Effects of chronic N-acetylcysteine treatment on the actions of peroxynitrite on aortic vascular reactivity in hypertensive rats.

BACKGROUND: Peroxynitrite (ONOO-), the product of superoxide and nitric oxide, seems to be involved in vascular alterations in hypertension. OBJECTIVES: To evaluate the effects of ONOO- on endothelium-dependent and independent aortic vascular responsiveness, oxidized/reduced glutathione balance (GSSG/GSH), malondialdehyde aortic content, and the formation of 3-nitrotyrosine (3-NT), a stable marker of ONOO-, in N-acetylcysteine (NAC)-treated normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). RESULTS: In SHR only, NAC significantly reduced heart rate and systolic, but not diastolic, blood pressure. It also improved endothelium-dependent aortic relaxation in SHR, but not after exposure to ONOO-. Endothelium-dependent and independent aortic relaxations were markedly impaired by ONOO- in both strains of rat. NAC partially protected SHR against the ONOO- -induced reduction in endothelium-independent relaxation. Aortic GSSG/GSH ratio and malondialdehyde, which were higher in SHR than in WKY rats, showed a greater increase in SHR after exposure to ONOO-. NAC decreased GSSG/GSH and malondialdehyde in both strains of rat before and after exposure to ONOO-. The 3-NT concentration, which was similar in both strains of rat under basal conditions, was greater in SHR than in WKY rats after the addition of ONOO-, with a reduction only in NAC-treated SHR. CONCLUSIONS: These findings suggest an increased vulnerability of SHR aortas to the effects of ONOO- as compared with those of WKY rats. The selective improvements produced by NAC, in systolic arterial pressure, heart rate, aortic endothelial function, ONOO- -induced impairment of endothelium-independent relaxation, aortic GSSG/GSH balance, malondialdehyde content and 3-NT formation in SHR suggest that chronic administration of NAC may have a protective effect against aortic vascular dysfunction in the SHR model of hypertension.     

Reversal of endothelial nitric oxide synthase uncoupling and up-regulation of endothelial nitric oxide synthase expression lowers blood pressure in hypertensive rats.

OBJECTIVES: We sought to examine the hypothesis that a pharmacologic up-regulation of endothelial nitric oxide synthase (eNOS) combined with a reversal of eNOS uncoupling provides a protective effect against cardiovascular disease. BACKGROUND: Many cardiovascular diseases are associated with oxidant stress involving protein kinase C (PKC) and uncoupling of eNOS. METHODS: Messenger ribonucleic acid (mRNA) expression was analyzed with RNase protection assay or quantitative real-time polymerase chain reaction, vascular nitric oxide (NO) with spin trapping, and reactive oxygen species (ROS) with dihydroethidium fluorescence. RESULTS: Aortas of spontaneously hypertensive rats (SHR) showed an elevated production of ROS when compared with aortas of Wistar-Kyoto rats (WKY). The aortic expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits (Nox1, Nox2, Nox4, and p22phox) was higher in SHR compared with WKY. In SHR, aortic production of ROS was reduced by the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), indicating eNOS "uncoupling" in hypertension. Oral treatment with the PKC inhibitor midostaurin reduced aortic Nox1 expression, diminished ROS production, and reversed eNOS uncoupling in SHR. Aortic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) were significantly reduced in SHR compared with WKY. Midostaurin normalized BH4 levels in SHR. In both WKY and SHR, midostaurin increased aortic expression of eNOS mRNA and protein, stimulated bioactive NO production, and enhanced relaxation of the aorta to acetylcholine. Midostaurin lowered blood pressure in SHR and, to a lesser extent, in WKY; the compound did not change blood pressure in WKY made hypertensive with L-NAME. CONCLUSIONS: Pharmacologic interventions that combine eNOS up-regulation and reversal of eNOS uncoupling can markedly increase bioactive NO in the vasculature and produce beneficial hemodynamic effects such as a reduction of blood pressure.     

Effects of chronic treatment with simvastatin on endothelial dysfunction in spontaneously hypertensive rats.

OBJECTIVE: To investigate the effects of chronic treatment with simvastatin (SV) on endothelium-dependent relaxation and ouabain-induced contractions in aortic rings from spontaneously hypertensive rats (SHR), comparing with normotensive Wistar-Kyoto rats (WKY). METHODS: After a 12-week period of administration of 1 or 2 mg/kg SV to SHR and WKY, systolic blood pressure (SBP) and vascular reactivity in endothelium-intact aortic rings were assessed. RESULTS: Relaxation in response to acetylcholine (ACh) in WKY remained unaltered, but in SHR treated with 1 mg/kg SV, enhanced ACh-induced relaxation (P<0.05 versus untreated SHR) reached values observed in untreated WKY. The 2 mg/kg treatment also improved ACh relaxation (P<0.01 and P<0.05 versus untreated SHR and WKY respectively). Inhibiting cyclo-oxygenase (COX) with indomethacin (INDO) improved ACh relaxation in SHR (P<0.05) but not in WKY, independent of treatment with SV. Inhibition of nitric oxide synthase (NOS) with N(G)-nitro-L-arginine (L-NOARG) abolished ACh relaxations in all cases (P<0.001). The result was unaltered when combining INDO plus L-NOARG. SV treatment also decreased ouabain-induced contractions in endothelium-intact aortic rings from SHR, diminishing the percentage effect of contraction from 64.56+/-2.95 (untreated SHR) to 26.98+/-7.06 and 38.10+/-8.21 (1 and 2 mg/kg treated SHR respectively). Response to ouabain in WKY was not significantly affected by SV treatment CONCLUSIONS: Chronic treatment of SHR with SV improves endothelium-dependent ACh relaxation of the aortic rings, probably by an NO-involving mechanism more than by inhibiting contractile COX-derived factors. An improvement in endothelial modulation of ouabain-induced contractions was also observed after treatment with SV in SHR, which might be due to an inhibition of a calcium-sodium exchanger.     

Dimethylarginine dimethylaminohydrolase inhibition and asymmetric dimethylarginine accumulation contribute to endothelial dysfunction in rats exposed to glycosylated protein: effects of aminoguanidine

OBJECTIVES: To determine whether alterations of endogenous asymmetric dimethylarginine (ADMA) concentration and dimethylarginine dimethylaminohydrolase (DDAH) activity are involved in endothelial dysfunction induced by glycosylated bovine serum albumin (GBSA) in rats and effects of aminoguanidine on them. METHODS: Endothelium-dependent relaxation of aortic rings from Sprague-Dawley rats after treatment with GBSA in vitro and in vivo was tested. Serum concentrations of ADMA, nitrite/nitrate, and activities of aortic DDAH, nitric oxide synthase (NOS) and superoxide dismutase were measured in GBSA-treated rats. Moreover, serum contents of glycosylated serum protein, and malondialdehyde were also assayed. RESULTS: Endothelium-dependent relaxation was significantly impaired either by incubation of aortic rings with GBSA (1.70mmol/l) in vitro for 60min or by injection of GBSA (35mg/kg/d, i.v.) to normal rats for 4 weeks, and serum ADMA levels were remarkably elevated in GBSA-treated rats, which was accompanied by decreases of nitrite/nitrate concentrations, NOS and DDAH activities. Furthermore, elevated glycosylated serum protein, malondialdehyde levels, and reduced superoxide dismutase activity were also observed in GBSA-treated rats. Treatment with aminoguanidine not only improved impairment of endothelium-dependent relaxation but also prevented elevation of endogenous ADMA, which were concomitant with increases of nitrite/nitrate concentration, NOS and DDAH activity. Serum levels of glycosylated serum protein, malondialdehyde, and vascular superoxide dismutase activity were also normalized after aminoguanidine treatment. CONCLUSIONS: Decreased DDAH activity and elevated endogenous ADMA is implicated in endothelial dysfunction of rats exposed to GBSA. Aminoguanidine can protect endothelium of rat aorta against injury induced by GBSA both in vitro and in vivo.     

Methylglyoxal production in vascular smooth muscle cells from different metabolic precursors

Methylglyoxal (MG), a metabolic by-product, reacts with certain proteins to yield irreversible advanced glycation end products (AGEs) and increases oxidative stress that causes the pathophysiological changes in diabetes, hypertension, and aging. Although MG production from glucose has been well documented, the contribution of other intermediates of different metabolic pathways to MG formation is far less known. Our aim was to determine and compare the formation of MG, MG-induced AGE, N(epsilon)-carboxyethyl-lysine (CEL), inducible nitric oxide synthase (iNOS), nitric oxide, and peroxynitrite from different metabolic precursors in cultured rat aortic vascular smooth muscle cells (VSMCs). High-performance liquid chromatography was used to determine MG levels, whereas nitrite + nitrate, indicators of nitric oxide production, and peroxynitrite levels were measured with specific assay kits. The CEL and iNOS were detected using immunocytochemistry. There was a concentration-dependent increase in MG levels in VSMCs after 3-hour incubation with 5, 15, and 25 mmol/L of D-glucose, fructose, or aminoacetone. Aminoacetone produced a 7-fold increase in MG levels above the basal value followed by fructose (3.9-fold), D-glucose (3.5-fold), acetol (2.8-fold), and sucrose (2.3-fold) after a 3-hour incubation with 25 mmol/L of each precursor. L-Glucose, 3-O-methylglucose, and mannitol had no effect on MG production. All precursors, except l-glucose, 3-O-methylglucose and mannitol, increased CEL. Aminoacetone, D-glucose, and fructose significantly increased iNOS, nitrite/nitrate, and peroxynitrite levels. In conclusion, aminoacetone is the most potent precursor of MG production in VSMCs, followed by fructose and d-glucose. This could have important implications in relation to high dietary fructose and protein intake.     

Attenuation of hypertension development by scavenging methylglyoxal in fructose-treated rats

OBJECTIVES: Methylglyoxal is a reactive dicarbonyl intermediate of metabolism produced in the body. It reacts with certain proteins and forms damaging advanced glycation endproducts (AGEs) such as N epsilon-carboxyethyl-lysine (CEL) and N epsilon-carboxymethyl-lysine (CML). Increased methylglyoxal levels are found in diabetes mellitus and associated with hypertension development in the spontaneously hypertensive rats (SHR). The purpose of this study was to investigate whether increased endogenous formation of methylglyoxal and methylglyoxal-induced AGEs caused hypertension development in normotensive Sprague Dawley rats. METHODS: The rats were fed chronically for 16 weeks with fructose, a known precursor of methylglyoxal formation. One group of rats was cotreated with fructose and metformin, an AGEs formation inhibitor. Methylglyoxal and reduced glutathione (GSH) were measured by high performance liquid chromatography, whereas hydrogen peroxide was measured by a dicholorofluorescin assay. Immunohistochemistry was performed for endothelial nitric oxide synthase (eNOS), CEL and CML. RESULTS: Fructose-fed rats had elevated blood pressure, serum methylglyoxal and triglycerides and reduced serum levels of GSH. Methylglyoxal, hydrogen peroxide and CEL were increased in the aorta, whereas eNOS was reduced. CEL and CML were also increased in the mesenteric artery endothelium along with media/lumen ratio, signifying structural remodelling. All the harmful changes in fructose-fed rats were attenuated in metformin and fructose cotreated rats. CONCLUSION: Increased methylglyoxal, AGEs, oxidative stress and reduced eNOS along with structural remodeling of the vessel wall in the aorta and mesenteric artery likely play a role in the pathogenesis of hypertension.     

Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation

OBJECTIVE: Enhanced oxidative stress is involved in mediating the endothelial dysfunction associated with hypertension. The aim of this study was to investigate the relative contributions of pro-oxidant and anti-oxidant enzymes to the pathogenesis of endothelial dysfunction in genetic hypertension. METHODS: Dilator responses to endothelium-dependent and endothelium-independent agents such as acetylcholine (ACh) and sodium nitroprusside were measured in the thoracic aortas of 28-week-old spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar Kyoto rats (WKY). The activity and expression (mRNA and protein levels) of endothelial nitric oxide synthase (eNOS), p22-phox, a membrane-bound component of NAD(P)H oxidase, and antioxidant enzymes, namely, superoxide dismutases (CuZn- and Mn-SOD), catalase and glutathione peroxidase (GPx), were also investigated in aortic rings. RESULTS: Relaxant responses to ACh were attenuated in phenylephrine-precontracted SHR aortic rings, despite a 2-fold increase in eNOS expression and activity. Although the activity and/or expression of SODs, NAD(P)H oxidase (p22-phox) and GPx were elevated in SHR aorta, catalase activity and expression remained unchanged compared to WKY. Pretreatment of SHR aortic rings with the inhibitor of xanthine oxidase, allopurinol, and the inhibitor of cyclooxygenase, indomethacin, significantly potentiated ACh-induced relaxation. Pretreatment of SHR rings with catalase and Tiron, a superoxide anion (O(2)(-)) scavenger, increased the relaxant responses to the levels observed in WKY rings whereas pyrogallol, a O(2)(-)-generator, abolished relaxant responses to ACh. CONCLUSION: These data demonstrate that dysregulation of several enzymes, resulting in oxidative stress, contributes to the pathogenesis of endothelial dysfunction in SHR and indicate that the antioxidant enzyme catalase is of particular importance in the reversal of this defect.     

Pomace olive oil improves endothelial function in spontaneously hypertensive rats by increasing endothelial nitric oxide synthase expression

BACKGROUND: The effect of dietary pomace olive oil, which has the same concentration of oleic acid but a higher proportion of oleanolic acid (OA) than olive oil, was examined on animal models of hypertension for the first time. METHODS: During 12 weeks, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were fed with either a control 2% corn oil diet (BD), or high-fat diets containing 15% of refined olive oil (OL), pomace olive oil (POM), or pomace olive oil supplemented in OA (up to 800 ppm) (POMO). Then, vascular reactivity and endothelial nitric oxide (NO) synthase (eNOS) expression were studied in aortic rings. Plasma nitrite + nitrate levels were also determined. RESULTS: Diets had no effects on blood pressure (BP). In contrast to the BD and OL dietary groups, POM intake improved relaxation evoked by acetylcholine in SHR aorta. The POMO intake increased vasodilatation to acetylcholine and attenuated phenylephrine-induced contractions in both strains of rats associated with a major NO participation revealed by inhibition of NOS. The enhanced relaxation shown in POM and POMO SHR aorta was attributed to an increased eNOS protein expression. Plasma nitrite levels were also increased in these groups. Although olive and pomace oils used in diets contained similar fatty acid composition, beneficial effects on endothelial function were absent in the OL group. Therefore, these effects must be associated with some minor components from pomace olive oil such as OA. CONCLUSIONS: Chronic intake of diets rich in pomace olive oil improves endothelial dysfunction in SHR aorta by mechanisms associated with enhanced eNOS expression. Important evidence is provided regarding the effects of pomace olive oil and OA on endothelial function in hypertensive animals.     

Wine polyphenols improve endothelial function in large vessels of female spontaneously hypertensive rats

Red wine polyphenols (RWPs) have been reported to prevent hypertension and endothelial dysfunction. Several individual RWPs exert estrogenic effects. We analyzed the possible in vivo protective effects on blood pressure and endothelial function of RWPs in female spontaneously hypertensive rats (SHR) and its relationship with ovarian function. RWPs (40 mg/kg by gavage) were orally administered for 5 weeks. Ovariectomized rats showed both increased isoprostaglandin F(2alpha) excretion and aortic superoxide production and reduced relaxant response to acetylcholine and contraction to the endothelial nitric oxide synthase (eNOS) inhibitor l-NAME measured in the aorta but similar blood pressure, as compared with sham-operated rats. Moreover, in ovariectomized rats aortic eNOS expression was unchanged, whereas caveolin-1, angiotensin II receptor (AT)-1, and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22(phox) and p47(phox) expression was increased compared with sham-operated rats. In both ovariectomized and sham-operated SHR, RWPs reduced systolic blood pressure, urinary isoprostaglandin F(2alpha) excretion, and aortic O(2)(-) production, improving the endothelium-dependent relaxant response to acetylcholine in SHR. These changes were associated with unchanged aortic eNOS expression, whereas caveolin-1 was increased and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22(phox) and p47(phox) expression was reduced. RWPs had no effect on the AT-1 overexpression found in ovariectomized animals. All these results suggest that a chronic treatment with RWPs reduces hypertension and vascular dysfunction through reduction in vascular oxidative stress in female SHR in a manner independent of the ovarian function.     

Inhibitor for advanced glycation end products formation attenuates hypertension and oxidative damage in genetic hypertensive rats

OBJECTIVE : A recent study demonstrated that free radicals were involved in the maintenance of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). Advanced glycation end-products (AGEs) accumulate progressively in the vasculature with ageing, and have been identified to be relevant mediators for various vascular complications. To elucidate the role of AGEs in genetic hypertension, we investigated the effect of OPB-9195, a novel inhibitor of AGEs, on hypertension and oxidative damage in SHRSP. METHODS : Five-week-old male SHRSP were divided into a control group, fed a control diet and two, OPB-9195, (+/-)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide, treatment groups, fed a diet supplemented with OPB-9195 at the concentration of 0.5 (OPB-L) or 2 mg/g (OPB-H) mixed chow for 10 weeks. RESULTS : The plasma of OPB-9195-treated SHRSP had lower levels of glycated albumin as compared with that of control SHRSP. OPB-9195 lowered the systolic blood pressure (SBP) by the fourth week of administration, and this effect was maintained throughout the study. We also confirmed SBP and diastolic blood pressure (DBP) rhythms, monitored by telemetry, were significantly lower in the OPB-H group than in the control group. Urinary nitric oxide (NO) excretion as well as the expression of endothelial NO synthase (eNOS) mRNA, and eNOS activity in the aorta were significantly increased in OPB-9195-treated groups compared with the control group. The levels of 8-hydroxydeoxyguanosine (8-OHdG), produced from deoxyguanosine under conditions of oxidative stress, in the urine of OPB-9195-treated SHRSP was significantly lower than in the control SHRSP. We also confirmed that the expression of glutathione peroxidase in the aorta was significantly increased in OPB-9195 treated SHRSP. CONCLUSIONS : Because long-term administration of a AGEs inhibitor reduces blood pressure and oxidative damage in SHRSP, this study suggests a role for AGEs in the progression or maintenance of hypertension and related diseases in genetic hypertension.     

Chronic ethanol ingestion increases aortic endothelial nitric oxide synthase expression and nitric oxide production in the rat

Background:  Chronic alcohol consumption perturbs cellular function in a variety of organ systems. Previous studies have suggested that moderate alcohol consumption reduces vascular disease, whereas heavier alcohol consumption may worsen it. The mechanisms for these vascular effects of chronic alcohol ingestion continue to be defined and constitute the focus of this study.
Methods:  Male Sprague Dawley rats were fed an isocaloric, Lieber-Decarli liquid diet containing either ethanol (36% calories) or Maltose–Dextrin (substituted for ethanol) for 6 weeks. Telemetric blood pressure measurements were taken before and after ethanol feeding. After the rats were killed, the aortas were analyzed for endothelial nitric oxide (NO) synthase expression and NO production.
Results:  Chronic ethanol ingestion decreased mean arterial pressure and increased aortic NO production as demonstrated by direct ex vivo measurements using iron diethyldithio-carbamic acid as well as analysis of nitrosyl-hemoglobin (NO-Hb) levels. Consistent with these assays of vascular NO production, endothelium-dependent relaxation responses to acetycholine (Ach) were enhanced in ethanol-fed animals. Aortic endothelial nitric oxide synthase expression was also increased by chronic ethanol ingestion.
Conclusions:  These findings demonstrate that a regimen of chronic alcohol ingestion in the rat produced generally salutary effects in the systemic vasculature following a 6-week treatment regimen. These findings extend previous in vitro studies to demonstrate that alcohol has potent effects on vascular endothelial nitric oxide synthase expression, NO production, and vascular function. Consistent with previous reports, these findings confirm that alcohol-induced alterations in the production of reactive nitrogen species play an important role in the pathogenesis of alcohol-mediated tissue effects.     

Quercetin downregulates NADPH oxidase, increases eNOS activity and prevents endothelial dysfunction in spontaneously hypertensive rats

BACKGROUND AND OBJECTIVE: Several studies have found that chronic treatment with the dietary flavonoid quercetin lowers blood pressure and restores endothelial dysfunction in hypertensive animal models. We hypothesized that increased endothelial nitric oxide synthase (eNOS) and/or decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase protein expression and activity, and reduced reactive oxygen species might be involved in the improvement of endothelial function induced by quercetin in spontaneously hypertensive rats (SHR). DESIGN AND METHODS: Male SHR and Wistar-Kyoto (WKY) rats (5 weeks old) were treated with quercetin (10 mg/kg) or vehicle for 13 weeks. Changes in vascular expression of eNOS, caveolin-1 and p47 were analysed by Western blot, eNOS activity by conversion of [H]arginine to L-[H]citrulline, and NADPH oxidase activity by NADPH-enhanced chemoluminescence of lucigenin. RESULTS: In SHR, quercetin reduced the increase in blood pressure and heart rate and enhanced the endothelium-dependent aortic vasodilation induced by acetylcholine, but had no effect on the endothelium-independent response induced by nitroprusside. However, quercetin had no effect on endothelium-dependent vasoconstriction and aortic thromboxane B2 production. Compared to WKY, SHR showed upregulated eNOS and p47 protein expression, downregulated caveolin-1 expression, increased NADPH-induced superoxide production but, paradoxically, eNOS activity was reduced. Chronic quercetin treatment prevented all these changes in SHR. In WKY, quercetin had no effect on blood pressure, endothelial function or the expression or activity of the proteins analysed. CONCLUSIONS: Enhanced eNOS activity and decreased NADPH oxidase-mediated superoxide anion (O2) generation associated with reduced p47 expression appear to be essential mechanisms for the improvement of endothelial function and the antihypertensive effects of chronic quercetin.     

Arginase inhibition reduces endothelial dysfunction and blood pressure rising in spontaneously hypertensive rats

OBJECTIVE: A decrease in nitric oxide (NO) bioavailability has been proposed to contribute to endothelial dysfunction and increased peripheral resistances during essential arterial hypertension. Given that arginine is a substrate for both arginase and NO synthase, arginase activity may be a critical factor in NO bioavailability. To test this hypothesis, we evaluated the effects of the arginase inhibitor alpha-difluoromethylornithine (DFMO) in spontaneously hypertensive rats (SHR). METHODS: Vascular reactivity experiments were performed on thoracic aortic rings from 10-week-old SHR and their normotensive counterparts, Wistar-Kyoto (WKY) rats. Blood pressure was measured by the tail-cuff method. DFMO treatment (30 mg/kg daily in drinking water) was started in 5-week-old SHR and maintained for 5 weeks. Aortic arginase I and arginase II expression as well as arginase activity were evaluated by western blotting and the spectrophotometric method, respectively. RESULTS: DFMO (1.2 x 10 mol/l) enhanced the vascular response to acetylcholine both in SHR (+24%, P < 0.01) and WKY rats (+12%, P < 0.01), and reversed the effects of the NO synthase inhibitor N-nitro-L-arginine-methyl-ester. The vasorelaxant response to sodium nitroprusside on endothelium-denuded rings was not affected by DFMO, neither in SHR nor in WKY rats. In SHR, DFMO prevented the increase in blood pressure and improved the response of aortic rings to acetylcholine. Finally, as compared with WKY rats, SHR exhibited increased expression of vascular arginase I (+72%, P < 0.05) and arginase II (+91%, P < 0.05) as well as increased arginase activity (+26%, P < 0.05). CONCLUSIONS: Our results showed that arginase inhibition reduced endothelial dysfunction and blood pressure rising in SHR.     

Uncoupled endothelial nitric oxide synthase and oxidative stress in a rat model of pregnancy-induced hypertension

BACKGROUND: Preeclampsia is a human pregnancy-associated syndrome associated with hypertension, proteinuria, and endothelial dysfunction. We tested whether increased reactive oxygen species (superoxide and peroxynitrite) production and decreased bioavailability of the endothelial nitric oxide (NO) synthase (eNOS) cofactor tetrahydrobiopterin (BH4) contributes to maternal endothelial dysfunction in rats with pregnancy-induced hypertension and several characteristics of preeclampsia. METHODS: Nonpregnant (DS) and pregnant (PDS) rats were treated with deoxycorticosterone acetate and 0.9% saline for approximately 3 weeks and nonpregnant (Con) and pregnant (P) rats received tap water. Blood pressure, urinary protein levels, mesenteric vascular reactivity, aortic protein expression, and aortic reactive oxygen species levels were compared between the four groups. RESULTS: The PDS rats had significantly decreased mesenteric endothelium-dependent relaxation responses and aortic NO production compared to Con, DS, and P rats despite increased aortic eNOS expression. Aortic superoxide and peroxynitrite levels were increased in PDS rats compared with Con, DS, and P rats. Scavenging of reactive oxygen species or increasing tetrahydrobiopterin levels normalized mesenteric endothelium-dependent relaxation responses, aortic NO production, and aortic superoxide and peroxynitrite levels in PDS rats. CONCLUSIONS: These data suggest that increased superoxide production by NADPH oxidase, peroxynitrite degradation of BH4, and uncoupled eNOS contribute to endothelial dysfunction in a rat model of pregnancy-induced hypertension.     

Simvastatin improves endothelial function in spontaneously hypertensive rats through a superoxide dismutase mediated antioxidant effect

BACKGROUND : Hydroxymethylglutaryl coenzyme A (HMGCoA) reductase inhibitors have beneficial effects beyond their cholesterol-lowering properties. The antioxidant mechanism of HMGCoA reductase inhibitors is not completely understood. OBJECTIVES : To elucidate the antioxidant effect of simvastatin. METHODS : We studied the influence of simvastatin treatment on the development of hypertension, modification of antioxidant systems, and reactivity of aortic rings in Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. RESULTS : Simvastatin had no effect on blood pressure (BP). Simvastatin treatment (either 1 or 2 mg/kg body weight for 12 or 20 weeks) increased superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in SHR rats compared with untreated control SHR rats. Carbachol-induced relaxation of aortic rings was impaired in control SHR rats and was restored by simvastatin treatment. Addition of SOD improved the response in control SHR rats and did not have any effect in treated SHR rats. Addition of diethyldithiocarbamic acid, a selective inhibitor of SOD, produced a mild non-significant impairment in carbachol-induced relaxation in control SHR rats, suggesting a deficient antioxidant system in these animals. However, in treated SHR and in WKY rats, impairment of the relaxation was marked, implying that SOD activity in these animals was important to maintain endothelial function. In aortic rings without endothelium from SHR rats, contraction induced by free radicals was substantially higher than in WKY rats. This effect was attenuated in 1-mg-treated rats and abolished in 2-mg-treated rats. CONCLUSIONS : Simvastatin promotes intracellular antioxidant systems, fundamentally SOD, restoring endothelial function but not having any effect on blood pressure.     

Catechin Averts Experimental Diabetes Mellitus-Induced Vascular Endothelial Structural and Functional Abnormalities

Diabetes mellitus is associated with an induction of vascular endothelial dysfunction (VED), an initial event that could lead to the pathogenesis of atherosclerosis and hypertension. Previous studies showed that catechin, a key component of green tea, possesses vascular beneficial effects. We investigated the effect of catechin hydrate in diabetes mellitus-induced experimental vascular endothelial abnormalities (VEA). Streptozotocin (50 mg/kg, i.p., once) administration to rats produced diabetes mellitus, which subsequently induced VEA in 8 weeks by markedly attenuating acetylcholine-induced endothelium-dependent relaxation in the isolated aortic ring preparation, decreasing aortic and serum nitrite/nitrate concentrations and impairing aortic endothelial integrity. These abnormalities in diabetic rats were accompanied with elevated aortic superoxide anion generation and serum lipid peroxidation in addition to hyperglycemia. Catechin hydrate treatment (50 mg/kg/day p.o., 3 weeks) markedly prevented diabetes mellitus-induced VEA and vascular oxidative stress. Intriguingly, in vitro incubation of L-NAME (100 μM), an inhibitor of nitric oxide synthase, or Wortmannin (100 nM), a selective inhibitor of phosphatidylinositol 3-kinase (PI3K), markedly prevented catechin hydrate-induced improvement in acetylcholine-provoked endothelium-dependent relaxation in the diabetic rat aorta. Moreover, catechin hydrate treatment considerably reduced the elevated level of serum glucose in diabetic rats. In conclusion, catechin hydrate treatment prevents diabetes mellitus-induced VED through the activation of endothelial PI3K signal and subsequent activation of eNOS and generation of nitric oxide. In addition, reduction in high glucose, vascular oxidative stress, and lipid peroxidation might additionally contribute to catechin hydrate-associated prevention of diabetic VEA.     

Inhibiting long chain fatty Acyl CoA synthetase increases basal and agonist-stimulated NO synthesis in endothelium

OBJECTIVES: Endothelial nitric oxide synthase (eNOS) activation/deactivation is associated with cyclic depalmitoylation/repalmitoylation of specific Cys residues. The mechanism of depalmitoylation has been identified recently, but repalmitoylation remains undefined. We hypothesized that long chain fatty acyl CoA synthetase (LCFACoAS) modulates endothelial nitric oxide synthase repalmitoylation by limiting palmitoyl CoA availability. METHODS: Human coronary endothelial cells were treated with triacsin-C, an inhibitor of long chain fatty acyl CoA synthetase, for 24 h. Media nitrite accumulation, eNOS activity, and eNOS palmitoylation were measured. Methacholine-induced NO synthesis or vascular relaxation were measured in endothelium-intact rat aortae in the presence and absence of triacsin-C. RESULTS: Triacsin-C significantly reduced incorporation of [3H] palmitate into immunoreactive endothelial nitric oxide synthase and over a concentration range of 0.1 to 10 microM, increased media nitrite accumulations 2- to 2.5-fold over baseline. Total in vitro catalytic activity of nitric oxide synthase in triacsin-C treated cells did not differ significantly from control. Triacsin-C significantly increased methacholine-induced NO synthesis in the isolated rat aorta, and significantly enhanced methacholine-induced relaxation of rat aortic rings. CONCLUSIONS: These data are consistent with the interpretation that inhibition of palmitoylation increases endothelial nitric oxide synthase activity without changing endothelial nitric oxide synthase expression, suggesting that inhibiting palmitoylation increases the catalytically active fraction of endothelial nitric oxide synthase.     

Ferulic acid restores endothelium-dependent vasodilation in aortas of spontaneously hypertensive rats

BACKGROUND: Ferulic acid (FA), a phytochemical constituent, has antihypertensive effects, but a detailed understanding of its effects on vascular function remains unclear. The vasoreactivity of FA was assessed using aortic rings isolated from normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). METHODS: The effects of FA (10(-5) to 10(-3) mol/L) on vasodilatory responses were evaluated based on contractile responses induced by phenylephrine (10(-6) mol/L) in thoracic aortic rings from male WKY rats and SHR. Basal nitric oxide (NO) bioavailability in the aorta was determined from the contractile response induced by the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) mol/L). The effects of FA on the production of NADPH-dependent superoxide anion were examined in SHR aortas. The impact of hydroxyhydroquinone, a generator of superoxide anions, on the FA-induced enhancement in acetylcholine-stimulated vasodilation was also investigated. RESULTS: The FA (10(-3) mol/L)-induced relaxation was partially blocked by removal of the endothelium or by pretreating SHR aortas with L-NAME. FA increased NO bioavailability, and decreased NADPH-dependent superoxide anion levels in SHR aortas. Ferulic acid improved acetylcholine-induced endothelium-dependent vasodilation in SHR, but not in WKY. Furthermore, the simultaneous addition of hydroxyhydroquinone significantly inhibited the increase in acetylcholine-induced vasodilation by FA. CONCLUSIONS: Ferulic acid restores endothelial function through enhancing the bioavailability of basal and stimulated NO in SHR aortas. The results explain, in part, the mechanisms underlying the effects of FA on blood pressure (BP) in SHR.     

Endothelial dysfunction: Possible mechanisms and ways of correction

BACKGROUND: Endothelial function is impaired in atherosclerosis, hypertension, diabetes and aging, and this may be associated with an attenuated ability of endothelial cells to generate nitric oxide (NO). OBJECTIVES: To evaluate possible alterations in endothelium-dependent relaxation in rat aortic rings, the activity of constitutive NO synthase and endothelial electrical responses to acetylcholine (Ach) in rat aorta, and the effect of one month of treatment with the angiotensin-converting enzyme inhibitor enalapril on endothelial function. METHODS: Endothelial membrane potential was measured in excised rat aorta using the perforated patch-clamp technique. Enzyme activity was determined by measuring the rate of formation of L-citrulline from L-arginine. RESULTS: In old rats and rats with experimental diabetes, the relaxation response to Ach and the activity of constitutive NO synthase were significantly depressed compared with control rats, and the endothelial resting membrane potential was significantly depolarized (-32.7+/-0.8 mV and -28.4+/-3.1 mV, respectively) compared with the control rats (-42.9+/-0.6 mV). The membrane potential attained during peak hyper-polarization to Ach in the arteries of diabetic and old rats (-57.6+/-1.1 mV and -55.7+/-2.1 mV, respectively) did not reach the level attained in the arteries of control rats (-63.1+/-0.6 mV). Enalapril treatment restored the relaxation response to Ach and increased the activity of constitutive NO synthase in aortic rings from diabetic and old rats. CONCLUSIONS: Altered electrical properties of endothelial cells and attenuated NO synthase activity underpin the suppressed relaxation to Ach in aging and experimental diabetes. Enalapril treatment improves endothelium-dependent relaxation and the activity of constitutive NO synthase.