Richard A Cohen

Whitaker Wellness Institute, Newport Beach, California, United States

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Publications (91)500.72 Total impact

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    ABSTRACT: Oxidative stress in the myocardium plays an important role in the pathophysiology of hemodynamic overload. The mechanism by which reactive oxygen species (ROS) in the cardiac myocyte mediate myocardial failure in hemodynamic overload is not known. Accordingly, our goals were to test whether myocyte-specific overexpression of peroxisomal catalase (pCAT) that localizes in the sarcoplasm a) protects mice from hemodynamic overload-induced failure and b) prevents oxidation and inhibition of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), an important sarcoplasmic protein. Chronic hemodynamic overload was caused by ascending aortic constriction (AAC) for 12 weeks in mice with myocyte-specific transgenic expression of pCAT. AAC caused LV hypertrophy and failure associated with a generalized increase in myocardial oxidative stress and specific oxidative modifications of SERCA at cysteine 674 and tyrosine 294/5. pCAT overexpression ameliorated myocardial hypertrophy and apoptosis, decreased pathological remodeling and prevented the progression to heart failure. Likewise, pCAT prevented oxidative modifications of SERCA and increased SERCA activity without changing SERCA expression. Thus, cardiac myocyte-restricted expression of pCAT a) effectively ameliorated the structural and functional consequences of chronic hemodynamic overload, and b) increased SERCA activity via a post-translational mechanism, most likely by decreasing inhibitory oxidative modifications. In pressure overload-induced heart failure cardiac myocyte cytosolic ROS play a pivotal role in mediating key pathophysiologic events including hypertrophy, apoptosis and decreased SERCA activity.
    AJP Heart and Circulatory Physiology 03/2014; · 3.63 Impact Factor
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    ABSTRACT: Glutaredoxin-1 (Glrx) is a cytosolic enzyme that regulates diverse cellular function by removal of GSH adducts from S-glutathionylated proteins including signaling molecules and transcription factors. Glrx is up-regulated during inflammation and diabetes. Glrx overexpression inhibits VEGF-induced endothelial cell (EC) migration. The aim was to investigate the role of up-regulated Glrx in EC angiogenic capacities and in vivo revascularization in the setting of hind limb ischemia. Glrx overexpressing EC from Glrx transgenic mice (TG) showed impaired migration and network formation and secreted higher level of soluble VEGF receptor 1 (sFlt), an antagonizing factor to VEGF. After hind limb ischemia surgery Glrx TG mice demonstrated impaired blood flow recovery, associated with lower capillary density and poorer limb motor function compared to wild type littermates. There were also higher levels of anti-angiogenic sFlt expression in the muscle and plasma of Glrx TG mice after surgery. Non-canonical Wnt5a is known to induce sFlt. Wnt5a was highly expressed in ischemic muscles and EC from Glrx TG mice, and exogenous Wnt5a induced sFlt expression and inhibited network formation in human microvascular EC. Adenoviral Glrx-induced sFlt in EC was inhibited by a competitive Wnt5a inhibitor. Furthermore, Glrx overexpression removed GSH adducts on p65 in ischemic muscle and EC, and enhanced nuclear factor kappa B (NF-kB) activity which was responsible for Wnt5a-sFlt induction. Taken together, up-regulated Glrx induces sFlt in EC via NF-kB -dependent Wnt5a, resulting in attenuated revascularization in hind limb ischemia. The Glrx-induced sFlt may be a part of mechanism of redox regulated VEGF signaling.
    Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
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    ABSTRACT: Using a novel cysteine thiol labeling strategy coupled with mass spectrometric analysis, we identified and quantified the changes in global reversible cysteine oxidation of proteins in the left ventricle of hearts from mice with metabolic syndrome-associated diastolic dysfunction. This phenotype was induced by feeding a high-fat, high-sucrose, type-2 diabetogenic diet to C57BL/6J mice for 8 mo. The extent of reversible thiol oxidation in relationship to the total available (free and reducible) level of each cysteine could be confidently determined for 173 proteins, of which 98 contained cysteines differentially modified ≥1.5-fold by the diet. Our findings suggest that the metabolic syndrome leads to potentially deleterious changes in the oxidative modification of metabolically active proteins. These alterations may adversely regulate energy substrate flux through glycolysis, β-oxidation, citric acid (TCA) cycle, and oxidative phosphorylation (oxphos), thereby contributing to maladaptive tissue remodeling that is associated with, and possibly contributing to, diastolic left ventricular dysfunction.-Behring, J. B., Kumar, V., Whelan, S. A., Chauhan, P., Siwik, D. A., Costello, C. E., Colucci, W. S., Cohen, R. A., McComb M. E., Bachschmid, M. M. Does reversible cysteine oxidation link the Western diet to cardiac dysfunction?
    The FASEB Journal 01/2014; · 5.70 Impact Factor
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    ABSTRACT: Sirtuin-1 (SirT1), a member of the NAD+-dependent class III histone deacetylase family, is inactivated in vitro by oxidation of critical cysteine thiols. In a model of metabolic disease, SirT1 activation attenuated apoptosis of hepatocytes and improved liver function including insulin and lipid metabolism. We show in SirT1 overexpressing HepG2 cells that oxidants (nitrosocysteine or hydrogen peroxide) or metabolic stress (high palmitate and high glucose) inactivate SirT1 by reversible oxidative post-translational modifications (OPTM) on cysteines. Mutating these oxidation-sensitive cysteines to serine preserves SirT1 activity and abolishes reversible OPTMs. Overexpressed mutant SirT1 maintains deacetylase activity and attenuates proapoptotic signaling, while overexpressed wild type SirT1 is less protective in metabolically or oxidant stressed cells. To prove that OPTMs of SirT1 are glutathione (GSH) adducts, glutaredoxin-1 (Glrx) was overexpressed to remove this modification. Glrx overexpression maintains endogenous SirT1 activity and prevents proapoptotic signaling in metabolically stressed HepG2 cells. The in vivo significance of oxidative inactivation of SirT1 was investigated in livers of high fat diet-fed C57/B6J mice. SirT1 deacetylase activity was decreased in the absence of changes in SirT1 expression and associated with a marked increase in OPTMs. These results indicate that glutathione adducts on specific SirT1 thiols may be responsible for dysfunctional SirT1 associated with liver disease in metabolic syndrome.
    Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
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    ABSTRACT: Diet-induced obesity and metabolic syndrome are important contributors to cardiovascular diseases. The decreased nitric oxide (NO) bioactivity in endothelium and the impaired response of smooth muscle cell (SMC) to NO significantly contribute to vascular pathologies, including atherosclerosis and arterial restenosis after angioplasty. Sarco/endoplasmic reticulum Ca2 + ATPase (SERCA) is an important mediator of NO function in both endothelial cells and SMCs, and its irreversible oxidation impair its stimulation by NO. We used C57BL/6 J mice fed a high fat, high sucrose diet (HFHSD) to study the role of SMC SERCA in diet-induced obesity and metabolic syndrome. We found that HFHSD upregulated Nox2 based NADPH oxidase, induced inflammation, increased irreversible SERCA oxidation, and suppressed the response of aortic SERCA to NO. Cultured aortic SMCs from mice fed HFHSD showed increased reactive oxygen species production, Nox2 upregulation, irreversible SERCA oxidation, inflammation, and a decreased ability of NO to inhibit SMC migration. Overexpression of wild type SERCA2b or downregulation of Nox2 restored NO-mediated inhibition of migration in SMCs isolated from HFHSD-fed mice. In addition, tumor necrosis factor alpha (TNFα) increased Nox2 which induced SERCA oxidation and inflammation. Taken together, Nox2 induced by HFHSD plays significant roles in controlling SMC responses to NO and TNFα-mediated inflammation, which may contribute to the development of cardiovascular diseases in diet-induced obesity and metabolic syndrome.
    Journal of Molecular and Cellular Cardiology 01/2014; · 5.15 Impact Factor
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    ABSTRACT: The endoplasmic reticulum (ER) plays a pivotal role in lipid and protein biosynthesis as well as calcium store regulation, which determines its essential role in cell function. Hypoxia, nutrient deprivation, perturbation of redox status and aberrant calcium regulation can all trigger the ER stress response, which is mediated through three main sensors, namely inositol requiring element-1 (IRE-1), protein kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6). This review explores the interaction of ER stress and ER stress-associated pathological processes, including inflammation, apoptosis, aberrant autophagy, mitochondrial dysfunction and hypoxic responses. In addition, the correlation of ER stress with lipid and calcium homeostasis and dysregulation, and its role in disease development is also presented. Improved understanding of ER stress and its cofactors in pathological processes may provide new perspective on disease development and control.
    Journal of pharmacological & biomedical analysis. 11/2013; 1(2):1000107.
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    ABSTRACT: Stiffening of conduit arteries is a risk factor for cardiovascular morbidity. Aortic wall stiffening increases pulsatile hemodynamic forces that are detrimental to the microcirculation in highly perfused organs, such as the heart, brain, and kidney. Arterial stiffness is associated with hypertension but presumed to be due to an adaptive response to increased hemodynamic load. In contrast, a recent clinical study found that stiffness precedes and may contribute to the development of hypertension although the mechanisms underlying hypertension are unknown. Here, we report that in a diet-induced model of obesity, arterial stiffness, measured in vivo, develops within 1 month of the initiation of the diet and precedes the development of hypertension by 5 months. Diet-induced obese mice recapitulate the metabolic syndrome and are characterized by inflammation in visceral fat and aorta. Normalization of the metabolic state by weight loss resulted in return of arterial stiffness and blood pressure to normal. Our findings support the hypothesis that arterial stiffness is a cause rather than a consequence of hypertension.
    Hypertension 09/2013; · 6.87 Impact Factor
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    ABSTRACT: The goal of this study was to identify the cellular mechanisms responsible for cardiac dysfunction in endotoxemic mice. We aimed to differentiate the roles of cGMP (produced by soluble guanylyl cyclase, sGC) vs. oxidative post-translational modifications (OPTM) of calcium (Ca(2+)) transporters. C57BL/6 mice (wild type, WT) were administered lipopolysaccharide (LPS, 25 μg/g, intraperitoneal) and euthanized 12 h later. Cardiomyocyte sarcomere shortening and Ca(2+) transients (ΔCai) were depressed in LPS-challenged mice vs. baseline. The time constant (τCa) of Ca(2+) decay was prolonged, and sarcoplasmic reticulum (SR) Ca(2+) load (CaSR) was depressed in LPS-challenged mice (vs. baseline), indicating decreased activity of SR Ca(2+) ATP-ase (SERCA). L-Type Ca(2+) channel (LTCC) current (ICa,L) was also decreased after LPS challenge, while Na(+)/Ca(2+) exchange activity, ryanodine receptors leak flux or myofilament sensitivity for Ca(2+) were unchanged. All Ca(2+) handling abnormalities induced by LPS (the decrease in sarcomere shortening, ΔCai, CaSR, ICa,L and τCa prolongation) were more pronounced in mice deficient in sGC main isoform (sGCα1(-/-)) vs. WT. LPS did not alter protein expression of SERCA and phospholamban (PLB) in either genotype. After LPS, PLB phosphorylation at Ser-16 and Thr-17 was unchanged in WT mice, and was increased in sGCα1(-/-) mice. LPS caused sulphonylation of SERCA cysteine-674 (as measured immunohistochemically and supported by iodoacetamide labeling), which was greater in sGCα1(-/-) vs. WT mice. Taken together, these results suggest that cardiac Ca(2+) dysregulation in endotoxemic mice is mediated by a decrease in LTCC function and OPTM of SERCA cysteine-674, the later (at least) being opposed by sGC-released cGMP.
    AJP Heart and Circulatory Physiology 08/2013; · 3.63 Impact Factor
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    ABSTRACT: Glutathionylation of the Na(+)-K(+) pump's β1 subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na(+)-K(+) pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β subunit isoform expression and receptor-coupled pathways. As Na(+)-K(+) pump activity is an important determinant of vascular tone through effects on [Ca(2+)]i, we have examined the role of oxidative regulation of the Na(+)-K(+) pump in mediating Angiotensin II (Ang II)-induced increase in vascular reactivity. β1 subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs) and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na(+)-K(+)ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K(+)-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na(+)-K(+) ATPase and decrease in K(+)-induced relaxation were reversed by pre-incubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β1 subunit. Knock-out of FXYD1 dramatically decreased K(+)-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β1 subunit glutathionylation, and enhanced K(+)-induced vasorelaxation. Ang II inhibits the Na(+)-K(+) pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump's β1 subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na(+)-K(+) pump and may have an important protective role in the vasculature under conditions of oxidative stress.
    Free radical biology & medicine 06/2013; · 5.42 Impact Factor
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    ABSTRACT: Unfavorable metabolic conditions (metabolic disorders) associated with obesity and diabetes are major causes for cardiovascular disease. Early detection of the adverse effects of metabolic disease remain elusive. We believe that nonspecific changes which occur in plasma proteins, indicators of inflammation and oxidants, may act as evidence of systemic metabolic disease. Here we elucidate potential biomarkers of CVD including both protein changes and changes in post-translational modifications (PTMs). Label-free mass spectrometry-based proteomics was used to interrogate changes in differential protein and PTM expression in plasma samples from mouse and human models. Label-free LCMS/MS typically yielded >1,000 features ( p<0.05, >2fold). A number of cardiovascular disease related proteins were observed. Up regulated proteins were: haptoglobin, a known biomarker related to inflammation, low mannose binding protein associated with inflammation and CVD in type 2 diabetes, superoxide dismutase and extracellular matrix protein both implicated in type 2 diabetes. We also observed different PTMs associated with oxidative stress including lipid peroxidation products such as hydroxynonenal and multiple forms of oxidation such as cysteine sulfonic acid. Development of a metabolic disorder/CVD-specific protein panel will afford the first step in biomarker panel development such that disease diagnosis and progression may be performed directly at the molecular level. This project was funded by NIH-NCRR grants P41 RR010888/ GM104603, S10 RR015942, S10 RR020946, S10 RR025082 and NIH-NHLBI contract N01 HV00239.
    Experimental Biology FASEB J April 9, 2013 27:663.10, Boston, MA; 04/2013
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    ABSTRACT: Unfavorable metabolic conditions (metabolic disorders) associated with obesity, diabetes, and hyperlipidemia are major causes for cardiovascular disease (CVD). One major environmental cause of this may be attributed to poor diet, aka the American diet. The early detection and monitoring of the adverse effects of metabolic disease on the heart and vasculature, although well studied, remain elusive. Our hypothesis is that poor diet causes unfavorable metabolic conditions in heart tissue resulting in inflammation and oxidative stress reflected in protein changes. Here we apply label-free proteomics to elucidate potential biomarkers of CVD including both protein changes and changes in post-translational modifications (PTMs). Heart tissue was from control mice and mice fed a high fat high sucrose diet (HFHS). MS/MS data was analyzed with Proteome Discoverer and Mascot software, using both variable-modification and error-tolerant search modes. Label-free quantification was conducted using Scaffold and Progenesis; typically yielding >1,000 features. Using IPA software revealed a number of cardiovascular disease related proteins were observed. This is the first step in biomarker panel development for disease diagnosis and progression. This project was funded by NIH-NCRR grants P41 RR010888/GM104603, S10 RR015942, S10 RR020946, S10 RR025082 and NIH-NHLBI contract N01 HV00239.
    Experimental Biology FASEB J April 9, 2013 27:794.17, Boston, MA; 04/2013
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    ABSTRACT: Global changes in reversible cysteine oxidation of cardiac proteins from mice fed a high fat, high sucrose (HFHS) diet for 8 months were quantified using novel 6-plex Iodo-based isobaric labels in a switch assay and high resolution mass spectrometry. 400 reversibly oxidized proteins were identified, 56 with cysteines exhibiting more than 1.5-fold change in oxidation by HFHS diet. As an improvement on "fold-change", our design also defined the percentage of oxidized cysteine at each site, or "site occupancy". Nearly 50% of the proteins localized to mitochondria, consistent with this organelle being the primary target of metabolic stress resulting in mitochondrial oxidant formation. Multiple proteins in each of the critical metabolic pathways, including: the electron transport chain, tricarboxylic acid cycle, acyl-carnitine shuttle, beta-oxidation, and ketolysis, were reversibly oxidized. This suggests that substrate utilization pathways are dysfunctional. The condition further worsens as arterial stiffness increases energy demand of an overworked heart. All of these factors likely contribute to cardiac diastolic dysfunction and hypertrophy that occurs in the mice. Identifying and quantifying reversible oxidative protein changes will elucidate novel signaling mechanisms for switches in metabolism; these modifications could potentially serve as biomarkers for diet-induced metabolic disease. NHLBI HHSN268201000031C
    Experimental Biology FASEB J April 9, 2013 27:558.3, Boston, MA; 04/2013
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    ABSTRACT: We demonstrate for the first time that endomembrane-delimited HRas mediates VEGF-induced activation of eNOS and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total HRas is S-palmitoylated, tethering the small GTPase to the plasma membrane, but leaving the function of the large majority of endomembrane-localized HRas unexplained. Knock-down of HRas blocked VEGF-induced PI3K-dependent Akt (S473) and eNOS (S1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of HRas. Activation of endogenous HRas led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous HRas was fully restored by modest over-expression of an endomembrane-delimited HRas palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized HRas in mediating nitric oxide-dependent pro-angiogenic signaling.
    Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
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    ABSTRACT: Aberrantly elevated sterol regulatory element binding protein (SREBP), the lipogenic transcription factor, contributes to the development of fatty liver and insulin resistance in animals. Our recent studies have discovered that AMP-activated protein kinase (AMPK) phosphorylates SREBP at Ser-327 and inhibits its activity, represses SREBP-dependent lipogenesis, and thereby ameliorates hepatic steatosis and atherosclerosis in insulin-resistant LDLR(-/-) mice. Chronic inflammation and activation of NLRP3 inflammasome have been implicated in atherosclerosis and fatty liver disease. However, whether SREBP is involved in vascular lipid accumulation and inflammation in atherosclerosis remains largely unknown. The preclinical study with aortic pouch biopsy specimens from humans with atherosclerosis and diabetes shows intense immunostaining for SREBP-1 and the inflammatory marker VCAM-1 in atherosclerotic plaques. The cleavage processing of SREBP-1 and -2 and expression of their target genes are increased in the well-established porcine model of diabetes and atherosclerosis, which develops human-like, complex atherosclerotic plaques. Immunostaining analysis indicates an elevation in SREBP-1 that is primarily localized in endothelial cells and in infiltrated macrophages within fatty streaks, fibrous caps with necrotic cores, and cholesterol crystals in advanced lesions. Moreover, concomitant suppression of NAD-dependent deacetylase SIRT1 and AMPK is observed in atherosclerotic pigs, which leads to the proteolytic activation of SREBP-1 by diminishing the deacetylation and Ser-372 phosphorylation of SREBP-1. Aberrantly elevated NLRP3 inflammasome markers are evidenced by increased expression of inflammasome components including NLPR3, ASC, and IL-1β. The increase in SREBP-1 activity and IL-1β production in lesions is associated with vascular inflammation and endothelial dysfunction in atherosclerotic pig aorta, as demonstrated by the induction of NF-κB, VCAM-1, iNOS, and COX-2, as well as by the repression of eNOS. These translational studies provide in vivo evidence that the dysregulation of SIRT1-AMPK-SREBP and stimulation of NLRP3 inflammasome may contribute to vascular lipid deposition and inflammation in atherosclerosis.
    PLoS ONE 01/2013; 8(6):e67532. · 3.73 Impact Factor
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    ABSTRACT: A hallmark of aging of the cardiac myocyte is impaired sarcoplasmic reticulum (SR) calcium uptake and relaxation due to decreased SR calcium ATPase (SERCA) activity. We tested the hypothesis that H2O2-mediated oxidation of SERCA contributes to impaired myocyte relaxation in aging. Young (5-month-old) and senescent (21-month-old) FVB wild-type (WT) or transgenic mice with myocyte-specific overexpression of catalase were studied. In senescent mice, myocyte-specific overexpression of catalase (1) prevented oxidative modification of SERCA as evidenced by sulfonation at Cys674, (2) preserved SERCA activity, (3) corrected impaired calcium handling and relaxation in isolated cardiac myocytes, and (4) prevented impaired left ventricular relaxation and diastolic dysfunction. Nitroxyl, which activates SERCA via S-glutathiolation at Cys674, failed to activate SERCA in freshly isolated ventricular myocytes from senescent mice. Finally, in adult rat ventricular myocytes in primary culture, adenoviral overexpression of SERCA in which Cys674 is mutated to serine partially preserved SERCA activity during exposure to H2O2. Oxidative modification of SERCA at Cys674 contributes to decreased SERCA activity and impaired myocyte relaxation in the senescent heart. Strategies to decrease oxidant levels and/or protect target proteins such as SERCA may be of value to preserve diastolic function in the aging heart.
    Journal of the American Heart Association. 01/2013; 2(4):e000184.
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    ABSTRACT: Endothelial cell (EC) migration in response to VEGF is a critical step in both physiological and pathological angiogenesis. Although VEGF signaling has been extensively studied, the mechanisms by which VEGF-dependent reactive oxygen species (ROS) production affects EC signaling are not well-understood. The aim of this study was to elucidate the involvement of Nox2- and Nox4-dependent ROS in VEGF-mediated EC Ca(2+) regulation and migration. VEGF induced migration of human aortic EC into a scratch wound over 6 hours that was inhibited by overexpression of either catalase or SOD. EC stimulation by micromolar concentrations of H(2)O(2) was inhibited by catalase, but also unexpectedly by SOD. Both VEGF and H(2)O(2) increased S-glutathiolation of SERCA2b and increased Ca(2+) influx into EC, and these events could be blocked by overexpression of catalase or overexpression of SERCA2b in which the reactive cysteine-674 was mutated to a serine. In determining the source of VEGF-mediated ROS production, our studies show that specific knock down of either Nox2 or Nox4 inhibited VEGF-induced S-glutathiolation of SERCA, Ca(2+) influx, and EC migration. Treatment with H(2)O(2) induced S-glutathiolation of SERCA and EC Ca(2+) influx, overcoming the knockdown of Nox4, but not Nox2, and Amplex Red measurements indicated that Nox4 is the source of H(2)O(2). These results demonstrate that VEGF stimulates EC migration through increased S-glutathiolation of SERCA and Ca(2+) influx in a Nox4- and H(2)O(2)-dependent manner, requiring Nox2 downstream.
    Free radical biology & medicine 10/2012; · 5.42 Impact Factor
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    ABSTRACT: Unfavorable metabolic conditions (metabolic disorders) associated with obesity and diabetes are major causes for cardiovascular disease. One major cause may be attributed to poor diet, aka the American diet model. Early detection of the adverse effects of metabolic disease remain elusive. We believe that nonspecific changes which occur in plasma proteins, indicators of inflammation and oxidants, may act as evidence of systemic metabolic disease. Here we use an American diet mouse model to elucidate potential biomarkers of CVD including both protein changes and changes in post-translational modifications (PTMs). Label-free mass spectrometry-based proteomics was used to interrogate changes in differential protein and PTM expression in our model. Plasma was from control mice and mice fed a high fat high sucrose diet. LC-MS/MS was performed on LTQ-Orbitrap or Q Exactive mass spectrometers, coupled with Waters NanoAcquity HPLCs. Proteome Discoverer (Thermo-Fisher), Mascot (Matrix Science) and Progenesis LCMS (Nonlinear Dynamics) were used for analysis. Meta-analysis was conducted using the Trans Proteomic Pipeline (ISB), Scaffold (Proteome Software), and STRAP, STRAPPTM (in-house) software. Label-free LCMS/MS yielded more than 700 features ( p<0.05, >2fold). A number of cardiovascular disease related proteins were observed. Up regulated proteins were: haptoglobin, a known biomarker related to inflammation, low mannose binding protein associated with inflammation and CVD in type 2 diabetes, superoxide dismutase and extracellular matrix protein both implicated in type 2 diabetes. We also observed different PTMs associated with oxidative stress including lipid peroxidation products such as hydroxynonenal and multiple forms of oxidation such as cysteine sulfonic acid. Development of a metabolic disorder/CVD-specific protein panel will afford the first step in biomarker panel development such that disease diagnosis and progression may be performed directly at the molecular level. This project was funded by NIH-NCRR grants P41 RR010888/ GM104603, S10 RR015942, S10 RR020946, S10 RR025082 and NIH-NHLBI contract N01 HV00239.
    The 11th HUPO World Congress, Boston, MA; 09/2012
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    ABSTRACT: AIMS: Vascular endothelial growth factor (VEGF) increases angiogenesis by stimulating endothelial cell (EC) migration. VEGF-induced nitric oxide ((•)NO) release from (•)NO synthase plays a critical role, but the proteins and signaling pathways that may be redox-regulated are poorly understood. The aim of this work was to define the role of (•)NO-mediated redox regulation of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) in VEGF-induced signaling and EC migration. RESULTS: VEGF-induced EC migration was prevented by the (•)NO synthase inhibitor, N (G)-nitro-L-arginine methyl ester (LNAME). Either VEGF or (•)NO stimulated endoplasmic reticulum (ER) (45)Ca(2+) uptake, a measure of SERCA activity, and knockdown of SERCA2 prevented VEGF-induced EC migration and (45)Ca(2+) uptake. S-glutathione adducts on SERCA2b, identified immunochemically, were increased by VEGF, and were prevented by LNAME or overexpression of glutaredoxin-1 (Glrx-1). Furthermore, VEGF failed to stimulate migration of ECs overexpressing Glrx-1. VEGF or (•)NO increased SERCA S-glutathiolation and stimulated migration of ECs in which wild-type (WT) SERCA2b was overexpressed with an adenovirus, but did neither in those overexpressing a C674S SERCA2b mutant, in which the reactive cysteine-674 was mutated to a serine. Increased EC Ca(2+) influx caused by VEGF or (•)NO was abrogated by overexpression of Glrx-1 or the C674S SERCA2b mutant. ER store-emptying through the ryanodine receptor (RyR) and Ca(2+) entry through Orai1 were also required for VEGF- and (•)NO-induced EC Ca(2+) influx. INNOVATION AND CONCLUSIONS: These results demonstrate that (•)NO-mediated activation of SERCA2b via S-glutathiolation of cysteine-674 is required for VEGF-induced EC Ca(2+) influx and migration, and establish redox regulation of SERCA2b as a key component in angiogenic signaling.
    Antioxidants & Redox Signaling 04/2012; 17(8):1099-108. · 8.20 Impact Factor
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    ABSTRACT: Activation of thromboxane receptors (TPr) may promote atherosclerosis by enhancing oxidative stress and inflammation. This study examined the role of Nox1, an NADPH-oxidase subunit, in the enhancement of interleukin (IL)-1β-induced monocyte adhesion by TPr. In cultured rat aortic vascular smooth muscle cells (VSMCs), U46619, a stable thromboxane A(2) mimetic, together with interleukin-1β significantly enhanced Nox1 mRNA expression, as well as adhesion of THP-1 monocytes. Activation of TPr also enhanced IL-1β-induced vascular cell adhesion molecule (VCAM)-1 expression, but inhibited inducible nitric oxide synthase (iNOS) expression. Silencing Nox1 expression by siRNA prevented the U46619 enhancement of IL-1β-induced monocyte adhesion, but had no significant effect on VCAM-1 or iNOS expression. Furthermore, monocyte adhesion was inhibited by superoxide dismutase, enhanced by a specific iNOS inhibitor, l-N(6)-(1-iminoethyl)-lysine, but not influenced by catalase. U46619 inhibited IL-1β-induced cyclic GMP production, and the inhibition was partially prevented by superoxide dismutase. In conclusion, activation of TPr enhances IL-1β-induced Nox1 expression in VSMCs, which is responsible for the up-regulation of monocyte adhesion. The effect of Nox1 is independent of the changes in VCAM-1 and iNOS expression, but depends on the inactivation of nitric oxide via generation of superoxide anion.
    Free radical biology & medicine 03/2012; 52(9):1760-6. · 5.42 Impact Factor
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    ABSTRACT: Diet-induced obesity is associated with metabolic heart disease characterized by left ventricular hypertrophy and diastolic dysfunction. Polyphenols such as resveratrol and the synthetic flavonoid derivative S17834 exert beneficial systemic and cardiovascular effects in a variety of settings including diabetes mellitus and chronic hemodynamic overload. We characterized the structural and functional features of a mouse model of diet-induced metabolic syndrome and used the model to test the hypothesis that the polyphenols prevent myocardial hypertrophy and diastolic dysfunction. Male C57BL/6J mice were fed a normal diet or a diet high in fat and sugar (HFHS) with or without concomitant treatment with S17834 or resveratrol for up to 8 months. HFHS diet-fed mice developed progressive left ventricular hypertrophy and diastolic dysfunction with preservation of systolic function in association with myocyte hypertrophy and interstitial fibrosis. In HFHS diet-fed mice, there was increased myocardial oxidative stress with evidence of oxidant-mediated protein modification via tyrosine nitration and 4-OH-2-nonenol adduction. HFHS diet-fed mice also exhibited increases in plasma fasting glucose, insulin, and homeostasis model assessment of insulin resistance indicative of insulin resistance. Treatment with S17834 or resveratrol prevented left ventricular hypertrophy and diastolic dysfunction. For S17834, these beneficial effects were associated with decreases in oxidant-mediated protein modifications and hyperinsulinemia and increased plasma adiponectin. Resveratrol and S17834 administered concurrently with a HFHS diet prevent the development of left ventricular hypertrophy, interstitial fibrosis, and diastolic dysfunction. Multiple mechanisms may contribute to the beneficial effects of the polyphenols, including a reduction in myocardial oxidative stress and related protein modifications, amelioration of insulin resistance, and increased plasma adiponectin. The polyphenols resveratrol and S17834 may be of value in the prevention of diet-induced metabolic heart disease.
    Circulation 03/2012; 125(14):1757-64, S1-6. · 15.20 Impact Factor

Publication Stats

3k Citations
194 Downloads
500.72 Total Impact Points

Institutions

  • 2001–2014
    • Whitaker Wellness Institute
      Newport Beach, California, United States
  • 2013
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 2006–2013
    • Boston Medical Center
      Boston, Massachusetts, United States
  • 2012
    • University of Massachusetts Boston
      Boston, Massachusetts, United States
  • 2002–2012
    • Boston University
      • • Department of Medicine
      • • Cardiovascular Proteomics Center
      Boston, MA, United States
    • University of Saskatchewan
      • College of Medicine
      Saskatoon, Saskatchewan, Canada
  • 2003
    • University of Tennessee
      • Department of Surgery
      Knoxville, TN, United States