Alteration of Endothelin 1, MCP-1 and Chromogranin A in patients with atrial fibrillation undergoing pulmonary vein isolation.

BACKGROUND: The relation between arrhythmias and stress is known. The aim of our current study was to elucidate whether plasma levels of previously described stress parameters are altered in highly symptomatic patients with atrial fibrillation (AF) per se and in patients undergoing ablation therapy by pulmonary vein isolation (PVI). METHODS: 96 patients with AF undergoing PVI were recruited. Plasma levels of Endothelin-1 (ET-1), MCP-1 and Chromogranin-A (CGA) were measured before and three months after ablation completed with clinical follow-up with respect to AF recurrence. Additionally, we examined 40 healthy age- and sex-matched volunteers as a reference. RESULTS: Symptomatic AF patients showed increased levels of ET-1 compared to healthy controls (2.62pg/ml vs. 1.57pg/ml; p<0.01). Baseline levels of ET-1 were higher in patients presenting with AF after PVI (2.96pg/ml vs. 2.57pg/ml;p = 0.02). The temporal comparison revealed decreased ET-1 levels in patients without (2.57pg/ml vs. 2.33pg/ml; p<0.01) and unchanged ET-1 levels in patients with AF after PVI. Baseline MCP-1 was increased in AF patients vs. controls (268pg/ml vs. 227 pg/ml; p = 0.03). Both groups, with and without AF after PVI, showed an increase of MCP-1 compared to baseline (268pg/ml vs. 349pg/ml;p<0.01; 281pg/ml vs. 355pg/ml;p = 0.03). CGA was lower in AF patients compared to healthy controls (13.8ng/ml vs. 25.6ng/ml;p<0.01). Over time patients without AF after PVI showed an increase of CGA (14.2ng/ml vs. 20.7ng/ml;p<0.01). No change was observed in patients with AF after PVI. CONCLUSION: Our study demonstrated dysregulated levels of ET-1, MCP-1 and CGA in symptomatic AF patients. We could demonstrate an association between ET-1 to presence or absence of AF. Furthermore, we could show that a decrease of ET-1 as well as an increase of CGA after PVI, representing a trend towards control cohort levels, were both associated with restoration of sinus rhythm. These results provide new insights into the role of stress-related biomarkers in AF and AF treatment by ablation therapy.

Influence of High Polyphenol Beverage on Stress-Induced Platelet Activation.

OBJECTIVES: Platelets are playing a crucial role in acute cardiovascular events. We investigated if physical stress activates platelets and whether this activation can be inhibited by a polyphenol-enriched diet. METHODS: Blood samples were taken from a total of 103 athletes three weeks before, one day before, immediately as well as 24 hours and 72 hours after a marathon run. Participants were randomized, double-blinded and divided into two groups. One group received a polyphenol-rich beverage the other the same beverage without polyphenols. Besides analysis of platelet counts and impedance-aggregometric-measurement of platelet activity, soluble P-selectin and Endothelin-A measurements were performed. RESULTS: In the control group, runners showed a 2.2-fold increased platelet aggregation directly after completing a marathon and within the following three days when compared with baseline values (p<0.01). In accordance, significant increases in sP-selectin (57.52ng/ml vs. 94.86ng/ml;p<0.01) were detectable. In contrast, for the group consuming a beverage with increased polyphenol content (upper quartile of study beverage intake) we did not find any increase of platelet aggregation. DISCUSSION: Physical stress causes a significant increase in platelet activity. Our results demonstrate that a diet enriched in polyphenols is capable of preventing platelet activation. These findings might indicate a diminished cardiovascular stress-reaction following pre-exposition to polyphenol-enriched diet.

Extreme exercise enhances chromogranin A levels correlating with stress levels but not with cardiac burden.

OBJECTIVE: Stress and heart failure are associated with increased systemic levels of chromogranin A (CGA). Here we analyzed the effects of marathon running on systemic CGA levels and the association with cardiac burden and stress. METHODS: We recruited 47 lean and obese runners for a 10week training program aiming at running a marathon. Heart rates, individual fitness and marathon finishing times were monitored. CGA, proBNP and troponin T levels were analyzed by ELISA. RESULTS: We found a significant increase of CGA (+51%; p<0.01) in lean runners after marathon. The obese group showed the highest troponin T (0.22ng/ml; p<0.01) and proBNP (176.6ng/ml; p<0.01) levels. There were no correlations between proBNP, troponin T and CGA. An inverse correlation (r=-0.45; p<0.01) was found between CGA and finishing times. CONCLUSION: Marathon running is associated with increased CGA levels. However, this does not seem to reflect cardiac burden but rather marathon induced stress.

Mortality due to myocardial infarction in the Bavarian population during World Cup Soccer 2006.

BACKGROUND: Previously, we had demonstrated that the World Cup Soccer 2006 provoked levels of emotional stress sufficient to increase the incidence of acute cardiovascular events. We sought to assess whether mortality was also increased as a result. METHOD: We analyzed daily data on mortality due to myocardial infarction (MI) and total mortality using data from the Bavarian State Office for Statistics. We retrospectively assessed study periods from 2006, 2005 and 2003. Quasi-Poisson regression with a log link to model the number of daily deaths was used. To be able to account for a possible delay, we also fitted a cubic distributed lag quasi-Poisson model for both 1 and 2 weeks post-exposure. RESULTS: A total of 6,699 deaths due to MI were investigated. No increase in death was found on days of World Cup matches either with or without German participation compared to the matched control periods. In addition, none of the analyses showed a significant effect of the (lagged) exposure to the risk period. Likewise, total mortality rates remained unchanged over the entire period of our analysis. CONCLUSION: During World Cup Soccer, the number of deaths due to myocardial infarction was not measurably increased compared to a matched control period. Thus, we could not demonstrate a translation of a stress-induced increase of cardiac morbidity into a noticeable increase in mortality. However, our findings are based on a public mortality registry, which may be flawed in many ways, regarding ascertainment of causes of death, in particular.

Modification of endothelial biology by acute and chronic stress hormones.

OBJECTIVE: An increasing number of studies have examined the role of emotional stress and coronary heart disease; the underlying pathophysiology is still poorly understood. The present study was designed to evaluate the relationship between acute (epi- and norepinephrine) and chronic stress hormones (dexamethasone, beta-endorphin, corticotropin releasing hormone) and endothelial dysfunction. METHODS: Human microvascular endothelial cells were incubated with stress hormones for 6 and 24 h. ET-1 release and ADMA were quantified via ELISA, NO release by using cell permeable 4.5-diaminofluorescein diacetate (DAF2-DA), oxidative stress fluometrically by the ROS-sensitive carboxy-H2-DCFDA method, mitochondrial metabolic activity by using the colorimetric assay WST-1, ET-1 receptor type A (ET(A)R) protein expression by Western blot, and cell proliferation activity was assessed by the colorimetric assay BrdU. RESULTS: With respect to analysed acute and chronic stress hormones, ET-1 release was significantly increased. Likewise, protein expression was enhanced after long term incubation (24 h) with norepinephrine and dexamethasone. In contrast, endothelial NO-levels were only influenced by short term stimulation of dexamethasone (upregulation of NO release) and norepinephrine (downregulation of NO release), whereas modified NO concentration mimics altered mitochondrial metabolic activity. Unexpectedly, both oxidative stress and cell proliferation were not modified by stress hormones. CONCLUSION: Results suggest that acute and chronic stress hormones induce a significant ET-1 release whereas NO release remained mainly unchanged. The imbalance of pro- and antiatherosclerotic factors may play a pivotal role in the initiation of stress-related endothelial dysfunction up to myocardial infarction.

Effects of beta-endorphin on endothelial/monocytic endothelin-1 and nitric oxide release mediated by mu1-opioid receptors: a potential link between stress and endothelial dysfunction?

Observations have been made linking the presence of psychosocial factors associated with elevated beta-endorphin concentrations with atherosclerosis. In this study, the authors assume an important role of the stress hormone beta-endorphin in several mechanisms that contribute to a dysbalance of human endothelial and monocytic endothelin (ET)-1 and nitric oxide (NO) release, mediated by mu1-opioid receptors. ET-1 and NO release were quantified via enzyme-linked immunosorbent assay (ELISA) or fluorometrically. mu1-Opioid receptors were identified by polymerase chain reaction (PCR) after stimulation with beta-endorphin. beta-Endorphin significantly increased endothelial and monocytic ET-1 release. The effect was mediated by mu1-opioid receptors and abolished by naloxonazine, a selective mu1-opioid receptor antagonist. In contrast, NO release was decreased under the influence of beta-endorphin. mu1-Opioid receptors on human monocytes and endothelial cells mediated a beta-endorphin-induced stimulation of ET-1 release, whereas NO release was decreased. Thus, the authors hypothesize a role of beta-Endorphin in the pathogenesis of stress-induced endothelial dysfunction through peripherally circulating beta-endorphin, which may offset the balance of vasoactive mediators, leading to an unopposed vasoconstriction. The data may also provide a new concept of mu1-opioid receptor antagonists, preventing beta-endorphin-induced disorders of vascular biology.

Female sex hormones decrease constitutive endothelin-1 release via endothelial sigma-1/cocaine receptors: an action independent of the steroid hormone receptors.

Cardiovascular disease is rare in premenopausal women compared to men. The authors investigate sex hormone-induced endothelin-1 (ET-1) release and the involvement of classic sex hormone receptors as well as the ability of sigma-1/cocaine receptors to respond to sex hormones. ET-1 release was measured in the supernatant of endothelial cells after treatment with beta-estradiol, progesterone, testosterone, or combined with their antagonists, and with the sigma-1 receptor ligand ditolylguanidine (DTG), or haloperidol, a sigma-1 receptor antagonist. Binding assays were performed using 2.5 x 10(-8) M [3H]DTG. Female sex hormones decreased ET-1 release whereas testosterone increased it, sex hormone antagonists only slightly attenuated or had no effect on the respective hormone's effect. DTG totally blocked the female sex hormone-induced inhibition on ET-1 release, whereas testosterone-induced stimulation was not affected. However, haloperidol blocked both. [3H]DTG binding was displaced by beta -estradiol but not by testosterone. DTG-binding sites account for 513 +/- 114 per cell, KD 8.79 nM. These data suggest that besides classic steroid hormone receptors, sigma-1/cocaine receptors mediate the effects of female sex hormones on ET-1 release, an up to now unknown signalling pathway. Results also suggest that female and male sex hormones may bind to different sites on sigma-1 receptors, exerting opposite pharmacological effects.

Coronary vasomotor dysfunction in the cardiac allograft: impact of different immunosuppressive regimens.

Immunosuppression may have an important impact on early graft coronary endothelial injury. We investigated functional and morphologic coronary alterations, myocardial expression, and cardiac release of possible mediators of allograft vasculopathy within 6 months after cardiac transplantation with respect to different immunosuppressive regimens. Epicardial and microvascular endothelium-dependent and endothelium-independent vasomotor function and epicardial intimal thickening were measured in 8 transplant recipients treated with cyclosporin A (CyA), azathioprine, and prednisone (group 1), 9 transplant recipients treated with tacrolimus (TKL), azathioprine, and prednisone (group 2), and 14 patients treated with TKL, mycophenolate mofetil (MMF), and prednisone (group 3). The gene expressions of inducible and endothelial nitric oxide synthase (iNOS and eNOS), endothelin-1, prostacyclinsynthase, and thromboxansynthase were analyzed in endomyocardial biopsy specimens using semiquantitative reverse transcription polymerase chain reaction. Transcardiac cytokine release, endothelin-1, and nitrate-release were determined from plasma samples. Epicardial endothelial dysfunction (vasoconstriction to acetylcholine > 10%) and microvascular smooth muscle cell dysfunction (flow velocity increase to adenosine and nifedipine < 2.0) were enhanced in heart transplant recipients immunosuppressed with TKL, azathioprine, and prednisone. The prevalence of epicardial dysfunction was 78% in group 2 versus 44% and 46% in group 1 and 3 (p < 0.05), respectively. The prevalence of microvascular dysfunction was 56% in group 2 versus 13% and 7% in group 1 and 3 (p < 0.02), respectively. Coronary vasomotor dysfunction was associated with increased myocardial iNOS expression (p < 0.05), decreased eNOS expression (p < 0.05), and enhanced cardiac immunoreactive interleukin-6 (p < 0.01). Coronary intimal thickening was not different between the groups. The combination of TKL and MMF appears to be superior to TKL and azathioprine (and comparable to CyA and azathioprine) concerning preservation of early coronary vasomotor function, eNOS expression, iNOS suppression as well as cardiac interleukin-6 release. This may have an important impact on subsequent development of transplant coronary atherosclerosis.

Endothelin in coronary endothelial dysfunction early after human heart transplantation.

BACKGROUND: Cytokines and growth factors released as part of the immune response to alloantigenic stimuli are capable of regulating endothelin-1 expression in the allograft. Endothelin plays a significant role as a modulator of coronary vascular reactivity in the early stages of atherosclerosis and may be important as a participant in and marker for cardiac allograft vasculopathy. METHODS: We characterized a possible relationship between morphological and functional coronary changes, transcardiac plasma endothelin level and myocardial endothelin-mRNA expression in 33 cardiac transplant recipients in the early, stable phase 5+/-3 months after orthotopic heart transplantation. Coronary microvascular function was determined as endothelium-dependent with acetylcholine and endothelium-independent with adenosine using intracoronary Doppler-FloWire. The percentage of the epicardial diameter changes was measured using quantitative coronary angiography. Intravascular ultrasound was performed to quantify intimal hyperplasia. Cardiac endothelin uptake or release was determined by measuring plasma endothelin levels in the coronary sinus and aorta. Myocardial endothelin-gene expression was determined using semiquantitative RT-PCR. RESULTS: The aortic endothelin levels were significantly increased in transplant recipients compared to nontransplanted patients (11.8+/-2.2 vs 7.2+/-0.9 fmol/mL; P < 0.001). Endothelin uptake was noticed in the majority of patients, and the amount of endothelin uptake was correlated to microvascular (r = 0.37; P < 0.05) and epicardial (r = 0.41; P < 0.03) endothelium-dependent vasodilatation. High mRNA signal intensity was associated with significantly reduced coronary flow response to acetylcholine compared to patients with low myocardial gene expression (coronary flow reserve 2.4+/-0.9 vs 3.4+/-0.8, respectively; P < 0.005). Morphological coronary changes early after transplantation were not correlated to endothelin plasma levels or myocardial gene expression. CONCLUSION: Coronary endothelial vasomotor dysfunction after cardiac transplantation is associated with an increased myocardial endothelin mRNA expression and decreased endothelin-uptake by the heart. We postulate that early activation in the endothelin system may have a pivotal role in the acceleration of the atherosclerotic process in transplant patients.

Cocaine increases the endothelial release of immunoreactive endothelin and its concentrations in human plasma and urine: reversal by coincubation with sigma-receptor antagonists.

BACKGROUND: Cocaine-associated vascular events are not completely explained by adrenergic stimulation. The purposes of this study were to investigate whether vasoconstrictive endothelin-1 is released by cocaine and to elucidate the mechanisms involved. METHODS AND RESULTS: Endothelin-1 was measured by radioimmunoassay and high-performance liquid chromatography (1) in the supernatant of porcine aortic endothelial cells after treatment with cocaine (10(-7) to 10(-4) mol/L) and a sigma-receptor antagonist, haloperidol (10(-6) mol/L) or ditolylguanidine (10(-5) mol/L) and (2) in plasma and urine of 12 cocaine-intoxicated patients and 13 healthy control subjects. Radioligand binding assays were performed on endothelial membrane preparations. In cell culture, cocaine significantly increased endothelin accumulation above baseline at 3 to 24 hours; endothelin release rates per hour increased dose-dependently, reaching a plateau of 175+/-23% of control at hour 4 to 5. Coincubation of cocaine with haloperidol or ditolylguanidine abolished or reduced cocaine-induced endothelin release. Endothelial membrane preparations specifically and displaceably bound the highly selective sigma-ligand [3H]ditolylguanidine (25x10(-9) mol/L), with 1400 binding sites estimated per cell. Endothelin-1 levels in plasma (22.7+/-5.6 versus 7.3+/-0.8 pmol/L) and urine (41.5+/-10.1 versus 12.7+/-3.8 pmol/L) of cocaine-intoxicated patients were significantly increased compared with control values. CONCLUSIONS: The data suggest that cocaine increases the endothelin-1 release in vitro and in vivo. The cocaine-induced vasoconstriction/vasospasm may therefore be facilitated by the release of endothelin-1. Cocaine appears to be an exogenous stimulator at endothelial sigma-receptors. The endogenous ligands of this antiopioid system may prove to play a role in vasospastic angina, acute myocardial infarction, and sudden cardiac death.

Endothelium-dependent microvascular vasomotion and its correlation with vasoactive mediators early after cardiac transplantation in humans.

Endothelial dysfunction precedes and predicts transplant vasculopathy. We investigated the relationship between endothelial dysfunction and the vasoactive mediators nitric oxide and endothelin, 33.7 +/- 2.0 days after heart transplantation. Coronary flow was measured in 18 patients to determine the endothelial microvascular vasomotor response to acetylcholine. Endomyocardial biopsies were taken to determine the levels of gene expression of isozymes of endothelin and nitric oxide synthases (NOS). Blood samples from the coronary sinus and aorta were withdrawn for measurement of endothelin, nitrite and cytokines. Five patients (30%) showed an impaired coronary flow reserve response to acetylcholine, significantly higher inducible NOS gene expression and significant transcardiac nitrite production. Plasma nitrite correlated with tumour necrosis factor-alpha levels in coronary sinus and a transcardiac net extraction of endothelin was noted in all patients. In conclusion, 30% of patients develop endothelial dysfunction early after heart transplantation; this correlates with the expression and activation of vasoactive and immunomodulatory mediators, which may predict the development of transplant vasculopathy.

Prevention of hyperacute rejection by human decay accelerating factor in xenogeneic perfused working hearts.

As a potential source of organs for xenotransplantation, pigs that are transgenic for human decay accelerating factor (DAF) have been bred in order to overcome hyperacute rejection. We investigated the protective effect of human DAF in a porcine working heart model perfused by human blood. Hearts of normal landrace pits served as controls. The following parameters were measured: stroke work index, coronary flow and arteriovenous oxygen consumption, 6-keto prostaglandin F1alpha and prostaglandin E2 as markers of endothelial cell activation; creatine phosphokinase and lactate dehydrogenase for evaluation of the extent of myocardial damage; TNFalpha and IL-6 as markers of mononuclear cell activation. Histological and ultrastructural investigations from myocardial tissue sections were done at the end of perfusion. Human (h) DAF appeared to inhibit complement-mediated endothelial cell activation of transgenic pig hearts successfully. This was in contrast to landrace pig hearts, which had a sixfold increase of prostaglandin levels during perfusion with human blood. The cardiac weight increase during perfusion time due to interstitial edema tended to be less in the hDAF group. Myocardial damage was minimal in transgenic hearts, whereas normal pig hearts produced a threefold increase of creatine phosphokinase and lactate dehydrogenase levels. In these hearts, electron microscopy revealed single cell necrosis of myocytes and vacuolization of mitochondria with cristae rupture. According to the results obtained in the working heart model, the breeding of pigs that are transgenic for hDAF represents a promising step to making heart xenotransplantation a clinical reality in the future.

Bidirectional effects of endogenous opioid peptides on endothelin release rates in porcine aortic endothelial cell culture: mediation by delta opioid receptor and opioid receptor antagonist-insensitive mechanisms.

The effects of opioid peptides on immunoreactive endothelin (ir ET) release from cultured porcine aortic endothelial cells over a 1-hr period (4-5 or 23-24 hr) were determined by radioimmunoassay and high-performance liquid chromatography after treatment for either 4 or 23 hr. Endogenous opioids, the synthetic delta opioid [D-Pen2,5]enkephalin and, for comparison, atrial and brain natriuretic peptides were added to the culture medium in concentrations ranging from 10(-12) to 10(-7) M. Thrombin (0.1-10 U/ml) served as a stimulatory reference. 1) Brain natriuretic peptide displayed only insignificant effects on ir ET release at 5 hr, but strongly inhibited ir ET release at 24 hr. 2) Opioids modulated release rates at 5 hr but did not display significant effects at 24 hr: metorphamide with predominant mu/kappa and weak delta opioid receptor activity stimulated release in a dose-dependent manner, whereas [Met5]enkephalin-Arg6-Phe7 with mu/delta activity and the delta agonists [Leu5]enkephalin, sulfated [Leu5]enkephalin and [D-Pen2,5]enkephalin decreased release rates; [Leu5]enkephalin was the most potent of the latter drugs. 3) Coincubation with either the nonselective opioid receptor antagonist naloxone (10(-5) M) or the delta receptor-selective antagonist ICI-174,864 (N,N-bisallyl-Tyr-D-Ala-Aib-Aib-Phe-Leu-OH) (10(-5) M) abolished all opioid-induced inhibitory effects, but rather potentiated or unmasked stimulatory effects of opioid peptides on ir ET release rates at 5 hr and also at 24 hr in the case of the delta agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased endothelin plasma concentrations in patients with coronary artery disease or hyperlipoproteinemia without coronary events.

Endothelins are made by endothelial cells, macrophages, and vascular smooth muscle cells, among others, and are the most potent endogenous vasoconstrictors yet discovered, with additional growth-promoting properties. A locally increased endothelin production in coronary artery disease or other atherosclerotic diseases may increase circulating endothelin plasma concentrations before symptoms of disease are manifest. We determined endothelin plasma concentrations (1) in 43 patients suffering from coronary artery disease (CAD); (2) in 43 patients with hyperlipoproteinemia without coronary artery disease (HLP); (3) in 29 healthy control subjects (C), by means of a novel extraction procedure and radioimmunoassay followed by chromatographic separation. Plasma concentrations in HLP and C overlapped, but were still significantly different (29 +/- 10 vs 21 +/- 8 fmol/ml, ANOVA and Duncan's test). Significantly increased plasma concentrations were also found in patients with CAD, with the highest levels in a subgroup of 8 patients presenting with unstable angina (43 +/- 12 vs 53 +/- 15 fmol/ml). There were no statistically significant differences between CAD groups with (n = 28) or without hyperlipoproteinemia (n = 15) (42 +/- 14 vs 41 +/- 16 fmol/ml; n.s.). Likewise there was no relationship between endothelin plasma concentration in any of the patients studied and lipid fractions in serum. Increased endothelin plasma concentrations in HLP patients without evidence of coronary artery disease are thus not related to the hyperlipidemic state per se, but may rather indicate presence of an increased vasoconstrictor tonus, pre-clinical or silent atherosclerotic disease.