Neelam Khaper

Hydrogen Sulfide Protects Against Cellular Senescence via S-Sulfhydration of Keap1 and Activation of Nrf2

AIMS H2S, a third member of gasotransmitter family along with nitric oxide and carbon monoxide, exerts a wide range of cellular and molecular actions in our body. Cystathionine gamma-lyase (CSE) is a major H2S-generating enzyme in our body. Aging at the cellular level, known as cellular senescence, can result from increases in oxidative stress. The aim of this study was to investigate how H2S attenuates oxidative stress and delays cellular senescence. RESULTS Here we showed that mouse embryonic fibroblasts isolated from CSE knockout mice (CSE KO-MEFs) display increased oxidative stress and accelerated cellular senescence in comparison with MEFs from wild-type mice (WT-MEFs). The protein expression of p53 and p21 was significantly increased in KO-MEFs, and knockdown of p53 or p21 reversed CSE deficiency-induced senescence. Incubation of the cells with NaHS (a H2S donor) significantly increased the glutathione (GSH) level and rescued KO-MEFs from senescence. Nrf2 is a master regulator of the antioxidant response, and Keap1 acts as a negative regulator of Nrf2. NaHS S-sulfhydrated Keap1 at cysteine-151, induced Nrf2 dissociation from Keap1, enhanced Nrf2 nuclear translocation, and stimulated mRNA expression of Nrf2-targeted downstream genes, such as glutamate-cysteine ligase and GSH reductase. INNOVATION These results provide a mechanistic insight into how H2S signaling mediates cellular senescence induced by oxidative stress. CONCLUSION H2S protects against cellular aging via S-sulfhydration of Keap1 and Nrf2 activation in association with oxidative stress.

Disturbed Diurnal Rhythm Alters Gene Expression and Exacerbates Cardiovascular Disease With Rescue by Resynchronization

Day/night rhythms are recognized as important to normal cardiovascular physiology and timing of adverse cardiovascular events; however, their significance in disease has not been determined. We demonstrate that day/night rhythms play a critical role in compensatory remodeling of cardiovascular tissue, and disruption exacerbates disease pathophysiology. We use a murine model of pressure overload cardiac hypertrophy (transverse aortic constriction) in a rhythm-disruptive 20-hour versus 24-hour environment. Echocardiography reveals increased left ventricular end-systolic and -diastolic dimensions and reduced contractility in rhythm-disturbed transverse aortic constriction animals. Furthermore, cardiomyocytes and vascular smooth muscle cells exhibit reduced hypertrophy, despite increased pressure load. Microarray and real-time PCR demonstrate altered gene cycling in transverse aortic constriction myocardium and hypothalamic suprachiasmatic nucleus. With rhythm disturbance, there is a consequent altered cellular clock mechanism (per2 and bmal), whereas key genes in hypertrophic pathways (ANF, BNP, ACE, and collagen) are downregulated paradoxical to the increased pressure. Phenotypic rescue, including reversal/attenuation of abnormal pathology and genes, only occurs when the external rhythm is allowed to correspond with the animals’ innate 24-hour internal rhythm. Our study establishes the importance of diurnal rhythm as a vital determinant in heart disease. Disrupted rhythms contribute to progression of organ dysfunction; restoration of normal diurnal schedules appears to be important for effective treatment of disease.

Cystathionine gamma-lyase deficiency and overproliferation of smooth muscle cells

AIMS Cystathionine gamma-lyase (CSE)-derived H2S plays an important role in regulating cell growth. Lack of CSE expression results in development of hypertension. The current study compared proliferation of smooth muscle cells derived from CSE gene knockout mice (SMCs-KO) with that of wild-type mice (SMCs-WT). METHODS AND RESULTS Cell proliferation was assessed by bromodeoxyuridine incorporation. Gene expression was analysed by western blotting, real-time PCR, and microarray analysis. Enhanced cell proliferation was detected in SMCs-KO and in the media of the aorta from CSE KO mice. SMCs-KO underwent significantly more apoptosis than SMCs-WT when treated with exogenous H2S (100 microM). CSE KO mice showed much lower level of phosphorylated extracellular signal-regulated kinase (ERK1/2) in mesentery arteries compared with those of WT mice, and exogenous H2S induced more phosphorylation of ERK1/2 in SMCs-KO compared with that in SMCs-WT. Decreased p21(Cip/WAF-1) but increased cyclin D1 expression was observed in isolated SMCs and vascular tissues from CSE KO mice, and exogenous H2S caused more increase in p21(Cip/WAF-1) expression and more decrease in cyclin D1 expression in SMCs-KO than in SMCs-WT. The transcriptional expression of calcitonin receptor-like, intergrin beta 1, and heparin-binding epidermal growth factor-like growth factor was also significantly increased in the aorta of CSE KO mice. CONCLUSION SMCs-KO display an increased proliferation rate in vitro and in vivo, and these cells are more susceptible to apoptosis induced by exogenous H2S at physiologically relevant concentrations. These cellular effects of H2S are mediated by phosphorylation of ERK1/2 and altered expression of cyclin D1 and p21(Cip/WAF-1).

Targeting the Vicious Inflammation–Oxidative Stress Cycle for the Management of Heart Failure

Oxidative stress and inflammation are each implicated independently in the development and progression of heart failure. Their interaction, however, is also evident throughout the process from initial injury to cardiac remodeling and failure. In the failing heart, the linkage between excessive reactive oxygen species (ROS) and the cytokine elaboration is manifested in shared elements and cross-promotion within downstream signaling pathways. In spite of this, the failure of anticytokine immunotherapy and antioxidant therapy, which had previously shown promise, suggests that a more complete perspective of ROS-cytokine interaction is required. The present review focuses on two of the major cytokines that are demonstrably connected to oxidative stress--the pro-inflammatory tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory interleukin-10 (IL-10)--and their interactions in cardiac remodeling and failure. It is proposed that an optimal balance between TNF-alpha and IL-10 may be of crucial importance in mitigating both inflammation and oxidative stress processes leading to heart failure.

Metformin Induces Apoptosis and Cell Cycle Arrest Mediated by Oxidative Stress, AMPK and FOXO3a in MCF-7 Breast Cancer Cells

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration- dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G0-G1 phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRβ, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H2O2 increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role.

The Primary Benefits of Angiotensin-Converting Enzyme Inhibition on Cardiac Remodeling Occur During Sleep Time in Murine Pressure Overload Hypertrophy

OBJECTIVES Our objective was to test the hypothesis that there is a significant diurnal variation for the therapeutic benefit of angiotensin-converting enzyme (ACE) inhibitors on pressure-overload cardiovascular hypertrophy. BACKGROUND Physiological and molecular processes exhibit diurnal rhythms that may affect efficacy of disease treatment (chronotherapy). Evidence suggests that the heart primarily remodels during sleep. Although a growing body of clinical and epidemiological evidence suggests that the timing of therapy, such as ACE inhibition, alters diurnal blood pressure patterns in patients with hypertension, the benefits of chronotherapy on myocardial and vascular remodeling have not been studied. METHODS We examined the effects of the short-acting ACE inhibitor, captopril, on the structure and function of cardiovascular tissue subjected to pressure overload by transverse aortic constriction (TAC) in mice. Captopril (15 mg/kg intraperitoneally) or placebo was administered at either murine sleep time or wake time for 8 weeks starting 1 week after surgery. RESULTS TAC mice given captopril at sleep time had improved cardiac function and significantly decreased heart: body weight ratios, myocyte cross-sectional areas, intramyocardial vascular medial wall thickness, and perivascular collagen versus TAC mice given captopril or placebo during wake time. Captopril induced similar drops in blood pressure at sleep or wake time, suggesting that time-of-day differences were not attributable to blood pressure changes. These beneficial effects of captopril were correlated with diurnal changes in ACE mRNA expression in the heart. CONCLUSIONS The ACE inhibitor captopril benefited cardiovascular remodeling only when administered during sleep; wake-time captopril ACE inhibition was identical to that of placebo. These studies support the hypothesis that the heart (and vessels) remodel during sleep time and also illustrate the importance of diurnal timing for some cardiovascular therapies.

The role of redox signaling in cardiac hypertrophy induced by experimental hyperthyroidism

This study was conducted to test whether oxidative stress activates the intracellular protein kinase B (AKT1) signaling pathway, which culminates with cardiac hypertrophy in experimental hyperthyroidism. Male Wistar rats were divided into four groups: control, vitamin E, thyroxine (T(4)), and T(4)+vitamin E. Hyperthyroidism was induced by T(4) administration (12 mg/l in drinking water for 28 days). Vitamin E treatment was given during the same period via s.c. injections (20 mg/kg per day). Morphometric and hemodynamic parameters were evaluated at the end of the 4-week treatment period. Protein oxidation, redox state (reduced glutathione, GSH/glutathione dissulfide, GSSG), vitamin C, total radical-trapping antioxidant potential (TRAP), hydrogen peroxide (H2O2), and nitric oxide metabolites (NO(X)) were measured in heart homogenates. The p-AKT1/AKT1 ratio, p-glycogen-synthase kinase (GSK)3B/GSK3B ratio, FOS, and JUN myocardial protein expression were also quantified by western blot after 4 weeks. Increases in biochemical parameters, such as protein oxidation (41%), H2O2 (62%), and NO(X) (218%), and increase in the left ventricular end-diastolic pressure were observed in the T(4) group. T(4) treatment also caused a decrease in GSH/GSSG ratio (83%), vitamin C (34%), and TRAP (55%). These alterations were attenuated by vitamin E administration to the hyperthyroid rats. Expression of p-AKT1/AKT1, p-GSK3B/GSK3B, FOS, and JUN were elevated in the T(4) group (by 69, 37, 130, and 33% respectively), whereas vitamin E administration promoted a significant reduction in their expression. These results indicate that oxidative stress plays an important role in cardiac hypertrophy, and suggest redox activation of AKT1 and JUN/FOS signaling pathways with H2O2 acting as a possible intracellular mediator in this adaptive response to experimental hyperthyroidism.

Bone marrow derived cells decrease inflammation but not oxidative stress in an experimental model of acute myocardial infarction

AIMS Bone marrow cell (BMC) therapy is thought to exert beneficial effects on the infarcted heart. We assessed cardiac function and its correlation with redox status and inflammation in cardiac tissue early post-AMI in rats treated with BMC. MAIN METHODS Male Wistar rats (8-week-old) were randomized into four groups: Sham-operated (S); AMI; S+treatment (ST) and AMI+treatment (AMIT). Therapy with BMC was carried out immediately post-experimental left anterior coronary artery ligation induced-AMI, and assessments made 48h later. Cardiac function and morphometrics were evaluated by echocardiographyc parameters in vivo. Cardiac tissue tumor necrosis factor (TNF)-α and interleukin (IL)-6 were measured by Western Blot. Oxidative stress parameters including reduced (GSH) and oxidized (GSSG) glutathione ratio, hydrogen peroxide level, lipid and protein oxidation, superoxide dismutase, catalase and glutathione peroxidase activities were measured spectrophotometrically. KEY FINDINGS Ejection fraction was lower in infarcted groups and did not improve in BMC-treated animals: AMI (51±5%) vs. S (74±7%) and AMIT (56±10%) vs. ST groups (73±3%). Both TNF-α and IL-6 myocardial expression increased post-AMI and were reduced following BMC therapy. Nonetheless, there was a decrease in GSH/GSSG ratio in infarcted groups which was greater in BMC-treated groups: AMI (8.21±3.8) vs. S (14.61±3.4) and AMIT (2.1±0.7) vs. ST (4.7±1.5). SIGNIFICANCE The data suggest that BMC promoted a redox status favorable to the oxidation of the pro-inflammatory cytokines in the myocardium, exerting an anti-inflammatory-like effect.

Effects of a chronic exercise training protocol on oxidative stress and right ventricular hypertrophy in monocrotaline-treated rats.

1 In the present study, we investigated the effects of exercise training on cardiac hypertrophy and oxidative stress in a monocrotaline (MCT)‐induced cor pulmonale model. Male Wistar rats were assigned to one of three groups: sedentary control (SC); sedentary MCT (SM); or trained MCT (TM). 2 Right ventricular hypertrophy (RVH) was induced by a single injection of MCT (60 mg/kg, i.p.). Exercise training consisted of running on a treadmill (five times a week, during Weeks 3, 4 and 5). Systemic oxidative stress was evaluated in erythrocytes by chemiluminescence (CL) and the activity of the anti‐oxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S‐transferase (GST) was determined. 3 At Week 3, MCT‐treated animals exhibited RVH, lung congestion, decreased SOD activity and increased CAT activity. Exercise training reduced MCT‐induced RVH and increased GST activity. At Week 4, MCT‐induced RVH was accompanied by an increase in CL and GST activity. However, in TM animals there was a decrease in CL and augmented SOD activity. At Week 5, there were no survivors in the SM group, whereas GST activity was elevated in TM rats compared with SC rats. There was no difference in GPx activity throughout the experimental protocol between the groups. 4 Taken together, our results indicate that exercise training is able to ameliorate RVH and improve survival, which is associated with a reduction in oxidative stress in MCT‐treated rats.

Reactive oxygen and nitrogen species balance in the determination of thyroid hormones-induced cardiac hypertrophy mediated by renin–angiotensin system

Role of reactive oxygen species (ROS)/nitric oxide (NO) balance and renin-angiotensin system in mediating cardiac hypertrophy in hyperthyroidism was evaluated in an in vivo and in vitro experimental model. Male Wistar rats were divided into four groups: control, thyroid hormone, vitamin E (or Trolox, its hydrosoluble analogue), thyroid hormone+vitamin E. Angiotensin II receptor (AT1/AT2) gene expression, immunocontent of AT1/AT2 receptors, angiotensinogen, NADPH oxidase (Nox2), and nitric oxide synthase isoforms, as well as ROS concentration (hydrogen peroxide and superoxide anion) were quantified in myocardium. Thyroid hormone increased ROS and NO metabolites, iNOS, nNOS and eNOS isoforms and it was accompanied by cardiac hypertrophy. AT1/AT2 expression and the immunocontent of angiotensinogen and Nox2 were enhanced by thyroid hormone. Antioxidants reduced ROS levels, Nox2, AT1/AT2, NOS isoforms and cardiac hypertrophy. In conclusion, ROS/NO balance may play a role in the control of thyroid hormone-induced cardiac hypertrophy mediated by renin-angiotensin system.