Endothelial Nitric Oxide Synthase Levels Research Articles

Elevated luteinizing hormone contributes to atherosclerosis formation by inhibiting nitric oxide synthesis via PI3K/Akt pathway

BackgroundThe contentious effects of estrogen therapy on the risk of postmenopausal cardiovascular disease (CVD) indicate that this type of atherosclerosis is not solely induced by estrogen deficiency. Other sex hormones such as elevated luteinizing hormone (LH) may also affect CVD risk in this population. We therefore explored the relationship between LH and atherosclerosis in ovariectomized (OVX) female mice. MethodsAortic atherosclerotic lesions were assessed in OVX ApoE knock out (ApoE−/−) female mice administered with LH. Human umbilical vascular endothelial cells (HUVECs) were cultured as cell model. The influence of LH on NO release, phosphorylated endothelial nitric oxide synthase (eNOS) and Akt levels were evaluated. Immunoprecipitation and lentiviral particle transfection were applied to assess the role of Gαq on PI3K activity. ResultsLH increased the atherosclerotic lesion area and carotid artery intima-media thickness (IMT) in OVX ApoE−/− female mice. High levels of LH attenuated vasodilation induced by Ach and inhibited NO release from HUVECs. These effects were related to the findings that LH enhanced interaction between Gαq and p110α, which subsequently inhibited PI3K activity and suppressed the phosphorylation of Akt and eNOS. ConclusionsElevated LH promotes atherosclerosis formation in OVX ApoE−/− female mice. This effect may be mediated by inhibiting endothelial NO synthesis via PI3K/Akt signaling pathway.

Berberine exhibits antioxidative effects and reduces apoptosis of the vaginal epithelium in bacterial vaginosis.

Bacterial vaginosis (BV) is a common type of vaginitis. Berberine is a natural alkaline product that reduces oxidative stress and apoptosis in cells. The aim of the present study was to investigate the effects of berberine on oxidative stress and apoptotic rates of BV. Vaginal epithelial and discharge samples were obtained from 60 healthy individuals and 180 patients with BV before and after one month of berberine treatment. Clinical observation was documented for all patients before and after treatment for comparison. Additionally, an in vitro study was performed; the samples were divided into groups the following groups: Control, model (H2O2-treated), LT (low-dose berberine), MT (medium-dose berberine) and HT (high-dose berberine). Expression levels of the oxidative stress related proteins were detected by western blotting. Clinical symptoms of patients with BV significantly improved following berberine treatment. Oxidative stress in vaginal discharge was significantly lower following treatment, indicated by the increased activity of superoxide dismutase (SOD) and catalase, as well as the reduced levels of malondialdehyde and H2O2. Apoptosis of the vaginal epithelial cells was also reduced, which was indicated by the reduced expression of apoptosis proteins caspase-3, cytochrome C, capase-12 and Bax, and increased expression of Bcl-2. The results of the in vitro experiments demonstrated a dose-dependent decrease in apoptosis with berberine treatment compared with levels before treatment. Oxidative stress relief was demonstrated by the reduced reactive oxygen species level and increased SOD and endothelial nitric oxide synthase levels, whereas suppression of apoptosis was further supported by the reduction in apoptotic proteins, as well as a decreased Bax/Bcl-2 ratio. Berberine exhibited effects on lowering oxidative stress in vaginal discharge and reducing oxidative damage, as well as apoptosis of the vaginal epithelium, which are beneficial to patients with bacterial vaginosis.

Ac2-26 ameliorates lung ischemia-reperfusion injury via the eNOS pathway

BackgroundLung ischemia-reperfusion injury (LIRI) is a major complication after lung transplantation. Annexin A1 (AnxA1) ameliorates inflammation in various injured organs. This study aimed to determine the effects and mechanism of AnxA1 on LIRI after lung transplantation. MethodsThirty-two rats were randomized into sham, saline, Ac2-26 and Ac2-26/L groups. Rats in the saline, Ac2-26 and Ac2-26/L groups underwent left lung transplantation and received saline, Ac2-26, and Ac2-26/L-NIO, respectively. After 24 h of reperfusion, serum and transplanted lung tissues were examined. ResultsThe partial pressure of oxygen (PaO2) was increased in the Ac2-26 group compared to that in the saline group but was decreased by L-NIO treatment. In the Ac2-26 group, the wet-to-dry (W/D) weight ratios, total protein concentrations, proinflammatory factors and inducible nitric oxide synthase levels were notably decreased, but the concentrations of anti-inflammatory factors and endothelial nitric oxide synthase levels were significantly increased. Ac2-26 attenuated histological injury and cell apoptosis, and this improvement was reversed by L-NIO. ConclusionsAc2-26 reduced LIRI and improved alveoli-capillary permeability by inhibiting oxygen stress, inflammation and apoptosis. The protective effect of Ac2-26 on LIRI largely depended on the endothelial nitric oxide synthase pathway.

Impaired l-arginine metabolism marks endothelial dysfunction in CD73-deficient mice

Changes in the ecto-5′-nucleotidase activity—an extracellular nucleotide catabolic enzyme may lead to the inflammation and endothelial dysfunction. We investigated the effect of CD73 deletion on the endothelial function and l-arginine metabolism in various age groups of mice. 1-,3-,6-, and 12-month-old, male C57BL/6 J wild type (WT) and C57BL/6 J CD73−/− (CD73−/−) mice were used. Blood samples were used for the analysis of adenine nucleotide concentrations. Serum samples were analyzed for the concentration of amino acids, Interleukin 6 (IL-6), Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), and endothelial nitric oxide synthase (eNOS) level. Serum and aortic nitrate/nitrite, as well as aortic arginase and NOS activity in endothelial cells (EC) were evaluated. CD73 deletion led to age-dependent increase in IL-6, ICAM-1, and VCAM-1 concentration compared to WT. All CD73−/− mice age groups were characterized by reduced l-Arginine concentration and eNOS level. Significantly lower NOS activity was noticed in EC isolated from CD73−/− mice lungs in comparison to EC isolated from WT lungs. The l-Arginine/ADMA ratio in the CD73−/− decreased in age-dependent manner in comparison to WT. The nitrate/nitrite ratio was reduced in serum and in aortas of 6-month-old CD73−/− mice as compared to WT. The ornithine/arginine and ornithine/citrulline ratios were increased in CD73−/− compared to controls. Blood (erythrocyte) Adenosine-5′-triphosphate and Adenosine-5′-diphosphate levels were reduced in favor to higher blood Adenosine-5′-monophosphate concentration in CD73−/− mice in comparison to WT. The CD73 deletion leads to the development of age-dependent endothelial dysfunction in mice, associated with impaired l-arginine metabolism. CD73 activity seems to protect endothelium.

Premature Myocardial Infarction: Genetic Variations in SIRT1 Affect Disease Susceptibility.

Objectives Premature myocardial infarction (PMI) is an uncommon disease, and its incidence varies between 2% and 10%, rising, depending on genetic susceptibility under the influence of lifestyle. The purpose of this study was to investigate the association between SIRT1 single nucleotide polymorphisms (SNPs), SIRT1, and eNOS (endothelial nitric oxide synthase) protein expressions, total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) in young patients with premature ST-elevation myocardial infarction (STEMI). Methods Genotyping of the three single-nucleotide polymorphisms (rs7895833 A > G in the promoter region, rs7069102 C > G in intron 4, and rs2273773 C > T in exon 5) in SIRT1 gene was performed in 108 consecutive patients (87.0% were men with a mean age of 40.74 ± 3.82 years) suffering from ST-elevation myocardial infarction at the age of ≤45 and 91 control subjects. Results The risk for myocardial infarction was increased by 2.31 times in carriers of CC or CG genotypes. SIRT1 protein levels were enhanced and endothelial nitric oxide synthase levels were diminished in ST-elevation myocardial infarction patients regardless of the underlying gene variant. There was no correlation between SIRT1 expression and the amount of endothelial nitric oxide synthase, total antioxidant status, total oxidant status, and oxidative stress index levels in patients and in the control group either. Conclusions SIRT1 single-nucleotide polymorphisms were associated with premature myocardial infarction, which affected the SIRT1 and endothelial nitric oxide synthase protein expression, irrespective of the underlying SIRT1 genotype.

Retraction Note: Association between T-786C polymorphism of endothelial nitric oxide synthase gene and level of the vessel dilation factor in patients with coronary artery disease

Retraction Note: Association between T-786C polymorphism of endothelial nitric oxide synthase gene and level of the vessel dilation factor in patients with coronary artery disease

Clinical value of anti-oxidative stress biomarkers for diagnosing in-stent restenosis and in-stent reocclusion after coronary stent implantation in advanced-aged patients

Objective To study the clinical value of anti-oxidative stress biomarkers for diagnosing in-stent restenosis and in-reocclusion after coronary stent implantation in aged patients. Methods A total of 72 advanced-aged patients with in-stent restenosis and in-stent reocclusion after coronary stent implantation were successively recruited in this retrospective study from February 2010 to November 2017.Changes in serum superoxide dismutase 3(SOD3), nitric oxide(NO), endothelial cell nitric oxide synthase(eNOS)and malondialdehyde(MDA)levels were measured. Results Serum 1evels of SOD3, NO and eNOS decreased and serum MDA levels were elevated in advanced-aged patients with in-stent restenosis.There were significant differences in serum levels of SOD3, NO, eNOS and MDA between the advanced-aged patients without in-stent restenosis and the advanced-aged patients with multivessel in-stent restenosis or reocclusion[(20.0±3.2)×103U/L vs.(10.9±3.9)×103U/L, (61.2±14.2)μmol/L vs.(28.3±17.2)μmol/L, (75.9±24.7)ng/L vs.(33.0±19.6)ng/L, (2.2±1.4)nmol/L vs.(11.7±3.1)nmol/L, respectively, P<0.01]. Patients with 50-69% restenosis had higher serum levels of SOD3, NO and eNOS and lower levels of MDA than patients with 100% restenosis[(21.3±2.9)×103U/L vs.(10.3±4.0)×103U/L, (59.7±16.7)μmol/L vs.(38.3±16.3)μmol/L, (74.5±21.1)ng/L vs.(41.9±26.8)ng/L, (2.6±3.9 nmol/L)vs.(10.1±3.1)nmol/L, respectively, P<0.01]. Patients with left ventricular ejection fraction(LVEF)≥55% had higher serum levels of SOD3, NO and eNOS and lower levels of MDA than patients with LVEF<30%[(21.0±4.1)×103U/L vs.(5.3±1.9)×103U/L, (60.1±14.2)μmol/L vs.(29.0±13.2)μmol/L, (74.7±25.1)ng/L vs.(39.3±20.3)ng/L, (2.3±1.5)nmol/L vs.(10.0±3.9)nmol/L, respectively, P<0.01]. Serum levels of SOD3, NO and eNOS were higher and MDA levels were lower in patients with New York Heart Association(NYHA)Class I than in patients with NYHA Class IV[(22.1±3.5)×103U/L vs.(9.7±2.9)×103U/L, (62.9±13.9)μmol/L vs.(24.9±13.3)μmol/L, (76.7±26.7)ng/L vs.(41.9±21.5)ng/L, (2.7±1.9)nmol/L vs.(8.7±3.8)nmol/L, respectively, P<0.01]. Conclusions Serum level changes of anti-oxidative stress biomarkers such as SOD3, NO and eNOS may have clinical value in diagnosing in-stent restenosis and in-reocclusion after coronary stent implantation in aged patients. Key words: Coronary restenosis; Superoxide dismutase; Nitric oxide Synthase

Glucose-6-phosphate dehydrogenase deficiency increases cell adhesion molecules and activates human monocyte-endothelial cell adhesion: Protective role of l-cysteine

Glucose-6-phosphate dehydrogenase is a major enzyme that supplies the reducing agent nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), which is required to recycle oxidized/glutathione disulfide (GSSH) to reduced glutathione (GSH). G6PD-deficient cells are susceptible to oxidative stress and a deficiency of GSH. Endothelial dysfunction is characterized by the loss of nitric oxide (NO) bioavailability, which regulates leukocyte adhesion to endothelium. G6PD-deficient endothelial cells (EC) demonstrate reduced expression of endothelial nitric oxide synthase (eNOS) and NO levels along with reduced GSH. Whether G6PD deficiency plays any role in EC dysfunction is unknown. The chronic inflammation commonly seen in those with metabolic syndrome, characterized by elevated levels of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1), provided an incentive for investigation of these cytokines as well. A GSH/G6PD-deficient model was created using human umbilical vein endothelial cells (HUVEC) treated with either buthionine sulfoximine (BSO), a pharmacological inhibitor of the rate-limiting enzyme of GSH biosynthesis (γ-glutamylcysteine synthetase), or with 6-aminonicotinamide (6-AN), an inhibitor of G6PD or G6PD siRNA. Normal and G6PD-deficient cells were also treated with pro-atherosclerotic stimuli such as high glucose, TNF, and MCP-1. After inhibiting or knocking down G6PD/GSH, the capacity of endothelial cells for monocyte recruitment was assessed by determining the expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), which was upregulated by G6PD deficiency and accompanied by the presence of the oxidative stress markers NADPH oxidase 4 (NOX4), inducible nitric oxide synthase (iNOS), and reactive oxygen species (ROS). Treatment with the inhibitors BSO and 6-AN caused increased levels of adhesion molecule mRNA and monocyte-EC adhesion. Following treatment with high glucose, G6PD-deficient cells showed an increase in levels of ICAM-1 and VCAM-1 mRNA, as well as monocyte-EC adherence, compared with results seen in control cells. Treatment with l-cysteine (a precursor of GSH) protected endothelial cells by increasing GSH and attenuating ROS, ICAM-1, VCAM-1, and monocyte-EC adhesion. These results suggest that G6PD/GSH deficiency plays a role in endothelial dysfunction and that supplementation with l-cysteine can restore GSH levels and reduce the EC activation markers in G6PD-deficient conditions.

Combined Effects of Low-Dose Proton Radiation and Simulated Microgravity on the Mouse Retina and the Hematopoietic System.

The purpose of the current study was to characterize the effects of simulated microgravity and radiation-induced changes in retina and retinal vasculature, and to assess the accompanying early changes in immune cells and hematological parameters. To better understand the effects of spaceflight, we used a combination of treatments designed to simulate both the radiation and low-gravity aspects of space conditions. To simulate the broad energy spectrum of a large solar particle event (SPE) and galactic cosmic ray (GCR) radiation, male C57BL/6J mice were exposed to whole-body irradiation using fully modulated beams of 150-MeV protons containing particles of energy from 0 to 150 MeV and a uniform dose-vs.-depth profile. The mice were also hindlimb-unloaded (HLU) by tail suspension. Mice were unloaded for 7 days, exposed to 50 cGy, unloaded for an additional 7 days and then sacrificed for tissue isolation at days 4 and 30 after the combined treatments. Increases in the number of apoptotic cells were observed in the endothelial cells of mice that received radiation alone or with HLU compared to controls at both days 4 and 30 (P < 0.05). Endothelial nitric oxide synthase (eNOS) levels were significantly elevated in the retina after irradiation only or combined with HLU compared to controls at the 30-day time point (P < 0.05). The most robust changes were observed in the combination group, suggesting a synergistic response to radiation and unloading. For hematopoietic parameters, our analysis indicated the main effects for time and radiation at day 4 after treatments (day 11 postirradiation) (P < 0.05), but a smaller influence of HLU for both white blood cell and lymphocyte counts. The group treated with both radiation and HLU showed greater than 50% reduction in lymphocyte counts compared to controls. Radiation-dependent differences were also noted in specific lymphocyte subpopulations (T, B, natural killer cells). This study shows indications of an early effect of low-dose radiation and spaceflight conditions on retina and immune populations.

Environmental tobacco smoke exposure during pregnancy has limited effect on infant birthweight and umbilical vein endothelial nitric oxide synthase

Women who smoke, deliver significantly smaller infants. These infants have reduced levels of the vasodilator endothelial nitric oxide synthase (eNOS) levels in the umbilical vessels, which may reduce fetal growth. Serum cotinine, the degradation product of nicotine, can be used to determine the level of tobacco exposure. Newborns of environmental smokers are suggested to be smaller and shorter in weight, length, and head circumference. eNOS levels have not yet been studied in these infants. We investigated the existence of a relation between maternal environmental tobacco smoke exposure, eNOS activity, concentration, and birthweight. We included 263 healthy singleton pregnancies categorized into three groups according to measured cotinine levels: 175 nonsmokers, 38 smokers, and 50 environmental smokers. Cotinine was quantified by mass spectrometry with a detection limit of .2ng/mL; eNOS activity and concentration were measured in endothelial cells (ECs) of the umbilical vein. Infants born to environmental smokers had similar weights to infants born to nonsmokers (47g heavier, P=.48). Cotinine concentrations were .06/.09/.12ng/mL (quartiles) in infants born to nonsmokers, .27/.37/.81ng/mL in infants born to women exposed to environmental tobacco smoke, and 43.0/63.8/108.1ng/mL in infants born to smokers. The eNOS concentration was 1.65±.92ng/106 ECs (mean±SD) in nonsmokers and 1.71±1.00ng/106 ECs in environmental smokers. The eNOS activity was 52.0±20.6pmol l-citrulline/min/106 ECs in nonsmokers and 48.7±19.8pmol l-citrulline/min/106 ECs in environmental smokers. Infants born to environmental smokers, as judged by umbilical serum cotinine levels close to .2ng/mL, are not associated with lower birthweight or reduced eNOS activity, or concentration in the fetal vascular bed.

Exendin‑4 reverses endothelial dysfunction in mice fed a high‑cholesterol diet by a GTP cyclohydrolase‑1/tetrahydrobiopterin pathway.

The present study examined whether exendin-4 (Ex4) can improve the endothelial dysfunction of apolipoprotein E knockout (APOE-KO) mice fed a high-cholesterol diet and the potential mechanism by which it acts. Genetically wild-type (WT) C57BL/6 mice and APOE-KO mice of C57BL/6 background, were each randomly assigned to receive either Ex4 treatment (Ex4-treated, for 8 weeks) or not (control). The 4 groups were fed the same high-cholesterol diet for 8 weeks. The following were measured at the end of the eighth week: Endothelium-dependent vasodilation of the arteries; plasma nitric oxide (NO) and metabolic index; levels of endothelial NO synthase (eNOS); phosphorylated eNOS (p-eNOS; Ser-1,177); guanosine triphosphate cyclohydrolase-1 (GCH1); and tetrahydrobiopterin (THB). Ex4 treatment was associated with higher p-eNOS levels in the WT group and in the APOE-KO group, and higher vascular expression of GCH1 and higher arterial THB content, compared with baseline values. The results of the present study suggested that Ex4 may exert cardioprotective effects by reversing high-cholesterol diet-induced endothelial dysfunction in APOE-KO mice. The protective mechanism is probably associated with the promotion of the expression levels of GCH1 protein and THB that maintain the normal function of eNOS.

Beneficial effects of cherry consumption as a dietary intervention for metabolic, hepatic and vascular complications in type 2 diabetic rats

BackgroundOxidative stress (OS) plays an important role in type 2 diabetes (T2D) pathogenesis and its complications. New therapies target natural antioxidants as an alternative and/or supplemental strategy to prevent and control them. Our previous chemical and biological studies highlighted the important antioxidant activities of cherries, among other fruits and vegetables, thus we aimed to determine in vivo effects of 2-month long cherry consumption using a high-fat/high-fructose (HFHF) model of diabetic-rats (Lozano et al. in Nutr Metab 13:15, 2016).MethodsAfter 2 months of HFHF, male Wistar rats were divided into: HFHF and HFHF enriched in cherry (nutritional approach) or standard diet ND (lifestyle measures) and ND plus cherry during 2 months. Metabolic, lipidic, oxidative parameters were quantified. Tissues (liver, pancreas and vessels) OS were assessed and hepatic (steatosis, fibrosis, inflammation) and vascular (endothelial dysfunction) complications were characterized.ResultsT2D was induced after 2 months of HFHF diet, characterized by systemic hyperglycaemia, hyperinsulinemia, glucose intolerance, dyslipidaemia, hyperleptinemia, and oxidative stress associated with endothelial dysfunction and hepatic complications. Cherry consumption for 2 months, in addition to lifestyle measures, in T2D-rats decreased and normalized the systemic disturbances, including oxidative stress complications. Moreover, in the vessel, cherry consumption decreased oxidative stress and increased endothelial nitric oxide (NO) synthase levels, thus increasing NO bioavailability, ensuring vascular homeostasis. In the liver, cherry consumption decreased oxidative stress by inhibiting NADPH oxidase subunit p22phox expression, nuclear factor erythroid-2 related factor 2 (Nrf2) degradation and the formation of reactive oxygen species. It inhibited the activation of sterol regulatory element-binding proteins (1c and 2) and carbohydrate-responsive element-binding protein, and thus decreased steatosis as observed in T2D rats. This led to the improvement of metabolic profiles, together with endothelial and hepatic function improvements.ConclusionCherry consumption normalized vascular function and controlled hepatic complications, thus reduced the risk of diabetic metabolic disorders. These results demonstrate that a nutritional intervention with a focus on OS could prevent and/or delay the onset of vascular and hepatic complications related to T2D.

Unexpected role of the human cytomegalovirus contribute to essential hypertension in the Kazakh Chinese population of Xinjiang.

Human cytomegalovirus (HCMV) infection, chronic inflammation and oxidative stress, the renin–angiotensin system (RAS), endothelial function, and DNA methylation play roles in the pathogenesis of essential hypertension (EH); however, the mechanism by which HCMV predisposes patients to hypertension remain unclear. Our group previously demonstrated an association between EH and HCMV infection in Kazakh Chinese. Here, we investigated the relationship between HCMV infection and other clinicopathological features in 720 Kazakh individuals with or without hypertension (n=360 each; age: 18–80). Multiple linear and logistic regression analyses were used to determine the associations between HCMV infection, clinical characteristics, and EH. Notably, patients with EH, particularly those with HCMV infection, exhibited a marked increase in tumor necrosis factor-α (TNF-α) and 8-hydroxy-2-deoxyguanosine (8-OHDG) levels, but a decrease in endothelial nitric oxide synthase (eNOS) and renin levels. Similarly, elevated TNF-α and 8-OHDG levels were independent predictors of increased HCMV antibody titers, whereas eNOS and renin were negatively correlated with the latter. Moreover, serum angiotensin-converting enzyme (sACE, ACE) methylation was increased, whereas 11-β hydroxysteroid dehydrogenase 2 (HSD11β2; HSD3B2) methylation was decreased in patients with EH who were also infected with HCMV. A positive correlation between HSD3B2 methylation and HCMV IgG titer and blood pressure was additionally observed, whereas angiotensin-converting enzyme (ACE) methylation was inversely correlated with blood pressure. Collectively, these data indicate that HCMV may contribute to EH development in the Kazakh Chinese by increasing TNF-α and 8-OHDG levels, suppressing eNOS and renin, and manipulating HSD3B2 and ACE methylation.

Effect of Diallyl Trisulfide on Ischemic Tissue Injury and Revascularization in a Diabetic Mouse Model.

Background and Objective:Allitridin [diallyl trisulfide (DATS)] is an extract from garlic (Allium sativum) that putatively improves endothelial function and is protective against cardiovascular diseases. Endothelial dysfunction after tissue ischemia in diabetic patients is partially due to poor angiogenic response. This study investigated whether DATS may improve angiogenesis in a diabetic mouse model with hind limb ischemia.Methods:Streptozotocin was administered by intraperitoneal injection to establish the model of diabetes in male C57BL/6 mice. After 14 days, nondiabetic and diabetic mice (n = 24, each) underwent unilateral hind limb ischemia by femoral artery ligation. The mice were apportioned to 4 groups: nondiabetic treated (or not) with DATS and diabetic treated (or not) with DATS. DATS treatment consisted of a single daily intraperitoneal injection of 500 μg·kg−1·d−1 for 14 days, beginning on the day of induced ischemia. Ischemia was scored by standard criteria. Blood perfusion was determined using thermal infrared imaging. Tissue capillary density and oxidative stress levels were measured by immunohistochemistry and immunofluorescence, respectively. Serum lipids were measured by enzymatic colorimetric assay. Fasting serum insulin was detected using an insulin enzyme-linked immunosorbent assay kit. Nitric oxide (NO) metabolites and protein carbonyls in tissues were determined by enzyme-linked immunosorbent assay. Targeted protein concentrations were measured by western blotting.Results:At 14 days after ligation, the ischemic skeletal muscle of the streptozotocin-induced diabetic mice had lower levels of endothelial NO synthase, phosphorylated endothelial NO synthase, and vascular endothelial growth factor compared with nondiabetic group. In addition, the hind limb blood perfusion, capillary density, and NO bioactivity were lower in the diabetic group, whereas oxidative stress and protein carbonyl levels were higher. These changes were ameliorated by DATS treatment.Conclusions:DATS treatment of diabetic mice promoted revascularization in ischemic tissue.

Lipopolysaccharide Induces Subacute Cerebral Microhemorrhages with Involvement of Nitric Oxide Synthase in Rats

Cerebral microhemorrhage (CMH) is a neuropathological term that could be easily found in cerebral amyloid angiopathy, intracerebral hemorrhages, etc. CMHs could be detected clearly in vivo by magnetic resonance imaging (MRI)-susceptibility-weighted imaging or MRI T2* scan. This terminology is now accepted in the area of neuroimaging. CMHs are quite common in elderly patients and are associated with several other neuropsychiatric disorders. The causes of CMHs are complicated, and neuroinflammation is considered as one of the well-accepted mechanical factors. This study investigated whether lipopolysaccharide (LPS)-induced CMHs occur through the regulation of nitric oxide synthase (NOS) isoforms and reveals the exact underlying mechanism of LPS-induced CMHs. Our work successfully developed a subacute model of CMHs in rats. LPS was intraperitoneally injected into rats at 0, 6, and 24 hours, which induced typical CMH features 7 days after the injection. These could be detected on the brain surface or parenchyma by hematoxylin and eosin staining and MRI. LPS-treated rats showed significant activation of astrocytes and microglia, as well as loss of pericytes and disruption of blood-brain barrier. Meanwhile, both astrocytes and microglia were positively correlated with CMH numbers. Furthermore, the expressions of NOS isoforms were also examined, and the levels of neuronal NOS and endothelial NOS were found to be elevated. These results implied that the NOS isoforms might be involved in the subacute model of CMHs in rats induced by LPS.

Effects Of Endothelin-1 On Intracellular Tetrahydrobiopterin Levels In Vascular Tissue

Objective: Tetrahydrobiopterin (BH4) is the essential cofactor of endothelial nitric oxide synthase (eNOS) and intracellular levels of BH4 is regulated by oxidative stress. The aim of this paper was to describe the influence of exogenous endothelin-1 on intracellular BH4 and its oxidation products dihydrobiopterin (BH2) and biopterin (B) in a wide range of vascular tissue.Design: Segments of internal mammary artery (IMA) and human saphenous vein (SV) from 41 patients undergoing elective surgery were incubated in ET-1 (0.1 μM). Aorta and lung from transgenic mice overexpressing ET-1 in the endothelium (ET-TG) were analysed with regards to intracellular biopterin levels. Human umbilical vein endothelial cells (HUVEC) were incubated in ET-1 (0.1 μM) and intracellular biopterin levels were analysed. From 6 healthy women undergoing caesarean section, subcutaneous fat was harvested and the resistance arteries in these biopsies were tested for ET-mediated endothelial dysfunction.Results: In HUVEC, exogenous ET-1 (0.1 μM) did not significantly change intracellular BH4, 1.54 ± 1.7 vs 1.68 ± 1.8 pmol/mg protein; p = .8. In IMA and SV, exogenous ET-1(0.1 μM) did not change intracellular BH4 n = 10, p = .4. In aorta from wild type vs ET-TG mice there was no significant difference in intracellular BH4 between the groups: 1.3 ± 0.49 vs 1.23 ± 0.3 pmol/mg protein; p = .6. In resistance arteries (n = 6) BH4 together with DTE (an antioxidant) was not able to prevent ET-mediated endothelial dysfunction.Conclusion: ET-1 did not significantly alter intracellular tetrahydrobiopterin levels in IMA, SV, HUVEC or aorta from ET-TG mice. These findings are important for future research in ET-1 mediated superoxide production and endothelial dysfunction.

The protective effects of Astragaloside Ⅳ on diastolic function of rat thoracic aortic rings impaired by microvesicles

To investigate the effects of Astragaloside IV (AST) on diastolic function of rat thoracic aorta rings which was injured by microvesicles derived from hypoxia/reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs), and the mechanism of AST. H/R-induced endothelial microvesicles (H/R-EMVs) were generated from cultured HUVECs in vitro under the condition of hypoxia for 12 hour/Reoxygenation for 4 hour, H/R-EMVs were stored in D-Hank's solution. Male Wistar rats were underwent thoracotomy, the thoracic aorta with intact endothelium were carefully removed and cut into 3~4 mm rings. The experiment was divided into six groups. H/R-EMVs group:thoracic aortic rings of rats were incubated in culture medium and treated with H/R-EMVs in a final concentration of 10μg/ml; different doses of AST groups:thoracic aortic rings of rats were treated with 10, 20, 40, 60 mg/L AST co-incubated with 10μg/ml H/R-EMVs respectively; control group were treated with the same volume of D-Hank's solution. Duration of incubation was 4 h, each group was tested in five replicate aortic rings. Effects of AST on endothelium-dependent relaxation were detected. The production of nitric oxide (NO) and the level of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS, Ser-1177), serine/threonine kinase (Akt), phosphorylated Akt (p-Akt, Ser-473), extracellular regulated protein kinases (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2, Thr202/Tyr204) of rat thoracic aortic rings were detected. Tenμg/ml H/R-EMVs could impaire the relaxation of rat thoracic aortic rings significantly (P<0.01). Compared with H/R-EMVs group, relaxation of rat thoracic aortic rings was increased by 20, 40 and 60 mg/L AST in a concentration-dependent manner (P<0.01), the level of NO production was also enhanced (P<0.05, P<0.01). The level of t-eNOS, t-Akt and ERK1/2 was not changed, but the level of p-eNOS, p-Akt and p-ERK1/2 increased by the treatment with AST (P<0.01). AST could effectively ameliorate endotheliumdependent relaxation of rat thoracic aortic rings impaired by H/R-EMVs in a concentration-dependent manner, the mechanism might involve the increase in production of NO, and the protein level of p-eNOS, p-Akt and p-ERK1/2.

Exercise training induces eNOS coupling and restores relaxation in coronary arteries of heart failure rats.

Exercise training (ET) has emerged as a nonpharmacological therapy for cardiovascular diseases because of its helpful milieu for improving vascular function. The aim of the present study was to assess whether ET reverses the alterations in vascular reactivity observed in heart failure (HF)-related coronary arteries and to elucidate the molecular mechanisms involved in these adjustments. Male Wistar rats were subjected to either coronary artery ligation or sham operation. Four weeks after the surgery, rats were divided into two groups: untrained HF (UHF) and exercise-trained HF (THF). ET was conducted on a treadmill for 8 wk. An untrained SO group was included in the study as a normal control. ET restored the impaired acetylcholine (ACh)- and sodium nitroprusside-induced relaxation in coronary arteries to levels of the control. Oxidative stress and reduced nitric oxide (NO) production were observed in UHF, whereas ET restored both parameters to the levels of the control. Expression levels of endothelial NO synthase (eNOS) and soluble guanylyl cyclase subunits were increased in coronary arteries of UHF rats but reduced in THF rats. Tetrahydrobiopterin restored ACh-induced NO production in the UHF group, indicating that eNOS was uncoupled. ET increased the eNOS dimer-to-monomer ratio and expression of GTP cyclohydrolase 1, thus increasing NO bioavailability. Taken together, these findings demonstrate that ET reverses the dysfunction of the NO/soluble guanylyl cyclase pathway present in coronary arteries of HF rats. These effects of ET are associated with increased GTP cyclohydrolase 1 expression, restoration of NO bioavailability, and reduced oxidative stress through eNOS coupling. NEW & NOTEWORTHY The present study provides a molecular basis for the exercise-induced improvement in coronary arteries function in heart failure. Increasing the expression of GTP cyclohydrolase 1, the rate-limiting enzyme in the de novo biosynthesis of tetrahydrobiopterin, exercise training couples endothelial nitric oxide synthase, reduces oxidative stress, and increases nitric oxide bioavailability and sensitivity in coronary arteries of heart failure rats.

Pancreatic kininogenase improves erectile function in streptozotocin-induced type 2 diabetic rats with erectile dysfunction.

Erectile dysfunction (ED) associated with type 2 diabetes is a severe problem that requires effective treatment. Pancreatic kininogenase (PK) has the potential to improve the erectile function of ED patients. This study aims to investigate the effect of PK on erectile function in streptozotocin-induced type 2 diabetic ED rats. To achieve this goal, we divided male Sprague–Dawley rats into five groups. One group was not treated, and the other four groups were treated with saline, sildenafil, PK or sildenafil, and PK, respectively, for 4 weeks after the induction of type 2 diabetic ED. Then, intracavernous pressure under cavernous nerve stimulation was measured, and penile tissue was collected for further study. Endothelial nitric oxide synthase levels, smooth muscle content, endothelium content, cyclic guanosine monophosphate (cGMP) levels in the corpus cavernosum, and neuronal nitric oxide synthase levels in the dorsal penile nerve were measured. Improved erectile function and endothelium and smooth muscle content in the corpus cavernosum were observed in diabetic ED rats. When treating diabetic ED rats with PK and sildenafil at the same time, a better therapeutic effect was achieved. These data demonstrate that intraperitoneal injection of PK can improve erectile function in a rat model of type 2 diabetic ED. With further research on specific mechanisms of erectile function improvement, PK may become a novel treatment for diabetic ED.

Sulodexide recovers endothelial function through reconstructing glycocalyx in the balloon-injury rat carotid artery model.

Disruption of endothelial cell function is a principle event in cardiovascular disease. Accordingly, therapies have mostly focused on repairing the endothelium, but little attention has been paid to the reconstruction of glycocalyx, which covers the endothelium and protects the function of endothelial cells. Sulodexide has a similar glycosaminoglycan structure to glycocalyx, so it is assumed to be effective in remodeling the glycocalyx following damage. We assessed the effect of sulodexide on glycocalyx remodeling and endothelial function in the balloon-injury rat carotid artery model. Electron micrographs showed that sulodexide (2mg/kg, administered by intraperitoneal injection for seven days after injury) could reconstruct the endothelial glycocalyx and recover the clear cytoarchitecture. With regard to endothelial function, sulodexide increased endothelial nitric oxide synthase level, attenuated endothelial hyperplasia, and inhibited platelet aggregation that benefitted from glycocalyx reforming. Sulodexide decreased the glycocalyx damage related expression of CD31 and intercellular cell adhesion molecule-1 in endothelium, accompanying by the downregulation of leukocyte counts and C-reactive protein levels. The levels of the atherosclerosis-related factors, osteopontin and vascular cell adhesion molecule-1, which increased in activated endothelial cells lacking glycocalyx, were normalized by sulodexide. Along with the benefit of glycocalyx reconstruction, sulodexide reversed the dyslipidemia. Moreover, sulodexide prevented CD68-positive inflammatory cells infiltration into the vascular wall, presumably as a result of glycocalyx reconstruction. In summary, sulodexide treatment reconstructed glycocalyx which therefore preserved endothelial function and attenuated the expression of inflammatory factors, and decreased the blood coagulation and lipid metabolism, all of which are important for vascular healing.