Louise McCann

Publications of Louise McCann

• Therapeutic targeting of mitochondrial superoxide in hypertension.

  Authors: Anna E Dikalova, Alfiya T Bikineyeva, Klaudia Budzyn, Rafal R Nazarewicz, Louise McCann, William Lewis, David G Harrison, Sergey I Dikalov

  Circulation research. 05/2010; 107(1):106-16.

  Superoxide (O2(-) ) has been implicated in the pathogenesis of many human diseases including hypertension; however, commonly used antioxidants have proven ineffective in clinical trials. It isSuperoxide (O2(-) ) has been implicated in the pathogenesis of many human diseases including hypertension; however, commonly used antioxidants have proven ineffective in clinical trials. It is possible that these agents are not adequately delivered to the subcellular sites of superoxide production. Because the mitochondria are important sources of reactive oxygen species, we postulated that mitochondrial targeting of superoxide scavenging would have therapeutic benefit. In this study, we found that the hormone angiotensin (Ang II) increased endothelial mitochondrial superoxide production. Treatment with the mitochondria-targeted antioxidant mitoTEMPO decreased mitochondrial O2(-), inhibited the total cellular O2(-), reduced cellular NADPH oxidase activity, and restored the level of bioavailable NO. These effects were mimicked by overexpressing the mitochondrial MnSOD (SOD2), whereas SOD2 depletion with small interfering RNA increased both basal and Ang II-stimulated cellular O2(-). Treatment of mice in vivo with mitoTEMPO attenuated hypertension when given at the onset of Ang II infusion and decreased blood pressure by 30 mm Hg following establishment of both Ang II-induced and DOCA salt hypertension, whereas a similar dose of nontargeted TEMPOL was not effective. In vivo, mitoTEMPO decreased vascular O2(-), increased vascular NO production and improved endothelial-dependent relaxation. Interestingly, transgenic mice overexpressing mitochondrial SOD2 demonstrated attenuated Ang II-induced hypertension and vascular oxidative stress similar to mice treated with mitoTEMPO. These studies show that mitochondrial O2(-) is important for the development of hypertension and that antioxidant strategies specifically targeting this organelle could have therapeutic benefit in this and possibly other diseases.

• Role of SOD3 in circumventricular organs versus the vascular smooth muscle in control of blood pressure

  Authors: Heinrich E Lob, Paul J Marvar, Louise McCann, Stefan Offermanns, Frank Gordon, David G Harrison

  Experimental Biology 2009, New Orleans; 04/2009

  We have shown that mice lacking the extracellular superoxide dismutase (SOD3) have augmented ang II-induced hypertension. To determine where SOD3 exerts this protective effect, we flanked the SOD3We have shown that mice lacking the extracellular superoxide dismutase (SOD3) have augmented ang II-induced hypertension. To determine where SOD3 exerts this protective effect, we flanked the SOD3 coding region with loxP sites. To examine a role of central SOD3, we performed intracerebroventricular (ICV) injection of an adenovirus encoding cre-recombinase (AdCre) and used a red-fluorescent protein adenovirus (AdRFP) as a control. Immunostaining confirmed SOD3 expression in the circumventricular organs (CVO) and its complete deletion by ICV AdCre. Deletion of CVO SOD3 increased basal blood pressure from 114 ± 3 to 133 ± 2 mmHg. Infusion of low-dose ang II (0.2 mg/kg/day) minimally affected pressure in AdRFP injected mice but caused severe hypertension (155 ± 9 mmHG) following CVO SOD3 deletion. In other studies, we deleted SOD3 in vascular smooth muscle (VSM) by using mice transgenic for an inducible vascular smooth muscle myosin heavy chain promoter-driven Cre. Deletion of VSM SOD3 had no effect on blood pressure at baseline or in response to ang II. Interestingly, deletion of SOD3 in either the CVO or VSM caused a vascular inflammatory infiltrate as determined by FACS, however this was predominantly T cells following CVO deletion and macrophages following VSM deletion. These studies show that SOD3 in the CVO modulates blood pressure while the VSM SOD3 predominantly protects against vascular inflammation.

• Role of extracellular superoxide dismutase in hypertension.

  Authors: Maria Carolina Gongora, Zhenyu Qin, Karine Laude, Ha Won Kim, Louise McCann, J Rodney Folz, Sergey Dikalov, Tohru Fukai, David G Harrison

  Hypertension. 09/2006; 48(3):473-81.

  We previously found that angiotensin II-induced hypertension increases vascular extracellular superoxide dismutase (ecSOD), and proposed that this is a compensatory mechanism that blunts theWe previously found that angiotensin II-induced hypertension increases vascular extracellular superoxide dismutase (ecSOD), and proposed that this is a compensatory mechanism that blunts the hypertensive response and preserves endothelium-dependent vasodilatation. To test this hypothesis, we studied ecSOD-deficient mice. ecSOD(-/-) and C57Blk/6 mice had similar blood pressure at baseline; however, the hypertension caused by angiotensin II was greater in ecSOD(-/-) compared with wild-type mice (168 versus 147 mm Hg, respectively; P<0.01). In keeping with this, angiotensin II increased superoxide and reduced endothelium-dependent vasodilatation in small mesenteric arterioles to a greater extent in ecSOD(-/-) than in wild-type mice. In contrast to these findings in resistance vessels, angiotensin II paradoxically improved endothelium-dependent vasodilatation, reduced intracellular and extracellular superoxide, and increased NO production in aortas of ecSOD(-/-) mice. Whereas aortic expression of endothelial NO synthase, Cu/ZnSOD, and MnSOD were not altered in ecSOD(-/-) mice, the activity of Cu/ZnSOD was increased by 80% after angiotensin II infusion. This was associated with a concomitant increase in expression of the copper chaperone for Cu/ZnSOD in the aorta but not in the mesenteric arteries. Moreover, the angiotensin II-induced increase in aortic reduced nicotinamide-adenine dinucleotide phosphate oxidase activity was diminished in ecSOD(-/-) mice as compared with controls. Thus, during angiotensin II infusion, ecSOD reduces hypertension, minimizes vascular superoxide production, and preserves endothelial function in resistance arterioles. We also identified novel compensatory mechanisms involving upregulation of copper chaperone for Cu/ZnSOD, increased Cu/ZnSOD activity, and decreased reduced nicotinamide-adenine dinucleotide phosphate oxidase activity in larger vessels. These compensatory mechanisms preserve large vessel function when ecSOD is absent in hypertension.

• The role of the multidrug resistance protein-1 in modulation of endothelial cell oxidative stress.

  Authors: Cornelius F H Mueller, Julian D Widder, Joseph S McNally, Louise McCann, Dean P Jones, David G Harrison

  Circulation research. 10/2005; 97(7):637-44.

  Glutathione (GSH) is the major source of intracellular sulfhydryl groups. Oxidized GSH (GSSG) can be recycled to GSH by the GSH reductase or exported from the cell. The mechanism by which GSSG isGlutathione (GSH) is the major source of intracellular sulfhydryl groups. Oxidized GSH (GSSG) can be recycled to GSH by the GSH reductase or exported from the cell. The mechanism by which GSSG is exported and the consequence of its export from endothelial cells has not been defined previously. We found that human endothelial cells express the multidrug resistance protein-1 (MRP1) and use this as their major exporter of GSSG. Oscillatory shear stress, which is known to stimulate endothelial cell production of reactive oxygen species, decreased intracellular GSH. In contrast, laminar shear significantly increased intracellular GSH. Oscillatory shear also caused a robust export of GSSG that was prevented by the MRP1 inhibitor MK571 and by MRP1 small interfering RNA. MRP1 inhibition prevented the decline in intracellular GSH, preserved the intracellular GSH Nernst potential, and reduced apoptosis caused by oscillatory shear. In aortas of hypertensive mice, endothelial disulfide export was doubled, and this was prevented by MK571 and was not observed in aortas of hypertensive MRP1-/- mice. Further, the altered endothelium-dependent vasodilatation caused by hypertension was ameliorated in MRP1-/- mice. GSSG export by MRP1 leads to a perturbation of endothelial redox state and ultimately endothelial cell apoptosis. Endothelial MRP1 may provide a novel therapeutic target for prevention of vascular disease.

• Hemodynamic and biochemical adaptations to vascular smooth muscle overexpression of p22phox in mice.

  Authors: Karine Laude, Hua Cai, Bruno Fink, Nyssa Hoch, David S Weber, Louise McCann, Georg Kojda, Tohru Fukai, Harald H H W Schmidt, Sergey Dikalov, Santhini Ramasamy, Graciela Gamez, Kathy K Griendling, David G Harrison

  American journal of physiology. Heart and circulatory physiology. 02/2005; 288(1):H7-12.

  Protein levels and polymorphisms of p22(phox) have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determineProtein levels and polymorphisms of p22(phox) have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determine whether increasing p22(phox) expression would alter vascular ROS production and hemodynamics by targeting p22(phox) expression to smooth muscle in transgenic (Tg) mice. Aortas of Tg(p22smc) mice had increased p22(phox) and Nox1 protein levels and produced more superoxide and H(2)O(2). Surprisingly, endothelium-dependent relaxation and blood pressure in Tg(p22smc) mice were normal. Aortas of Tg(p22smc) mice produced twofold more nitric oxide (NO) at baseline and sevenfold more NO in response to calcium ionophore as detected by electron spin resonance. Western blot analysis revealed a twofold increase in endothelial NO synthase (eNOS) protein expression in Tg(p22smc) mice. Both eNOS expression and NO production were normalized by infusion of the glutathione peroxidase mimetic ebselen or by crossing Tg(p22smc) mice with mice overexpressing catalase. We have previously found that NO stimulates extracellular superoxide dismutase (ecSOD) expression in vascular smooth muscle. In keeping with this, aortic segments from Tg(p22smc) mice expressed twofold more ecSOD, and chronic treatment with the NOS inhibitor N(G)-nitro-L-arginine methyl ester normalized this, suggesting that NO regulates ecSOD protein expression in vivo. These data indicate that chronic oxidative stress caused by excessive H(2)O(2) production evokes a compensatory response involving increased eNOS expression and NO production. NO in turn increases ecSOD protein expression and counterbalances increased ROS production leading to the maintenance of normal vascular function and hemodynamics.

• Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay.

  Authors: Bruno Fink, Karine Laude, Louise McCann, Abdul Doughan, David G Harrison, Sergey Dikalov

  American journal of physiology. Cell physiology. 11/2004; 287(4):C895-902.

  Recently, it was demonstrated that superoxide oxidizes dihydroethidium to a specific fluorescent product (oxyethidium) that differs from ethidium by the presence of an additional oxygen atom in itsRecently, it was demonstrated that superoxide oxidizes dihydroethidium to a specific fluorescent product (oxyethidium) that differs from ethidium by the presence of an additional oxygen atom in its molecular structure. We have adapted this new HPLC-based assay to quantify this product as a tool to estimate intracellular superoxide in intact tissues. Ethidium and oxyethidium were separated using a C-18 column and quantified using fluorescence detection. Initial cell-free experiments with potassium superoxide and xanthine oxidase confirmed the formation of oxyethidium from dihydroethidium. The formation of oxyethidium was inhibited by superoxide dismutase but not catalase and did not occur upon the addition of H(2)O(2), peroxynitrite, or hypochlorous acid. In bovine aortic endothelial cells (BAEC) and murine aortas, the redox cycling drug menadione increased the formation of oxyethidium from dihydroethidium ninefold (0.4 nmol/mg in control vs. 3.6 nmol/mg with 20 microM menadione), and polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) significantly inhibited this effect. Treatment of BAEC with angiotensin II caused a twofold increase in oxyethidium formation, and this effect also was reduced by PEG-SOD (0.5 nmol/mg). In addition, in the aortas of mice with angiotensin II-induced hypertension and DOCA-salt hypertension, the formation of oxyethidium was increased in a manner corresponding to superoxide production estimated on the basis of cytochrome c reduction. Detection of oxyethidium using HPLC represents a new, convenient, quantitative method for the detection of superoxide in intact cells and tissues.

• Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension.

  Authors: Ulf Landmesser, Sergey Dikalov, S Russ Price, Louise McCann, Tohru Fukai, Steven M Holland, William E Mitch, David G Harrison

  The Journal of clinical investigation. 05/2003; 111(8):1201-9.

  Tetrahydrobiopterin is a critical cofactor for the NO synthases, and in its absence these enzymes become "uncoupled," producing reactive oxygen species (ROSs) rather than NO. In aortas of mice withTetrahydrobiopterin is a critical cofactor for the NO synthases, and in its absence these enzymes become "uncoupled," producing reactive oxygen species (ROSs) rather than NO. In aortas of mice with deoxycorticosterone acetate-salt (DOCA-salt) hypertension, ROS production from NO synthase is markedly increased, and tetrahydrobiopterin oxidation is evident. Using mice deficient in the NADPH oxidase subunit p47(phox) and mice lacking either the endothelial or neuronal NO synthase, we obtained evidence that hypertension produces a cascade involving production of ROSs from the NADPH oxidase leading to oxidation of tetrahydrobiopterin and uncoupling of endothelial NO synthase (eNOS). This decreases NO production and increases ROS production from eNOS. Treatment of mice with oral tetrahydrobiopterin reduces vascular ROS production, increases NO production as determined by electron spin resonance measurements of nitrosyl hemoglobin, and blunts the increase in blood pressure due to DOCA-salt hypertension. Endothelium-dependent vasodilation is only minimally altered in vessels of mice with DOCA-salt hypertension but seems to be mediated by hydrogen peroxide released from uncoupled eNOS, since it is inhibited by catalase. Tetrahydrobiopterin oxidation may represent an important abnormality in hypertension. Treatment strategies that increase tetrahydrobiopterin or prevent its oxidation may prove useful in preventing vascular complications of this common disease.

• Role of c-Src in regulation of endothelial nitric oxide synthase expression during exercise training.

  Authors: Michael E Davis, Hua Cai, Louise McCann, Tohru Fukai, David G Harrison

  American journal of physiology. Heart and circulatory physiology. 04/2003; 284(4):H1449-53.

  We have shown that c-Src plays a role in shear stress stimulation of endothelial nitric oxide synthase (eNOS) expression in cultured cells. To examine the role of c-Src in vivo, we exercised C57Blk/6We have shown that c-Src plays a role in shear stress stimulation of endothelial nitric oxide synthase (eNOS) expression in cultured cells. To examine the role of c-Src in vivo, we exercised C57Blk/6 and c-Src heterozygous (c-Src(+/-)) mice on a treadmill for 3 wk. Western analysis demonstrated that c-Src(+/-) mice express less than one-half the normal amount of c-Src. Exercise increased heart rate and blood pressure to identical levels in both strains as determined using radiotelemetry. Exercise training increased eNOS protein >2-fold in the aorta and 1.7-fold in the heart in C57Blk/6 mice but had no effect on eNOS protein levels in c-Src(+/-) mice. In contrast to exercise, treatment of mice with mevastatin, which stimulates expression of eNOS posttranscriptionally, increased eNOS protein in both strains. Training also increased aortic extracellular superoxide dismutase protein expression, which is regulated by nitric oxide, in C57Blk/6 mice but not in c-Src(+/-)mice. These data indicate that c-Src has an important role in modulating vascular adaptations to exercise training, in particular increasing eNOS and extracellular superoxide dismutase protein expression.

• Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II.

  Authors: Ulf Landmesser, Hua Cai, Sergey Dikalov, Louise McCann, Jinah Hwang, Hanjoong Jo, Steven M Holland, David G Harrison

  Hypertension. 10/2002; 40(4):511-5.

  Hypertension caused by angiotensin II is dependent on vascular superoxide (O2*-) production. The nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase is a major source of vascular O2*- andHypertension caused by angiotensin II is dependent on vascular superoxide (O2*-) production. The nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase is a major source of vascular O2*- and is activated by angiotensin II in vitro. However, its role in angiotensin II-induced hypertension in vivo is less clear. In the present studies, we used mice deficient in p47(phox), a cytosolic subunit of the NADPH oxidase, to study the role of this enzyme system in vivo. In vivo, angiotensin II infusion (0.7 mg/kg per day for 7 days) increased systolic blood pressure from 105+/-2 to 151+/-6 mm Hg and increased vascular O2*- formation 2- to 3-fold in wild-type (WT) mice. In contrast, in p47(phox-/-) mice the hypertensive response to angiotensin II infusion (122+/-4 mm Hg; P<0.05) was markedly blunted, and there was no increase of vascular O2*- production. In situ staining for O2*- using dihydroethidium revealed a marked increase of O2*-production in both endothelial and vascular smooth muscle cells of angiotensin II-treated WT mice, but not in those of p47(phox-/-) mice. To directly examine the role of the NAD(P)H oxidase in endothelial production of O2*-, endothelial cells from WT and p47(phox-/-) mice were cultured. Western blotting confirmed the absence of p47(phox) in p47(phox-/-) mice. Angiotensin II increased O2*- production in endothelial cells from WT mice, but not in those from p47(phox-/-) mice, as determined by electron spin resonance spectroscopy. These results suggest a pivotal role of the NAD(P)H oxidase and its subunit p47(phox) in the vascular oxidant stress and the blood pressure response to angiotensin II in vivo.

• Peroxidase properties of extracellular superoxide dismutase: role of uric acid in modulating in vivo activity.

  Authors: H Ulrich Hink, Nalini Santanam, Sergey Dikalov, Louise McCann, Andrew D Nguyen, Sampath Parthasarathy, David G Harrison, Tohru Fukai

  Arteriosclerosis, thrombosis, and vascular biology. 10/2002; 22(9):1402-8.

  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 ofOBJECTIVE: 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 approximately 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.