Endothelial Nitric Oxide Synthase Dysfunction Research Articles

Cardiovascular characterization of the novel organic mononitrate NDIBP in rats

Organic nitrates are widely used to restore endogenous nitric oxide (NO) levels reduced by endothelial nitric oxide synthase dysfunction. However, these drugs are associated with undesirable side effects, including tolerance. This study aims to investigate the cardiovascular effects of the new organic nitrate 1,3-diisobutoxypropan-2-yl nitrate (NDIBP). Specifically, we assessed its effects on blood pressure, vascular reactivity, acute toxicity, and the ability to induce tolerance. In vitro and ex vivo techniques showed that NDIBP released NO both in a cell-free system and in isolated mesenteric arteries preparations through a process catalyzed by xanthine oxidoreductase. NDIBP also evoked endothelium-independent vasorelaxation, which was significantly attenuated by 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, 300 μM), a nitric oxide scavenger; 1-H-[1,2,4] oxadiazolo–[4,3-a]quinoxalin-1-one (ODQ, 10 μM), a soluble guanylyl cyclase inhibitor; tetraethylammonium (TEA, 3 mM), a potassium channel blocker; febuxostat (500 nM), a xanthine oxidase inhibitor; and proadifen (10 μM), an inhibitor of cytochrome P450 enzyme. Furthermore, this organic nitrate did not induce tolerance in isolated vessels and presented low toxicity following acute oral administration. In vivo changes on cardiovascular parameters were assessed using normotensive and renovascular hypertensive rats. NDIBP evoked a reduction of blood pressure that was significantly higher in hypertensive animals. Our results suggest that NDIBP acts as a NO donor, inducing blood pressure reduction without having the undesirable effects of tolerance. Those effects seem to be mediated by activation of NO-sGC-cGMP pathway and positive modulation of K+ channels in vascular smooth muscle.

Metastatic Melanoma Progression Is Associated with Endothelial Nitric Oxide Synthase Uncoupling Induced by Loss of eNOS:BH4 Stoichiometry.

Melanoma is the most aggressive type of skin cancer due to its high capability of developing metastasis and acquiring chemoresistance. Altered redox homeostasis induced by increased reactive oxygen species is associated with melanomagenesis through modulation of redox signaling pathways. Dysfunctional endothelial nitric oxide synthase (eNOS) produces superoxide anion (O2−•) and contributes to the establishment of a pro-oxidant environment in melanoma. Although decreased tetrahydrobiopterin (BH4) bioavailability is associated with eNOS uncoupling in endothelial and human melanoma cells, in the present work we show that eNOS uncoupling in metastatic melanoma cells expressing the genes from de novo biopterin synthesis pathway Gch1, Pts, and Spr, and high BH4 concentration and BH4:BH2 ratio. Western blot analysis showed increased expression of Nos3, altering the stoichiometry balance between eNOS and BH4, contributing to NOS uncoupling. Both treatment with L-sepiapterin and eNOS downregulation induced increased nitric oxide (NO) and decreased O2• levels, triggering NOS coupling and reducing cell growth and resistance to anoikis and dacarbazine chemotherapy. Moreover, restoration of eNOS activity impaired tumor growth in vivo. Finally, NOS3 expression was found to be increased in human metastatic melanoma samples compared with the primary site. eNOS dysfunction may be an important mechanism supporting metastatic melanoma growth and hence a potential target for therapy.

L-citrulline increases arginase II protein levels and arginase activity in hypoxic piglet pulmonary artery endothelial cells.

The L-arginine precursor, L-citrulline, re-couples endothelial nitric oxide synthase, increases nitric oxide production, and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs. L-arginine can induce arginase, which, in turn, may diminish nitric oxide production. Our major purpose was to determine if L-citrulline increases arginase activity in hypoxic piglet pulmonary arterial endothelial cells, and if so, concomitantly impacts the ability to increase endothelial nitric oxide synthase re-coupling and nitric oxide production. Piglet pulmonary arterial endothelial cells were cultured in hypoxic conditions with L-citrulline (0–3 mM) and/or the arginase inhibitor S-(2-boronoethyl)-L-cysteine. We measured arginase activity and nitric oxide production. We assessed endothelial nitric oxide synthase coupling by measuring endothelial nitric oxide synthase dimers and monomers. L-citrulline concentrations ≥0.5 mM increased arginase activity in hypoxic pulmonary arterial endothelial cells. L-citrulline concentrations ≥0.1 mM increased nitric oxide production and concentrations ≥0.5 mM elevated endothelial nitric oxide synthase dimer-to-monomer ratios. Co-treatment with L-citrulline and S-(2-boronoethyl)-L-cysteine elevated endothelial nitric oxide synthase dimer-to-monomer ratios more than sole treatment. Despite inducing arginase, L-citrulline increased nitric oxide production and endothelial nitric oxide synthase coupling in hypoxic piglet pulmonary arterial endothelial cells. However, these dose-dependent findings raise the possibility that there could be L-citrulline concentrations that elevate arginase to levels that negate improvements in endothelial nitric oxide synthase dysfunction. Moreover, our findings suggest that combining an arginase inhibitor with L-citrulline merits evaluation as a treatment for chronic hypoxia-induced pulmonary hypertension.

Abstract 16286: Absence of Fxyd1 is Associated With a Female-specific Pro-inflammatory and Hypercholesterolemic Environment: Implications for Atherosclerosis

Introduction: Bioavailability of nitric oxide (NO) is often compromised in early atherosclerosis, driving disease onset. This is usually due to dysfunctional endothelial nitric oxide synthase (eNOS). We have found that the transmembrane protein, FXYD1, is highly concentrated in the caveolae sub-cellular region and is closely associated with eNOS. Our preliminary data suggest that FXYD1 protects eNOS from redox-dependent inactivation. So, we hypothesise that FXYD1 may protect against atherosclerosis. Hypothesis: We aimed to test the role of FXYD1 in a murine model of atherosclerosis Methods: We produced a novel mutant mouse line by cross-breeding FXYD1 heterozygote (FXYD1 +/- ) and apolipoprotein E knock out (ApoE -/- ) mice. FXYD1 -/- /ApoE -/- and FXYD1 +/+ /ApoE -/- littermates were fed a high fat/high cholesterol diet from 6-8 weeks of age for 16 weeks. At sacrifice, blood was taken, and plasma was separated for analysis of cholesterol and pro-inflammatory cytokines using ELISA. The thoracic aorta was isolated and formalin-fixed to assess atherosclerotic plaque development using oil red O stain. Immunoblotting for eNOS expression was performed in heart lysates. Results: Plasma total cholesterol was significantly increased in FXYD1-/-/ApoE-/- female mice (n = 14 compared to FXYD1+/+/ApoE-/- mice (n = 21; p<0.05)). Circulating IL-1β levels were also significantly increased in female FXYD1-/-/ApoE-/- vs. FXYD1+/+/ApoE-/- mice. Despite this, there was no effect on plaque size as determined by oil red O stain of fixed aortae. eNOS protein expression was decreased in heart tissue from FXYD1-/-/ApoE-/- vs. FXYD1+/+/ApoE-/- female mice which may lead to lower NO generation. All of the FXYD1-mediated changes were specific to female mice as we detected no differences in these parameters in littermate males. Conclusions: FXYD1-/- atherosclerotic female mice have higher circulating lipids and pro-inflammatory cytokines, but this does not appear to alter plaque development in the short term. Ongoing studies will determine the effect on plaque stability and role of FXYD1 in lipidomics. FXYD1 appears to have an anti-inflammatory role and may also be involved in cholesterol metabolism or handling. This could provide a novel therapeutic angle for females.

Baicalein Alleviates Erectile Dysfunction Associated With Streptozotocin-Induced Type I Diabetes by Ameliorating Endothelial Nitric Oxide Synthase Dysfunction, Inhibiting Oxidative Stress and Fibrosis

BackgroundManagement of diabetes mellitus induced-erectile dysfunction (DMED) is challenging because of its poor responses to phosphodiesterase type 5 inhibitors. Increasingly important roles of 12-lipoxygenase (12-LOX) have been proven in diabetes mellitus. AimTo investigate 12-LOX activity and therapeutic effect of its inhibitor, baicalein (BE), on DMED. MethodsIntraperitoneal streptozotocin injection was used to induce type I DM, and an apomorphine test was used to evaluate erectile function. In experiment A, we assessed 12-LOX expression alteration in the corpus cavernosum (CC) of rats with DMED of different levels of severity. In experiment B, rats with DMED were intraperitoneally injected with BE for 4 weeks, and control rats were injected with vehicles. The erectile function was tested by cavernous nerve stimulation before penile tissue was harvested. We performed Western blot, immunohistochemistry, immunofluorescence, Masson trichrome staining, and enzyme-linked immunosorbent assays to measure related proteins in CC. Main Outcome MeasureThe main outcome measures included rectile response, histologic examination, and expression alteration of related proteins. Results12-LOX upregulation was associated with the progression of type I DMED. After 4 weeks treatment, compared with the DMED group, the DMED + BE group showed better erectile responses to cavernous nerve stimulation. In the DMED + BE group, significantly enhanced endothelial nitric oxide synthase/nitric oxide/cyclic guanosine monophosphate pathway, reduced 12-LOX expression, and inhibited p38 mitogen-activated protein kinase/arginase II/L-arginine pathway were showed in CC relative to the DMED group. In addition, overactivated oxidative stress and fibrosis in the DMED group were both partially ameliorated in the DMED + BE group. Clinical ImplicationsBE may be considered as an effective therapy for DMED, but needs to be verified in future human investigations. Strengths & LimitationsThe role of 12-LOX and its inhibitor, BE, is firstly demonstrated in rats with type I DMED. However, the experimental data are derived from animal models with without evidences from cellular-based experiments. Conclusion12-LOX might serve as an important factor in the pathogenesis of type I DMED. BE alleviated erectile dysfunction in rats with type I DMED probably by inhibiting 12-LOX expression, ameliorating endothelial nitric oxide synthase dysfunction, as well as suppressing oxidative stress and fibrosis.Chen Y, Zhou B, Yu Z, et al. Baicalein Alleviates Erectile Dysfunction Associated With Streptozotocin-Induced Type I Diabetes by Ameliorating Endothelial Nitric Oxide Synthase Dysfunction, Inhibiting Oxidative Stress and Fibrosis. J Sex Med 2020;17:1434–1447.

Role of the eNOS Uncoupling and the Nitric Oxide Metabolic Pathway in the Pathogenesis of Autoimmune Rheumatic Diseases.

Atherosclerosis and its clinical complications constitute the major healthcare problems of the world population. Due to the central role of endothelium throughout the atherosclerotic disease process, endothelial dysfunction is regarded as a common mechanism for various cardiovascular (CV) disorders. It is well established that patients with rheumatic autoimmune diseases are characterized by significantly increased prevalence of cardiovascular morbidity and mortality compared with the general population. The current European guidelines on cardiovascular disease (CVD) prevention in clinical practice recommend to use a 1,5-factor multiplier for CV risk in rheumatoid arthritis as well as in other autoimmune inflammatory diseases. However, mechanisms of accelerated atherosclerosis in these diseases, especially in the absence of traditional risk factors, still remain unclear. Oxidative stress plays the major role in the endothelial dysfunction and recently is strongly attributed to endothelial NO synthase dysfunction (eNOS uncoupling). Converted to a superoxide-producing enzyme, uncoupled eNOS not only leads to reduction of the nitric oxide (NO) generation but also potentiates the preexisting oxidative stress, which contributes significantly to atherogenesis. However, to date, there are no systemic analyses on the role of eNOS uncoupling in the excess CV mortality linked with autoimmune rheumatic diseases. The current review paper addresses this issue.

Vitamin D3, L-Arginine, L-Citrulline, and antioxidant supplementation enhances nitric oxide bioavailability and reduces oxidative stress in the vascular endothelium – Clinical implications for cardiovascular system

Background: Nitric oxide (NO) is a crucial signaling molecule which regulates the blood flow and prevents the adhesion of blood components to the vascular wall. A deficiency in bioavailable NO concentration is associated with the dysfunction of endothelial NO synthase (eNOS) and/or an increase in oxidative stress. The deficiency of bioavailable NO is a common denominator of several cardiovascular diseases, including diabetes, atherosclerosis, and hypertension. Materials and Methods: We used a nanomedical technology to elucidate the balance between bioavailable NO and oxidative stress (peroxynitrite ONOO−) in human umbilical vein endothelial cells (HUVECs) treated with a supplement containing L-arginine, L-citrulline, Vitamin D3, and antioxidants. Nanosensors, with a diameter of 200–300 nm, are capable of measuring in situ NO and peroxynitrite (ONOO−) concentrations produced by single endothelial cells. Results: The ratio of the concentration of cytoprotective NO [NO] to the concentration of cytotoxic peroxynitrite [ONOO−] was used to estimate the efficiency of eNOS. HUVECs incubated with L-citrulline, L-arginine, and Vitamin D3increased the [NO]/[ONOO−] ratio by 25%, while in the presence of antioxidants, the increase was 15%. The synergistic effect between the mix of L-arginine, L-citrulline, Vitamin D3, and antioxidants was a favorable increase of the overall [NO]/[ONOO−] ratio by 50%. Conclusion: The findings of the study presented here clearly indicate that L-arginine, L-citrulline, and Vitamin D3can significantly alter the function of the endothelium and NO production, in a favorable manner, while pointedly reducing ONOO− – the main component of oxidative stress. This effect can be significantly potentiated in the presence of antioxidants.

Abstract 402: Adiponectin Receptor 3 is Associated With Endothelial Nitric Oxide Synthase Dysfunction and Predicts Insulin Resistance in South Asians

Type 2 diabetes (T2DM) is a global epidemic affecting over 400 million people and causes significant morbidity and mortality. T2DM has a strong association with cardiovascular disease (CVD), the number one cause of death globally with 17.5 million deaths per year. A precursor of T2DM, insulin resistance is central to the development of T2DM and is a risk factor for CVD. Insulin resistance is difficult to diagnose and individuals are often untreated prior to the onset of T2DM or CVD. South Asians are more likely to have insulin resistance, diabetes and cardiovascular disease when compared to age matched European cohorts. The molecular mechanisms of why South Asians are predisposed to insulin resistance and consequently cardiovascular disease were investigated using induced pluripotent stem cells (iPSCs) derived endothelial cells (iPSC-EC). Endothelial cells line blood vessels of the cardiovascular system. Unlike previous models, iPSC-EC are unique because they contain the individual's genetic information and the environmental influences retained in epigenetic marks are removed via reprogramming and differentiation. iPSC-ECs from insulin resistant South Asians show evidence of impaired insulin signaling as evidenced by decreased Akt phosphorylation, and paradoxically overexpression of eNOS and adiponectin receptor 3. In the cellular milieu of prediabetes, insulin resistance iPSC-ECs show impaired tubule formation and nitric oxide release. When adiponectin receptor 3 expression is suppressed using siRNA, eNOS expression decreases and expression of components of the insulin signaling cascade are improved to levels observed in control iPSC-ECs. Multiple linear regression modeling of clinical characteristics and gene and cellular phenotype was used to develop a scoring system that predicts a patient’s risk of developing insulin resistance and hence subsequently diabetes and cardiovascular complications. To our knowledge, this is the first iPSC-derived endothelial cell risk calculator with the potential to identify South Asian patients at risk for developing insulin resistance and cardiovascular disease before disease onset, which would allow for the early implementation of interventions that prevent morbidity and mortality.

Acute and Chronic Sleep Deprivation-Related Changes in N-methyl-D-aspartate Receptor-Nitric Oxide Signalling in the Rat Cerebral Cortex with Reference to Aging and Brain Lateralization.

Aging and chronic sleep deprivation (SD) are well-recognized risk factors for Alzheimer’s disease (AD), with N-methyl-D-aspartate receptor (NMDA) and downstream nitric oxide (NO) signalling implicated in the process. Herein, we investigate the impact of the age- and acute or chronic SD-dependent changes on the expression of NMDA receptor subunits (NR1, NR2A, and NR2B) and on the activities of NO synthase (NOS) isoforms in the cortex of Wistar rats, with reference to cerebral lateralization. In young adult controls, somewhat lateralized seasonal variations in neuronal and endothelial NOS have been observed. In aged rats, overall decreases in NR1, NR2A, and NR2B expression and reduction in neuronal and endothelial NOS activities were found. The age-dependent changes in NR1 and NR2B significantly correlated with neuronal NOS in both hemispheres. Changes evoked by chronic SD (dysfunction of endothelial NOS and the increasing role of NR2A) differed from those evoked by acute SD (increase in inducible NOS in the right side). Collectively, these results demonstrate age-dependent regulation of the level of NMDA receptor subunits and downstream NOS isoforms throughout the rat brain, which could be partly mimicked by SD. As described herein, age and SD alterations in the prevalence of NMDA receptors and NOS could contribute towards cognitive decline in the elderly, as well as in the pathobiology of AD and the neurodegenerative process.

Modulatory influence of sex hormones on vascular aging.

This review summarizes a presentation given during the "Countermeasures to Cardiovascular Aging Symposium" that was part of the American Physiological Society Conference on Cardiovascular Aging: New Frontiers and Old Friends held in Westminster, CO, in August 2017. Endothelial dysfunction, a characteristic of vascular aging, is a major risk factor for age-associated cardiovascular diseases. In women, the decline in endothelial function is attenuated until menopause, whereafter the rate of decline accelerates to match that seen in men. Sex differences in the decline in endothelial function have been attributed to changes in sex hormones with aging. Women have a progressive impairment in endothelial function across the stages of the menopause transition, related in part to declining estradiol levels. In contrast to women, little is known about the impact of declining testosterone levels on endothelial function in men. Some evidence suggests greater endothelial dysfunction in men with low testosterone compared with men with higher testosterone. The underlying causes of endothelial dysfunction with sex hormone deficiency are unknown but may be related to endothelial nitric oxide synthase dysfunction and oxidative stress. Lifestyle behaviors, including habitual endurance exercise, attenuates and reverses the age-associated decline in endothelial function in older men. However, in older women, these exercise adaptations are diminished or absent, possibly related to estrogen deficiency. Understanding how declines in sex hormones contribute to the vascular aging process in both women and men will inform effective sex-specific intervention strategies to preserve vascular health and prevent cardiovascular diseases.

Cigarette smoke constituents cause endothelial nitric oxide synthase dysfunction and uncoupling due to depletion of tetrahydrobiopterin with degradation of GTP cyclohydrolase

Cigarette smoking (CS) is a well-established risk factor for cardiovascular disease (CVD). Endothelial dysfunction (ED) with loss of nitric oxide (NO) production is a central mechanism leading to the advent of CVD. Despite many prior studies of this major health problem, the exact mechanism by which CS induces ED is not well understood. This study examines the mechanism by which CS induces ED with altered endothelial NO synthase (eNOS) function in aortic endothelial cells (AECs). Exposure of AECs to cigarette smoke extract (CSE) resulted in a marked decrease in NO production with concomitant increase in superoxide (O2.-) generation and accumulation of 4-hydroxy-2-nonenal protein adducts. CSE exposure led to depletion of the essential eNOS cofactor tetrahydrobiopterin (BH4) as well as total biopterin levels and decreased the expression level of guanosine triphosphate cyclohydrolase (GTPCH), the rate limiting enzyme in BH4 biosynthesis. Moreover, exposure of AECs to CSE increased the level of ubiquitinated proteins and increased 26 S proteasomal activity in a concentration-dependent manner. Pre-treatment with MG132, a 26 S proteasome inhibitor, partially prevented CSE-induced loss of BH4, total biopterin, GTPCH, and increased NO production following CSE exposure, indicating a role of the ubiquitin-proteasome system in CSE-induced eNOS dysfunction. In conclusion, CSE-induced eNOS dysfunction and uncoupling occurs due to BH4 depletion with BH4de novo synthesis limited by diminished GTPCH expression.

Endothelial Nitric Oxide Synthase-Induced Hypertrophy and Vascular Dysfunction Contribute to the Left Ventricular Dysfunction in Caveolin-1−/− Mice

BackgroundCaveolin-1 (Cav1)−/− mice display impaired development of left ventricular pressure and increased left ventricular wall thickness but no dilated ventricle; these are typical findings in patients with heart failure with preserved ejection fraction (HfpEF). Aiming to clarify if dysfunctional endothelial nitric oxide synthase (eNOS) influences cardiomyocyte contractility, cardiac conduction system, or afterload/vascular resistance, we studied Cav1−/−/eNOS−/− mice. MethodsCardiac function was assessed in vivo by pressure-volume-catheterization of the left ventricle, echocardiography and electrocardiography. In addition, isolated tissue experiments were performed to evaluate cardiomyocyte contractility (atria) and vessel morphology and function (aorta). Histology, immunoblotting and quantitative polymerase chain reaction were applied to characterise radical formation and oxidative stress in the heart. ResultsCardiac hypertrophy was completely reversed in Cav1−/−/eNOS−/− mice. The impaired pump function in Cav1−/− mice was significantly improved in Cav1−/−/eNOS−/− mice, but no complete alignment with eNOS−/− controls was achieved, indicating an additional eNOS-independent mechanism contributing to HFpEF in Cav1−/− mice. It is unlikely that frequently occurring arrhythmias contributed to HFpEF in Cav1−/− mice. In contrast, numerous eNOS-dependent and eNOS-independent vascular abnomalities could explain the cardiac phenotypes of Cav1−/− mice. ConclusionsSynergistic effects between eNOS-related cardiac hypertrophy and vascular hypercontractility appear to underlie the left ventricular dysfunction in Cav1−/−mice. These findings provide insights relevant to the poorly understood pathophysiology of HFpEF.

(-)-Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase.

(-)-Epicatechin increases indicators associated with mitochondrial biogenesis in endothelial cells and myocardium. We investigated endothelial nitric oxide synthase involvement on (-)-epicatechin-induced increases in indicators associated with mitochondrial biogenesis in human coronary artery endothelial cells cultured in normal-glucose and high-glucose media, as well as to restore indicators of cardiac mitochondria from the effects of simulated diabetes. Here, we demonstrate the role of endothelial nitric oxide synthase on (-)-epicatechin-induced increases in mitochondrial proteins, transcription factors and sirtuin 1 under normal-glucose conditions. In simulated diabetes endothelial nitric oxide synthase function, mitochondrial function-associated and biogenesis-associated indicators were adversely impacted by high glucose, effects that were reverted by (-)-epicatechin. As an animal model of type 2 diabetes, 2-month old C57BL/6 mice were fed a high-fat diet for 16 weeks. Fasting and fed blood glucose levels were increased and NO plasma levels decreased. High-fat-diet-fed mice myocardium revealed endothelial nitric oxide synthase dysfunction, reduced mitochondrial activity and markers of mitochondrial biogenesis. The administration of 1 mg/kg (-)-epicatechin for 15 days by oral gavage shifted these endpoints towards control mice values. Results suggest that endothelial nitric oxide synthase mediates (-)-epicatechin-induced increases of indicators associated with mitochondrial biogenesis in endothelial cells. (-)-Epicatechin also counteracts the negative effects that high glucose or simulated type 2 diabetes has on endothelial nitric oxide synthase function.

Chronic Co-Administration of Sepiapterin and L-Citrulline Ameliorates Diabetic Cardiomyopathy and Myocardial Ischemia/Reperfusion Injury in Obese Type 2 Diabetic Mice.

Diabetic heart disease is associated with tetrahydrobiopterin oxidation and high arginase activity, leading to endothelial nitric oxide synthase dysfunction. Sepiapterin (SEP) is a tetrahydrobiopterin precursor, and L-citrulline (L-Cit) is converted to endothelial nitric oxide synthase substrate, L-arginine. Whether SEP and L-Cit are effective at reducing diabetic heart disease is not known. The present study examined the effects of SEP and L-Cit on diabetic cardiomyopathy and ischemia/reperfusion injury in obese type 2 diabetic mice. Db/db and C57BLKS/J mice at 6 to 8 weeks of age received vehicle, SEP, or L-Cit orally alone or in combination for 8 weeks. Cardiac function was evaluated with echocardiography. Db/db mice displayed hyperglycemia, obesity, and normal blood pressure and cardiac function compared with C57BLKS/J mice at 6 to 8 weeks of age. After vehicle treatment for 8 weeks, db/db mice had reduced ejection fraction, mitral E/A ratio, endothelium-dependent relaxation of coronary arteries, tetrahydrobiopterin concentrations, ratio of endothelial nitric oxide synthase dimers/monomers, and nitric oxide levels compared with vehicle-treated C57BLKS/J mice. These detrimental effects of diabetes mellitus were abrogated by co-administration of SEP and L-Cit. Myocardial infarct size was increased, and coronary flow rate and ± dP/dt were decreased during reperfusion in vehicle-treated db/db mice subjected to ischemia/reperfusion injury compared with control mice. Co-administration of SEP and L-Cit decreased infarct size and improved coronary flow rate and cardiac function in both C57BLKS/J and db/db mice. Co-administration of SEP and L-Cit limits diabetic cardiomyopathy and ischemia/reperfusion injury in db/db mice through a tetrahydrobiopterin/endothelial nitric oxide synthase/nitric oxide pathway.

Mechanisms and consequences of endothelial nitric oxide synthase dysfunction in hypertension.

Reduced nitric oxide bioavailability contributes to endothelial dysfunction and hypertension. The endothelial isoform of nitric oxide synthase (eNOS) is responsible for the production of nitric oxide within the endothelium. Loss of eNOS cofactor tetrahydrobiopterin to initial increase in oxidative stress leads to uncoupling of eNOS, in which the enzyme produces superoxide anion rather than nitric oxide, further substantiating oxidative stress to induce vascular pathogenesis. The current review focuses on recent advances on the molecular mechanisms and consequences of eNOS dysfunction in hypertension, and potential novel therapeutic strategies restoring eNOS function to treat hypertension.

Aging, arterial stiffness, and hypertension.

Although the etiology of essential hypertension remains unknown, it is clear that multiple factors may contribute to the pathogenesis of hypertension. Hypertension is an outcome of the interaction of multiple genetic and environmental factors. Several epidemiological studies indicated that the incidence of arterial stiffness and hypertension and related cardiovascular disease (stroke, myocardial infarction) is higher in the aged than in the young population.1–4 The prevalence of arterial stiffening and hypertension increases with age.1,5 Based on an epidemiological study,6 the prevalence of hypertension is more than doubled in the elderly than in the young population. More than two-thirds of individuals after 65 years of age experience hypertension according to the Seventh Report of the Joint National Committee (JNC-7).7 Therefore, it is generally thought that hypertension is an aging disorder. In recent years, metabolic syndrome and hypertension are increasingly seen in the middle-aged and young populations. In these subpopulations, insulin resistance and overproduction of adipokines impair endothelial and heart function leading to early and accelerated cardiovascular aging. It was reported that premature aging (progeria) is associated with accelerated vascular stiffening or vascular aging.8 Aging is defined as the age-related decline in physiological function essential for survival and fertility. Cardiovascular aging is an important factor that determines life span. The wall of large conduit arteries, especially aorta, thicken and lose elasticity over time, and this process results in an increase in pulse wave velocity, an important and reliable measure of arterial stiffness. The increased arterial stiffness, whatever its underlying causes, would reduce the reservoir/buffering function of the conduit arteries near the heart and increase pulse wave velocity, both of which increase systolic and pulse pressure. Therefore, aging-related hypertension is characterized by a significant increase in systolic blood pressure with no change or even a …

Fibroblast Growth Factor 21 Protects Against High Glucose Induced Cellular Damage and Dysfunction of Endothelial Nitric-Oxide Synthase in Endothelial Cells

Aims: Fibroblast growth factor 21 (FGF21) is a powerful endocrine hormone modulating glucose and lipid metabolism and represents a promising drug for type 2 diabetes. The present study was to determine the effect of FGF21 on high glucose-induced damage and dysfunction in endothelial cells. Methods: The protein expression of β-klotho was examined in human umbilical vein endothelial cell (HUVECs) using immunofluorescence and Western blotting. HUVECs were cultured in medium with normal glucose (NG), high glucose (HG) and HG + FGF21 (30 nM). Cell viability, migration, reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase, nitric oxide (NO) production, intracellular cyclic guanosine monophosphate (cGMP) and endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177/Ser-633 sites were measured. Results: β-klotho, the anchor protein of FGF21, is expressed in HUVECs. Administration of FGF21 prevented HG-induced impairment of cell viability, migration, oxidant stress, NO production and intracellular cGMP levels in HUVECs. FGF21 also rescued HG-induced decrease of eNOS phosphorylation at Ser-1177 and Ser-633. HG and FGF21 had no effects on eNOS phosphorylation at Ser-617 and Thr-495. Inhibition of AMP-activated protein kinase (AMPK), but not Akt or Ca²⁺/calmodulin-dependent protein kinase II, abolished the protective effect of FGF21 on eNOS phosphorylation at Ser-1177. The protective effect of FGF21 on eNOS phosphorylation at Ser-633 was also abolished by inhibition of AMPK but not by Akt or cAMP-dependent protein kinase A. Conclusion: Our results provide the first evidence that FGF21 protects against high glucose induced cell damage and eNOS dysfunction in an AMPK-dependent manner in HUVECs, and suggest that FGF21 may be a promoting therapeutic agent for vascular complications in diabetes.

The smoking-associated oxidant hypothiocyanous acid induces endothelial nitric oxide synthase dysfunction

Smokers have an elevated risk of cardiovascular disease but the origin(s) of this increased risk are incompletely defined. Considerable evidence supports an accumulation of the oxidant-generating enzyme MPO (myeloperoxidase) in the inflamed artery wall, and smokers have high levels of SCN(-), a preferred MPO substrate, with this resulting in HOSCN (hypothiocyanous acid) formation. We hypothesized that this thiol-specific oxidant may target the Zn(2+)-thiol cluster of eNOS (endothelial nitric oxide synthase), resulting in enzyme dysfunction and reduced formation of the critical signalling molecule NO•. Decreased NO• bioavailability is an early and critical event in atherogenesis, and HOSCN-mediated damage to eNOS may contribute to smoking-associated disease. In the present study it is shown that exposure of isolated eNOS to HOSCN or MPO/H2O2/SCN(-) decreased active dimeric eNOS levels, and increased inactive monomer and Zn(2+) release, compared with controls, HOCl (hypochlorous acid)- or MPO/H2O2/Cl(-)-treated samples. eNOS activity was increasingly compromised by MPO/H2O2/Cl(-) with increasing SCN(-) concentrations. Exposure of HCAEC (human coronary artery endothelial cell) lysates to pre-formed HOSCN, or MPO/H2O2/Cl(-) with increasing SCN(-), increased eNOS monomerization and Zn(2+) release, and decreased activity. Intact HCAECs exposed to HOCl and HOSCN had decreased eNOS activity and NO2(-)/NO3(-) formation (products of NO• decomposition), and increased free Zn(2+). Exposure of isolated rat aortic rings to HOSCN resulted in thiol loss, and decreased eNOS activity and cGMP levels. Overall these data indicate that high SCN(-) levels, as seen in smokers, can increase HOSCN formation and enhance eNOS dysfunction in human endothelial cells, with this potentially contributing to increased atherogenesis in smokers.

Cigarette smoke extract causes endothelial nitric oxide synthase dysfunction through stimulation of ubiquitin proteasome system

Cigarette smoking (CS) is a major risk factor for endothelial dysfunction (ED), a prognostic predictor for the risk of future cardiovascular events. Endothelial nitric oxide synthase (eNOS) dysfunction has been linked to CS‐induced ED; however, the exact mechanism is not fully understood. This study reports the role of the ubiquitin proteasome system (UPS) in CS‐ induced eNOS dysfunction and ED. Exposure of bovine aortic endothelial cells (BAECs) to cigarette smoke extract (CSE) resulted in a concentration‐dependent decrease in nitric oxide (NO) production, accompanied by an increase in superoxide generation and formation of 4‐hydroxy‐2‐nonenal protein adducts. CSE depleted tetrahydrobiopterin (BH4). total biopterin, and decreased the expression of eNOS and guanosine triphosphate cyclohydrolase‐1 (GTPCH‐1). More importantly, exposure of BAECs to CSE led to accumulation of ubiqutinylated protein and increased the 26S proteasomal activity in a concentration‐and time‐dependent manner. Pre‐treatment of BAECs with MG132, an inhibitor of 26S proteasome, restored the levels of eNOS, BH4, GTPCH‐1, and enhanced NO production in BAECs following CS exposure. Our data reveal that CS‐induced stimulation of the UPS resulted in degradation of eNOS and GTPCH‐1 that led to eNOS dysfunction and uncoupling. Thus, controlled interference with the UPS pathway could be a novel modality in the prevention of CS‐induced ED. (NIH # HL38324 to JLZ; Egyptian government)

Cigarette smoke extract causes endothelial nitric oxide synthase dysfunction through S‐glutathionylation

Dysfunction of endothelial nitric oxide synthase (eNOS) occurs in a wide range of cardiovascular disease and has been linked to cigarette smoking (CS)‐induced endothelial dysfunction (ED). Glutathionylation has recently been reported as a novel mechanism for eNOS uncoupling in ED. However, the role of eNOS glutathionylation in CS‐induced ED remains to be investigated. Bovine aortic endothelial cells (BAECs) were exposed to cigarette smoke extract (CSE) at different concentrations for two hours. Nitric oxide (NO) production, superoxide (O2• −) generation and glutathione level were assessed. Formation of glutathionylated proteins was determined in the lysate or after pull down of eNOS following exposure to CSE with or without N‐acetyl cysteine (NAC). We show that exposure of BAECs to CSE decreased NO production with a concomitant increase in O2•− generation and depletion of cellular reduced glutathione. More importantly, formation of protein glutathionylation was significantly higher in CSE‐exposed cells. Pre‐incubation with NAC enhanced eNOS function, decreased eNOS glutathionylation and increased reduced glutathione in CSE‐exposed cells. Our data indicate that CS‐induced ED could be explained, at least in part, through eNOS glutathionylation. The role of CS‐induced eNOS glutathionylation, in the process of ED, and reversal of such modifications in animal models or human subjects warrants further investigation.(NIH# HL38324 to JLZ; and Egyptian government)