Rajat S. Barua

Reactive Oxygen Species Are Involved in Smoking-Induced Dysfunction of Nitric Oxide Biosynthesis and Upregulation of Endothelial Nitric Oxide Synthase An In Vitro Demonstration in Human Coronary Artery Endothelial Cells

Background—Our group has previously shown that human umbilical vein endothelial cells exposed to smokers’ serum decreased nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in the presence of increased eNOS expression. In the present study, we examined whether these observations extended to human coronary artery endothelial cells (HCAECs). In addition, the role of reactive oxygen species in the observed alterations was examined. Methods and Results—HCAECs were incubated with serum from 10 nonsmokers and 15 smokers for 12 hours with or without the addition of either polyethylene glycol-superoxide dismutase (PEG-SOD, 300 U/mL), PEG-SOD+PEG-catalase (1000 U/mL), chelerythrine (3 &mgr;mol/L), or tetrahydrobiopterin (20 &mgr;mol/L). At the end of incubation, NO, eNOS protein, and eNOS activity were measured from the same culture. HCAECs incubated with smokers’ serum alone showed significantly lower NO production (P <0.05) and eNOS activity (P <0.005) but higher eNOS expression (P <0.005) compared with nonsmokers. In smokers, addition of PEG-SOD, PEG-SOD+PEG-catalase, or tetrahydrobiopterin significantly (P <0.05) improved NO levels and eNOS activity. Interestingly, in the same smokers, a significant decrease in eNOS expression was only seen with the addition of PEG-SOD+PEG-catalase (P <0.05) and treatment with PEG-SOD alone insignificantly increased eNOS expression. Conclusions—The present study indicates that in vitro, HCAECs show similar changes in NO biosynthesis as human umbilical vein endothelial cells when exposed to smokers’ serum and also confirms that oxidative stress plays a central role in smoking-mediated dysfunction of NO biosynthesis in endothelial cells. Furthermore, these data support other studies suggesting a role for hydrogen peroxide in the upregulation of eNOS.

Normalization of testosterone level is associated with reduced incidence of myocardial infarction and mortality in men

AIMS There is a significant uncertainty regarding the effect of testosterone replacement therapy (TRT) on cardiovascular (CV) outcomes including myocardial infarction (MI) and stroke. The aim of this study was to examine the relationship between normalization of total testosterone (TT) after TRT and CV events as well as all-cause mortality in patients without previous history of MI and stroke. METHODS AND RESULTS We retrospectively examined 83 010 male veterans with documented low TT levels. The subjects were categorized into (Gp1: TRT with resulting normalization of TT levels), (Gp2: TRT without normalization of TT levels) and (Gp3: Did not receive TRT). By utilizing propensity score-weighted Cox proportional hazard models, the association of TRT with all-cause mortality, MI, stroke, and a composite endpoint was compared between these groups. The all-cause mortality [hazard ratio (HR): 0.44, confidence interval (CI) 0.42-0.46], risk of MI (HR: 0.76, CI 0.63-0.93), and stroke (HR: 0.64, CI 0.43-0.96) were significantly lower in Gp1 (n = 43 931, median age = 66 years, mean follow-up = 6.2 years) vs. Gp3 (n = 13 378, median age = 66 years, mean follow-up = 4.7 years) in propensity-matched cohort. Similarly, the all-cause mortality (HR: 0.53, CI 0.50-0.55), risk of MI (HR: 0.82, CI 0.71-0.95), and stroke (HR: 0.70, CI 0.51-0.96) were significantly lower in Gp1 vs. Gp2 (n = 25 701, median age = 66 years, mean follow-up = 4.6 years). There was no difference in MI or stroke risk between Gp2 and Gp3. CONCLUSION In this large observational cohort with extended follow-up, normalization of TT levels after TRT was associated with a significant reduction in all-cause mortality, MI, and stroke.

Impaired mitochondrial function induced by serum from septic shock patients is attenuated by inhibition of nitric oxide synthase and poly(ADP-ribose) synthase.

ObjectiveThe purpose of this study was to determine the role of nitric oxide and poly(ADP-ribose) synthase on impaired mitochondrial function in septic shock. DesignHuman umbilical vein endothelial cells were incubated with serum from ten healthy controls, 20 patients with septic shock, and seven critically ill patients who were not septic. The experiment was repeated after pretreatment with 3-aminobenzamide, a poly(ADP-ribose) synthase inhibitor, or NG-methyl-l-arginine, a nonspecific nitric oxide synthase inhibitor. MeasurementsMitochondrial respiration was measured using a modified MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay. SettingResearch laboratory. Main ResultEndothelial cell mitochondrial respiration was significantly depressed by septic serum and averaged 61% ± 6% of control values (p < .05). Incubation with septic serum as compared with control serum also significantly decreased cellular adenosine triphosphate levels (6.7 ± 1.2 nM vs. 13.5 ± 1.9 nM, p < .01). The level of mitochondrial respiration in endothelial cells exposed to septic serum did not correlate with arterial lactate concentration but was correlated with both cardiac output (rs = .52, p < .05) and mixed venous oxygen saturation (rs = .61, p < .05). Pretreatment with NG-methyl-l-arginine significantly increased mitochondrial respiration in endothelial cells treated with septic serum from 63% ± 6% of normal to 88% ± 6% (p < .05) of normal values. Similarly, pretreatment with 3-aminobenzamide increased mitochondrial respiration in endothelial cells treated with septic serum from 64% ± 6% to 100% ± 4% (p < .01) of normal values. Endothelial cells incubated with serum from nonseptic critically ill patients did not demonstrate a significant decrease in mitochondrial respiration. ConclusionIn vitro mitochondrial respiration was significantly depressed by septic serum. The addition of NG-methyl-l-arginine, a nitric oxide synthase inhibitor, and 3-aminobenzamide, a blocker of the poly(ADP-ribose) synthase pathway, significantly attenuated this suppression. These data suggest that nitric oxide and poly(ADP-ribose) synthase activation may play an important role in the inhibition of mitochondrial respiration in septic shock.

Mechanisms of Coronary Thrombosis in Cigarette Smoke Exposure

Acute rupture or erosion of a coronary atheromatous plaque and subsequent coronary artery thrombosis cause the majority of sudden cardiac deaths and myocardial infarctions. Cigarette smoking is a major risk factor for acute coronary thrombosis. Indeed, a majority of sudden cardiac deaths attributable to acute thrombosis are in cigarette smokers. Both active and passive cigarette smoke exposure seem to increase the risk of coronary thrombosis and myocardial infarctions. Cigarette smoke exposure seems to alter the hemostatic process via multiple mechanisms, which include alteration of the function of endothelial cells, platelets, fibrinogen, and coagulation factors. This creates an imbalance of antithrombotic/prothrombotic factors and profibrinolytic/antifibrinolytic factors that support the initiation and propagation of thrombosis.

Effects of Cigarette Smoke Exposure on Clot Dynamics and Fibrin Structure An Ex Vivo Investigation

Objectives—The purpose of this study was to examine the effect of cigarette smoke exposure (CSE) on clot dynamics and fibrin architecture and to isolate the relative contribution of platelets and fibrinogen to clot dynamics. Methods and Results—From young healthy males smokers (n=34) and nonsmokers (n=34) a baseline blood was drawn, and smokers had another blood draw after smoking 2 regular cigarettes. Using thromboelastography (TEG) the degree of platelet-fibrin interaction was measured. In additional experiments, abciximab (20 &mgr;g/mL) was added to the smokers samples (n=27) to reduce the effects of platelet function from the TEG parameters. The maximum clot strength (G) obtained with abciximab measured mainly the contribution of fibrinogen to clot strength (GF). By subtracting GF from G, the contribution of platelets to clot strength (GP) was presumed. A significant difference was found for all TEG parameters between nonsmokers versus postsmoking and pre- versus postsmoking samples. Postsmoking both GF and GP were significantly higher as compared to presmoking. On electron microscopy and turbidity analysis, postsmoking fibrin clots were significantly different compared to presmoking and nonsmoking samples. Conclusions—Acute CSE changes clot dynamics and alters fibrin architecture. Both functional changes in fibrinogen and platelets appear to contribute to heightened thrombogenicity after acute CSE.

Mechanisms of platelet-neutrophil interactions and effects on cell filtration in septic shock

We examined the mechanisms and the adhesive molecules mediating platelet-neutrophil adhesion in patients with septic shock. Neutrophils, platelets, and platelet poor plasma (NPPP) were isolated from 12 normal volunteers. Platelets and neutrophils were stimulated with platelet poor plasma (SPPP) removed from 12 patients in septic shock. Cell adhesion was assessed by filtration through 5-&mgr;m pore filters and by flow cytometry. Blocking monoclonal antibodies were used against the platelet and neutrophil surface receptors glycoprotein complex IIb/IIIa, P-selectin, ICAM-2, CD11a, CD11b, and CD18. The filtration pressure (Pi) of cells suspended in SPPP was significantly greater than that of cells suspended in NPPP (24 ± 1.0 mmHg vs. 14 ± 1.0 mmHg;P < 0.05). The difference between the Pi of cells suspended in SPPP or NPPP (&Dgr;Pi SPPP-NPPP) in the presence of monoclonal antibodies anti-CD41, anti-CD62P, abciximab, anti-CD11a, anti-CD11b, and anti-CD18 was significantly less than the &Dgr;Pi SPPP-NPPP of cell suspensions without the addition of these monoclonal antibodies (P < 0.01). The greatest reduction in Pi occurred when platelet receptor P-selectin was blocked simultaneously with the CD11b receptor on the neutrophil as compared to all other single blocking monoclonal antibodies or combinations of monoclonal antibodies. The mean fluorescence of activated platelet CD63-PE binding to neutrophils suspended in SPPP was significantly greater than that of cells suspended in NPPP (780 ± 130 lfu vs. 295 ± 35 lfu;P < 0.05). The greatest attenuation in mean fluorescence occurred by blocking the P-selectin receptor on the platelet simultaneously with CD11b receptor on the neutrophil. We conclude that platelet-neutrophil aggregation is increased in septic shock. This aggregation is mediated by the interaction of multiple platelet and neutrophil surface receptors. The platelet receptor P-selectin and the neutrophil receptor CD11b/CD18 appear to play the most important role in these interactions.

Methamphetamine-Associated Acute Left Ventricular Dysfunction: A Variant of Stress-Induced Cardiomyopathy

This brief report describes a case of transient left ventricular dysfunction in a 42-year-old woman associated with methamphetamine abuse. Transient (stress-induced) left ventricular dysfunction has been described previously, usually in postmenopausal women following emotional stress and also severe medical illness. This is the first reported case associated with methamphetamine abuse.

Monophosphoryl lipid A stimulated up-regulation of reactive oxygen intermediates in human monocytes in vitro

The production of reactive oxygen and nitrogen intermediates is a common response to infectious challenge in vivo. These agents have been implicated in the modulation of cytokine responses and are produced in large amounts in response to endotoxins produced by a number of infectious agents. The antigen‐presenting cell activation caused by these lipopolysacchardies (LPS) has been exploited in the use of these agents as adjuvants. In recent years, less‐toxic derivatives have been sought. One such agent, monophosphoryl lipid A (MPL), has been used increasingly in vivo as an adjuvant and as a modulator of the inflammatory process. It is known that this agent modulates the inflammatory response and cytokine production. In addition, we have shown its effect on the production of reactive nitrogen intermediates. In this paper, we show that MPL stimulates the release of high levels of superoxide (O2−) and hydrogen peroxide (H2O2), the latter being greater than that seen with LPS and appearing to be related to the inability of MPL to stimulate catalase activity. When cells were pretreated with LPS or MPL and subsequently challenged with LPS, the production of O2− and H2O2 was inhibited significantly by LPS and MPL. The concentration of MPL required to induce significant hyporesponsiveness to subsequent LPS challenge was 10 times lower than that of LPS. Hyporesponsiveness was greatest when induced by 10 μg/ml MPL, the same concentration that induced the maximum release of H2O2 in primary stimulation. In addition, we have shown that following MPL pretreatment, LPS stimulation does not cause the loss of cytoplasmic IκBα, which occurs when human monocytes are cultured with LPS. From our results, we propose a model for the reduced toxicity of MPL.

Cigarette Smoke Amplifies Inflammatory Response and Atherosclerosis Progression Through Activation of the H1R-TLR2/4-COX2 Axis

Emerging evidence suggests that infection and persistent inflammation are key players in the pathogenesis of atherosclerotic cardiovascular disease (CVD). Although it is well established that cigarette smoke (CS) promotes atherosclerotic CVD, very little is known about the potential impact of the collective effects of CS and intermittent or chronic subclinical infection on atherosclerosis. Our previous studies demonstrated that mast cell-derived histamine and lipopolysaccharide (LPS) synergistically enhance endothelial cell inflammatory response. We further noted that the synergy between histamine and LPS was due to reciprocal upregulation of histamine receptor and Toll-like receptor 4 (TLR4) expression and functions. These results suggest that the combined and persistent effects of mast cell mediators and bacterial agents on the vasculature are risk factors of CVD. Our recent data demonstrated that CS extract enhances histamine- and LPS-induced expression of cyclooxygenase-2 (COX-2) in endothelial cells, suggesting that CS and mast cell mediators may collectively amplify inflammatory response in the vessel wall. We hypothesize that CS enhances histamine-mediated upregulation of TLR2/TLR4 signaling in the endothelium and promotes progression of atherosclerosis. This article presents our perspective on the modulatory effects of CS and nicotine on the “histamine-TLR-COX-2 axis.”

Normalization of Testosterone Levels After Testosterone Replacement Therapy Is Associated With Decreased Incidence of Atrial Fibrillation

Background Atrial fibrillation (AF) is the most common cardiac dysrhythmia associated with significant morbidity and mortality. Several small studies have reported that low serum total testosterone (TT) levels were associated with a higher incidence of AF. In contrast, it is also reported that anabolic steroid use is associated with an increase in the risk of AF. To date, no study has explored the effect of testosterone normalization on new incidence of AF after testosterone replacement therapy (TRT) in patients with low testosterone. Methods and Results Using data from the Veterans Administrations Corporate Data Warehouse, we identified a national cohort of 76 639 veterans with low TT levels and divided them into 3 groups. Group 1 had TRT resulting in normalization of TT levels (normalized TRT), group 2 had TRT without normalization of TT levels (nonnormalized TRT), and group 3 did not receive TRT (no TRT). Propensity score–weighted stabilized inverse probability of treatment weighting Cox proportional hazard methods were used for analysis of the data from these groups to determine the association between post‐TRT levels of TT and the incidence of AF. Group 1 (40 856 patients, median age 66 years) had significantly lower risk of AF than group 2 (23 939 patients, median age 65 years; hazard ratio 0.90, 95% CI 0.81–0.99, P=0.0255) and group 3 (11 853 patients, median age 67 years; hazard ratio 0.79, 95% CI 0.70–0.89, P=0.0001). There was no statistical difference between groups 2 and 3 (hazard ratio 0.89, 95% CI 0.78– 1.0009, P=0.0675) in incidence of AF. Conclusions These novel results suggest that normalization of TT levels after TRT is associated with a significant decrease in the incidence of AF.