Richard A Cohen

Whitaker Wellness Institute, Newport Beach, California, United States

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Publications (107)638.46 Total impact

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    ABSTRACT: Vascular endothelial cells (ECs) are responsible for post-ischemic angiogenesis, a process that is regulated by reactive oxygen species. Recent studies indicate that endothelial Nox4 based NADPH oxidase may have a key role. This study examines the role of endothelial Nox4 in ischemia-induced angiogenesis and explores the potential mechanisms involved. Mouse lines overexpressing human Nox4 wild type (EWT) or its dominant negative form P437H (EDN) specifically in the endothelium were used. Non-transgenic littermate mice (NTg) were used as controls. Following hind limb ischemia, blood flow recovery was enhanced in EWT and was impaired in EDN compared with NTg. The critical angiogenesis regulating genes vascular endothelial growth factor receptor 2 (VEGFR2), endothelial nitric oxide synthase (eNOS) and transforming growth factor β1 (TGFβ1) were upregulated in EWT both in the ischemic muscle and in heart ECs, while TGFβ1 was downregulated in EDN ECs. In EC, both VEGFA and TGFβ1 stimulated EC proliferation, migration, and capillary-like network formation in EWT but failed to do so in EDN. Application of TGFβ1 increased both VEGFR2 and eNOS expression levels, whereas blocking TGFβ1 or addition of catalase inhibited the phosphorylation of VEGFR2 and eNOS, indicating H2O2 and TGFβ1 signaling downstream of Nox4 is critical to maintain EC angiogenic functions. Use of cell specific transgenic mice with both upregulation and downregulation of endothelial Nox4 indicate several mechanisms linked to Nox4 play a role in angiogenesis. Endothelial Nox4 regulates ischemia-induced angiogenesis, likely through H2O2- and TGFβ1-mediated activation of cell signaling pathways essential for endothelial function.
    Biochimica et biophysica acta. 10/2014;
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    ABSTRACT: Ischemia is a complex phenomenon modulated by the concerted action of several cell types. We have identified that sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase 2 (SERCA 2) cysteine 674 (C674) S-glutathiolation is essential for ischemic angiogenesis, vascular endothelial growth factor (VEGF)-mediated endothelial cell (EC) migration and network formation. A heterozygote SERCA 2 C674S knockin (SKI) mouse shows impaired ischemic blood flow recovery after femoral artery ligation, and its EC show depleted endoplasmic reticulum (ER) Ca(2+) stores and impaired angiogenic behavior. Here we studied the role of SERCA 2 C674 in the interaction between ECs and macrophages in the context of ischemia and discovered the involvement of the ER stress response protein, ER oxidoreductin-1α (ERO1). In wild type (WT) mice, expression of ERO1 was increased in the ischemic hind limb in vivo, as well as in ECs and macrophages exposed to hypoxia in vitro. The increase in ERO1 to ischemia/hypoxia was less in SKI mice. In WT ECs, both vascular cell adhesion molecule 1 (VCAM1) expression and bone marrow-derived macrophage adhesion to ECs were increased by hypoxia, and both were attenuated in SKI ECs. In WT ECs, ERO1 siRNA blocked hypoxia-induced VCAM1 expression and macrophage adhesion. In WT macrophages, hypoxia also stimulated both ERO1 and VEGF expression, and both were less in SKI macrophages. Compared with conditioned media of hypoxic SKI macrophages, conditioned media from WT macrophages had a greater effect on EC angiogenic behavior, and was blocked by VEGF neutralizing antibody. Taken together, under hypoxic conditions, SERCA 2 C674 and ERO1 enable increased VCAM1 expression and macrophage adhesion to ECs, as well as macrophage VEGF production that, in turn, promote angiogenesis. This study highlights the hitherto unrecognized interaction of two ER proteins, SERCA 2 C674 and ERO1, which mediate the EC and macrophage angiogenic response to ischemia/hypoxia.
    Journal of Molecular and Cellular Cardiology 09/2014; · 5.15 Impact Factor
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    ABSTRACT: Background: Diet-induced obesity leads to metabolic heart disease (MHD) characterized by increased oxidative stress that may cause oxidative post-translational modifications (OPTM) of cardiac mitochondrial proteins. The functional consequences of OPTM of cardiac mitochondrial proteins in MHD are unknown. Our objective was to determine whether cardiac mitochondrial dysfunction in MHD due to diet-induced obesity is associated with cysteine OPTM. Methods and results: Male C57BL/6J mice were fed either a high-fat, high-sucrose (HFHS) or control diet for 8months. Cardiac mitochondria from HFHS-fed mice (vs. control diet) had an increased rate of H2O2 production, a decreased GSH/GSSG ratio, a decreased rate of complex II substrate-driven ATP synthesis and decreased complex II activity. Complex II substrate-driven ATP synthesis and complex II activity were partially restored ex-vivo by reducing conditions. A biotin switch assay showed that HFHS feeding increased cysteine OPTM in complex II subunits A (SDHA) and B (SDHB). Using iodo-TMT multiplex tags we found that HFHS feeding is associated with reversible oxidation of cysteines 89 and 231 in SDHA, and 100, 103 and 115 in SDHB. Conclusions: MHD due to consumption of a HFHS "Western" diet causes increased H2O2 production and oxidative stress in cardiac mitochondria associated with decreased ATP synthesis and decreased complex II activity. Impaired complex II activity and ATP production are associated with reversible cysteine OPTM of complex II. Possible sites of reversible cysteine OPTM in SDHA and SDHB were identified by iodo-TMT tag labeling. Mitochondrial ROS may contribute to the pathophysiology of MHD by impairing the function of complex II. This article is part of a Special Issue entitled 'Mitochondria'.
    Journal of Molecular and Cellular Cardiology 08/2014; · 5.15 Impact Factor
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    ABSTRACT: The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) is key to Ca2+ homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca2+ uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. In order to determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared to wild type (WT). Ca2+ studies showed decreased nitric oxide (NO)-induced 45Ca2+ uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca2+ stores and decreased vascular endothelial growth factor (VEGF)- and NO-induced Ca2+ influx. Adenoviral overexpression of calreticulin, an ER Ca2+ binding protein, increased ionomycin-releasable stores, VEGF-induced Ca2+ influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive C674 thiol on SERCA 2 is required for normal endothelial cell Ca2+ homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca2+ stores.
    The Journal of biological chemistry. 06/2014;
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    ABSTRACT: Autophagy is a highly conserved degradation process by which intracellular components, including soluble macromolecules (e.g. nucleic acids, proteins, carbohydrates, and lipids) and dysfunctional organelles (e.g. mitochondria, ribosomes, peroxisomes, and endoplasmic reticulum) are degraded by the lysosome. Autophagy is orchestrated by the autophagy related protein (Atg) composed protein complexes to form autophagosomes, which fuse with lysosomes to generate autolysosomes where the contents are degraded to provide energy for cell survival in response to environmental and cellular stress. Autophagy is an important player in cardiovascular disease development such as atherosclerosis, cardiac ischemia/reperfusion, cardiomyopathy, heart failure and hypertension. Autophagy in particular contributes to cardiac ischemia, hypertension and diabetes by interaction with reactive oxygen species generated in endoplasmic reticulum and mitochondria. This review highlights the dual role of autophagy in cardiovascular disease development. Full recognition of autophagy as an adaptive or maladaptive response would provide potential new strategies for cardiovascular disease prevention and management. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.
    Biochimica et biophysica acta. 05/2014;
  • Francesca Seta, Richard A Cohen
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    ABSTRACT: For over 30 years the endothelium has assumed greater and greater importance in our understanding of the development of vascular pathology. This includes the discoveries that the endothelium releases powerful vasodilator and antiplatelet mediators, prostacyclin and nitric oxide, as well as its role in governing permeability, inflammation, and monocyte/macrophage infiltration of the blood vessel. In this issue of Circulation, Fan and colleagues(1) show that boosting endothelial-derived oxidants in the mouse aorta by overexpression of the NADPH oxidase isoform, Nox2, during prolonged angiotensin II-induced hypertension, results in a high incidence of infra-renal aortic dissection.
    Circulation 05/2014; · 15.20 Impact Factor
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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; · 4.01 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: Autophagy is a highly conserved degradation process by which intracellular components, including soluble macromolecules (e.g. nucleic acids, proteins, carbohydrates, and lipids) and dysfunctional organelles (e.g. mitochondria, ribosomes, peroxisomes, and endoplasmic reticulum) are degraded by the lysosome. Autophagy is orchestrated by the autophagy related protein (Atg) composed protein complexes to form autophagosomes, which fuse with lysosomes to generate autolysosomes where the contents are degraded to provide energy for cell survival in response to environmental and cellular stress. Autophagy is an important player in cardiovascular disease development such as atherosclerosis, cardiac ischemia/reperfusion, cardiomyopathy, heart failure and hypertension. Autophagy in particular contributes to cardiac ischemia, hypertension and diabetes by interaction with reactive oxygen species generated in endoplasmic reticulum and mitochondria. This review highlights the dual role of autophagy in cardiovascular disease development. Full recognition of autophagy as an adaptive or maladaptive response would provide potential new strategies for cardiovascular disease prevention and management. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.
    Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 01/2014;
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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; · 4.01 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 and Medicine 06/2013; · 5.27 Impact Factor
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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: 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: 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

Publication Stats

4k Citations
638.46 Total Impact Points

Institutions

  • 1999–2014
    • Whitaker Wellness Institute
      Newport Beach, California, United States
  • 2013
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 2000–2013
    • Boston Medical Center
      Boston, Massachusetts, United States
  • 1996–2012
    • University of Massachusetts Boston
      Boston, Massachusetts, United States
  • 1989–2012
    • Boston University
      • • Department of Medicine
      • • Cardiovascular Proteomics Center
      • • Department of Urology
      Boston, MA, United States
  • 2003
    • University of Tennessee
      • Department of Surgery
      Knoxville, TN, United States
  • 2002
    • University of Saskatchewan
      • College of Medicine
      Saskatoon, Saskatchewan, Canada