David R. Pimental

Inhibition of Copper-Zinc Superoxide Dismutase Induces Cell Growth, Hypertrophic Phenotype, and Apoptosis in Neonatal Rat Cardiac Myocytes In Vitro

Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that inhibition of endogenous antioxidant enzymes can regulate the phenotype of cardiac myocytes. Neonatal rat ventricular myocytes in vitro were exposed to diethyldithiocarbamic acid (DDC), an inhibitor of cytosolic (Cu, Zn) and extracellular superoxide dismutase (SOD). DDC inhibited SOD activity and increased intracellular superoxide in a concentration-dependent manner. A low concentration (1 micromol/L) of DDC stimulated myocyte growth, as demonstrated by increases in protein synthesis, cellular protein, prepro-atrial natriuretic peptide, and c-fos mRNAs and decreased sarcoplasmic reticulum Ca(2+)ATPase mRNA. These actions were all inhibited by the superoxide scavenger Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid). Higher concentrations of DDC (100 micromol/L) stimulated myocyte apoptosis, as evidenced by DNA laddering, characteristic nuclear morphology, in situ terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL), and increased bax mRNA expression. DDC-stimulated apoptosis was inhibited by the SOD/catalase mimetic EUK-8. The growth and apoptotic effects of DDC were mimicked by superoxide generation with xanthine plus xanthine oxidase. Thus, increased intracellular superoxide resulting from inhibition of SOD causes activation of a growth program and apoptosis in cardiac myocytes. These findings support a role for oxidative stress in the pathogenesis of myocardial remodeling and failure.

Uncoupling of Endothelial Nitric Oxidase Synthase by Hypochlorous Acid: Role of NAD(P)H Oxidase–Derived Superoxide and Peroxynitrite

Objective—The aim of the present study is to determine whether hypochlorous acid (HOCl), the major oxidant of leukocyte-derived myeloperoxidase (MPO), oxidizes the zinc-thiolate center of endothelial nitric oxide synthase (eNOS) and uncouples the enzyme. Methods and Results—Exposure of purified recombinant eNOS to HOCl (≥100 &mgr;mol/L) released zinc and disrupted the enzyme-active eNOS dimers. In parallel with increased detections of both O2·− and ONOO−, clinically relevant concentrations of HOCl disrupted eNOS dimers in cultured human umbilical vein endothelial cells (HUVEC) at concentration 10- to 100-fold lower than those required for recombinant eNOS. In HUVEC, HOCl increased the translocation of both p67phox and p47phox of NAD(P)H oxidase and the phosphorylation of atypical protein kinase C-&zgr;. Further, genetic or pharmacological inhibition of either NAD(P)H oxidase–derived O2·− or PKC-&zgr; or NOS abolished the effects of HOCl on eNOS dimers. Consistently, HOCl increased both O2·− and ONOO− and eNOS dimer oxidation in isolated mouse aortas from C57BL/6 but less in those of gp91phox knock-out mice. Finally, in human carotid atherosclerotic arteries, eNOS predominantly existed as monomers in parallel with increased staining of both MPO and 3-nitrotyrosine. Conclusions—We conclude that HOCl uncouples eNOS by ONOO− generated from PKC-&zgr;–dependent NAD(P)H oxidase.

S-glutathiolation by peroxynitrite of p21ras at cysteine-118 mediates its direct activation and downstream signaling in endothelial cells

The highly reactive species, peroxynitrite, is produced in endothelial cells in pathological states in which the production of superoxide anion and NO is increased. Here, we show that peroxynitrite added exogenously or generated endogenously in response to exposure to an NO donor or oxidized low‐density lipoproteins (oxLDL) increases p21ras activity in bovine aortic endothelial cells. The activation is not dependent on upstream elements but rather is due to direct targeting of p21ras by reversible S‐glutathiolation of cysteine thiols as demonstrated by biotin‐labeling techniques. The time course of p21ras S‐glutathiolation following peroxynitrite corresponds to the increase in its Raf‐1 binding activity and translocation to the membrane. Moreover, p21ras S‐glutathiolation and activation can be reversed by dithiothreitol, confirming the importance of a disulfide bond. S‐glutathiolation also promoted guanine nucleotide exchange of recombinant p21ras. In addition, the oxidant‐induced activation of Mek/Erk and PI3 kinase/Akt was abrogated by dominant‐negative and Cys‐118 p21ras mutants, and the latter also prevented S‐glutathiolation of p21ras. These results indicate that peroxynitrite arising from NO donors or pathological stimuli such as oxLDL triggers direct S‐glutathiolation of p21ras Cys118, which increases p21ras activity and mediates downstream signaling.

Vascular aging: Chronic oxidative stress and impairment of redox signaling—consequences for vascular homeostasis and disease

Characteristic morphological and molecular alterations such as vessel wall thickening and reduction of nitric oxide occur in the aging vasculature leading to the gradual loss of vascular homeostasis. Consequently, the risk of developing acute and chronic cardiovascular diseases increases with age. Current research of the underlying molecular mechanisms of endothelial function demonstrates a duality of reactive oxygen and nitrogen species in contributing to vascular homeostasis or leading to detrimental effects when formed in excess. Furthermore, changes in function and redox status of vascular smooth muscle cells contribute to age-related vascular remodeling. The age-dependent increase in free radical formation causes deterioration of the nitric oxide signaling cascade, alters and activates prostaglandin metabolism, and promotes novel oxidative posttranslational protein modifications that interfere with vascular and cell signaling pathways. As a result, vascular dysfunction manifests. Compensatory mechanisms are initially activated to cope with age-induced oxidative stress, but become futile, which results in irreversible oxidative modifications of biological macromolecules. These findings support the ‘free radical theory of aging’ but also show that reactive oxygen and nitrogen species are essential signaling molecules, regulating vascular homeostasis.

Regulation of angiotensin II-stimulated osteopontin expression in cardiac microvascular endothelial cells: role of p42/44 mitogen-activated protein kinase and reactive oxygen species.

Using spontaneously hypertensive and aortic banded rats, we have shown that expression of myocardial osteopontin, an extracellular matrix protein, coincides with the development of heart failure and is inhibited by captopril, suggesting a role for angiotensin II (ANG II). This study tested whether ANG II induces osteopontin expression in adult rat ventricular myocytes and cardiac microvascular endothelial cells (CMEC), and if so, whether induction is mediated via activation of mitogen‐activated protein kinases (p42/44 MAPK) and involves reactive oxygen species (ROS). ANG II (1 μM, 16 h) increased osteopontin expression (fold increase 3.3±0.34, n = 12, P < 0.01) in CMEC as measured by northern analysis, but not in ARVM. ANG II stimulated osteopontin expression in CMEC in a time‐ (within 4 h) and concentration‐dependent manner, which was prevented by the AT1 receptor antagonist, losartan. ANG II elicited robust phosphorylation of p42/44 MAPK as measured using phospho‐specific antibodies, and increased superoxide production as measured by cytochrome c reduction and lucigenin chemiluminescence assays. These effects were blocked by diphenylene iodonium (DPI), an inhibitor of the flavoprotein component of NAD(P)H oxidase. PD98059, an inhibitor of p42/44 MAPK pathway, and DPI each inhibited ANG II‐stimulated osteopontin expression. Northern blot analysis showed basal expression of p22phox, a critical component of NADH/NADPH oxidase system, which was increased 40–60% by exposure to ANG II. These results suggest that p42/44 MAPK is a critical component of the ROS‐sensitive signaling pathways activated by ANG II in CMEC and plays a key role in the regulation of osteopontin gene expression. Published 2001 Wiley‐Liss, Inc.

Homocysteine induces cardiomyocyte dysfunction and apoptosis through p38 MAPK-mediated increase in oxidant stress.

Elevated plasma homocysteine (Hcy) is a risk factor for cardiovascular disease. While Hcy has been shown to promote endothelial dysfunction by decreasing the bioavailability of nitric oxide and increasing oxidative stress in the vasculature, the effects of Hcy on cardiomyocytes remain less understood. In this study we explored the effects of hyperhomocysteinemia (HHcy) on myocardial function ex vivo and examined the direct effects of Hcy on cardiomyocyte function and survival in vitro. Studies with isolated hearts from wild type and HHcy mice (heterozygous cystathionine-beta synthase deficient mice) demonstrated that HHcy mouse hearts had more severely impaired cardiac relaxation and contractile function and increased cell death following ischemia reperfusion (I/R). In isolated cultured adult rat ventricular myocytes, exposure to Hcy for 24 h impaired cardiomyocyte contractility in a concentration-dependent manner, and promoted apoptosis as revealed by terminal dUTP nick-end labeling and cleaved caspase-3 immunoblotting. These effects were associated with activation of p38 MAPK, decreased expression of thioredoxin (TRX) protein, and increased production of reactive oxygen species (ROS). Inhibition of p38 MAPK by the selective inhibitor SB203580 (5 μM) prevented all of these Hcy-induced changes. Furthermore, adenovirus-mediated overexpression of TRX in cardiomyocytes significantly attenuated Hcy-induced ROS generation, apoptosis, and impairment of myocyte contractility. Thus, Hcy may increase the risk for CVD not only by causing endothelial dysfunction, but also by directly exerting detrimental effects on cardiomyocytes.

Redox regulation of SERCA2 is required for vascular endothelial growth factor-induced signaling and endothelial cell migration.

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.

Hexokinase regulates Bax-mediated mitochondrial membrane injury following ischemic stress

Hexokinase (HK), the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. Since HK isoforms I and II have mitochondrial targeting sequences, we attempted to separate the protective effects of HK on cell metabolism from those on apoptosis. We exposed renal epithelial cells to metabolic stress causing ATP depletion in the absence of glucose and found that this activated glycogen synthase kinase 3β (GSK3β) and Bax caused mitochondrial membrane injury and apoptosis. ATP depletion led to a progressive HK II dissociation from mitochondria, released mitochondrial apoptosis inducing factor and cytochrome c into the cytosol, activated caspase-3, and reduced cell survival. Compared with control, adenoviral-mediated HK I or II overexpression improved cell survival following stress, but did not prevent GSK3β or Bax activation, improve ATP content, or reduce mitochondrial fragmentation. HK I or HK II overexpression increased mitochondria-associated isoform-specific HK content, and decreased mitochondrial membrane injury and apoptosis after stress. In vivo, HK II localized exclusively to the proximal tubule. Ischemia reduced total renal HK II content and dissociated HK II from proximal tubule mitochondria. In cells overexpressing HK II, Bax and HK II did not interact before or after stress. While the mechanism by which HK antagonizes Bax-mediated apoptosis is unresolved by these studies, one possible scenario is that the two proteins compete for a common binding site on the outer mitochondrial membrane.

Oxidation of HRas cysteine thiols by metabolic stress prevents palmitoylation in vivo and contributes to endothelial cell apoptosis

Here we demonstrate a new paradigm in redox signaling, whereby oxidants resulting from metabolic stress directly alter protein palmitoylation by oxidizing reactive cysteine thiolates. In mice fed a high‐fat, high‐sucrose diet and in cultured endothelial cells (ECs) treated with high palmitate and high glucose (HPHG), there was decreased HRas palmitoylation on Cys181/184 (61±24% decrease for cardiac tissue and 38±7.0% in ECs). This was due to oxidation of Cys181/184, detected using matrix‐assisted laser desorption/ionization time of flight (MALDI TOF)‐TOF. Decrease in HRas palmitoylation affected its compartmentalization and Ras binding domain binding activity, with a shift from plasma membrane tethering to Golgi localization. Loss of plasma membrane‐bound HRas decreased growth factor‐stimulated ERK phosphorylation (84±8.6% decrease) and increased apoptotic signaling (24±6.5‐fold increase) after HPHG treatment that was prevented by overexpressing wild‐type but not C181/184S HRas. The essential role of HRas in metabolic stress was made evident by the similar effects of expressing an inactive dominant negative N17‐HRas or a MEK inhibitor. Furthermore, the relevance of thiol oxidation was demonstrated by overexpressing manganese superoxide dismutase, which improved HRas palmitoylation and ERK phosphorylation, while lessening apoptosis in HPHG treated ECs.—Burgoyne, J. R., Haeussler, D. J., Kumar, V., Ji, Y., Pimental, D. R., Zee, R. S., Costello, C. E., Lin, C., McComb, M. E., Cohen, R. A., Bachschmid, M. M. Oxidation of HRas cysteine thiols by metabolic stress prevents palmitoylation in vivo and contributes to endothelial cell apoptosis. FASEB J. 26, 832–841 (2012). www.fasebj.org

Checkpoints in Adenoviral Production: Cross-Contamination and E1A

Adenoviruses are widely used for overexpressing proteins in primary mammalian cells. Incorporation of the early viral gene, E1A, or viral cross-contamination can occur during amplification, and identification of these products is crucial as the transcription of unwanted genetic material can impact cell function and compromise data interpretation. Here we report methods for evaluation of contaminating adenovirus and E1 viral DNA.