Cross-Talk Between Mitochondria and NADPH Oxidase: Effects of Mild Mitochondrial Dysfunction on Angiotensin II-Mediated Increase in Nox Isoform Expression and Activity in Vascular Smooth Muscle Cells

Mitochondria and NADPH oxidase activation are concomitantly involved in pathogenesis of many vascular diseases. However, possible cross-talk between those ROS-generating systems is unclear. We induced mild mitochondrial dysfunction due to mitochondrial DNA damage after 24 h incubation of rabbit aortic smooth muscle (VSMC) with 250 ng/mL ethidium bromide (EtBr). VSMC remained viable and had 29% less oxygen consumption, 16% greater baseline hydrogen peroxide, and unchanged glutathione levels. Serum-stimulated proliferation was unaltered at 24 h. Although PCR amplification of several mtDNA sequences was preserved, D-Loop mtDNA region showed distinct amplification of shorter products after EtBr. Such evidence for DNA damage was further enhanced after angiotensin-II (AngII) incubation. Remarkably, the normally observed increase in VSMC membrane fraction NADPH oxidase activity after AngII was completely abrogated after EtBr, together with failure to upregulate Nox1 mRNA expression. Conversely, basal Nox4 mRNA expression increased 1.6-fold, while being unresponsive to AngII. Similar loss in AngII redox response occurred after 24 h antimycin-A incubation. Enhanced Nox4 expression was unassociated with endoplasmic reticulum stress markers. Protein disulfide isomerase, an NADPH oxidase regulator, exhibited increased expression and inverted pattern of migration to membrane fraction after EtBr. These results unravel functionally relevant cross-talk between mitochondria and NADPH oxidase, which markedly affects redox responses to AngII. Antioxid Redox Signal 11, 1265-1278.

Heparin affects the interaction of kininogen on endothelial cells

In the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PM) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. In the presence of Zn(+2), HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PM binding to cell- or ECM-bound-HK and PM activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in the fluid phase, was augmented by this glycosaminoglycan in the presence of Zn(2+). Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. In conclusion, glycosaminoglycans that accumulated in inflammatory fluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment. (C) 2011 Elsevier Masson SAS. All rights reserved.

Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity

Obesity has been shown to impair myocardial performance. Nevertheless, the mechanisms underlying the participation of calcium (Ca(2+)) handling on cardiac dysfunction in obesity models remain unknown. L-type Ca(2+) channels and sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a), may contribute to the cardiac dysfunction induced by obesity. The purpose of this study was to investigate whether myocardial dysfunction in obese rats is related to decreased activity and/or expression of L-type Ca(2+) channels and SERCA2a. Male 30-day-old Wistar rats were fed standard (C) and alternately four palatable high-fat diets (Ob) for 15 weeks. Obesity was determined by adiposity index and comorbidities were evaluated. Myocardial function was evaluated in isolated left ventricle papillary muscles under basal conditions and after inotropic and lusitropic maneuvers. L-type Ca(2+) channels and SERCA2a activity were determined using specific blockers, while changes in the amount of channels were evaluated by Western blot analysis. Phospholamban (PLB) protein expression and the SERCA2a/PLB ratio were also determined. Compared with C rats, the Ob rats had increased body fat, adiposity index and several comorbidities. The Ob muscles developed similar baseline data, but myocardial responsiveness to post-rest contraction stimulus and increased extracellular Ca(2+) was compromised. The diltiazem promoted higher inhibition on developed tension in obese rats. In addition, there were no changes in the L-type Ca(2+) channel protein content and SERCA2a behavior (activity and expression). In conclusion, the myocardial dysfunction caused by obesity is related to L-type Ca(2+) channel activity impairment without significant changes in SERCA2a expression and function as well as L-type Ca(2+) protein levels. J. Cell. Physiol. 226: 2934-2942, 2011. (C) 2011 Wiley-Liss, Inc.

Exercise training reduces cardiac angiotensin II levels and prevents cardiac dysfunction in a genetic model of sympathetic hyperactivity-induced heart failure in mice

The role of exercise training (ET) on cardiac renin-angiotensin system (RAS) was investigated in 3-5 month-old mice lacking alpha(2A-) and alpha(2C-)adrenoceptors (alpha(2A)/alpha(2C)ARKO) that present heart failure (HF) and wild type control (WT). ET consisted of 8-week running sessions of 60 min, 5 days/week. In addition, exercise tolerance, cardiac structural and function analysis were made. At 3 months, fractional shortening and exercise tolerance were similar between groups. At 5 months, alpha(2A)/alpha(2C)ARKO mice displayed ventricular dysfunction and fibrosis associated with increased cardiac angiotensin (Ang) II levels (2.9-fold) and increased local angiotensin-converting enzyme activity (ACE 18%). ET decreased alpha(2A)/alpha(2C)ARKO cardiac Ang II levels and ACE activity to age-matched untrained WT mice levels while increased ACE2 expression and prevented exercise intolerance and ventricular dysfunction with little impact on cardiac remodeling. Altogether, these data provide evidence that reduced cardiac RAS explains, at least in part, the beneficial effects of ET on cardiac function in a genetic model of HF.

Exercise training delays cardiac dysfunction and prevents calcium handling abnormalities in sympathetic hyperactivity-induced heart failure mice

Exercise training (ET) is a coadjuvant therapy in preventive cardiology. It delays cardiac dysfunction and exercise intolerance in heart failure (HF); however, the molecular mechanisms underlying its cardioprotection are poorly understood. We tested the hypothesis that ET would prevent Ca2+ handling abnormalities and ventricular dysfunction in sympathetic hyperactivity-induced HF mice. A cohort of male wildtype (WT) and congenic (alpha 2A/alpha 2C)-adrenoceptor knockout ((alpha 2A/alpha 2C)ARKO) mice with C57BL6/J genetic background (3-5 mo of age) were randomly assigned into untrained and exercise-trained groups. ET consisted of 8-wk swimming session, 60 min, 5 days/wk. Fractional shortening (FS) was assessed by two-dimensional guided M-mode echocardiography. The protein expression of ryanodine receptor (RyR), phospho-Ser(2809)-RyR, sarcoplasmic reticulum Ca2+ ATPase (SERCA2), Na+/Ca2+ exchanger (NCX), phospholamban (PLN), phospho-Ser(16)-PLN, and phospho-Thr(17)-PLN were analyzed by Western blotting. At 3 mo of age, no significant difference in FS and exercise tolerance was observed between WT and (alpha 2A/alpha 2C)ARKO mice. At 5 mo, when cardiac dysfunction is associated with lung edema and increased plasma norepinephrine levels, (alpha 2A/alpha 2C)ARKO mice presented reduced FS paralleled by decreased SERCA2 (26%) and NCX (34%). Conversely, (alpha 2A/alpha 2C)ARKO mice displayed increased phospho-Ser(16)-PLN (76%) and phospho-Ser(2809)-RyR (49%). ET in (alpha 2A/alpha 2C)ARKO mice prevented exercise intolerance, ventricular dysfunction, and decreased plasma norepinephrine. ET significantly increased the expression of SERCA2 (58%) and phospho-Ser(16)-PLN (30%) while it restored the expression of phospho-Ser(2809)-RyR to WT levels. Collectively, we provide evidence that improved net balance of Ca2+ handling proteins paralleled by a decreased sympathetic activity on ET are, at least in part, compensatory mechanisms against deteriorating ventricular function in HF.

Baroreflex Sensitivity Impairment Is Associated With Cardiac Diastolic Dysfunction in Rats

Background: Studies have shown that the autonomic dysfunction accompanied by impaired baroreflex sensitivity was associated with higher mortality. However, the influence of decreased baroreflex sensitivity on cardiac function, especially in diastolic function, is not well understood. This study evaluated the morpho-functional changes associated with baroreflex impairment induced by chronic sinoaortic denervation (SAD). Methods and Results: Animals were divided into sinoaortic denervation (SAD) and control (C) groups. Baroreflex sensitivity was evaluated by tachycardic and bradycardic responses, induced by vasoactive drugs. Cardiac function was studied by echocardiography and by left ventricle (LV) catheterization. LV collagen content and the expression of regulatory proteins involved in intracellular Ca(2+) homeostasis were quantified. Results showed higher LV mass in SAD versus C animals. Furthermore, an increase in deceleration time of E-wave in the SAD versus the C group (2.14 +/- 0.07 ms vs 1.78 +/- 0.03 ms) was observed. LV end-diastolic pressure was increased and the minimum dP/dt was decreased in the SAD versus the C group (12 +/- 1.5 mm Hg vs 5.3 +/- 0.2 mm Hg and 7,422 +/- 201 vs 4,999 +/- 345 mm Hg/s, respectively). SERCA/NCX ratio was lower in SAD than in control rats. The same was verified in SERCA/PLB ratio. Conclusions: The results suggest that baroreflex dysfunction is associated with cardiac diastolic dysfunction independently of the presence of other risk factors. (J Cardiac Fail 2011;17:519-525)

Endothelial nitric oxide synthase polymorphisms and adaptation of parasympathetic modulation to exercise training

SILVA, B. M., F. J. NEVES, M. V. NEGRÃO, C. R. ALVES, R. G. DIAS, G. B. ALVES, A. C. PEREIRA, M. Urbana A. RONDON, J. E. KRIEGER, C. E. NEGRÃO, and A. C. DA NOBREGA. Endothelial Nitric Oxide Synthase Polymorphisms and Adaptation of Parasympathetic Modulation to Exercise Training. Med. Sci. Sports Exerc., Vol. 43, No. 9, pp. 1611-1618, 2011. Purpose: There is a large interindividual variation in the parasympathetic adaptation induced by aerobic exercise training, which may be partially attributed to genetic polymorphisms. Therefore, we investigated the association among three polymorphisms in the endothelial nitric oxide gene (-786T>C, 4b4a, and 894G>T), analyzed individually and as haplotypes, and the parasympathetic adaptation induced by exercise training. Methods: Eighty healthy males, age 20-35 yr, were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis, and haplotypes were inferred using the software PHASE 2.1. Autonomic modulation (i.e., HR variability and spontaneous baroreflex sensitivity) and peak oxygen consumption ((V) over dotO(2peak)) were measured before and after training (running, moderate to severe intensity, three times per week, 60 min.day(-1), during 18 wk). Results: Training increased (V) over dotO(2peak) (P < 0.05) and decreased mean arterial pressure (P < 0.05) in the whole sample. Subjects with the -786C polymorphic allele had a significant reduction in baroreflex sensitivity after training (change: wild type (-786TT) = 2% +/- 89% vs polymorphic (-786TC/CC) = -28% +/- 60%, median +/- quartile range, P = 0.03), and parasympathetic modulation was marginally reduced in subjects with the 894T polymorphic allele (change: wild type (894GG) = 8% +/- 67% vs polymorphic (894GT/TT) = -18% +/- 59%, median +/- quartile range, P = 0.06). Furthermore, parasympathetic modulation percent change was different between the haplotypes containing wild-type alleles(-786T/4b/894G) and polymorphic alleles at positions -786 and 894 (-786C/4b/894T) (-6% +/- 56% vs -41% +/- 50%, median T quartile range, P = 0.04). Conclusions: The polymorphic allele at position -786 and the haplotype containing polymorphic alleles at positions -786 and 894 in the endothelial nitric oxide gene were associated with decreased parasympathetic modulation after exercise training.

Timing and Dose of Statin Therapy Define Its Impact on Inflammatory and Endothelial Responses During Myocardial Infarction

Objective-Clinical trials of statins during myocardial infarction (MI) have differed in their therapeutic regimes and generated conflicting results. This study evaluated the role of the timing and potency of statin therapy on its potential mechanisms of benefit during MI. Methods and Results-ST-elevation MI patients (n = 125) were allocated into 5 groups: no statin; 20, 40, or 80 mg/day simvastatin starting at admission; or 80 mg/day simvastatin 48 hours after admission. After 7 days, all patients switched their treatment to 20 mg/day simvastatin for an additional 3 weeks and then underwent flow-mediated dilation in the brachial artery. As of the second day, C-reactive protein (CRP) differed between non-statin users (12.0 +/- 4.1 mg/L) and patients treated with 20 (8.5 +/- 4.0 mg/L), 40 (3.8 +/- 2.5 mg/L), and 80 mg/day (1.4 +/- 1.5 mg/L), and the daily differences remained significant until the seventh day (P < 0.0001). The higher the statin dose, the lower the elevation of interleukin-2 and tumor necrosis factor-alpha, the greater the reduction of 8-isoprostane and low-density lipoprotein(-), and the greater the increase in nitrate/nitrite levels during the first 5 days (P < 0.001). Later initiation of statin was less effective than its early introduction in relation to attenuation of CRP, interleukin-2, tumor necrosis factor-alpha, 8-isoprostane, and low-density lipoprotein(-), as well as in increase in nitrate/nitrite levels (P < 0.0001). At the 30th day, there was no longer a difference in lipid profile or CRP between groups; the flow-mediated dilation, however, was proportional to the initial statin dose and was higher for those who started the treatment early (P = 0.001). Conclusion-This study demonstrates that the timing and potency of statin treatment during MI are key elements for their main mechanisms of benefit.

Lopap: A non-inflammatory and cytoprotective molecule in neutrophils and endothelial cells

Lopap (Lonomia obliqua prothrombin activator protease) is a member of the lipocalin family isolated from the extract of L obliqua bristles. Lopap displays serine protease-like activities, including coagulation disturbance, cytokine secretion and antiapoptotic activity in human cultured endothelial cells. Here, we have investigated the effects of the recombinant protein (rLopap) on the inflammatory and apoptotic processes of neutrophils and endothelial cells from male Wistar rats. We found that rLopap did not induce in vivo leukocyte-endothelial interactions in the microvasculature, initial steps of leukocyte recruitment during inflammation. Incubation of rLopap with neutrophils or endothelial cells prevented apoptosis evoked by serum deprivation and induced nitric oxide (NO) production in both cell types, and increased the expression of ICAM-1 by endothelial cells. Simultaneous incubation of endothelial cells or neutrophils with rLopap and N(omega)-nitro-L-arginine methyl ester (L-NAME), a non-specific inhibitor of NO synthases, inhibited NO production and impaired the protection on apoptosis. Differently, incubation of endothelial cells with monoclonal antibody anti ICAM-1 did not change the protection on apoptosis evoked by rLopap. Together, these results indicate that rLopap does not display inflammatory properties in vivo but inhibits apoptosis of neutrophils and endothelial cells depending, at least in part, on NO production. (C) 2009 Elsevier Ltd. All rights reserved.

Cytotoxicity and Endothelial Cell Adhesion of Lyophilized and Irradiated Bovine Pericardium Modified With Silk Fibroin and Chitosan

Grafts of biological tissues have been used since the 1960s as an alternative to the mechanical heart prostheses. Nowadays, the most consolidated treatment to bovine pericardial (BP) bioprostheses is the crosslinking with glutaraldehyde (GA), although GA may induce calcification in vivo. In previous work, our group demonstrated that electron beam irradiation applied to lyophilized BP in the absence of oxygen promoted crosslinks among collagen fibers of BP tissue. In this work, the incorporation of silk fibroin (SF) and chitosan (CHIT) in the BP not treated with GA was studied. The samples were irradiated and then analyzed for their cytotoxicity and the ability of adhesion and growth of endothelial cells. Initially, all samples showed cytotoxicity. However, after a few washing cycles, the cytotoxicity due to acetic acid and ethanol residues was removed from the biomaterial making it suitable for the biofunctional test. The samples modified with SF/CHIT and electron beam irradiated favored the adhesion and growth of endothelial cells throughout the tissue.

Budlein A from Viguiera robusta inhibits leukocyte-endothelial cell interactions, adhesion molecule expression and inflammatory mediators release

Budlein A has been reported to exert some analgesic and anti-inflammatory properties. In this study, we have evaluated its effect on LPS-induced leukocyte recruitment in vivo and the mechanisms involved in its anti-inflammatory activity. In vivo, intravital videomicroscopy was used to determine the effects of budlein A on LPS-induced leukocyte-endothelial cell interactions in the murine cremasteric microcirculation. In vitro, the effects of budlein A on LPS-induced cytokine, chemokine and nitrites release, T-cell proliferative response as well as cell adhesion molecule expression (CAM) were evaluated. In vivo, intraperitoneal administration of budlein A (2.6 mM/kg) caused a significant reduction of LPS-induced leukocyte rolling flux, adhesion and emigration by 84, 92 and 96% respectively. In vitro, T-cell proliferative response was also affected by budlein A. When murine J774 macrophages were incubated with the sesquiterpene lactone, LPS-induced IL-1 beta, tumor necrosis factor-alpha (TNF-alpha) and keratinocyte-derived chemokine (KC) release were concentration-dependently inhibited. In human umbilical vein endothelial cells (HUVECs), budlein A also reduced the production of TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), IL-8, nitrites and CAM expression elicited by LPS. Budlein A is a potent inhibitor of LPS-induced leukocyte accumulation in vivo. This effect appears to be mediated through inhibition of cytokine and chemokine release and down-regulation of CAM expression. Thus, it has potential therapeutic interest for the control of leukocyte recruitment that occurs in different inflammatory disorders. (C) 2009 Elsevier GrnbH. All rights reserved.

Bcl-2 in endothelial cells is increased by vitamin E and alpha-lipoic acid supplementation but not exercise training

Atherosclerotic plaque contains apoptotic endothelial cells with oxidative stress implicated in this process. Vitamin E and a-lipoic acid are a potent antioxidant combination with the potential to prevent endothelial apoptosis. Regular exercise is known to increase myocardial protection, however, little research has investigated the effects of exercise on the endothelium. The purpose of these studies was to investigate the effects of antioxidant supplementation and/or exercise training on proteins that regulate apoptosis in endothelial cells. Male rats received a control or antioxidant-supplemented diet (vitamin E and alpha-lipoic acid) and were assigned to sedentary or exercise-trained groups for 14 weeks. Left ventricular endothelial cells (LVECs) were isolated and levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were measured. Antioxidant supplementation caused a fourfold increase in Bcl-2 (P < 0.05) with no change in Bax (P > 0.05). Bcl-2:Bax was increased sixfold with antioxidant supplementation compared to non-supplemented animals (P < 0.05). Exercise training had no significant effect on Bcl-2, Bax or Bcl-2:Bax either alone or combined with antioxidant supplementation (P > 0.05) compared to non-supplemented animals. However, Bax was significantly lower (P < 0.05) in the supplemented trained group compared to non-supplemented trained animals. Cultured bovine endothelial cells incubated for 24 h with vitamin E and/or a-lipoic acid showed the combination of the two antioxidants increased Bcl-2 to a greater extent than cells incubated with the vehicle alone. In summary, vitamin E and a-lipoic acid increase endothelial cell Bcl-2, which may provide increased protection against apoptosis. (c) 2005 Elsevier Ltd. All rights reserved

Evidence for persistent dysfunction of wild-type aldosterone synthase gene in glucocorticoid-treated familial hyperaldosteronism type I

Background In familial hyperaldosteronism type I (FH-I), glucocorticoid treatment suppresses adrenocorticotrophic hormone-regulated hybrid gene expression and corrects hyperaldosteronism. Objective To determine whether the wild-type aldosterone synthase genes, thereby released from chronic suppression, are capable of functioning normally. Methods We compared mid-morning levels of plasma potassium, plasma aldosterone, plasma renin activity (PRA) and aldosterone : PRA ratios, measured with patients in an upright position, and responsiveness of aldosterone levels to infusion of angiotensin II (AII), for 11 patients with FH-I before and during long-term (0.8-14.3 years) treatment with 0.25-0.75 mg/day dexamethasone or 2.5-10 mg/day prednisolone. Results During glucocorticoid treatment, hypertension was corrected in all. Potassium levels, which had been low (< 3.5 mmol/l) in two patients before treatment, were normal in all during treatment (mean 4.0 +/- 0.1 mmol/l, range 3.5-4.6). Aldosterone levels during treatment [13.2 +/- 2.1 ng/100 ml (mean +/- SEM)] were lower than those before treatment (20.1 +/- 2.5 ng/100 ml, P < 0.05). PRA levels, which had been suppressed before treatment (0.5 +/- 0.2 ng/ml per h), were unsuppressed during treatment (5.1 +/- 1.5 ng/ml per h, P < 0.01) and elevated (> 4 ng/ml per h) in six patients. Aldosterone : PRA ratios, which had been elevated (> 30) before treatment (101.1 +/- 25.9), were much lower during treatment (4.1 +/- 1.0, P < 0.005) and below normal (< 5) in eight patients. Surprisingly, aldosterone level, which had not been responsive (< 50% rise) to infusion of AII for all 11 patients before treatment, remained unresponsive for 10 during treatment. Conclusions Apparently regardless of duration of glucocorticoid treatment in FH-I, aldosterone level remains poorly responsive to AII, with a higher than normal PRA and a low aldosterone : PRA ratio. This is consistent with there being a persistent defect in functioning of wild-type aldosterone synthase gene. (C) Rapid Science Publishers ISSN 0263-6352.

Erectile dysfunction in general medicine practice: prevalence and clinical correlates

Erectile dysfunction (ED) is a common problem in general medical practice affecting especially the elderly and those with cardiovascular disease and diabetes mellitus, A study was undertaken by questionnaire distributed to consecutive adult male attendees at 62 general medical practices. 1240 completed questionnaires were available for analysis. The mean age of participants was 56.4 y (range 18 - 91 y). 488 men (39.4%) reported ED: 119 (9.6%) 'occasionally', 110 (8.9%) 'often', and 231 (18.6%) 'all the time' (complete ED). Among 707 men aged 40-69 y 240 (33.9%) reported ED and 84 (11.9%) had complete ED. The prevalence of complete ED increased with age, rising from 2.0% in the 40-49 y age group to 44.9% in the 70-79 y age group. Only 11.6% of men with ED had received treatment. Hypertension, ischaemic heart disease, peripheral vascular disease and diabetes mellitus were frequently associated with ED. 40% of diabetic men aged 60 y or older had ED all the time.