Nitric Oxide Bioavailability In Endothelial Cells Research Articles

NAD+-boosting compounds enhance nitric oxide production and prevent oxidative stress in endothelial cells exposed to plasma from patients with COVID-19

SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), induces vascular endothelial dysfunction, but the mechanisms are unknown. We tested the hypothesis that the “circulating milieu” (plasma) of patients with COVID-19 would cause endothelial cell dysfunction (characterized by lower nitric oxide (NO) production), which would be linked to greater reactive oxygen species (ROS) bioactivity and depletion of the critical metabolic co-substrate, nicotinamide adenine dinucleotide (NAD+). We also investigated if treatment with NAD+-boosting compounds would prevent COVID-19-induced reductions in endothelial cell NO bioavailability and oxidative stress. Human aortic endothelial cells (HAECs) were exposed to plasma from men and women (age 18–85 years) who were hospitalized and tested positive (n = 34; 20 M) or negative (n = 13; 10 M) for COVID-19. HAECs exposed to plasma from patients with COVID-19 also were co-incubated with NAD+ precursors nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN). Acetylcholine-stimulated NO production was 27% lower and ROS bioactivity was 54% higher in HAECs exposed to plasma from patients with COVID-19 (both p < 0.001 vs. control); these responses were independent of age and sex. NAD+ concentrations were 30% lower in HAECs exposed to plasma from patients with COVID-19 (p = 0.001 vs. control). Co-incubation with NR abolished COVID-19-induced reductions in NO production and oxidative stress (both p > 0.05 vs. control). Co-treatment with NMN produced similar results. Our findings suggest the circulating milieu of patients with COVID-19 promotes endothelial cell dysfunction, characterized by lower NO bioavailability, greater ROS bioactivity, and NAD+ depletion. Supplementation with NAD+ precursors may exert a protective effect against COVID-19-evoked endothelial cell dysfunction and oxidative stress.

Eicosapentaenoic acid (EPA) reduces pulmonary endothelial dysfunction and inflammation due to changes in protein expression during exposure to particulate matter air pollution

AimsInhalation of air pollution small particle matter (PM) is a leading cause of cardiovascular (CV) disease. Exposure to PMs causes endothelial cell (EC) dysfunction as evidenced by nitric oxide (NO) synthase uncoupling, vasoconstriction and inflammation. Eicosapentaenoic acid (EPA) has been shown to mitigate PM-induced adverse cardiac changes in patients receiving omega-3 fatty acid supplementation. We set out to determine the pro-inflammatory effects of multiple PMs (urban and fine) on pulmonary EC NO bioavailability and protein expression, and whether EPA restores EC function under these conditions. Methods and resultsWe pretreated pulmonary ECs with EPA and then exposed them to urban or fine air pollution PMs. LC/MS-based proteomic analysis to assess relative expression levels. Expression of adhesion molecules was measured by immunochemistry. The ratio of NO to peroxynitrite (ONOO–) release, an indication of eNOS coupling, was measured using porphyrinic nanosensors following calcium stimulation. Urban/fine PMs also modulated 9/12 and 13/36 proteins, respectively, linked to platelet and neutrophil degranulation pathways and caused > 50% (p < 0.001) decrease in the stimulated NO/ONOO– release ratio. EPA treatment altered expression of proteins involved in these inflammatory pathways, including a decrease in peroxiredoxin-5 and an increase in superoxide dismutase-1. EPA also increased expression of heme oxygenase-1 (HMOX1), a cytoprotective protein, by 2.1-fold (p = 0.024). EPA reduced elevations in sICAM-1 levels by 22% (p < 0.01) and improved the NO/ONOO– release ratio by > 35% (p < 0.05). ConclusionThese cellular changes may contribute to anti-inflammatory, cytoprotective and lipid changes associated with EPA treatment during air pollution exposure.

Mitochondrial-targeted antioxidant supplementation for improving age-related vascular dysfunction in humans: A study protocol.

Background: Cardiovascular disease (CVD) is the leading cause of death worldwide and aging is the primary risk factor for the development of CVD. The increased risk of CVD with aging is largely mediated by the development of vascular dysfunction. Excessive production of mitochondrial reactive oxygen species (mtROS) is a key mechanism of age-related vascular dysfunction. Therefore, establishing the efficacy of therapies to reduce mtROS to improve vascular function with aging is of high biomedical importance. Previously, in a small, randomized, crossover-design pilot clinical trial, our laboratory obtained initial evidence that chronic oral supplementation with the mitochondrial-targeted antioxidant MitoQ improves vascular function in healthy older adults. Here, we describe the protocol for an ongoing R01-funded phase IIa clinical trial to establish the efficacy of MitoQ as a therapy to improve vascular function in older adults (ClinicalTrials.gov Identifier: NCT04851288). Outcomes: The primary outcome of the study is nitric oxide (NO)-mediated endothelium-dependent dilation (EDD) as assessed by brachial artery flow-mediated dilation (FMDBA). Secondary outcomes include mtROS-mediated suppression of EDD, aortic stiffness as measured by carotid-femoral pulse wave velocity, carotid compliance and β-stiffness index, and intima media thickness. Other outcomes include the assessment of endothelial mitochondrial health and oxidative stress in endothelial cells obtained by endovascular biopsy; the effect of altered circulating factors following MitoQ treatment on endothelial cell NO bioavailability and whole cell and mitochondrial reactive oxygen species production ex vivo; and circulating markers of oxidative stress, antioxidant status, and inflammation. Methods: We are conducting a randomized, placebo-controlled, double-blind, parallel group, phase IIa clinical trial in 90 (45/group) healthy older men and women 60 years of age or older. Participants complete baseline testing and are then randomized to either 3 months of oral MitoQ (20 mg; once daily) or placebo supplementation. Outcome measures are assessed at the midpoint of treatment, i.e., 6 weeks, and again at the conclusion of treatment. Discussion: This study is designed to establish the efficacy of chronic supplementation with the mitochondrial-targeted antioxidant MitoQ for improving vascular endothelial function and reducing large elastic artery stiffness in older adults, and to investigate the mechanisms by which MitoQ supplementation improves endothelial function.

EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia.

Blood clot formation initiates ischemic events, but coagulation roles during postischemic tissue repair are poorly understood. The endothelial protein C receptor (EPCR) regulates coagulation, as well as immune and vascular signaling, by protease activated receptors (PARs). Here, we show that endothelial EPCR-PAR1 signaling supports reperfusion and neovascularization in hindlimb ischemia in mice. Whereas deletion of PAR2 or PAR4 did not impair angiogenesis, EPCR and PAR1 deficiency or PAR1 resistance to cleavage by activated protein C caused markedly reduced postischemic reperfusion in vivo and angiogenesis in vitro. These findings were corroborated by biased PAR1 agonism in isolated primary endothelial cells. Loss of EPCR-PAR1 signaling upregulated hemoglobin expression and reduced endothelial nitric oxide (NO) bioavailability. Defective angiogenic sprouting was rescued by the NO donor DETA-NO, whereas NO scavenging increased hemoglobin and mesenchymal marker expression in human and mouse endothelial cells. Vascular specimens from patients with ischemic peripheral artery disease exhibited increased hemoglobin expression, and soluble EPCR and NO levels were reduced in plasma. Our data implicate endothelial EPCR-PAR1 signaling in the hypoxic response of endothelial cells and identify suppression of hemoglobin expression as an unexpected link between coagulation signaling, preservation of endothelial cell NO bioavailability, support of neovascularization, and prevention of fibrosis.

Empagliflozin restores chronic kidney disease–induced impairment of endothelial regulation of cardiomyocyte relaxation and contraction

Chronic kidney disease (CKD) promotes development of cardiac abnormalities and is highly prevalent in patients with heart failure, particularly in those with preserved ejection fraction. CKD is associated with endothelial dysfunction, however, whether CKD can induce impairment of endothelium-to-cardiomyocyte crosstalk leading to impairment of cardiomyocyte function is not known. The sodium-glucose co-transporter 2 inhibitor, empagliflozin, reduced cardiovascular events in diabetic patients with or without CKD, suggesting its potential as a new treatment for heart failure with preserved ejection fraction. We hypothesized that uremic serum from patients with CKD would impair endothelial control of cardiomyocyte relaxation and contraction, and that empagliflozin would protect against this effect. Using a co-culture system of human cardiac microvascular endothelial cells with adult rat ventricular cardiomyocytes to measure cardiomyocyte relaxation and contraction, we showed that serum from patients with CKD impaired endothelial enhancement of cardiomyocyte function which was rescued by empagliflozin. Exposure to uremic serum reduced human cardiac microvascular endothelial cell nitric oxide bioavailability, and increased mitochondrial reactive oxygen species and 3-nitrotyrosine levels, indicating nitric oxide scavenging by reactive oxygen species. Empagliflozin attenuated uremic serum-induced generation of endothelial mitochondrial reactive oxygen species, leading to restoration of nitric oxide production and endothelium-mediated enhancement of nitric oxide levels in cardiomyocytes, an effect largely independent of sodium-hydrogen exchanger-1. Thus, empagliflozin restores the beneficial effect of cardiac microvascular endothelial cells on cardiomyocyte function by reducing mitochondrial oxidative damage, leading to reduced reactive oxygen species accumulation and increased endothelial nitric oxide bioavailability.

Metabolites of flavonoid compounds preserve indices of endothelial cell nitric oxide bioavailability under glucotoxic conditions

We hypothesized that metabolites of dietary flavonoids attenuate impairments in nitric oxide (NO) bioavailability evoked by glucotoxic conditions mimicking Type 1 or 2 diabetes. To test this, human aortic endothelial cells were treated with either vehicle control, quercetin-3-O-glucoronide, piceatannol or 3-(3-hydroxyphenyl)propionoic acid for 24 h. These are metabolites of quercetin, resveratrol and proanthocyanidin, respectively. Next, cells were exposed to control (5 mM) or high (25 mM) glucose conditions for 48 h, followed by insulin treatment (100 nM, 10 min) to stimulate NO production. In control glucose conditions NO production, phosphorylated to total endothelial nitric oxide synthase (p-eNOSser1177: eNOS), and phosphorylated to total Akt (p-AktSer473: Akt) were all increased by insulin stimulation. This response was abolished during high glucose conditions. Pretreatment of cells with flavonoid metabolites prior to high glucose challenge preserved insulin stimulated increases in NO production, p-AktSer473: Akt and p-eNOSSer1177: eNOS. These effects may be secondary to oxidative stress as pretreatment with all flavonoid metabolites prevented elevations in reactive oxygen and nitrogen species in response to high glucose. These data support the hypothesis that beneficial effects of flavonoids on endothelial cell function in the context of glucotoxicity, at least in part, are secondary to their metabolites.

PPARδ Is Required for Exercise to Attenuate Endoplasmic Reticulum Stress and Endothelial Dysfunction in Diabetic Mice.

Physical activity has profound benefits on health, especially on cardiometabolic wellness. Experiments in rodents with trained exercise have shown that exercise improves vascular function and reduces vascular inflammation by modulating the balance between nitric oxide (NO) and oxidative stress. However, the upstream regulator of exercise-induced vascular benefits is unclear. We aimed to investigate the involvement of peroxisome proliferator-activated receptor δ (PPARδ) in exercise-induced vascular functional improvement. We show that PPARδ is a crucial mediator for exercise to exert a beneficial effect on the vascular endothelium in diabetic mice. In db/db mice and high-fat diet-induced obese mice, 4 weeks of treadmill exercise restored endothelium-dependent vasodilation of aortas and flow-mediated vasodilation in mesenteric resistance arteries, whereas genetic ablation of Ppard abolished such improvements. Exercise induces AMPK activation and subsequent PPARδ activation, which help to reduce endoplasmic reticulum (ER) and oxidative stress, thus increasing NO bioavailability in endothelial cells and vascular tissues. Chemical chaperones 4-phenylbutyric acid and tauroursodeoxycholic acid decrease ER stress and protect against endothelial dysfunction in diabetic mice. The results demonstrate that PPARδ-mediated inhibition of ER stress contributes to the vascular benefits of exercise and provides potentially effective targets for treating diabetic vasculopathy.

Metabolites of polyphenols preserve indices of endothelial cell nitric oxide bioavailability under glucotoxic conditions (372.6)

We hypothesized that biological metabolites of quercetin, resveratrol, and grape seed extract previously identified in human plasma can prevent impairment of nitric oxide (NO) bioavailability due to glucotoxic conditions (e.g. Type 1 or 2 diabetes).Human aortic endothelial cells were treated for 24 h with 2μΜ Quercetin‐3‐O‐glucoronide, 5μΜ Piceatannol, or 1μΜ 3‐Hydroproponoic acid. Cells were next exposed to normal (5mM) or high (25mM) glucose for 48h, then treated with insulin (100nM, 10 min) to stimulate NO production.In the absence of polyphenols, insulin stimulation increased (P&lt;0.05) indices of NO production, phosphorylated to total Akt (p‐AktSer473:Akt), and endothelial nitric oxide synthase (p‐eNOSser1177:eNOS) in cells grown in 5mM but not 25mM glucose. Pretreatment of cells with polyphenol metabolites prior to 25mM glucose exposure preserved insulin stimulated increases (P&lt;0.05) in NO, p‐AktSer473:Akt and p‐eNOSser1177:eNOS. These effects may be secondary to oxidative stress as elevations (P&lt;0.05) in reactive oxygen and nitrogen species in cells treated with 25mM glucose were completely prevented by all polyphenol metabolites.These data indicate that biological metabolites of quercetin, resveratrol, and grape seed extract protected against gluocotoxic impairment of insulin dependent NO bioavailability, and preserved the insulin ‐ Akt ‐ eNOS signaling axis.

A Endothelial cell nitric oxide bioavailability and insulin sensitivity are regulated by IGF-1 and insulin receptor levels

BackgroundIn a similar manner to insulin, the growth promoting hormone Insulin-like Growth Factor-1 (IGF-1), may be an important regulator of endothelial nitric oxide (NO) bioavailability. We have previously reported evidence...

Cryptotanshinone AttenuatesIn VitrooxLDL-Induced Pre-Lesional Atherosclerotic Events

Development of early stage atherosclerosis involves the activation of endothelial cells by oxidized low-density lipoprotein (oxLDL) with subsequent increases in endothelial permeability and expression of adhesion molecules favoring the adherence of monocytes to the endothelium. Cryptotanshinone (CTS), a major compound derived from the Chinese herb Salvia miltiorrhiza, is known for its protective effects against cardiovascular diseases. The aim of this study was to determine whether CTS could prevent the oxLDL-induced early atherosclerotic events. OxLDL (100 µg/mL) was used to increase endothelial permeability and induce monocyte-endothelial cell adhesion in human umbilical vein endothelial cells (HUVECs). Endothelial nitric oxide (NO) concentrations, a permeability-regulating molecule, and expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were measured. Results show that a) endothelial hyperpermeability was suppressed by 94 % (p < 0.005), b) monocyte adhesion by 105 % (p < 0.01), and c) ICAM-1 and VCAM-1 expressions by 90 % (p < 0.01) and 150 % (p < 0.005), respectively, when CTS was applied. In contrast, CTS increased NO levels by 129 % (p < 0.01) and was found to be noncytotoxic in the concentrations between 1-10 µM. These findings indicate that CTS suppresses an increase in endothelial permeability, likely due to the restoration of NO bioavailability in endothelial cells. They also indicate that CTS may attenuate monocyte adhesion to endothelial cells through the inhibition of adhesion molecules' expression. Thus, CTS may play an important role in the prevention of early or pre-lesional stage of atherosclerosis.

Role of endothelial dysfunction in modulating the plasma redox homeostasis in visceral leishmaniasis

BackgroundEvidence in the literature suggests that down-regulation of nitric oxide (NO) is associated with the pathophysiological conditions during visceral leishmaniasis (VL). Here we have investigated the mechanism that leads to the down regulation of systemic NO in the infected condition. Moreover, we have determined whether down regulation of NO is associated with increased generation of reactive oxygen species (ROS) during this disease. Therapeutic strategy targeting signaling molecules of these events was evaluated. MethodsPlasma protein-nitrotyrosine was examined by ELISA kit. Generation of superoxides and peroxynitrites was investigated by flow cytometry. NO bioavailability in endothelial cells was evaluated using DAF-2DA fluorescence. Ceramide contents were evaluated using FACS analysis, HPTLC and HPLC. ResultsL. donovani infected reticulo-endothelial cells regulated the activity of eNOS and NAD(P)H oxidase in the endothelial cells through the generation of intercellular messenger, ceramide. Activation of SMases played an important role in the generation of ceramide in animals during chronic infection. These events led to generation of ROS within endothelial cells. Modulation of redox status of plasma and accumulation of ROS in endothelial cells were critically involved in the regulation of NO bioavailability in plasma of the infected animal. Endothelial dysfunction and decline of NO were resulted from an increased production of superoxide where upregulation of eNOS expression appeared as an ineffective compensatory event. Inhibition of ceramide generation increased NO bioavailability, prevented endothelial dysfunction and concomitant oxidative stress. Conclusion and general significanceDecreased NO bioavailability and endothelial dysfunction were the downstream of ceramide signaling cascade. ROS accumulation promoted peroxynitrite generation and reduced NO bioavailability. Inhibition of ceramide generation may be a potential therapeutic option in preventing the co-morbidity associated with VL.

Dietary flavonoid quercetin stimulates vasorelaxation in aortic vessels

Considerable epidemiological evidence indicates that dietary consumption of moderate levels of polyphenols decreases both the incidence of cardiovascular disease and the mortality associated with myocardial infarction. Molecular mechanisms of this cardiovascular protection remain uncertain but can involve changes in rates of nitric oxide (NO) generation by endothelial nitric oxide synthase (eNOS). We examined the vascular responses to quercetin using a combination of biochemical and vessel function criteria. Quercetin treatment for 30 min enhanced relaxation of rat aortic ring segments. Moreover, the addition of L-NAME (100 μM) or charybdotoxin (ChTx) blocked quercetin-mediated vasorelaxation thus demonstrating the effect was partially dependent on NOS and endothelium-derived hyperpolarizing factor (EDHF). Additionally, bovine aortic endothelial cells (BAEC) treated with quercetin showed a rapid increase of intracellular Ca 2+ concentrations as well as a dose- and time-dependent stimulation of eNOS phosphorylation with a concomitant increase in NO production. These results demonstrate that quercetin-mediated stimulation of eNOS phosphorylation increases NO bioavailability in endothelial cells and can thus play a role in the vascular protective effects associated with improved endothelial cell function.

Abstract 1432: Nitric Oxide Bioavailability is Lower in Endothelium of Healthy Mexican American Donors as Compared to Non-Hispanic White Donors: Effect of Nebivolol

Background: Epidemiologic studies indicate that Mexican Americans (MA) have a higher prevalence of CV risk factors and disease as compared with non-Hispanic whites (NHW). This increase in CV risk may be due, in part, to differences in endothelial function. In this study, we measured endothelial function in cells from normotensive, age-matched MA and NHW donors, as well as the effects of treatment with nebivolol, a new β 1 -selective blocker with vasodilating properties. Methods: Endothelial nitric oxide (NO) and peroxynitrite (ONOO − ) release in human umbilical vein endothelial cells (HUVECs) from age-matched MA and NHW donors were measured simultaneously using a nanosensor array. The effects of nebivolol on NO and ONOO − release were evaluated following pretreatment (24 h) with a calcium ionophore (CaI) as a receptor-independent stimulus. Endothelial NO synthase (eNOS) levels were measured by Western blot analysis, and drug-membrane interactions were determined by small angle x-ray diffraction approaches. Results: NO bioavailability in endothelial cells of MA donors was 30% lower than that of cells from NHW donors (383 ± 10 nM versus 543 ± 8 nM, n=6) following stimulation with CaI (1.0 μM). Pretreatment with nebivolol (1.0 μM) eliminated these interracial differences and enhanced NO release disproportionately in MA cells (57%) versus NHW cells (20%). Nebivolol also reduced ONOO − levels in MA endothelium by 75% (746 ± 12 nM to 195 ± 10 nM) and by 50% in NHW cells (416 ± 7 nM to 191 ± 13 nM). The ratio of NO to ONOO − , an indicator of eNOS coupling, increased more than 5-fold in MA cells following nebivolol treatment. In addition, eNOS levels were 40% lower in MA endothelium compared to NHW, but increased 2-fold with nebivolol treatment. These effects were not observed with atenolol, a hydrophilic β 1 -selective antagonist. Conclusion: We observed differences between Mexican Americans and non-Hispanic whites in endothelial NO bioavailability and nitroxidative stress–factors that may contribute to increased CV risk. Treatment with nebivolol, but not atenolol, enhanced both the expression and coupling efficiency of eNOS in Mexican American endothelium.

Nebivolol Reduces Nitroxidative Stress and Restores Nitric Oxide Bioavailability in Endothelium of Black Americans

Alterations in endothelial function may contribute to increased susceptibility of black Americans to cardiovascular disease. The ability to pharmacologically reverse endothelial dysfunction in blacks was tested with nebivolol, a beta1-selective agent with vasodilating and antioxidant properties. The effects of nebivolol on endothelial nitric oxide (NO), superoxide (O2-), and peroxynitrite concentration (ONOO-) release were studied in human umbilical vein endothelial cells and iliac artery endothelial cells isolated from age-matched black and white donors. Kinetics and concentrations of NO/O2-/ONOO- were measured simultaneously with nanosensors from single cells and shown to have significant interracial differences. The rate of NO release was &5 times slower in blacks than in whites (94 versus 505 nmol . L(-1).s(-1)), whereas the rates of release were faster by &2 times for O2- and &4 times for ONOO- (22.1 versus 9.4 nmol.L(-1).s(-1) for O2- and 810 versus 209 nmol.L(-1).s(-1) for ONOO-). Pretreatment with 1.0 to 5.0 micromol/L nebivolol restored NO bioavailability in endothelial cells from black donors with concurrent reductions in O2- and ONOO- release, similar to levels in the endothelium of whites. The effects of nebivolol were dose-dependent and not observed with atenolol; similar effects were observed with apocynin, an NAD(P)H oxidase inhibitor. Reduced endothelial NO bioavailability in American blacks is mainly due to excessive O2- and ONOO- generation by NAD(P)H and uncoupled endothelial NO synthase. Nebivolol decreased O2- and ONOO- concentrations and restored NO bioavailability in blacks to the level recorded in cells from whites, independently of beta1-selective blockade.

Nitric oxide and endothelial cell aging

Nitric oxide (NO) is a crucial factor for the integrity and function of the endothelium. Besides its role in blood pressure regulation, NO acts antithrombotically and antiapoptotically. The laminar flow in the blood vessel, called shear stress, is the most potent endogenous protective force against endothelial cell apoptosis, mostly by increasing the expression of the endothelial NO synthase (eNOS) and thereby increasing NO bioavailability in endothelial cells. However, during the process of endothelial cell aging, shear stress is unable to induce eNOS expression and protect against apoptosis induction. Moreover, apoptosis induction is correlated with aging in vivo, suggesting a link between NO bioavailability, aging, and apoptosis. Moreover, cellular aging is accompanied with an increase in reactive oxygen species (ROS), which results in an imbalance of the redox status of the cell. Thioredoxin is an important redox regulator in endothelial cells and can “bind” NO. S-nitrosylation of thioredoxin increases its enzymatic activity, which in turn leads to reduced intracellular ROS and apoptosis, suggesting that thioredoxin may play an important role in NO bioavailability. One crucial step in the process of cellular aging is the telomerase activity that is reduced in aged endothelial cells. NO can inhibit the decrease in telomerase activity and thereby delay the onset of replicative senescence. Thus, the reduction in NO bioavailability is a crucial factor for endothelial cell aging and apoptosis.

Protective effect of chronic vitamin C treatment on endothelial function of apolipoprotein E-deficient mouse carotid artery.

Endothelium-dependent relaxations are impaired in carotid artery of apolipoprotein E-deficient (apoE-/-) mice. This impairment seems to be due to increased formation of superoxide anions and inactivation of endothelial nitric oxide (NO). In the present study, we tested hypothesis that chronic treatment with vitamin C may prevent endothelial dysfunction by increasing release of NO from endothelial cells. C57BL/6 and apoE-/- mice were treated for 26 weeks with Western-type fat diet with and without 1% vitamin C. Vasomotor function of isolated carotid arteries was studied by video dimension analyzer. Expression of endothelial NO synthase (eNOS) and platelet-endothelial cell adhesion molecule-1 (PECAM-1) protein were evaluated by Western blotting. Levels of cGMP and cAMP were measured by radioimmunoassay. In apoE-/- mice, vitamin C significantly augmented relaxations to acetylcholine (10-9-10-5 mol/l), but did not affect relaxations to NO donor diethylammonium-(Z)-1-(N,N-diethylamino) diazen-1-1,2-diolate (DEA-NONOate; 10-9-10-5 mol/l). In contrast, vitamin C reduced relaxations to acetylcholine and DEA-NONOate in C57BL/6 mice. Interestingly, vitamin C significantly increased basal cGMP levels in C57BL/6 mice but did not affect cGMP formation in apoE-/-. Vitamin C treatment did not affect expression of eNOS protein, whereas elevated expression of PECAM-1 protein in apoE-/- mice was returned to normal level. Our findings demonstrate that chronic treatment with vitamin C prevents endothelial dysfunction of carotid artery induced by hypercholesterolemia. This effect seems to be mediated by preservation of NO bioavailability in endothelial cells.