Anna Dikalova

Publications of Anna Dikalova

• Anti-inflammatory activity of Chios mastic gum is associated with inhibition of TNF-alpha induced oxidative stress.

  Authors: Angelike Triantafyllou, Alfiya Bikineyeva, Anna Dikalova, Rafal Nazarewicz, Stamatios Lerakis, Sergey Dikalov

  Nutrition journal. 06/2011; 10:64.

  Gum of Chios mastic (Pistacia lentiscus var. chia) is a natural antimicrobial agent that has found extensive use in pharmaceutical products and as a nutritional supplement. The molecular mechanismsGum of Chios mastic (Pistacia lentiscus var. chia) is a natural antimicrobial agent that has found extensive use in pharmaceutical products and as a nutritional supplement. The molecular mechanisms of its anti-inflammatory activity, however, are not clear. In this work, the potential role of antioxidant activity of Chios mastic gum has been evaluated. Scavenging of superoxide radical was investigated by electron spin resonance and spin trapping technique using EMPO spin trap in xanthine oxidase system. Superoxide production in endothelial and smooth muscle cells stimulated with TNF-α or angiotensin II and treated with vehicle (DMSO) or mastic gum (0.1-10 μg/ml) was measured by DHE and HPLC. Cellular H2O2 was measured by Amplex Red. Inhibition of protein kinase C (PKC) with mastic gum was determined by the decrease of purified PKC activity, by inhibition of PKC activity in cellular homogenate and by attenuation of superoxide production in cells treated with PKC activator phorbol 12-myristate 13-acetate (PMA). Spin trapping study did not show significant scavenging of superoxide by mastic gum itself. However, mastic gum inhibited cellular production of superoxide and H2O2 in dose dependent manner in TNF-α treated rat aortic smooth muscle cells but did not affect unstimulated cells. TNF-α significantly increased the cellular superoxide production by NADPH oxidase, while mastic gum completely abolished this stimulation. Mastic gum inhibited the activity of purified PKC, decreased PKC activity in cell homogenate, and attenuated superoxide production in cells stimulated with PKC activator PMA and PKC-dependent angiotensin II in endothelial cells. We suggest that mastic gum inhibits PKC which attenuates production of superoxide and H2O2 by NADPH oxidases. This antioxidant property may have direct implication to the anti-inflammatory activity of the Chios mastic gum.

• NADPH oxidase 4 mediates TGF-β-induced smooth muscle α-actin via p38MAPK and serum response factor.

  Authors: Abel Martin-Garrido, David I Brown, Alicia N Lyle, Anna Dikalova, Bonnie Seidel-Rogol, Bernard Lassègue, Alejandra San Martín, Kathy K Griendling

  Free radical biology & medicine. 11/2010; 50(2):354-62.

  In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle geneIn contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle α-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-β (TGF-β) as a differentiating agent, we examined Nox4-dependent signaling. TGF-β increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-β-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-β-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2. Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-β. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-β-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-β-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-β.

• Reactive oxygen species-selective regulation of aortic inflammatory gene expression in Type 2 diabetes.

  Authors: Alejandra San Martín, Pingfeng Du, Anna Dikalova, Bernard Lassègue, María Aleman, María Carolina Góngora, Kathryn Brown, Giji Joseph, David G Harrison, W Robert Taylor, Hanjoong Jo, Kathy K Griendling

  American journal of physiology. Heart and circulatory physiology. 06/2007; 292(5):H2073-82.

  Vascular diseases are a major complication of diabetes mellitus (DM), although their etiology is poorly understood. NADPH oxidase-derived reactive oxygen species (ROS) production and inflammation areVascular diseases are a major complication of diabetes mellitus (DM), although their etiology is poorly understood. NADPH oxidase-derived reactive oxygen species (ROS) production and inflammation are potential mediators of DM-associated vascular diseases. Using db/db mice as a Type 2 diabetes model, we examined the relationship between NADPH oxidase-derived ROS and vascular inflammation. When compared with control m+/+ mice, aortas from 4- and 12-wk-old db/db mice had higher NADPH oxidase activity and increased superoxide levels, leading to NADPH oxidase-dependent impaired vasodilation at 12 wk. Diabetes progression from 4 to 12 wk led to increased Nox1, Nox4, and p22(phox) subunit mRNAs and induced the expression of a group of matrix remodeling-related cytokines: connective tissue growth factor (CTGF), bone morphogenetic protein 4 (BMP-4), and osteopontin (OPN). After 8 wk of treatment with the superoxide scavenger Tempol, 12-wk-old db/db mice had lower superoxide production, reduced plasma glucose and lipids, and lower BMP-4 and OPN protein expression when compared with nontreated mice. No changes were observed with Tempol in CTGF or m+/+ mice. The ability of Tempol to reverse ROS production as well as OPN and BMP-4, but not CTGF, induction suggests that DM-induced vascular inflammation involves both ROS-sensitive and -insensitive pathways.

• Vitamin E reduces glomerulosclerosis, restores renal neuronal NOS, and suppresses oxidative stress in the 5/6 nephrectomized rat.

  Authors: You-Lin Tain, Gary Freshour, Anna Dikalova, Kathy Griendling, Chris Baylis

  American journal of physiology. Renal physiology. 06/2007; 292(5):F1404-10.

  Chronic kidney disease is accompanied by nitric oxide (NO) deficiency and oxidative stress, which contribute to progression. We investigated whether the antioxidant vitamin E could preserve renalChronic kidney disease is accompanied by nitric oxide (NO) deficiency and oxidative stress, which contribute to progression. We investigated whether the antioxidant vitamin E could preserve renal function and NO bioavailability and reduce oxidative stress in the 5/6th nephrectomy (NX) rat model. We studied the following three groups of male Sprague-Dawley rats: sham (n = 6), 5/6 NX control (n = 6), and 5/6 NX treated with vitamin E (5,000 IU/kg chow; n = 5). The 5/6 NX group showed increased severity of glomerulosclerosis vs. sham, and this was ameliorated by vitamin E therapy. Both 5/6 NX groups showed similar elevations in plasma creatinine and proteinuria and decreased 24-h creatinine clearance compared with sham. There was increased NADPH-dependent superoxide production in 5/6 NX rats vs. sham that was prevented by vitamin E. Total NO production was similarly reduced in both 5/6 NX groups. There was unchanged abundance of endothelial nitric oxide synthesis (NOS) in renal cortex and medulla and neuronal (n) NOS in medulla. However, in kidney cortex, 5/6 NX rats had lower nNOS abundance than sham, which was restored by vitamin E. An increased plasma asymmetric dimethylarginine occurred with 5/6 NX associated with decreased renal dimethylarginine dimethylaminohydrolase activity and increased type 1 protein arginine methyltransferase expression.

• Lack of long-term protective effect of antioxidant/anti-inflammatory therapy in transplant-induced ischemia/reperfusion injury.

  Authors: You-Lin Tain, Veronika Muller, Attila Szabo, Anna Dikalova, Kathy Griendling, Chris Baylis

  American journal of nephrology. 02/2006; 26(3):213-7.

  BACKGROUND: Alloantigen-independent factors contribute to long-term damage in renal transplant recipients, likely due to ischemia/reperfusion (I/R) injury at transplantation (Tx). I/R injury promotesBACKGROUND: Alloantigen-independent factors contribute to long-term damage in renal transplant recipients, likely due to ischemia/reperfusion (I/R) injury at transplantation (Tx). I/R injury promotes oxidative stress and inflammation resulting in endothelial injury. METHODS: In this study we investigated the long-term efficacy (22 weeks) of short-term (10 day) endothelial protection therapy (EP) in 'optimal' donor kidneys using the male Fisher 344 rat isograft (ISO) model. ISO-EP kidneys were compared to untreated ISO (ISO-UN) kidneys. EP involved dexamethasone to donor, ex vivo treatment of the kidney with deferoxamine and tempol, and administration to the recipient of L-arginine and tempol for 10 days. Rats were sacrificed 22 weeks following Tx and compared to age-matched, normal controls. RESULTS: Both groups of ISO Tx rats developed similar renal dysfunction and structural damage and renal NADPH-oxidase-dependent O2- production was similarly elevated in ISO-UN and ISO-EP groups vs. controls. In vitro renal cortex NO synthase (NOS) activity was also similar in ISO-UN and ISO-EP rats, despite lower nNOS and eNOS protein abundance in ISO-EP. CONCLUSION: I/R injury-induced late graft dysfunction occurs even when optimal donors are used and when short-term EP treatment is given. Increased renal superoxide production is not prevented by short-term EP therapy.

• Nox1 overexpression potentiates angiotensin II-induced hypertension and vascular smooth muscle hypertrophy in transgenic mice.

  Authors: Anna Dikalova, Roza Clempus, Bernard Lassègue, Guangjie Cheng, James McCoy, Sergey Dikalov, Alejandra San Martin, Alicia Lyle, David S Weber, Daiana Weiss, W Robert Taylor, Harald H H W Schmidt, Gary K Owens, J David Lambeth, Kathy K Griendling

  Circulation. 11/2005; 112(17):2668-76.

  BACKGROUND: Reactive oxygen species (ROS) have been implicated in the development of cardiovascular pathologies. NAD(P)H oxidases (Noxes) are major sources of reactive oxygen species in the vesselBACKGROUND: Reactive oxygen species (ROS) have been implicated in the development of cardiovascular pathologies. NAD(P)H oxidases (Noxes) are major sources of reactive oxygen species in the vessel wall, but the importance of individual Nox homologues in specific layers of the vascular wall is unclear. Nox1 upregulation has been implicated in cardiovascular pathologies such as hypertension and restenosis. METHODS AND RESULTS: To investigate the pathological role of Nox1 upregulation in vascular smooth muscle, transgenic mice overexpressing Nox1 in smooth muscle cells (TgSMCnox1) were created, and the impact of Nox1 upregulation on the medial hypertrophic response during angiotensin II (Ang II)-induced hypertension was studied. These mice have increased expression of Nox1 protein in the vasculature, which is accompanied by increased superoxide production. Infusion of Ang II (0.7 mg/kg per day) into these mice for 2 weeks led to a potentiation of superoxide production compared with similarly treated negative littermate controls. Systolic blood pressure and aortic hypertrophy were also markedly greater in TgSMCnox1 mice than in their littermate controls. To confirm that this potentiation of vascular hypertrophy and hypertension was due to increased ROS formation, additional groups of mice were coinfused with the antioxidant Tempol. Tempol decreased the level of Ang II-induced aortic superoxide production and partially reversed the hypertrophic and hypertensive responses in these animals. CONCLUSIONS: These data indicate that smooth muscle-specific Nox1 overexpression augments the oxidative, pressor, and hypertrophic responses to Ang II, supporting the concept that medial Nox1 participates in the development of cardiovascular pathologies.

• Vascular hypertrophy in angiotensin II-induced hypertension is mediated by vascular smooth muscle cell-derived H2O2.

  Authors: Yong Zhang, Kathy K Griendling, Anna Dikalova, Gary K Owens, W Robert Taylor

  Hypertension. 10/2005; 46(4):732-7.

  Angiotensin II induces the development of vascular hypertrophy and hypertension. An increasing number of studies have demonstrated that reactive oxygen species are involved in many of the vascularAngiotensin II induces the development of vascular hypertrophy and hypertension. An increasing number of studies have demonstrated that reactive oxygen species are involved in many of the vascular responses to angiotensin II. However, the role of specific cell types and the precise identity of the functionally relevant reactive oxygen species remain unclear. In this study, we established a line of transgenic mice with vascular smooth muscle cell (SMC)-specific overexpression of the human catalase gene to explicitly test the functional role of vascular smooth muscle-derived hydrogen peroxide in the hypertensive and hypertrophic responses to angiotensin II in vivo. Catalase overexpression was confirmed by increased expression of catalase mRNA and protein, as well as by an increase in catalase enzymatic activity. The catalase transgenic mice were viable, had no change in basal hydrogen peroxide release (0.36+/-0.03 versus 0.37+/-0.14 micromol/L), and showed no overt developmental abnormality. In response to angiotensin II treatment, catalase transgenic mice exhibited lower hydrogen peroxide release compared with control animals. There was no effect on the hypertensive response to angiotensin II (147+/-10 versus 148+/-12 mm Hg). However, angiotensin II-induced aortic wall hypertrophy was dramatically attenuated in the catalase transgenic mice (wall thickness 32.4+/-2.0 versus 43.2+/-7.6 microm; P<0.001). These results demonstrate that vascular SMC-derived hydrogen peroxide plays an important role in angiotensin II-induced hypertrophy of the arterial wall.

• NAD(P)H oxidase 4 mediates transforming growth factor-beta1-induced differentiation of cardiac fibroblasts into myofibroblasts.

  Authors: Ioan Cucoranu, Roza Clempus, Anna Dikalova, Patrick J Phelan, Srividya Ariyan, Sergey Dikalov, Dan Sorescu

  Circulation research. 10/2005; 97(9):900-7.

  Human cardiac fibroblasts are the main source of cardiac fibrosis associated with cardiac hypertrophy and heart failure. Transforming growth factor-beta1 (TGF-beta1) irreversibly converts fibroblastsHuman cardiac fibroblasts are the main source of cardiac fibrosis associated with cardiac hypertrophy and heart failure. Transforming growth factor-beta1 (TGF-beta1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle alpha-actin (SM alpha-actin) de novo and produce extracellular matrix. We hypothesized that TGF-beta1-stimulated conversion of fibroblasts to myofibroblasts requires reactive oxygen species derived from NAD(P)H oxidases (Nox). We found that TGF-beta1 potently upregulates the contractile marker SM alpha-actin mRNA (7.5+/-0.8-fold versus control). To determine whether Nox enzymes are involved, we first performed quantitative real time polymerase chain reaction and found that Nox5 and Nox4 are abundantly expressed in cardiac fibroblasts, whereas Nox1 and Nox2 are barely detectable. On stimulation with TGF-beta1, Nox4 mRNA is dramatically upregulated by 16.2+/-0.8-fold (n=3, P<0.005), whereas Nox5 is downregulated. Small interference RNA against Nox4 downregulates Nox4 mRNA by 80+/-5%, inhibits NADPH-driven superoxide production in response to TGF-beta1 by 65+/-7%, and reduces TGF-beta1-induced expression of SM alpha-actin by 95+/-2% (n=6, P<0.05). Because activation of small mothers against decapentaplegic (Smads) 2/3 is critical for myofibroblast conversion in response to TGF-beta1, we also determined whether Nox4 affects Smad 2/3 phosphorylation. Depletion of Nox4 but not Nox5 inhibits baseline and TGF-beta1 stimulation of Smad 2/3 phosphorylation by 75+/-5% and 68+/-3%, respectively (n=7, P<0.0001). We conclude that Nox 4 mediates TGF-beta1-induced conversion of fibroblasts to myofibroblasts by regulating Smad 2/3 activation. Thus, Nox4 may play a critical role in the pathological activation of cardiac fibroblasts in cardiac fibrosis associated with human heart failure.

• Functional association of nox1 with p22phox in vascular smooth muscle cells.

  Authors: Ibrahim R Hanna, Lula L Hilenski, Anna Dikalova, Yoshihiro Taniyama, Sergey Dikalov, Alicia Lyle, Mark T Quinn, Bernard Lassègue, Kathy K Griendling

  Free radical biology & medicine. 12/2004; 37(10):1542-9.

  The vascular NAD(P)H oxidases constitute important sources of ROS in the vessel wall and have been implicated in vascular disease. Vascular smooth muscle cells (VSMCs) from conduit arteries expressThe vascular NAD(P)H oxidases constitute important sources of ROS in the vessel wall and have been implicated in vascular disease. Vascular smooth muscle cells (VSMCs) from conduit arteries express two gp91phox homologs, Nox1 and Nox4, of which Nox1 is agonist-sensitive. Because p22phox has been shown to be functionally important in vascular cells stimulated with vasoactive hormones, the relationship of Nox1 and p22phox was investigated in VSMCs from rat and human aortas. Coimmunoprecipitation studies demonstrated that p22phox and hemagglutinin-tagged Nox1 associate in unstimulated VSMCs. These findings were confirmed by confocal microscopy, showing colocalization of the two proteins in their native states in the plasma membrane and submembrane areas of the cell. NADPH-driven superoxide production, as measured by electron spin resonance using 1-hydroxy-3-carboxypyrrolidine as a spin probe, is dependent on the coexpression of both subunits, suggesting the importance of the association for the functional integrity of the enzyme. These results indicate that in contrast to the neutrophil enzyme, VSMCs can use Nox1 rather than gp91phox as a catalytic center in the p22phox-based oxidase and that these two proteins are preassembled at or near the plasma membrane and submembrane vesicular structures in unstimulated cells.

• Functional association of nox1 with p22phox in vascular smooth muscle cells

  Authors: Ibrahim R. Hanna, Lula L. Hilenski, Anna Dikalova, Yoshihiro Taniyama, Sergey Dikalov, Alicia Lyle, Mark T. Quinn, Bernard Lassègue, Kathy K. Griendling

  Free Radical Biology and Medicine.

  The vascular NAD(P)H oxidases constitute important sources of ROS in the vessel wall and have been implicated in vascular disease. Vascular smooth muscle cells (VSMCs) from conduit arteries expressThe vascular NAD(P)H oxidases constitute important sources of ROS in the vessel wall and have been implicated in vascular disease. Vascular smooth muscle cells (VSMCs) from conduit arteries express two gp91phox homologs, Nox1 and Nox4, of which Nox1 is agonist-sensitive. Because p22phox has been shown to be functionally important in vascular cells stimulated with vasoactive hormones, the relationship of Nox1 and p22phox was investigated in VSMCs from rat and human aortas. Coimmunoprecipitation studies demonstrated that p22phox and hemagglutinin-tagged Nox1 associate in unstimulated VSMCs. These findings were confirmed by confocal microscopy, showing colocalization of the two proteins in their native states in the plasma membrane and submembrane areas of the cell. NADPH-driven superoxide production, as measured by electron spin resonance using 1-hydroxy-3-carboxypyrrolidine as a spin probe, is dependent on the coexpression of both subunits, suggesting the importance of the association for the functional integrity of the enzyme. These results indicate that in contrast to the neutrophil enzyme, VSMCs can use Nox1 rather than gp91phox as a catalytic center in the p22phox-based oxidase and that these two proteins are preassembled at or near the plasma membrane and submembrane vesicular structures in unstimulated cells.