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Peroxidase Properties of Extracellular Superoxide Dismutase: Role of Uric Acid in Modulating In Vivo Activity
- H. Ulrich Hink,
- Nalini Santanam,
- Sergey Dikalov,
- Louise McCann,
- Andrew D. Nguyen,
- Sampath Parthasarathy,
- David G. Harrison and
- Tohru Fukai
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 22, Issue 91 September 2002

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Objective— The cytosolic form of Cu/Zn-containing superoxide dismutase (SOD1) has peroxidase activity, with H₂O₂ used as a substrate to oxidize other molecules. We examined peroxidase properties of the extracellular form of SOD (SOD3), a major isoform of SOD in the vessel wall, by using recombinant SOD3 and an in vivo model of atherosclerosis.Methods and Results— In the presence of HCO₃⁻, SOD3 reacted with H₂O₂ to produce a hydroxyl radical adduct of the spin trap 5-diethoxyphosphoryl-5methyl-1-pyrroline N-oxide (DEMPO). SOD1 and SOD3 were inactivated by H₂O₂ in a dose- and time-dependent fashion, and this was prevented by physiological levels of uric acid. To examine the in vivo role of uric acid on SOD1 and SOD3, control and apolipoprotein E-deficient (ApoE^−/−) mice were treated with oxonic acid, which inhibits urate metabolism. This treatment increased plasma levels of uric acid in control and ApoE^−/− mice by ≈3-fold. Although increasing uric acid levels did not alter aortic SOD1 and SOD3 protein expression, aortic SOD1 and SOD3 activities were increased by 2- to 3-fold in aortas from ApoE^−/− mice but not in aortas from control mice.Conclusions— These studies show that SOD1 and SOD3 are partially inactivated in atherosclerotic vessels of ApoE^−/− mice and that levels of uric acid commonly encountered in vivo may regulate vascular redox state by preserving the activity of these enzymes.
Electron Spin Resonance Detection of Hydrogen Peroxide as an Endothelium-Derived Hyperpolarizing Factor in Porcine Coronary Microvessels
- Tetsuya Matoba,
- Hiroaki Shimokawa,
- Keiko Morikawa,
- Hiroshi Kubota,
- Ikuko Kunihiro,
- Lemmy Urakami-Harasawa,
- Yasushi Mukai,
- Yoji Hirakawa,
- Takaaki Akaike and
- Akira Takeshita
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 23, Issue 71 July 2003

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Objective— Endothelium-derived hyperpolarizing factor (EDHF) plays an important role in modulating vascular tone, especially in microvessels, although its nature has yet to be elucidated. This study was designed to examine whether hydrogen peroxide (H₂O₂) is an EDHF in porcine coronary microvessels with use of an electron spin resonance (ESR) method to directly detect H₂O₂ production from the endothelium.Methods and Results— Isometric tension and membrane-potential recordings demonstrated that bradykinin and substance P caused EDHF-mediated relaxations and hyperpolarizations of porcine coronary microvessels in the presence of indomethacin and N^ω-nitro-l-arginine. The contribution of H₂O₂ to the EDHF-mediated responses was demonstrated by the inhibitory effect of catalase and by the relaxing and hyperpolarizing effects of exogenous H₂O₂. Endothelial production of H₂O₂ was quantified in bradykinin- or substance P–stimulated intact blood vessels by ESR spectroscopy. Tiron, a superoxide scavenger that facilitates H₂O₂ formation, enhanced bradykinin-induced production of H₂O₂, as well as the EDHF-mediated relaxations and hyperpolarizations. By contrast, cytochrome P-450 inhibitors (sulfaphenazole or 17-octadecynoic acid) or a gap junction inhibitor (18α-glycyrrhetinic acid) failed to inhibit the EDHF-mediated relaxations. Involvement of endothelium-derived K⁺ was not evident in experiments with ouabain plus Ba²⁺ or exogenous K⁺.Conclusion— These results provide ESR evidence that H₂O₂ is an EDHF in porcine coronary microvessels.
p47phox Associates With the Cytoskeleton Through Cortactin in Human Vascular Smooth Muscle Cells: Role in NAD(P)H Oxidase Regulation by Angiotensin II
- R.M. Touyz,
- G. Yao,
- M.T. Quinn,
- P.J. Pagano and
- E.L. Schiffrin
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 25, Issue 31 March 2005

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Objective— We tested the hypothesis that p47phox associates with the actin cytoskeleton, enabling site-directed activation of NAD(P)H oxidase, and assessed whether these actions influence reactive oxygen species (ROS) generation and signaling by angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs) from human resistance and coronary arteries.Methods and Results— Electroporation of anti-p47phox antibody into VSMCs abrogated Ang II-mediated O₂ generation, establishing the requirement for p47phox in this response. Immunfluorescence confocal microscopy demonstrated a cytosolic distribution of p47phox in basal conditions. After Ang II stimulation, p47phox rearranged in a linear fashion, colocalizing with F-actin. Co-immunoprecipitation studies confirmed an association between p47phox and actin and demonstrated an interaction with the actin-binding protein cortactin. Cytoskeletal disruption with cytochalasin prevented p47phox:actin interaction and attenuated ROS formation and p38MAP kinase and Akt phosphorylation by Ang II. Intracellular ROS generation in response to LY83583 (O₂ generator) or exogenous H₂O₂ and Ang II-induced ERK1/2 activation were unaltered by cytochalasin.Conclusions— The p47phox:actin interaction, through cortactin, plays an important role in Ang II-mediated site-directed assembly of functionally active NAD(P)H oxidase, ROS generation, and activation of redox-sensitive p38MAP kinase and Akt, but not ERK1/2. These findings demonstrate the importance of an intact actin-cytoskeleton in NAD(P)H oxidase regulation and redox signaling by Ang II in human VSMCs.We demonstrate that p47phox:actin interaction, through cortactin, is involved in Ang II-mediated site-directed assembly of NAD(P)H oxidase, ROS generation, and activation of redox-sensitive p38MAPK and Akt, but not ERK1/2. These findings demonstrate the importance of an intact actin-cytoskeleton in NAD(P)H oxidase regulation and redox-signaling by Ang II in human VSMCs.
Regulation of Xanthine Oxidoreductase Protein Expression by Hydrogen Peroxide and Calcium
- J. Scott McNally,
- Archana Saxena,
- Hua Cai,
- Sergey Dikalov and
- David G. Harrison
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 25, Issue 81 August 2005

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Objective— We have previously demonstrated that endothelial xanthine oxidase (XO) levels are dependent on the NADPH oxidase. We postulated that H₂O₂ may modulate the irreversible conversion of xanthine dehydrogenase (XDH) to XO and sought to examine mechanisms involved.Methods and Results— H₂O₂ (100 μmol/L) decreased bovine aortic endothelial cell (BAEC) XDH protein expression, and metabolic labeling studies indicated that H₂O₂ stimulated conversion of XDH to XO. The decline in XDH was mimicked by the reactive oxygen species (ROS) generating compounds SIN-1 and Menadione, as well as by stimulating BAECs with angiotensin II (200 nmol/L). BAPTA-AM prevented the decline in XDH by H₂O₂, indicating that it was calcium-dependent. In keeping with calcium acting downstream of H₂O₂, the calcium ionophore A23187 (1 μmol/L) caused XDH-to-XO conversion, and this was not prevented by the antioxidants. In addition, XDH-to-XO conversion was blocked by 2-APB and NO donors and induced by thapsigargin and M-3M3FBS, implicating phospholipase C and endoplasmic reticulum calcium stores in this process.Conclusions— Endothelial XO and XDH expression are strongly dependent on H₂O₂ and calcium. Stimulation of XDH conversion to XO may represent a feed-forward mechanism whereby H₂O₂ can stimulate further production of ROS.In this study we found that H₂O₂-dependent calcium release from the ER caused conversion of XDH to XO, and that this was prevented by nitric oxide. This may represent a feed-forward mechanism in many disease processes whereby H₂O₂ can stimulate further ROS production through XDH-to-XO conversion.
Upregulation of Arginase by H₂O₂ Impairs Endothelium-Dependent Nitric Oxide-Mediated Dilation of Coronary Arterioles
- Naris Thengchaisri,
- Travis W. Hein,
- Wei Wang,
- Xin Xu,
- Zhenbo Li,
- Theresa W. Fossum and
- Lih Kuo
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 26, Issue 91 September 2006

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Objective— Overproduction of reactive oxygen species such as hydrogen peroxide (H₂O₂) has been implicated in various cardiovascular diseases. However, mechanism(s) underlying coronary vascular dysfunction induced by H₂O₂ is unclear. We studied the effect of H₂O₂ on dilation of coronary arterioles to endothelium-dependent and endothelium-independent agonists.Methods and Results— Porcine coronary arterioles were isolated and pressurized without flow for in vitro study. All vessels developed basal tone and dilated dose-dependently to activators of nitric oxide (NO) synthase (adenosine and ionomycin), cyclooxygenase (arachidonic acid), and cytochrome P450 monooxygenase (bradykinin). Intraluminal incubation of vessels with H₂O₂ (100 μmol/L, 60 minutes) did not alter basal tone but inhibited vasodilations to adenosine and ionomycin in a manner similar as that by NO synthase inhibitor L-NAME. H₂O₂ affected neither endothelium-dependent responses to arachidonic acid and bradykinin nor endothelium-independent dilation to sodium nitroprusside. The inhibited adenosine response was not reversed by removal of H₂O₂ but was restored by excess L-arginine. Inhibition of L-arginine consuming enzyme arginase by α-difluoromethylornithine or N^ω-hydroxy-nor-l-arginine also restored vasodilation. Administering deferoxamine, an inhibitor of hydroxyl radical production, prevented the H₂O₂-induced impairment of vasodilation to adenosine. Western blot and reverse-transcription polymerase chain reaction results indicated that arginase I was upregulated after treating vessels with H₂O₂.Conclusions— H₂O₂ specifically impairs endothelium-dependent NO-mediated dilation of coronary microvessels by reducing L-arginine availability through upregulation of arginase. The formation of hydroxyl radicals from H₂O₂ may contribute to this process.Overproduction of reactive oxygen species such as hydrogen peroxide (H₂O₂) has been implicated in various cardiovascular diseases. Treatment of isolated coronary arterioles with H₂O₂ specifically attenuated endothelium-dependent NO-mediated dilation through the upregulation of arginase. Activation of this pathway may contribute to vascular dysfunction associated with oxidative stress.
Exercise Training Restores Coronary Arteriolar Dilation to NOS Activation Distal to Coronary Artery Occlusion: Role of Hydrogen Peroxide
- Naris Thengchaisri,
- Robert Shipley,
- Yi Ren,
- Janet Parker and
- Lih Kuo
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 27, Issue 41 April 2007

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Objective— Exercise training has been shown to restore vasodilation to nitric oxide synthase (NOS) activation in arterioles distal to coronary artery occlusion. Because reactive oxygen species are generated during NOS uncoupling and the production of vasodilator H₂O₂ is increased during exercise in patients with coronary disease, we proposed that H₂O₂ may contribute to the restoration of vasodilation in porcine coronary occlusion model.Methods and Results— Left circumflex (LCX) coronary artery of miniature swine was progressively occluded for 8 weeks followed by exercise training (EX; 5 days/wk treadmill) or sedentary (SED) protocols for 12 weeks. Arterioles were isolated from distal LCX and nonoccluded left anterior descending (LAD) artery for in vitro study. Vasodilation to NOS activators adenosine and ionomycin was impaired in SED LCX, but not LAD, arterioles. This impairment was restored by L-arginine. NO production induced by adenosine was also reduced in SED LCX arterioles. EX had no effect on LAD arterioles but improved NO production and restored dilation of LCX arterioles. NOS blockade (L-NAME) inhibited vasodilation to NOS activators in LAD (SED & EX) arterioles but was ineffective in SED LCX arterioles. In EX LCX arterioles, vasodilation to NOS activators was slightly inhibited by L-NAME but abolished by catalase. H₂O₂ production was markedly increased by adenosine in EX LCX arterioles.Conclusions— This study demonstrates that endothelium-dependent NO-mediated dilation is impaired in SED LCX arterioles and that EX training restores the impaired function. It appears that H₂O₂, in addition to NO, contributes significantly to EX-induced restoration of endothelium-dependent dilation of coronary arterioles distal to occlusion.This study demonstrates that endothelium-dependent NO-mediated dilation is impaired in SED LCX arterioles and that EX training restores the impaired function. It appears that H₂O₂, in addition to NO, contributes significantly to EX-induced restoration of endothelium-dependent dilation of coronary arterioles distal to occlusion.
Overexpression of Human Catalase Gene Decreases Oxidized Lipid-Induced Cytotoxicity in Vascular Smooth Muscle Cells
- Nalini Santanam,
- Nathalie Augé,
- Mimi Zhou,
- Channa Keshava and
- Sampath Parthasarathy
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 19, Issue 81 August 1999

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Abstract—Reactive oxygen metabolites such as hydrogen peroxide (H₂O₂) and oxidized fatty acids are proinflammatory and are involved in the pathophysiology of various diseases including atherosclerosis. The effects of these oxidants could be inhibited by the external addition of an antioxidant, suggesting the promotion or propagation of further oxidation. In this study, we describe the stable overexpression of human catalase in smooth muscle cells and the resistance of these cells to cytotoxicity induced not only by the addition of H₂O₂ but also by the addition of 13-hydroperoxyoctadecadienoic acid (13-HPODE). The results pose an intriguing possibility of the generation of H₂O₂ from a peroxidized fatty acid. Accordingly, incubation of cells with both 13-HPODE and 13-hydroxyoctadecadienoic acid resulted in the generation of intracellular H₂O₂. To explain the observed results by which catalase could overcome the effects of 13-HPODE, we propose that oxidized fatty acids are degraded in the cellular peroxisomes, resulting in the generation of H₂O₂. In other words, the cellular effects of peroxidized fatty acids could be attributed to the generation of H₂O₂.
Angiotensin II Stimulates Endothelial Vascular Cell Adhesion Molecule-1 via Nuclear Factor-κB Activation Induced by Intracellular Oxidative Stress
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 20, Issue 31 March 2000

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Abstract—The recruitment of monocytes via the endothelial expression of vascular cell adhesion molecule-1 (VCAM-1) is a key step in the formation of the initial lesion in atherosclerosis. Because angiotensin (Ang) II may be involved in this process, we investigated its role on the signaling cascade leading to VCAM-1 expression in endothelial cells. Ang II stimulates mRNA and protein expression of VCAM-1 in these cells via the AT₁ receptor. This effect was enhanced by N^G-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor, and blocked by pyrrolidinedithiocarbamate, an antioxidant molecule. Ang II activated the redox-sensitive transcription factor nuclear factor-κB and stimulated the degradation of both inhibitor of κB (IκB)α and IκBβ with different kinetics. The degradation of IκBs induced by Ang II was not modified by incubation with exogenous superoxide dismutase and catalase, suggesting that this effect was not mediated by the extracellular production of O₂⁻. In contrast, rotenone and antimycin, 2 inhibitors of the mitochondrial respiratory chain, inhibited the Ang II–induced IκB degradation, showing that generation of reactive oxygen species in the mitochondria is involved on Ang II action. BXT-51702, a glutathione peroxidase mimic, inhibited the effect of Ang II, and aminotriazole, an inhibitor of catalase, enhanced it, suggesting a role for H₂O₂ in IκB degradation. This is confirmed by experiments showing that Ang II stimulates the intracellular production of H₂O₂ in endothelial cells. These results demonstrate that Ang II induced an intracellular oxidative stress in endothelial cells, which stimulates IκB degradation and nuclear factor-κB activation. This activation enhances the expression of VCAM-1 and probably other genes involved in the early stages of atherosclerosis.
Enhanced H₂O₂-Induced Cytotoxicity in “Epithelioid” Smooth Muscle Cells : Implications for Neointimal Regression
- Wei-Gen Li,
- Francis J. Miller,
- Michael R. Brown,
- Papri Chatterjee,
- Gregory R. Aylsworth,
- Jianqiang Shao,
- Arthur A. Spector,
- Larry W. Oberley and
- Neal L. Weintraub
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 20, Issue 61 June 2000

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Abstract—Vascular smooth muscle cells (SMCs) are phenotypically diverse. Although most medial SMCs can be classified as “fusiform,” others are of the “epithelioid” phenotype. Proliferation and apoptosis of epithelioid SMCs may contribute importantly to neointimal formation and regression, respectively. Because reactive oxygen species (ROS) are increased in vascular injury and can induce apoptosis of SMCs, we compared the effects of ROS on epithelioid and fusiform SMCs. Epithelioid and fusiform SMC lines were clonally isolated from rat aortic media and studied under similar conditions and passage numbers. H₂O₂ produced dose- and time-dependent cytotoxicity that was enhanced in epithelioid compared with fusiform cells. After 24-hour exposure to 50 μmol/L H₂O₂, epithelioid cell numbers were reduced by 34±5% versus a 3±5% (P<0.05) reduction in fusiform cell numbers. Similar results were obtained whether H₂O₂ was administered to growth-arrested or growing cells or when epithelioid and fusiform cells were exposed to extracellular O₂^.−. To investigate whether apoptosis contributed to enhanced ROS-induced cytotoxicity in epithelioid SMCs, terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) staining was performed. The incidence of TUNEL positivity was 5-fold increased in epithelioid versus fusiform SMCs after treatment with 50 μmol/L H₂O₂ (19±1% epithelioid versus 5±1% fusiform, P<0.05). Enhanced H₂O₂-induced apoptosis in epithelioid SMCs was confirmed by DNA laddering. Furthermore, when balloon-injured aortas were exposed to H₂O₂ ex vivo, enhanced apoptosis was observed in neointimal compared with medial SMCs. These results suggest that epithelioid SMCs exhibit enhanced sensitivity to ROS-induced apoptosis, which may play an important role in neointimal regression.
Reactive Oxygen Species Mediate Endothelium-Dependent Relaxations in Tetrahydrobiopterin-Deficient Mice
- Francesco Cosentino,
- Jane E. Barker,
- Michael P. Brand,
- Simon J. Heales,
- Ernst R. Werner,
- John R. Tippins,
- Nick West,
- Keith M. Channon,
- Massimo Volpe and
- Thomas F. Lüscher
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 21, Issue 41 April 2001

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Abstract—(6R)-5,6,7,8-Tetrahydro-biopterin (H₄B) is essential for the catalytic activity of all NO synthases. The hyperphenylalaninemic mouse mutant (hph-1) displays 90% deficiency of the GTP cyclohydrolase I, the rate-limiting enzyme in H₄B synthesis. A relative shortage of H₄B may shift the balance between endothelial NO synthase (eNOS)-catalyzed generation of NO and reactive oxygen species. Therefore, the hph-1 mouse represents a unique model to assess the effect of chronic H₄B deficiency on endothelial function. Aortas from 8-week-old hph-1 and wild-type mice (C57BL×CBA) were compared. H₄B levels were determined by high-performance liquid chromatography and NO synthase activity by [³H]citrulline assay in homogenized tissue. Superoxide production by the chemiluminescence method was measured. Isometric tension was continuously recorded. The intracellular levels of H₄B as well as constitutive NO synthase activity were significantly lower in hph-1 compared with wild-type mice. Systolic blood pressure was increased in hph-1 mice. However, endothelium-dependent relaxations to acetylcholine were present in both groups and abolished by inhibition of NO synthase with N^G-nitro-l-arginine methyl ester as well. Only in hph-1 mice were the relaxations inhibited by catalase and enhanced by superoxide dismutase. After incubation with exogenous H₄B, the differences between the 2 groups disappeared. Our findings demonstrate that H₄B deficiency leads to eNOS dysfunction with the formation of reactive oxygen species, which become mediators of endothelium-dependent relaxations. A decreased availability of H₄B may favor an impaired activity of eNOS and thus contribute to the development of vascular diseases.
NO or H₂O₂ for Endothelium-Dependent Vasorelaxation : Tetrahydrobiopterin Makes the Difference
- Victor W. M. van Hinsbergh
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 21, Issue 51 May 2001

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Estrogen Prevents Oxidative Stress–Induced Endothelial Cell Apoptosis in Rats
- Noriko Sudoh,
- Kenji Toba,
- Masahiro Akishita,
- Junya Ako,
- Masayoshi Hashimoto,
- Katsuya Iijima,
- Seungbum Kim,
- Yi-Qiang Liang,
- Yumiko Ohike,
- Tokumitsu Watanabe,
- Ieharu Yamazaki,
- Masao Yoshizumi,
- Masato Eto and
- Yasuyoshi Ouchi
Circulation Volume 103, Issue 56 February 2001

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Background—Estrogen replacement attenuates the increased risk of cardiovascular disease in postmenopausal women. Recent studies using an in vitro culture system have shown that estrogen inhibits endothelial cell (EC) apoptosis. The in vivo relevance of this finding, however, is not defined. To do so, we have developed a rat vascular injury model in which EC apoptosis induced by hydrogen peroxide plays a role.Methods and Results—Intracarotid arterial administration of 0.01 mmol/L hydrogen peroxide for 5 minutes evoked EC apoptosis after 6 to 24 hours, determined by nuclear staining with Hoechst 33342, terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling, and electron microscopy. Apoptosis was associated with EC loss and was followed by EC regeneration at 72 hours and neointima formation at 1 to 2 weeks. Estradiol replacement in ovariectomized female Wistar rats decreased the rate of apoptotic ECs by ≈50%, assayed by nuclear morphology of en face specimens, resulting in increased remaining ECs and decreased neointima formation. Progesterone did not influence the effects of estradiol on EC apoptosis.Conclusions—These results provide new insight into the cardioprotective action of estrogen as well as a paradigm of the response-to-injury hypothesis.
Novel Nitric Oxide Synthase–Dependent Mechanism of Vasorelaxation in Small Arteries From Hypertensive Rats
- Kyu-Tae Kang,
- Jennifer C. Sullivan,
- Jennifer M. Sasser,
- John D. Imig and
- Jennifer S. Pollock
Hypertension Volume 49, Issue 41 April 2007

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To determine the mechanism(s) involved in vasorelaxation of small arteries from hypertensive rats, normotensive (NORM), angiotensin II-infused (ANG), high-salt (HS), ANG high-salt (ANG/HS), placebo, and deoxycorticosterone acetate-salt rats were studied. Third-order mesenteric arteries from ANG or ANG/HS displayed decreased sensitivity to acetylcholine (ACh)-induced vasorelaxation compared with NORM or HS, respectively. Maximal relaxations were comparable between groups. Blockade of Ca²⁺-activated K⁺ channels had no effect on ANG versus blunting relaxation in NORM (log EC₅₀: −6.8±0.1 versus −7.2±0.1 mol/L). NO synthase (NOS) inhibition abolished ACh-mediated relaxation in small arteries from ANG, ANG/HS, and deoxycorticosterone acetate-salt versus blunting relaxation in NORM, HS, and placebo (% maximal relaxation: ANG: 2.7±1.8; ANG/HS: 7.2±3.2; NORM: 91±3.1; HS: 82.1±13.3; deoxycorticosterone acetate-salt: 35.2±17.7; placebo: 79.3±10.3), indicating that NOS is the primary vasorelaxation pathway in these arteries from hypertensive rats. We hypothesized that NO/cGMP signaling and NOS-dependent H₂O₂ maintains vasorelaxation in small arteries from ANG. ACh increased NOS-dependent cGMP production, indicating that NO/cGMP signaling is present in small arteries from ANG (55.7±6.9 versus 30.5±5.1 pmol/mg), and ACh stimulated NOS-dependent H₂O₂ production (ACh: 2.8±0.2 μmol/mg; N_ω-nitro-l-arginine methyl ester hydrochloride+ACh: 1.8±0.1 μmol/mg) in small arteries from ANG. H₂O₂ induced vasorelaxation and catalase blunted ACh-mediated vasorelaxation. In conclusion, Ca²⁺-activated K⁺ channel–mediated relaxation is dysfunctional in small mesenteric arteries from hypertensive rats, and the NOS pathway compensates to maintain vasorelaxation in these arteries through NOS-mediated cGMP and H₂O₂ production.
Oxidative Stress in the Dahl Hypertensive Rat
- Allen Swei,
- Fred Lacy,
- Frank A. DeLano and
- Geert W. Schmid-Schönbein
Hypertension Volume 30, Issue 61 December 1997

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Abstract Enhanced production of oxygen free radicals may play a role in hypertension by affecting vascular smooth muscle contraction, resistance to blood flow, and organ damage. The aim of this study was to determine whether oxygen free radicals are involved in the development of salt-induced hypertension. Dahl salt-sensitive (Dahl-S) and salt-resistant (Dahl-R) rats were fed either a high salt (6.0% NaCl) or low salt (0.3% NaCl) diet for 4 weeks. The high salt diet caused the development of severe hypertension in Dahl-S animals and had no effect on blood pressure in Dahl-R animals. A tetranitroblue tetrazolium dye was used to detect superoxide radicals in microvessels of the mesentery. Light absorption measurements revealed enhanced staining along the endothelium of arterioles and venules in hypertensive Dahl-S animals, with significantly lower values in normotensive animals. In addition, a Clark electrochemical electrode was used to measure hydrogen peroxide levels in fresh plasma. Hypertensive Dahl-S animals had a higher plasma hydrogen peroxide concentration compared with their normotensive counterparts (2.81±0.43 versus 2.10±0.41 μmol/L), while no difference was detected between high- and low salt–treated Dahl-R animals (1.70±0.35 versus 1.56±0.51 μmol/L). The plasma hydrogen peroxide levels of all groups correlated with mean arterial pressure (r=.77). These findings demonstrate an enhanced production of oxygen free radicals in the microvasculature of hypertensive Dahl-S rats.
Role of NADH/NADPH Oxidase–Derived H₂O₂ in Angiotensin II–Induced Vascular Hypertrophy
- A. Maziar Zafari,
- Masuko Ushio-Fukai,
- Marjorie Akers,
- Qiqin Yin,
- Aalok Shah,
- David G. Harrison,
- W. Robert Taylor and
- Kathy K. Griendling
Hypertension Volume 32, Issue 31 September 1998

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Abstract—Recent evidence suggests that oxidative mechanisms may be involved in vascular smooth muscle cell (VSMC) hypertrophy. We previously showed that angiotensin II (Ang II) increases superoxide production by activating an NADH/NADPH oxidase, which contributes to hypertrophy. In this study, we determined whether Ang II stimulation of this oxidase results in H₂O₂ production by studying the effects of Ang II on intracellular H₂O₂ generation, intracellular superoxide dismutase and catalase activity, and hypertrophy. Ang II (100 nmol/L) significantly increased intracellular H₂O₂ levels at 4 hours. Neither superoxide dismutase activity nor catalase activity was affected by Ang II; the SOD present in VSMCs is sufficient to metabolize Ang II–stimulated superoxide to H₂O₂, which accumulates more rapidly than it is degraded by catalase. This increase in H₂O₂ was inhibited by extracellular catalase, diphenylene iodonium, an inhibitor of the NADH/NADPH oxidase, and the AT₁ receptor blocker losartan. In VSMCs stably transfected with antisense p22phox, a critical component of the NADH/NADPH oxidase in which oxidase activity was markedly reduced, Ang II–induced production of H₂O₂ was almost completely inhibited, confirming that the source of Ang II–induced H₂O₂ was the NADH/NADPH oxidase. Using a novel cell line that stably overexpresses catalase, we showed that this increased H₂O₂ is a critical step in VSMC hypertrophy, a hallmark of many vascular diseases. Inhibition of intracellular superoxide dismutase by diethylthiocarbamate (1 mmol/L) also resulted in attenuation of Ang II–induced hypertrophy (62±2% inhibition). These data indicate that AT₁ receptor–mediated production of superoxide generated by the NADH/NADPH oxidase is followed by an increase in intracellular H₂O₂, suggesting a specific role for these oxygen species and scavenging systems in modifying the intracellular redox state in vascular growth.
Reactive Oxygen Species Are Critical in the Oleic Acid–Mediated Mitogenic Signaling Pathway in Vascular Smooth Muscle Cells
- Gang Lu,
- Eddie L. Greene,
- Toshi Nagai and
- Brent M. Egan
Hypertension Volume 32, Issue 61 December 1998

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Abstract—Obese hypertensive patients with cardiovascular risk factor clustering have increased plasma nonesterified fatty acid levels and are at high risk for atherosclerotic events. Our previous studies demonstrated that oleic acid induces a mitogenic response in rat aortic smooth muscle cells (RASMCs) through protein kinase C (PKC)– and extracellular signal–regulated kinase (ERK)–dependent pathways. In the present study we investigated the possibility that the generation of reactive oxygen species (ROS) constitutes a critical component of the oleic acid–induced mitogenic signaling pathway in RASMCs. We studied the effect(s) of oleic acid on the generation of ROS using the oxidant-sensitive fluoroprobe 2′,7′-dichlorofluorescin diacetate. Relative fluorescence intensity and fluorescent images were obtained with laser confocal scanning microscopy from 1 to 5 minutes, since preliminary studies demonstrated that the peak fluorescence intensity occurred within 5 minutes. Oleic acid (100 μmol/L) induced a time-dependent increase of cell fluorescence that was >8-fold of that seen in control cells at 5 minutes. This was blocked by catalase, which suggests that H₂O₂ was the principal ROS. The oleic acid–induced increases in H₂O₂ were blocked when PKC was inhibited with the use of bisindolylmaleimide and when PKC activity was downregulated by exposing RASMCs to phorbol 12–myristate 13–acetate for 24 hours. Stearic and elaidic acids, which are weak PKC activators, did not significantly increase H₂O₂ production. The increase of H₂O₂ in response to oleic acid was inhibited by the antioxidant N-acetylcysteine. N-Acetylcysteine also completely blocked ERK activation and the increase of thymidine incorporation in response to oleic acid. The data suggest that generation of H₂O₂ in RASMCs exposed to oleic acid is PKC dependent. Moreover, H₂O₂ production emerges as a critical intermediary event in the oleic acid–mediated mitogenic signaling pathway between the activation of PKC and ERK. These observations raise the possibility that the elevated plasma nonesterified fatty acids, including oleic acid, in obese hypertensive patients contribute to vascular growth and remodeling by a PKC-dependent mechanism to generate ROS that subsequently activate ERK.
O₂ Sensing in the Human Ductus Arteriosus: Regulation of Voltage-Gated K⁺ Channels in Smooth Muscle Cells by a Mitochondrial Redox Sensor
- Evangelos D. Michelakis,
- Ivan Rebeyka,
- XiChen Wu,
- Ali Nsair,
- Bernard Thébaud,
- Kyoko Hashimoto,
- Jason R.B. Dyck,
- Al Haromy,
- Gwyneth Harry,
- Amy Barr and
- Stephen L. Archer
Circulation Research Volume 91, Issue 620 September 2002

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Functional closure of the human ductus arteriosus (DA) is initiated within minutes of birth by O₂ constriction. It occurs by an incompletely understood mechanism that is intrinsic to the DA smooth muscle cell (DASMC). We hypothesized that O₂ alters the function of an O₂ sensor (the mitochondrial electron transport chain, ETC) thereby increasing production of a diffusible redox-mediator (H₂O₂), thus triggering an effector mechanism (inhibition of DASMC voltage-gated K⁺ channels, Kv). O₂ constriction was evaluated in 26 human DAs (12 female, aged 9±2 days) studied in their normal hypoxic state or after normoxic tissue culture. In fresh, hypoxic DAs, 4-aminopyridine (4-AP), a Kv inhibitor, and O₂ cause similar constriction and K⁺ current inhibition (I_K). Tissue culture for 72 hours, particularly in normoxia, causes ionic remodeling, characterized by decreased O₂ and 4-AP constriction in DA rings and reduced O₂- and 4-AP–sensitive I_K in DASMCs. Remodeled DAMSCs are depolarized and express less O₂-sensitive channels (including Kv2.1, Kv1.5, Kv9.3, Kv4.3, and BK_Ca). Kv2.1 adenoviral gene-transfer significantly reverses ionic remodeling, partially restoring both the electrophysiological and tone responses to 4-AP and O₂. In fresh DASMCs, ETC inhibitors (rotenone and antimycin) mimic hypoxia, increasing I_K and reversing constriction to O₂, but not phenylephrine. O₂ increases, whereas hypoxia and ETC inhibitors decrease H₂O₂ production by altering mitochondrial membrane potential (ΔΨm). H₂O₂, like O₂, inhibits I_K and depolarizes DASMCs. We conclude that O₂ controls human DA tone by modulating the function of the mitochondrial ETC thereby varying ΔΨm and the production of H₂O₂, which regulates DASMC Kv channel activity and DA tone.
Hypoxic Pulmonary Vasoconstriction: Ups and Downs of Reactive Oxygen Species
- James S.K. Sham
Circulation Research Volume 91, Issue 818 October 2002

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Stretch Enhances Contraction of Bovine Coronary Arteries via an NAD(P)H Oxidase–Mediated Activation of the Extracellular Signal–Regulated Kinase Mitogen-Activated Protein Kinase Cascade
- Richard A. Oeckler,
- Pawel M. Kaminski and
- Michael S. Wolin
Circulation Research Volume 92, Issue 110 January 2003

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This study examines the effects of an increase in passive stretch in endothelium-removed bovine coronary artery on oxidant-induced changes in force generation. Increasing passive stretch on the arterial segments from 5 to 20 g for 20 minutes caused a subsequent increase (P<0.05) in force generation to 30 mmol/L KCl or 0.1 μmol/L serotonin compared with the prestretch control response. Also associated with the passive stretch were increases in superoxide detection by lucigenin and a selective increase in extracellular signal–regulated kinase (ERK) mitogen-activated protein (MAP) kinase phosphorylation measured by Western analysis. The stretch-induced increase in force generation was eliminated by inhibition of the ERK pathway by the MEK inhibitor PD98059 but not by inhibitors of the p38 MAP kinase pathway (SB202190) or c-Jun N-terminal protein kinase pathway (SP200169). Additionally, stretch-induced increases in both ERK phosphorylation and force generation were attenuated by inhibition of tyrosine kinases (genistein), src (PP2), and specific sites on the epidermal growth factor receptor (EGFR) (AG1478). Probes for oxidant signaling, including NAD(P)H oxidase inhibitors (diphenyliodonium and apocynin) or enhancement of peroxide consumption (ebselen) but not inhibition of xanthine oxidase (allopurinol), attenuated the effects of stretch on both ERK phosphorylation and force generation. Furthermore, stretch caused an increase in EGFR phosphorylation and cytosolic to membrane translocation of the p47phox NAD(P)H oxidase subunit. Hydrogen peroxide also elicited contraction through EGFR phosphorylation and ERK. In summary, stretch seems to enhance force generation via ERK signaling through an EGFR/src-dependent mechanism activated by peroxide derived from a stretch-mediated activation of the NAD(P)H oxidase, a response that may contribute to hypertensive alterations in vascular reactivity.
Origins of Serum Semicarbazide-Sensitive Amine Oxidase
- Craig M. Stolen,
- Gennady G. Yegutkin,
- Riikka Kurkijärvi,
- Petri Bono,
- Kari Alitalo and
- Sirpa Jalkanen
Circulation Research Volume 95, Issue 19 July 2004

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Semicarbazide-sensitive amine oxidases (SSAO) are enzymes that are capable of deaminating primary amines to produce aldehyde, ammonia, and hydrogen peroxide. This activity has been associated with vascular adhesion protein-1 (VAP-1) and is found in the serum, endothelium, adipose, and smooth muscle of mammals. Circulating SSAO activity is increased in congestive heart failure, diabetes, and inflammatory liver diseases. To investigate the origin of circulating SSAO activity, two transgenic mouse models were created with full-length human VAP-1 (hVAP-1) expressed on either endothelial (mTIEhVAP-1) or adipose tissues (aP2hVAP-1), with tie-1 and adipocyte P2 promoters, respectively. Under normal conditions a circulating form of hVAP-1 was found at high levels in the serum of mice with endothelium-specific expression and at low levels in the serum of mice with adipose specific expression. The level of circulating hVAP-1 in the transgenic mice varied with gender, transgene zygosity, diabetes, and fasting. Serum SSAO activity was absent from VAP-1 knockout mice and endothelial cell–specific expression of human VAP-1 restored SSAO activity to the serum of VAP-1 knockout mice. Together, these experiments show that in the mouse VAP-1 is the only source of serum SSAO, that under physiological conditions vascular endothelial cells can be a major source of circulating VAP-1 protein and SSAO, and that serum VAP-1 can originate from both endothelial cells and adipocytes during experimental diabetes. An increased endothelial cell capacity for lymphocyte binding and altered expression of redox-sensitive proteins was also associated with the mTIEhVAP-1 transgene.

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