Sergey Dikalov

Publications of Sergey Dikalov

• Folate Receptor-Targeted Antioxidant Therapy Ameliorates Renal Ischemia-Reperfusion Injury.

  Authors: Sarah F Knight, Kousik Kundu, Giji Joseph, Sergey Dikalov, Daiana Weiss, Niren Murthy, W Robert Taylor

  Journal of the American Society of Nephrology : JASN. 01/2012;

  Antioxidant therapy can protect against ischemic injury, but the inability to selectively target the kidney would require extremely high doses to achieve effective local concentrations of drug. Here,Antioxidant therapy can protect against ischemic injury, but the inability to selectively target the kidney would require extremely high doses to achieve effective local concentrations of drug. Here, we developed a directed therapeutic that specifically targets an antioxidant to renal proximal tubule cells via the folate receptor. Because a local increase in superoxide contributes to renal ischemic injury, we created the folate-antioxidant conjugate 4-hydroxy-Tempo (tempol)-folate to target folate receptors, which are highly expressed in the proximal tubule. Dihydroethidium high-performance liquid chromatography demonstrated that conjugated tempol retained its efficacy to scavenge superoxide in proximal tubule cells. In a mouse model of renal ischemia-reperfusion injury, tempol-folate reduced renal superoxide levels more effectively than tempol alone. Furthermore, electron spin resonance revealed the successful targeting of the tempol-folate conjugate to the kidney and other tissues expressing folate receptors. Administration of tempol-folate protected the renal function of mice after ischemia-reperfusion injury and inhibited infiltration of macrophages. In conclusion, kidney-specific targeting of an antioxidant has therapeutic potential to prevent renal ischemic injury. Conjugation of other pharmaceuticals to folate may also facilitate the development of treatments for other kidney diseases.

• Redox signaling in an in vivo murine model of low magnitude oscillatory wall shear stress.

  Authors: Nick J Willett, Kousik Kundu, Sarah F Knight, Sergey Dikalov, Niren Murthy, W Robert Taylor

  Antioxidants & redox signaling. 09/2011; 15(5):1369-78.

  Wall Shear Stress (WSS) has been identified as an important factor in the pathogenesis of atherosclerosis. We utilized a novel murine aortic coarctation model to acutely create a region of lowWall Shear Stress (WSS) has been identified as an important factor in the pathogenesis of atherosclerosis. We utilized a novel murine aortic coarctation model to acutely create a region of low magnitude oscillatory WSS in vivo. We employed this model to test the hypothesis that acute changes in WSS in vivo induce upregulation of inflammatory proteins, mediated by reactive oxygen species (ROS). Superoxide generation and VCAM-1 expression both increased in regions of low magnitude oscillatory WSS. WSS-dependent superoxide formation was attenuated by tempol treatment, but was unchanged in p47 phox knockout (ko) mice. However, in both the p47 phox ko mice and the tempol-treated mice, low magnitude oscillatory WSS produced an increase in VCAM-1 expression comparable to control mice. Additionally, this same VCAM-1 expression was observed in ebselen-treated mice and catalase overexpressing mice. These results suggest that although the redox state is important to the overall pathogenesis of atherosclerosis, the initial WSS-dependent inflammatory response leading to lesion localization is not dependent on ROS.

• 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.

• Role of increased guanosine triphosphate cyclohydrolase-1 expression and tetrahydrobiopterin levels upon T cell activation.

  Authors: Wei Chen, Li Li, Torben Brod, Omar Saeed, Salim Thabet, Thomas Jansen, Sergey Dikalov, Cornelia Weyand, Jorg Goronzy, David G Harrison

  The Journal of biological chemistry. 02/2011; 286(16):13846-51.

  Tetrahydrobiopterin (BH(4)) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O(2)(·-)) rather than NO. The rate-limiting enzyme forTetrahydrobiopterin (BH(4)) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O(2)(·-)) rather than NO. The rate-limiting enzyme for BH(4) production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH(4) levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH(4). Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH(4) levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH(4). Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH(4) accumulation, reduced NO production, and increased T cell O(2)(·-) production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH(2) polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH(4) in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH(4) levels, NO production, and modulating T cell cytokine production.

• 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.

• Uncoupled cardiac nitric oxide synthase mediates diastolic dysfunction.

  Authors: Gad A Silberman, Tai-Hwang M Fan, Hong Liu, Zhe Jiao, Hong D Xiao, Joshua D Lovelock, Beth M Boulden, Julian Widder, Scott Fredd, Kenneth E Bernstein, Beata M Wolska, Sergey Dikalov, David G Harrison, Samuel C Dudley

  Circulation. 02/2010; 121(4):519-28.

  Heart failure with preserved ejection fraction is 1 consequence of hypertension and is caused by impaired cardiac diastolic relaxation. Nitric oxide (NO) is a known modulator of cardiac relaxation.Heart failure with preserved ejection fraction is 1 consequence of hypertension and is caused by impaired cardiac diastolic relaxation. Nitric oxide (NO) is a known modulator of cardiac relaxation. Hypertension can lead to a reduction in vascular NO, in part because NO synthase (NOS) becomes uncoupled when oxidative depletion of its cofactor tetrahydrobiopterin (BH(4)) occurs. Similar events may occur in the heart that lead to uncoupled NOS and diastolic dysfunction. In a hypertensive mouse model, diastolic dysfunction was accompanied by cardiac oxidation, a reduction in cardiac BH(4), and uncoupled NOS. Compared with sham-operated animals, male mice with unilateral nephrectomy, with subcutaneous implantation of a controlled-release deoxycorticosterone acetate pellet, and given 1% saline to drink were mildly hypertensive and had diastolic dysfunction in the absence of systolic dysfunction or cardiac hypertrophy. The hypertensive mouse hearts showed increased oxidized biopterins, NOS-dependent superoxide production, reduced NO production, and dephosphorylated phospholamban. Feeding hypertensive mice BH(4) (5 mg/d), but not treating with hydralazine or tetrahydroneopterin, improved cardiac BH(4) stores, phosphorylated phospholamban levels, and diastolic dysfunction. Isolated cardiomyocyte experiments revealed impaired relaxation that was normalized with short-term BH(4) treatment. Targeted cardiac overexpression of angiotensin-converting enzyme also resulted in cardiac oxidation, NOS uncoupling, and diastolic dysfunction in the absence of hypertension. Cardiac oxidation, independently of vascular changes, can lead to uncoupled cardiac NOS and diastolic dysfunction. BH(4) may represent a possible treatment for diastolic dysfunction.

• Regulation of endothelial cell tetrahydrobiopterin pathophysiological and therapeutic implications.

  Authors: David G Harrison, Wei Chen, Sergey Dikalov, Li Li

  Advances in pharmacology (San Diego, Calif.). 01/2010; 60:107-32.

  Tetrahydrobiopterin (BH(4)) is a critical cofactor for the nitric oxide synthases. In the absence of BH(4), these enzymes become uncoupled, fail to produce nitric oxide, and begin to produceTetrahydrobiopterin (BH(4)) is a critical cofactor for the nitric oxide synthases. In the absence of BH(4), these enzymes become uncoupled, fail to produce nitric oxide, and begin to produce superoxide and other reactive oxygen species (ROS). BH(4) levels are modulated by a complex biosynthetic pathway, salvage enzymes, and by oxidative degradation. The enzyme GTP cyclohydrolase-1 catalyzes the first step in the de novo synthesis of BH(4) and new evidence shows that this enzyme is regulated by phosphorylation, which reduces its interaction with its feedback regulatory protein (GFRP). In the setting of a variety of common diseases, such as atherosclerosis, hypertension, and diabetes, reactive oxygen species promote oxidation of BH(4) and inhibit expression of the salvage enzyme dihydrofolate reductase (DHFR), promoting accumulation of BH(2) and NOS uncoupling. There is substantial interest in therapeutic approaches to increasing tissue levels of BH(4), largely by oral administration of this agent. BH(4) treatment has proved effective in decreasing atherosclerosis, reducing blood pressure, and preventing complications of diabetes in experimental animals. While these basic studies have been very promising, there are only a few studies showing any effect of BH(4) therapy in humans in treatment of these common problems. Whether BH(4) or related agents will be useful in treatment of human diseases needs additional study.

• The delivery of superoxide dismutase encapsulated in polyketal microparticles to rat myocardium and protection from myocardial ischemia-reperfusion injury.

  Authors: Gokulakrishnan Seshadri, Jay C Sy, Milton Brown, Sergey Dikalov, Stephen C Yang, Niren Murthy, Michael E Davis

  Biomaterials. 11/2009;

  Oxidative stress is increased in the myocardium following infarction and plays a significant role in death of cardiac myocytes, leading to cardiac dysfunction. Levels of the endogenous antioxidantOxidative stress is increased in the myocardium following infarction and plays a significant role in death of cardiac myocytes, leading to cardiac dysfunction. Levels of the endogenous antioxidant Cu/Zn-superoxide dismutase (SOD1) decrease following myocardial infarction. While SOD1 gene therapy studies show promise, trials with SOD1 protein have had little success due to poor pharmacokinetics and thus new delivery vehicles are needed. In this work, polyketal particles, a recently developed delivery vehicle, were used to make SOD1-encapsulated-microparticles (PKSOD). Our studies with cultured macrophages demonstrated that PKSOD treatment scavenges both intracellular and extracellular superoxide, suggesting efficient delivery of SOD1 protein to the inside of cells. In a rat model of ischemia/reperfusion (IR) injury, injection of PKSOD, and not free SOD1 or empty particles was able to scavenge IR-induced excess superoxide 3 days following infarction. In addition, only PKSOD treatment significantly reduced myocyte apoptosis. Further, PKSOD treatment was able to improve cardiac function as measured by acute changes in fractional shortening from baseline echocardiography, suggesting that sustained delivery of SOD1 is critical during the early phase of cardiac repair. These data demonstrate that delivery of SOD1 with polyketals is superior to free SOD1 protein therapy and may have potential clinical implications.

• Targeted detoxification of selected reactive oxygen species in the vascular endothelium.

  Authors: Vladimir V Shuvaev, Melpo Christofidou-Solomidou, Faiz Bhora, Karine Laude, Hua Cai, Sergey Dikalov, Evguenia Arguiri, Charalambos C Solomides, Steven M Albelda, David G Harrison, Vladimir R Muzykantov

  The Journal of pharmacology and experimental therapeutics. 09/2009;

  Oxidative stress underlies diverse vascular diseases, but its management remains elusive, in part due to our inability to selectively detoxify reactive oxygen species (ROS) in pathological sites andOxidative stress underlies diverse vascular diseases, but its management remains elusive, in part due to our inability to selectively detoxify reactive oxygen species (ROS) in pathological sites and limited understanding which species need to be eliminated. The antioxidant enzymes (AOE) superoxide dismutase (SOD) and catalase (which decompose O2(.-) and H2O2, respectively) conjugated with an antibody to Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1) bind to endothelial cells and alleviate oxidative stress in cell culture models. Here, we studied the effects of these antioxidant conjugates in mouse models of vascular oxidative stress. Anti-PECAM/catalase and anti-PECAM/SOD conjugates, in contrast to control IgG/AOE conjugates, accumulated in the lungs and vascularized organs after intravenous injection in wild type, but not PECAM KO mice. Anti-PECAM/catalase, but not anti-PECAM/SOD, protected mice from lung injury induced by H2O2 produced by glucose oxidase deposited in the pulmonary vasculature. Anti-PECAM/catalase also reduced alveolar edema and attenuated decline in arterial oxygen in mice that underwent unilateral lung ischemia/reperfusion, while anti-PECAM/SOD was not effective, implying key role of H2O2 in tissue damage in this pathology. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase prevented oxidation of tetrahydrobiopterin (BH4) and normalized vasoreactivity in the vessels of mice rendered hypertensive by pre-treatment with angiotensin-II. This outcome agrees with reports implicating superoxide and peroxynitrite in altered endothelium-dependent vasodilatation in hypertension. Therefore, the use of endothelial cell-targeted antioxidants identifies the key specific species of ROS involved in various forms of vascular disease and holds promise for the mechanistically-tailored treatment of these pathologies.

• Long-Term Exposure to AZT, but not d4T, Increases Endothelial Cell Oxidative Stress and Mitochondrial Dysfunction.

  Authors: Erik Kline, Leda Bassit, Brenda Hernandez-Santiago, Mervi Detorio, Bill Liang, Dean Kleinhenz, Erik Walp, Sergey Dikalov, Dean Jones, Raymond Schinazi, Roy Sutliff

  Cardiovascular toxicology. 01/2009;

  Nucleoside reverse transcriptase inhibitors (NRTIs), such as zidovudine (AZT) and stavudine (d4T), cause toxicities to numerous tissues, including the liver and vasculature. While much is known aboutNucleoside reverse transcriptase inhibitors (NRTIs), such as zidovudine (AZT) and stavudine (d4T), cause toxicities to numerous tissues, including the liver and vasculature. While much is known about hepatic NRTI toxicity, the mechanism of toxicity in endothelial cells is incompletely understood. Human aortic endothelial and HepG2 liver cells were exposed to 1 muM AZT or d4T for up to 5 weeks. Markers of oxidative stress, mitochondrial function, NRTI phosphorylation, mitochondrial DNA (mtDNA) levels, and cytotoxicity were monitored over time. In endothelial cells, AZT significantly oxidized glutathione redox potential, increased total cellular and mitochondrial-specific superoxide, decreased mitochondrial membrane potential, increased lactate release, and caused cell death from weeks 3 through 5. Toxicity occurred in the absence of di- and tri-phosphorylated AZT and mtDNA depletion. These data show that oxidative stress and mitochondrial dysfunction in endothelial cells occur with a physiologically relevant concentration of AZT, and require long-term exposure to develop. In contrast, d4T did not induce endothelial oxidative stress, mitochondrial dysfunction, or cytotoxicity despite the presence of d4T-triphosphate. Both drugs depleted mtDNA in HepG2 cells without causing cell death. Endothelial cells are more susceptible to AZT-induced toxicity than HepG2 cells, and AZT caused greater endothelial dysfunction than d4T because of its pro-oxidative effects.

• Calcium-Dependent NOX5 Nicotinamide Adenine Dinucleotide Phosphate Oxidase Contributes to Vascular Oxidative Stress in Human Coronary Artery Disease.

  Authors: Tomasz J Guzik, Wei Chen, Maria C Gongora, Bartlomiej Guzik, Heinrich E Lob, Deepa Mangalat, Nyssa Hoch, Sergey Dikalov, Pawel Rudzinski, Boguslaw Kapelak, Jerzy Sadowski, David G Harrison

  Journal of the American College of Cardiology. 12/2008; 52(22):1803-9.

  OBJECTIVES: This study sought to examine the expression and activity of the calcium-dependent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in human atherosclerotic coronaryOBJECTIVES: This study sought to examine the expression and activity of the calcium-dependent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in human atherosclerotic coronary arteries. BACKGROUND: The NOX-based NADPH oxidases are major sources of reactive oxygen species (ROS) in human vessels. Several NOX homologues have been identified, but their relative contribution to vascular ROS production in coronary artery disease (CAD) is unclear; NOX5 is a unique homolog in that it is calcium dependent and thus could be activated by vasoconstrictor hormones. Its presence has not yet been studied in human vessels. METHODS: Coronary arteries from patients undergoing cardiac transplantation with CAD or without CAD were studied; NOX5 was quantified and visualized using Western blotting, immunofluorescence, and quantitative real-time polymerase chain reaction. Calcium-dependent NADPH oxidase activity, corresponding greatly to NOX5 activity, was measured by electron paramagnetic resonance. RESULTS: Both Western blotting and quantitative real-time polymerase chain reaction indicated a marked increase in NOX5 protein and messenger ribonucleic acid (mRNA) in CAD versus non-CAD vessels. Calcium-dependent NADPH-driven production of ROS in vascular membranes, reflecting NOX5 activity, was increased 7-fold in CAD and correlated significantly with NOX5 mRNA levels among subjects. Immunofluorescence showed that NOX5 was expressed in the endothelium in the early lesions and in vascular smooth muscle cells in the advanced coronary lesions. CONCLUSIONS: These studies identify NOX5 as a novel, calcium-dependent source of ROS in atherosclerosis.

• Sustained release of a p38 inhibitor from non-inflammatory microspheres inhibits cardiac dysfunction.

  Authors: Jay C Sy, Gokulakrishnan Seshadri, Stephen C Yang, Milton Brown, Teresa Oh, Sergey Dikalov, Niren Murthy, Michael E Davis

  Nature materials. 11/2008;

  Cardiac dysfunction following acute myocardial infarction is a major cause of death in the world and there is a compelling need for new therapeutic strategies. In this report we demonstrate that aCardiac dysfunction following acute myocardial infarction is a major cause of death in the world and there is a compelling need for new therapeutic strategies. In this report we demonstrate that a direct cardiac injection of drug-loaded microparticles, formulated from the polymer poly(cyclohexane-1,4-diylacetone dimethylene ketal) (PCADK), improves cardiac function following myocardial infarction. Drug-delivery vehicles have great potential to improve the treatment of cardiac dysfunction by sustaining high concentrations of therapeutics within the damaged myocardium. PCADK is unique among currently used polymers in drug delivery in that its hydrolysis generates neutral degradation products. We show here that PCADK causes minimal tissue inflammatory response, thus enabling PCADK for the treatment of inflammatory diseases, such as cardiac dysfunction. PCADK holds great promise for treating myocardial infarction and other inflammatory diseases given its neutral, biocompatible degradation products and its ability to deliver a wide range of therapeutics.

• Honokiol is a potent scavenger of superoxide and peroxyl radicals.

  Authors: Sergey Dikalov, Tanya Losik, Jack L Arbiser

  Biochemical pharmacology. 09/2008; 76(5):589-96.

  Honokiol, a compound extracted from Magnolia officinalis, has antitumor and antiangiogenic properties in several tumor models in vivo. Among the downstream pathways inhibited by honokiol is nuclearHonokiol, a compound extracted from Magnolia officinalis, has antitumor and antiangiogenic properties in several tumor models in vivo. Among the downstream pathways inhibited by honokiol is nuclear factor kappa beta (NFkappabeta). A prime physiologic stimulus of NFkappabeta is reactive oxygen species. The chemical structure of honokiol suggests that it may be an effective scavenger of reactive oxygen species. In this work, we have studied the reactions of honokiol with superoxide and peroxyl radicals in cell-free and cellular systems using electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques. Honokiol efficiently scavenged superoxide radicals in xanthine oxidase and cytochrome P-450 cell-free systems with the rate constant 3.2x10(5)M(-1)s(-1), which is similar to reactivity of ascorbic acid but 20-times higher than reactivity of vitamin E analog trolox. Honokiol potently scavenged intracellular superoxide within melanoma cells. In addition, honokiol scavenged peroxyl radicals generated by 2,2'-azo-bis(2-amidinopropane hydrochloride) (AAPH). The rate constant of the reaction of honokiol with peroxyl radicals (1.4x10(6)M(-1)s(-1)) was calculated from the competition with spin trap 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO), and was found close to reactivity of trolox (2.5x10(6)M(-1)s(-1)). Therefore, honokiol is an effective scavenger of both superoxide and peroxyl radicals, which may be important for physiological activity of honokiol.

• Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration.

  Authors: Erik R Kline, Dean J Kleinhenz, Bill Liang, Sergey Dikalov, David M Guidot, C Michael Hart, Dean P Jones, Roy L Sutliff

  American journal of physiology. Heart and circulatory physiology. 07/2008; 294(6):H2792-804.

  Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports haveHuman immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.

• Role of the T cell in the genesis of angiotensin II induced hypertension and vascular dysfunction.

  Authors: Tomasz J Guzik, Nyssa E Hoch, Kathryn A Brown, Louise A McCann, Ayaz Rahman, Sergey Dikalov, Jorg Goronzy, Cornelia Weyand, David G Harrison

  The Journal of experimental medicine. 11/2007; 204(10):2449-60.

  Hypertension promotes atherosclerosis and is a major source of morbidity and mortality. We show that mice lacking T and B cells (RAG-1-/- mice) have blunted hypertension and do not developHypertension promotes atherosclerosis and is a major source of morbidity and mortality. We show that mice lacking T and B cells (RAG-1-/- mice) have blunted hypertension and do not develop abnormalities of vascular function during angiotensin II infusion or desoxycorticosterone acetate (DOCA)-salt. Adoptive transfer of T, but not B, cells restored these abnormalities. Angiotensin II is known to stimulate reactive oxygen species production via the nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase in several cells, including some immune cells. Accordingly, adoptive transfer of T cells lacking the angiotensin type I receptor or a functional NADPH oxidase resulted in blunted angiotensin II-dependent hypertension and decreased aortic superoxide production. Angiotensin II increased T cell markers of activation and tissue homing in wild-type, but not NADPH oxidase-deficient, mice. Angiotensin II markedly increased T cells in the perivascular adipose tissue (periadventitial fat) and, to a lesser extent the adventitia. These cells expressed high levels of CC chemokine receptor 5 and were commonly double negative (CD3+CD4-CD8-). This infiltration was associated with an increase in intercellular adhesion molecule-1 and RANTES in the aorta. Hypertension also increased T lymphocyte production of tumor necrosis factor (TNF) alpha, and treatment with the TNFalpha antagonist etanercept prevented the hypertension and increase in vascular superoxide caused by angiotensin II. These studies identify a previously undefined role for T cells in the genesis of hypertension and support a role of inflammation in the basis of this prevalent disease. T cells might represent a novel therapeutic target for the treatment of high blood pressure.

• Regulation of tetrahydrobiopterin biosynthesis by shear stress.

  Authors: Julian D Widder, Wei Chen, Li Li, Sergey Dikalov, Beat Thöny, Kazuyuki Hatakeyama, David G Harrison

  Circulation research. 11/2007; 101(8):830-8.

  An essential cofactor for the endothelial NO synthase is tetrahydrobiopterin (H4B). In the present study, we show that in human endothelial cells, laminar shear stress dramatically increases H4BAn essential cofactor for the endothelial NO synthase is tetrahydrobiopterin (H4B). In the present study, we show that in human endothelial cells, laminar shear stress dramatically increases H4B levels and enzymatic activity of GTP cyclohydrolase (GTPCH)-1, the first step of H4B biosynthesis. In contrast, protein levels of GTPCH-1 were not affected by shear. Shear did not change protein expression or activity of the downstream enzymes 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase and decreased protein levels of the salvage enzyme dihydrofolate reductase. Oscillatory shear only modestly affected H4B levels and GPTCH-1 activity. We also demonstrate that laminar, but not oscillatory shear stress, stimulates phosphorylation of GTPCH-1 on serine 81 and that this is mediated by the alpha prime (alpha') subunit of casein kinase 2. The increase in H4B caused by shear is essential in allowing proper function of endothelial NO synthase because GPTCH-1 blockade with 2,4-diamino-6-hydroxypyrimidine during shear inhibited dimer formation of endothelial NO synthase, increased endothelial cell superoxide production, and prevented the increase in NO production caused by shear. Thus, shear stress not only increases endothelial NO synthase levels but also stimulates production of H4B by markedly enhancing GTPCH-1 activity via casein kinase 2-dependent phosphorylation on serine 81. These findings illustrate a new function of casein kinase 2 in the endothelium and provide insight into regulation of GTPCH-1 activity.

• Measurement of reactive oxygen species in cardiovascular studies.

  Authors: Sergey Dikalov, Kathy K Griendling, David G Harrison

  Hypertension. 05/2007; 49(4):717-27.

• Detection of reactive oxygen species and nitric oxide in vascular cells and tissues: comparison of sensitivity and specificity.

  Authors: Hua Cai, Sergey Dikalov, Kathy K Griendling, David G Harrison

  Methods in molecular medicine. 02/2007; 139:293-312.

  Reactive oxygen and nitrogen species are thought to contribute to pathogenesis of many cardiovascular diseases including hypertension, atherosclerosis, restenosis, heart failure, and diabeticReactive oxygen and nitrogen species are thought to contribute to pathogenesis of many cardiovascular diseases including hypertension, atherosclerosis, restenosis, heart failure, and diabetic vascular complications. Some of these reactive oxygen species also play an important role in vascular signaling. In this chapter, we describe various techniques that we have successfully employed to reliably measure superoxide and hydrogen peroxide. Because reactive oxygen species are capable of rapidly inactivating nitric oxide and because endothelial function characterized by nitric oxide bioavailability is an important indicator of vascular health, we have also included novel techniques capable of directly measuring nitric oxide radical from vascular cells and tissues.

• Angiotensinergic stimulation of vascular endothelium in mice causes hypotension, bradycardia, and attenuated angiotensin response.

  Authors: Ramaswamy Ramchandran, Takanobu Takezako, Yasser Saad, Linda Stull, Bruno Fink, Hirotsugu Yamada, Sergey Dikalov, David G Harrison, Christine Moravec, Sadashiva S Karnik

  Proceedings of the National Academy of Sciences of the United States of America. 01/2007; 103(50):19087-92.

  It is not clear whether endothelial cell (EC) activation by the hormone angiotensin II (Ang II) modulates contraction of vascular smooth muscle cells (VSMCs) in the vasculature and whether impairmentIt is not clear whether endothelial cell (EC) activation by the hormone angiotensin II (Ang II) modulates contraction of vascular smooth muscle cells (VSMCs) in the vasculature and whether impairment of this regulation in vivo contributes to hypertension. Delineation of the actions of Ang II through the type 1 receptor (AT1R) on ECs in the blood vessels has been a challenging problem because of the predominance of the AT1R functions in VSMCs that lie underneath the endothelium. We have obviated this limitation by generating transgenic (TG) mice engineered to target expression of the constitutively active N111G mutant AT1R only in ECs. In these TG mice, the enhanced angiotensinergic signal in ECs without infusion of Ang II resulted in hypotension and bradycardia. The pressor response to acute infusion of Ang II was significantly reduced. Increased expression of endothelial nitric oxide synthase and production of hypotensive mediators, nitric oxide and cyclic guanosine monophosphate, cause these phenotypes. Hypotension and bradycardia observed in the TG mice could be rescued by treatment with an AT1R-selective antagonist. Our results imply that the Ang II action by means of EC-AT1R is antagonistic to vasoconstriction in general, and it may moderate the magnitude of functional response to Ang II in VSMCs. This control mechanism in vivo most likely is a determinant of altered hemodynamic regulation involved in endothelial dysfunction in hypertensive cardiovascular disease.

• 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.