Search

Background Tibetan pigs have exhibited unique characteristics from low‐altitudes pigs and adapted well to the Qinghai‐Tibet Plateau. Objectives The current study was undertaken to investigate the hypoxic adaptation of heart in Tibetan pigs. Methods The hearts of Tibetan pigs and Landrace pigs raised at high or low altitudes were compared using 3D casting technology, scanning electron microscopy and real‐time quantitative PCR (qRT‐PCR). Results We found that the ratio of the major axis to the minor axis and the density of the heart were significantly higher in Tibetan pigs than in Landrace pigs (p, Tibetan pigs have exhibited unique characteristics from low‐altitudes pigs and have adapted well to the Qinghai‐Tibet Plateau, but how this occurs is incompletely understood. We found that Tibetan pigs had larger diameters and higher densities of arterioles than Landrace pigs (p

Background. C-reactive protein (CRP) is an independent biomarker of systemic inflammation and a predictor of future cardiovascular disease (CVD). More than just a pure bystander, CRP directly interacts with endothelial cells to decrease endothelial nitric oxide synthase (eNOS) expression and bioactivity, decrease nitric oxide (NO) production, and increase the release of vasoconstrictors and adhesion molecules. Race is significantly associated with CRP levels and CVD risks. With aerobic exercise, the vessel wall is exposed to chronic high laminar shear stress (HiLSS) that shifts the endothelium phenotype towards an anti-inflammatory, antioxidant, antiapoptotic, and antiproliferative environment. Thus, the purpose of this study was to assess the racial differences concerning the CRP-induced effects in endothelial cells and the potential role of HiLSS in mitigating these differences. Methods. Human umbilical vein endothelial cells (HUVECs) from four African American (AA) and four Caucasian (CA) donors were cultured and incubated under the following conditions: (1) static control, (2) CRP (10 μg/mL, 24 hours), (3) CRP receptor (FcγRIIB) inhibitor followed by CRP stimulation, (4) HiLSS (20 dyne/cm2, 24 hours), and (5) HiLSS followed by CRP stimulation. Results. AA HUVECs had significantly higher FcγRIIB receptor expression under both basal and CRP incubation conditions. Blocking FcγRIIB receptor significantly attenuated the CRP-induced decrements in eNOS expression only in AA HUVECs. Finally, HiLSS significantly counteracted CRP-induced effects. Conclusion. Understanding potential racial differences in endothelial function is important to improve CVD prevention. Our results shed light on FcγRIIB receptor as a potential contributor to racial differences in endothelial function in AA.

Nitric oxide (NO) is a key factor in inflammation. Endothelial nitric oxide synthase (eNOS), whose activity increases after stimulation with proinflammatory cytokines, produces NO in endothelium. NO activates two pathways: 1) soluble guanylate cyclase-protein kinase G and 2) S-nitrosylation (NO-induced modification of free-thiol cysteines in proteins). S-nitrosylation affects phosphorylation, localization, and protein interactions. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce a fast leukocyte adhesion, which suggests that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium in vitro and in vivo using tumor necrosis factor-α (TNF-α) as proinflammatory agonist. ICAM-1 changes were evaluated by immunofluorescence, subcellular fractionation, immunoprecipitation, and fluorescence recovery after photobleaching (FRAP). Protein kinase Cζ (PKCζ) activity and S-nitrosylation were evaluated by Western blot analysis and biotin switch method, respectively. TNF-α, at short times of stimulation, activated the eNOS S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-α-induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-α-induced NO also produced S-nitrosylation and phosphorylation of PKCζ, association of PKCζ with ICAM-1, and ICAM-1 phosphorylation. The inhibition of PKCζ blocked leukocyte adhesion induced by TNF-α. Mass spectrometry analysis of purified PKCζ identified cysteine 503 as the only S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS S-nitrosylation-dependent mechanism that induces leukocyte adhesion and suggests that S-nitrosylation of PKCζ may be an important regulatory step in early leukocyte adhesion in inflammation. NEW & NOTEWORTHY Contrary to the well-established inhibitory role of NO in leukocyte adhesion, we demonstrate a positive role of nitric oxide in this process. We demonstrate that NO induced by eNOS after TNF-α treatment induces early leukocyte adhesion activating the S-nitrosylation pathway. Our data suggest that PKCζ S-nitrosylation may be a key step in this process.

Dietary supplementation with 0.4 or 0.8% l-arginine (Arg) to gilts between days 14 and 25 of gestation enhances embryonic survival and vascular development in placentae; however, the underlying mechanisms are largely unknown. This study tested the hypothesis that Arg supplementation stimulated placental expression of mRNAs and proteins that enhance angiogenesis, including endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), placental growth factor (PGF), GTP cyclohydrolase-I (GTP-CH1), ornithine decarboxylase (ODC1), and vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2). Beginning on the day of breeding, gilts were fed daily 2 kg of a corn–soybean meal-based diet supplemented with 0.0 (control), 0.4, or 0.8% Arg. On day 25 of gestation, gilts were hysterectomized to obtain uteri and conceptuses for histochemical and biochemical analyses. eNOS and VEGFR1 proteins were localized to endothelial cells of maternal uterine blood vessels and to the uterine luminal epithelium, respectively. Compared with the control, dietary supplementation with 0.4 or 0.8% Arg increased (P

Purpose Endothelial dysfunction and the subsequent decrease in endothelium-dependent vascular relaxation of small arteries are major features of hypertension. Artemisinin, a well-known antimalarial drug, has been shown to exert protecting roles against endothelial cell injury in cardiac and pulmonary vascular diseases. The current study aimed to investigate the effects of artemisinin on endothelium-dependent vascular relaxation and arterial blood pressure, as well as the potential signalling pathways in spontaneously hypertensive rats (SHRs). Methods In this study, acetylcholine (ACh)-induced dose-dependent relaxation assays were performed to evaluate vascular endothelial function after treatment with artemisinin. Artemisinin was administered to the rats by intravenous injection or to arteries by incubation for the acute exposure experiments, and it was administered to rats by intraperitoneal injection for 28 days for the chronic experiments. Results Both acute and chronic administration of artemisinin decreased the heart rate and improved ACh-induced endothelium-dependent relaxation but negligibly affected the arterial blood pressure in SHRs. Incubation with artemisinin decreased basal vascular tension, NAD(P)H oxidase activity and reactive oxygen species (ROS) levels, but it also increased endothelial nitric oxide (NO) synthase (eNOS) activity and NO levels in the mesenteric artery, coronary artery, and pulmonary artery of SHRs. Artemisinin chronic administration to SHRs increased the protein expression of eNOS and decreased the protein expression of the NAD(P)H oxidase subunits NOX-2 and NOX-4 in the mesenteric artery. Conclusion These results indicate that treatment with artemisinin has beneficial effects on reducing the heart rate and basal vascular tension and improving endothelium-dependent vascular relaxation in hypertension, which might occur by increasing eNOS activation and NO release and inhibiting NAD(P)H oxidase derived ROS production., Graphical Abstract

Peroxynitrite is a reactive intermediate formed in vivo through uncatalysed reaction of superoxide and nitric oxide radicals. Despite significant interest in detecting peroxynitrite in vivo and understanding its production, little attention has been given to the evolutionary origins of peroxynitrite signalling. Herein we focus on two enzymes that are key to the biosynthesis of superoxide and nitric oxide, NADPH oxidase 5 (NOX5) and endothelial nitric oxide synthase (eNOS), respectively. Multiple sequence alignments of both enzymes including homologues from all domains of life, coupled with a phylogenetic analysis of NOX5, suggest eNOS and NOX5 are present in animals as the result of horizontal gene transfer from ancestral cyanobacteria to ancestral eukaryotes. Therefore, biochemical studies from other laboratories on a NOX5 homologue in Cylindrospermum stagnale and an eNOS homologue in Synechococcus sp. PCC 7335 are likely to be of relevance to human NOX5 and eNOS and to the production of superoxide, nitric oxide and peroxynitrite in humans.

Age-related cerebral small-vessel disease (CSVD) is a major cause of stroke and dementia. Despite a widespread acceptance of small-vessel arteriopathy, lacunar infarction, diffuse white matter injury, and cognitive impairment as four cardinal features of CSVD, a unifying pathologic mechanism of CSVD remains elusive. Herein, we introduce partial endothelial nitric oxide synthase (eNOS)-deficient mice as a model of age-dependent, spontaneous CSVD. These mice developed cerebral hypoperfusion and blood-brain barrier leakage at a young age, which progressively worsened with advanced age. Their brains exhibited elevated oxidative stress, astrogliosis, cerebral amyloid angiopathy, microbleeds, microinfarction, and white matter pathology. Partial eNOS-deficient mice developed gait disturbances at middle age, and hippocampus-dependent memory deficits at older ages. These mice also showed enhanced expression of bone morphogenetic protein 4 (BMP4) in brain pericytes before myelin loss and white matter pathology. Because BMP4 signaling not only promotes astrogliogenesis but also blocks oligodendrocyte differentiation, we posit that paracrine actions of BMP4, localized within the neurovascular unit, promote white matter disorganization and neurodegeneration. These observations point to BMP4 signaling pathway in the aging brain vasculature as a potential therapeutic target. Finally, because studies in partial eNOS-deficient mice corroborated recent clinical evidence that blood-brain barrier disruption is a primary cause of white matter pathology, the mechanism of impaired nitric oxide signaling-mediated CSVD warrants further investigation.

Background Preventing pulmonary vascular remodeling is a key strategy for pulmonary hypertension (PH). Causes of PH include pulmonary vasoconstriction and inflammation. This study aimed to determine whether cilostazol (CLZ), a phosphodiesterase-3 inhibitor, prevents monocrotaline (MCT)- and chronic hypoxia (CH)-induced PH development in rats. Methods Fifty-one male Sprague–Dawley rats were fed rat chow with (0.3% CLZ) or without CLZ for 21 days after a single injection of MCT (60 mg/kg) or saline. Forty-eight rats were fed rat chow with and without CLZ for 14 days under ambient or hypobaric (air at 380 mmHg) CH exposure. The mean pulmonary artery pressure (mPAP), the right ventricle weight-to-left ventricle + septum weight ratio (RV/LV + S), percentages of muscularized peripheral pulmonary arteries (%Muscularization) and medial wall thickness of small muscular arteries (%MWT) were assessed. Levels of the endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (peNOS), AKT, pAKT and IκB proteins in lung tissue were measured using Western blotting. Monocyte chemotactic protein (MCP)-1 mRNA in lung tissue was also assessed. Results mPAP [35.1 ± 1.7 mmHg (MCT) (n = 9) vs. 16.6 ± 0.7 (control) (n = 9) (P P P P P P Conclusions We found model differences in the effect of CLZ on PH development. CLZ might exert a preventive effect on PH development in an inflammatory PH model but not in a vascular structural change model of PH preceded by vasoconstriction. Thus, the preventive effect of CLZ on PH development might depend on the PH etiology.

Yorkshire swine were fed standard diet (n = 7) or standard diet containing applesauce rich in caffeic acid with Lactobacillus plantarum (n = 7) for 3 wk. An ameroid constrictor was next placed around the left coronary circumflex artery, and the dietary regimens were continued. At 14 wk, cardiac function, myocardial perfusion, vascular density, and molecular signaling in ischemic myocardium were evaluated. The L. plantarum-applesauce augmented NF-E2-related factor 2 (Nrf2) in the ischemic myocardium and induced Nrf2-regulated antioxidant enzymes heme oxygenase-1 (HO-1), NADPH dehydrogenase quinone 1 (NQO-1), and thioredoxin reductase (TRXR-1). Improved left ventricular diastolic function and decreased myocardial collagen expression were seen in animals receiving the L. plantarum-applesauce supplements. The expression of endothelial nitric oxide synthase (eNOS) was increased in ischemic myocardial tissue of the treatment group, whereas levels of asymmetric dimethyl arginine (ADMA), hypoxia inducible factor 1α (HIF-1α), and phosphorylated MAPK (pMAPK) were decreased. Collateral-dependent myocardial perfusion was unaffected, whereas arteriolar and capillary densities were reduced as determined by α-smooth muscle cell actin and CD31 immunofluorescence in ischemic myocardial tissue. Dietary supplementation with L. plantarum-applesauce is a safe and effective method of enhancing Nrf2-mediated antioxidant signaling cascade in ischemic myocardium. Although this experimental diet was associated with a reduction in hypoxic stimuli, decreased vascular density, and without any change in collateral-dependent perfusion, the net effect of an increase in antioxidant activity and eNOS expression resulted in improvement in diastolic function. NEW & NOTEWORTHY Colonization of the gut microbiome with certain strains of L. Plantarum has been shown to convert caffeic acid readily available in applesauce to 4-vinyl-catechol, a potent activator of the Nrf2 antioxidant defense pathway. In this exciting study, we show that simple dietary supplementation with L. Plantarum-applesauce-mediated Nrf2 activation supports vascular function, ameliorates myocardial ischemic diastolic dysfunction, and upregulates expression of eNOS.

Although arginase primarily participates in the last reaction of the urea cycle, we have previously demonstrated that arginase II is an important cytosolic calcium regulator through spermine production in a p32-dependent manner. Here, we demonstrated that rhaponticin (RPT) is a novel medicinal-plant arginase inhibitor and investigated its mechanism of action on Ca2+-dependent endothelial nitric oxide synthase (eNOS) activation. RPT was uncompetitively inhibited for both arginases I and II prepared from mouse liver and kidney. It also inhibited arginase activity in both aorta and human umbilical vein endothelial cells (HUVECs). Using both microscope and FACS analyses, RPT treatments induced increases in cytosolic Ca2+ levels using Fluo-4 AM as a calcium indicator. Increased cytosolic Ca2+ elicited the phosphorylations of both CaMKII and eNOS Ser1177 in a time-dependent manner. RPT incubations also increased intracellular L-arginine (L-Arg) levels and activated the CaMKII/AMPK/Akt/eNOS signaling cascade in HUVECs. Treatment of L-Arg and ABH, arginase inhibitor, increased intracellular Ca2+ concentrations and activated CaMKII-dependent eNOS activation in ECs of WT mice, but, the effects were not observed in ECs of inositol triphosphate receptor type 1 knockout (IP3R1-/-) mice. In the aortic endothelium of WT mice, RPT also augmented nitric oxide (NO) production and attenuated reactive oxygen species (ROS) generation. In a vascular tension assay using RPT-treated aortic tissue, cumulative vasorelaxant responses to acetylcholine (Ach) were enhanced, and phenylephrine (PE)-dependent vasoconstrictive responses were retarded, although sodium nitroprusside and KCl responses were not different. In this study, we present a novel mechanism for RPT, as an arginase inhibitor, to increase cytosolic Ca2+ concentration in a L-Arg-dependent manner and enhance endothelial function through eNOS activation. [BMB Reports 2021; 54(10): 516-521].

Rationale: Nitric oxide (NO) produced by eNOS (endothelial NO synthase) protects against myocardial ischemia-reperfusion injury (I/R). However, reperfusion of myocardium results in superoxide (O 2 •− ) generation, which promotes eNOS glutathionylation that produces O 2 •− instead of NO. It is unclear whether SG-eNOS (glutathionylated eNOS) continues to produce O 2 •− indefinitely or undergoes a time-dependent degradation. Objective: To determine whether SG-eNOS continues to produce O 2 •− in I/R for a prolonged period causing accentuated I/R injury or it undergoes a time-dependent degradation. Methods and Results: Since SG-eNOS produces significant O 2 •− instead of NO, we sought to determine the time-course of SG-eNOS levels in the human coronary artery endothelial cell in hypoxia/reoxygenation by Western analysis and immunoprecipitation. SG-eNOS was degraded by chaperone-mediated autophagy (CMA), as inhibitors of CMA rescued eNOS expression. We further confirmed CMA by high-resolution confocal and electron microscopy. We showed that SG-eNOS is targeted by HSC70 chaperone via its interaction with glutathionylated-cysteine 691 and 910. Glutathionylation of cysteine 691 residue in hypoxia/reoxygenation exposes 735QRYRL739 motif for interaction with HSC70, and consequent transportation to LAMP2A (lysosome-associated membrane protein 2) vesicle, where it is degraded by lysosomal proteases. Mutagenesis of these residues in eNOS inhibited its CMA. Using contrast echocardiography and electron paramagnetic resonance spectrometry, we found that Trx-Tg mice show improved myocardial perfusion and decreased myocardial apoptosis in I/R due to deglutathionylation of SG-eNOS and restoration of NO generation. Further, WT mice treated with rhTrx (recombinant human thioredoxin) were protected against eNOS CMA and restored NO production with improved myocardial perfusion and decreased I/R injury. Conclusions: SG-eNOS undergoes degradation via CMA, following prolonged retention in the cytosol. CMA of SG-eNOS terminates O 2 •− generation preventing further tissue damage but causes irreversible loss of eNOS and NO availability. Prompt deglutathionylation of SG-eNOS prevents CMA, promotes NO production, and improved myocardial perfusion, resulting in amelioration of reperfusion injury.

Fine particulate matter (PM2.5 )-induced detrimental cardiovascular effects have been widely concerned, especially for endothelial cells, which is the first barrier of the cardiovascular system. Among potential mechanisms involved, reactive oxidative species take up a crucial part. However, source of oxidative stress and its relationship with inflammatory response have been rarely studied in PM2.5 -induced endothelial injury. Here, as a key oxidase that catalyzes redox reactions, NADPH oxidase (NOX) was investigated. Human umbilical vein endothelial cells (EA.hy926) were exposed to Standard Reference Material 1648a of urban PM2.5 for 24 h, which resulted in NOX-sourced oxidative stress, endothelial dysfunction, and inflammation induction. These are manifested by the up-regulation of NOX, increase of superoxide anion and hydrogen peroxide, elevated endothelin-1 (ET-1) and asymmetric dimethylarginine (ADMA) level, reduced nitric oxide (NO) production, and down-regulation of phosphorylation of endothelial NO synthase (eNOS) with increased levels of inducible NO synthase, as well as the imbalance between tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1), and changes in the levels of pro-inflammatory and anti-inflammatory factors. However, administration of NOX1/4 inhibitor GKT137831 alleviated PM2.5 -induced elevated endothelial dysfunction biomarkers (NO, ET-1, ADMA, iNOS, and tPA/PAI-1), inflammatory factors (IL-1β, IL-10, and IL-18), and adhesion molecules (ICAM-1, VCAM-1, and P-selectin) and also passivated NOX-dependent AKT and eNOS phosphorylation that involved in endothelial activation. In summary, PM2.5 -induced NOX up-regulation is the source of ROS in EA.hy926, which activated AKT/eNOS/NO signal response leading to endothelial dysfunction and inflammatory damage in EA.hy926 cells.

Endothelial dysfunction is related to the reduced bioavailability of nitric oxide (NO) and plays a significant role in developing hypertension. The intake of a diet rich in antioxidants decreases t...

Background The non-cancerous functions of Akt in the airway are understudied. In some tissues, Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO) that has anti-inflammatory effects. NO production has antibacterial and antiviral effects in the airway, and increasing NO may be a useful anti-pathogen strategy. Akt also stimulates the nuclear factor erythroid 2–related factor 2 (Nrf-2) transcription factor, which transcribes antioxidant genes. Therefore, we hypothesized that activation of the Akt/eNOS pathway, which also activates Nrf-2, may have protective effects in human airway cells against injury. Methods To directly test the effects of Akt signaling in the airway, we treated A549 and 16HBE cells as well as primary bronchial, nasal, and type II alveolar epithelial cells with small molecule Akt activator SC79. We examined the effects of SC79 on eNOS activation, NO production, Nrf-2 target levels, and interleukin-8 (IL-8) transcription during exposure to TNF-α or Pseudomonas flagellin (TLR5 agonist). Additionally, air–liquid interface bronchial cultures were treated with cadmium, an oxidative stressor that causes airway barrier breakdown. Results SC79 induced a ~ twofold induction of p-eNOS and Nrf-2 protein levels blocked by PI3K inhibitor LY294002. Live cell imaging revealed SC79 increased acute NO production. Quantitative RT-PCR showed a ~ twofold increase in Nrf-2 target gene transcription. TNF-α or flagellin-induced IL-8 levels were also significantly reduced with SC79 treatment. Moreover, the transepithelial electrical resistance decrease observed with cadmium was ameliorated by SC79, likely by an acute increase in tight junction protein ZO-1 levels. Conclusions Together, the data presented here demonstrate SC79 activation of Akt induces potentially anti-pathogenic NO production, antioxidant gene transcription, reduces IL-8 transcription, and may protect against oxidative barrier dysfunction in a wide range of airway epithelial cells.

Background Uric acid (UA) transporters mediate the uptake and outflow of UA, and are greatly involved in the control of UA concentrations. Glucose transporter 9 (GLUT9), one of the UA transporters, has been confirmed to be expressed in human umbilical vein endothelial cells (HUVECs). This study aimed to characterize GLUT9’s effect on intracellular UA accumulation in HUVECs in a high-UA environment and to explore the mechanism of cellular dysfunction. Methods and results HUVECs were treated with UA to establish a model of cellular dysfunction. Then, UA uptake, GLUT9 expression and endothelial nitric oxide synthase (eNOS) and reactive oxygen species (ROS) amounts were measured. UA uptake was concentration- and time-dependent, and UA treatment significantly reduced nitric oxide (NO) levels and eNOS activity. UA also upregulated pro-inflammatory molecules and GLUT9, and increased intracellular ROS amounts in HUVECs. GLUT9 knockdown reduced UA uptake and ROS content, but antioxidant treatment did not reduce GLUT9 expression. To assess the function of JAK2/STAT3 signaling, HUVECs were treated with UA, and the phosphorylation levels of JAK2, STAT3, IL-6 and SOCS3 were increased by a high concentration of UA. In addition, GLUT9 knockdown reduced the phosphorylation of JAK2/STAT3 intermediates and increased p-eNOS amounts. Conclusions GLUT9 mediated the effects of high UA levels on HUVECs by increasing the cellular uptake of UA, activating JAK2/STAT3 signaling, and reduced the production of active eNOS and NO in HUVECs.

Doxorubicin (DOX) is an anthracycline antibiotic that exhibits high heart toxicity. Human-induced pluripotent stem cell-derived ventricular cardiomyocytes (hiPSC-vCMs) are important in vitro models for testing drug cardiotoxicity. Photobiomodulation therapy (PBMT) is a non-invasive therapy that stimulates cells growth and self-repair using light irradiation. This study aimed to investigate the in vitro effects of PBMT preconditioning on cardiotoxicity induced by DOX. HiPSC-vCMs were treated with PBMT for 500 s, followed by the addition of 2 μM DOX. LED irradiation preconditioning parameters were at 660 nm with an irradiance of 10 mW/cm2, performing 5 J/cm2, followed by 24-h DOX exposure (2 μM). Human iPSC-vCMs treated with 2 μM DOX or irradiated with PBMT composed the second and third groups, respectively. The control group did neither receive PBMT preconditioning nor DOX and was irradiated with a white standard lamp. Cells from all groups were collected to perform mRNA and miRNA expressions quantification. PBMT, when applied before the DOX challenge, restored the viability of hiPSC-vCMs and reduced ROS levels. Although downregulated by DOX, myocardial UCP2 mRNA expression presented marked upregulation after PBMT preconditioning. Expression of eNOS and UCP2 mRNA and NO production were decreased after DOX exposure, and PBMT preconditioning before the DOX challenge reversed these changes. Moreover, our data indicated that PBMT preconditioning lowered the miR-24 expression. Our data suggested that PBMT preconditioning ameliorated in vitro DOX-induced cardiotoxicity on transcription level, restoring NO levels and reducing oxidative stress.

Cancer is a lethal disease that is characterized by uncontrolled cell division and proliferation, and it results in death in many organisms. Doxorubicin (DOX) is a therapeutic agent used for treatment of many cancer types, but it induces serious hepatotoxicity. In this study, we aimed to determine possible hepato-therapeutic effects of thymoquinone (THQ) on DOX-induced hepatotoxicity in rats. Rats were divided into five groups (n = 8): Control, THQ (10 mg/kg/day/i.p for 14 days), Olive Oil (equal volume with THQ for 14 days), DOX (single dose, 15 mg/kg/i.p on 7th day) and DOX + THQ (10 mg/kg/day/i.p and DOX 15 mg/kg/i.p on 7th day). At the end of the experiment, liver tissues were extracted and evaluated histopathologically. eNOS, iNOS and Cas-3 immunostaining were performed to determine the expression levels. TUNEL method was used to determine apoptotic index. Furthermore, liver tissue total antioxidant status (TAS), total oxidant status (TOS), TNF-alpha and TGF-beta levels were measured by ELISA assay. The DOX group showed histopathological deterioration compared to Control group. Moreover, apoptotic index, eNOS, iNOS and Cas-3 expressions increased in DOX group. While TAS level of the DOX group decreased, TOS level increased. TNF-alpha and TGF-beta levels increased in DOX group. However, there was improvement in DOX + THQ group compared to DOX group. Moreover, apoptotic cell number, eNOS, iNOS and Cas-3 expressions decreased in DOX + THQ group compared to DOX group. We concluded that thymoquinone can be used as a phytotherapeutic for reducing DOX-induced liver damage.

The association of the T894G endothelial nitric oxide synthase (eNOS) gene polymorphism with certain indicators of endothelial function in patients with hypothyroidism and concomitant chronic non-calculous cholecystitis was investigated. Significant increase by 33.1 % (P = 0.01) of desquamated endothelial cells number in the blood was found in patients with T-allele compared with that of in GG-genotype carriers. Also, the concentration of total nitric oxide stable metabolites was 10.4 % (P = 0.04) higher and the level of nitrate anions in the blood was 10.8 % (P = 0.02) higher than in comparison group. Among patients with hypothyroidism and concomitant chronic cholecystitis, the content of vascular endothelial growth factor was 27.7 % (P = 0.03) higher in T-allele carriers as compared with patients with GG-genotype.

A characteristic aspect of the robust, systemic inflammatory state in sickle cell disease is dysfunction of endothelial nitric oxide synthase (eNOS). We identify ten aberrant endothelial cell inputs, present in the specific sickle context, that are known to have the ability to cause eNOS dysfunction. These are: endothelial arginase depletion, asymmetric dimethylarginine, complement activation, endothelial glycocalyx degradation, free fatty acids, inflammatory mediators, microparticles, oxidized low density lipoproteins, reactive oxygen species, and Toll-like receptor 4 signaling ligands. The effect of true eNOS dysfunction on clinical testing using flow-mediated dilation can be simulated by two known examples of endothelial dysfunction mimicry (hemoglobin consumption of NO; and oxidation of smooth muscle cell soluble guanylate cyclase). This lends ambiguity to interpretation of such clinical testing. The presence of these multiple perturbing factors argues that a therapeutic approach targeting only a single injurious endothelial input (or either example of mimicry) would not be sufficiently efficacious. This would seem to argue for identifying therapeutics that directly protect eNOS function or application of multiple therapeutic approaches. This article is protected by copyright. All rights reserved.

We have shown that endothelial-specific DHFR (dihydrofolate reductase) deficiency underlies eNOS (endothelial NO synthase) uncoupling and formation of abdominal aortic aneurysm (AAA). Here, we examined a novel role of microRNA-192-5p in mediating NOX (NADPH oxidase)-dependent DHFR deficiency and AAA formation. microRNA-192-5p is predicted to target DHFR. Intriguingly, homo sapiens–microRNA-192-5p expression was substantially upregulated in human patients with AAA. In human aortic endothelial cells exposed to hydrogen peroxide (H 2 O 2 ), homo sapiens–microRNA-192-5p expression was significantly upregulated. This was accompanied by a marked downregulation in DHFR mRNA and protein expression, which was restored by homo sapiens–microRNA-192-5p–specific inhibitor. Of note, microRNA-192-5p expression was markedly upregulated in Ang II (angiotensin II)–infused hph-1 (hyperphenylalaninemia 1) mice, which was attenuated in hph-1–NOX1, hph-1–NOX2, hph-1–neutrophil cytosol factor 1, and hph-1–NOX4 double mutant mice where AAA incidence was also abrogated, indicating a downstream effector role of microRNA-192-5p following NOX activation. In vivo treatment with mus musculus–microRNA-192-5p inhibitor attenuated expansion of abdominal aortas in Ang II–infused hph-1 mice as defined by ultrasound and postmortem inspection. It also reversed features of vascular remodeling including matrix degradation, adventitial hypertrophy, and formation of intraluminal thrombi. These animals had restored DHFR mRNA and protein expression, attenuated superoxide production, recoupled eNOS, and preserved NO bioavailability. In conclusion, our data for the first time demonstrate a critical role of microRNA-192-5p in mediating NOX-dependent DHFR deficiency and AAA formation, inhibition of which is robustly effective in attenuating development of AAA. Since the mouse and human microRNA-192-5p sequences are identical, the microRNA-192-5p inhibitors may be readily translatable into novel therapeutics for the treatment of AAA.

Background Ischemia-reperfusion injury (IRI) is the major cause of delayed graft function (DGF) during the post-transplantation period. Estradiol (E2) prevents IRI-induced kidney dysfunction and tissue injury. However, many side-effects limit E2's in vivo application. Recent evidence uncovers E2's expanded use in the field of transplantation. We aimed to study if and how E2 exerts protective activity during the period of kidney organ preservation. Methods The autologous kidney transplant model in rats was first established. Rats were divided into 5 groups, which were normal group (N), sham group (Sham), static cold storage (SCS) 4 h group (Control), SCS 4 h + ethanol (1 µl/mL) group (Solvent), and SCS 4 h + ethanol (1 µl/mL) + E2 (1000 ng/mL) group (E2). ERα expression under hypothermia was measured by western blotting. Moreover, biochemical analysis of plasma levels of Creatinine, BUN, Estradiol, and testosterone were examined. Among all groups, kidney tissues were collected and processed for further western blot analysis about ERα, eNOS, Bcl-2, and Bax expression, histological analyses such as H&E staining to evaluate pathological severity. In addition, a TUNEL assay is performed to evaluate apoptosis. Results E2 co-preservation up-regulated ERα expression under hypothermia. Moreover, E2 co-preservation reduced levels of Creatinine and BUN in plasma but without affecting estradiol and testosterone. Further, E2 co-preservation increased expression of eNOS and anti-apoptotic Bcl-2, and decreases expression of pro-apoptotic Bax. E2 co-preservation significantly inhibited IRI-induced apoptosis and evidently improved pathological severity in the kidney of rats. Conclusions E2 co-preservation exerts protective activity against IRI-induced pro-inflammatory and pro-apoptotic effects in kidneys during organ preservation time and improves transplanted kidney function.

GATA2 is a transcription factor with key roles in hematopoiesis. Germline GATA2 gene variants have been associated with several inherited and acquired hematologic disorders, including myelodysplastic syndromes. Among the spectrum of GATA2 deficiency- associated manifestations thrombosis has been reported in 25% of patients, but the mechanisms are unknown. GATA2 was shown to be involved in endothelial nitric oxide synthase (eNOS) regulation and vascular development. We assessed eNOS expression and angiogenesis in patients with GATA2 deficiency. Platelets and blood outgrowth endothelial cells (BOEC) from GATA2 variant carriers showed impaired NO production and reduction of eNOS mRNA and protein expression and of eNOS activity. GATA2 binding to the eNOS gene was impaired in BOEC from GATA2-deficient patients, differently from control BOEC. GATA2 deficiency BOEC showed also defective angiogenesis, which was completely restored by treatment with the NO-donor Snitroso- N-acetylpenicillamine (SNAP). Atorvastatin, but not resveratrol, largely restored eNOS expression, NO biosynthesis and neoangiogenesis in GATA2-deficient BOEC by a mechanism involving increased expression of the eNOS transcription factor AP-1/c-JUN, replacing GATA2 when the latter is inactive. Our results unravel a possible thrombogenic mechanism of GATA2 mutations, definitely establish the regulation of eNOS by GATA2 in endothelial cells and show that endothelial angiogenesis is strictly dependent on the eNOS/NO axis. Given the ability of atorvastatin to restore NO production and angiogenesis by GATA2-deficient endothelial cells, the preventive effect of atorvastatin on thrombotic events and possibly on other clinical manifestations of the syndrome related to deranged angiogenesis should be explored in patients with GATA2 deficiency in an ad hoc designed clinical trial.

Objective To study the therapeutic effect of Huoxue Tongluo Decoction (HXTLD) on erectile dysfunction caused by ischemic stroke and identify the mechanisms involved. Methods Network pharmacology was used to predict the key active ingredients and targets of HXTLD. Surgical methods were used to create a rat model of ischemic stroke. The rats were then given a suspension of HXTLD by ig administration. Erectile function was evaluated by Apomorphine (APO) induction. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (Real-time PCR) and Western blotting were used to detect the expression of related mRNAs and proteins in rat penile corpus cavernous tissue and brain tissue. Hematoxylin & Eosin (HE) staining was used to investigate structural changes in the penile cavernous tissue. Results Network pharmacology showed that tumor necrosis factor (TNF), nitric oxide synthase 3 (eNOS), and vascular endothelial growth factor (VEGF) were the key targets of HXTLD in the treatment of erectile dysfunction caused by ischemic stroke. Experimental studies showed that HXTLD improved erectile dysfunction caused by ischemic stroke. HE results showed that HXTLD improved the structure of the corpus cavernosa. HXTLD also inhibited the expression of TNF and VEGF proteins in penile tissue (P Conclusion HXTLD improved the erectile function of rats with erectile dysfunction caused by ischemic stroke by regulating the mRNA and protein levels of TNF, eNOS and VEGF.

Introduction. Nitric oxide (NO) produced by NO synthases (NOS) is involved in the regulation of vital physiological functions. At the same time, NO and NOS are involved in events associated with the tumor process: mutagenesis, proliferation, apoptosis, angiogenesis, etc., exerting a multidirectional effect on the tumor.Objectives – analyze and summarize literature data concerning the role of NO and endothelial NOS (eNOS) in the initiation and progression of tumors, as well as in the inhibition of tumor growth.Materials and methods. In preparing the review, publications of information bases of biomedical literature were used: SciVerse Scopus (538), PubMed (1327), Web of Science (905), Russian Science Citation Index (125).Results. The molecular mechanisms of the action of NO and its derivatives on the initiation and progression of carcinogenesis have been explored. Numerous factors and conditions regulating the activity of eNOS in health and tumor growth have been analyzed. The molecular signaling pathways through which the pro-tumor effects of NO and eNOS, stimulating angiogenesis, lymphangiogenesis, are realized, including through the mobilization of stem cells, are considered.Conclusion. Nitric oxide produced by activated eNOS promotes tumor progression by increasing the proliferation of tumor cells, enhancing the action of pro-angiogenic factors, stimulating angiogenesis, lymphangiogenesis, and metastasis. Selective inhibition of increased eNOS activity may be a promising therapeutic approach aimed at reducing metastasis and tumor growth.

Background and aims The apolipoprotein A-I mimetic peptide D-4F, among its anti-atherosclerotic effects, improves vasodilation through mechanisms not fully elucidated yet. Methods Low-density lipoprotein (LDL) receptor null (LDLr−/−) mice were fed Western diet with or without D-4F. We then measured atherosclerotic lesion formation, endothelial nitric oxide synthase (eNOS) phosphorylation and its association with heat shock protein 90 (HSP90), nitric oxide (NO) and superoxide anion (O2•-) production, and tetrahydrobiopterin (BH4) and GTP-cyclohydrolase 1 (GCH-1) concentration in the aorta. Human umbilical vein endothelial cells (HUVECs) and aortas were treated with oxidized LDL (oxLDL) with or without D-4F; subsequently, BH4 and GCH-1 concentration, NO and O2•- production, eNOS association with HSP90, and endothelium-dependent vasodilation were measured. Results Unexpectedly, eNOS phosphorylation, eNOS-HSP90 association, and O2•- production were increased, whereas BH4 and GCH-1 concentration and NO production were reduced in atherosclerosis. D-4F significantly inhibited atherosclerosis, eNOS phosphorylation, eNOS-HSP90 association, and O2•- generation but increased NO production and BH4 and GCH-1 concentration. OxLDL reduced NO production and BH4 and GCH-1 concentration but enhanced O2•- generation and eNOS association with HSP90, and impaired endothelium-dependent vasodilation. D-4F inhibited the overall effects of oxLDL. Conclusions Hypercholesterolemia enhanced uncoupled eNOS activity by decreasing GCH-1 concentration, thereby reducing BH4 levels. D-4F reduced uncoupled eNOS activity by increasing BH4 levels through GCH-1 expression and decreasing eNOS phosphorylation and eNOS-HSP90 association. Our findings elucidate a novel mechanism by which hypercholesterolemia induces atherosclerosis and D-4F inhibits it, providing a potential therapeutic approach.

Background Although high doses of proton pump inhibitors can elicit an anticancer effect, this strategy may impair vascular biology. In particular, their effects on endothelial Ca2⁺ signaling and production of endothelium-derived relaxing factor (EDRF) are unknown. To this end, we investigated the effects of high dosages of omeprazole on endothelial Ca2⁺ responses and EDRF production in primary cultured porcine aortic endothelial cells.Methods and Results Omeprazole (10–1000μM) suppressed both bradykinin (BK)- and thapsigargin-induced endothelial Ca2⁺ response in a dose-dependent manner. Furthermore, omeprazole slightly attenuated Ca2⁺ mobilization from the endoplasmic reticulum, whereas no inhibitory effects on endoplasmic reticulum Ca2⁺-ATPase were observed. Omeprazole decreased BK-induced phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 and tended to decrease BK-induced nitric oxide production. Production of prostaglandin I₂ metabolites, especially 6-keto-prostaglandin 1α, also tended to be reduced by omeprazole.Conclusion Our results are the first to indicate that high doses of omeprazole may suppress both store-operated Ca2⁺ channels and partially the G protein-coupled receptor/phospholipase C/inositol 1,4,5-triphosphate pathway, and decreased BKinduced, Ca2⁺-dependent phosphorylation of eNOS(Ser1177). Thus, high dosages of omeprazole impaired EDRF production by attenuating intracellular Ca2⁺ signaling.

Sirtuins are regulators of eNOS and endothelial function; however, no studies have examined the influence of exercise on sirtuin regulation of endothelial function. Effects of the novel sirtuin inhibitor, salermide, on vascular reactivity in rat aortas were investigated following exercise training of different durations. Male Wistar rats (8–9 months old) were divided into four groups (n = 10–12/group): sedentary (SED), 1 day (1D), 2 weeks (2WK), or 6 weeks (6WK) of exercise. Exercise consisted of running on a motor-driven treadmill at 15 m/min, 15% grade, for 40 min (1D) increased up to 1 h at the end of 2 weeks (2WK) and sustained for an additional 4 weeks (6WK). Dose responses to phenylephrine, sodium nitroprusside, and acetylcholine in the presence or absence of salermide (30 µM) were analyzed. SIRT1 and eNOS protein expression as well as nitrotyrosine levels were determined by immunoblotting. Superoxide dismutase activity was determined by colorimetric assay. Sirtuin inhibition significantly impaired acetylcholine-induced vasorelaxtion in aortas in SED, 1D, and 2WK endurance trained rats but not in 6WK. eNOS expression significantly increased ~ 2.0-fold in 1D, 2WK, and 6WK groups. SIRT1 expression and 3-nitrotyrosine levels were significantly increased in 1D and 2WK but were not significantly elevated in 6WK. SOD levels were significantly elevated in 6WK. These data suggest that chronic endurance training diminishes the role of sirtuins in regulating endothelium-dependent relaxation and appears to be related to changes in SIRT1 expression as well as redox status.

More than 30 years have passed since endothelial nitric oxide synthesis was described using the umbilical artery and vein endothelium. That seminal report set the cornerstone for unveiling the molecular aspects of endothelial function. In parallel, the understanding of placental physiology has gained growing interest, due to its crucial role in intrauterine development, with considerable long-term health consequences. This review discusses the evidence for nitric oxide (NO) as a critical player of placental development and function, with a special focus on endothelial nitric oxide synthase (eNOS) vascular effects. Also, the regulation of eNOS-dependent vascular responses in normal pregnancy and pregnancy-related diseases and their impact on prenatal and postnatal vascular health are discussed. Recent and compelling evidence has reinforced that eNOS regulation results from a complex network of processes, with novel data concerning mechanisms such as mechano-sensing, epigenetic, posttranslational modifications, and the expression of NO- and l-arginine-related pathways. In this regard, most of these mechanisms are expressed in an arterial-venous-specific manner and reflect traits of the fetal systemic circulation. Several studies using umbilical endothelial cells are not aimed to understand placental function but general endothelial function, reinforcing the influence of the placenta on general knowledge in physiology.

BACKGROUND Prenatal exposure to alcohol leads to a greater incidence of many cardiovascular-related diseases, presumably via a mechanism that may involve increased oxidative stress. An agonist of peroxisome proliferator-activated receptor gamma (PPARγ; rosiglitazone) has been shown to suppress alcohol-induced neuroinflammation and oxidative stress. The goal of this study was to determine whether acute and chronic treatment with rosiglitazone could restore or prevent impaired nitric oxide synthase (NOS)-dependent responses of cerebral arterioles in male and female adult (14-16 weeks old) rats exposed to alcohol in utero. METHODS We fed Sprague-Dawley dams a liquid diet with or without 3% ethanol for the duration of their pregnancy (21-23 days). In the first series of studies, we examined the reactivity of cerebral arterioles to eNOS- (ADP), nNOS-dependent (NMDA), and NOS-independent agonists in male and female adult rats before and during acute (1 hour) topical application of rosiglitazone (1 µM). In a second series of studies, we examined the influence of chronic treatment with rosiglitazone (3 mg/kg/day in drinking water for 2-3 weeks) on the responses of cerebral arterioles in male and female adult rats exposed to alcohol in utero. RESULTS We found that in utero exposure to alcohol similarly reduced responses of cerebral arterioles to ADP and NMDA, but not to nitroglycerin in male and female adult rats. In addition, acute treatment of the male and female adult rats with rosiglitazone similarly restored this impairment in cerebral vascular function to that observed in controls. We also found that chronic treatment with rosiglitazone prevented impaired vascular function in male and female adult rats that were exposed to alcohol in utero. CONCLUSIONS PPARγ activation may be an effective and relevant treatment to reverse or prevent cerebral vascular abnormalities associated with prenatal exposure to alcohol.

Symptoms of COVID-19 range from asymptomatic/mild symptoms to severe illness and death, consequence of an excessive inflammatory process triggered by SARS-CoV-2 infection. The diffuse inflammation leads to endothelium dysfunction in pulmonary blood vessels, uncoupling eNOS activity, lowering NO production, causing pulmonary physiological alterations and coagulopathy. On the other hand, iNOS activity is increased, which may be advantageous for host defense, once NO plays antiviral effects. However, overproduction of NO may be deleterious, generating a pro-inflammatory effect. In this review, we discussed the role of endogenous NO as a protective or deleterious agent of the respiratory and vascular systems, the most affected in COVID-19 patients, focusing on eNOS and iNOS roles. We also reviewed the currently available NO therapies and pointed out possible alternative treatments targeting NO metabolism, which could help mitigate health crises in the present and future CoV’s spillovers., Graphical abstract Image 1

Nitric oxide (NO) production by eNOS (endothelial NO synthase) is critical for vascular health. Oxidative stress-induced uncoupling of eNOS leads to decreased NO bioavailability, compounded by increased superoxide generation. FXYD1 (FXYD domain containing ion transport regulator 1), a caveolar protein, protects against oxidative inhibition of the Na + -K + -ATPase. We hypothesized that FXYD1 may afford a similar inhibition of oxidative dysregulation of eNOS, providing a broader protection within caveolae. FXYD1-eNOS colocalization was demonstrated by co-immunoprecipitation in heart protein and by proximity ligation assay in human umbilical vein endothelial cells. The functional nature of this partnership was shown by silencing FXYD1 in human umbilical vein endothelial cells, where 50% decreased NO and 2-fold augmented superoxide was shown. Three-dimensional cocultured cardiac spheroids generated from FXYD1 knockout mice were incapable of acetylcholine-induced NO production. Overexpression of FXYD1 in HEK293 cells revealed a possible mechanism, where FXYD1 protected against redox modification of eNOS cysteines. In vivo, vasodilation in response to increasing doses of bradykinin was impaired in knockout mice, and this was rescued in mice by delivery of FXYD1 protein packaged in exosomes. Bloods vessels extracted from knockout mice exhibited increased oxidative and nitrosative stress with evidence of reduce eNOS phosphorylation. Impaired vascular function and augmented superoxide generation were also evident in diabetic knockout mice. Despite this, blood pressure was similar in wildtype and knockout mice, but after chronic angiotensin II infusion, knockout of FXYD1 was associated with a heightened blood pressure response. FXYD1 protects eNOS from dysregulated redox signaling and is protective against both hypertension and diabetic vascular oxidative stress.

Cyclosporine (CsA) is a widely used immunosuppressive agent that incurs marked nephrotoxicity in the clinical setting. Thus, there is a need for finding safe/effective agents that can attenuate CsA‐induced kidney injury. Meanwhile, the underlying mechanisms for CsA‐associated nephrotoxicity are inadequately investigated, in particular, the AKT/eNOS/NO pathway. Here, the present work aimed to explore the potential of camel milk, a natural product with distinguished antioxidant/anti‐inflammatory actions, to ameliorate CsA‐induced nephrotoxicity in rats. The molecular mechanisms related to renal oxidative aberrations and apoptosis were studied, including Nrf2/HO‐1 and AKT/eNOS/NO pathways. The kidney tissues were inspected using histopathology, ELISA, Western blotting, and immunohistochemistry. The present findings demonstrated that camel milk (10 ml/kg) significantly lowered creatine, BUN, and NGAL nephrotoxicity markers and the aberrant histopathology, with similar efficacy to the reference quercetin. Moreover, camel milk suppressed the renal oxidative stress, as evidenced by significantly lowering NOX‐1 and lipid peroxides and significantly augmenting the renal antioxidant moieties (GSH, GPx, and SOD), thereby, driving the restoration of Nrf2/HO‐1 pathway. Meanwhile, camel milk counteracted the pro‐apoptotic reactions by significantly lowering Bax protein expression, caspase‐3 activity/cleavage, and PARP cleavage, alongside significantly increasing the expression of the proliferation signal PCNA. Regarding the anti‐apoptotic AKT/eNOS/NO pathway, camel milk activated its signaling by significantly increasing the protein expression of PI3Kp110, p‐AKT(Ser473)/total AKT, and p‐eNOS (Ser1177)/total eNOS besides significantly boosting the renoprotective NO levels. In conclusion, these findings reveal that camel milk may be a promising candidate for the alleviation of CsA‐induced nephrotoxicity., Camel milk lowers creatinine, BUN, and NGAL nephrotoxicity markers. It suppresses oxidative stress, NOX‐1 expression, and lipid peroxides and activates Nrf2/HO‐1 pathway, and boosts GSH, GPx, and SOD antioxidant moieties. It curtails renal apoptosis markers (Bax, PARP, and caspase‐3) and upregulates PCNA, and activates the renal PI3K/Akt/eNOS/NO pathway.

Supplemental Digital Content is available in the text., Objective: Remote ischemic preconditioning (RIPC) is an intervention process where the application of multiple cycles of short ischemia/reperfusion (I/R) in a remote vascular bed provides protection against I/R injury. However, the identity of the specific RIPC factor and the mechanism by which RIPC alleviates I/R injury remains unclear. Here, we have investigated the identity and the mechanism by which the RIPC factor provides protection. Approach and ResultS: Using fluorescent in situ hybridization and immunofluorescence, we found that RIPC induces Nrg1β expression in the endothelial cells, which is secreted into the serum. Whereas, RIPC protected against myocardial apoptosis and infarction, treatment with neutralizing-Nrg1 antibodies abolished the protective effect of RIPC. Further, increased superoxide anion generated in RIPC is required for Nrg1 expression. Improved myocardial perfusion and nitric oxide production were achieved by RIPC as determined by contrast echocardiography and electron spin resonance. However, treatment with neutralizing-Nrg1β antibody abrogated these effects, suggesting Nrg1β is a RIPC factor. ErbB2 (Erb-B2 receptor tyrosine kinase 2) is not expressed in the adult murine cardiomyocytes, but expressed in the endothelial cells of heart which is degraded in I/R. RIPC-induced Nrg1β interacts with endothelial ErbB2 and thereby prevents its degradation. Mitochondrial Trx2 (thioredoxin) is degraded in I/R, but rescue of ErbB2 by Nrg1β prevents Trx-2 degradation that decreased myocardial apoptosis in I/R. Conclusions: Nrg1β is a RIPC factor that interacts with endothelial ErbB2 and prevents its degradation, which in turn prevents Trx2 degradation due to phosphorylation and inactivation of ATG5 (autophagy-related 5) by ErbB2. Nrg1β also restored loss of eNOS (endothelial nitric oxide synthase) function in I/R via its interaction with Src.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex disease characterized by unexplained debilitating fatigue. Although the etiology is unknown, evidence supports immunological abnormalities, such as persistent inflammation and immune-cell activation, in a subset of patients. Since the interplay between inflammation and vascular alterations is well-established in other diseases, endothelial dysfunction has emerged as another player in ME/CFS pathogenesis. Endothelial nitric oxide synthase (eNOS) generates nitric oxide (NO) that maintains endothelial homeostasis. eNOS is activated by silent information regulator 1 (Sirt1), an anti-inflammatory protein. Despite its relevance, no study has addressed the Sirt1/eNOS axis in ME/CFS. The interest in circulating microRNAs (miRs) as potential biomarkers in ME/CFS has increased in recent years. Accordingly, we analyze a set of miRs reported to modulate the Sirt1/eNOS axis using plasma from ME/CFS patients. Our results show that miR-21, miR-34a, miR-92a, miR-126, and miR-200c are jointly increased in ME/CFS patients compared to healthy controls. A similar finding was obtained when analyzing public miR data on peripheral blood mononuclear cells. Bioinformatics analysis shows that endothelial function-related signaling pathways are associated with these miRs, including oxidative stress and oxygen regulation. Interestingly, histone deacetylase 1, a protein responsible for epigenetic regulations, represented the most relevant node within the network. In conclusion, our study provides a basis to find endothelial dysfunction-related biomarkers and explore novel targets in ME/CFS.

Chronic Chlorella intake and aerobic exercise training reduce arterial stiffness and increase circulating nitric oxide (NO) levels, which has beneficial effects. This study aimed to clarify the combined aortic NO-mediated effects of chronic Chlorella intake and aerobic exercise training on endothelial vasorelaxation in aged mice. In this study, 38-week-old male senescence-accelerated mouse prone 1 (SAMP1) mice were divided into aged sedentary control (Con), aerobic exercise training (AT; voluntary wheel running for 12 weeks), Chlorella intake (CH; 0.5% Chlorella powder in normal diet), and AT and CH combined (AT+CH) groups. Endothelium-dependent vasorelaxation by addition of acetylcholine to the isolated mouse aortic rings was significantly higher in the AT, CH, and AT+CH groups than in the Con group; a significantly greater effect was seen in the AT+CH group than in the AT and CH groups. Similarly, plasma and arterial nitrite/nitrate levels and arterial endothelial NO synthase phosphorylation were significantly higher in the AT, CH, and AT+CH groups than in the Con group; the AT+CH group had higher values than the AT and CH groups. Thus, chronic Chlorella intake combined with aerobic exercise training had pronounced effects on endothelial vasorelaxation in aged mice via an additive increase in arterial NO production. Novelty: Endothelium-dependent vasorelaxation was improved by Chlorella intake and exercise. Chlorella intake and exercise increased arterial Akt/eNOS/NO signaling. This combination approach further improved vasorelaxation via arterial NO production.

Patients with rheumatoid arthritis (RA), a chronic and disabling autoimmune condition that is characterized by articular and extra-articular manifestations and a pro-inflammatory and pro-oxidant state, suffer from premature atherosclerosis and excessive cardiovascular disease burden. A key step in the pathogenesis of atherosclerosis is impaired synthesis of the endogenous messenger nitric oxide (NO) by endothelial cells which, in turn, alters local homeostatic mechanisms and favors vascular damage and plaque deposition. While the exact mechanisms of endothelial dysfunction in RA remain to be established, there is good evidence that RA patients have relatively high circulating concentrations of the methylated arginine asymmetric dimethylarginine (ADMA), a potent endogenous inhibitor of endothelial NO synthase (eNOS). This review discusses the biological and pathophysiological role of ADMA, the interplay between ADMA, inflammation and oxidative stress, and the available evidence on the adverse impact of ADMA on endothelial function and atherosclerosis and potential ADMA-lowering therapies in RA patients.

Dyslipidemia and hyperglycemia portray “cause-and-consequence” of type 2 diabetes mellitus (T2DM). They are linked to malondialdehyde (MDA) and methylglyoxal (MGO) generation that result from membrane lipid peroxidation and oxidative glucose conversions. We compared the effects of exogenous MDA and MGO on human umbilical vein endothelial cells and found that MDA but not MGO impairs insulin activation of PI3-kinase pathway, NO production, and endothelial barrier capacity. MDA abolished insulin activation of Akt and eNOS but not that of IRS. These results substantiate the hypothesis that MDA may be involved in endothelial dysfunction as an early event in the development of T2DM.

Preeclampsia is a pregnancy-related hypertensive disorder with unclear mechanisms. While hypersensitivity to angiotensin II via vasoconstrictive angiotensin type-1 receptor (AT1R) is observed in preeclampsia, the importance of vasodilatory angiotensin type-2 receptor (AT2R) in the control of vascular dysfunction is less clear. We assessed whether AT1R, AT2R, and endothelial nitric oxide synthase (eNOS) expression are altered in placental vessels of preeclamptic women and tested if ex vivo incubation with AT2R agonist Compound 21 (C21; 1 µM) could restore AT1R, AT2R, and eNOS balance. Further, using a rat model of gestational hypertension induced by elevated testosterone, we examined whether C21 (1 µg/kg/day, oral) could preserve AT1R and AT2R balance and improve blood pressure, uterine artery blood flow, and vascular function. Western blots revealed that AT1R protein level was higher while AT2R and eNOS protein were reduced in preeclamptic placental vessels, and AT2R agonist C21 decreased AT1R and increased AT2R and eNOS protein levels in preeclamptic vessels. In testosterone dams, blood pressure was higher, and uterine artery blood flow was reduced, and C21 treatment reversed these levels similar to those in controls dams. C21 attenuated the exaggerated Ang II contraction and improved endothelium-dependent vasorelaxation in uterine arteries of testosterone dams. These C21-mediated vascular effects were associated with decreased AT1R and increased AT2R and eNOS protein levels. C21 also increased serum nitrate/nitrite and bradykinin production in testosterone dams and attenuated the fetoplacental growth restriction. Thus, AT1R upregulation and AT2R downregulation are observed in preeclampsia and testosterone model, and increasing AT2R activity could help restore AT1R and AT2R balance and improve gestational vascular function.Summary sentenceAT1R/AT2R balance is tilted toward vasoconstrictive AT1R in preeclampsia; restoring this balance using AT2R agonist mitigates vascular dysfunction in hypertensive pregnant rats, providing a new approach in managing gestational vascular dysfunction.

Diabetic vascular complications are the leading causes of death and disability in patients with diabetes. Alpha‐mangostin has been reported to have anti‐diabetic capacity in recent years. Here, we investigated the protective function of alpha‐mangostin on endothelium in vitro and in vivo experiments. We also observed that alpha‐mangostin improved impaired endothelium‐dependent vasodilation (EDV) of diabetic animals while it limited the aSMase/ceramide pathway and up‐regulated eNOS/NO pathway in aortas from diabetic mice. Meanwhile, alpha‐mangostin inhibited elevated aSMase/ceramide pathway and reversed impaired EDV induced by high glucose in isolated mouse aortas. In addition, alpha‐mangostin increased phosphorylation of eNOS and NO production in high glucose‐treated aortas. Alpha‐mangostin normalized high glucose‐induced activation of aSMase/ceramide pathway and improved eNOS/NO pathway in endothelial cells with high glucose. In conclusion, alpha‐mangostin regulates eNOS/NO pathway and improves EDV in aortas of diabetic mice through inhibiting aSMase activity and endogenous ceramide accumulation.

Hypercholesterolemia is one of the risk factors associated with increased morbidity and mortality in cardiovascular disorders. Chrysin (Chy) is reported to exhibit anti-inflammatory, anti-cancerous, anti-oxidative, anti-aging, and anti-atherogenic properties. In the present study, we aimed to investigate whether Chy would mediate the cardioprotective effect against hypercholesterolemia-triggered myocardial oxidative stress. Male Sprague Dawley rats were divided into different groups as control and fed with high-fat diet (HFD) followed by oral administration of Chy (100 mg/kg b.wt), atorvastatin (Atv) (10 mg/kg b.wt), and L-NAME (10 mg/kg b.wt) for 30 days. At the end of the experimental period, the rats were sacrificed and tissues were harvested. Biochemical results showed a significant increase of cardiac disease marker enzymes (ALT, AST, and CKMB), lipid peroxidation, and lipid profile (TC, TG, LDL, and VLDL) in HFD-fed rat tissues when compared to control, whereas oral administration of Chy significantly reduced the activities of these marker enzymes and controlled the lipid profile. qRT-PCR studies revealed that Chy administration significantly increased the expression of endothelial nitric oxide synthase (eNOS), and Nrf2 target genes such as SOD, catalase, and GCL3 in left ventricular heart tissue of HFD-challenged rats. Immunohistochemistry results also showed that Chy treatment increased myocardial protein expression of eNOS and Nrf2 in HFD-challenged rats. Concluding the results of the present study, the Chy could mediate the cardioprotective effect through the activation of eNOS and Nrf2 signaling against hypercholesterolemia-induced oxidative stress. Thus, the administration of Chy would provide a promising therapeutic strategy for the prevention of HFD-induced oxidative stress-mediated myocardial complications.

Purpose Obesity is associated with the development of risk factors for cardiovascular disease (CVD) and polyphenols have been shown to possess ameliorative effects against obesity-induced CVD risk factors. Rooibos (Aspalathus linearis) is rich in polyphenols, therefore we investigated the cardioprotective effects of aspalathin-rich green rooibos (GRT) on obesity-induced CVD risk factors in obese Wistar rats. Methods Adult male Wistar rats (n = 20 per group) were fed a control or a high-fat diet (HFD) for 16 weeks and treated with GRT (60 mg/kg/day) for six weeks. Blood pressure was monitored throughout. Vascular reactivity was measured and Western blots of cell-signalling proteins (eNOS, AMPK and PKB) were performed in aortic tissues. Effects on oxidative stress were determined by measuring antioxidant enzyme activity and thiobarbituric reactive substance (TBARS) levels in the liver. Results HFD animals had (1) increased blood pressure, (2) impaired vasodilation, (3) attenuated PKB and AMPK expression, (4) decreased antioxidant enzyme activity, (5) increased malondialdehyde (MDA) levels, and (6) increased phosphorylated eNOS levels. Treatment with GRT extract significantly alleviated these obesity-induced CVD risk factors. Conclusions Supplementation with GRT extract alleviated cardiovascular risk factors in the HFD animals, suggesting a therapeutic potential for GRT in obesity-induced cardiovascular risk.

Background and Objective: All three isoforms of nitric oxide (NO) synthase (NOS) are targets for thyroid hormones in the cardiovascular system. The aim of this study was to assess the effects of hypo- and hyperthyroidism on inducible (iNOS), endothelial (eNOS), and neural (nNOS) NOS levels in aorta and heart tissues of male rats. Methods: Rats were divided into control, hypothyroid, and hyperthyroid groups; hypo- and hyperthyroidism were induced by adding propylthiouracil (500 mg/L) and L-thyroxine (12 mg/L) to drinking water for a period of 21 days. On day 21, systolic blood pressure, heart rate, left ventricular developed pressure (LVDP), peak rate of positive and negative (±dp/dt) changes in left ventricular pressure as well as NO metabolites (NOx) and iNOS, eNOS, and nNOS protein levels in aorta and heart, were all measured. Results: Compared to controls, LVDP and ±dp/dt were lower in both hypo- and hyperthyroid rats. Compared to controls, heart rate and systolic blood pressure were lower in hypothyroid and higher in hyperthyroid rats. NOx levels in the heart of hypothyroid rats were lower (53%), whereas that in hyperthyroid rats were higher (56% and 40%) than controls. Compared to controls, hypothyroid rats had lower levels of eNOS, iNOS, and nNOS in the aorta (16%, 34%, and 15%, respectively) and lower iNOS and higher nNOS in heart tissue (27% and 46%). In hyperthyroid rats, eNOS levels were lower (54% and 30%) and iNOS were higher (63%, and 35%) in the aorta and heart while nNOS was lower in the aorta (18%). Conclusion: Hypothyroidism increased while hyperthyroidism decreased the ratio of eNOS/iNOS in aorta and heart; these changes of NOS levels were associated with impaired cardiovascular function.

Low levels of ascorbate (Asc) are observed in cardiovascular and neurovascular diseases. Asc has therapeutic potential for the treatment of endothelial dysfunction, which is characterized by a reduction in nitric oxide (NO) bioavailability and increased oxidative stress in the vasculature. However, the potential mechanisms remain poorly understood for the Asc mitigation of endothelial dysfunction. In this study, we developed an endothelial cell based computational model integrating endothelial cell nitric oxide synthase (eNOS) biochemical pathway with downstream reactions and interactions of oxidative stress, tetrahydrobiopterin (BH4) synthesis and biopterin ratio ([BH4]/[TBP]), Asc and glutathione (GSH). We quantitatively analyzed three Asc mediated mechanisms that are reported to improve/maintain endothelial cell function. The mechanisms include the reduction of •BH3 to BH4, direct scavenging of superoxide (O2•−) and peroxynitrite (ONOO−) and increasing eNOS activity. The model predicted that Asc at 0.1–100 μM concentrations improved endothelial cell NO production, total biopterin and biopterin ratio in a dose dependent manner and the extent of cellular oxidative stress. Asc increased BH4 availability and restored eNOS coupling under oxidative stress conditions. Asc at concentrations of 1–10 mM reduced O2•− and ONOO− levels and could act as an antioxidant. We predicted that glutathione peroxidase and peroxiredoxin in combination with GSH and Asc can restore eNOS coupling and NO production under oxidative stress conditions. Asc supplementation may be used as an effective therapeutic strategy when BH4 levels are depleted. This study provides detailed understanding of the mechanism responsible and the optimal cellular Asc levels for improvement in endothelial dysfunction.

Background Korean Red ginseng extract (KRGE) has beneficial effects on the cardiovascular system by improving endothelial cell function. However, its pharmacological effect on endothelial cell senescence has not been clearly elucidated. Therefore, we examined the effect and molecular mechanism of KRGE on the senescence of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were grown in normal or KRGE-supplemented medium. Furthermore, they were transfected with heme oxygenase-1 (HO-1) gene or treated with its inhibitor, a NF-κB inhibitor, and a miR-155-5p mimic or inhibitor. Senescence-associated characteristics of endothelial cells were determined by biochemical and immunohistochemical analyses. Results Treatment of HUVECs with KRGE resulted in delayed onset and progression of senescence-associated characteristics, such as increased lysosomal acidic β-galactosidase and decreased telomerase activity, angiogenic dysfunction, and abnormal cell morphology. KRGE preserved the levels of anti-senescent factors, such as eNOS-derived NO, MnSOD, and cyclins D and A: however, it decreased the levels of senescence-promoting factors, such as ROS, activated NF-κB, endothelial cell inflammation, and p21 expression. The beneficial effects of KRGE were due to the induction of HO-1 and the inhibition of NF-κB-dependent biogenesis of miR-155-5p that led to the downregulation of eNOS. Moreover, treatment with inhibitors of HO-1, NF-κB, and miR-155-5p abolished the anti-senescence effects of KRGE. Conclusion KRGE delayed or prevented HUVEC senescence through a signaling cascade involving the induction of HO-1, the inhibition of NF-κB-dependent miR-155-5p biogenesis, and the maintenance of the eNOS/NO axis activity, suggesting that it may protect against vascular diseases associated with endothelial senescence.

Background and purpose It is well established that both smokers and patients with COPD are at a significantly heightened risk of cardiovascular disease (CVD), although the mechanisms underpinning the onset and progression of co-morbid CVD are largely unknown. Here, we explored whether cigarette smoke (CS) exposure impairs vascular function in mice and given the well-known pathological role for oxidative stress in COPD, whether the antioxidant compound ebselen prevents CS-induced vascular dysfunction in mice. Experimental approach Male BALB/c mice were exposed to either room air (sham) or CS generated from nine cigarettes per day, 5 days a week for 8 weeks. Mice were treated with ebselen (10 mg·kg-1 , oral gavage once daily) or vehicle (5% w/v CM cellulose in water) 1 h prior to the first CS exposure of the day. Upon killing, bronchoalveolar lavage fluid (BALF) was collected to assess pulmonary inflammation, and the thoracic aorta was excised to investigate vascular endothelial and smooth muscle dilator responses ex vivo. Key results CS exposure caused a significant increase in lung inflammation which was reduced by ebselen. CS also caused significant endothelial dysfunction in the thoracic aorta which was attributed to a down-regulation of eNOS expression and increased vascular oxidative stress. Ebselen abolished the aortic endothelial dysfunction seen in CS-exposed mice by reducing the oxidative burden and preserving eNOS expression. Conclusion and implications Targeting CS-induced oxidative stress with ebselen may provide a novel means for treating the life-threatening pulmonary and cardiovascular manifestations associated with cigarette smoking and COPD.

Carbon monoxide (CO) poisoning is a leading cause of morbidity and mortality worldwide. The delayed encephalopathy occurs a period after poisoning in patients without effective treatment. Simvastatin (Sim), a lipid-lowering drug, was reported to exert endothelial protective effects and inhibit inflammatory response. This research focused on the effects of Sim on delayed encephalopathy caused by CO poisoning. The acute CO poisoning model was established by exposing rats to 2500 ppm CO gas for 40 min, then 3000 ppm for 30 min or until they lost consciousness. Rats in the treatment group were given Sim (20 mg/kg/day, ig.). The behavioral tests included the Morris water maze test and shuttle box. The pathological changes were evaluated by H and E staining. The inflammatory mediators were analyzed by ELISA. The expression levels of eNOS, iNOS and the NF-κB-related proteins were analyzed by Western blot. The results showed that Sim could alleviate CO-induced behavioral disorders and the hippocampal nerve cells apoptosis. Sim administration reversed the effects of CO on oxidative stress-related molecules. Sim could also inhibit the production of the inflammatory mediators induced by CO. The level of eNOS was decreased after CO exposure, while iNOS was increased. Sim could significantly inhibit the effects of CO. Furthermore, Sim inhibited the phosphorylation of IκBα (an NF-κB inhibitory protein), i.e., the activation of NF-κB, which indicated that Simvastatin reduced the inflammatory response induced by CO poisoning partially through inhibiting the activation of NF-κB signaling pathway. To sum up, our research indicated that Sim could attenuate the delayed encephalopathy induced by CO poisoning via regulating oxidative stress, inflammation and NF-κB pathway.

MARCH5 is a critical regulator of mitochondrial dynamics, apoptosis and mitophagy. However, its role in cardiovascular system remains poorly understood. This study aimed to investigate the role of MARCH5 in endothelial cell (ECs) injury and the involvement of the Akt/eNOS signalling pathway in this process. Rat models of myocardial infarction (MI) and human cardiac microvascular endothelial cells (HCMECs) exposed to hypoxia (1% O2) were used in this study. MARCH5 expression was significantly reduced in ECs of MI hearts and ECs exposed to hypoxia. Hypoxia inhibited the proliferation, migration and tube formation of ECs, and these effects were aggravated by knockdown of MARCH5 but antagonized by overexpressed MARCH5. Overexpression of MARCH5 increased nitric oxide (NO) content, p‐eNOS and p‐Akt, while MARCH5 knockdown exerted the opposite effects. The protective effects mediated by MARCH5 overexpression on ECs could be inhibited by eNOS inhibitor L‐NAME and Akt inhibitor LY294002. In conclusion, these results indicated that MARCH5 acts as a protective factor in ischaemia/hypoxia‐induced ECs injury partially through Akt/eNOS pathway.

Background: Endothelial nitric oxide synthase (eNOS) plays a major role in the response of anti-hypercholesterol statin drugs. Genetic polymorphisms in the eNOS gene affect the activity of eNOS thereby modulating the statin response. Objectives: This study investigated the influence of major functional eNOS gene polymorphisms (rs2070744, rs1799983, and rs61722009) on the lipid profile of type 2 diabetes mellitus (T2DM) Jordanian patients treated with atorvastatin. Methods: The sample comprised 103 T2DM patients who attended the diabetes clinic of Jordan University Hospital. The T2DM patients had regularly been taking 20 mg atorvastatin. The atorvastatin response was calculated by measuring the lipid profile before and after three months of atorvastatin treatment. The eNOS genotypes of the subjects were analyzed using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) assay. Results: No significant association was found between eNOS genetic polymorphisms and the response to atorvastatin (ANOVA, p > 0.05). In addition, no significant difference in the frequency of eNOS genotypes was found between T2DM patients and healthy subjects. However, patients with eNOS rs1799983, 4a/4a, and rs61722009 G/G genotypes showed significantly lower levels of baseline total cholesterol (TC) and low density lipoprotein (LDL) than did patients carrying the rs1799983 4b/4b or rs61722009 T/T genotype (p < 0.05). The eNOS rs1799983 and rs61722009 polymorphisms were in complete linkage disequilibrium (D' = 1). Conclusion: Although no association was found between eNOS genetic polymorphisms and atorvastatin response, there was a significant association between the rs1799983 and rs61722009 genotypes and baselines levels of TC and LDL in Jordanian T2DM patients. These genetic variants affect cholesterol levels and may play a role in the susceptibility to cardiovascular diseases in T2DM patients. Further studies are needed to validate these findings.

BACKGROUND The explicit mechanism of erectile dysfunction caused by low androgen status is unknown. It was reported that eNOS was expressed in extracellular vesicles (EVs). Androgen may regulate erectile function by affect the release of EVs from endothelial cells. OBJECTIVES To investigate whether androgen affects the production of EVs and nitric oxide (NO) in endothelial cells of rat penile corpus cavernosum. MATERIALS AND METHODS Endothelial cells of rat penile corpus cavernosum were isolated and purified from 6-week-old healthy male Sprague Dawley (SD) rats. Endothelial cells were treated with different concentrations of dihydrotestosterone (DHT) in a cell culture medium as follows: no-androgen group (NA group, DHT 0 nmol/L), very-low androgen group (VLA group, DHT 0.1 nmol/L), low androgen group (LA group, DHT 1 nmol/L), and physiological concentrations androgen group (PA group, DHT 10 nmol/L). After 24 h, EVs of supernatant in each group were isolated and identified. The content of EVs and NO in the supernatant and the expression of CD9, CD63, TSG101, and eNOS in EVs were detected. RESULTS Positive expression of CD9, CD63, TSG101, and eNOS was found in isolated EVs. The concentration of EVs was lower in the NA group compared with other groups (p

An increasing number of studies have shown that nicotinamide mononucleotide (NMN) can inhibit not only ageing but also oxidative stress and inflammatory reactions by improving energy metabolism. However, the role of NMN in regulating the anti‐apoptotic, antioxidative stress and inflammatory responses of brain microvascular endothelial cells is still unknown. Therefore, here we studied the effects of NMN on H2O2‐induced oxidative damage of bEnd.3 cells. In this study, we found that NMN could inhibit the NF‐κBp65 inflammatory signalling pathway and increase the expression of the enzymes NAMPT, VEGF and eNOS, alleviating H2O2‐induced apoptosis in bEnd.3 cells. Taken together, these results suggest that NMN reduces H2O2‐induced oxidative stress and apoptosis and improves cell functions by inhibiting the NF‐κBp65 inflammatory pathway and increasing NAMPT expression., Nicotinamide mononucleotide (NMN) has antioxidant and anti‐apoptosis effects in diabetes and cerebrovascular disease models. We verified that NMN can alleviate the apoptosis of bEnd.3 cells induced by oxidative stress, and we further found that the anti‐oxidation and anti‐apoptosis effects of NMN were related to the regulation of NF‐KBp65 pathway and NAMPT enzyme.