Anna E Dikalova

Publications of Anna E Dikalova

• Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling.

  Authors: Anna E Dikalova, María Carolina Góngora, David G Harrison, J David Lambeth, Sergey Dikalov, Kathy K Griendling

  American journal of physiology. Heart and circulatory physiology. 09/2010; 299(3):H673-9.

  Recent work has made it clear that oxidant systems interact. To investigate potential cross talk between NADPH oxidase (Nox) 1 upregulation in vascular smooth muscle and endothelial function,Recent work has made it clear that oxidant systems interact. To investigate potential cross talk between NADPH oxidase (Nox) 1 upregulation in vascular smooth muscle and endothelial function, transgenic mice overexpressing Nox1 in smooth muscle cells (Tg(SMCnox1)) were subjected to angiotensin II (ANG II)-induced hypertension. As expected, NADPH-dependent superoxide generation was increased in aortas from Nox1-overexpressing mice. Infusion of ANG II (0.7 mg x kg(-1) x day(-1)) for 2 wk potentiated NADPH-dependent superoxide generation and hydrogen peroxide production compared with similarly treated negative littermate controls. Endothelium-dependent relaxation was impaired in transgenic mice, and bioavailable nitric oxide was markedly decreased. To test the hypothesis that eNOS uncoupling might contribute to endothelial dysfunction, the diet was supplemented with tetrahydrobiopterin (BH(4)). BH(4) decreased aortic superoxide production, partially restored bioavailable nitric oxide in aortas of ANG II-treated Tg(SMCnox1) mice, and significantly improved endothelium-dependent relaxation in these mice. Western blot analysis revealed less dimeric eNOS in Tg(SMCnox1) mice compared with the wild-type mice; however, total eNOS was equivalent. Pretreatment of mouse aortas with the eNOS inhibitor N(G)-nitro-L-arginine methyl ester decreased ANG II-induced superoxide production in Tg(SMCnox1) mice compared with wild-type mice, indicating that uncoupled eNOS is also a significant source of increased superoxide in transgenic mice. Thus overexpression of Nox1 in vascular smooth muscle leading to enhanced production of reactive oxygen species in response to ANG II causes eNOS uncoupling and a decrease in nitric oxide bioavailability, resulting in impaired vasorelaxation.

• 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.

• Mechanisms of Vascular Smooth Muscle NADPH Oxidase 1 (Nox1) Contribution to Injury-Induced Neointimal Formation.

  Authors: Moo Yeol Lee, Alejandra San Martin, Puja K Mehta, Anna E Dikalova, Abel Martin Garrido, Erin Lyons, Karl-Heinz Krause, Botond Banfi, J David Lambeth, Bernard Lassègue, Kathy K Griendling

  Arteriosclerosis, thrombosis, and vascular biology. 02/2009;

  OBJECTIVE: Vascular NADPH oxidases (Noxes) have been implicated in cardiovascular diseases; however, the importance of individual Nox homologues remains unclear. Here, the role of the vascular smoothOBJECTIVE: Vascular NADPH oxidases (Noxes) have been implicated in cardiovascular diseases; however, the importance of individual Nox homologues remains unclear. Here, the role of the vascular smooth muscle cell (VSMC) Nox1 in neointima formation was studied using genetically modified animal models. METHODS AND RESULTS: Wire injury-induced neointima formation in the femoral artery, along with proliferation and apoptosis, was reduced in Nox1(y/-) mice, but there was little difference in Tg(SMCnox1) mice compared with wild-type (WT) mice. Proliferation and migration were reduced in cultured Nox1(y/-) VSMCs and increased in Tg(SMCnox1) cells. Tg(SMCnox1) cells exhibited increased fibronectin secretion, but neither collagen I production nor cell adhesion was affected by alteration of Nox1. Using antibody microarray and Western blotting analysis, increased cofilin phosphorylation and mDia1 expression and decreased PAK1 expression were detected in Nox1(y/-) cells. Overexpression of S3A, a constitutively active cofilin mutant, partially recovered reduced migration of Nox1(y/-) cells, suggesting that reduction in cofilin activity contributes to impaired migration of Nox1(y/-) VSMCs. CONCLUSIONS: These results indicate that Nox1 plays a critical role in neointima formation by mediating VSMC migration, proliferation, and extracellular matrix production, and that cofilin is a major effector of Nox1-mediated migration. Inhibition of Nox1 may be an efficient strategy to suppress neointimal formation.

• Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production.

  Authors: Sergey I Dikalov, Anna E Dikalova, Alfiya T Bikineyeva, Harald H H W Schmidt, David G Harrison, Kathy K Griendling

  Free radical biology & medicine. 09/2008;

  NADPH oxidases are major sources of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation andNADPH oxidases are major sources of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation and differentiation, while ROS overproduction has been implicated in hypertension and atherosclerosis. It is known that the heme-containing catalytic subunits Nox1 and Nox4 are responsible for oxygen reduction in vascular smooth muscle cells from large arteries. However, the exact mechanism of ROS production by NADPH oxidases is not completely understood. We hypothesized that Nox1 and Nox4 play distinct roles in basal and angiotensin II (AngII)-stimulated production of O(2)(-) and H(2)O(2). Nox1 and Nox4 expression in rat aortic smooth muscle cells (RASMCs) was selectively reduced by treatment with siNox4 or antisense Nox1 adenovirus. Production of O(2)(-) and H(2)O(2) in intact RASMCs was analyzed by dihydroethidium and Amplex Red assay. Activity of NADPH oxidases was measured by NADPH-dependent O(2)(-) and H(2)O(2) production using electron spin resonance (ESR) and 1-hydroxy-3-carboxypyrrolidine (CPH) in the membrane fraction in the absence of cytosolic superoxide dismutase. It was found that production of O(2)(-) by quiescent RASMC NADPH oxidases was five times less than H(2)O(2) production. Stimulation of cells with AngII led to a 2-fold increase of O(2)(-) production by NADPH oxidases, with a small 15 to 30% increase in H(2)O(2) formation. Depletion of Nox4 in RASMCs led to diminished basal H(2)O(2) production, but did not affect O(2)(-) or H(2)O(2) production stimulated by AngII. In contrast, depletion of Nox1 in RASMCs inhibited production of O(2)(-) and AngII-stimulated H(2)O(2) in the membrane fraction and intact cells. Our data suggest that Nox4 produces mainly H(2)O(2), while Nox1 generates mostly O(2)(-) that is later converted to H(2)O(2.) Therefore, Nox4 is responsible for basal H(2)O(2) production, while O(2)(-) production in nonstimulated and AngII-stimulated cells depends on Nox1. The difference in the products generated by Nox1 and Nox4 may help to explain the distinct roles of these NADPH oxidases in cell signaling. These findings also provide important insight into the origin of H(2)O(2) in vascular cells, and may partially account for the limited pharmacological effect of antioxidant treatments with O(2)(-) scavengers that do not affect H(2)O(2).

• Nox5 mediates PDGF-induced proliferation in human aortic smooth muscle cells.

  Authors: Desmond B Jay, Christopher A Papaharalambus, Bonnie Seidel-Rogol, Anna E Dikalova, Bernard Lassègue, Kathy K Griendling

  Free radical biology & medicine. 09/2008; 45(3):329-35.

  The proliferation of vascular smooth muscle cells is important in the pathogenesis of many vascular diseases. Reactive oxygen species (ROS) produced by NADPH oxidases in smooth muscle cells have beenThe proliferation of vascular smooth muscle cells is important in the pathogenesis of many vascular diseases. Reactive oxygen species (ROS) produced by NADPH oxidases in smooth muscle cells have been shown to participate in signaling cascades regulating proliferation induced by platelet-derived growth factor (PDGF), a powerful smooth muscle mitogen. We sought to determine the role of Nox5 in the regulation of PDGF-stimulated human aortic smooth muscle cell (HASMC) proliferation. Cultured HASMC were found to express four isoforms of Nox5. When HASMC stimulated with PDGF were pretreated with N-acetyl cysteine (NAC), proliferation was significantly reduced. Proliferation induced by PDGF was also heavily dependent on JAK/STAT activation, as the JAK inhibitor, AG490, was able to completely abolish PDGF-stimulated HASMC growth. Specific knockdown of Nox5 with a siRNA strategy reduced PDGF-induced HASMC ROS production and proliferation. Additionally, siRNA to Nox5 inhibited PDGF-stimulated JAK2 and STAT3 phosphorylation. ROS produced by Nox5 play an important role in PDGF-induced JAK/STAT activation and HASMC proliferation.

• Nox4 is required for maintenance of the differentiated vascular smooth muscle cell phenotype.

  Authors: Roza E Clempus, Dan Sorescu, Anna E Dikalova, Lily Pounkova, Patricia Jo, George P Sorescu, Harald H H Schmidt, Bernard Lassègue, Kathy K Griendling

  Arteriosclerosis, thrombosis, and vascular biology. 02/2007; 27(1):42-8.

  OBJECTIVE: The mechanisms responsible for maintaining the differentiated phenotype of adult vascular smooth muscle cells (VSMCs) are incompletely understood. Reactive oxygen species (ROS) have beenOBJECTIVE: The mechanisms responsible for maintaining the differentiated phenotype of adult vascular smooth muscle cells (VSMCs) are incompletely understood. Reactive oxygen species (ROS) have been implicated in VSMC differentiation, but the responsible sources are unknown. In this study, we investigated the role of Nox1 and Nox4-derived ROS in this process. METHODS AND RESULTS: Primary VSMCs were used to study the relationship between Nox homologues and differentiation markers such as smooth muscle alpha-actin (SM alpha-actin), smooth muscle myosin heavy chain (SM-MHC), heavy caldesmon, and calponin. We found that Nox4 and differentiation marker genes were downregulated from passage 1 to passage 6 to 12, whereas Nox1 was gradually upregulated. Nox4 co-localized with SM alpha-actin-based stress fibers in differentiated VSMC, and moved into focal adhesions in de-differentiated cells. siRNA against nox4 reduced NADPH-driven superoxide production in serum-deprived VSMCs and downregulated SM-alpha actin, SM-MHC, and calponin, as well as SM-alpha actin stress fibers. Nox1 depletion did not decrease these parameters. CONCLUSIONS: Nox4-derived ROS are critical to the maintenance of the differentiated phenotype of VSMCs. These findings highlight the importance of identifying the specific source of ROS involved in particular cellular functions when designing therapeutic interventions.

• Noninvasive diagnostic tool for inflammation-induced oxidative stress using electron spin resonance spectroscopy and an extracellular cyclic hydroxylamine.

  Authors: Sergey I Dikalov, Anna E Dikalova, Ronald P Mason

  Archives of biochemistry and biophysics. 07/2002; 402(2):218-26.

  Inflammation is one of the leading causes of the many pathological states associated with oxidative stress. A crucial role in the development of inflammation-induced oxidative stress is played byInflammation is one of the leading causes of the many pathological states associated with oxidative stress. A crucial role in the development of inflammation-induced oxidative stress is played by reactive oxidant species (ROS), which are very difficult to detect in vivo. One of the most sensitive and definitive methods in the detection of ROS is electron spin resonance, especially as used in conjunction with spin trapping. Unfortunately, the commonly used nitrone spin traps have a very low efficacy for trapping superoxide radicals, and their radical adducts are not stable. To address this deficiency, we have developed negatively charged cyclic hydroxylamines such as 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H) for the detection of reactive oxidant species as a diagnostic tool for extracellular inflammation-induced oxidative stress. We used inflammation induced by a bacterial endotoxin lipopolysaccharide (LPS) as a model. ROS formation was tested in cultured macrophages, in blood and in vivo. PP-H reacts with reactive oxidant species generating the stable nitroxide radical 4-phosphonooxy-TEMPO. It was shown that a 5-h treatment of macrophages with LPS (1 microg/ml) leads to a threefold increase in superoxide formation as demonstrated using superoxide dismutase. Formation of reactive oxidant species 5 h after LPS (1 mg/kg) treatment of Fischer rats was analyzed in arterial blood; formation of reactive oxidant species in LPS-treated animals increased by a factor of 2.2 and was dependent upon the LPS dose. Diphenyleneiodonium (0.1 mM) inhibited formation of LPS-stimulated reactive oxidant species by 80%. We suggest that this test could be used as a noninvasive diagnostic tool for inflammation-induced oxidative stress.

• Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production

  Authors: Sergey I. Dikalov, Anna E. Dikalova, Alfiya T. Bikineyeva, Harald H.H.W. Schmidt, David G. Harrison, Kathy K. Griendling

  Free Radical Biology and Medicine.

  NADPH oxidases are major sources of superoxide (O2●−) and hydrogen peroxide (H2O2) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation andNADPH oxidases are major sources of superoxide (O2●−) and hydrogen peroxide (H2O2) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation and differentiation, while ROS overproduction has been implicated in hypertension and atherosclerosis. It is known that the heme-containing catalytic subunits Nox1 and Nox4 are responsible for oxygen reduction in vascular smooth muscle cells from large arteries. However, the exact mechanism of ROS production by NADPH oxidases is not completely understood. We hypothesized that Nox1 and Nox4 play distinct roles in basal and angiotensin II (AngII)-stimulated production of O2●− and H2O2. Nox1 and Nox4 expression in rat aortic smooth muscle cells (RASMCs) was selectively reduced by treatment with siNox4 or antisense Nox1 adenovirus. Production of O2●− and H2O2 in intact RASMCs was analyzed by dihydroethidium and Amplex Red assay. Activity of NADPH oxidases was measured by NADPH-dependent O2●− and H2O2 production using electron spin resonance (ESR) and 1-hydroxy-3-carboxypyrrolidine (CPH) in the membrane fraction in the absence of cytosolic superoxide dismutase. It was found that production of O2●− by quiescent RASMC NADPH oxidases was five times less than H2O2 production. Stimulation of cells with AngII led to a 2-fold increase of O2●− production by NADPH oxidases, with a small 15 to 30% increase in H2O2 formation. Depletion of Nox4 in RASMCs led to diminished basal H2O2 production, but did not affect O2●− or H2O2 production stimulated by AngII. In contrast, depletion of Nox1 in RASMCs inhibited production of O2●− and AngII-stimulated H2O2 in the membrane fraction and intact cells. Our data suggest that Nox4 produces mainly H2O2, while Nox1 generates mostly O2●− that is later converted to H2O2. Therefore, Nox4 is responsible for basal H2O2 production, while O2●− production in nonstimulated and AngII-stimulated cells depends on Nox1. The difference in the products generated by Nox1 and Nox4 may help to explain the distinct roles of these NADPH oxidases in cell signaling. These findings also provide important insight into the origin of H2O2 in vascular cells, and may partially account for the limited pharmacological effect of antioxidant treatments with O2●− scavengers that do not affect H2O2.