Vascular Superoxide Production Research Articles

Vascular xanthine oxidoreductase contributes to the antihypertensive effects of sodium nitrite in l-NAME hypertension

Nitrate and nitrite have emerged as an important novel source of nitric oxide (NO). We have previously demonstrated that sodium nitrite is an antihypertensive compound that exerts antioxidant effects in experimental hypertension. These unpredicted antioxidant effects of nitrite raised the question whether the beneficial effects found were caused by its conversion to NO or simply due to reversal of endothelial dysfunction as a consequence of its antioxidant effects. Here, we evaluated the antihypertensive effects of a daily dose of sodium nitrite for 4 weeks in L-NAME-induced hypertension in rats. We studied the effects of nitrite on markers of NO bioavailability, vascular oxidative stress, and expression of xanthine oxidoreductase. Moreover, we tested if xanthine oxidoreductase inhibition could attenuate the acute hypotensive effects of sodium nitrite in L-NAME hypertensive rats. We found that a single pharmacological dose of sodium nitrite exerts antihypertensive effects in L-NAME-induced hypertension. While the beneficial antihypertensive properties of nitrite were associated with increased levels of NO metabolites, hypertension increased vascular xanthine oxidoreductase expression by approximately 40%, with minor increases in vascular superoxide production. The inhibition of xanthine oxidoreductase by oxypurinol attenuated the acute hypotensive effects of nitrite. Taken together, our results show that nitrite exerts antihypertensive effects in L-NAME hypertensive rats and provide evidence that xanthine oxidoreductase plays an important role in this antihypertensive effect.

Is antioxidant therapy effective for advanced hypertension and renal injury?

nappropriate augmentation of reactive oxygen species(ROS) production reduces the bioavailability of anti-oxidant defenses [1,2] and plays a pivotal role in thepathophysiology of hypertension and renal injury [3–5].Experimental studies have indicated that enhanced ROSproduction is involved in vascular remodeling and endo-thelium dysfunction during the development of hyperten-sion [6]. Earlier clinical studies have also suggested a strongassociation between lower antioxidant levels and cardio-vascular risks [7].To date, much effort has been directed toward thebenefits of antioxidant therapy. Clinical studies, includingthe Cambridge Heart Antioxidant Study [8] and the Anti-oxidant Supplementation in Atherosclerosis PreventionStudy [9], have shown the beneficial effects of antioxidanttherapy for cardiovascular events. Large epidemiologicalstudies have also reported that dietary intake of anti-oxidants inverselycorrelateswithhypertension[9,10].Simi-larly, treatment with ascorbic acid significantly improvedSBP and DBP in mild-to-moderate hypertensive patients[11]. In experimental models of hypertension, vitamin Calone or in combination with vitamin E increased thesynthesis of nitric oxide and reduced blood pressure[12,13]. Furthermore, an antioxidant rich diet was shownto relieve hypertension and reduces renal immune cellinfiltration [14]. By contrast, many clinical trials failed todemonstrate any beneficial effects of antioxidant therapyon blood pressure [15–17] and cardiovascular events[18–20]. For example, the Heart Protection Study Collabo-rative Group studied high-risk individuals and showed thatalthoughantioxidantvitaminsupplementationsubstantiallyincreased blood vitamin concentrations, it did not produceany significant reductions in the incidence of any type ofcardiovascular disease [19].In this issue of Journal of Hypertension, the article‘Superoxide dismutase mimetic, tempol, aggravates renalinjury in advanced-stage stroke-prone spontaneouslyhypertensive rats’ by Sugama et al. [21] shows that anti-oxidanttherapywithasuperoxidemimetic,tempol,didnotreduce blood pressure in salt-loaded old stroke pronespontaneously hypertensive rats (SHRSP). The authors alsoshowed that although tempol effectively reduced oxidativestress in the kidney, it actually exacerbated the progressionof proteinuria and histological damage, such as glomerulo-sclerosis and interstitial fibrosis. These data suggestthat alternative pathways that are ROS-independent areinvolved in the pathogenesis of the advanced stage ofhypertension and renal injury in SHRSP. It is also possiblethat oxidative stress is merely a consequence of the acti-vation of these alternative pathways. However, Park et al.[22] had performed similar experiments in SHRSP andshown that long-term treatment with tempol from earlystages of hypertension did attenuate the progression ofhypertension through the prevention of vascular remodel-ing, and that this was associated with decreased vascularsuperoxide production and increased plasma antioxidantlevels.Thus, thequestion arisesastowhyantioxidanttherapiesshow such conflicting results in clinical and experimentalstudies [23]. Importantly, it is clear from the clinical studiesthatdosedifferencesdonotseemtoberesponsibleforsuchdiscrepant results [23]. One possible explanation for thediscrepancy in these studies is the different stage of hyper-tension and renal injury. In both clinical and experimentalstudies,beneficialeffectsofantioxidantsweremostlyfoundwhen interventions were started at an early stage. Forexample, Park et al. [22] administered tempol from an earlyage and showed an attenuation in the development ofhypertension in SHRSP, whereas Sugama et al. [21] failedto observe a blood pressure-lowering effect of tempolif treatment started at an advanced hypertensive stage.Inatomi et al. [24] have also shown that in patients withmild cardioembolic stroke, treatment with an antioxidant,edaravone, resulted in early functional improvement, butthese effects were not observed in late stages.Thefindingthatantioxidanttherapieshavenobeneficialeffects on advanced hypertension, as well as cardiovas-cular and renal diseases, suggest a potentialinvolvement of

Asiatic Acid Alleviates Hemodynamic and Metabolic Alterations via Restoring eNOS/iNOS Expression, Oxidative Stress, and Inflammation in Diet-Induced Metabolic Syndrome Rats

Asiatic acid is a triterpenoid isolated from Centella asiatica. The present study aimed to investigate whether asiatic acid could lessen the metabolic, cardiovascular complications in rats with metabolic syndrome (MS) induced by a high-carbohydrate, high-fat (HCHF) diet. Male Sprague-Dawley rats were fed with HCHF diet with 15% fructose in drinking water for 12 weeks to induce MS. MS rats were treated with asiatic acid (10 or 20 mg/kg/day) or vehicle for a further three weeks. MS rats had an impairment of oral glucose tolerance, increases in fasting blood glucose, serum insulin, total cholesterol, triglycerides, mean arterial blood pressure, heart rate, and hindlimb vascular resistance; these were related to the augmentation of vascular superoxide anion production, plasma malondialdehyde and tumor necrosis factor-alpha (TNF-α) levels (p < 0.05). Plasma nitrate and nitrite (NOx) were markedly high with upregulation of inducible nitric oxide synthase (iNOS) expression, but dowregulation of endothelial nitric oxide synthase (eNOS) expression (p < 0.05). Asiatic acid significantly improved insulin sensitivity, lipid profiles, hemodynamic parameters, oxidative stress markers, plasma TNF-α, NOx, and recovered abnormality of eNOS/iNOS expressions in MS rats (p < 0.05). In conclusion, asiatic acid improved metabolic, hemodynamic abnormalities in MS rats that could be associated with its antioxidant, anti-inflammatory effects and recovering regulation of eNOS/iNOS expression.

Vascular and antioxidant effects of an aqueous Mentha cordifolia extract in experimental N(G)-nitro-L-arginine methyl ester-induced hypertension.

The effect of an aqueous Mentha cordifolia (MC) extract on the haemodynamic status, vascular remodeling, function, and oxidative status in NG-nitro-L-arginine methyl ester (L-NAME)-induced hypertension was investigated. Male Sprague-Dawley rats were given L-NAME [50 mg/(kg body weight (BW) d)] in their drinking water for 5 weeks and were treated by intragastric administration with the MC extract [200 mg/(kgBWd)] for 2 consecutive weeks. Quercetin [25 mg/(kg BW d)] was used as a positive control. The effects of the MC extract on the haemodynamic status, thoracic aortic wall thickness, and oxidative stress markers were determined, and the vasorelaxant activity of the MC extract was tested in isolated mesenteric vascular beds in rats. Significant increases in the mean arterial pressure (MAP), heart rate (HR), hind limb vascular resistance (HVR), wall thickness, and cross-sectional area of the thoracic aorta, as well as oxidative stress markers were found in the L-NAME-treated group compared to the control (P < 0.05). MAP, HVR, wall thickness, cross-sectional area of the thoracic aorta, plasma malondialdehyde (MDA), and vascular superoxide anion production were significantly reduced in L-NAME hypersensitive rats treated with the MC extract or quercetin. Furthermore, the MC extract induced vasorelaxation in the pre-constricted mesenteric vascular bed with intact and denuded endothelium of normotensive and hypertensive rats. Our results suggest that the MC extract exhibits an antihypertensive effect via its antioxidant capacity, vasodilator property, and reduced vascular remodeling.

Aortic Superoxide Production at the Early Hyperglycemic Stage in a Rat Type 2 Diabetes Model and the Effects of Pravastatin

Endothelium-derived superoxide induces vascular dysfunctions. The aim of this study was to examine the activity of protein kinase C (PKC) isoforms and endothelial nitric oxide synthase (eNOS), which leads to vascular superoxide production in type 2 diabetes, in addition to the effects of pravastatin. We studied these mechanisms in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (type 2 diabetes model) at the early hyperglycemic stage (vs. non-diabetic Long-Evans Tokushima Otsuka [LETO] rats). Superoxide production and catalase activity were measured in aortas, as were the protein expressions of PKCδ and phospho-Ser(1177) eNOS. Superoxide production was increased in OLETF rats, and this increase was inhibited by the selective conventional PKC (cPKC) inhibitor Gö6976 and by the non-selective cPKC and novel PKC inhibitor GF109203X. Phospho-Ser(1177) eNOS was significantly increased in OLETF rats, whereas the protein expressions of PKCδ and phosopho-Thr(505) PKCδ and catalase activity were all greatly reduced. Pravastatin administration to OLETF rats in vivo had normalizing effects on all of these variables. The increment in superoxide production seen in OLETF rats (but not the production in pravastatin-treated OLETF rats) was abolished by high concentration of N(ω)-nitro-L-arginine methyl ester (electron transport inhibitor of eNOS), by sepiapterin (precursor of tetrahydrobiopterin), and by LY294002 (phosphatidylinositol 3-kinase [PI3-kinase] inhibitor). In OLETF rats at the early hyperglycemic stage, aortic superoxide production is increased owing to activation of uncoupled eNOS through phosphorylation by PI3-kinase/Akt. This may be related to the observed reduction in PKCδ/catalase activities. Pravastatin inhibited endothelial superoxide production via normalization of PKCδ/catalase activities.

Dissecting the genetic predisposition to albuminuria and endothelial dysfunction in a genetic rat model

The Munich Wistar Frömter (MWF) rat develops progressive spontaneous albuminuria largely attributable to quantitative trait loci on rat chromosome (RNO)6 and RNO8, respectively. We tested the hypothesis whether quantitative trait loci on these chromosomes are linked to both albuminuria and endothelial dysfunction. Experiments were performed in male 12-week-old MWF, Wistar Kyoto (WKY), spontaneously hypertensive rat (SHR) and consomic MWF-6 and MWF-8 rats, in which RNO6 or RNO8 was replaced by the respective SHR chromosome (n = 10 per strain). Vascular function was assessed in aorta and vascular superoxide anion production was determined by confocal microscopy. Acetylcholine potency to induce relaxation was significantly reduced in MWF (6.2 ± 0.1) compared with WKY (7.1 ± 0.1) or SHR (7.3 ± 0.1; P < 0.01). N-nitro-L-arginine methyl ester abolished relaxation to acetylcholine in all three strains, whereas indomethacin exhibited no effect in WKY and MWF. Contractions to noradrenaline and superoxide production were significantly higher in MWF compared with SHR and WKY (P < 0.05, respectively). In consomic MWF-6 and MWF-8 rats albuminuria was markedly suppressed (-85 and -92%, P < 0.005 compared with MWF, respectively). Interestingly, relaxation to acetylcholine and contraction to noradrenaline were restored to normal WKY levels only in MWF-8, but not in MWF-6, due to an increase in stimulated and basal nitric oxide availability, respectively. These data demonstrate the partial genetic independence of albuminuria and endothelial dysfunction in this model. From two known albuminuria quantitative trait loci one locus affects both albuminuria and endothelial dysfunction. Whether this observation is based on a common genetic mechanism related to NO availability warrants further investigation.

Hydrogen sulfide protects endothelial nitric oxide function under conditions of acute oxidative stress in vitro.

The aim of this study was to examine the ability of H2S, released from NaHS to protect vascular endothelial function under conditions of acute oxidative stress by scavenging superoxide anions (O2(-)) and suppressing vascular superoxide anion production. O2(-) was generated in Krebs' solution by reacting hypoxanthine with xanthine oxidase (Hx-XO) or with the O2(-) generator pyrogallol to model acute oxidative stress in vitro. O2(-) generation was measured by lucigenin-enhanced chemiluminescence. Functional responses in mouse aortic rings were assessed using a small vessel myograph. NaHS scavenged O2(-) in a concentration-dependent manner. Isolated aortic rings exposed to either Hx-XO or pyrogallol displayed significantly attenuated maximum vasorelaxation responses to the endothelium-dependent vasodilator acetylcholine, and significantly reduced NO bioavailability, which was completely reversed if vessels were pre-incubated with NaHS (100 μM). NADPH-stimulated aortic O2(-) production was significantly attenuated by the NADPH oxidase inhibitor diphenyl iodonium. Prior treatment of vessels with NaHS (100 nM-100 μM; 30 min) inhibited NADPH-stimulated aortic O2(-) production in a concentration-dependent manner. This effect persisted when NaHS was washed out prior to measuring NADPH-stimulated O2(-) production. These data show for the first time that NaHS directly scavenges O2(-) and suppresses vascular NADPH oxidase-derived O2(-) production in vitro. Furthermore, these properties protect endothelial function and NO bioavailability in an in vitro model of acute oxidative stress. These results suggest that H2S can elicit vasoprotection by both scavenging O2(-) and by reducing vascular NADPH oxidase-derived O2(-) production.

Add Another Killer for Interfered Vascular Function

The predominant physiological role of the renin–angiotensin system is to regulate blood pressure and water/sodium homeostasis. Excessive angiotensin II (Ang II), the major bioactive angiotensin peptide of this system, leads to vascular dysfunction that contributes to many cardiovascular diseases. Increasing evidence supports a concept that overactivation of the renin–angiotensin system disrupts normal immune/inflammatory responses. Although this overview of adverse responses to Ang II is widely accepted, how alterations in inflammation contribute to vascular dysfunction is not completely understood. See accompanying article on page 1313 Alterations in immune responses to vascular function were reported >4 decades ago but less advancement in understanding how immune-mediated or inflammation-induced responses have been identified.1 Recently, Guzik et al2 reported that RAG-1−/− mice, lacking both functional T and B lymphocytes, were protected from Ang II–induced loss of endothelium-dependent vasodilatation. Conversely, adoptive transfer of T lymphocytes led to vascular dysfunction that were attributed to impaired endothelium-dependent vasodilatation and an increase in vascular superoxide production in response to Ang II. The authors interpreted these findings as one mechanism of Ang II–induced high blood pressure. After this initial report, other groups have confirmed contribution of immune/inflammation to Ang II–induced vascular dysfunction beyond …

Abstract 357: Hepatic ERK2 Suppresses Endoplasmic Reticulum Stress, Leading to Protection from Oxidative Stress and Endothelial Dysfunction

Background Insulin signaling comprises 2 major cascades, the IRS/PI3K/Akt and Ras/Raf/MEK/ERK pathways. Many studies on the tissue-specific effects of the former pathway had been conducted, however, the role of the latter cascade in tissue-specific insulin resistance had not been investigated. High glucose/fatty acid toxicity, inflammation and oxidative stress, all of which are associated with insulin resistance, can activate ERK. Liver plays a central role of metabolism and hepatosteatosis (HST) is associated with vascular diseases. The aim of this study is to elucidate the role of hepatic ERK2 in HST, metabolic remodeling and endothelial dysfunction. Methods Serum biomarkers of vascular complications in human were compared between subjects with and without HST diagnosed by echography for regular medical checkup. Next, we created liver-specific ERK2 knockout mice (LE2KO) and fed them with a high-fat/high-sucrose diet (HFHSD) for 20 weeks. The histological analysis, the expression of hepatic sarco/endoplasmic reticulum (ER) Ca 2+ -ATPase 2 (SERCA2) and glucose-tolerance/insulin-sensitivity (GT/IS) were tested. Vascular superoxide production and endothelial function were evaluated with dihydroethidium staining and isometric tension measurement of aorta. Results The presence of HST significantly increased HOMA-IR, an indicator of insulin resistance or atherosclerotic index in human. HFHSD-fed LE2KO revealed a marked exacerbation in HST and metabolic remodeling represented by the impairment of GT/IS, elevated serum free fatty acid and hyperhomocysteinemia without changes in body weight, blood pressure and serum cholesterol/triglyceride levels. In the HFHSD-fed LE2KO, mRNA and protein expressions of hepatic SERCA2 were significantly decreased, which resulted in hepatic ER stress. Induction of vascular superoxide production and remarkable endothelial dysfunction were also observed in them. Conclusions Hepatic ERK2 revealed the suppression of hepatic ER stress and HST in vivo , which resulted in protection from vascular oxidative stress and endothelial dysfunction. HST with hepatic ER stress can be a prominent risk of vascular complications by metabolic remodeling and oxidative stress in obese-related diseases.

Experimental periodontitis promotes transient vascular inflammation and endothelial dysfunction

ObjectivesThis study aimed to evaluate the systemic inflammatory response and cardiovascular changes induced by experimental periodontitis in rats. DesignExperimental periodontitis was induced by placing a cotton ligature around the cervix of both sides of mandibular first molars and maxillary second molars in each male rat. Sham-operated rats had the ligature removed immediately after the procedure. Seven, 14 or 28 days after procedure, the effects of acetylcholine, sodium nitroprusside and phenylephrine were evaluated on blood pressure, aortic rings and isolated and perfused mesenteric bed. The blood was obtained for plasma Interleukin-6 (IL-6), C-reactive protein (CRP) and lipid evaluation. The mesenteric vessels were obtained to evaluate superoxide production and nitric oxide synthase 3 (NOS-3) expression. ResultsLigature induced periodontitis reduced endothelium-dependent vasodilatation, a hallmark of endothelial dysfunction. This effect was associated with an increase in systemic inflammatory markers (IL-6 and CRP), worsens on lipid profile, increased vascular superoxide production and reduced NOS-3 expression. It is interesting to note that many of these effects were transitory. ConclusionPeriodontitis induced a transient systemic and vascular inflammation which leads to endothelial dysfunction, an initial step for cardiovascular diseases. Moreover, the animal model of periodontitis used here may represent a valuable tool for studying the relationship between periodontitis and endothelial dysfunction.

Mechanisms of oxidative stress in human aortic aneurysms--association with clinical risk factors for atherosclerosis and disease severity.

Aortic abdominal aneurysms (AAA) are important causes of cardiovascular morbidity and mortality. Oxidative stress may link multiple mechanisms of AAA including vascular inflammation and increased metalloproteinase activity. However, the mechanisms of vascular free radical production remain unknown. Accordingly, we aimed to determine sources and molecular regulation of vascular superoxide (O2•−) production in human AAA.Methods and resultsAAA segments and matched non-dilated aortic samples were obtained from 40 subjects undergoing AAA repair. MDA levels (determined by HPLC/MS) were greater in plasma of AAA subjects (n = 16) than in risk factor matched controls (n = 16). Similarly, superoxide production, measured by lucigenin chemiluminescence and dihydroethidium fluorescence, was increased in aneurysmatic segments compared to non-dilated aortic specimens. NADPH oxidases and iNOS are the primary sources of O2•− in AAA. Xanthine oxidase, mitochondrial oxidases and cyclooxygenase inhibition had minor or no effect. Protein kinase C inhibition had no effect on superoxide production in AAA. NADPH oxidase subunit mRNA levels for p22phox, nox2 and nox5 were significantly increased in AAAs while nox4 mRNA expression was lower. Superoxide production was higher in subjects with increased AAA repair risk Vanzetto score and was significantly associated with smoking, hypercholesterolemia and presence of CAD in AAA cohort. Basal superoxide production and NADPH oxidase activity were correlated to aneurysm size.ConclusionsIncreased expression and activity of NADPH oxidases are important mechanisms underlying oxidative stress in human aortic abdominal aneurysm. Uncoupled iNOS may link oxidative stress to inflammation in AAA. Oxidative stress is related to aneurysm size and major clinical risk factors in AAA patients.

SIRT1 inhibits NADPH oxidase activation and protects endothelial function in the rat aorta: Implications for vascular aging

Vascular aging is characterized by up-regulation of NADPH oxidase, oxidative stress and endothelial dysfunction. Previous studies demonstrate that the activity of the evolutionarily conserved NAD+-dependent deacetylase SIRT1 declines with age and that pharmacological activators of SIRT1 confer significant anti-aging cardiovascular effects. To determine whether dysregulation of SIRT1 promotes NADPH oxidase-dependent production of reactive oxygen species (ROS) and impairs endothelial function we assessed the effects of three structurally different inhibitors of SIRT1 (nicotinamide, sirtinol, EX527) in aorta segments isolated from young Wistar rats. Inhibition of SIRT1 induced endothelial dysfunction, as shown by the significantly reduced relaxation to the endothelium-dependent vasodilators acetylcholine and the calcium ionophore A23187. Endothelial dysfunction induced by SIRT1 inhibition was prevented by treatment of the vessels with the NADPH oxidase inhibitor apocynin or superoxide dismutase. Inhibition of SIRT1 significantly increased vascular superoxide production, enhanced NADPH oxidase activity, and mRNA expression of its subunits p22phox and NOX4, which were prevented by resveratrol. Peroxisome proliferator-activated receptor-α (PPARα) activation mimicked the effects of resveratrol while PPARα inhibition prevented the effects of this SIRT1 activator. SIRT1 co-precipitated with PPARα and nicotinamide increased the acetylation of the PPARα coactivator PGC-1α, which was suppressed by resveratrol. In conclusion, impaired activity of SIRT1 induces endothelial dysfunction and up-regulates NADPH oxidase-derived ROS production in the vascular wall, mimicking the vascular aging phenotype. Moreover, a new mechanism for controlling endothelial function after SIRT1 activation involves a decreased PGC-1α acetylation and the subsequent PPARα activation, resulting in both decreased NADPH oxidase-driven ROS production and NO inactivation.

Relevance of vascular peroxisome proliferator‐activated receptor γ coactivator‐1α to molecular alterations in atherosclerosis

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is emerging as a novel factor that plays a critical role in integrating signalling pathways in the control of cellular and systemic metabolism. We investigated the role of vascular expression of PGC-1α and related factors, such as sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ (PPARγ) and adiponectin, during the atherosclerotic process. Endothelial function, vascular superoxide anion production and inflammatory mediators were also evaluated. This study was carried out in male New Zealand rabbits fed a diet containing 0.5% cholesterol and 14% coconut oil for 8 weeks. Animals developed mixed dyslipidaemia and atherosclerotic lesions, which were associated with endothelial dysfunction, aortic overproduction of superoxide anions and inflammation. Expression of PGC-1α, SIRT1, PPARγ and adiponectin was reduced (P<0.05) in aorta from atherosclerotic rabbits. Levels of PGC-1α were correlated negatively (P<0.05) with total cholesterol levels, aortic superoxide anion production and tumour necrosis factor-α expression, and positively (P<0.05) with maximal relaxation in response to acetylcholine. The observed results suggest that PGC-1α could be considered to be a link between the main atherosclerotic processes (endothelial dysfunction, oxidation and inflammation) and alterations of other factors involved in vascular wall integrity, such as SIRT1, PPARγ and adiponectin.

Developmental Programming of eNOS Uncoupling and Enhanced Vascular Oxidative Stress in Adult Rats After Transient Neonatal Oxygen Exposure

The authors have previously shown that neonatal hyperoxic stress leads to high blood pressure, impaired endothelium-mediated vasodilatation, and increased vascular production of superoxide anion by NAD(P)H oxidase in adulthood. However, it is unknown whether changes in nitric oxide (NO) production and/or bioinactivation prevail and whether NO synthase (NOS) is also a source of superoxide. The purpose of this study was to evaluate whether adult animals exposed to neonatal hyperoxic stress have impaired vascular NO production associated with NOS uncoupling participating to vascular superoxide production and vascular dysfunction. In adult male rats exposed to 80% oxygen from day 3 to 10 of life (H, n = 6) versus room air controls (CTRL, n = 6), vascular (aorta) NO production is decreased at baseline (CTRL: 21 ± 1 vs. H: 16 ± 2 4,5-diaminofluorescein diacetate fluorescence intensity arbitrary units; P < 0.05) and after carbachol stimulation (acetylcholine analog; CTRL: 26 ± 2 vs. H: 18±2; P < 0.05). Pretreatment with L-arginine (CTRL: 32 ± 4 vs. H: 31 ± 5) and L-sepiapterine [analog of key NOS cofactor tetrahydro-L-biopterin (BH4)] (CTRL: 30 ± 3 vs. H: 29 ± 3) normalizes NO production after carbachol. L-Sepiapterine also normalizes impaired vasodilatation to carbachol. Vascular endothelial NO synthase (eNOS) immunostaining is reduced, whereas total eNOS protein expression is increased in H (CTRL: 0.76 ± 0.08 vs. H: 1.76± 0.21; P < 0.01). The significantly higher superoxide generation (CTRL: 20 ± 2 vs. H: 28 ± 3 hydroethidine fluorescence intensity arbitrary units; P < 0.05) is prevented by pretreatment with the eNOS inhibitor N-nitro-L-arginine methyl ester (CTRL: 21 ± 4 vs. H: 22 ± 4). Taken together, the current data indicate a role for eNOS uncoupling in enhanced vascular superoxide, impaired endothelium-mediated vasodilatation, and decreased NO production in adult animals with programmed elevated blood pressure after a brief neonatal oxygen exposure.

Abstract 514: Caspase-1 Activation Contributes to Angiotensin-II-induced Vascular Dysfunction and Hypertension

Caspase-1 (casp-1) regulates key steps in microbial and sterile inflammatory responses. Hypertension is associated with inflammation of the vascular wall, but it is not known whether casp-1 mediates this phenomenon. We hypothesized that casp-1 contributes to vascular dysfunction in angiotensin II (AngII)-induced hypertension. Male C57BL/6 wild type (WT) and casp-1 knockout (casp-1 -/- ) mice were infused with AngII (90 ng/min, 14 days, osmotic mini-pumps). Control mice were sham-operated. Systolic blood pressure (SBP, mmHg) was significantly increased in AngII-WT mice (162±7.9 vs. 138±2.9 WT-sham, p&lt;0.05), however this elevation was less in AngII-casp-1 -/- mice (148±13 vs. 140±6.2 casp-1 -/- -sham, p&gt;0.05). AngII-WT mice exhibited cardiac hypertrophy (mg/g: 10±0.36 vs. 4.9±0.19 WT-sham, p&lt;0.05), which was not observed in AngII-casp-1 -/- mice (mg/g: 7.6±0.70 vs. 5.6±0.68 casp-1 -/- -sham, p&gt;0.05). Severe endothelial dysfunction [acetylcholine (ACh)-dependent dilation, second-order mesenteric arteries] was observed in AngII-WT mice, but this was attenuated in AngII-casp-1 -/- mice (inserted Figure). Vascular superoxide anion production (dihydroethidium fluorescence, arbitrary units) augmented in AngII-WT (6.2±0.60 vs. 4.9±0.48 WT-sham, p&lt;0.05), but not in AngII-casp-1 -/- (4.9±0.48 vs. 4.0±0.64 casp-1 -/- -sham, p&gt;0.05) mice. Preliminary data also show that AngII in vivo and in vitro induces casp-1 activation (western blotting for cleaved casp-1). These data provide evidence that activation of casp-1 plays a role in vascular dysfunction associated with AngII-hypertension.

Role of Copper Transport Protein Antioxidant 1 in Angiotensin II–Induced Hypertension

Extracellular superoxide dismutase (SOD3) is a secretory copper enzyme involved in protecting angiotensin II (Ang II)–induced hypertension. We found previously that Ang II upregulates SOD3 expression and activity as a counterregulatory mechanism; however, underlying mechanisms are unclear. Antioxidant 1 (Atox1) is shown to act as a copper-dependent transcription factor, as well as a copper chaperone, for SOD3 in vitro, but its role in Ang II–induced hypertension in vivo is unknown. Here we show that Ang II infusion increases Atox1 expression, as well as SOD3 expression and activity, in aortas of wild-type mice, which are inhibited in mice lacking Atox1. Accordingly, Ang II increases vascular superoxide production, reduces endothelium-dependent vasodilation, and increases vasoconstriction in mesenteric arteries to a greater extent in Atox1 −/− than in wild-type mice. This contributes to augmented hypertensive response to Ang II in Atox1 −/− mice. In cultured vascular smooth muscle cells, Ang II promotes translocation of Atox1 to the nucleus, thereby increasing SOD3 transcription by binding to Atox1-responsive element in the SOD3 promoter. Furthermore, Ang II increases Atox1 binding to the copper exporter ATP7A, which obtains copper from Atox1, as well as translocation of ATP7A to plasma membranes, where it colocalizes with SOD3. As its consequence, Ang II decreases vascular copper levels, which is inhibited in Atox1 −/− mice. In summary, Atox1 functions to prevent Ang II–induced endothelial dysfunction and hypercontraction in resistant vessels, as well as hypertension, in vivo by reducing extracellular superoxide levels via increasing vascular SOD3 expression and activity.

Systemic and Vascular Oxidation Limits the Efficacy of Oral Tetrahydrobiopterin Treatment in Patients With Coronary Artery Disease

The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) plays a pivotal role in maintaining endothelial function in experimental vascular disease models and in humans. Augmentation of endogenous BH4 levels by oral BH4 treatment has been proposed as a potential therapeutic strategy in vascular disease states. We sought to determine the mechanisms relating exogenous BH4 to human vascular function and to determine oral BH4 pharmacokinetics in both plasma and vascular tissue in patients with coronary artery disease. Forty-nine patients with coronary artery disease were randomized to receive low-dose (400 mg/d) or high-dose (700 mg/d) BH4 or placebo for 2 to 6 weeks before coronary artery bypass surgery. Vascular function was quantified by magnetic resonance imaging before and after treatment, along with plasma BH4 levels. Vascular superoxide, endothelial function, and BH4 levels were determined in segments of saphenous vein and internal mammary artery. Oral BH4 treatment significantly augmented BH4 levels in plasma and in saphenous vein (but not internal mammary artery) but also increased levels of the oxidation product dihydrobiopterin (BH2), which lacks endothelial nitric oxide synthase cofactor activity. There was no effect of BH4 treatment on vascular function or superoxide production. Supplementation of human vessels and blood with BH4 ex vivo revealed rapid oxidation of BH4 to BH2 with predominant BH2 uptake by vascular tissue. Oral BH4 treatment augments total biopterin levels in patients with established coronary artery disease but has no net effect on vascular redox state or endothelial function owing to systemic and vascular oxidation of BH4. Alternative strategies are required to target BH4-dependent endothelial function in established vascular disease states.

Endothelial-specific Nox2 overexpression increases vascular superoxide and macrophage recruitment in ApoE−/− mice

AimsVascular disease states are associated with endothelial dysfunction and increased production of reactive oxygen species derived from NADPH oxidases. However, it remains unclear whether a primary increase in superoxide production specifically in the endothelium alters the initiation or progression of atherosclerosis.Methods and resultsMice overexpressing Nox2 specifically in the endothelium (Nox2-Tg) were crossed with ApoE−/− mice to produce Nox2-Tg ApoE−/− mice and ApoE−/− littermates. Endothelial overexpression of Nox2 in ApoE−/− mice did not alter blood pressure, but significantly increased vascular superoxide production compared with ApoE−/− littermates, measured using both lucigenin chemiluminescence and 2-hydroxyethidium production (ApoE−/−, 19.9 ± 6.3 vs. Nox2-Tg ApoE−/−, 47.0 ± 7.0 nmol 2-hydroxyethidium/aorta, P< 0.05). Increased endothelial superoxide production increased endothelial levels of vascular cell adhesion protein 1 and enhanced macrophage recruitment in early lesions in the aortic roots of 9-week-old mice, indicating increased atherosclerotic plaque initiation. However, endothelial-specific Nox2 overexpression did not alter native or angiotensin II-driven atherosclerosis in either the aortic root or the descending aorta.ConclusionEndothelial-targeted Nox2 overexpression in ApoE−/− mice is sufficient to increase vascular superoxide production and increase macrophage recruitment possible via activation of endothelial cells. However, this initial increase in macrophage recruitment did not alter the progression of atherosclerosis. These results indicate that Nox-mediated reactive oxygen species signalling has important cell-specific and distinct temporal roles in the initiation and progression of atherosclerosis.

ENOS Protects from Atherosclerosis Despite Relevant Superoxide Production by the Enzyme in apoE−/− Mice

BackgroundAll three nitric oxide synthase (NOS) isoforms are expressed in atherosclerotic plaques. NOS enzymes in general catalyse NO production. However, under conditions of substrate and cofactor deficiency, the enzyme directly catalyse superoxide formation. Considering this alternative chemistry, the effects of NOS on key events in spontaneous hyperlipidemia driven atherosclerosis have not been investigated yet. Here, we evaluate how endothelial nitric oxide synthase (eNOS) modulates leukocyte/endothelial- (L/E) and platelet/endothelial- (P/E) interactions in atherosclerosis and the production of nitric oxide (NO) and superoxide by the enzyme.Principal FindingsIntravital microscopy (IVM) of carotid arteries revealed significantly increased L/E-interactions in apolipoproteinE/eNOS double knockout mice (apoE−/−/eNOS−/−), while P/E-interactions did not differ, compared to apoE−/−. eNOS deficiency increased macrophage infiltration in carotid arteries and vascular cell adhesion molecule-1 (VCAM-1) expression, both in endothelial and smooth muscle cells. Despite the expression of other NOS isoforms (inducible NOS, iNOS and neuronal NOS, nNOS) in plaques, Electron Spin Resonance (ESR) measurements of NO showed significant contribution of eNOS to total circulating and vascular wall NO production. Pharmacological inhibition and genetic deletion of eNOS reduced vascular superoxide production, indicating uncoupling of the enzyme in apoE−/− vessels.ConclusionOvert plaque formation, increased vascular inflammation and L/E- interactions are associated with significant reduction of superoxide production in apoE−/−/eNOS−/− vessels. Therefore, lack of eNOS does not cause an automatic increase in oxidative stress. Uncoupling of eNOS occurs in apoE−/− atherosclerosis but does not negate the enzyme's strong protective effects.

Exacerbation of endothelial dysfunction during the progression of diabetes: role of oxidative stress

To test the deterioration of endothelial function during the progression of diabetes, shear stress-induced dilation (SSID; 10, 20, and 40 dyn/cm(2)) was determined in isolated mesenteric arteries (80-120 μm in diameter) of 6-wk (6W), 3-mo (3M), and 9-mo (9M)-old male db/db mice and their wild-type (WT) controls. Nitric oxide (NO)-mediated SSID was comparable in 6W WT and db/db mice, but the dilation was significantly reduced in 3M db/db mice and declined further in 9M db/db mice. Vascular superoxide production was progressively increased in 3M and 9M db/db mice, associated with an increased expression of NADPH oxidase. Inhibition of NADPH oxidase significantly improved NO-mediated SSID in arteries of 3M, but not in 9M, db/db mice. Although endothelial nitric oxide synthase (eNOS) expression was comparable in all groups, a progressive reduction in shear stress-induced eNOS phosphorylation existed in vessels of 3M and 9M db/db mice. Moreover, inducible NOS (iNOS) that was not detected in WT, nor in 6W and 3M db/db mice, was expressed in vessels of 9M db/db mice. A significantly increased expression of nitrotyrosine in total protein and immunoprecipitated eNOS was also found in vessels of 9M db/db mice. Thus, impaired NO bioavailability plays an essential role in the endothelial dysfunction of diabetic mice, which becomes aggravated when endothelial nitrosative stress is further activated via perhaps, an additional iNOS-mediated pathway during the progression of diabetes.