eNOS Levels Research Articles

Abstract 10099: Loss of CD70 Impairs Nitric Oxide Homeostasis and Augments Intracellular Oxidative Stress in Endothelial Cells

Introduction: TNF superfamily ligands exert multiple effects on endothelial cell (EC) function, metabolism, and redox status. CD70 is a TNF superfamily member classically expressed on antigen-presenting cells and drives T memory cell polarization. Despite evidence of expression by ECs, the role of CD70 in endothelial biology has not been defined. We hypothesized that CD70 affects EC function through regulation of nitric oxide (NO) generation and cellular oxidative stress. Methods: Human aortic and pulmonary artery ECs (HAECs and HPAECs, respectively) were transfected with control and CD70-directed siRNA (siCD70). NO levels of individual cells were monitored in real-time after stimulation with ATP using the intracellular fluorescent biosensor c-geNOp. Similarly, intracellular hydrogen peroxide was assayed in real-time using the intracellular fluorescent biosensor Hyper7 following stimulation by auranofin and histamine. For both studies, 70-100 individual cells, averaged over 4 experiments, were analyzed using confocal microscopy. Control and siCD70-treated cell lysates (n = 4-6) were analyzed by Western blot and quantitative RT-PCR, as well as for glutathione peroxidase (GPx) enzyme activity. Results: CD70 knockdown was associated with a reduction in agonist-stimulated NO levels in HAECs and HPAECs by 30% and 40%, respectively. Protein levels of eNOS decreased after CD70 knockdown, with a reduction of 50% in HAECs and 75% in HPAECs. Phosphorylation at Ser1177 of eNOS in response to EC stimulation with ATP was reduced after siCD70 treatment, and expression of the key eNOS-stimulatory protein Hsp90 was decreased. Intracellular hydrogen peroxide was significantly increased (20% in HAECs and 80% in HPAECs) following ROS stimulation in siCD70-treated versus control cells. This change in redox status was associated with a decrease in mitochondrial superoxide dismutase protein and an increase in GPx-1 expression and enzyme activity. Conclusions: Loss of CD70 in ECs is associated with reduced eNOS expression and bioactivity as well as increased susceptibility to intracellular oxidative stress, potentially from a mitochondrial source. These results suggest an important homeostatic role for CD70 in endothelial function and redox status.

Ziziphus Oxyphylla hydro-methanolic extract ameliorates hypertension in L-NAME induced hypertensive rats through NO/cGMP pathway and suppression of oxidative stress related inflammatory biomarkers

Ethnopharmacological relevanceZiziphus Oxyphylla belongs to family Ziziphus and has been used traditionally in hypertension. It is enriched with quercetin and kaempferol derivatives, catechin and cyclopeptide alkaloids. AimThe current research evaluates the antihypertensive potential of aqueous methanolic extract of Z. oxyphylla (AMEZO) in NG-nitro-L-arginine methyl ester (LNAME) induced hypertension in rats. Material and methodologyPhytochemical analysis of AMEZO was carried out using high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS/MS). Antihypertensive activities of AMEZO (200 and 400 mg/kg) and Kaempferol were assessed in L-NAME (185 μmol/kg, intraperitoneal) injected hypertensive rats. In normotensive rats, blood pressure was assessed using Power Lab data system. Serum and tissue samples were preserved for estimation of nitric oxide (NO), Cyclic guanosine monophosphate (cGMP), interleukin-6 (IL-6), tumor necrosis factor (TNF- α) and oxidative stress markers respectively. mRNA levels of eNOS, ACE, COX-2 and NF-kB genes were assessed through qPCR. ResultsThe HPLC and ESI-MS/MS identified kaempferol, quercetin, catechin, ceanothic acid, zizybernalic acid and oxyphylline F. Chronic administration of AMEZO and kaempferol in L-NAME induced hypertensive rats significantly (p < 0.001) reduced systolic, diastolic and mean blood pressure. AMEZO and kaempferol caused meaningfully improved (p < 0.001) serum NO and cGMP levels. AMEZO administration also noticeably decrease the elevated IL-6 and TNF- α concentration in hypertensive animals. Administration of AMEZO and kaempferol also improved oxidative stress markers (MDA, CAT, SOD, GSH). The antihypertensive activity of AMEZO also resulted in upregulation of eNOS and downregulation of ACE. ConclusionThese data depict that AMEZO and kaempferol showed antihypertensive activity in LNAME induced hypertensive rats possibly mediated through improvement in NO and cGMP levels, modulation of mRNA expression of eNOS, ACE, COX-2 and NF-kB and suppression of oxidative stress related inflammatory markers, proposing a defensive role in cardiovascular diseases.

MiR-1226-3p Promotes eNOS Expression of Pulmonary Arterial Endothelial Cells to Mitigate Hypertension in Rats via Targeting Profilin-1.

In pulmonary arterial hypertension (PAH), microRNAs (miRNAs) are related with dysfunction of pulmonary arterial endothelial cells. miR-1226-3p was found to be downregulated in the serum of PAH patients, while few studies have illustrated the regulation mechanism of miR-1226-3p on PAH. In this study, we aimed to systematically investigate the role of miR-1226-3p in PAH. Sprague-Dawley (SD) rats were treated with monocrotaline (MCT) to establish the PAH models. The right ventricular systolic pressure (RVSP), ratio of the right ventricle to the left ventricle with septum (RV/(LV+S) ratio), and nitric oxide (NO) content were used to reflect the symptom of the rats. The rat models were used to observe the regulation mechanism of miR-1226-3p on PAH, and dual-luciferase reporter assay was used to verify the binding effect of miR-1226-3p to Pfn1. Besides, the qRT-PCR and western blot were used to measure the expression levels of miR-1226-3p and some keys proteins such as eNOS and Pfn1, respectively. The results showed that the PAH models were established successfully. The RVSP levels and the RV/(LV+S) ratio of the PAH rats were higher than those indexes in normal rats, while the NO content showed the opposite trends. Besides, the decreased miR-1226-3p and eNOS were, respectively, found in the PAH rats and rPAECs, and overexpressed miR-1226-3p could reverse the disadvantages of the PAH rats including increased RVSP, high RV/(LV+S) ratio, and decreased NO content. Furthermore, miR-1226-3p could directly target the 3′-UTR of Profilin-1 (Pfn1). Overexpressed Pfn1 led to decreased eNOS, while miR-1226-3p could partly inhibit the expression of Pfn1 and increase the expression level of eNOS in rPAECs. In summary, this study suggests miR-1226-3p as a protector to increase eNOS, improve NO content in rPAECs of the PAH rats via targeting Pfn, and finally protect the rats from the injury induced by PAH.

The impact of cardiomotor rehabilitation on endothelial function in elderly patients with chronic heart failure

BackgroundTo explore the effects of cardiac exercise rehabilitation on peripheral blood endothelial progenitor cells (EPC) in elderly patients with chronic heart failure.Methods80 elderly patients with chronic heart failure were selected from March 2017 to March 2019 and randomly divided into two groups (N = 40). The control group was treated routinely and walked freely for 30–60 min every day. The patients in the exercise rehabilitation group developed a cardiac exercise rehabilitation plan. Then, cardiac function and peripheral blood B-natriuretic peptide (BNP) levels in the two groups were compared. The cell viability, proliferation, apoptosis, and invasion ability of EPCs were detected. The levels of the PI3K/AKT pathway and eNOS and VEGF were compared.ResultsThere were no significant differences in all indexes between the two groups before treatment (P > 0.05), and both improved significantly after treatment (P < 0.05). After treatment, LVEF and LVFS in the exercise rehabilitation group were significantly higher than those in the control group (P < 0.05), and LVEDD and LVESD were significantly lower than those in the control group (P < 0.05). The BNP level in the exercise rehabilitation group was significantly lower than that in the control group (P < 0.05). The cell viability, proliferation, invasion ability of EPC, and the levels of PI3K, AKT, eNOS, and VEGF mRNA and protein in the exercise rehabilitation group were significantly higher than those in the control group. Apoptosis rate was significantly lower than those in the control group (P < 0.05).ConclusionsVisceral exercise rehabilitation can improve cardiac ejection and myocardial function in elderly patients with chronic heart failure, and can promote the vitality, proliferation, and invasion of peripheral blood EPC, and promote the expression of eNOS and VEGF by upregulating the PI3K/AKT pathway to promote angiogenesis and endothelial function.

Вплив реперфузійної терапії на рівні ендотеліальної синтази оксиду азоту у пацієнтів з гострим інфарктом міокарда та цукровим діабетом 2-го типу

The purpose of the study was to investigate the effect of reperfusion therapy on the levels of endothelial nitric oxide synthase in patients with acute myocardial infarction and concomitant type 2 diabetes mellitus. Materials and methods. The study involved patients with acute myocardial infarction, who were divided into groups depending on the presence of type 2 diabetes mellitus and the therapy performed for them: group 1 – 58 patients with acute myocardial infarction and diabetes mellitus who underwent percutaneous coronary intervention; group 2 – 15 patients with acute myocardial infarction and diabetes mellitus who underwent exclusively conservative treatment; group 3 – 40 patients with acute myocardial infarction without diabetes who underwent percutaneous coronary intervention; group 4 – 17 patients with acute myocardial infarction without diabetes, who underwent exclusively conservative treatment. Results and discussion. According to the obtained results, in patients with concomitant type 2 diabetes mellitus, significantly lower levels of endothelial nitric oxide synthase were revealed, compared with patients without disturbances in carbohydrate metabolism, which indicates deeper impairments of endothelial function against the background of hyperglycemia and insulin resistance. Analysis of the effect of reperfusion therapy showed a statistically significant increase in the level of endothelial nitric oxide synthase both in the group of patients with concomitant disorders of carbohydrate metabolism and without them, which indicates an improvement in the processes of endothelium-dependent dilation, regardless of the presence or absence of type 2 diabetes mellitus. The obtained results testify to the positive effect of percutaneous coronary intervention at the level of eNOS, which, in our opinion, will help to improve the vasodilating properties of vascular endothelium and prevent a complicated course of acute myocardial infarction. Conclusion. The patients with acute myocardial infarction and concomitant type 2 diabetes mellitus have significantly lower levels of endothelial nitric oxide synthase levels than patients without type 2 diabetes mellitus that indicates deep violations of endothelial function against background of metabolic changes associated with insulin resistance and hyperglycemia. Reperfusion therapy accelerates its restoration regardless of the presence or absence of type 2 diabetes mellitus

Stable Gastric Pentadecapeptide BPC 157 as a Therapy for the Disable Myotendinous Junctions in Rats.

(1) Aim: The stable gastric pentadecapeptide BPC 157 is known to heal transected muscle, tendon, and ligament. Thereby, in this study, we investigated the effect of BPC 157 on the dissection of the quadriceps tendon from the quadriceps muscle in rats. (2) Materials and Methods: Myotendinous junction defect, which cannot heal spontaneously in rats, as evidenced with consistent macro/microscopic, biomechanical, functional assessments, eNOS, and COX-2 mRNA levels and oxidative stress and NO-levels in the myotendinous junctions. BPC 157 (10 µg/kg, 10 ng/kg) regimen was given (i) intraperitoneally, first application immediately after surgery, last 24 h before sacrifice; (ii) per-orally, in drinking water (0.16 µg/mL, 0.16 ng/mL, 12 mL/rat/day), till the sacrifice at 7, 14, 28 and 42 postoperative days. (3) Results: These BPC 157 regimens document prominent therapy effects (macro/microscopic, biomechanical, functional much like eNOS and COX-2 mRNA levels and counteracted oxidative stress and NO-levels in the myotendinous junctions), while controls have a poor presentation. Especially, in rats with the disabled myotendinous junction, along with full functional recovery, BPC 157 counteracts muscle atrophy that is regularly progressive and brings muscle presentation close to normal. Accordingly, unlike the perilous course in controls, those rats, when receiving BPC 157 therapy, exhibit a smaller defect, and finally defects completely disappear. Microscopically, there are no more inflammatory infiltrate, well-oriented recovered tissue of musculotendon junction appears in BPC 157 treated rats at the 28 days and 42 days. (4) Conclusions: BPC 157 restores myotendinous junction in accordance with the healing of the transected muscle, tendon, and ligament.

Nitric Oxide Is Locally Produced in the Human Middle Ear and Is Reduced by Acquired Cholesteatoma.

To find out if nitric oxide (NO) can be locally produced in the middle ear and if chronic otitis media (COM) and acquired cholesteatoma affect the production. Case-control study. Two tertiary-referral hospitals. Gaseous NO from 11 patients with unilateral perforations or grommet openings was measured with chemiluminescence. Middle ear mucosa from 48 patients with COM and 26 patients with cholesteatoma was investigated. Detection of NO. Expression of nitric oxide synthase (NOS) mRNA, in mucosa from COM, cholesteatoma and healthy controls, quantified using polymerase chain reaction. The gaseous NO from ears with a unilateral tympanic membrane perforation or a grommet was higher (9 ± 3 ppb, n = 11) than among the controls (4 ± 1 ppb, n = 11, p = 0.04). Lower levels of eNOS (2.64 ± 0.86 mol/100,000 mol ACTB) were detected in the pooled samples from the COM group (n = 48), compared with the control group (140.48 ± 92 mol/100,000 mol ACTB, n = 45, p = 0.010). In the cholesteatoma group (n = 26), a lower expression of nNOS (5.78 × 10-6 ± 1.13 × 10-6 ΔCt) was found in comparison with the controls (1.23 × 10-4 ± 3.18 × 10-5 ΔCt, n = 15, p = 0.011). NO is likely a natural and permanent part of the gas mixture in the human middle ear. The presence of NOS enzymes in the middle ear mucosa indicates an ongoing NO production and the reduction of NOS in ears with cholesteatoma, and pooled samples from ears with COM, suggest a role for locally produced NO in middle ear disease.

Fig (Ficus exasperata and Ficus asperifolia)-Supplemented diet improves sexual function, endothelial nitric oxide synthase and suppresses tumour necrosis factor-alpha genes in hypertensive rats.

We measured the effect of varieties of Fig leaves, Ficus exasperata Vahl. (FE) and Ficus asperifolia Miq (FA), commonly found in Sub-sahara Africa for managing hypertension on sexual performance in hypertensive rats, which is unknown. Hypertensive rats experienced erectogenic damage after exposure to 40mgkg-1 bw-1 , N(G)-nitro-l-arginine-methyl-ester (L-NAME). Experimental rats were grouped into eight groups (n=6) namely: control rats, hypertensive rats, hypertensive treatment groups with atenolol (10mgkg-1 day-1 ) and sildenafil (5.0mg/kg), rats treated with FE- and FA-formulated biscuits at proportions of 2.5g and 5.0g respectively. Furthermore, we measured the level of sexual performance (Intromission number, latency, Mounting number, and latency), hormonal levels, phosphodiesterase-5 enzyme (PDE-5) activity and genes expressed using real-time quantitative polymerase chain reaction (RT-qPCR) in hypertensive rats. The result deduced revealed that treated hypertensive rats showed significantly reduced follicle-stimulating hormone, luteinising hormone levels, PDE-5 enzyme activity, tumour necrosis factor-alpha (TNF-α) expression while having a marked increase in testosterone level, sexual performance and endothelial nitric oxide synthase (eNOS) expression. Therefore, this study revealed the comparative dietary enhancing effect of FE- and FA-formulated biscuit on sexual behaviour activity, hormonal levels and the level of eNOS and TNF-α genes expressed in hypertensive rats.

Intermittent High Glucose Elevates Nuclear Localization of EZH2 to Cause H3K27me3-Dependent Repression of KLF2 Leading to Endothelial Inflammation

Epigenetic mechanisms have emerged as one of the key pathways promoting diabetes-associated complications. Herein, we explored the role of enhancer of zeste homolog 2 (EZH2) and its product histone 3 lysine 27 trimethylation (H3K27me3) in high glucose-mediated endothelial inflammation. To examine this, we treated cultured primary endothelial cells (EC) with different treatment conditions—namely, constant or intermittent or transient high glucose. Intermittent high glucose maximally induced endothelial inflammation by upregulating transcript and/or protein-level expression of ICAM1 and P-selectin and downregulating eNOS, KLF2, and KLF4 protein levels. We next investigated the underlining epigenetic mechanisms responsible for intermittent hyperglycemia-dependent endothelial inflammation. Compared with other high glucose treatment groups, intermittent high glucose-exposed EC exhibited an increased level of H3K27me3 caused by reduction in EZH2 threonine 367 phosphorylation and nuclear retention of EZH2. Intermittent high glucose also promoted polycomb repressive complex-2 (PRC2) assembly and EZH2′s recruitment to histone H3. Abrupt enrichment of H3K27me3 on KLF2 and KLF4 gene promoters caused repression of these genes, further supporting endothelial inflammation. In contrast, reducing H3K27me3 through small molecule and/or siRNA-mediated inhibition of EZH2 rescued KLF2 level and inhibited endothelial inflammation in intermittent high glucose-challenged cultured EC and isolated rat aorta. These findings indicate that abrupt chromatin modifications cause high glucose-dependent inflammatory switch of EC.

Gestational Intermittent Hypoxia Induces Sex-Specific Impairment in Endothelial Mechanisms and Sex Steroid Hormone Levels in Male Rat Offspring.

Obstructive sleep apnea (OSA) is highly prevalent during gestation and is linked with adverse fetal outcomes. We examined whether gestational intermittent hypoxia (GIH), the main feature of OSA, leads to sex-specific alterations in cardiovascular function and vascular mechanisms in the offspring. Pregnant rats exposed to intermittent hypoxia or ambient air from gestation days 10 to 21 and their offspring were used for the study. GIH exposure did not affect water and food intake in dams. Compared to controls, the male and female offspring born to GIH dams were smaller in weight by 14% and 12%, respectively, and exhibited catch-up growth. Cardiac function was not affected in either GIH males or females. At 12weeks of age, blood pressure was increased in GIH males, but not GIH females, compared to their control counterparts. While mesenteric arterial contractile responses to phenylephrine and endothelin were unaffected in GIH males and females, relaxation response to acetylcholine was reduced in GIH males but not GIH females. Relaxation to sodium nitroprusside was unaffected in both GIH males and females. Total eNOS expression was not affected, but phospho(Ser1177)-eNOS levels were decreased in GIH males. eNOS expression and its phosphorylation status were unaffected in GIH females. Serum testosterone and estradiol levels were higher in GIH males but were unaltered in GIH females. Together, these findings suggest that GIH leads to a sex-specific increase in blood pressure in adult male offspring with blunted endothelium-mediated relaxation, decreased eNOS activity, and elevated sex steroid hormone levels.

Knockdown of miR-423-5p simultaneously upgrades the eNOS and VEGFa pathways in ADSCs and improves erectile function in diabetic rats.

This study aimed to explore the possibility of miR‐423‐5p modified adipose‐derived stem cell (ADSCs) therapy on streptozotocin (STZ)‐induced diabetes mellitus erectile dysfunction (DMED) rats. MiR‐423‐5p was knocked down in ADSCs. ADSCs, NC‐miR‐ADSCs and miR‐ADSCs were co‐cultured with human umbilical vein endothelial cells (HUVECs). Normal and high glucose media were supplemented. The supernatant and HUVECs were collected for assessment of eNOS and VEGFa expression, cell proliferation, and apoptosis. HUVECs co‐cultured with ADSCs or miR‐ADSCs exhibited higher eNOS and VEGFa protein expression levels compared to DM groups. MiR‐ADSCs enhanced HUVEC proliferation compared to the ADSCs and NC‐miR‐ADSCs. Lower apoptotic rates were observed when HUVECs were co‐cultured with miR‐ADSCs, compared to ADSCs and NC‐miR‐ADSCs. Fifteen male Sprague‐Dawley (SD) rats aged 12 weeks were induced to develop diabetes mellitus by intraperitoneal injection with STZ, and five healthy SD rats were used as normal controls. Eight weeks after developing diabetes, the rats received ADSCs and miR‐ADSCs via injection into the corpora cavernosa, whereas normal controls and DM controls were injected with saline. Erectile function and histological assessment of penile tissues were performed 8 weeks after injection. The ICP/MAP indicated that erectile function was impaired in the DM rats compared with the normal group. Injection of ADSCs and miR‐ADSCs improved erectile function significantly and was associated with the overexpression of eNOS and VEGFa. MiR‐423‐5p knockdown in ADSCs ameliorated high glucose‐mediated damage to HUVECs and improved erectile function in DM rats by inducing eNOS and VEGFa overexpression, indicating that miR‐423‐5p may be a potential target in the treatment of DMED.

3′-Oxo-tabernaelegantine A (OTNA) selectively relaxes pulmonary arteries by inhibiting AhR

BackgroundPulmonary arterial hypertension (PAH), characterized by pulmonary artery constriction and vascular remodeling, has a high mortality rate. New drugs for the treatment of PAH urgently need to be developed. PurposeThis study was designed to investigate the vasorelaxant activity of OTNA in isolated pulmonary arteries, and explore its molecular mechanism. MethodsPulmonary arteries and thoracic aortas were isolated from mice, and vascular tone was tested with a Wire Myograph System. Nitric oxide levels were determined with DAF-FM DA and DAX-J2™ Red. Cellular thermal shift assays, microscale thermophoresis, and molecular docking were used to identify the interaction between OTNA and aryl hydrocarbon receptor (AhR). The levels of PI3K, p-PI3K, Akt, p-Akt, eNOS, p-eNOS, and AhR were analyzed by Western blotting. ResultsOTNA selectively relaxed the isolated pulmonary artery rings in an endothelium-dependent manner. Mechanistic study showed that OTNA induced NO production through activation of the PI3K/Akt/eNOS pathway in endothelial cells. Furthermore, we also found that OTNA directly bound to AhR and activated the PI3K/Akt/eNOS pathway to dilate pulmonary arteries by inhibiting AhR. ConclusionsOTNA relaxes pulmonary arteries by antagonizing AhR. This study provides a new natural antagonist of AhR as a promising lead compound for PAH treatment.

The serum levels of testosterone in coronary artery disease patients; relation to NO, eNOS, endothelin-1, and disease severity.

The changes in testosterone level and its correlation with the endothelial nitric oxide systems balance in patients with coronary artery disease (CAD) remains uncertain. Therefore, in our study, we aimed to evaluate the levels of testosterone, endothelin-1 (ET-1), nitric oxide (NO), and endothelial NOS (eNOS) in CAD patients, and control group to find the relationship between these parameters and disease severity. Forty-four patients as CAD group with significant (≥50%) stenosis confirmed by angiography was included in the study, and 40 healthy men were included as the control group. According to the number of vessels obstruction, CAD severity was determined. The serum indicated parameters were assessed to discriminate between patients and controls. It was found that testosterone levels in the CDA group were significantly lower than those of the control group (p<0.05). In addition, the level of ET-1 in the CAD group was higher than that in the control group, but levels of NO and eNOS in observation were significantly lower than those in the control group (p<0.05). The correlation analysis revealed that testosterone was passivity correlated with serum NO levels (r=0.550, p=0.001). The current study reports that serum levels of testosterone are closely related to endothelial NO levels and might be of relevance to the pathogenesis of endothelial dysfunction and disease severity in CAD patients.

Diagnostic accuracy of maternal serum endothelial-derived nitric oxide synthase (eNOS) and its correlation with birth outcomes, in preeclampsia - a case-control study

Introduction and Aim: Pre­eclampsia (PE) results from impaired placentation, leading to placental hypoxia and dysfunction and abnormalities in the endothelial nitric oxide synthase (eNOS)-nitric oxide pathway. The present study was undertaken to estimate and compare maternal serum eNOS concentrations in women with PE and normal healthy pregnant women, to know the correlation of eNOS level with severity of PE, to find the best cut-off value for the diagnosis of PE and to study its association of eNOS levels with the birth outcome.&#x0D; Material and Methods: This was a case-control study, conducted at a tertiary care hospital. The study was approved by the Institutional ethics committee. Informed consent was obtained from all the participants. Primigravida (singleton) pregnant women aged 18-35 years, 20 or more weeks of gestation, diagnosed as PE and classified as mild/severe PE based on America college of obstetricians and gynecologists guidelines were selected for the study. eNOS was estimated by the ELISA method. All the participants were visited again to note the outcomes. &#x0D; Results:The maternal serum eNOS in PE was significantly lower than the control group (p=0.019). The best cut-off value was 187.25pg/ml to diagnose PE, area under the ROC curve (AUC) 0.61, sensitivity 95.1%, specificity 37.5% and diagnostic accuracy was 66,67%. Birth outcomes did not have a significant correlation with eNOS.&#x0D; Conclusion: eNOS was significantly lowerer in PE, it can be used as a diagnostic marker with the best cut of value of 187.25 pg/ml.&#x0D;

Effects of different enantiomers of amlodipine on lipid profiles and vasomotor factors in atherosclerotic rabbits.

This research aimed to describe the functions of vascular endothelial cells (VECs) in protecting target organs and the anti-atherosclerotic effects of different enantiomers of amlodipine on a rabbit model of atherosclerosis. Thirty male New Zealand white rabbits were randomly allocated to four groups (nA = 9, nB = 7, nC = 7, and nD = 7 rabbits): rabbits in group-A (control group) were fed a high-fat diet, group-B rabbits were fed a high-fat diet plus 2.5 mg/kg/day S-amlodipine, group-C rabbits were fed a high-fat diet plus 2.5 mg/kg/day R-amlodipine, and group-D rabbits were fed a high-fat diet plus 5 mg/kg/day racemic amlodipine. Different enantiomers of amlodipine did not influence lipid profiles and serum level of eNOS in the rabbit atherosclerosis model but decreased ET-1 expression to some extent. The serum NO and iNOS levels in the drug intervention groups were significantly reduced. No significant differences in the rabbits’ body weights were observed. At the 4th and 8th weeks, the serum lipid profiles significantly increased in high cholesterol diet groups. The serum ET-1 level was significantly increased in each group of rabbits at the 8th week. Both S-amlodipine and R-amlodipine may protect the endothelium by reducing the serum ET-1 level, downregulating iNOS expression.

Features of cognitive impairment in patients with chronic kidney disease stages 3A-5D: biological value of endothelial nitric oxide synthase.

THE AIM:a comprehensive assessment of the frequency, severity, and composition of cognitive impairments (CI) in patients with CKD stages 3A-5D, as well as to study the relationship between the levels of endothelial nitric oxide synthase (eNOS) and these impairments. PATIENTS AND METHODS:The study included 80 patients with CKD aged 26 to 79 years (mean age 58.9 ± 1.4 years), among them 43 women (mean age 60.1 ± 1.9 years) and 37 men (mean age 57.4 ± 2.3 years). All patients were divided into 2 groups: group 1 (pre-dialysis) included 40 patients (28 women and 12 men) with CKD 3A-5 (mean age 59.9 ± 2.1 years), group 2 (on dialysis) included 40 patients (18 women and 22 men) with CKD 5D (mean age 58.1 ± 2.1 years). Sarcopenia was verified according to the criteria of the European Working Group on Sarcopenia in Older People (EWGSOP). The presence and severity of CI were determined using the short mental status assessment scale (MMSE) and the Montreal cognitive assessment test (MoCA). To identify endothelial dysfunction, all patients underwent a test with endothelium-dependent vasodilation of the brachial artery, as well as determination of the level of eNOS (ELISA Kit, USA) in serum. RESULTS:The prevalence of sarcopenia in the 1st group was 12.5 %, and in the 2nd group, 42.5 %. The average age of patients with sarcopenia was 66.1 ± 2.1 years. The prevalence of CI according to the MoCA scale in the general cohort was 70 %, while in the 1st group – 67.5 %, in the second – 72.5 %, in the subgroup with sarcopenia – 76.2 %. CIs, determined by the MMSE scale, were on average in 67.5 % of the surveyed, and the quantitative prevalence in the groups was identical, however, the qualitative composition of CIs in the comparative analysis differed in the severity of manifestations. In a comparative assessment of the prevalence of CI (according to the MMSE scale) in patients with sarcopenia, it was found that this indicator was significantly higher than in the subgroup without sarcopenia and amounted to 90.5 % and 59.3 %, respectively. In patients with sarcopenia, the level of eNOS was lower than the mean values compared with the general sample, patients of the 1st group and the subgroup without sarcopenia (0.75 ± 0.1 ng / ml, 0.88 ± 0.1 ng / ml, 1 ± 0.1 ng / ml and 0.92 ± 0.2 ng / ml, respectively (p = 0.02)). According to the results of the test with endothelium-dependent vasodilation, the prevalence of endothelial dysfunction in the total cohort of patients was 48.8 % (in the 1st group – 27.5 %, and in the 2nd group – 70 % (p = 0.001)), in the subgroup of patients with sarcopenia – 57.1 %. CONCLUSION:A high prevalence of CI was found in patients with CKD. The progression of CKD is associated with the formation of endothelial dysfunction and the development of CI. The latter significantly impairs the quality and life expectancy of patients. The main mechanism of their development is increasing endothelial dysfunction, and eNOS plays a key role in this process. Sarcopenia is associated with an increased risk of CI, regardless of the studied population and the criteria for the diagnosis of sarcopenia, and plays an important prognostic value.

Y-016. Interaction between previous preeclampsia and inter-pregnancy chronic hypertension development on preeclampsia risk in the subsequent pregnancy

Our recent study demonstrates the beneficial effect of a combined supplementation of vitamin B12, folic acid, and docosahexaenoic acid in reducing the severity of pregnancy induced hypertension (PIH). It is also known to be associated with angiogenic imbalance and inflammation. The current study examines whether the individual/combined supplementation of folic acid, vitamin B12 and omega-3 fatty acid during pregnancy can ameliorate the inflammatory markers and restore the angiogenic balance in a rat model of PIH.There were total of six groups, control and five treatment groups: PIH Induced; PIH+vitamin B12; PIH+folic acid; PIH+Omega-3 fatty acids and PIH+combined micronutrient supplementation (vitamin B12+folic acid+omega-3 fatty acids). Hypertension during pregnancy was induced using L- Nitroarginine methylester (L-NAME; 50 mg/kg body weight/day). Dams were dissected at d20 of gestation and placental tissues were collected for further analysis.Animals from the PIH induced group demonstrated lower (p<0.01 for both) IL-10 and VEGF levels as compared to control. However, PIH induction did not alter the protein levels of eNOS, IL-6, Flt and mRNA levels of VEGF and VEGFR-1/ Flt-1. Individual micronutrient supplementation of vitamin B12 and folate did not offer benefit. In contrast individual omega-3 fatty acid as well as combined micronutrient supplementation showed IL-10 and VEGF levels comparable to that of control.Omega 3 fatty acid supplementation plays a key role in reducing inflammation in pregnancy induced hypertension.

Comparison of the antiremodeling effects of losartan and mirabegron in a rat model of uremic cardiomyopathy

Uremic cardiomyopathy is characterized by diastolic dysfunction (DD), left ventricular hypertrophy (LVH), and fibrosis. Angiotensin-II plays a major role in the development of uremic cardiomyopathy via nitro-oxidative and inflammatory mechanisms. In heart failure, the beta-3 adrenergic receptor (β3-AR) is up-regulated and coupled to endothelial nitric oxide synthase (eNOS)-mediated pathways, exerting antiremodeling effects. We aimed to compare the antiremodeling effects of the angiotensin-II receptor blocker losartan and the β3-AR agonist mirabegron in uremic cardiomyopathy. Chronic kidney disease (CKD) was induced by 5/6th nephrectomy in male Wistar rats. Five weeks later, rats were randomized into four groups: (1) sham-operated, (2) CKD, (3) losartan-treated (10 mg/kg/day) CKD, and (4) mirabegron-treated (10 mg/kg/day) CKD groups. At week 13, echocardiographic, histologic, laboratory, qRT-PCR, and Western blot measurements proved the development of uremic cardiomyopathy with DD, LVH, fibrosis, inflammation, and reduced eNOS levels, which were significantly ameliorated by losartan. However, mirabegron showed a tendency to decrease DD and fibrosis; but eNOS expression remained reduced. In uremic cardiomyopathy, β3-AR, sarcoplasmic reticulum ATPase (SERCA), and phospholamban levels did not change irrespective of treatments. Mirabegron reduced the angiotensin-II receptor 1 expression in uremic cardiomyopathy that might explain its mild antiremodeling effects despite the unchanged expression of the β3-AR.

Valsartan Prevented Neointimal Hyperplasia and Inhibited SRSF1 Expression and the TLR4-iNOS-ERK-AT1 Receptor Pathway in the Balloon-injured Rat Aorta.

Valsartan has the potential to attenuate neointimal hyperplasia and to suppress the inflammatory response. This study aimed to evaluate the role of valsartan in neointimal hyperplasia and the toll-like receptor 4 (TLR4)-nitric oxide synthase (NOS) pathway in the balloon-injured rat aorta.Forty-eight Wistar rats were randomly allocated to three groups: sham control (control), balloon-injured group (surgery), and balloon-injured+valsartan-treated group (valsartan). Rats were killed at 14 and 28 days after balloon-injury, and then the aortic tissues were collected for morphometric analysis as well as for measurements of the mRNA or protein expression of angiotensin II, angiotensin II type 1 (AT1) receptor, angiotensin II type 2 (AT2) receptor, TLR4, endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), serine/arginine-rich splicing factor 1(SRSF1) and extracellular signal regulated kinase (ERK). Valsartan at a dose of 20 mg/kg/day markedly decreased neointimal hyperplasia in the aorta of balloon-injured rats, and significantly reduced the mRNA or protein expression of TLR4, AT1 receptor, SRSF1 and phosphorylated-ERK (p-ERK) as well as the aortic levels of iNOS (all p < 0.05). Moreover, valsartan increased the eNOS level and AT2 receptor mRNA and protein expression levels (all p < 0.05). Valsartan prevented neointimal hyperplasia and inhibited SRSF1 expression and the TLR4-iNOS-ERK-AT1 receptor pathway in the balloon-injured rat aorta.

C1q/tumor necrosis factor-related protein-3 improves microvascular endothelial function in diabetes through the AMPK/eNOS/NO· signaling pathway

The repair of vascular endothelial cell dysfunction is an encouraging approach for the treatment of vascular complications associated with diabetes. It has been demonstrated that members of C1q/tumor necrosis factor-related protein (CTRP) family may improve endothelial function. Nevertheless, the protective properties of CTRPs in diabetic microvascular complications continue to be mostly unknown. Here, we demonstrate that the C1q-like globular domain of CTRP3, CTRP5, and CTRP9 (gCTRP3, 5, 9) exerted a vasorelaxant effect on the microvasculature, of which gCTRP3 was the most powerful one. In a murine model of type 2 diabetes mellitus, serum gCTRP3 level and endothelial function decreased markedly compared with controls. Two weeks of gCTRP3 treatment (0.5 μg/g/d) enhanced endothelium-dependent relaxation in microvessels, increased nitric oxide (NO·) production, and reduced retinal vascular leakage. In addition, Western blotting in human retinal microvascular endothelial cells indicated that gCTRP3 triggered AMP-activated protein kinase-α (AMPKα), hence increasing the endothelial NO synthase (eNOS) level and NO· production. In addition, incubation with gCTRP3 in vitro ameliorated the endothelial dysfunction induced by high glucose in the branch of the mesenteric artery. Blockade of either eNOS or AMPKα completely abolished the effects of gCTRP3 described above. Taken together, we demonstrate for the first time that gCTRP3 improves impaired vasodilatation of microvasculature in diabetes by ameliorating endothelial cell function through the AMPK/eNOS/NO· signaling pathway. This finding may suggest an effective intervention against diabetes-associated microvascular complications.