Vascular Reactive Oxygen Species Production Research Articles

Endothelin-1 Overexpression Exaggerates Diabetes-Induced Endothelial Dysfunction by Altering Oxidative Stress.

Increased endothelin (ET)-1 expression causes endothelial dysfunction and oxidative stress. Plasma ET-1 is increased in patients with diabetes mellitus. Since endothelial dysfunction often precedes vascular complications in diabetes, we hypothesized that overexpression of ET-1 in the endothelium would exaggerate diabetes-induced endothelial dysfunction. Diabetes was induced by streptozotocin treatment (55mg/kg/day, i.p.) for 5 days in 6-week-old male wild type (WT) mice and in mice overexpressing human ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Small mesenteric artery (MA) endothelial function and vascular remodeling by pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining and mRNA expression by reverse transcription/quantitative PCR were determined. Endothelium-dependent vasodilatory responses to acetylcholine of MA were reduced 24% by diabetes in WT ( P < 0.05), and further decreased by 12% in eET-1 ( P < 0.05). Diabetes decreased MA media/lumen in WT and eET-1 ( P < 0.05), whereas ET-1 overexpression increased MA media/lumen similarly in diabetic and nondiabetic WT mice ( P < 0.05). Vascular ROS production was increased 2-fold by diabetes in WT ( P < 0.05) and further augmented 1.7-fold in eET-1 ( P < 0.05). Diabetes reduced endothelial nitric oxide synthase (eNOS, Nos3 ) expression in eET-1 by 31% ( P < 0.05) but not in WT. Induction of diabetes caused a 52% ( P < 0.05) increase in superoxide dismutase 1 ( Sod1 ) and a 32% ( P < 0.05) increase in Sod2 expression in WT but not in eET-1. Increased expression of ET-1 exaggerates diabetes-induced endothelial dysfunction. This may be caused by decrease in eNOS expression, increase in vascular oxidative stress, and decrease in antioxidant capacity.

Role of UCP2 in the protective effects of PPARβ/δ activation on lipopolysaccharide-induced endothelial dysfunction

Bacterial endotoxin lipopolysaccharide (LPS) activates inflammatory pathways, induces cytokine expression in the endothelium, augments reactive oxygen species (ROS) production in the vascular wall, and induces endothelial dysfunction. The aim of the present study was to analyze the effects of peroxisome proliferator-activated receptor (PPAR)β/δ activation on LPS-induced inflammation, oxidative stress and endothelial dysfunction and to determine whether uncoupling protein-2 (UCP2) plays a role in these effects. In vivo, the PPARβ/δ agonist GW0742 treatment prevented the LPS-induced reduction in aortic relaxation, the increase in vascular ROS production, the upregulation of NOX1, NOX2, p47phox, and p22phox mRNA levels, and the endoplasmic reticulum (ER) stress markers in mice. We show that in mouse aortic endothelial cells (MAECs), GW0742 prevented the decreased A23187-stimulated nitric oxide (NO) production, and the increased intracellular ROS levels caused by exposure to LPS in vitro. The PPARβ/δ antagonist GSK0660 abolished all these in vivo and in vitro protective effects induced by GW0742. This agonist also restored the reduced expression of UCP2 and mitofusin-2 induced by LPS. The effects of GW0742 on NO and ROS production in MAEC exposed to LPS were abolished by the UCP2 inhibitor genipin or by siRNA targeting UCP-2. Genipin also suppressed the expressional changes on NADPH oxidase and ER stress markers induced by GW0742. In conclusion, PPARβ/δ activation restored the LPS-induced endothelial dysfunction by upregulation of UCP2, with the subsequent alleviation of ER stress and NADPH oxidase activity, thus reducing intracellular ROS production and increasing NO bioavailability.

Tempol improves xanthine oxidoreductase-mediated vascular responses to nitrite in experimental renovascular hypertension

Upregulation of xanthine oxidoreductase (XOR) increases vascular reactive oxygen species (ROS) levels and contributes to nitroso-redox imbalance. However, XOR can generate nitric oxide (NO) from nitrite, and increased superoxide could inactivate NO formed from nitrite. This study tested the hypothesis that XOR contributes to the cardiovascular effects of nitrite in renovascular hypertension, and that treatment with the antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) improves XOR-mediated effects of nitrite. Blood pressure was assessed weekly in two-kidney one-clip (2K1C) and control rats. After six weeks of hypertension, the relaxing responses to nitrite were assessed in aortic rings in the presence of the XOR inhibitor oxypurinol (or vehicle), either in the absence or in the presence of tempol. Moreover, in vivo hypotensive responses to nitrite were also examined in the presence of oxypurinol (or vehicle) and tempol (or vehicle). Aortic XOR activity and expression were evaluated by fluorescence and Western blot, respectively. Vascular ROS production was assessed by the dihydroethidium assay. 2K1C hypertensive rats showed increased aortic XOR activity and vascular ROS production compared with control rats. Oxypurinol shifted the nitrite concentration–response curve to the right in aortic rings from 2K1C rats (but not in controls). Oxypurinol also attenuated the hypotensive responses to nitrite in 2K1C rats (but not in controls). These functional findings agree with increased aortic and plasma XOR activity found in 2K1C rats. Tempol treatment enhanced oxypurinol-induced shift of the nitrite concentration–response curve to the right. However, antioxidant treatment did not affect XOR-mediated hypotensive effects of nitrite. Our results show that XOR is important to the cardiovascular responses to nitrite in 2K1C hypertension, and XOR inhibitors commonly used by patients may cancel this effect. This finding suggests that nitrite treatment may not be effective in patients being treated with XOR inhibitors. Moreover, while tempol may improve the vascular responses to nitrite, antihypertensive responses are not affected.

Downregulation of Nuclear Factor Erythroid 2-Related Factor and Associated Antioxidant Genes Contributes to Redox-Sensitive Vascular Dysfunction in Hypertension.

Oxidative stress is implicated in vascular dysfunction in hypertension. Although mechanisms regulating vascular pro-oxidants are emerging, there is a paucity of information on antioxidant systems, particularly nuclear factor erythroid 2-related factor (Nrf2), a master regulator of antioxidants enzymes. We evaluated the vascular regulatory role of Nrf2 in hypertension and examined molecular mechanisms, whereby Nrf2 influences redox signaling in small arteries and vascular smooth muscle cells from Wistar Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Cells were stimulated with angiotensin II in the absence/presence of Nrf2 activators (bardoxolone/L-sulforaphane). Increased vascular reactive oxygen species production (chemiluminescence and amplex red) was associated with reduced Nrf2 activity in arteries (18%) and vascular smooth muscle cells (48%) in SHRSP (P<0.05 versus WKY). Expression of antioxidant enzymes, including superoxide dismutase-1 (64%), catalase (60%), peroxiredoxin 1 (75%), and glutathione peroxidase (54%), was reduced in SHRSP. L-sulforaphane reversed these effects. Angiotensin II increased nuclear accumulation of Nrf2 in vascular smooth muscle cells from WKY (197% versus vehicle), with blunted effects in SHRSP (44% versus vehicle). These responses were associated with increased antioxidant expression (superoxide dismutase-1, 32%; catalase, 42%; thioredoxin, 71%; peroxiredoxin, 1%-90%; quinone oxidoreductase, 84%; P<0.05 versus vehicle) and increased activity of superoxide dismutase-1, catalase, and thioredoxin in WKY but not in SHRSP, which exhibited increased Bach1 expression. Nrf2 activators blocked angiotensin II-induced reactive oxygen species generation. Vascular function demonstrated increased contractility (Emax WKY 113.4±5.6 versus SHRSP 159.0±8.3) and decreased endothelial-dependent relaxation (Emax WKY 88.6±3.1 versus SHRSP 74.6±3.2, P<0.05) in SHRSP, effects corrected by L-sulforaphane. Our findings suggest that Nrf2 downregulation contributes to redox-sensitive vascular dysfunction in hypertension.

Early treatment with hydroxychloroquine prevents the development of endothelial dysfunction in a murine model of systemic lupus erythematosus.

IntroductionAccelerated atherosclerosis is one of the major causes of morbidity in patients with systemic lupus erythematosus (SLE). Endothelial dysfunction (ED) is considered an early marker of atherosclerosis. It is a reversible alteration, thus representing an attractive target for prevention strategies against cardiovascular disease. Studies have shown that ED occurs in patients with SLE even in the absence of severe, active disease. Hydroxychloroquine (HCQ) is widely used in SLE to control disease activity, but its use is also associated with an improvement in long-term prognosis. Beyond the beneficial effect in well-established disease, our hypothesis is that treatment with HCQ might have a beneficial impact on ED prevention in SLE. The aim of this study was to assess the impact of early treatment with HCQ on ED in a murine model of SLE.MethodsTwelve-week-old NZB/W F1 (NZ) and C57BL/6 J mice (controls) were allocated to receive HCQ or vehicle for 6, 12, or 18 weeks. Proteinuria and anti–double-stranded DNA autoantibodies were determined. ED was assessed in mesenteric arteries (pressurized myography). Nitric oxide (NO) availability and reactive oxygen species (ROS) production were evaluated. Vascular ROS production was measured with dihydroethidium (DHE) fluorescent dye.ResultsStarting from 18 weeks of age, NZ mice showed a progressive reduction in NO availability, which was normalized by ascorbic acid and apocynin in the up to 24-week-old group, and partly ameliorated in older animals. HCQ administration normalized the NO availability in the up to 24-week-old group, with a partial amelioration in the 30-week-old group. DHE analysis revealed a progressive increment of vascular ROS generation among NZ groups, which was prevented by apocynin. Similarly, in the NZ HCQ-treated group, vascular ROS production was abrogated.ConclusionsThe ED that characterizes this mouse model of SLE is caused by the nicotinamide adenine dinucleotide phosphate oxidase–driven ROS excess. Very early treatment with HCQ is able to exert vascular protection via an antioxidant effect.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0790-3) contains supplementary material, which is available to authorized users.

Role of perivascular adipose tissue on vascular reactive oxygen species in type 2 diabetes: a give-and-take relationship.

A major complication of type 2 diabetes (T2D) is atherosclerotic vascular disease, which develops earlier and more rapidly in patients with T2D than in subjects without diabetes (1). One of the characteristic features of T2D is excessive generation of reactive oxygen species (ROS) in the artery wall and the resultant oxidative stress, which contributes to the development of endothelial dysfunction and atherosclerosis (2–5). Moreover, activity of NADPH oxidase, the primary ROS-generating enzyme in vascular cells, has been shown to be increased in T2D (2–5). Notably, behavioral and pharmacological interventions that reduce vascular NADPH oxidase expression and activity demonstrate improvements in endothelial function and reduced atherogenesis (6–8). Furthermore, mice lacking components of the NADPH oxidase subunits are protected against hypertension, and when crossed to the apoE−/− background, they have a marked reduction in vascular ROS production, enhanced nitric oxide bioavailability, and reduced atherosclerotic lesion formation (9,10), thus demonstrating that excessive NADPH oxidase–derived ROS is detrimental to vascular health. Although the recognition that increased vascular NADPH oxidase is an important contributor to vascular complications in T2D, the mechanisms regulating its enzyme activity remain poorly understood. Recent studies implicate adipose tissue adjacent to the artery wall (i.e., perivascular adipose tissue [PVAT]) as playing an important role in the pathogenesis of vascular diseases (11–13). The PVAT serves not …

165 Activation of the G-protein Coupled Receptor 43 Attenuates High Glucose-induced Vascular Oxidative Stress

Vascular oxidative stress has been found to be a prominent feature underlying the vascular abnormalities of patients with insulin-resistance and type-2 diabetes. Short chain fatty acids (SCFA) i.e. acetate (NaA),...

Endothelial cell-specific reactive oxygen species production increases susceptibility to aortic dissection.

Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II-mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45(+) inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II-induced vascular smooth muscle cell ROS production. These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II-mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

Inhibition of calpain reduces oxidative stress and attenuates endothelial dysfunction in diabetes.

AimsThe present study was to investigate the role of calpain in reactive oxygen species (ROS) production in endothelial cells and endothelium-dependent vascular dysfunction under experimental conditions of diabetes.Methods and resultsExposure to high glucose activated calpain, induced apoptosis and reduced nitric oxide (NO) production without changing eNOS protein expression, its phosphorylation and dimers formation in primary human umbilical vein endothelial cells (HUVECs). These effects of high glucose correlated with intracellular ROS production and mitochondrial superoxide generation. Selectively scavenging mitochondrial superoxide increased NO production in high glucose-stimulated HUVECs. Inhibition of calpain using over-expression of calpastatin or pharmacological calpain inhibitor prevented high glucose-induced ROS production, mitochondrial superoxide generation and apoptosis, which were concurrent with an elevation of NO production in HUVECs. In mouse models of streptozotocin-induced type-1 diabetes and OVE26 type-1 diabetic mice, calpain activation correlated with an increase in ROS production and peroxynitrite formation in aortas. Transgenic over-expression of calpastatin reduced ROS production and peroxynitrite formation in diabetic mice. In parallel, diabetes-induced reduction of endothelium-dependent relaxation in aortic ring was reversed by over-expression of calpastatin in mouse models of diabetes. However, the protective effect of calpastatin on endothelium-dependent relaxation was abrogated by eNOS deletion in diabetic mice.ConclusionsThis study suggests that calpain may play a role in vascular endothelial cell ROS production and endothelium-dependent dysfunction in diabetes. Thus, calpain may be an important therapeutic target to overcome diabetes-induced vascular dysfunction.

Vascular oxidative stress promotes aortic stiffening (867.2)

Oxidative stress correlates with arterial stiffness in clinical studies. Reactive oxygen species (ROS) contribute to blood pressure elevation, vascular smooth muscle hypertrophy and vascular dysfunction in hypertension. We hypothesized that vascular ROS induce aortic stiffening, which in turn promotes systolic hypertension with aging. We created mice overexpressing p22phox in smooth muscle (tgsm/p22phox mice), which have a constitutive increase in vascular ROS production. Six month old tgsm/p22phox mice were normotensive at baseline (118±6 systolic, mmHg), but had increased aortic stiffness as indicated by the leftward shift on stress‐strain curves. Aortic collagen level, as detected by hydroxyproline content, was increased in tgsm/p22phox mice compared to age‐matched C57Bl/6 mice (76.3±11.9 vs. 43.2±7.2 ng/μg total protein). At 9 months of age, aortic stiffening did not further increase but these mice developed mild hypertension (137±11 systolic), demonstrating that excessive ROS first promotes aortic stiffening and subsequently hypertension. Tempol, a superoxide dismutase mimetic, normalized blood pressure (117±10 systolic), reduced vascular collagen deposition (37.5±9.5 ng/μg) and prevented aortic stiffening in these mice, suggesting a role of vascular ROS in aging‐induced aortic stiffening. ROS oxidize arachidonic acid to produce isoketals, known to cross‐link lysines and promote protein cross‐linking, which could lead to vascular stiffening. Immunohistochemistry indicated a marked increase of isoketals in the aorta and peri‐aortic fat of tgsm/p22phox mice, which was prevented by 2‐hydroxybenzylamine (2‐HOBA), a scavenger of isoketals. 2‐HOBA also reduced blood pressure, prevented collagen deposition and aortic stiffening in tgsm/p22phox mice treated from 3 months to 9 months of age. We conclude that ROS promote aortic stiffening and that this might in part be due to isoketal formation and protein crosslinking.Grant Funding Source: Supported by NIH R01 grant HL105294

Pharmacological inhibition of NOX reduces atherosclerotic lesions, vascular ROS and immune–inflammatory responses in diabetic Apoe −/− mice

Enhanced vascular inflammation, immune cell infiltration and elevated production of reactive oxygen species (ROS) contribute significantly to pro-atherogenic responses in diabetes. We assessed the immunomodulatory role of NADPH oxidase (NOX)-derived ROS in diabetes-accelerated atherosclerosis. Diabetes was induced in male Apoe(-/-) mice with five daily doses of streptozotocin (55 mg kg(-1) day(-1)). Atherosclerotic plaque size, markers of ROS and immune cell accumulation were assessed in addition to flow cytometric analyses of cells isolated from the adjacent mediastinal lymph nodes (meLNs). The role of NOX-derived ROS was investigated using the NOX inhibitor, GKT137831 (60 mg/kg per day; gavage) administered to diabetic and non-diabetic Apoe(-/-) mice for 10 weeks. Diabetes increased atherosclerotic plaque development in the aortic sinus and this correlated with increased lesional accumulation of T cells and CD11c(+) cells and altered T cell activation in the adjacent meLNs. Diabetic Apoe(-/-) mice demonstrated an elevation in vascular ROS production and expression of the proinflammatory markers monocyte chemoattractant protein 1, vascular adhesion molecule 1 and IFNγ. Blockade of NOX-derived ROS using GKT137831 prevented the diabetes-mediated increase in atherosclerotic plaque area and associated vascular T cell infiltration and also significantly reduced vascular ROS as well as markers of inflammation and plaque necrotic core area. Diabetes promotes pro-inflammatory immune responses in the aortic sinus and its associated lymphoid tissue. These changes are associated with increased ROS production by NOX. Blockade of NOX-derived ROS using the NOX inhibitor GKT137831 is associated with attenuation of these changes in the immune response and reduces the diabetes-accelerated development of atherosclerotic plaques in Apoe(-/-) mice.

Abstract 319: Vascular Oxidative Stress Promotes Aortic Stiffening

Clinical studies have shown a relationship between markers of oxidative stress and parameters of arterial stiffness. We have previously found that vascular reactive oxygen species (ROS) contribute to aortic hypertrophy in hypertension. In the present study, we hypothesize that vascular oxidative stress induces aortic stiffening by causing oxidative injury to the vessels. To test this hypothesis, we first studied isolated aortas from tg sm/p22phox mice, which have a constitutive increase in vascular ROS production. Six month old tg sm/p22phox mice were normotensive at baseline (118±6 systolic, 89±5 diastolic, mean±S.E.M. mmHg), but had increased aortic stiffness as indicated by the leftward shift on stress-strain curves (p&lt;0.05 vs. wild type, one way ANOVA). While treatment with angiotensin II increased aortic stiffness in wild-type mice, it did not further increase stiffness in the tg sm/p22phox mice. In order to understand the nature of ROS involved in aortic stiffening, we deleted extracellular superoxide dismutase (ecSOD) in vascular smooth muscle using cre-loxp technology. Deletion of ecSOD would increase O 2 ·- , but not H 2 O 2 . Surprisingly, we found no difference in aortic stiffness or blood pressure in these mice compared to wild-type mice, either at baseline or in response to angiotensin II. These data indicate that O 2 ·- is unlikely involved in increasing aortic stiffness. It is therefore likely that other ROS, such as H 2 O 2 , modulate this response. In this regard, we have performed additional studies in which mice were treated with salicylamine, a scavenger of isoketals, which are oxidized products of arachidonic acid. These are known to cross-link lysines and could promote cross-linking of cellular components leading to vascular stiffening. Amazingly, this drug markedly reduced blood pressure (133±8 vs. 168±9 systolic; 95±6 vs. 125±7 diastolic, salicylamine vs. angiotensin II alone, p&lt;0.01) and aortic stiffening in response to angiotensin II. In conclusion, our data strongly suggest that vascular ROS promotes aortic stiffening by inducing oxidative injury to the vessels.

Endothelin-1 Overexpression Exacerbates Atherosclerosis and Induces Aortic Aneurysms in Apolipoprotein E Knockout Mice

Endothelin (ET)-1 plays a role in vascular reactive oxygen species production and inflammation. ET-1 has been implicated in human atherosclerosis and abdominal aortic aneurysm (AAA) development. ET-1 overexpression exacerbates high-fat diet-induced atherosclerosis in apolipoprotein E(-/-) (Apoe(-/-)) mice. ET-1-induced reactive oxygen species and inflammation may contribute to atherosclerosis progression and AAA development. Eight-week-old male wild-type mice, transgenic mice overexpressing ET-1 selectively in endothelium (eET-1), Apoe(-/-) mice, and eET-1/Apoe(-/-) mice were fed high-fat diet for 8 weeks. eET-1/Apoe(-/-) had a 45% reduction in plasma high-density lipoprotein (P<0.05) and presented ≥ 2-fold more aortic atherosclerotic lesions compared with Apoe(-/-) (P<0.01). AAAs were detected only in eET-1/Apoe(-/-) (8/21; P<0.05). Reactive oxygen species production was increased ≥ 2-fold in perivascular fat, media, or atherosclerotic lesions in the ascending aorta and AAAs of eET-1/Apoe(-/-) compared with Apoe(-/-) (P<0.05). Monocyte/macrophage infiltration was enhanced ≥ 2.5-fold in perivascular fat of ascending aorta and AAAs in eET-1/Apoe(-/-) compared with Apoe(-/-) (P<0.05). CD4(+) T cells were detected almost exclusively in perivascular fat (3/6) and atherosclerotic lesions (5/6) in ascending aorta of eET-1/Apoe(-/-) (P<0.05). The percentage of spleen proinflammatory Ly-6C(hi) monocytes was enhanced 26% by ET-1 overexpression in Apoe(-/-) (P<0.05), and matrix metalloproteinase-2 was increased 2-fold in plaques of eET-1/Apoe(-/-) (P<0.05) compared with Apoe(-/-). ET-1 plays a role in progression of atherosclerosis and AAA formation by decreasing high-density lipoprotein, and increasing oxidative stress, inflammatory cell infiltration, and matrix metalloproteinase-2 in perivascular fat, vascular wall, and atherosclerotic lesions.

Abstract 546: Negative Regulation of Reactive Oxygen Species From Nadph Oxidases by Caveolar Proteins

The importance of caveolar proteins, including caveolin-1 (Cav1) have been widely explored in cardiovascular disease. Cav1 binds to numerous signaling proteins via interactions with its scaffolding domain, and caveolin-2 (Cav2) and Cavin1 depend on Cav1 for stability and expression. The NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS) and oxidative stress. Loss of cav1 has been shown to increase vascular ROS production and can promote cardiovascular diseases such as pulmonary arterial hypertension. However, the ability of Cav1 to regulate Nox enzymes is not known. Here, we found that increased expression of Cav1 by gene transfer or by administration of its scaffolding domain peptide, potently inhibited ROS production from Nox1, Nox2, Nox4 and Nox5. In contrast, inhibition of endogenous Cav1 with small interfering RNA increased ROS production from human vascular cells. The Cav1 scaffolding domain mutant, Cav1 F16A, failed to inhibit Nox activity. In addition, Cav2 and Cavin-1 can also decreased ROS production from Nox enzymes. To further explore the underlying mechanism, we found Cav1 did not modify intracellular calcium but reduced ROS output in an isolated enzyme assay. Furthermore, we found increased Cav1, Cav2 and Cavin-1 expression robustly decreased the expression level of Nox proteins. Given the similar intracellular location of Cav1 and Nox and the presence of highly conserved Cav1 binding sequence in Nox enzymes, we predict that caveolin proteins may directly bind with Nox and promote their degradation. It is also possible that degradation is mediated by Cav1 regulated Nox protein ubiquitylation. Together, these results suggest that caveolin proteins negatively regulate NADPH oxidases to ameliorate cellular stress and are an important regulatory pathway in the cardiovascular system.

INCREASED PRODUCTION OF THE ARACHIDONIC ACID METABOLITE 20‐HETE CONTRIBUTES TO HYPERTENSION‐INDUCED CEREBROVASCULAR ALTERATIONS

Hypertension increases oxidative stress and inflammation in cerebral arteries and impairs vasomotor function. We tested the hypothesis that increased production of the arachidonic acid metabolite 20‐hydroxy‐5,8,11,14‐eicosatetraenoic acid (20‐HETE) contributes to hypertension‐induced cerebrovascular alterations.Male spontaneously hypertensive rats (SHR) were treated with HET0016, an inhibitor of 20‐HETE synthesis. In middle cerebral arteries (MCAs) of SHRs vasomotor responses were obtained and cellular production of reactive oxygen species (ROS), inflammatory cytokine expression and NF‐κB activation were assessed.Treated SHRs remained hypertensive, although their blood pressure was decreased compared to control SHRs. In MCAs of SHRs flow‐induced constriction was augmented and acetylcholine‐and ATP‐induced dilations were impaired. These alterations were reversed by HET0016. In MCAs of SHRs HET0016 decreased ROS level and inhibited inflammation and NF‐κB activation. Also, 20‐HETE significantly increased vascular production of ROS and promoted NF‐κB activation in cultured cerebromicrovascular endothelial cells.Inhibition of 20‐HETE synthesis in hypertension elicits reduction in blood pressure, anti‐oxidative, anti‐inflammatory effects and improve vasomotor function of cerebral arteries potentiating cerebrovascular protection.

Treatment with the cytochrome P450 ω‐hydroxylase inhibitor HET0016 attenuates cerebrovascular inflammation, oxidative stress and improves vasomotor function in spontaneously hypertensive rats

Hypertension increases cerebrovascular oxidative stress and inflammation and impairs vasomotor function. These pathological alterations lead to dysregulation of cerebral blood flow and exacerbate atherogenesis, increasing the morbidity of ischaemic cerebrovascular diseases and promoting vascular cognitive impairment. We aimed to test the hypothesis that increased production of the arachidonic acid metabolite 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) contributes to hypertension-induced cerebrovascular alterations. We treated male spontaneously hypertensive rats (SHR) with HET0016 (N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine), an inhibitor of 20-HETE synthesis. In middle cerebral arteries (MCAs) of SHRs, we focused on vasomotor responses and end points that are highly relevant for cellular reactive oxygen species (ROS) production, inflammatory cytokine expression and NF-κB activation. SHRs treated with HET0016 remained hypertensive (SHR + HET0016: 149 ± 8 mmHg, Wistar-Kyoto rat: 115 ± 4 mmHg; P < 0.05.), although their systolic blood pressure was decreased compared to untreated SHRs (191 ± 6 mmHg). In MCAs of SHRs, flow-induced constriction was increased, whereas ACh- and ATP-induced dilations were impaired. This functional impairment was reversed by treatment with HET0016. Treatment with HET0016 also significantly decreased oxidative stress in MCAs of SHRs (as shown by dihydroethidium staining and analysis of vascular 5-nitrotyrosine, 4-hydroxynonenal and carbonyl content) and inhibited cerebrovascular inflammation (shown by the reduced mRNA expression of TNFα, IL-1β and IL-6). Treatment of SHRs with HET0016 also attenuated vascular NF-κB activation. In vitro treatment with 20-HETE significantly increased vascular production of ROS and promoted NF-κB activation in cultured cerebromicrovascular endothelial cells. Taken together, treatment with HET0016 confers anti-oxidative and anti-inflammatory effects in the cerebral arteries of SHRs by disrupting 20-HETE-mediated autocrine/paracrine signalling pathways in the vascular wall. It is likely that HET0016-induced decreases in blood pressure also potentiate the cerebrovascular protective effects of the drug.

Increased Superoxide and Endothelial NO Synthase Uncoupling in Blood Vessels of Bmal1-Knockout Mice

Disruption of the circadian clock in mice produces vascular dysfunction as evidenced by impairments in endothelium-dependent signaling, vasomotion, and blood vessel remodeling. Although the altered function of endothelial NO synthase and the overproduction of reactive oxygen species are central to dysfunction of the endothelium, to date, the impact of the circadian clock on endothelial NO synthase coupling and vascular reactive oxygen species production is not known. The goals of the present study were to determine whether deletion of a critical component of the circadian clock, Bmal1, can influence endothelial NO synthase coupling and reactive oxygen species levels in arteries from Bmal1-knockout (KO) mice. Endothelial function was reduced in aortae from Bmal1-KO mice and improved by scavenging reactive oxygen species with polyethylene glycol-superoxide dismutase and nonselectively inhibiting cyclooxygenase isoforms with indomethacin. Aortae from Bmal1-KO mice exhibited enhanced superoxide levels as determined by electron paramagnetic resonance spectroscopy and dihydroethidium fluorescence, an elevation that was abrogated by administration of nitro-l-arginine methyl ester. High-performance liquid chromatography analysis revealed a reduction in tetrahydrobiopterin and an increase in dihydrobiopterin levels in the lung and aorta of Bmal1-KO mice, whereas supplementation with tetrahydrobiopterin improved endothelial function in the circadian clock KO mice. Furthermore, levels of tetrahydrobiopterin, dihydrobiopterin, and the key enzymes that regulate biopterin bioavailability, GTP cyclohydrolase and dihydrofolate reductase exhibited a circadian expression pattern. Having an established influence in the metabolic control of glucose and lipids, herein, we describe a novel role for the circadian clock in metabolism of biopterins, with a significant impact in the vasculature, to regulate coupling of endothelial NO synthase, production of superoxide, and maintenance of endothelial function.

Pioglitazone treatment increases COX‐2‐derived prostacyclin production and reduces oxidative stress in hypertensive rats: role in vascular function

PPARγ agonists, glitazones, have cardioprotective and anti-inflammatory actions associated with gene transcription interference. In this study, we determined whether chronic treatment of adult spontaneously hypertensive rats (SHR) with pioglitazone alters BP and vascular structure and function, and the possible mechanisms involved. Mesenteric resistance arteries from untreated or pioglitazone-treated (2.5 mg·kg⁻¹ ·day⁻¹ , 28 days) SHR and normotensive [Wistar Kyoto (WKY)] rats were used. Vascular structure was studied by pressure myography, vascular function by wire myography, protein expression by Western blot and immunohistochemistry, mRNA levels by RT-PCR, prostanoid levels by commercial kits and reactive oxygen species (ROS) production by dihydroethidium-emitted fluorescence. In SHR, pioglitazone did not modify either BP or vascular structural and mechanical alterations or phenylephrine-induced contraction, but it increased vascular COX-2 levels, prostacyclin (PGI₂) production and the inhibitory effects of NS 398, SQ 29,548 and tranylcypromine on phenylephrine responses. The contractile phase of the iloprost response, which was reduced by SQ 29,548, was greater in pioglitazone-treated and pioglitazone-untreated SHR than WKY. In addition, pioglitazone abolished the increased vascular ROS production, NOX-1 levels and the inhibitory effect of apocynin and allopurinol on phenylephrine contraction, whereas it did not modify eNOS expression but restored the potentiating effect of N-nitro-L-arginine methyl ester on phenylephrine responses. Although pioglitazone did not reduce BP in SHR, it increased COX-2-derived PGI₂ production, reduced oxidative stress, and increased NO bioavailability, which are all involved in vasoconstrictor responses in resistance arteries. These effects would contribute to the cardioprotective effect of glitazones reported in several pathologies.

The T Helper 1 Transcription Factor T Box Expressed in T Cells Is a Mediator of Angiotensin II Induced Inflammation, Vascular Dysfunction and Oxidative Stress in the Murine Vasculature

Recent studies described a protection from atherosclerosis by targeting of the Th1 key transcription factor T box expressed in T cells (T-bet). It remains to be established, whether T-bet is involved in mediating angiotensin II (ATII) induced vascular dysfunction and oxidative stress. ATII (1mg/kg/d) was administered by osmotic minipumps for one week and radiotelemetry was performed in T-bet-/- vs. control C57/BL6(WT) mice. Vascular dysfunction was assessed by isometric tension studies and reactive oxygen species (ROS) were detected by using DHE staining of aortic cryosections, aortic rings Lucigenin enhanced chemiluminescence (ECL) and L-012 enhanced blood oxidative burst. Protein expression was determined by western blot and mRNA expression by realtime PCR. CD4+ cytokines were measured by ELISA and myelomonocytic cells were analyzed by FACS and IHC. Aortic T-bet protein expression was increased in response to ATII in WT mice. In T-bet deficient mice AT II caused a similar increase of blood pressure as compared to WT. In contrast to WT, T-bet-/- showed an attenuated vascular dysfunction, ROS production and expression of NADPH oxidase subunits in response to AT II. Furthermore, iNOS mRNA expression and protein tyrosine nitration in the aorta was markedly reduced in T-bet-/- mice which showed a significant decrease of macrophages in the vascular wall caused by a reduced presence of pro-inflammatory cells after AT II treatment compared to WT. In a rescue experiment we transferred WT CD4+ T cells in T-bet deficient mice which resulted in increased vascular oxidative stress and dysfunction. We conclude, that T-bet mediates, at least in part, ATII induced vascular damage and might represent a novel target to treat vascular dysfunction and inflammation in arterial hypertension.

Abstract 17239: Mechanisms of Vascular Oxidative Stress Induction of Obesity

The present study investigates whether and how vascular reactive oxygen species (ROS) induce obesity and insulin resistance/metabolic syndrome. Epidemiologically, pathological conditions like hypertension and hypercholesterolemia associated with obesity, and these diseases promote vascular ROS production. It has been thought that obesity is causal in these conditions. We instead propose that vascular ROS may play a causal role in obesity by promoting inflammation, adipogenesis and exercise intolerance. Wild-type (WT) mice and mice having excessive vascular ROS production (smooth muscle-specific overexpression of p22phox, a key component of NADPH oxidase, tg sm/p22phox , body weight (BW) elevated modestly but significantly at baseline) were fed high-fat diet for 6 weeks. Intriguingly, tg sm/p22phox mice developed exaggerated obesity. The BW increased from 32.16±2.34 g in the 6 mo old tg sm/p22phox mice to 43.03±1.44 g (vs. 30.81±0.71 g to 37.89±1.16 g in the WT mice). The percentage of increase is 34±3.35% vs. 23±3.08% respectively (p&lt;0.01). Fat mass was also significantly further increased. Glucose tolerance tests indicate a more severe insulin resistance phenotype in the tg sm/p22phox mice, which was associated with reduced HDL cholesterol and increased leptin levels. These animals also had impaired spontaneous activity, increased ROS production and mitochondrial dysfunction in skeletal muscle. Oral administration with antioxidant ebselen prevented weight gain and insulin resistance in high-fat fed tg sm/p22phox mice. In mice having deficient vascular ROS production however (p22phox loxp/loxp crossed with mice expressing Cre recombinase driven by the tamoxifen-inducible smooth muscle myosin heavy chain promoter, tg smmhc/cre mice), high-fat feeding failed to induce weight gain and leptin resistance. These vascular p22phox knockout mice also had reduced T cell infiltration of perivascular fat. In conclusion, these data seem to provocatively implicate that vascular NADPH oxidase-derived ROS induce obesity and insulin resistance/metabolic syndrome. To our knowledge this represents the first evidence that vascular ROS play a causal role in the development of obesity and insulin resistance/metabolic syndrome.