Early and late-onset preeclampsia: effects of DDAH2 polymorphisms on ADMA levels and association with DDAH2 haplotypes.

Objective: To examine whether the DDAH2 promoter polymorphisms -1415G/A (rs2272592), -1151A/C (rs805304) and -449G/C (rs805305), and their haplotypes, are associated with PE compared with normotensive pregnant women, and whether they affect ADMA levels in these groups. Methods: A total of 208 pregnant women were included in the study and classified as early-onset (N=57) or late-onset PE (N =49), and as normotensive pregnant women (N = 102). Results: Pregnant with early-onset PE carrying the GC and GG genotypes for the DDAH2 -449G/C polymorphism had increased ADMA levels (P=0.01). No association of DDAH2 polymorphisms with PE in single-locus analysis was found. However, the G-C-G haplotype was associated with the risk for late-onset PE. Conclusion: It is suggested that DDAH2 polymorphisms could affect ADMA levels in PE, and that DDAH2 haplotypes may affect the risk for PE.

Near-Infrared II Photobiomodulation Preconditioning Ameliorates Stroke Injury via Phosphorylation of eNOS.

BACKGROUND: The current management of patients with stroke with intravenous thrombolysis and endovascular thrombectomy is effective only when it is timely performed on an appropriately selected but minor fraction of patients. The development of novel adjunctive therapy is highly desired to reduce morbidity and mortality with stroke. Since endothelial dysfunction is implicated in the pathogenesis of stroke and is featured with suppressed endothelial nitric oxide synthase (eNOS) with concomitant nitric oxide deficiency, restoring endothelial nitric oxide represents a promising approach to treating stroke injury. METHODS: This is a preclinical proof-of-concept study to determine the therapeutic effect of transcranial treatment with a low-power near-infrared laser in a mouse model of ischemic stroke. The laser treatment was performed before the middle cerebral artery occlusion with a filament. To determine the involvement of eNOS phosphorylation, unphosphorylatable eNOS S1176A knock-in mice were used. Each measurement was analyzed by a 2-way ANOVA to assess the effect of the treatment on cerebral blood flow with laser Doppler flowmetry, eNOS phosphorylation by immunoblot analysis, and stroke outcomes by infarct volumes and neurological deficits. RESULTS: Pretreatment with a 1064-nm laser at an irradiance of 50 mW/cm2 improved cerebral blood flow, eNOS phosphorylation, and stroke outcomes. CONCLUSIONS: Near-infrared II photobiomodulation could offer a noninvasive and low-risk adjunctive therapy for stroke injury. This new modality using a physical parameter merits further consideration to develop innovative therapies to prevent and treat a wide array of cardiovascular diseases.

Reversal of Pulmonary Hypertension in a Human-Like Model: Therapeutic Targeting of Endothelial DHFR.

BACKGROUND: Pulmonary hypertension (PH) is a progressive disorder characterized by remodeling of the pulmonary vasculature and elevated mean pulmonary arterial pressure, resulting in right heart failure. METHODS: Here, we show that direct targeting of the endothelium to uncouple eNOS (endothelial nitric oxide synthase) with DAHP (2,4-diamino 6-hydroxypyrimidine; an inhibitor of GTP cyclohydrolase 1, the rate-limiting synthetic enzyme for the critical eNOS cofactor tetrahydrobiopterin) induces human-like, time-dependent progression of PH phenotypes in mice. RESULTS: Critical phenotypic features include progressive elevation in mean pulmonary arterial pressure, right ventricular systolic blood pressure, and right ventricle (RV)/left ventricle plus septum (LV+S) weight ratio; extensive vascular remodeling of pulmonary arterioles with increased medial thickness/perivascular collagen deposition and increased expression of PCNA (proliferative cell nuclear antigen) and alpha-actin; markedly increased total and mitochondrial superoxide production, substantially reduced tetrahydrobiopterin and nitric oxide bioavailabilities; and formation of an array of human-like vascular lesions. Intriguingly, novel in-house generated endothelial-specific dihydrofolate reductase (DHFR) transgenic mice (tg-EC-DHFR) were completely protected from the pathophysiological and molecular features of PH upon DAHP treatment or hypoxia exposure. Furthermore, DHFR overexpression with a pCMV-DHFR plasmid transfection in mice after initiation of DAHP treatment completely reversed PH phenotypes. DHFR knockout mice spontaneously developed PH at baseline and had no additional deterioration in response to hypoxia, indicating an intrinsic role of DHFR deficiency in causing PH. RNA-sequencing experiments indicated great similarity in gene regulation profiles between the DAHP model and human patients with PH. CONCLUSIONS: Taken together, these results establish a novel human-like murine model of PH that has long been lacking in the field, which can be broadly used for future mechanistic and translational studies. These data also indicate that targeting endothelial DHFR deficiency represents a novel and robust therapeutic strategy for the treatment of PH.

Association between NOS3 gene polymorphisms and genetic susceptibility to congenital heart Disease: A systematic review and meta-analysis.

BACKGROUND: Endothelial nitric oxide (NO) produced by endothelial Nitric Oxide Synthase (eNOS) can promote the expression of pro-angiogenic cytokines and is favorable for angiogenesis. However, the relationship between NOS3 gene polymorphisms and genetic susceptibility to congenital heart disease (CHD) was still unclear. METHODS: We searched five databases including Pubmed, Cochrane Library, Embase, Web of Science, CNKI, and Wan Fang, to find all studies on NOS3 gene polymorphisms and CHD. Rstudio was used to merge the data included in the study to obtain OR, 95%CI, and forest plots. RESULTS: Five relevant literatures were included, including three sites of NOS3 gene, rs1799983 (G894T), rs2070744 (T-786C), and rs7830 (G10T). Several models including the homozygous model of rs1799983 (G894T) gene polymorphism (TT VS GG: OR = 1.602, 95%CI: 1.098 âˆ¼ 2.337, P = 0.027), rs7830 (G10T) gene polymorphism allele model (A VS C: OR = 1.171, 95%CI: 1.029 âˆ¼ 1.333, P = 0.017), homozygous model (AA VS CC: OR = 1.474, 95%CI: 1.122 âˆ¼ 1.936, P = 0.005) and implicit model (AA VS CC + AC: OR = 1.451, 95%CI: 1.133 âˆ¼ 1.859, P = 0.003) indicated that there was a correlation. The results of the combined analysis of each gene model of rs2070744 (T-786C) gene polymorphism sites were not statistically significant, and their P values were all>0.05. CONCLUSION: rs1799983 (G894T) and rs7830 (G10T) polymorphic sites might play a role in the susceptibility of sporadic congenital heart disease and increase the risk of CHD. Yet, it is still necessary to expand the sample size and conduct more prospective/retrospective studies to confirm whether the rs2070744 (T-786C) polymorphism tended to increase the incidence of CHD.

Early and late-onset preeclampsia: effects of DDAH2 polymorphisms on ADMA levels and association with DDAH2 haplotypes

Abstract Objective: To examine whether the DDAH2 promoter polymorphisms -1415G/A (rs2272592), -1151A/C (rs805304) and -449G/C (rs805305), and their haplotypes, are associated with PE compared with normotensive pregnant women, and whether they affect ADMA levels in these groups. Methods: A total of 208 pregnant women were included in the study and classified as early-onset (N=57) or late-onset PE (N =49), and as normotensive pregnant women (N = 102). Results: Pregnant with early-onset PE carrying the GC and GG genotypes for the DDAH2 -449G/C polymorphism had increased ADMA levels (P=0.01). No association of DDAH2 polymorphisms with PE in single-locus analysis was found. However, the G-C-G haplotype was associated with the risk for late-onset PE. Conclusion: It is suggested that DDAH2 polymorphisms could affect ADMA levels in PE, and that DDAH2 haplotypes may affect the risk for PE.

Association of endothelial nitric oxide synthase gene variants in pre-eclampsia: an updated systematic review and meta-analysis.

INTRODUCTION: Three common endothelial nitric oxide synthase (eNOS) gene variants are existed such as; G-894T, T-786C, and variable number tandem repeats in intron-4 (VNTR intron-4) which has been proposed to be linked with PE. However, there is still debate regarding the findings. To address this, a review was conducted to assess the potential association of eNOS gene variants at these positions with the risk of PE. METHODS: PubMed, Scopus, Science Direct, Hinari, and African Journal Online databases and Google Scholar search engines were utilized to search studies published in English-language until 30 January 2023. The Joanna Briggs Institute Meta-Analysis instrument was used for data extraction process and the Newcastle-Ottawa Scale was used to appraise the quality of the included studies. Meta-regression analysis was conducted using Stata 14 statistical software. The pooled odds ratios (ORs) of fixed and random effect models were utilized to evaluate the association of eNOS gene polymorphism with the risk of PE at 95% CI. Publication bias was assessed using Egger's test and a funnel plot. RESULTS: The study included 47 observational studies involving 13,795 pregnant women (6216 cases and 7579 controls). Pregnant women carrying TT and CC genotypes of eNOS gene at 894 and 786 positions were found to have a greater probability of developing PE as compared to GG and TT genotypes (OR = 1.54 vs. 1.43 and CI: 1.12 - 2.14 vs.1.02 - 2.00 at 95% CI), respectively. However, a significant association was not observed between aa genotype of eNOS gene in VNTR intron-4 region and risk of PE as compared to bb genotype (OR =1.26, 95% CI: 0.83 - 1.89). The allelic model of eNOS gene at all positions showed nonsignificant association with the risk of PE. CONCLUSIONS: The women having eNOS gene variants at 894 and 786 positions showed a significant association with the risk of PE. Yet, the women having eNOS gene variant at intron-4 region showed nonsignificant association with the risk of PE. Thus, this study suggests that eNOS gene variants may play a role in the development of PE, but large-scale studies are required to inaugurate concrete evidence on the roles of eNOS gene variants in PE pathogenesis.

Association between the Polymorphisms rs2070744, 4b/a and rs1799983 of the NOS3 Gene with Chronic Kidney Disease of Uncertain or Non-Traditional Etiology in Mexican Patients.

Background and Objectives: Chronic Kidney Disease of uncertain or non-traditional etiology (CKDnT) is a form of chronic kidney disease of undetermined etiology (CKDu) and is not associated with traditional risk factors. The aim of this study was to investigate the association of polymorphisms rs2070744, 4b/a and rs1799983 of the NOS3 gene with CKDnT in Mexican patients. Materials and Methods: We included 105 patients with CKDnT and 90 controls. Genotyping was performed by PCR-RFLP's, genotypic and allelic frequencies were determined and compared between the two groups using χ2 analysis, and differences were expressed as odd ratios with 95% confidence intervals (CI). Values of p < 0.05 were considered statistically significant. Results: Overall, 80% of patients were male. The rs1799983 polymorphism in NOS3 was found to be associated with CKDnT in the Mexican population (p = 0.006) (OR = 0.397; 95% CI, 0.192-0.817) under a dominant model. The genotype frequency was significantly different between the CKDnT and control groups (χ2 = 8.298, p = 0.016). Conclusions: The results of this study indicate that there is an association between the rs2070744 polymorphism and CKDnT in the Mexican population. This polymorphism can play an important role in the pathophysiology of CKDnT whenever there is previous endothelial dysfunction.

Calciprotein Particles Induce Endothelial Dysfunction by Impairing Endothelial Nitric Oxide Metabolism.

BACKGROUND: Calciprotein particles (CPPs) are associated with the development of vascular calcifications in chronic kidney disease. The role of endothelial cells (ECs) in this process is unknown. Here, we investigated the interaction of CPPs and ECs, thereby focusing on endothelial nitric oxide metabolism and oxidative stress. METHODS: CPPs were generated in calcium- and phosphate-enriched medium. Human umbilical vein endothelial cells were exposed to different concentrations of CPPs (0-100 µg/mL) for 24 or 72 hours. Ex vivo porcine coronary artery rings were used to measure endothelial cell-dependent vascular smooth muscle cell relaxation after CPP exposure. Serum samples from an early chronic kidney disease cohort (n=245) were analyzed for calcification propensity (measure for CPP formation) and nitrate and nitrite levels (NOx). RESULTS: CPP exposure for 24 hours reduced eNOS (endothelial nitric oxide synthase) mRNA expression and decreased nitrite production, indicating reduced nitric oxide bioavailability. Also, 24-hour CPP exposure caused increased mitochondria-derived superoxide generation, together with nitrotyrosine protein residue formation. Long-term (72 hours) exposure of human umbilical vein endothelial cells to CPPs induced eNOS uncoupling and decreased eNOS protein expression, indicating further impairment of the nitric oxide pathway. The ex vivo porcine coronary artery model showed a significant reduction in endothelial-dependent vascular smooth muscle cell relaxation after CPP exposure. A negative association was observed between NOx levels and calcification propensity (r=-0.136; P=0.049) in sera of (early) chronic kidney disease patients. CONCLUSIONS: CPPs cause endothelial cell dysfunction by impairing nitric oxide metabolism and generating oxidative stress. Our findings provide new evidence for direct effects of CPPs on ECs and pathways involved.

Gene Sequencing Identifies Perturbation in Nitric Oxide Signaling as a Nonlipid Molecular Subtype of Coronary Artery Disease.

BACKGROUND: A key goal of precision medicine is to disaggregate common, complex diseases into discrete molecular subtypes. Rare coding variants in the low-density lipoprotein receptor gene (LDLR) are identified in 1% to 2% of coronary artery disease (CAD) patients, defining a molecular subtype with risk driven by hypercholesterolemia. METHODS: To search for additional subtypes, we compared the frequency of rare, predicted loss-of-function and damaging missense variants aggregated within a given gene in 41 081 CAD cases versus 217 115 controls. RESULTS: Rare variants in LDLR were most strongly associated with CAD, present in 1% of cases and associated with 4.4-fold increased CAD risk. A second subtype was characterized by variants in endothelial nitric oxide synthase gene (NOS3), a key enzyme regulating vascular tone, endothelial function, and platelet aggregation. A rare predicted loss-of-function or damaging missense variants in NOS3 was present in 0.6% of cases and associated with 2.42-fold increased risk of CAD (95% CI, 1.80-3.26; P=5.50×10-9). These variants were associated with higher systolic blood pressure (+3.25 mm Hg; [95% CI, 1.86-4.65]; P=5.00×10-6) and increased risk of hypertension (adjusted odds ratio 1.31; [95% CI, 1.14-1.51]; P=2.00×10-4) but not circulating cholesterol concentrations, suggesting that, beyond lipid pathways, nitric oxide synthesis is a key nonlipid driver of CAD risk. CONCLUSIONS: Beyond LDLR, we identified an additional nonlipid molecular subtype of CAD characterized by rare variants in the NOS3 gene.

Endothelial NO synthase 786T/T polymorphism increases hemorrhagic transformation after endovascular thrombectomy.

BACKGROUND AND PURPOSE: This study examined whether the 786 NOS3 polymorphism is associated with the risk of hemorrhagic transformation (HT) in stroke patients with anterior large vessel occlusion (ALVO) treated using endovascular thrombectomy (EVT). METHODS: We performed an observational cohort study that included 118 patients with ALVO who underwent EVT. HT was assessed in follow-up CT and MRI. HT and non-HT patients were compared in terms of the 786 NOS3 polymorphism, flow mediated dilation (FMD) values within 3 days after the stroke, and collateral status based on three grading scales. Demographics, vascular risk factors, additional radiological data including ASPECT score, thrombus length and infarct size, and EVT procedure and outcome variables were also included. RESULTS: Radiological HT occurred in 55 (46.6%) patients and the 786T/T NOS3 polymorphism was associated with HT (unadjusted OR of 2.33, 95%CI: 1.05-5.20, adjusted OR of 3.14, 95%CI: 1.16-8.54). Collateral status and systemic endothelial function assessed by FMD were not mediators of this relationship as no differences were seen in the median FMD percentage values or collateral status between NOS3 genotypes. CONCLUSIONS: Our results suggest that genetic variations affecting the NO pathway, such as the 786 NOS3 polymorphism, may contribute to individual variability in the occurrence of HT and these results support involvement of this pathway in the pathogenesis of ischemia-reperfusion injury after EVT.

Adventitial Collagen Cross-Linking by Glutaraldehyde Reinforcing Human Saphenous Vein - Implication for Coronary Artery Bypass Grafting.

INTRODUCTION: A weak venous wall is one of the major reasons contributing to vein graft failure after coronary artery bypass grafting (CABG). We investigated whether adventitial collagen cross-linking by glutaraldehyde reinforces venous wall, preserving the endothelium of veins during high-pressure distention. METHODS: Human saphenous veins (SVs) were collected from 40 patients undergoing CABG, and adventitia cross-linking was performed with 0.3% glutaraldehyde for five minutes. The cross-linked SVs were accessed by biodegradation assay, immunofluorescent staining, and tensile test. Native SVs and cross-linked SVs from another 20 patients received the 200 mmHg pressure distention for two minutes. Pressure-induced injury of SVs were accessed by immunohistochemistry and electron microscopy. RESULTS: Time to digestion was 97±13 minutes for native SVs and 720±0 minutes for cross-linked SVs (P<0.05). After adventitial cross-linking, the collagen I fibres of the vein remarkably presented with compact and nonporous arrangement. In the high-stretch region (stretch ratio 1.4-1.8), the Young's elastic modulus of stress-stretch ratio curve in cross-linked SVs was larger than that in native SVs (13.88 vs. 5.83, P<0.05). The cross-linked SVs had a lower extent of endothelial denudation without fibre fracture during high-pressure distension than native SVs. Comparing with the non-cross-linked SVs, the percentage of endothelial nitric oxide synthase staining length on the endothelium of cross-linked SVs was significantly preserved after high-pressure distension (85.2% vs. 64.7%, P<0.05). CONCLUSION: Adventitial collagen cross-linking by glutaraldehyde reinforced venous wall by increasing stiffness and decreasing extensibility of SVs and mitigated the endothelial damage under high-pressure distension.

Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells.

BACKGROUND: Endothelial dysfunction is a critical component in the pathogenesis of cardiovascular diseases and is closely associated with nitric oxide (NO) levels and oxidative stress. Here, we report on novel findings linking endothelial expression of CD70 (also known as CD27 ligand) with alterations in NO and reactive oxygen species. METHODS: CD70 expression was genetically manipulated in human aortic and pulmonary artery endothelial cells. Intracellular NO and hydrogen peroxide (H2O2) were measured using genetically encoded biosensors, and cellular phenotypes were assessed. RESULTS: An unbiased phenome-wide association study demonstrated that polymorphisms in CD70 associate with vascular phenotypes. Endothelial cells treated with CD70-directed short-interfering RNA demonstrated impaired wound closure, decreased agonist-stimulated NO levels, and reduced eNOS (endothelial nitric oxide synthase) protein. These changes were accompanied by reduced NO bioactivity, increased 3-nitrotyrosine levels, and a decrease in the eNOS binding partner heat shock protein 90. Following treatment with the thioredoxin inhibitor auranofin or with agonist histamine, intracellular H2O2 levels increased up to 80% in the cytosol, plasmalemmal caveolae, and mitochondria. There was increased expression of NADPH oxidase 1 complex and gp91phox; expression of copper/zinc and manganese superoxide dismutases was also elevated. CD70 knockdown reduced levels of the H2O2 scavenger catalase; by contrast, glutathione peroxidase 1 expression and activity were increased. CD70 overexpression enhanced endothelial wound closure, increased NO levels, and attenuated the reduction in eNOS mRNA induced by TNFα. CONCLUSIONS: Taken together, these data establish CD70 as a novel regulatory protein in endothelial NO and reactive oxygen species homeostasis, with implications for human vascular disease.

Adventitial Collagen Cross-Linking by Glutaraldehyde Reinforcing Human Saphenous Vein - Implication for Coronary Artery Bypass Grafting

ABSTRACT Introduction: A weak venous wall is one of the major reasons contributing to vein graft failure after coronary artery bypass grafting (CABG). We investigated whether adventitial collagen cross-linking by glutaraldehyde reinforces venous wall, preserving the endothelium of veins during high-pressure distention. Methods: Human saphenous veins (SVs) were collected from 40 patients undergoing CABG, and adventitia cross-linking was performed with 0.3% glutaraldehyde for five minutes. The cross-linked SVs were accessed by biodegradation assay, immunofluorescent staining, and tensile test. Native SVs and cross-linked SVs from another 20 patients received the 200 mmHg pressure distention for two minutes. Pressure-induced injury of SVs were accessed by immunohistochemistry and electron microscopy. Results: Time to digestion was 97±13 minutes for native SVs and 720±0 minutes for cross-linked SVs (P<0.05). After adventitial cross-linking, the collagen I fibres of the vein remarkably presented with compact and nonporous arrangement. In the high-stretch region (stretch ratio 1.4-1.8), the Young's elastic modulus of stress-stretch ratio curve in cross-linked SVs was larger than that in native SVs (13.88 vs. 5.83, P<0.05). The cross-linked SVs had a lower extent of endothelial denudation without fibre fracture during high-pressure distension than native SVs. Comparing with the non-cross-linked SVs, the percentage of endothelial nitric oxide synthase staining length on the endothelium of cross-linked SVs was significantly preserved after high-pressure distension (85.2% vs. 64.7%, P<0.05). Conclusion: Adventitial collagen cross-linking by glutaraldehyde reinforced venous wall by increasing stiffness and decreasing extensibility of SVs and mitigated the endothelial damage under high-pressure distension.

Anti-hypertensive and endothelia protective effects of Fufang Qima capsule on primary hypertension via adiponectin/adenosine monophosphate activated protein kinase pathway.

OBJECTIVE: To investigate the potential mechanism of the vascular remodeling effect and provide additional information about anti-hypertension activity of Fufang Qima capsule. METHODS: Spontaneous hypertensive rats (SHRs) were used to study the underlying mechanism of the anti-hypertension activity of QM. In this study, SHRs were randomly divided into 5 groups: model group, Telmisartan group (7.2 mg/kg, p.o.), and three QM groups (0.9298, 1.8596, and 3.7192 g/kg, p.o.). Wistar Kyoto rats (WKY) were used as normal control group. Blood pressure (BP), aorta, perivascular adipose tissue (PVAT) histology were investigated to evaluate the effect of QM. Nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) phosphorylation were measured. Adiponectin (APN) secretion, as well as APN signal pathway proteins including APN, adiponectin receptors (R1 and R2) and adenosine 5'-monophosphate-activated protein kinase (AMPK) were all analyzed. RESULTS: QM significantly reduced BP and ameliorated the vascular pathological change, i.e. intima media thicken and collagen fiber hyperplasia. Meanwhile, QM increased concentration of NO and the phosphorylation of eNOS in the aorta. The anti-hypertensive and endothelia-protective effect of QM could be attributed to activating APN/ AMPK pathway by up-regulating the expression of APN in PVAT and APN Receptor 2, AMPKα and phosphorylated AMPKα in the aorta. CONCLUSION: The QM alleviation effect mechanism for primary hypertension was via modulating the APN/AMPK signal pathway.

In Vivo Function of Flow-Responsive Cis-DNA Elements of eNOS Gene: A Role for Chromatin-Based Mechanisms.

BACKGROUND: eNOS (endothelial nitric oxide synthase) is an endothelial cell (EC)-specific gene predominantly expressed in medium- to large-sized arteries where ECs experience atheroprotective laminar flow with high shear stress. Disturbed flow with lower average shear stress decreases eNOS transcription, which leads to the development of atherosclerosis, especially at bifurcations and curvatures of arteries. This prototypic arterial EC gene contains 2 distinct flow-responsive cis-DNA elements in the promoter, the shear stress response element (SSRE) and the KLF (Krüppel-like factor) element. Previous in vitro studies suggested their positive regulatory functions on flow-induced transcription of EC genes including eNOS. However, the in vivo function of these cis-DNA elements remains unknown. METHODS: Insertional transgenic mice with a mutation at each flow-responsive cis-DNA element were generated using a murine eNOS promoter-ß-galactosidase reporter by linker-scanning mutagenesis and compared with episomal-based mutations in vitro. DNA methylation at the eNOS proximal promoter in mouse ECs was assessed by bisulfite sequencing or pyrosequencing. RESULTS: Wild type mice with a functional eNOS promoter-reporter transgene exhibited reduced endothelial reporter expression in the atheroprone regions of disturbed flow (n=5). It is surprising that the SSRE mutation abrogated reporter expression in ECs and was associated with aberrant hypermethylation at the eNOS proximal promoter (n=7). Reporter gene silencing was independent of transgene copy number and integration position, indicating that the SSRE is a critical cis-element necessary for eNOS transcription in vivo. The KLF mutation demonstrated an integration site-specific decrease in eNOS transcription, again with marked promoter methylation (n=8), suggesting that the SSRE alone is not sufficient for eNOS transcription in vivo. In wild type mice, the native eNOS promoter was significantly hypermethylated in ECs from the atheroprone regions where eNOS expression was markedly repressed by chronic disturbed flow, demonstrating that eNOS expression is regulated by flow-dependent DNA methylation that is region-specific in the arterial endothelium in vivo. CONCLUSIONS: We report, for the first time, that the SSRE and KLF elements are critical flow sensors necessary for a transcriptionally permissive, hypomethylated eNOS promoter in ECs under chronic shear stress in vivo. Moreover, eNOS expression is regulated by flow-dependent epigenetic mechanisms, which offers novel mechanistic insight on eNOS gene regulation in atherogenesis.

Expressions and relationship of Krüppel-like factor 15 and endothelial nitric oxide synthase in experimental deep venous thrombosis.

BACKGROUND: Deep vein thrombosis (DVT) is an early postoperative complication. Thrombosis formation, which is potentially life-threatening, seriously affects the rehabilitation of patients after surgery. We aimed to establish a C57 mouse model of DVT and to examine the changes in the expression of Krüppel-like factor 15 (KLF15) and endothelial nitric oxide synthase (eNOS) in venous wall tissues, and we also investigated the regulatory relationship of KLF15 and eNOS in the thrombin-induced human umbilical vein endothelial cell (HUVEC) injury cell model. METHODS: The DVT model was established using the inferior vena cava (IVC) stenosis method. The expression levels of KLF15 and eNOS were analyzed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In cell experiments, the expression of KLF15 and eNOS was analyzed in the model of thrombin-induced HUVEC injury with KLF15 siRNA. RESULTS: Compared to the control and sham-operated groups, KLF15 in the DVT group was upregulated, while eNOS was downregulated. The results of cell experiments revealed that KLF15 was downregulated in the thrombin+KLF15 siRNA group compared with the thrombin group. Meanwhile, eNOS was upregulated in the thrombin+KLF15 siRNA group compared with the thrombin group. These findings suggested that KLF15 regulated the expression of eNOS in the DVT model. CONCLUSIONS: We successfully constructed a DVT mouse model. In the early stage of DVT formation, KLF15 regulated the expression and inhibited the antithrombotic effect of eNOS, resulting in thrombi formation.

The effect of nitric oxide, endothelial nitric oxide synthetase, and asymmetric dimethylarginine in hemorrhoidal disease

SUMMARY AIM The aim of this study was to examine the roles of nitric oxide (NOx), endothelial nitric oxide synthetase (eNOS), and asymmetric dimethylarginine (ADMA), which is the major endogenous inhibitor of nitric oxide synthases (NOS), in the pathophysiology of hemorrhoidal disease. METHODS This study included 54 patients with grades 3 and 4 internal hemorrhoidal disease and 54 patients without the disease who attended the General Surgery Clinic. NOx, eNOS, and ADMA levels were measured with the Enzyme-Linked ImmunoSorbent Assay (ELISA) method. RESULTS The patients had higher NO and eNOS levels and lower ADMA levels than the control subjects (p<0.001). A significant highly positive correlation was found between NO and eNOS (p<0.001). Nevertheless, there was a highly negative correlation between ADMA and NO-eNOS(p<0.001, p<0.001). CONCLUSION This preliminary study reveals that higher NOx and eNOS activities and lower ADMA levels in the rectal mucosa are observed in patients with hemorrhoidal disease than in those with normal rectal tissue. The imbalance between endothelium-derived relaxing factors, such as NO and endogenous competitive inhibitor of NOS, ADMA, may cause hemorrhoidal disease. Our study proposes that hemorrhoids display apparent vascular dilatation and present with bleeding or swelling. ADMA is an effective NOS inhibitor and may be a promising therapeutic option for hemorrhoidal disease.

RESUMO OBJETIVO O objetivo deste estudo foi examinar os papéis do óxido nítrico (NOx), do óxido nítrico sintetase endotelial (eNOS) e da dimetilarginina assimétrica (ADMA), que é o principal inibidor endógeno das óxido nítrico sintase (NOS) na fisiopatologia da doença hemorróida. MÉTODOS Este estudo incluiu 54 pacientes com doença hemorróida interna de grau 3 e 4 e 54 pacientes sem a doença que se inscreveram na Clínica Geral de Cirurgia. Os níveis de NOx, eNOS e ADMA foram medidos com o método de Ensaio Imuno absorvente ligado a enzima (ELISA). RESULTADOS Os pacientes têm níveis mais altos de NO e eNOS e níveis mais baixos de ADMA do que os indivíduos controle (p <0,001). Uma correlação altamente positiva significativa foi encontrada entre o NO-eNOS (p <0,001). No entanto, houve uma correlação negativa muito séria entre ADMA e NO-eNOS (p <0,001, p <0,001). CONCLUSÃO Este estudo preliminar revela que os pacientes com doença hemorróida têm atividades mais altas de NOx e eNOS e níveis mais baixos de ADMA na mucosa retal do que os pacientes com tecido retal normal. Desequilíbrio entre o fator relaxante derivado do endotélio, como; O NO e o inibidor competitivo endógeno da NOS, ADMA, podem causar doenças hemorróidas. Nosso estudo propõe que as hemorróidas exibam aparente dilatação vascular e apresentam sangramento ou inchaço, a ADMA é um inibidor eficaz da NOS e pode ser uma opção terapêutica promissora para a doença hemorróida.

Association of endothelial nitric oxide synthase gene (G894T) polymorphism and hypertension in diabetic Bataknese patients.

Aim To assess endothelial nitric oxide synthase (eNOS) gene (G894T) polymorphism and nitric oxide (NO) level in hypertensive diabetic Bataknese patients. Methods A hospital-based, case control study (hypertensive and normotensive diabetic patient) was conducted. Genotyping of eNOS gene (G894T) was done using polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP). Nitric oxide was quantified by sandwich enzyme-linked immunosorbent assay (Sandwich ELISA). Results GT polymorphism and T allele were higher in the hypertensive diabetic patients, 37.5% (p=0.6) and 54% (p=0.39), respectively. Nitric oxide level tended to be lower in the hypertensive diabetic patients (88.87µmol/L) comparing to the normotensive (95.42 µmol/L (p=0.54), as well as GT and TT polymorphism type (p=0.75). Conclusion eNOS gene (G894T) polymorphism is not associated with NO level and hypertension in the diabetic Bataknese patients.

Caveolin-1 Regulates Atherogenesis by Attenuating Low-Density Lipoprotein Transcytosis and Vascular Inflammation Independently of Endothelial Nitric Oxide Synthase Activation.

BACKGROUND: Atherosclerosis is driven by synergistic interactions between pathological, biomechanical, inflammatory, and lipid metabolic factors. Our previous studies demonstrated that absence of caveolin-1 (Cav1)/caveolae in hyperlipidemic mice strongly inhibits atherosclerosis, which was attributed to activation of endothelial nitric oxide (NO) synthase (eNOS) and increased production of NO and reduced inflammation and low-density lipoprotein trafficking. However, the contribution of eNOS activation and NO production in the athero-protection of Cav1 and the exact mechanisms by which Cav1/caveolae control the pathogenesis of diet-induced atherosclerosis are still not clear. METHODS: Triple-knockout mouse lacking expression of eNOS, Cav1, and Ldlr were generated to explore the role of NO production in Cav1-dependent athero-protective function. The effects of Cav1 on lipid trafficking, extracellular matrix remodeling, and vascular inflammation were studied both in vitro and in vivo with a mouse model of diet-induced atherosclerosis. The expression of Cav1 and distribution of caveolae regulated by flow were analyzed by immunofluorescence staining and transmission electron microscopy. RESULTS: We found that absence of Cav1 significantly suppressed atherogenesis in Ldlr-/-eNOS-/- mice, demonstrating that athero-suppression is independent of increased NO production. Instead, we find that the absence of Cav1/caveolae inhibited low-density lipoprotein transport across the endothelium and proatherogenic fibronectin deposition and disturbed flow-mediated endothelial cell inflammation. Consistent with the idea that Cav1/caveolae may play a role in early flow-dependent inflammatory priming, distinct patterns of Cav1 expression and caveolae distribution were observed in athero-prone and athero-resistant areas of the aortic arch even in wild-type mice. CONCLUSIONS: These findings support a role for Cav1/caveolae as a central regulator of atherosclerosis that links biomechanical, metabolic, and inflammatory pathways independently of endothelial eNOS activation and NO production.

Effect of miR-134 against myocardial hypoxia/reoxygenation injury by directly targeting NOS3 and regulating PI3K/Akt pathway

Abstract Purpose To reveal the function of miR-134 in myocardial ischemia. Methods Real-time PCR and western blotting were performed to measure the expression of miR-134, nitric oxide synthase 3 (NOS3) and apoptotic-associated proteins. Lactic dehydrogenase (LDH) assay, cell counting kit-8 (CCK-8), Hoechst 33342/PI double staining and flow cytometry assay were implemented in H9c2 cells, respectively. MiR-134 mimic/inhibitor was used to regulate miR-134 expression. Bioinformatic analysis and luciferase reporter assay were utilized to identify the interrelation between miR-134 and NOS3. Rescue experiments exhibited the role of NOS3. The involvement of PI3K/AKT was assessed by western blot analysis. Results MiR-134 was high regulated in the myocardial ischemia model, and miR-134 mimic/inhibitor transfection accelerated/impaired the speed of cell apoptosis and attenuated/exerted the cell proliferative prosperity induced by H/R regulating active status of PI3K/AKT signaling. LDH activity was also changed due to the different treatments. Moreover, miR-134 could target NOS3 directly and simultaneously attenuated the expression of NOS3. Co-transfection miR-134 inhibitor and pcDNA3.1-NOS3 highlighted the inhibitory effects of miR-134 on myocardial H/R injury. Conclusion This present work puts insights into the crucial effects of the miR-134/NOS3 axis in myocardial H/R injury, delivering a potential therapeutic technology in future.