M, TRPV1 I585V, NOS3 −786T>C and IL6 −174C>G polymorphisms and the response to locally applied articaine-epinephrine anesthetic.Methods: In this observational study, 114 oral cell samples of patients anesthetized with articaine-epinephrine (54 from men 60 from women), were collected from dental centers in Madrid (Spain). High molecular weight DNA was obtained from oral mucosa cells. The analysis of OPRM1 N40D (rs1799971), TRPV1 I316M (rs222747), TRPV1 I585V (rs8065080) and IL6 −174C>G polymorphism was performed through real-time PCR allelic discrimination using TaqMan probes. Polymorphism NOS3 −786T> C (rs2070744) was analyzed using RFLP-PCR.Results: The studied polymorphisms are involved neither in the response to the anesthetic, nor in the intensity of perceived dental pain. However, in a subset of female patients we found that TRPV1 I316M was associated with a delayed onset of anesthesia.Conclusions: There is no association among these polymorphisms and the time elapsed between the application of the anesthetic and the onset of its effect.Keywords: Pain, polymorphism, OPRM1, TRPV1, NOS3, IL6

Background:NOS3 (endothelial NOS, eNOS) is a member of the nitric oxide synthase (NOS) enzyme family, mainly participating in nitric oxide (NO) generation. NOS3 has been reported to inhibit apoptosis and promote angiogenesis, proliferation, and invasiveness. However, the expression pattern of NOS3 and its diagnostic and prognostic potential has not been investigated in a pan-cancer perspective.Methods: Data from the Genotype-Tissue Expression (GTEx), the Cancer Genome Atlas (TCGA), the Cancer Cell Line Encyclopedia (CCLE), and the Cancer Therapeutics Response Portal (CTRP) were employed and NOS3 expression was comprehensively analyzed in normal tissues, cancer tissues, and cell lines. Immunohistochemical staining of tissue sections were used to validate the prognostic role of NOS3 in gastric cancer patients. GSVA and GSEA analyses were performed to investigate signaling pathways related to NOS3 expression.Results: In normal tissues, NOS3 was expressed highest in the spleen and lowest in the blood. NOS3 expression was increased in stomach adenocarcinoma (STAD) and significantly associated with poor prognosis of patients. Immunohistochemical staining validated that NOS3 was an independent prognostic factor of gastric cancer. Several canonical cancer-related pathways were found to be correlated with NOS3 expression in STAD. The expression of NOS3 was related to the response to QS-11 and brivinib in STAD.Conclusions:NOS3 was an independent prognostic factor for patients with STAD. Increased expression of NOS3 influenced occurrence and development of STAD through several canonical cancer-related pathways. Drug response analysis reported drugs to suppress NOS3. NOS3 might be a novel target for gastric cancer treatment.

Abstract Recently we showed that homoarginine supplementation confers kidney protection in diabetic mouse models. In this study we tested whether the protective effect of homoarginine is nitric oxide synthase‐3 (NOS3)‐independent in diabetic nephropathy (DN). Experiments were conducted in NOS3 deficient (NOS3−/−) mice and their wild type littermate using multiple low doses of vehicle or streptozotocin and treated with homoarginine via drinking water for 24 weeks. Homoarginine supplementation for 24 weeks in diabetic NOS3−/− mice significantly attenuated albuminuria, increased blood urea nitrogen, histopathological changes and kidney fibrosis, kidney fibrotic markers, and kidney macrophage recruitment compared with vehicle‐treated diabetic NOS3−/− mice. Furthermore, homoarginine supplementation restored kidney mitochondrial function following diabetes. Importantly, there were no significant changes in kidney NOS1 or NOS2 mRNA expression between all groups. In addition, homoarginine supplementation improved cardiac function and reduced cardiac fibrosis following diabetes. These data demonstrate that the protective effect of homoarginine is independent of NOS3, which will ultimately change our understanding of the mechanism(s) by which homoarginine induce renal and cardiac protection in DN. Homoarginine protective effect in DN could be mediated via improving mitochondrial function.

We studied association of polymorphic markers Glu298Asp (rs1799983), C774T (rs1549758), and T786C (rs2070744) of the NOS3 gene with the risk of atopic dermatitis. It was found that T786C polymorphic marker of the NOS3 gene is associated with lower risk of erythematosquamous lichenoid atopic dermatitis. A significant association between the homozygous CC genotype in locus 786 of the NOS3 gene and the development of erythematosquamous atopic dermatitis with lichenification was revealed. The homozygous CC genotype can be considered as a risk factor of erythematosquamous atopic dermatitis with lichenification. [ABSTRACT FROM AUTHOR]

Background: NOS3 (endothelial NOS, eNOS) is a member of the nitric oxide synthase (NOS) enzyme family, mainly participating in nitric oxide (NO) generation. NOS3 has been reported to inhibit apoptosis and promote angiogenesis, proliferation, and invasiveness. However, the expression pattern of NOS3 and its diagnostic and prognostic potential has not been investigated in a pan-cancer perspective. Methods: Data from the Genotype-Tissue Expression (GTEx), the Cancer Genome Atlas (TCGA), the Cancer Cell Line Encyclopedia (CCLE), and the Cancer Therapeutics Response Portal (CTRP) were employed and NOS3 expression was comprehensively analyzed in normal tissues, cancer tissues, and cell lines. Immunohistochemical staining of tissue sections were used to validate the prognostic role of NOS3 in gastric cancer patients. GSVA and GSEA analyses were performed to investigate signaling pathways related to NOS3 expression. Results: In normal tissues, NOS3 was expressed highest in the spleen and lowest in the blood. NOS3 expression was increased in stomach adenocarcinoma (STAD) and significantly associated with poor prognosis of patients. Immunohistochemical staining validated that NOS3 was an independent prognostic factor of gastric cancer. Several canonical cancer-related pathways were found to be correlated with NOS3 expression in STAD. The expression of NOS3 was related to the response to QS-11 and brivinib in STAD. Conclusions: NOS3 was an independent prognostic factor for patients with STAD. Increased expression of NOS3 influenced occurrence and development of STAD through several canonical cancer-related pathways. Drug response analysis reported drugs to suppress NOS3. NOS3 might be a novel target for gastric cancer treatment. [ABSTRACT FROM AUTHOR]

Recently we showed that homoarginine supplementation confers kidney protection in diabetic mouse models. In this study we tested whether the protective effect of homoarginine is nitric oxide synthase‐3 (NOS3)‐independent in diabetic nephropathy (DN). Experiments were conducted in NOS3 deficient (NOS3−/−) mice and their wild type littermate using multiple low doses of vehicle or streptozotocin and treated with homoarginine via drinking water for 24 weeks. Homoarginine supplementation for 24 weeks in diabetic NOS3−/− mice significantly attenuated albuminuria, increased blood urea nitrogen, histopathological changes and kidney fibrosis, kidney fibrotic markers, and kidney macrophage recruitment compared with vehicle‐treated diabetic NOS3−/− mice. Furthermore, homoarginine supplementation restored kidney mitochondrial function following diabetes. Importantly, there were no significant changes in kidney NOS1 or NOS2 mRNA expression between all groups. In addition, homoarginine supplementation improved cardiac function and reduced cardiac fibrosis following diabetes. These data demonstrate that the protective effect of homoarginine is independent of NOS3, which will ultimately change our understanding of the mechanism(s) by which homoarginine induce renal and cardiac protection in DN. Homoarginine protective effect in DN could be mediated via improving mitochondrial function. [ABSTRACT FROM AUTHOR]

The interaction between genetic, epigenetic and environmental factors plays an important role in the aetiology of hypertension. GWAS and observational studies link the C677T polymorphism in methylenetetrahydrofolate reductase (MTHFR) with hypertension, while riboflavin, the MTHFR cofactor, has been shown to reduce blood pressure and global DNA methylation in homozygous (TT genotype) individuals. It is currently unclear whether riboflavin modulates DNA methylation of other hypertension-related genes. To compare DNA methylation of hypertension-related genes in adults stratified by MTHFR genotype and effect of riboflavin intervention in adults with the variant MTHFR 677TT genotype. Pyrosequencing was carried out for hypertension-related genes (ACE , AGTR1, GCK, GNA12 , IGF2, MMP9 and NOS3) in blood samples from participants in previous trials (CC, n = 40; TT, n = 40). The effect of intervention with riboflavin (1.6 mg/d for16 weeks) or placebo on DNA methylation was investigated in adults with the variant MTHFR 677TT genotype (n = 80). Individuals with the MTHFR 677TT v CC genotype had significantly higher average DNA methylation at NOS3 (+1.66%, P = 0.044). In response to riboflavin supplementation in TT individuals, there was an increase in average DNA methylation at IGF2 (+1.09%, P = 0.019) and a decrease at ACE (−0.44%, P = 0.021) in females only. Specific CpG sites were hypomethylated in GNA12 and hypermethylated in AGTR1. This study provides the first RCT evidence that riboflavin alters DNA methylation of hypertension-related genes in adults with the MTHFR 677TT genotype, providing some insight into mechanisms linking hypertension with the genotype-specific response of BP to riboflavin. • Riboflavin modulates DNA methylation in adults with the MTHFR 677TT genotype • First RCT evidence of epigenetic modulation of BP-related loci by riboflavin • NOS3 is hypermethylated in variant MTHFR 677TT compared to CC genotype • DNA methylation changes may underpin TT genotype-specific BP-lowering by riboflavin [ABSTRACT FROM AUTHOR]

Patients with a congenital bicuspid aortic valve (BAV), a valve with two instead of three aortic leaflets, have an increased risk of developing thoracic aneurysms and aortic dissection. The mechanisms underlying BAV-associated aortopathy are poorly understood. This study examined BAV-associated aortopathy in Nos3−/− mice, a model with congenital BAV formation. A combination of histological examination and in vivo ultrasound imaging was used to investigate aortic dilation and dissections in Nos3−/− mice. Moreover, cell lineage analysis and single-cell RNA sequencing were used to observe the molecular anomalies within vascular smooth muscle cells (VSMCs) of Nos3−/− mice. Spontaneous aortic dissections were found in ascending aortas located at the sinotubular junction in ∼13% of Nos3−/− mice. Moreover, Nos3−/− mice were prone to developing aortic dilations in the proximal and distal ascending aorta during early adulthood. Lower volumes of elastic fibres were found within vessel walls of the ascending aortas of Nos3−/− mice, as well as incomplete coverage of the aortic inner media by neural crest cell (NCC)-derived VSMCs. VSMCs of Nos3−/− mice showed downregulation of 15 genes, of which seven were associated with aortic aneurysms and dissections in the human population. Elastin mRNA was most markedly downregulated, followed by fibulin-5 expression, both primary components of elastic fibres. This study demonstrates that, in addition to congenital BAV formation, disrupted endothelial-mediated nitric oxide (NO) signalling in Nos3−/− mice also causes aortic dilation and dissection, as a consequence of inhibited elastic fibre formation in VSMCs within the ascending aorta.

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 μg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3−/− compared to Nos2−/− mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2−/− mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3−/− or Nos2−/−/Nos3−/− mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2−/− mice), as this beneficial effect was absent in Nos3−/− or Nos2−/−/Nos3−/− mice.

The interplay between lncRNAs and miRNAs was reported to be associated with cerebral ischemia reperfusion injury (CIRI). Besides, the lncRNA ZFAS1 was significantly downregulated in patients with ischemic stroke. However, the exact biological role of ZFAS1 and its mechanism in CIRI remain to be elucidated. The current study was designed to explore the protective effects of ZFAS1 on neuronal injury, inflammation, oxidative stress, and neuronal apoptosis in CIRI. ZFAS1 and miR-582-3p levels were assessed by RT-qPCR. Cerebral infarction was assessed by tetrazolium chloride (TTC) staining. Inflammation and oxidative stress were measured by ELISA and matched test kits. The apoptotic rate and apoptotic mediators in OGD/R-suffered PC12 cells were respectively detected by flow cytometry and western blot. NO production was measured by NO kit and western blot assay. The regulatory relationship between lncRNA ZFAS1 and miR-582-3p was analyzed using the luciferase reporter assay. ZFAS1 was downregulated in CIRI. ZFAS1 overexpression alleviated neurological function deficit and neuronal damage in a MCAO rat model. Besides, ZFAS1 upregulation or miR-582-3p downregulation could both inhibit the inflammation, oxidative stress, and apoptosis and enhance NO level in OGD/R-suffered PC12 cells. Reporter assay indicated that ZFAS1 served as a molecular sponge for miR-582-3p. In addition, ZFAS1 could negatively regulate miR-582-3p expression and release its effects in CIRI. Taken together, our study revealed that lncRNA ZFAS1 protected against neuronal damage and modulated the inflammation, oxidative stress, apoptosis, and NO level via regulating miR-582-3p in cerebral I/R injury. Therefore, lncRNA ZFAS1 might serve as a therapeutic application to suppress CIRI progression. [ABSTRACT FROM AUTHOR]

Acute coronary syndrome occurs when the heart muscle does not receive adequate oxygen and nutrients in a timely manner. Acute coronary syndromes are primarily due to atherosclerosis of the coronary arteries, i.e., coronary heart disease. Nitric oxide (NO) is synthesised from L-arginine in endothelial cells by the constitutive calcium-calmodulin-dependent enzyme, nitric oxide synthase (NOS), which mediates endothelium-dependent vasodilatation. Endothelial nitric oxide synthase (eNOS) is predominantly expressed in endothelial cells. Three NOS isoforms have been detected in different tissue: (1) neuronal NOS (nNOS) (NOS1), (2) eNOS (NOS2), and (3) inducible NOS (iNOS) (NOS3). These isoforms are encoded by three different genes. NOS3 is located on chromosome 7q35–36 and contains 26 exons. Previous studies have suggested that NOS3 polymorphisms may be associated with acute coronary syndromes. Therefore, the aim of the study was to examine the associations between NOS3 rs1799983 (894G/T)andrs2070744 (–786T/C) polymorphisms and unstable angina. This study included 246 patients with unstable angina, as confirmed by coronary angiography. We also included 189 healthy controls who were also assessed by this technique. There were no significant differences in genotype distributions of NOS3 rs1799983and rs2070744 polymorphisms in patients with unstable angina and healthy controls in both univariate and multivariate analyses. In patients with the NOS3 rs1799983 TT genotype, we observed a higher BMI (TT vs. GT + GG, p = 0.068), and in patients with the NOS3 rs2070744 TT genotype, we observed a higher waist circumference (TT vs. TC + CC, p = 0.023; TT vs. CC, p = 0.0053). These data suggest a lack of association between the NOS3 rs1799983andrs2070744 polymorphisms and unstable angina in our patient population. However, these polymorphisms may be associated with some obesity parameters, rs1799983 in females and rs2070744 in males. [ABSTRACT FROM AUTHOR]

The pathophysiology of varicocele remains to be unknown. Several genetic factors have been implicated in varicocele etiopathogenesis. We studied the relationship between NOS3 c.894G>T, c.786T>C and 4b/a polymorphisms to varicocele risk and their prognostic value as regards improvement of the post‐operative seminal parameters &/or seminal malonaldehyde levels. The three NOS3 polymorphisms were evaluated in 100 patients with varicocele and 100 healthy subjects by RT‐PCR. Seminal plasma MDA level was measured pre‐operatively and 3 months after varicocelectomy by the thiobarbituric acid method. The GT, TT, TC and bb genotypes of NOS3 polymorphism were more commonly observed in varicocele patients (30%, 9%, 28% and 70% respectively) compared to normal controls (12%, 0%, 10% and 50% respectively). The mean percentage of post‐varicocelectomy seminal MDA reduction was highest with the GT genotype (p <.001). Genotypes GT+TT, TC and bb were associated with varicocele occurrence in our patients. The T (c.894G>T), C (c.786T>C) and b (NOS3 intron 4 VNTR) alleles were significantly associated with varicocele occurrence in our cohort of patients. We also report a better response regarding the reduction of seminal MDA after varicocelectomy with the GT and ba genotypes. [ABSTRACT FROM AUTHOR]

The frequencies of genotypes and alleles for SNP of antioxidant genes (SOD1, SOD2, CAT, GPX4) and EDN1 and NOS3 were investigated in women with pregnancy loss. The 7958A allele of the SOD1 gene was associated with an increased risk of spontaneous abortion in the first trimester (OR = 2.17, 95% CI = 1.14–4.13). Women with the Asn/Asn genotype of the EDN1 gene have an increased risk of miscarriage in the first trimester (OR = 7.18, 95% CI = 1.4–36.75). An association with increased risk of pregnancy loss in the first trimester was found for the NOS3 –786T allele (OR = 1.68, 95% CI = 1.04–2.72). The significant models of gene-gene interaction changing the risk of miscarriage were revealed. [ABSTRACT FROM AUTHOR]

Abstract Background Essential hypertension (EH) is influenced by various environmental and genetic factors. Nitric oxide is important for the functional integrity of the vascular endothelium and is produced in endothelial cells by the enzyme endothelial nitric oxide synthase (eNOS). EH has a strong genetic component, and the NOS3 gene, which encodes eNOS, represents an interesting candidate for contribution to the phenotype. The most clinically relevant polymorphisms in the NOS3 gene are rs1799983 in exon 7 (encoding Glu298Asp), a variable number tandem repeat (VNTR) in intron 4, and rs2070744 (T-786C) in the promoter region. This study aims to investigate the association between these three polymorphisms in the NOS3 gene and EH in Sudanese patients. Methods Hypertensive patients (n = 157) > 18 years of age with established hypertension from various hospitals in Khartoum, and controls (n = 85) > 18 years of age and with blood pressure measurements 1% of the Sudanese population under study. Conclusion The results of this study indicated that the rs2070744 polymorphism in NOS3 may be a genetic susceptibility factor for EH in the Sudanese population. The c allele of intron 4 VNTR is not rare in the Sudanese population.

Aim. To assess the associations of polymorphism T-786C gene NOS3 with the severity of clinical course of coronary heart disease (CHD) and platelet aggregatability in the selection of patients receiving clopidogrel and acetylsalicylic acid (ASA) compounds after selective coronary intervention.Material and methods. In the study, 203 CHD males included, taking ASA and clopidogrel as double antiplatelet therapy for coronary intervention. The test performed, of induced platelet aggregation with adenosine diphosphate (ADP) (2,5 mcM and 5,0 mcM) and epinephrine (0,2 mcM). Genotyping was done with the allele specific polymerase chain reaction (“SNP-express”, SPF Litekh, Russia). Statistics was done with Mann-Whitney test, Kruskal-Wallis test and Pearson chisquare or bi-test by Fisher. Differences were taken as significant at p

Stroke significantly affects white matter in the brain by impairing axon function, which results in clinical deficits. Axonal mitochondria are highly dynamic and are transported via microtubules in the anterograde or retrograde direction, depending upon axonal energy demands. Recently, we reported that mitochondrial division inhibitor 1 (Mdivi-1) promotes axon function recovery by preventing mitochondrial fission only when applied during ischemia. Application of Mdivi-1 after injury failed to protect axon function. Interestingly, L-NIO, which is a NOS3 inhibitor, confers post-ischemic protection to axon function by attenuating mitochondrial fission and preserving mitochondrial motility via conserving levels of the microtubular adaptor protein Miro-2. We propose that preventing mitochondrial fission protects axon function during injury, but that restoration of mitochondrial motility is more important to promote axon function recovery after injury. Thus, Miro-2 may be a therapeutic molecular target for recovery following a stroke. [ABSTRACT FROM AUTHOR]