Substitution of the SERCA2 Cys674 reactive thiol accelerates atherosclerosis by inducing endoplasmic reticulum stress and inflammation.

BACKGROUND AND PURPOSE: The cysteine674 (C674) thiol of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 is easily and irreversibly oxidized under atherosclerotic conditions. However, the contribution of the C674 thiol redox status in the development of atherosclerosis remains unclear. Our goal was to elucidate the possible mechanism involved. EXPERIMENTAL APPROACH: Heterozygous SERCA2 C674S knock-in mice in which half of the C674 was substituted by serine (S674) were used to mimic the removal of the reactive C674 thiol, which occurs under pathological conditions. Bone marrow-derived macrophages (BMDMs) and cardiac endothelial cells (ECs) were used for intracellular Ca2+ , macrophage adhesion, and protein expression analysis. The whole aorta and aortic root were isolated for histological analysis. KEY RESULTS: Cell culture studies suggest the partial substitution of SERCA2 C674 increased intracellular Ca2+ levels and induced ER stress in both BMDMs and ECs. The release of proinflammatory factors and macrophage adhesion increased in SKI BMDMs. In ECs, overexpression of S674 induced endothelial inflammation and promoted macrophage recruitment. SKI mice developed more severe atherosclerotic plaque and macrophage accumulation. Additionally, 4-phenyl butyric acid, an ER stress inhibitor, suppressed ER stress and inflammatory responses in BMDMs and ECs, and alleviated atherosclerosis in SKI mice. CONCLUSIONS AND IMPLICATIONS: The substitution of SERCA2 C674 thiol accelerates the development of atherosclerosis by inducing ER stress and inflammation. Our findings highlight the importance of SERCA2 C674 redox state in the context of atherosclerosis and open up a novel therapeutic strategy to combat atherosclerosis.

BCL11B Regulates Arterial Stiffness and Related Target Organ Damage.

[Figure: see text].

Redox-Resistant SERCA [Sarco(endo)plasmic Reticulum Calcium ATPase] Attenuates Oxidant-Stimulated Mitochondrial Calcium and Apoptosis in Cardiac Myocytes and Pressure Overload-Induced Myocardial Failure in Mice.

BACKGROUND: SERCA [sarco(endo)plasmic reticulum calcium ATPase] is regulated by oxidative posttranslational modifications at cysteine 674 (C674). Because sarcoplasmic reticulum (SR) calcium has been shown to play a critical role in mediating mitochondrial dysfunction in response to reactive oxygen species, we hypothesized that SERCA oxidation at C674 would modulate the effects of reactive oxygen species on mitochondrial calcium and mitochondria-dependent apoptosis in cardiac myocytes. METHODS: Adult rat ventricular myocytes expressing wild-type SERCA2b or a redox-insensitive mutant in which C674 is replaced by serine (C674S) were exposed to H2O2 (100 µmol/Lµ). Free mitochondrial calcium concentration was measured in adult rat ventricular myocytes with a genetically targeted fluorescent probe, and SR calcium content was assessed by measuring caffeine-stimulated release. Mice with heterozygous knock-in of the SERCA C674S mutation were subjected to chronic ascending aortic constriction. RESULTS: In adult rat ventricular myocytes expressing wild-type SERCA, H2O2 caused a 25% increase in mitochondrial calcium concentration that was associated with a 50% decrease in SR calcium content, both of which were prevented by the ryanodine receptor inhibitor tetracaine. In cells expressing the C674S mutant, basal SR calcium content was decreased by 31% and the H2O2-stimulated rise in mitochondrial calcium concentration was attenuated by 40%. In wild-type cells, H2O2 caused cytochrome c release and apoptosis, both of which were prevented in C674S-expressing cells. In myocytes from SERCA knock-in mice, basal SERCA activity and SR calcium content were decreased. To test the effect of C674 oxidation on apoptosis in vivo, SERCA knock-in mice were subjected to chronic ascending aortic constriction. In wild-type mice, ascending aortic constriction caused myocyte apoptosis, LV dilation, and systolic failure, all of which were inhibited in SERCA knock-in mice. CONCLUSIONS: Redox activation of SERCA C674 regulates basal SR calcium content, thereby mediating the pathologic reactive oxygen species-stimulated rise in mitochondrial calcium required for myocyte apoptosis and myocardial failure.

Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.

Glutaredoxin-1 (Glrx) is a small cytosolic enzyme that removes S-glutathionylation, glutathione adducts of protein cysteine residues, thus modulating redox signaling and gene transcription. Although Glrx up-regulation prevented endothelial cell (EC) migration and global Glrx transgenic mice had impaired ischemic vascularization, the effects of cell-specific Glrx overexpression remained unknown. Here, we examined the role of EC-specific Glrx up-regulation in distinct models of angiogenesis; namely, hind limb ischemia and tumor angiogenesis. EC-specific Glrx transgenic (EC-Glrx TG) overexpression in mice significantly impaired EC migration in Matrigel implants and hind limb revascularization after femoral artery ligation. Additionally, ECs migrated less into subcutaneously implanted B16F0 melanoma tumors as assessed by decreased staining of EC markers. Despite reduced angiogenesis, EC-Glrx TG mice unexpectedly developed larger tumors compared with control mice. EC-Glrx TG mice showed higher levels of VEGF-A in the tumors, indicating hypoxia, which may stimulate tumor cells to form vascular channels without EC, referred to as vasculogenic mimicry. These data suggest that impaired ischemic vascularization does not necessarily associate with suppression of tumor growth, and that antiangiogenic therapies may be ineffective for melanoma tumors because of their ability to implement vasculogenic mimicry during hypoxia.-Yura, Y., Chong, B. S. H., Johnson, R. D., Watanabe, Y., Tsukahara, Y., Ferran, B., Murdoch, C. E., Behring, J. B., McComb, M. E., Costello, C. E., Janssen-Heininger, Y. M. W., Cohen, R. A., Bachschmid, M. M., Matsui, R. Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.

Improved mass spectrometry-based activity assay reveals oxidative and metabolic stress as sirtuin-1 regulators.

Sirtuin-1 (SirT1) catalyzes NAD+-dependent protein lysine deacetylation and is a critical regulator of energy and lipid metabolism, mitochondrial biogenesis, apoptosis, and senescence. Activation of SirT1 mitigates metabolic perturbations associated with diabetes and obesity. Pharmacologic molecules, cellular redox, and nutritional states can regulate SirT1 activity. Technical barriers against measuring endogenous SirT1 activity have limited characterization of SirT1 in disease and its activation by small molecules. Herein, we developed a relative quantitative mass spectrometry-based technique for measuring endogenous SirT1 activity (RAMSSAY/RelAtive Mass Spectrometry Sirt1 Activity assaY) in cell and tissue homogenates using a biotin-labeled, acetylated p53-derived peptide as a substrate. We demonstrate that oxidative and metabolic stress diminish SirT1 activity in the hepatic cell line HepG2. Moreover, pharmacologic molecules including nicotinamide and EX-527 attenuate SirT1 activity; purported activators of SirT1, the polyphenol S17834, the polyphenol resveratrol, or the non-polyphenolic Sirtris compound SRT1720, failed to activate endogenous SirT1 significantly. Furthermore, we provide evidence that feeding a high fat high sucrose diet (HFHS) to mice inhibits endogenous SirT1 activity in mouse liver. In summary, we introduce a robust, specific and sensitive mass spectrometry-based assay for detecting and quantifying endogenous SirT1 activity using a biotin-labeled peptide in cell and tissue lysates. With this assay, we determine how pharmacologic molecules and metabolic and oxidative stress regulate endogenous SirT1 activity. The assay may also be adapted for other sirtuin isoforms.

Smooth Muscle Nitric Oxide Responsiveness and Clinical Maturation of Hemodialysis Arteriovenous Fistulae.

The arteriovenous fistula is the preferred type of hemodialysis vascular access for patients with end-stage renal disease, but a high proportion of newly created fistulas fail to mature for use. Stenosis caused by neointimal hyperplasia often is present in fistulas with maturation failure, suggesting that local mechanisms controlling vascular smooth muscle cell (SMC) migration and proliferation are important contributors to maturation failure. SMCs cultured from explants of vein tissue obtained at the time of fistula creation from 19 patients with end-stage renal disease were studied to determine whether smooth muscle responsiveness to nitric oxide is associated with fistula maturation outcomes. Nitric oxide-induced inhibition of smooth muscle cell migration, but not proliferation, was greater in cells from patients with subsequent fistula maturation success than from patients with subsequent fistula maturation failure (mean inhibition percentage, 17 versus 5.7, respectively; P = 0.035). Impaired nitric oxide responsiveness was associated with oxidation of the calcium regulatory protein, sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and was reversed by overexpressing SERCA (1.8-fold increase in inhibition, P = 0.0128) or down-regulating Nox4-based NADPH oxidase (2.3-fold increase in inhibition; P = 0.005). Our data suggest that the nitric oxide responsiveness of SMC migration is associated with fistula maturation success and raises the possibility that therapeutic restoration of nitric oxide responsiveness through manipulation of local mediators may prevent fistula maturation failure.

Endothelial Nox4-based NADPH oxidase regulates atherosclerosis via soluble epoxide hydrolase.

Nox4-based NADPH oxidase is a major reactive oxygen species-generating enzyme in the vasculature, but its role in atherosclerosis remains controversial. OBJECTIVE: Our goal was to investigate the mechanisms of endothelial Nox4 in regulating atherosclerosis. APPROACH AND RESULTS: Atherosclerosis-prone conditions (disturbed blood flow, type I diabetes, and Western diet) downregulated endothelial Nox4 mRNA in arteries. To address whether the downregulated endothelial Nox4 was directly involved in the development of atherosclerosis, we generated mice carrying a human Nox4 P437H dominant negative mutation (Nox4DN), driven by the endothelial specific promoter Tie-2, on atherosclerosis-prone genetic background (ApoE deficient mice) to mimic the effect of decreased endothelial Nox4. Nox4DN significantly increased type I diabetes-induced aortic stiffness and atherosclerotic lesions. Gene analysis indicated that soluble epoxide hydrolase 2 (sEH) was significantly upregulated in Nox4DN endothelial cells (EC). Inhibition of sEH activity in Nox4DN EC suppressed inflammation and macrophage adhesion to EC. On the contrary, overexpression of endothelial wild type Nox4 suppressed sEH, ameliorated Western diet-induced atherosclerosis and decreased aortic stiffness. CONCLUSIONS: Atherosclerosis-prone conditions downregulated endothelial Nox4 to accelerate the progress of atherosclerosis, at least in part, by upregulating sEH to enhance inflammation.

Glutaredoxin-1 Deficiency Causes Fatty Liver and Dyslipidemia by Inhibiting Sirtuin-1.

AIMS: Nonalcoholic fatty liver (NAFL) is a common liver disease associated with metabolic syndrome, obesity, and diabetes that is rising in prevalence worldwide. Various molecular perturbations of key regulators and enzymes in hepatic lipid metabolism cause NAFL. However, redox regulation through glutathione (GSH) adducts in NAFL remains largely elusive. Glutaredoxin-1 (Glrx) is a small thioltransferase that removes protein GSH adducts without having direct antioxidant properties. The liver contains abundant Glrx but its metabolic function is unknown. RESULTS: Here we report that normal diet-fed Glrx-deficient mice (Glrx-/-) spontaneously develop obesity, hyperlipidemia, and hepatic steatosis by 8 months of age. Adenoviral Glrx repletion in the liver of Glrx-/- mice corrected lipid metabolism. Glrx-/- mice exhibited decreased sirtuin-1 (SirT1) activity that leads to hyperacetylation and activation of SREBP-1 and upregulation of key hepatic enzymes involved in lipid synthesis. We found that GSH adducts inhibited SirT1 activity in Glrx-/- mice. Hepatic expression of nonoxidizable cysteine mutant SirT1 corrected hepatic lipids in Glrx-/- mice. Wild-type mice fed high-fat diet develop metabolic syndrome, diabetes, and NAFL within several months. Glrx deficiency accelerated high-fat-induced NAFL and progression to steatohepatitis, manifested by hepatic damage and inflammation. INNOVATION: These data suggest an essential role of hepatic Glrx in regulating SirT1, which controls protein glutathione adducts in the pathogenesis of hepatic steatosis. CONCLUSION: We provide a novel redox-dependent mechanism for regulation of hepatic lipid metabolism, and propose that upregulation of hepatic Glrx may be a beneficial strategy for NAFL. Antioxid. Redox Signal. 27, 313-327.

The redox mechanism for vascular barrier dysfunction associated with metabolic disorders: Glutathionylation of Rac1 in endothelial cells.

BACKGROUND: Oxidative stress is implicated in increased vascular permeability associated with metabolic disorders, but the underlying redox mechanism is poorly defined. S-glutathionylation, a stable adduct of glutathione with protein sulfhydryl, is a reversible oxidative modification of protein and is emerging as an important redox signaling paradigm in cardiovascular physiopathology. The present study determines the role of protein S-glutathionylation in metabolic stress-induced endothelial cell permeability. METHODS AND RESULTS: In endothelial cells isolated from patients with type-2 diabetes mellitus, protein S-glutathionylation level was increased. This change was also observed in aortic endothelium in ApoE deficient (ApoE-/-) mice fed on Western diet. Metabolic stress-induced protein S-glutathionylation in human aortic endothelial cells (HAEC) was positively correlated with elevated endothelial cell permeability, as reflected by disassembly of cell-cell adherens junctions and cortical actin structures. These impairments were reversed by adenoviral overexpression of a specific de-glutathionylation enzyme, glutaredoxin-1 in cultured HAECs. Consistently, transgenic overexpression of human Glrx-1 in ApoE-/- mice fed the Western diet attenuated endothelial protein S-glutathionylation, actin cytoskeletal disorganization, and vascular permeability in the aorta. Mechanistically, glutathionylation and inactivation of Rac1, a small RhoGPase, were associated with endothelial hyperpermeability caused by metabolic stress. Glutathionylation of Rac1 on cysteine 81 and 157 located adjacent to guanine nucleotide binding site was required for the metabolic stress to inhibit Rac1 activity and promote endothelial hyperpermeability. CONCLUSIONS: Glutathionylation and inactivation of Rac1 in endothelial cells represent a novel redox mechanism of vascular barrier dysfunction associated with metabolic disorders.

Vascular Smooth Muscle Sirtuin-1 Protects Against Diet-Induced Aortic Stiffness.

Arterial stiffness, a major cardiovascular risk factor, develops within 2 months in mice fed a high-fat, high-sucrose (HFHS) diet, serving as a model of human metabolic syndrome, and it is associated with activation of proinflammatory and oxidant pathways in vascular smooth muscle (VSM) cells. Sirtuin-1 (SirT1) is an NAD(+)-dependent deacetylase regulated by the cellular metabolic status. Our goal was to study the effects of VSM SirT1 on arterial stiffness in the context of diet-induced metabolic syndrome. Overnight fasting acutely decreased arterial stiffness, measured in vivo by pulse wave velocity, in mice fed HFHS for 2 or 8 months, but not in mice lacking SirT1 in VSM (SMKO). Similarly, VSM-specific genetic SirT1 overexpression (SMTG) prevented pulse wave velocity increases induced by HFHS feeding, during 8 months. Administration of resveratrol or S17834, 2 polyphenolic compounds known to activate SirT1, prevented HFHS-induced arterial stiffness and were mimicked by global SirT1 overexpression (SirT1 bacterial artificial chromosome overexpressor), without evident metabolic improvements. In addition, HFHS-induced pulse wave velocity increases were reversed by 1-week treatment with a specific, small molecule SirT1 activator (SRT1720). These beneficial effects of pharmacological or genetic SirT1 activation, against HFHS-induced arterial stiffness, were associated with a decrease in nuclear factor kappa light chain enhancer of activated B cells (NFκB) activation and vascular cell adhesion molecule (VCAM-1) and p47phox protein expressions, in aorta and VSM cells. In conclusion, VSM SirT1 activation decreases arterial stiffness in the setting of obesity by stimulating anti-inflammatory and antioxidant pathways in the aorta. SirT1 activators may represent a novel therapeutic approach to prevent arterial stiffness and associated cardiovascular complications in overweight/obese individuals with metabolic syndrome.

Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization.

Reactive oxygen species (ROS) are increased in ischemic tissues and necessary for revascularization; however, the mechanism remains unclear. Exposure of cysteine residues to ROS in the presence of glutathione (GSH) generates GSH-protein adducts that are specifically reversed by the cytosolic thioltransferase, glutaredoxin-1 (Glrx). Here, we show that a key angiogenic transcriptional factor hypoxia-inducible factor (HIF)-1α is stabilized by GSH adducts, and the genetic deletion of Glrx improves ischemic revascularization. In mouse muscle C2C12 cells, HIF-1α protein levels are increased by increasing GSH adducts with cell-permeable oxidized GSH (GSSG-ethyl ester) or 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanyl thiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), an inhibitor of glutathione reductase. A biotin switch assay shows that GSSG-ester-induced HIF-1α contains reversibly modified thiols, and MS confirms GSH adducts on Cys(520) (mouse Cys(533)). In addition, an HIF-1α Cys(520) serine mutant is resistant to 2-AAPA-induced HIF-1α stabilization. Furthermore, Glrx overexpression prevents HIF-1α stabilization, whereas Glrx ablation by siRNA increases HIF-1α protein and expression of downstream angiogenic genes. Blood flow recovery after femoral artery ligation is significantly improved in Glrx KO mice, associated with increased levels of GSH-protein adducts, capillary density, vascular endothelial growth factor (VEGF)-A, and HIF-1α in the ischemic muscles. Therefore, Glrx ablation stabilizes HIF-1α by increasing GSH adducts on Cys(520) promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles.

Redox Regulation of Ischemic Angiogenesis　- Another Aspect of Reactive Oxygen Species.

Antioxidants are expected to improve cardiovascular disease (CVD) by eliminating oxidative stress, but clinical trials have not shown promising results in chronic CVD. Animal studies have revealed that reactive oxygen species (ROS) exacerbate acute CVDs in which high levels of ROS are observed. However, ROS are also necessary for angiogenesis after ischemia, because ROS not only damage cells but also stimulate the cell signaling required for angiogenesis. ROS affect signaling by protein modifications, especially of cysteine amino acid thiols. Although there are several cysteine modifications, S-glutathionylation (GSH adducts; -SSG), a reversible cysteine modification by glutathione (GSH), plays an important role in angiogenic signal transduction by ROS. Glutaredoxin-1 (Glrx) is an enzyme that specifically removes GSH adducts in vivo. Overexpression of Glrx inhibits, whereas deletion of Glrx improves revascularization after mouse hindlimb ischemia. These studies indicate that increased levels of GSH adducts in ischemic muscle are beneficial in promoting angiogenesis. The underlying mechanism can be explained by multiple targets of S-gluathionylation, which mediate the angiogenic effects in ischemia. Increments in the master angiogenic transcriptional factor, HIF-1α, reduction of the anti-angiogenic factor sFlt1, activation of the endoplasmic reticulum Ca(2+)pump, SERCA, and inhibition of phosphatases may occur as a consequence of enhanced S-glutathionylation in ischemic tissue. In summary, inducing S-glutathionylation by inhibiting Glrx may be a therapeutic strategy to improve ischemic angiogenesis in CVD. (Circ J 2016; 80: 1278-1284).

Endothelial Cell Redox Regulation of Ischemic Angiogenesis.

The endothelium produces and responds to reactive oxygen and nitrogen species (RONS), providing important redox regulation to the cardiovascular system in physiology and disease. In no other situation are RONS more critical than in the response to tissue ischemia. Here, tissue healing requires growth factor-mediated angiogenesis that is in part dependent on low levels of RONS, which paradoxically must overcome the damaging effects of high levels of RONS generated as a result of ischemia. Although the generation of endothelial cell RONS in hypoxia/reoxygenation is acknowledged, the mechanism for their role in angiogenesis is still poorly understood. During ischemia, the major low molecular weight thiol glutathione (GSH) reacts with RONS and protein cysteines, producing GSH-protein adducts. Recent data indicate that GSH adducts on certain proteins are essential to growth factor responses in endothelial cells. Genetic deletion of the enzyme glutaredoxin-1, which selectively removes GSH protein adducts, improves, whereas its overexpression impairs revascularization of the ischemic hindlimb of mice. Ischemia-induced GSH adducts on specific cysteine residues of several proteins, including p65 NF-kB and the sarcoplasmic reticulum calcium ATPase 2, evidently promote ischemic angiogenesis. Identifying the specific proteins in the redox response to ischemia has provided therapeutic opportunities to improve clinical outcomes of ischemia.

Pro-atherogenic role of smooth muscle Nox4-based NADPH oxidase.

UNLABELLED: Nox4-based NADPH oxidase is a major reactive oxygen species-generating enzyme in the vasculature, but its role in atherosclerosis remains controversial. OBJECTIVE: Our goal was to investigate the role of smooth muscle Nox4 in atherosclerosis. APPROACH AND RESULTS: Atherosclerosis-prone conditions (disturbed blood flow and Western diet) increased Nox4 mRNA level in smooth muscle of arteries. To address whether upregulated smooth muscle Nox4 under atherosclerosis-prone conditions was directly involved in the development of atherosclerosis, mice carrying a human Nox4 P437H dominant negative mutation (Nox4DN), specifically in smooth muscle, were generated on a FVB/N ApoE deficient genetic background to counter the effect of increased smooth muscle Nox4. Nox4DN significantly decreased aortic stiffness and atherosclerotic lesions, with no effect on blood pressure. Gene analysis indicated that soluble epoxide hydrolase 2 (sEH) was significantly downregulated in Nox4DN smooth muscle cells (SMC), at both mRNA and protein levels. Downregulation of sEH by siRNA decreased SMC proliferation and migration, and suppressed inflammation and macrophage adhesion to SMC. CONCLUSIONS: Downregulation of smooth muscle Nox4 inhibits atherosclerosis by suppressing sEH, which, at least in part, accounts for inhibition of SMC proliferation, migration and inflammation.

Mitochondrial Reactive Oxygen Species Mediate Cardiac Structural, Functional, and Mitochondrial Consequences of Diet-Induced Metabolic Heart Disease.

BACKGROUND: Mitochondrial reactive oxygen species (ROS) are associated with metabolic heart disease (MHD). However, the mechanism by which ROS cause MHD is unknown. We tested the hypothesis that mitochondrial ROS are a key mediator of MHD. METHODS AND RESULTS: Mice fed a high-fat high-sucrose (HFHS) diet develop MHD with cardiac diastolic and mitochondrial dysfunction that is associated with oxidative posttranslational modifications of cardiac mitochondrial proteins. Transgenic mice that express catalase in mitochondria and wild-type mice were fed an HFHS or control diet for 4 months. Cardiac mitochondria from HFHS-fed wild-type mice had a 3-fold greater rate of H2O2 production (P=0.001 versus control diet fed), a 30% decrease in complex II substrate-driven oxygen consumption (P=0.006), 21% to 23% decreases in complex I and II substrate-driven ATP synthesis (P=0.01), and a 62% decrease in complex II activity (P=0.002). In transgenic mice that express catalase in mitochondria, all HFHS diet-induced mitochondrial abnormalities were ameliorated, as were left ventricular hypertrophy and diastolic dysfunction. In HFHS-fed wild-type mice complex II substrate-driven ATP synthesis and activity were restored ex vivo by dithiothreitol (5 mmol/L), suggesting a role for reversible cysteine oxidative posttranslational modifications. In vitro site-directed mutation of complex II subunit B Cys100 or Cys103 to redox-insensitive serines prevented complex II dysfunction induced by ROS or high glucose/high palmitate in the medium. CONCLUSION: Mitochondrial ROS are pathogenic in MHD and contribute to mitochondrial dysfunction, at least in part, by causing oxidative posttranslational modifications of complex I and II proteins including reversible oxidative posttranslational modifications of complex II subunit B Cys100 and Cys103.

Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins.

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

Role of smooth muscle Nox4-based NADPH oxidase in neointimal hyperplasia.

UNLABELLED: Elevated levels of reactive oxygen species (ROS) in the vascular wall play a key role in the development of neointimal hyperplasia. Nox4-based NADPH oxidase is a major ROS generating enzyme in the vasculature, but its roles in neointimal hyperplasia remain unclear. OBJECTIVE: Our purpose was to investigate the role of smooth muscle cell (SMC) Nox4 in neointimal hyperplasia. APPROACH AND RESULTS: Mice overexpressing a human Nox4 mutant form, carrying a P437H dominant negative mutation (Nox4DN) and driven by SM22α promoter, to achieve specific expression in SMC, were generated in a FVB/N genetic background. After wire injury-induced endothelial denudation, Nox4DN had significantly decreased neointima formation compared with non-transgenic littermate controls (NTg). ROS production, serum-induced proliferation and migration, were significantly decreased in aortic SMCs isolated from Nox4DN compared with NTg. Both mRNA and protein levels of thrombospondin 1 (TSP1) were significantly downregulated in Nox4DN SMCs. Downregulation of TSP1 by siRNA decreased cell proliferation and migration in SMCs. Similar to Nox4DN, downregulation of Nox4 by siRNA significantly decreased TSP1 expression level, cell proliferation and migration in SMCs. CONCLUSIONS: Downregulation of smooth muscle Nox4 inhibits neointimal hyperplasia by suppressing TSP1, which in part can account for inhibition of SMC proliferation and migration.