Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration

Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.     

A fish oil-rich diet reduces vascular oxidative stress in apoE–/– mice

Abstract

Oxidative stress contributes to lipid peroxidation and decreases nitric oxide (NO) bioavailability in atherosclerosis. While long-chain (n-3) polyunsaturated fatty acids (PUFA) are easily oxidized in vitro, they improve endothelial function. Hence, this study postulates that long-chain (n-3) PUFA decrease atherogenic oxidative stress in vivo. To test this, apoE^–/– mice were fed a corn oil- or a fish oil (FO)-rich diet for 8, 14 or 20 weeks and parameters related to NO and superoxide (O₂^.–) plus markers of lipid peroxidation and protein oxidative damage in the aortic root were evaluated. The FO-rich diet increased NO production and endothelial NO synthase (NOS) expression and lowered inducible NOS, p22^phox expression and O₂^.–production after 14 and 20 weeks of diet. Protein lipoxidative damage (including 4-hydroxynonenal) was decreased after a long-term FO-diet. This supports the hypothesis that a FO-rich diet could counteract atherogenic oxidative stress, showing beneficial effects of long-chain (n-3) PUFA.     

Regional Heterogeneity of Cerebral Microvessels and Brain Susceptibility to Oxidative Stress

The hippocampus is one of the earliest and most affected regions in Alzheimer’s disease (AD), followed by the cortex while the cerebellum is largely spared. Importantly, endothelial dysfunction is a common feature of cerebral blood vessels in AD. In this study, we sought to determine if regional heterogeneity of cerebral microvessels might help explain the susceptibility of the hippocampus and cortex as compared to the cerebellum. We isolated microvessels from wild type mice from the cerebellum, cortex, and hippocampus to characterize their vascular phenotype. Superoxide anion was significantly higher in microvessels isolated from the cortex and hippocampus as compared to the cerebellum. Importantly, protein levels of NADPH oxidase (NOX)-2 and NOX-4 were significantly higher in the cortical and hippocampal microvessels as compared to microvessels from the cerebellum. In addition, expression of manganese superoxide dismutase protein was significantly lower in microvessels from the cortex and hippocampus as compared to cerebellum while other antioxidant enzymes were unchanged. There was no difference in eNOS protein expression between the microvessels of the three brain regions; however, bioavailability of tetrahydrobiopterin (BH₄), an essential cofactor for eNOS activity, was significantly reduced in microvessels from the hippocampus and cortex as compared to the cerebellum. Higher levels of superoxide and reduced tetrahydrobiopterin bioavailability may help explain the vulnerability of the hippocampus and cortical microvessels to oxidative stress and development of endothelial dysfunction.     

Oxidative stress and increased eNOS and NADPH oxidase expression in mouse microvessel endothelial cells

Elevated oxidative stress plays a key role in diabetes-associated vascular disease. In this study, we tested the hypothesis that high glucose-induced oxidative stress was associated with changes in the expression of NADPH oxidase, superoxide dismutase (SOD) and endothelial nitric oxide synthase (eNOS). Oxidative stress was assessed in cell cultures of mouse microvessel endothelial cells (MMECs) by fluorescence labelling with dihydroethidium, lucigenin-enhanced chemiluminescence and determining NADPH oxidase subunit and eNOS expression with real-time polymerase chain reaction protocol and Western blotting. Oxidative stress and expression of the NADPH oxidase subunit, p22phox, were both increased, SOD1 and 3 expression lowered and eNOS significantly elevated in MMECs treated with 40 mM glucose for 72 h compared to low glucose medium. Oxidative stress, p22phox mRNA, eNOS mRNA, and protein were lowered by concurrent incubation with sepiapterin. When eNOS protein expression in endothelial cells was significantly decreased by eNOS siRNA treatment, superoxide generation was significantly higher in the MMECs grown in low glucose, but reduced in those grown in high glucose for 72 h. Thus, exposure of MMECs to high glucose results in increased oxidative stress that is associated with increased eNOS and NADPH oxidase subunit expression, notably p22phox, and decreased expression of SOD1 and 3.     

Differential effects of eNOS uncoupling on conduit and small arteries in GTP-cyclohydrolase I-deficient hph-1 mice

In the present study, we used the hph-1 mouse, which displays GTP-cyclohydrolase I (GTPCH I) deficiency, to test the hypothesis that loss of tetrahydrobiopterin (BH(4)) in conduit and small arteries activates compensatory mechanisms designed to protect vascular wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and BH(4) levels were reduced in the aortas and small mesenteric arteries of hph-1 mice. However, the BH(4)-to-7,8-dihydrobiopterin ratio was significantly reduced only in hph-1 aortas. Furthermore, superoxide anion and 3-nitrotyrosine production were significantly enhanced in aortas but not in small mesenteric arteries of hph-1 mice. In contrast to the aorta, protein expression of copper- and zinc-containing superoxide dismutase (CuZnSOD) was significantly increased in small mesenteric arteries of hph-1 mice. Protein expression of catalase was increased in both aortas and small mesenteric arteries of hph-1 mice. Further analysis of endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) signaling demonstrated that protein expression of phosphorylated Ser(1177)-eNOS as well as basal cGMP levels and hydrogen peroxide was increased in hph-1 aortas. Increased production of hydrogen peroxide in hph-1 mice aortas appears to be the most likely mechanism responsible for phosphorylation of eNOS and elevation of cGMP. In contrast, upregulation of CuZnSOD and catalase in resistance arteries is sufficient to protect vascular tissue from increased production of reactive oxygen species generated by uncoupling of eNOS. The results of our study suggest that anatomical origin determines the ability of vessel wall to cope with oxidative stress induced by uncoupling of eNOS.     

Effects of selenium on endothelial dysfunction and metabolic profile in low dose streptozotocin induced diabetic rats fed a high fat diet

Endothelial dysfunction develops as a result of oxidative stress and is responsible for diabetic vascular complications. We investigated the effects of selenium on endothelial dysfunction and oxidative stress in type 2 diabetic rats. Male Wistar rats were divided into five groups: controls, untreated diabetics, and diabetics treated with 180, 300, 500 mcg/kg selenium each day. Diabetes was induced by a single intraperitoneal injection of low dose streptozotocin to rats fed a high fat diet. Endothelium-dependent and -independent relaxations were measured in the thoracic aorta. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and endothelial nitric oxide synthase (eNOS) mRNA expressions were analyzed using real-time polymerase chain reaction (RT-PCR). Fasting blood glucose, lipid profile, lipid oxidation, insulin and nitric oxide were measured in blood samples. Malondialdehyde, superoxide dismutase, catalase and glutathione peroxidase levels were measured in liver samples. RT-PCR showed that selenium reversed increased NADPH oxidase expression and decreased eNOS expression to control levels. Selenium also improved the impairment of endothelium-dependent vasorelaxation in the diabetic aorta. Selenium treatment significantly decreased blood glucose, cholesterol and triglyceride levels, and enhanced the antioxidant status in diabetic rats. Our findings suggest that selenium restores a normal metabolic profile and ameliorates vascular responses and endothelial dysfunction in diabetes by regulating antioxidant enzyme and nitric oxide release.     

Homocysteine induces endothelial dysfunction via inhibition of arginine transport.

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases. High levels of plasma homocysteine (HCY) increase oxidative stress and reduce endothelial-dependent relaxation. We determined whether hyperhomocysteinemia-induced endothelial dysfunction is mediated through inhibition of cellular transport of L-arginine. In endothelial cells, HCY had a biphasic effect on arginine transport. HCY treatment for 6 hr increased L-arginine uptake by 34%; however, uptake was decreased by 25% after 24 h. HCY caused membrane hyperpolarization during both 6 and 24 h incubation periods, indicating that the negative charge facilitating arginine uptake was maintained. HCY significantly reduced expression of cellular arginine transporter protein (CAT-1) after 24 h treatment; whereas endothelial nitric oxide synthase (eNOS) protein levels and basal eNOS activity were not altered. Nevertheless, nitric oxide (NO) formation was significantly decreased. The antioxidant ascorbic acid prevented the effect of HCY on arginine transport. HCY induced formation of the peroxynitrite biomarker nitrotyrosine, which was blocked by supplemental L-arginine. HCY treatment of aortic rings caused decreased vasorelaxation to acetylcholine, which was prevented by supplemental arginine. In conclusion, HCY decreased NO formation and induced endothelial dysfunction without altering protein level or basal activity of eNOS, but through decreases in function and protein expression of the CAT-1 transporter. Reduced arginine supply may lead to eNOS uncoupling and generation of superoxide, contributing to HCY-induced oxidative stress.     

N-3 PUFA improved post-menopausal depression induced by maternal separation and chronic mild stress through serotonergic pathway in rats—effect associated with lipid mediators

Early life maternal separation (MS) increases the vulnerability to depression in rats with chronic mild stress (CMS). N-3 polyunsaturated fatty acids (PUFA) improved depressive behaviors in rats with acute stress; however, their effects on rats with MS+CMS were not apparent. The purpose of the present study was to investigate the hypothesis that lifetime n-3 PUFA supplementation improves post-menopausal depression through the serotonergic and glutamatergic pathways while modulating n-3 PUFA-derived metabolites. Female rats were fed diets of either 0% n-3 PUFA during lifetime or 1% energy n-3 PUFA during pre-weaning, post-weaning, or lifetime periods. Rats were allocated to non-MS or MS groups and underwent CMS after ovariectomy. N-3 PUFA increased brain n-3 PUFA-derived endocannabinoid/oxylipin levels, and reversed depressive behaviors. N-3 PUFA decreased blood levels of adrenocorticotropic hormone and corticosterone, and brain expressions of corticotropin-releasing factor and miRNA-218, which increased the expression of the glucocorticoid receptor. N-3 PUFA decreased the expression of tumor necrosis factor-α, interleukin (IL)-6, IL-1β, and prostaglandin E₂, while increased the expression of miRNA-155. N-3 PUFA also increased brainstem serotonin levels and hippocampal expression of the serotonin-1A receptor, cAMP response element-binding protein (CREB), phospho-CREB, and brain-derived neurotrophic factor. However, n-3 PUFA did not affect brain expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subtype 1, N-methyl-D-aspartate receptor subtype 2B, or miRNA-132. Moreover, n-3 PUFA exposure during lifetime caused greater effects than pre- and post-weaning periods. The present study suggested that n-3 PUFA improved depressive behaviors through serotonergic pathway while modulating the metabolites of n-3 PUFA in post-menopausal depressed rats with chronic stress.     

Inhibition of renin-angiotensin system reverses endothelial dysfunction and oxidative stress in estrogen deficient rats

Background

Estrogen deficiency increases the cardiovascular risks in postmenopausal women. Inhibition of the renin-angiotensin system (RAS) and associated oxidative stress confers a cardiovascular protection, but the role of RAS in estrogen deficiency-related vascular dysfunction is unclear. The present study investigates whether the up-regulation of RAS and associated oxidative stress contributes to the development of endothelial dysfunction during estrogen deficiency in ovariectomized (OVX) rats.

Methodology/Principal Findings

Adult female rats were ovariectomized with and without chronic treatment with valsartan and enalapril. Isometric force measurement was performed in isolated aortae. The expression of RAS components was determined by immunohistochemistry and Western blotting method while ROS accumulation in the vascular wall was evaluated by dihydroethidium fluorescence. Ovariectomy increased the expression of angiotensin-converting enzyme (ACE), angiotensin II type 1 receptor (AT₁R), NAD(P)H oxidase, and nitrotyrosine in the rat aorta. An over-production of angiotensin II and ROS was accompanied by decreased phosphorylation of eNOS at Ser¹¹⁷⁷ in OVX rat aortae. These pathophysiological changes were closely coupled with increased oxidative stress and decreased nitric oxide bioavailability, culminating in markedly impaired endothelium-dependent relaxations. Furthermore, endothelial dysfunction and increased oxidative stress in aortae of OVX rats were inhibited or reversed by chronic RAS inhibition with enalapril or valsartan.

Conclusions/Significance

The novel findings highlight a significant therapeutic benefit of RAS blockade in the treatment of endothelial dysfunction-related vascular complications in postmenopausal states.     

Menadione induces endothelial dysfunction mediated by oxidative stress and arylation

Our previous studies showed that menadione causes endothelial dysfunction which results in decreased relaxation and increased contraction of blood vessels. This investigation examined the role of two possible mechanisms (oxidative stress and arylation) in menadione-induced endothelial dysfunction. Menadione increased superoxide anion generation in aortic rings in a dose-dependent manner. Superoxide dismutase (SOD), reversed the inhibitory effects of menadione on vascular relaxation. The relaxation induced by the NO donor, sodium nitroprusside, was inhibited by menadione pretreatment in a dose-dependent manner. Endothelial nitric oxide synthase activity (eNOS) was suppressed by menadione. Menadione resulted in a dose-dependent reduction of cGMP levels accumulated by acetylcholine. This reduction of cGMP levels was blocked by SOD treatment, suggesting that superoxide anion generated by menadione could play a role in the inhibition of the nitric oxide pathway. Evidence supporting a possible role for arylation in impaired vascular relaxation was suggested by the observation that benzoquinone, which does not induce oxidative stress in aortic rings, inhibited acetylcholine-induced vascular relaxation to the same extent as menadione. Collectively, these results suggest that menadione can cause endothelial dysfunction in blood vessels by the inhibition of the nitric oxide pathway via superoxide anion generation and that arylation activity may also be another important mechanism.     

Osteopontin-stimulated apoptosis in cardiac myocytes involves oxidative stress and mitochondrial death pathway: role of a pro-apoptotic protein BIK

Increased osteopontin (OPN) expression in the heart, specifically in myocytes, associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we provided evidence that OPN interacts with CD44 receptor, and induces myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. Here we tested the hypothesis that OPN induces oxidative stress in myocytes and the heart via the involvement of mitochondria and NADPH oxidase-4 (NOX-4). Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) increased oxidative stress as analyzed by protein carbonylation, and intracellular reactive oxygen species (ROS) levels as analyzed by ROS detection kit and dichlorohydrofluorescein diacetate staining. Pretreatment with NAC (antioxidant), apocynin (NOX inhibitor), MnTBAP (superoxide dismutase mimetic), and mitochondrial K_ATP channel blockers (glibenclamide and 5-hydroxydecanoate) decreased OPN-stimulated ROS production, cytosolic cytochrome c levels, and apoptosis. OPN increased NOX-4 expression, while decreasing SOD-2 expression. OPN decreased mitochondrial membrane potential as measured by JC-1 staining, and induced mitochondrial abnormalities including swelling and reorganization of cristae as observed using transmission electron microscopy. OPN increased expression of BIK, a pro-apoptotic protein involved in reorganization of mitochondrial cristae. Expression of dominant-negative BIK decreased OPN-stimulated apoptosis. In vivo, OPN expression in cardiac myocyte-specific manner associated with increased protein carbonylation, and expression of NOX-4 and BIK. Thus, OPN induces oxidative stress via the involvement of mitochondria and NOX-4. It may affect mitochondrial morphology and integrity, at least in part, via the involvement of BIK.

     

Protective Effects of PARP-1 Knockout on Dyslipidemia-Induced Autonomic and Vascular Dysfunction in ApoE?/? Mice: Effects on eNOS and Oxidative Stress

The aims of this study were to investigate the role of poly(ADP-ribose) polymerase (PARP)-1 in dyslipidemia-associated vascular dysfunction as well as autonomic nervous system dysregulation. Apolipoprotein (ApoE)^−/− mice fed a high-fat diet were used as a model of atherosclerosis. Vascular and autonomic functions were measured in conscious mice using telemetry. The study revealed that PARP-1 plays an important role in dyslipidemia-associated vascular and autonomic dysfunction. Inhibition of this enzyme by gene knockout partially restored baroreflex sensitivity in ApoE^−/− mice without affecting baseline heart-rate and arterial pressure, and also improved heart-rate responses following selective blockade of the autonomic nervous system. The protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction was associated with preservation of eNOS activity. Dyslipidemia induced PARP-1 activation was accompanied by oxidative tissue damage, as evidenced by increased expression of iNOS and subsequent protein nitration. PARP-1 gene deletion reversed these effects, suggesting that PARP-1 may contribute to vascular and autonomic pathologies by promoting oxidative tissue injury. Further, inhibition of this oxidative damage may account for protective effects of PARP-1 gene deletion on vascular and autonomic functions. This study demonstrates that PARP-1 participates in dyslipidemia-mediated dysregulation of the autonomic nervous system and that PARP-1 gene deletion normalizes autonomic and vascular dysfunctions. Maintenance of eNOS activity may be associated with the protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction.     

Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction

Background

We recently reported that ER stress plays a key role in vascular endothelial dysfunction during hypertension. In this study we aimed to elucidate the mechanisms by which ER stress induction and oxidative stress impair vascular endothelial function.

Methodology/principal findings

We conducted in vitro studies with primary endothelial cells from coronary arteries stimulated with tunicamycin, 1 μg/mL, in the presence or absence of two ER stress inhibitors: tauroursodeoxycholic acid (Tudca), 500 μg/mL, and 4-phenylbutyric acid (PBA), 5 mM. ER stress induction was assessed by enhanced phosphorylation of PERK and eIF2α, and increased expression of CHOP, ATF6 and Grp78/Bip. The ER stress induction increased p38 MAPK phosphorylation, Nox2/4 mRNA levels and NADPH oxidase activity, and decreased eNOS promoter activity, eNOS expression and phosphorylation, and nitrite levels. Interestingly, the inhibition of p38 MAPK pathway reduced CHOP and Bip expressions enhanced by tunicamycin and restored eNOS promoter activation as well as phosphorylation. To study the effects of ER stress induction in vivo, we used C57BL/6J mice and p47phox^−/− mice injected with tunicamycin or saline. The ER stress induction in mice significantly impaired vascular endothelium-dependent and independent relaxation in C57BL/6J mice compared with p47phox^−/− mice indicating NADPH oxidase activity as an intermediate for ER stress in vascular endothelial dysfunction.

Conclusion/significance

We conclude that chemically induced ER stress leads to a downstream enhancement of p38 MAPK and oxidative stress causing vascular endothelial dysfunction. Our results indicate that inhibition of ER stress could be a novel therapeutic strategy to attenuate vascular dysfunction during cardiovascular diseases.     

Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H₂O₂‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p16^INK4a and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked H₂O₂‐induced superoxide formation, NADPH oxidase levels and VCAM‐1, ICAM‐1, IL‐6 and miR‐34a synthesis. Kallistatin reversed H₂O₂‐mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)‐2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti‐senescence and anti‐oxidant effects were attributed to SIRT1‐mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up‐regulated Let‐7g, whereas Let‐7g inhibitor abolished kallistatin's effects on miR‐34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium‐specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild‐type mouse endothelial cells, and H₂O₂ treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let‐7g, SIRT1, eNOS, catalase and SOD‐1 mRNA levels, and elevated miR‐34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway.     

Anthocyanins attenuate endothelial dysfunction through regulation of uncoupling of nitric oxide synthase in aged rats

Endothelial dysfunction is one of the main age‐related arterial phenotypes responsible for cardiovascular disease (CVD) in older adults. This endothelial dysfunction results from decreased bioavailability of nitric oxide (NO) arising downstream of endothelial oxidative stress. In this study, we investigated the protective effect of anthocyanins and the underlying mechanism in rat thoracic aorta and human vascular endothelial cells in aging models. In vitro, cyanidin‐3‐rutinoside (C‐3‐R) and cyanidin‐3‐glucoside (C‐3‐G) inhibited the d‐galactose (d‐gal)‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p21, and p16^INK4a. Anthocyanins blocked d‐gal‐induced reactive oxygen species (ROS) formation and NADPH oxidase activity. Anthocyanins reversed d‐gal‐mediated inhibition of endothelial nitric oxide synthase (eNOS) serine phosphorylation and SIRT1 expression, recovering NO level in endothelial cells. Also, SIRT1‐mediated eNOS deacetylation was shown to be involved in anthocyanin‐enhanced eNOS activity. In vivo, anthocyanin‐rich mulberry extract was administered to aging rats for 8 weeks. In vivo, mulberry extract alleviated endothelial senescence and oxidative stress in the aorta of aging rats. Consistently, mulberry extract also raised serum NO levels, increased phosphorylation of eNOS, increased SIRT1 expression, and reduced nitrotyrosine in aortas. The eNOS acetylation was higher in the aging group and was restored by mulberry extract treatment. Similarly, SIRT1 level associated with eNOS decreased in the aging group and was restored in aging plus mulberry group. These findings indicate that anthocyanins protect against endothelial senescence through enhanced NO bioavailability by regulating ROS formation and reducing eNOS uncoupling.     

N-3 PUFAs Protect against Aortic Inflammation and Oxidative Stress in Angiotensin II-Infused Apolipoprotein E-/- Mice

Abdominal aortic aneurysm is associated with infiltration of inflammatory cells into the aortic wall. The inflammatory response is also evident in animal models, such as apolipoprotein E-deficient (ApoE^-/-) mice that have been infused with angiotensin II, prior to development of aortic aneurysm. Since omega-3 polyunsaturated fatty acids (n-3 PUFAs) and their metabolites have anti-inflammatory and pro-resolving activity, we hypothesised that dietary supplementation with n-3 PUFAs would protect against inflammatory processes in this mouse model. Twenty C57 and 20 ApoE^-/- 3-4 week old male mice were supplemented with a low (0.14%, n = 10/group) or high (0.70%, n = 10/group) n-3 PUFA diet for 8 weeks before 2-day infusion with 0.9% saline or angiotensin II (1000 ng/kg/min). Four ApoE^-/- mice on the low n-3 PUFA diet and none of the ApoE^-/- mice on the high n-3 PUFA diet showed morphological evidence of abdominal aortic dissection. The plasma concentration of the n-3 PUFA metabolite, resolvin D1 was higher in angiotensin II-infused ApoE^-/- mice fed the high, compared to the low n-3 PUFA diet. The number of neutrophils and macrophages infiltrating the abdominal aorta was elevated in ApoE^-/- mice on the low n-3 PUFA diet, and this was significantly attenuated in mice that were fed the high n-3 PUFA diet. Most neutrophils and macrophages were associated with dissected aortas. Immunoreactivity of the catalytic subunit of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, Nox2, and superoxide were elevated in ApoE^-/- mice that were fed the low n-3 PUFA diet, and this was also significantly attenuated in mice that were fed the high n-3 PUFA diet. Together, the findings indicate that supplementation of ApoE^-/- mice with a diet high in n-3 PUFA content protected the mice against pro-inflammatory and oxidative stress responses following short-term infusion with angiotensin II.     

Dual Role of Endothelial Nitric Oxide Synthase in Oxidized LDL-Induced,p66Shc-Mediated Oxidative Stress in Cultured Human Endothelial Cells

Background

The aging gene p66^Shc, is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66^Shc deletion increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B. However, the putative role of the NO pathway on p66^Shc activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66^Shc activation.

Methods and Results

Incubation of HAEC with oxidized low density lipoprotein (oxLDL) led to phosphorylation of p66^Shc at Ser-36, resulting in an enhanced production of superoxide anion (O₂ ^-). In the absence of oxLDL, inhibition of eNOS by small interfering RNA or L-NAME, induced p66^Shc phosphorylation, suggesting that basal NO production inhibits O₂ ^- production. oxLDL-induced, p66^Shc-mediated O₂- was prevented by eNOS inhibition, suggesting that when cells are stimulated with oxLDL eNOS is a source of reactive oxygen species. Endogenous or exogenous NO donors, prevented p66^Shc activation and reduced O_2- production. Treatment with tetrahydrobiopterin, an eNOS cofactor, restored eNOS uncoupling, prevented p66^Shc activation, and reduced O₂- generation. However, late treatment with tetrahydropterin did not yield the same result suggesting that eNOS uncoupling is the primary source of reactive oxygen species.

Conclusions

The present study reports that in primary cultured HAEC treated with oxLDL, p66^Shc-mediated oxidative stress is derived from eNOS uncoupling. This finding contributes novel information on the mechanisms of p66^Shc activation and its dual interaction with eNOS underscoring the importance eNOS uncoupling as a putative antioxidant therapeutical target in endothelial dysfunction as observed in cardiovascular disease.     

Chronic resveratrol treatment restores vascular responsiveness of cerebral arterioles in type 1 diabetic rats

Decreased dilation of cerebral arterioles via an increase in oxidative stress may be a contributing factor in the pathogenesis of diabetes-induced complications leading to cognitive dysfunction and/or stroke. Our goal was to determine whether resveratrol, a polyphenolic compound present in red wine, has a protective effect on cerebral arterioles during type 1 diabetes (T1D). We measured the responses of cerebral arterioles in untreated and resveratrol-treated (10 mg·kg(-1)·day(-1)) nondiabetic and diabetic rats to endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase (NOS)-dependent agonists and to a NOS-independent agonist. In addition, we harvested brain tissue from nondiabetic and diabetic rats to measure levels of superoxide under basal conditions. Furthermore, we used Western blot analysis to determine the protein expression of eNOS, nNOS, SOD-1, and SOD-2 in cerebral arterioles and/or brain tissue from untreated and resveratrol-treated nondiabetic and diabetic rats. We found that T1D impaired eNOS- and nNOS-dependent reactivity of cerebral arterioles but did not alter NOS-independent vasodilation. While resveratrol did not alter responses in nondiabetic rats, resveratrol prevented T1D-induced impairment in eNOS- and nNOS-dependent vasodilation. In addition, superoxide levels were higher in brain tissue from diabetic rats and resveratrol reversed this increase. Furthermore, eNOS and nNOS protein were increased in diabetic rats and resveratrol produced a further increased eNOS and nNOS proteins. SOD-1 and SOD-2 proteins were not altered by T1D, but resveratrol treatment produced a decrease in SOD-2 protein. Our findings suggest that resveratrol restores vascular function and oxidative stress in T1D. We suggest that our findings may implicate an important therapeutic potential for resveratrol in treating T1D-induced cerebrovascular dysfunction.     

SIRT1 regulates oxidant- and cigarette smoke-induced eNOS acetylation in endothelial cells: Role of resveratrol

Endothelial nitric oxide synthase (eNOS) plays a crucial role in endothelial cell functions. SIRT1, a NAD⁺-dependent deacetylase, is shown to regulate endothelial function and hence any alteration in endothelial SIRT1 will affect normal vascular physiology. Cigarette smoke (CS)-mediated oxidative stress is implicated in endothelial dysfunction. However, the role of SIRT1 in regulation of eNOS by CS and oxidants are not known. We hypothesized that CS-mediated oxidative stress downregulates SIRT1 leading to acetylation of eNOS which results in reduced nitric oxide (NO)-mediated signaling and endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) exposed to cigarette smoke extract (CSE) and H₂O₂ showed decreased SIRT1 levels, activity, but increased phosphorylation concomitant with increased eNOS acetylation. Pre-treatment of endothelial cells with resveratrol significantly attenuated the CSE- and oxidant-mediated SIRT1 levels and eNOS acetylation. These findings suggest that CS- and oxidant-mediated reduction of SIRT1 is associated with acetylation of eNOS which have implications in endothelial dysfunction.     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood–brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood–brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (−23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 μM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [³H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood–brain barrier, possibly via changes in GLUT1 protein expression and activity.