Objective-The goal of this study was to test the hypothesis that IL-6 mediates the increases in superoxide, vascular hypertrophy, and endothelial dysfunction in response to angiotensin II (Ang II). Methods and Results-Responses of carotid arteries from control and IL-6 -deficient mice were examined after acute (22-hour) incubation with Ang II (10 nmol/L) or chronic infusion of Ang II (1.4 mg/kg/d for 14 days). The hypertrophic response and endothelial dysfunction produced by Ang II infusion was markedly less in carotid arteries from IL-6 -deficient mice than that in control mice. IL-6 deficiency also protected against endothelial dysfunction in response to acute (local) Ang II treatment (eg, 100 mol/L acetylcholine produced 100Ϯ4 and 98Ϯ4% relaxation in vehicle-treated and 51Ϯ4 and 99Ϯ4% relaxation in Ang II-treated, control, and IL-6 -deficient vessels, respectively). Endothelial dysfunction could be reproduced in vessels from IL-6 -deficient mice with combined Ang II plus IL-6 (0.1 nmol/L) treatment. Increases in vascular superoxide and IL-6, as well as reductions in endothelial nitric oxide synthase mRNA expression, produced by Ang II were absent in IL-6 -deficient mice. Conclusions-These data demonstrate that IL-6 is essential for Ang II-induced increases in superoxide, endothelial dysfunction, and vascular hypertrophy. (Arterioscler Thromb Vasc Biol.

Abstract-Angiotensin II (Ang II) produces inflammation and endothelial dysfunction in blood vessels. We tested the hypothesis that interleukin 10 (IL-10), an antiinflammatory cytokine, protects against Ang II-induced vascular dysfunction. Responses of carotid arteries from wild-type and IL-10 -deficient mice (IL-10 Ϫ/Ϫ ) were examined in vitro after overnight incubation with vehicle or Ang II (1 nmol/L). In arteries from wild-type mice, acetylcholine (an endothelium-dependent agonist) produced relaxation that was not affected by Ang II. In contrast, relaxation to acetylcholine in arteries from IL-10 Ϫ/Ϫ mice was reduced by Ͼ50% by Ang II (PϽ0.05) and this effect was prevented by a scavenger of superoxide. Vascular superoxide increased Ϸ2-fold (PϽ0.05) after treatment with Ang II in IL-10

Abstract-The goal of the present study was to test the hypothesis that the CuZn isoform of superoxide dismutase (CuZnSOD) protects against angiotensin II (Ang II)-induced endothelial dysfunction. Vascular responses of carotid arteries from control, CuZnSOD-deficient (CuZnSOD ϩ/Ϫ ), and CuZnSOD transgenic mice were examined in vitro after overnight incubation with either vehicle or Ang II (1 or 10 nmol/L). In control mice, acetylcholine produced concentration-dependent relaxation that was not affected by 1 nmol/L Ang II. In contrast, relaxation to acetylcholine in arteries from CuZnSODϩ/Ϫ mice was markedly and selectively attenuated after incubation with 1 nmol/L Ang II (eg, 100 mol/L acetylcholine produced 93Ϯ6% and 44Ϯ15% relaxation in vehicle-and Ang II-treated arteries, respectively). A higher concentration of Ang II (10 nmol/L) selectively impaired relaxation to acetylcholine in arteries from control mice (eg, 100 mol/L acetylcholine produced 96Ϯ4% and 45Ϯ7% relaxation in vehicle-and Ang II-treated vessels, respectively). In contrast, 10 nmol/L Ang II had no effect on responses to acetylcholine in carotid arteries from CuZnSOD transgenic mice (or in control mice treated with the superoxide scavenger Tiron [1 mmol/L]). Superoxide levels in control mice were higher in aorta treated with Ang II than with vehicle and were markedly reduced in CuZnSOD transgenic mice. These findings provide the first direct evidence that CuZnSOD limits Ang II-mediated impairment of endothelial function and that loss of 1 copy of the CuZnSOD gene is sufficient to enhance Ang II-induced vascular dysfunction.

Angiotensin II (Ang II) is known to promote vascular disease and hypertension in part by formation of cytokines like interleukin-6 (IL-6). However, the role of signal transducer and activator of transcription 3 (STAT3) in these process and Ang II/IL-6 signaling is unclear. Using two models, we tested the hypothesis that STAT3 is essential for Ang II-induced vascular dysfunction and hypertension. Incubation of isolated carotid arteries from C57BL/6J mice with Ang II overnight increased superoxide ~2 fold and reduced vasodilator responses to the endothelium-dependent agonist acetylcholine (Ach) by ~50% vs controls (P<0.05). These effects were prevented by the addition of small molecular inhibitors of STAT3 activation (S3I-201 or STATTIC). In vivo, administration of Ang II (1.4 mg kg−1 day−1) using osmotic minipumps increased arterial pressure by ~40 mmHg at day 14 compared to vehicle treated mice and this effect was prevented by S3I-201 treatment (5 mg kg−1 IP, q.o.d.). Following systemic treatment with Ang II, dilator responses to acetylcholine were reduced by ~30-50% in carotid artery and basilar arteries whereas S3I-201 treatment prevented most of this impairment (P<0.05). In contrast to effects on vascular function and blood pressure, S31-201 did not prevent Ang II-induced hypertrophy in the carotid artery. These findings provide the first evidence that inhibitors of STAT3 activation protect against Ang II-induced oxidative stress, endothelial dysfunction, and hypertension. Because Ang II promotes vascular disease in the presence of multiple cardiovascular risk factors, these results suggest selective targeting STAT3 may have substantial therapeutic potential.

Abstract-The goal of this study was to test the hypothesis that loss of a single copy of the gene for CuZn superoxide dismutase (CuZnSOD) increases vascular superoxide levels and produces vascular dysfunction with aging. Responses of carotid arteries from young (7 months) and old (22 to 24 months of age) heterozygous CuZnSOD-deficient (CuZnSOD ϩ/Ϫ ) mice and their wild-type (CuZnSOD ϩ/ϩ ) littermates were examined in vitro. Total superoxide dismutase activity in aorta was reduced by Ϸ30% (PϽ0.05) in CuZnSOD ϩ/Ϫ mice compared with wild-type mice. Responses to acetylcholine (an endothelium-dependent agonist) produced relaxation that was similar (PϾ0.05) in carotid arteries from young wild-type, young CuZnSOD ϩ/Ϫ , and old wild-type mice. In contrast, relaxation to acetylcholine was markedly impaired in old CuZnSOD ϩ/Ϫ mice (eg, 100 mol/L acetylcholine produced 51Ϯ5% and 96Ϯ5% relaxation in vessels from old CuZnSOD ϩ/Ϫ and old wild-type mice, respectively). This effect was selective, because relaxation to nitroprusside (an endothelium-independent agonist) was not affected by either CuZnSOD genotype or aging. The impaired response to acetylcholine in old CuZnSOD ϩ/Ϫ mice was restored toward normal with either tempol (a scavenger of superoxide; 1 mmol/L) or PJ34 (an inhibitor of poly-ADP-ribose polymerase; 3 mol/L). Vascular superoxide levels were increased in aorta in old CuZnSOD ϩ/ϩ mice and increased further in CuZnSOD ϩ/Ϫ mice with aging. These findings provide the first direct evidence that normal CuZnSOD expression protects endothelial function and that deficiency in a single copy of the gene that encodes CuZnSOD produces increases in superoxide and marked impairment of endothelial function with aging.

Background and Purpose Obesity is an increasing epidemic worldwide; however little is known regarding effects of obesity produced by high-fat diet (HFD) on the cerebral circulation. The purpose of this study was to examine the functional and temporal effects of a HFD on carotid and cerebral vascular function and to identify mechanisms that contribute to such functional alterations. Methods Responses of cerebral arterioles (in vivo) and carotid arteries (in vitro) were examined in C57Bl/6 (wild-type) and Nox2-deficient (Nox2−/−) mice fed a control (10%) or a HFD (45 or 60% kcal from fat) for either 8, 12, 30, or 36 wks. RESULTS In wild-type mice HFD produced obesity and endothelial dysfunction by 12 and 36 wks in cerebral arterioles and carotid artery, respectively, in wild-type mice. Endothelial function could be significantly improved with Tempol (a superoxide scavenger) treatment in wild-type mice fed a HFD. Despite producing a similar degree of obesity in both wild-type and Nox2−/− mice, endothelial dysfunction was only observed in wild-type, but not in Nox2−/−, mice fed a HFD. Conclusions Endothelial dysfunction produced by a HFD occurs in a temporal manner and appears much earlier in cerebral arterioles than in carotid artery. Genetic studies revealed that Nox2-derived superoxide plays a major role in endothelial dysfunction produced by a HFD. Such functional changes may serve to predispose blood vessels to reduced vasodilator responses and thus may contribute to alterations in cerebral blood flow associated with obesity.

Abstract-Levels of reactive oxygen species, including hydrogen peroxide , increase in blood vessels during hypertension and in response to angiotensin II (Ang II). Although glutathione peroxidases are known to metabolize hydrogen peroxide, the role of glutathione peroxidase during hypertension is poorly defined. We tested the hypothesis that glutathione peroxidase-1 protects against Ang II-induced endothelial dysfunction. Responses of carotid arteries from Gpx1-deficient (Gpx1 ϩ/Ϫ and Gpx1 Ϫ/Ϫ ) and Gpx1 transgenic mice, and their respective littermate controls, were examined in vitro after overnight incubation with either vehicle or Ang II. Under control conditions, relaxation to acetylcholine (ACh; an endothelium-dependent agonist) was similar in control, Gpx1 ϩ/Ϫ , and Gpx1 transgenic mice, whereas in Gpx1 Ϫ/Ϫ mice, responses to ACh were impaired. In control mice, ACh-induced vasorelaxation was not affected by 1 nmol/L of Ang II. In contrast, relaxation to ACh in arteries from Gpx1 ϩ/Ϫ mice was inhibited by Ϸ60% after treatment with 1 nmol/L of Ang II, indicating that Gpx1 haploinsufficiency markedly enhances Ang II-induced endothelial dysfunction. A higher concentration of Ang II (10 nmol/L) selectively impaired relaxation to ACh in arteries from control mice, and this effect was prevented in arteries from Gpx1 transgenic mice or in arteries from control mice treated with polyethylene glycol-catalase (which degrades hydrogen peroxide). Thus, genetic and pharmacological evidence suggests a major role for glutathione peroxidase-1 and hydrogen peroxide in Ang II-induced effects on vascular function. (Hypertension. 2008;51:872-877.)

Carotid and cerebrovascular disease increase markedly with age contributing to stroke and cognitive impairment. Inflammation is a key element of vascular disease. In these studies, we tested the hypothesis that interleukin-10 (IL-10), a potent anti-inflammatory cytokine, protects against aging-induced endothelial dysfunction. Responses of carotid arteries from adult (5 ± 1 months) and old (22 ± 1 months) wild-type and IL-10-deficient mice were examined in vitro. Acetylcholine (an endothelium-dependent agonist) produced relaxation in arteries from adult wild-type that was not altered in old mice. In contrast, relaxation to acetylcholine in arteries from old IL-10-deficient mice was reduced by ∼50% (P < 0.05). Tempol, a scavenger of superoxide, did not affect responses in adult or old wild-type mice, but restored vasodilation to acetylcholine to normal in old IL-10-deficient mice. Responses of the carotid artery to nitroprusside (an endothelium-independent agonist) were not altered in any group. Vascular expression of IL-6 (a proinflammatory mediator of vascular disease) and components of NADPH oxidase (a major source of superoxide) was increased in old IL-10-deficient mice compared with wild-type (P < 0.05). These findings provide the first evidence that age-related and superoxide-mediated endothelial dysfunction occurs earlier with IL-10 deficiency. Our findings suggest a novel role for IL-10 to protect against age-related increases in expression of IL-6, oxidative stress, and endothelial dysfunction.