Endothelial Nitric Oxide Synthase Dysfunction Research Articles

Involvement of the Endothelial Nitric Oxide Pathway and Leukocyte Infiltration in Secondhand Smoke Exposure‐Induced Vascular Endothelial Dysfunction and Hypertension

Loss of nitric oxide (NO) production due to dysfunction of endothelial NO synthase (eNOS) occurs in a wide range of cardiovascular disease and has been linked to secondhand smoke (SHS)‐induced vascular endothelial dysfunction (VED). Our aim was to investigate the mechanisms of SHS‐induced VED. C57BL/6 mice were exposed for up to 48 weeks to SHS generated from 3R4F reference research cigarettes using the Teague smoking machine. SHS‐exposed (SHSE) mice showed persistent hypertension and impairment of acetylcholine‐induced endothelium‐dependent relaxation of thoracic aorta. Expression of NAD(P)H oxidase subunits p22phox and gp91phox increased in aortas of SHSE mice with concomitant superoxide production. H&E stained aortic sections showed evidence of leukocyte infiltration in SHSE mice. Plasma of SHSE mice showed significant depletion of tetrahydrobiopterin (BH4). Western blotting and immunohistochemistry showed a decrease in eNOS expression in SHSE mice aortas. Also, phosphorylation of eNOS and Aktwas decreased in SHSE mice. In conclusion, SHS induces leukocyte infiltration that triggers NAD(P)H oxidase over‐expression and ROS generation which leads to depletion of BH4 and eNOS uncoupling, resulting in VED and hypertension. Overall, our study provides important insights toward understanding contributions of SHS exposure to the genesis of cardiovascular disease. (NIH # HL38324 to JLZ)

Dissociation of Vasospasm and Secondary Effects of Experimental Subarachnoid Hemorrhage by Clazosentan

Endothelin receptor antagonists such as clazosentan decrease large-artery vasospasm after experimental and clinical subarachnoid hemorrhage. We used clazosentan to gain insight into the pathophysiology of subarachnoid hemorrhage by determining if decreasing vasospasm is associated with alleviation of other secondary complications of subarachnoid hemorrhage such as oxidative stress, endothelial nitric oxide synthase dysfunction, microthromboembolism, and neuronal injury. Mice were subjected to subarachnoid hemorrhage by injection of blood into the chiasmatic cistern. They were treated with clazosentan or vehicle by continuous intraperitoneal infusion for 48 hours. Middle cerebral artery vasospasm, superoxide anion radical, peroxynitrite, microthromboemboli, endothelial nitric oxide synthase uncoupling, cerebral blood flow, neuronal injury, and mortality were assessed. Clazosentan preserved cerebral blood flow, alleviated vasospasm, and decreased mortality but did not affect superoxide anion radical, peroxynitrite, or microthromboemboli in the brain. Endothelial nitric oxide synthase uncoupling and neuronal injury also were not reduced by clazosentan. This study shows large-artery vasospasm is pathophysiologically independent of some other effects of subarachnoid hemorrhage. The findings have implications for development of treatments for this disease.

Mechanisms of Diesel-Induced Endothelial Nitric Oxide Synthase Dysfunction in Coronary Arterioles

Background and objectiveIncreased air pollutants correlate with increased incidence of cardiovascular disease potentially due to vascular dysfunction. We have reported that acute diesel engine exhaust (DE) exposure enhances vasoconstriction and diminishes acetylcholine (ACh)-induced dilation in coronary arteries in a nitric oxide synthase (NOS)-dependent manner. We hypothesize that acute DE inhalation leads to endothelial dysfunction by uncoupling NOS.MethodsRats inhaled fresh DE (300 μg particulate matter/m3) or filtered air for 5 hr. After off-gassing, intraseptal coronary arteries were isolated and dilation to ACh recorded using videomicroscopy.ResultsArteries from DE-exposed animals dilated less to ACh than arteries from air-exposed animals. NOS inhibition did not affect ACh dilation in control arteries but increased dilation in the DE group, suggesting NOS does not normally contribute to ACh-induced dilation in coronary arteries but does contribute to endothelial dysfunction after DE inhalation. Cyclooxygenase (COX) inhibition did not affect ACh dilation in the DE group, but combined inhibition of NOS and COX diminished dilation in both groups and eliminated intergroup differences, suggesting that the two pathways interact. Superoxide scavenging increased ACh dilation in DE arteries, eliminating differences between groups. Tetrahydrobiopterin (BH4) supplementation with sepiapterin restored ACh-mediated dilation in the DE group in a NOS-dependent manner. Superoxide generation (dihydroethidium staining) was greater in DE arteries, and superoxide scavenging, BH4 supplementation, or NOS inhibition reduced the signal in DE but not air arteries.ConclusionAcute DE exposure appears to uncouple NOS, increasing reactive oxygen species generation and causing endothelial dysfunction, potentially because of depletion of BH4 limiting its bioavailability.

No arguments for increased endothelial nitric oxide synthase activity in migraine based on peripheral biomarkers

To assess whether migraine patients display a chronic nitric oxide synthase (NOS) hyperactivity by comparing the nitric oxide (NO) production before and following a loading dose of L-arginine between migraine patients (interictally) and matched healthy control subjects. In addition, we evaluated whether a loading dose of L-arginine triggers an acute migraine headache in migraineurs. Twenty healthy subjects and 20 migraine patients participated in a 2-period, randomised, double-blind, placebo-controlled study. Each subject received a 30-min infusion, by peripheral vein, of 30 g L-arginine hydrochloride or placebo (i.e. an equal volume of 0.9% saline solution). Meanwhile, biomarkers associated with the L-arginine-NO pathway (i.e. exhaled NO/nasal NO), plasma citrulline and urinary excretion of nitrite/nitrate and cGMP were assessed before and for 6 h following the start of the infusion. At baseline, exhaled NO and nasal NO were higher in migraineurs compared to healthy subjects (mean±95% confidence interval): 15.9 (8.8, 23.0) parts per billion (ppb) versus 10.8 (7.0, 14.5) ppb for exhaled NO (P=0.04) and 76.3 (61.2, 91.4) versus 61.6 (51.2, 72.0) ppb for nasal NO (P=0.03), respectively. The AUC0-6 in ppb for exhaled NO and nasal NO following L-arginine or saline infusion did not differ between both groups. The increase in L-citrulline, following L-arginine infusion, was smaller in migraine patients (15 (13, 18) µmol/l) compared to healthy volunteers (19 (16, 23) µmol/l; P=0.046). In healthy subjects, both nitrate and cGMP excretion were higher following L-arginine compared to placebo infusion: 132.63 (100.24, 165.02) versus 92.07 (66.33, 117.82) µmol/mmol creatinine for nitrate (P=0.014) and 50.53 (42.19, 58.87) versus 39.64 (33.94, 45.34) nmol/mmol creatinine for cGMP (P=0.0003), respectively. In migraineurs, excretion of these biomarkers was comparable following L-arginine or saline infusion. The results of the present study do not support the idea of a generalised increase in NO synthase activity in migraine patients outside of a migraine attack. The smaller increase in plasma L-citrulline, urinary nitrate and cGMP excretion following L-arginine infusion in migraine patients might indicate dysfunction of endothelial NO synthase.

Effects of telmisartan, a unique angiotensin receptor blocker with selective peroxisome proliferator-activated receptor-γ-modulating activity, on nitric oxide bioavailability and atherosclerotic change

Telmisartan is a unique angiotensin II (Ang II) receptor blocker (ARB) with selective peroxisome proliferator-activated receptor-gamma (PPAR gamma). We therefore investigated the effects of telmisartan on endothelial function and atherosclerotic change in genetically hyperlipidemic rabbits, compared with candesartan, an ARB without PPAR gamma activity. A total of 30 Watanabe heritable hyperlipidemic (WHHL) rabbits equally derived (n = 6 each) were treated with (1) vehicle (control), (2) GW9662, a PPAR gamma antagonist (0.5 mg/kg per day), (3) telmisartan (5 mg/kg per day), (4) telmisartan + GW9662, (5) candesartan (5 mg/kg per day) for 8 weeks. After treatment, acetylcholine (ACh)-induced nitric oxide production was measured as a surrogate for endothelium protective function, and vascular nitrotyrosine (a product of superoxide and nitric oxide) was measured for assessing dysfunctional endothelial nitric oxide synthase activity. Plaque area was quantified by histology. Telmisartan increased ACh-induced nitric oxide by 5.5 nmol/l, significantly more than control. Interestingly, cotreatment with GW9662 significantly attenuated telmisartan-induced ACh-induced nitric oxide almost to the levels observed with candesartan. Vascular nitrotyrosine concentration was 1.4 pmol/mg protein in the control group and significantly higher than that in the telmisartan or candesartan group. The lowest nitrotyrosine concentration was observed in the telmisartan group, which was significantly lower than that in the candesartan or telmisartan + GW9662 group. Histology of the thoracic aorta revealed that the plaque area was more significantly decreased in the telmisartan group than in the candesartan or telmisartan + GW9662 group. In addition to a class effect of ARBs, telmisartan may have additional effects on nitric oxide bioavailability and atherosclerotic change through its PPAR gamma-mediated effects in genetically hyperlipidemic rabbits.

Combined Effects of an 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitor and Angiotensin II Receptor Antagonist on Nitric Oxide Bioavailability and Atherosclerotic Change in Myocardial Infarction-Prone Watanabe Heritable Hyperlipidemic Rabbits

We investigated the effects of co-administration of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor and angiotensin II type 1 receptor blocker (ARB) on nitric oxide (NO) bioavailability in genetically hyperlipidemic rabbits with our newly developed NO sensor. A total of 36 myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits equally derived (n=6 per group) were treated with 1) vehicle (control), 2) hydralazine (15 mg/kg/d), 3) the HMG-CoA reductase inhibitor pitavastatin (P: 0.5 mg/kg/d), 4) the ARB valsartan (V: 5 mg/kg/d), and 5) pitavastatin+valsartan (P+V) together without or 6) with N(G)-nitro-L-arginine methyl ester (L-NAME) for 8 weeks. After treatment, acetylcholine (ACh)-induced NO production was measured as a surrogate for endothelium protective function, and vascular peroxynitrite (a product of superoxide and NO) was measured for assessing dysfunctional endothelial NO synthase activity. Plaque area was quantified by histology as well as optical coherence tomography (OCT). Intra-aortic infusion of ACh produced an increase in plasma NO concentration, which was significantly greater with all drug treatments than with the control. P+V increased ACh-induced NO by 4.1 nmol/L significantly more than either P or V singly. The vascular peroxynitrite concentration was 1.6 pmol/mg protein in the control group and significantly less than those in the P- and V-monotherapy-groups. The lowest peroxynitrite concentration was observed in the P+V group (0.4 pmol/mg protein), which was significantly lower than those in the P- and the V-monotherapy-groups. OCT and histology of the thoracic aorta revealed that the plaque area decreased significantly more with the combination than with the monotherapy. In conclusion, the combined treatment with an HMG-CoA reductase inhibitor and an ARB may have additive protective effects on endothelial function as well as atherosclerotic change.

Deficiency of Endothelial Nitric-Oxide Synthase Confers Susceptibility to Diabetic Nephropathy in Nephropathy-Resistant Inbred Mice

Recent studies have implicated dysfunctional endothelial nitric-oxide synthase (eNOS) as a common pathogenic pathway in diabetic vascular complications. However, functional consequences are still incompletely understood. To determine the role of eNOS-derived nitric oxide (NO) in diabetic nephropathy, we induced diabetes in eNOS knockout (KO) and wild-type (WT) mice on the C57BL6 background, using low-dose streptozotocin injection, and we investigated their glomerular phenotype at up to 20 weeks of diabetes. Although the severity of hyperglycemia in diabetic eNOS KO mice was similar to diabetic WT mice, diabetic eNOS KO mice developed overt albuminuria, hypertension, and glomerular mesangiolysis, whereas diabetic WT and nondiabetic control mice did not. Glomerular hyperfiltration was also significantly reduced in diabetic eNOS KO mice. Electron micrographs from diabetic eNOS KO mice revealed an injured endothelial morphology, thickened glomerular basement membrane, and focal foot process effacement. Furthermore, the anionic sites at glomerular endothelial barrier estimated by cationic ferritin binding were reduced in diabetic eNOS KO mice. In aggregate, these results demonstrate that deficiency of eNOS-derived NO causes glomerular endothelial injury in the setting of diabetes and results in overt albuminuria and glomerular mesangiolysis in nephropathy-resistant inbred mice. The findings indicate a vital role for eNOS-derived NO in the pathogenesis of diabetic nephropathy.

The puzzle of endothelial nitric oxide synthase dysfunction in portal hypertension: The missing piece?

The puzzle of endothelial nitric oxide synthase dysfunction in portal hypertension: The missing piece?

D‐4F, an apolipoprotein A‐1 mimetic, inhibit endothelium‐derived microparticles‐induced endothelial nitric oxide synthase dysfunction

Endothelium-derived microparticles (EMP) have recently been identified in patients with cardiovascular diseases and are suggested as markers of endothelial dysfunction. D-4F, an apolipoprotein A-1 mimetic, has been reported to improve endothelium- and endothelial nitric oxide synthase (eNOS) dependent vasodilation in a variety of vascular diseases. However, whether D-4F can preserve eNOS function in EMP-treated endothelial cells remains unknown. Bovine aortic endothelial cells (BAEC) were pretreated with EMP (2x106/ml) derived from plasminogen activated inhibitor-1 stimulated human umbilical vein endothelial cells for 30 min. Superoxide anion (O2·–) and nitric oxide (NO) generation were determined. EMP significantly increased O2·– generation where L-nitroargininemethylester partially inhibit it, and decreased NO generation. Western blots show EMP decreased phosphorylation of eNOS with unaltered eNOS expression. However, when BAEC were pretreated with EMP + D-4F 10 μg/ml, D-4F partially restored phosphorylation of eNOS. Our findings indicate D-4F can restore in part, eNOS function in EMP-treated endothelial cell cultures and may provide a new therapeutic approach for treating endothelial dysfunction.

The clinical significance of endothelial dysfunction

Endothelial dysfunction is thought to play a pivotal role in the development, progression, and clinical complications of atherosclerosis. Several recent studies have addressed the clinical implications of endothelial dysfunction for cardiovascular events, atherosclerosis, restenosis, and heart failure. Novel findings with respect to endothelial progenitor cells and their alteration by cardiovascular risk factors are characterized and potential therapeutic interventions to improve endothelial and endothelial progenitor cell function are discussed. Over the past 5 years evidence has accumulated from clinical studies for a close association of the degree of endothelial dysfunction and clinical cardiovascular events in patients with cardiovascular risk factors, coronary disease, acute coronary syndrome, or heart failure. Understanding of the mechanisms leading to endothelial dysfunction has improved, including the notion that dysfunctional endothelial nitric oxide synthase, in part due to deficiency of the endothelial nitric oxide synthase cofactor tetrahydrobiopterin, likely plays an important role. Major progress has been made in understanding the role of endothelial progenitor cells, which likely contribute to both ischemia-induced neovascularization and endothelial regeneration after injury. Endothelial progenitor cell function is altered in patients with cardiovascular risk factors. Recent research on endothelial and endothelial progenitor cell dysfunction supports their clinical significance and has led to important insights in the pathophysiology of cardiovascular disease and at the same time provides an important opportunity to develop novel therapeutic approaches. Endothelial function represents a valuable surrogate endpoint to assess the impact of therapeutic interventions.

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation

Impaired nitric oxide (NO) bioactivity and increased superoxide (SO) production are characteristics of vascular endothelial dysfunction in diabetes. The underlying mechanisms remain unknown. In this regard, we investigated the role of tetrahydrobiopterin (BH4) bioavailability in regulating endothelial nitric oxide synthase (eNOS) activity, dimerisation and SO production in streptozotocin-induced diabetic mice. Mouse aortas were used for assays of the following: (1) aortic function by isometric tension; (2) NO by electronic paramagnetic resonance; (3) SO by lucigenin-enhanced chemiluminescence and dihydroethidine fluorescence; (4) total biopterin and BH4 by high-performance liquid chromatography; and (5) eNOS protein expression and dimerisation by immunoblotting. In diabetic mouse aortas, relaxations to acetylcholine and NO levels were significantly decreased, but SO production was increased, in association with reductions in total biopterins and BH4. Although total eNOS levels were increased in diabetes, the protein mainly existed in monomeric form. Conversely, specifically augmented BH4 in diabetic endothelium preserved eNOS dimerisation, but the expression remained unchanged. Our results demonstrate that BH4 plays an important role in regulating eNOS activity and its functional protein structure, suggesting that increasing endothelial BH4 and/or protecting it from oxidation may be a rational therapeutic strategy to restore eNOS function in diabetes.

1136-82 Tetrahydrobiopterin depletion leads to endothelial nitric oxide synthase dysfunction in ischemic hearts

1136-82 Tetrahydrobiopterin depletion leads to endothelial nitric oxide synthase dysfunction in ischemic hearts

153 Tetrahydrobiopterin depletion leads to endothelial nitric oxide synthase dysfunction in ischaemic hearts

153 Tetrahydrobiopterin depletion leads to endothelial nitric oxide synthase dysfunction in ischaemic hearts

Native low-density lipoprotein induces endothelial nitric oxide synthase dysfunction: role of heat shock protein 90 and caveolin-1

Although native LDL (n-LDL) is well recognized for inducing endothelial cell (EC) dysfunction, the mechanisms remain unclear. One hypothesis is n-LDL increases caveolin-1 (Cav-1), which decreases nitric oxide ( NO) production by binding endothelial nitric oxide synthase (eNOS) in an inactive state. Another is n-LDL increases superoxide anion (O 2 •−), which inactivates NO. To test these hypotheses, EC were incubated with n-LDL and then analyzed for NO, O 2 •−, phospho-eNOS (S1179), eNOS, Cav-1, calmodulin (CaM), and heat shock protein 90 (hsp90). n-LDL increased NOx by more than 4-fold while having little effect on A23187-stimulated nitrite production. In contrast, n-LDL decreased cGMP under basal and A23187-stimulated conditions and increased O 2 •− by a mechanism that could be inhibited by L-nitroargininemethylester (L-NAME) and BAPTA/AM. n-LDL increased phospho-eNOS by 149%, eNOS by ∼34%, and Cav-1 by 28%, and decreased the association of hsp90 with eNOS by 49%. n-LDL did not appear to alter eNOS distribution between membrane fractions (∼85%) and cytosol (∼15%). Only 3–6% of eNOS in membrane fractions was associated with Cav-1. These data support the hypothesis that n-LDL increases O 2 •−, which scavenges NO, and suggest that n-LDL uncouples eNOS activity by decreasing the association of hsp90 as an initial step in signaling eNOS to generate O 2 •−.

Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase.

Increased superoxide production contributes to reduced vascular nitric oxide (NO) bioactivity and endothelial dysfunction in experimental models of diabetes. We characterized the sources and mechanisms underlying vascular superoxide production in human blood vessels from diabetic patients with coronary artery disease compared with nondiabetic patients. Vascular superoxide production was quantified in both saphenous veins and internal mammary arteries from 45 diabetic and 45 matched nondiabetic patients undergoing coronary artery bypass surgery. NAD(P)H-dependent oxidases were important sources of vascular superoxide in both diabetic and nondiabetic patients, but both the activity of this enzyme system and the levels of NAD(P)H oxidase protein subunits (p22phox, p67phox, and p47phox) were significantly increased in diabetic veins and arteries. In nondiabetic vessels, endothelial NO synthase produced NO that scavenged superoxide. However, in diabetic vessels, the endothelium was an additional net source of superoxide production because of dysfunctional endothelial NO synthase that was corrected by intracellular tetrahydrobiopterin supplementation. Furthermore, increased superoxide production in diabetes was abrogated by the protein kinase C inhibitor chelerythrine. These observations suggest important roles for NAD(P)H oxidases, endothelial NO synthase uncoupling, and protein kinase C signaling in mediating increased vascular superoxide production and endothelial dysfunction in human diabetes mellitus.

Working under pressure: the vascular endothelium in arterial hypertension

The vascular endothelium synthesizes and releases a spectrum of vasoactive substances like nitric oxide (NO) and endothelin (ET). In hypertension, the delicate balance of endothelium-derived factors is disturbed. ET acts as the natural counterpart to endothelium-derived NO, which exerts vasodilating, antithrombotic, and antiproliferative effects, and inhibits leukocyte-adhesion to the vascular wall. Besides its blood pressure rising effect also in man, ET induces vascular and myocardial hypertrophy, which are independent risk factors for cardiovascular morbidity and mortality. The derangement of endothelial function in hypertension is likely to be caused in part by genetic factors, but also due to elevated blood pressure itself. Due to its position between blood pressure and smooth muscle cells responsible for peripheral resistance, the endothelium is thought to be both target and mediator of arterial hypertension. Oxidative stress plays an important role in the pathogenesis of hypertension. Superoxide anions, ie, oxygen radicals produced in part by angiotensin II-activated NAD(P)H oxidase, can scavenge NO to form peroxynitrite, which can nitrosylate membrane proteins and oxidize lipids. Another source of superoxide is cyclooxygenase. Paradoxically, dysfunctional endothelial NO synthase may also be a source of superoxide anions. Surprisingly and in contrast to animal experiments, not all antihypertensive treatments consistently restore endothelium-dependent vasodilation in patients with arterial hypertension. Endothelial dysfunction in hypertension is crucial both for the development of the disease process in the vasculature and an important therapeutic target.

Inhibition of tetrahydrobiopterin biosynthesis impairs endothelium-dependent relaxations in canine basilar artery.

Tetrahydrobiopterin is an essential cofactor in biosynthesis of nitric oxide. The present study was designed to determine the effect of decreased intracellular tetrahydrobiopterin levels on endothelial function of isolated cerebral arteries. Blood vessels were incubated for 6 h in minimum essential medium (MEM) in the presence or absence of a GTP cyclohydrolase I inhibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP, 10(-2) M). Rings with and without endothelium were suspended for isometric force recording in the presence of a cyclooxygenase inhibitor, indomethacin (10(-5) M). In arteries with endothelium, DAHP significantly reduced intracellular levels of tetrahydrobiopterin. DAHP in combination with a precursor of the salvage pathway of tetrahydrobiopterin biosynthesis, sepiapterin (10(-4) M), not only restored but increased levels of tetrahydrobiopterin above control values. In DAHP-treated arteries, endothelium-dependent relaxations to bradykinin (10(-10)-10(-6) M) or calcium ionophore A23187 (10(-9)-10(-6) M) were significantly reduced, whereas endothelium-independent relaxations to a nitric oxide donor, 3-morpholinosydnonimine (10(-9)-10(-4) M), were not affected. When DAHP-treated arteries with endothelium were incubated with sepiapterin (10(-4) M) or superoxide dismutase (150 U/ml), relaxations to bradykinin and A23187 were restored to control levels. In contrast, superoxide dismutase did not affect endothelium-dependent relaxations in arteries incubated in MEM. A nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (10(-4) M), abolished relaxations to bradykinin or A23187 in control arteries and in DAHP-treated arteries. These studies demonstrate that in cerebral arteries, decreased intracellular levels of tetrahydrobiopterin can reduce endothelium-dependent relaxations. Production of superoxide anions during activation of dysfunctional endothelial nitric oxide synthase appears to be responsible for the impairment of endothelial function.