Sean P. Didion

Increased Superoxide and Vascular Dysfunction in CuZnSOD-Deficient Mice

Abstract— Increased superoxide is thought to play a major role in vascular dysfunction in a variety of disease states. Superoxide dismutase (SOD) limits increases in superoxide; however, the functional significance of selected isoforms of SOD within the vessel wall are unknown. We tested the hypothesis that selective loss of CuZnSOD results in increased superoxide and altered vascular responsiveness in CuZnSOD-deficient (CuZnSOD−/−) mice compared with wild-type (CuZnSOD+/+) littermates. Total SOD activity was reduced (P <0.05) by approximately 60% and CuZnSOD protein was absent in aorta from CuZnSOD−/− as compared with wild-type mice. Vascular superoxide levels, measured using lucigenin (5 &mgr;mol/L)-enhanced chemiluminescence and hydroethidine (2 &mgr;mol/L)-based confocal microscopy, were increased (approximately 2-fold;P <0.05) in CuZnSOD−/− mice as compared with wild-type mice. Relaxation of the carotid artery in response to acetylcholine and authentic nitric oxide was impaired (P <0.05) in CuZnSOD−/− mice. For example, maximal relaxation to acetylcholine (100 &mgr;mol/L) was 50±6% and 69±5% in CuZnSOD−/− and wild-type mice, respectively. Contractile responses of the carotid artery were enhanced (P <0.05) in CuZnSOD−/− mice in response to phenylephrine and serotonin, but not to potassium chloride or U46619. In vivo, dilatation of cerebral arterioles (baseline diameter=31±1 &mgr;m) to acetylcholine was reduced by approximately 50% in CuZnSOD−/− mice as compared with wild-type mice (P <0.05). These findings provide the first direct insight into the functional importance of CuZnSOD in blood vessels and indicate that this specific isoform of SOD limits increases in superoxide under basal conditions. CuZnSOD-deficiency results in altered responsiveness in both large arteries and microvessels.

PPARγ Agonist Rosiglitazone Improves Vascular Function and Lowers Blood Pressure in Hypertensive Transgenic Mice

Abstract—The peroxisome proliferator activated receptor (PPAR&ggr;) agonist rosiglitazone has been reported to yield cardiovascular benefits in patients by a mechanism that is not completely understood. We tested whether oral rosiglitazone (25 mg/kg per day, 21 days) treatment improves blood pressure and vascular function in a transgenic mouse expressing both human renin and human angiotensinogen transgenes (R+A+). Rosiglitazone decreased systolic (138±5 versus 128±5 mm Hg) and mean blood pressure (145±5 versus 126±7 mm Hg) of R+A+ mice as measured by tail-cuff and indwelling carotid catheters, respectively. Relaxation of carotid arteries to acetylcholine and authentic nitric oxide, but not papaverine, was impaired in R+A+ mice when compared with littermate controls (RA−). There were no effects of rosiglitazone on RA− mice; however, relaxation to acetylcholine (49±10 versus 82±9% at 100 &mgr;mol/L) and nitric oxide (51±11 versus 72±6% at 10 &mgr;mol/L) was significantly improved in treated R+A+ mice. Rosiglitazone treatment of R+A+ mice did not alter the expression of genes, including endothelial nitric oxide synthase (eNOS), angiotensin 1 receptors, and preproendothelin-1, nor did it alter the levels of eNOS or soluble guanylyl cyclase protein. In separate studies, carotid arteries from R+A+ and RA− mice relaxed in a concentration-dependent manner to rosiglitazone, suggesting possible PPAR&ggr;-independent effects in the vasculature. This response was not inhibited with the nitric oxide synthase inhibitor N&ohgr;-nitro-l-arginine methyl ester (200 &mgr;mol/L) or the PPAR&ggr; antagonist bisphenol A diglycidyl ether; 4,4′-isopropylidenediphenol diglycidyl ether (100 &mgr;mol/L). These data suggest that in addition to potential genomic regulation caused by PPAR&ggr; activation, the direct effect of rosiglitazone in blood vessels may contribute to the improved blood pressure and vessel function.

IL-6 Deficiency Protects Against Angiotensin II–Induced Endothelial Dysfunction and Hypertrophy

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 &mgr;mol/L acetylcholine produced 100±4 and 98±4% relaxation in vehicle-treated and 51±4 and 99±4% 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.

Glutamate-Induced Disruption of the Blood-Brain Barrier in Rats Role of Nitric Oxide

BACKGROUND AND PURPOSE The first goal of this study was to determine the effect of glutamate on permeability and reactivity of the cerebral microcirculation. The second goal of this study was to determine a possible role for nitric oxide in the effects of glutamate on the cerebral microcirculation. METHODS We examined the pial microcirculation in rats with intravital microscopy. Permeability of the blood-brain barrier was quantified by the clearance of fluorescent-labeled dextran (molecular weight, 10 000 D; FITC-dextran-10K) before and during application of glutamate (0.1 and 1.0 mmol/L). In addition, we examined the permeability of the blood-brain barrier during application of a nitric oxide donor, S-nitroso-acetyl-penicillamine (SNAP; 10 mumol/L). Diameter of pial arterioles was measured before and during application of glutamate or SNAP. To determine a potential role for nitric oxide in glutamate-induced effects on the cerebral microcirculation, we examined the effects of NG-monomethyl-L-arginine (10 mumol/L). RESULTS In control rats, clearance of FITC-dextran-10K from pial vessels was minimal, and the diameter of pial arterioles remained constant during the experimental period. Topical application of glutamate (0.1 and 1.0 mmol/L) and SNAP (10 mumol/L) produced an increase in clearance of FITC-dextran-10K and in diameter of pial arterioles. In addition, NG-monomethyl-L-arginine (10 mumol) attenuated glutamate-induced increases in permeability of the blood brain barrier and glutamate-induced dilatation of cerebral arterioles. CONCLUSIONS The findings of the present study suggest that glutamate, a major neurotransmitter in the brain, increases permeability of the blood-brain barrier to low-molecular-weight molecules and dilates cerebral arterioles via a nitric oxide-dependent mechanism.

Impaired Endothelium-Dependent Responses and Enhanced Influence of Rho-Kinase in Cerebral Arterioles in Type II Diabetes

Background and Purpose— Although the incidence of type II diabetes is increasing, very little is known regarding vascular responses in the cerebral circulation in this disease. The goals of this study were to examine the role of superoxide in impaired endothelium-dependent responses and to examine the influence of Rho-kinase on vascular tone in the cerebral microcirculation in type II diabetes. Methods— Diameter of cerebral arterioles (29±1 &mgr;m; mean±SE) was measured in vivo using a cranial window in anesthetized db/db and control mice. Results— Dilatation of cerebral arterioles in response to acetylcholine (ACh; 1 and 10 &mgr;mol/L), but not to nitroprusside, was markedly reduced in db/db mice (eg, 10 &mgr;mol/L ACh produced 29±1% and 9±1% in control and db/db mice, respectively). Superoxide levels were increased (P<0.05) in cerebral arterioles from db/db mice (n=6) compared with controls (n=6). Vasodilatation to ACh in db/db mice was restored to normal by polyethylene glycol-superoxide dismutase (100 U/mL). Y-27632 (1 to 100 &mgr;mol/L; a Rho-kinase inhibitor) produced modest vasodilatation in control mice but much greater responses in db/db mice. NG-nitro-l-arginine (100 &mgr;mol/L; an inhibitor of NO synthase) significantly enhanced Y-27632–induced dilatation in control mice to similar levels as observed in db/db mice. Conclusions— These findings provide the first evidence for superoxide-mediated impairment of endothelium-dependent responses of cerebral vessels in any model of type II diabetes. In addition, the influence of Rho-kinase on resting tone appears to be selectively enhanced in the cerebral microcirculation in this genetic model of type II diabetes.

Endogenous Interleukin-10 Inhibits Angiotensin II–Induced Vascular Dysfunction

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−/−) 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−/− mice was reduced by >50% by Ang II (P<0.05) and this effect was prevented by a scavenger of superoxide. Vascular superoxide increased ≈2-fold (P<0.05) after treatment with Ang II in IL-10−/− mice but not in wild-type. After systemic administration of Ang II (1.4 mg/kg per day for 10 days), Ang II produced modest impairment of endothelial function in wild-type mice but marked impairment in IL-10−/− mice (P<0.05) that was reversed by a superoxide scavenger. Increases in arterial pressure in response to Ang II were similar in wild-type and IL-10−/− mice. These findings provide the first evidence that endogenous IL-10 limits Ang II-mediated oxidative stress and vascular dysfunction both in vitro and in vivo suggesting that at least some of the protective effects of IL-10 may occur within the vessel wall.

Cerebral vascular effects of angiotensin II: new insights from genetic models.

Very little is known regarding the mechanisms of action of angiotensin II (Ang II) or the consequences of Ang II-dependent hypertension in the cerebral circulation. We tested the hypothesis that Ang II produces constriction of cerebral arteries that is mediated by activation of AT1A receptors and Rho-kinase. Basilar arteries (baseline diameter ~130 µm) from mice were isolated, cannulated and pressurized to measure the vessel diameter. Angiotensin II was a potent constrictor in arteries from male, but not female, mice. Vasoconstriction in response to Ang II was prevented by an inhibitor of Rho-kinase (Y-27632) in control mice, and was reduced by ~85% in mice deficient in expression of AT1A receptors. We also examined the chronic effects of Ang II using a model of Ang II-dependent hypertension, mice which overexpress human renin (R+) and angiotensinogen (A+). Responses to the endothelium-dependent agonist acetylcholine were markedly impaired in R+ A+ mice (P < 0.01) compared with controls, but were restored to normal by a superoxide scavenger (PEG-SOD). A-23187 (another endothelium-dependent agonist) produced vasodilation in control mice, but no response or vasoconstriction in R+ A+ mice. In contrast, dilation of the basilar artery in response to a NO donor (NONOate) was similar in R+ A+ mice and controls. Thus, Ang II produces potent constriction of cerebral arteries via activation of AT1A receptors and Rho-kinase. There are marked gender differences in cerebral vascular responses to Ang II. Endothelial function is greatly impaired in a genetic model of Ang II-dependent hypertension via a mechanism that involves superoxide.

Role of oxidative stress and AT1 receptors in cerebral vascular dysfunction with aging.

Vascular dysfunction occurs with aging. We hypothesized that oxidative stress and ANG II [acting via ANG II type 1 (AT(1)) receptors] promotes cerebral vascular dysfunction with aging. We studied young (5-6 mo), old (17-19 mo), and very old (23 +/- 1 mo) mice. In basilar arteries in vitro, acetylcholine (an endothelium-dependent agonist) produced dilation in young wild-type mice that was reduced by approximately 60 and 90% (P < 0.05) in old and very old mice, respectively. Similar effects were seen using A23187, a second endothelium-dependent agonist. The vascular response to acetylcholine in very old mice was almost completely restored with tempol (a scavenger of superoxide) and partly restored by PJ34, an inhibitor of poly(ADP-ribose) polymerase (PARP). We used mice deficient in Mn-SOD (Mn-SOD(+/-)) to test whether this form of SOD protected during aging but found that age-induced endothelial dysfunction was not altered by Mn-SOD deficiency. Cerebral vascular responses were similar in young mice lacking AT(1) receptors (AT(1)(-/-)) and wild-type mice. Vascular responses to acetylcholine and A23187 were reduced by approximately 50% in old wild-type mice (P < 0.05) but were normal in old AT(1)-deficient mice. Thus, aging produces marked endothelial dysfunction in the cerebral artery that is mediated by ROS, may involve the activation of PARP, but was not enhanced by Mn-SOD deficiency. Our findings suggest a novel and fundamental role for ANG II and AT(1) receptors in age-induced vascular dysfunction.

Angiotensin II–Induced Vascular Dysfunction Is Mediated by the AT1A Receptor in Mice

Abstract—Many of the actions of angiotensin II (Ang II) are mediated by angiotensin type 1 receptors (AT1), of which there are 2 pharmacologically indistinguishable subtypes (AT1A and AT1B). The purpose of this study was to evaluate the effect of an AT1A homozygous deletion (AT1A −/−) on vascular reactivity. AT1A −/− mice and control littermates (AT1A+/+) were infused with vehicle (saline) or Ang II (1000 ng · kg−1 · min−1) for 7 days by osmotic pumps. Systolic pressure was increased in AT1A+/+ mice (Δ45±8 mm Hg, P <0.0001) but unchanged in AT1A −/− mice (Δ5±3 mm Hg, P >0.013) on day 7. The carotid artery response to the vasodilators acetylcholine (Ach), nitroprusside, and papaverine and to the vasoconstrictors phenylephrine, U46619, 5-hydroxytryptamine (5-HT), and KCl were not different between vehicle-infused AT1A+/+ AT1A −/− animals. Carotid relaxation to ACh was impaired and contraction to 5-HT was increased in Ang II–infused AT1A+/+ mice. Ang II did not affect carotid responses in AT1A −/− mice. Superoxide, after vehicle or Ang II infusion, suggesting that it was not contributing to the altered Ach and 5-HT responses. The Rho-kinase inhibitor Y-27632 (1 μmol/L) attenuated the 5-HT response in both vehicle- and Ang II-infused AT1A+/+ mice. Moreover, concentration-dependent relaxation to Y-27632 and RhoA protein expression were not different in vehicle- or Ang II–infused AT1A+/+. These data demonstrate that the AT1A receptor is required for Ang II–induced changes in carotid artery function.

Effect of Aging, MnSOD Deficiency, and Genetic Background on Endothelial Function: Evidence for MnSOD Haploinsufficiency

Objective—The goal of this study was to compare vascular function, superoxide levels, and MnSOD protein expression in young (4 to 7 months) and old (22 to 24 months) MnSOD+/+ and MnSOD-deficient (MnSOD+/−) mice. Methods and Results—Relaxation of aorta in vitro to the endothelium-dependent dilator acetylcholine (ACh) was similar in young MnSOD+/+ (n=9) and young MnSOD+/− (n=6) mice. This response was impaired in old MnSOD+/+ (n=8) mice and old MnSOD+/− mice (n=14), with dysfunction being greater in old MnSOD-deficient mice (eg, 100 &mgr;mol/L ACh produced 77±3% [mean±SE], 77±3%, 70±4%, and 57±4% relaxation in young MnSOD+/+, young MnSOD+/−, old MnSOD+/+, and old MnSOD+/− mice, respectively). The endothelial dysfunction was similar in mice on both C57BL/6 and CD-1 genetic backgrounds. In contrast to ACh, responses to the endothelium-independent dilator sodium nitroprusside were enhanced in old MnSOD+/+ and MnSOD+/− mice compared with both groups of young mice (P<0.05). Superoxide levels, as measured using lucigenin-enhanced chemiluminescence, were increased more than 2-fold in old MnSOD+/− mice compared with old MnSOD+/+ and young mice (P<0.05). Conclusions—These data provide the first direct evidence that MnSOD halpoinsufficiency results in increased vascular oxidative stress and endothelial dysfunction with aging.