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Background: Enhanced aortic stiffness and blood pressure variability (BPV) are independent risk factors for cardiovascular disease and all-cause mortality in man. They are also correlated with increased blood pressure (BP) and/or arterial remodeling. However, the interplay between BP and BPV on the stiffening process is still unclear. Our objectives were to determine the temporal evolution of both BPV and pulse wave velocity (PWV), a surrogate measure of arterial stiffness, using an animal model of remodeling-dependent aortic stiffening., Method: We thus, developed a new telemetric technique allowing continuous measurement of PWV in conscious, unrestrained rats. Studies were performed in spontaneously hypertensive rats (SHR) treated for 2 weeks with N-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor (SHR-LN). BPV was evaluated conventionally or with a new device composed of two pressure transducers in two different sets of rats. This allowed a continuous monitoring of telemetered PWV, systolic (SPV), diastolic (DPV), and pulse pressure variability (PPV). Aortic structure was then characterized by immunohistochemical analysis., Results: SPV, DPV, and PPV were increased in SHR-LN, when calculated by 24-h SD or using average real variability a parameter used to assess short-term variability in man. We observed rapid and simultaneous increases in BP, SPV, and PWV. Interestingly, PPV was the most increased parameter resulting mainly from different time course of SPV and DPV. Structural alterations of the aortic wall were observed, with a eutrophic inward remodeling and accumulation of fibronectin and its two main receptors (α5 and αv integrins)., Conclusion: This offers unequivocal evidence of a significant relationship between PWV, BPV, and arterial structure.

Background: Lipocalin-2 is a proinflammatory adipokine upregulated in obese humans and animals. A pathogenic role of lipocalin-2 in hypertension has been suggested. Mice lacking lipocalin-2 are protected from dietary obesity-induced cardiovascular dysfunctions. Administration of lipocalin-2 causes abnormal vasodilator responses in mice on a high-fat diet (HFD)., Methods and Results: Wild-type and lipocalin-2 knockout mice were fed with standard chow or HFD. Immunoassays were performed for evaluating the circulating and tissue contents of lipocalin-2. The relaxation and contraction of arteries were studied using a wire myograph. Blood pressure was monitored with implantable radio telemetry. Dietary obesity promoted the accumulation of lipocalin-2 protein in blood and arteries. Deficiency of this adipokine protected mice from dietary obesity-induced elevation of blood pressure. Mass spectrometry analysis revealed that human and murine lipocalin-2 were modified by polyamination. Polyaminated lipocalin-2 was rapidly cleared from the circulation. Adipose tissue was a major site for lipocalin-2 deamidation. The circulating levels and the arterial accumulation of deamidated lipocalin-2 were significantly enhanced by treatment with linoleic acid (18:2n-6), which bound to lipocalin-2 with high affinity and prevented its interactions with matrix metalloproteinase 9 (MMP9). Combined administration of linoleic acid with lipocalin-2 caused vascular inflammation and endothelial dysfunction and raised the blood pressure of mice receiving standard chow. A human lipocalin-2 mutant with cysteine 87 replaced by alanine (C87A) contained less polyamines and exhibited a reduced capacity to form heterodimeric complexes with MMP9. After treatment, C87A remained in the circulation for a prolonged period of time and evoked endothelial dysfunction in the absence of linoleic acid., Conclusions: Polyamination facilitates the clearance of lipocalin-2, whereas the accumulation of deamidated lipocalin-2 in arteries causes vascular inflammation, endothelial dysfunction, and hypertension.

Generous donors: The dithioperoxyanhydrides (CH3 COS)2 , (PhCOS)2 , CH3 COSSCO2 Me and PhCOSSCO2 Me act as thiol-activated hydrogen sulfide donors in aqueous buffer solution. The most efficient donor (CH3 COS)2 can induce a biological response in cells, and advantageously replace hydrogen sulfide in ex vivo vascular studies., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Alterations in RyR2 function have been proposed as a major pathophysiological mechanism of arrhythmias and heart failure (HF). Cardiac FKBP12.6 overexpression protects against myocardial infarction-induced HF and catecholamine-promoted ventricular arrhythmias. We tested the hypothesis that FKBP12.6 overexpression protects against maladaptive LVH and triggered ventricular arrhythmias following transverse aorta constriction (TAC) in the mouse. The TAC-associated mortality rate was significantly lower in male transgenic (DT) than in Ctr mice (p < 0.05). TAC-associated maladaptive hypertrophy was blunted in DT mice especially 1 month post-TAC and their SERCA2a/PLB ratio remained unchanged 1 and 2 months post-TAC. Two months after TAC, trains of 30 stimuli (burst pacing) performed following isoproterenol injection (0.2 mg/kg, ip), induced VT in 50% of the TAC-Ctr and in none of the TAC-DT mice (p = 0.022). The increase in myocyte shortening and Ca(2+) spark frequency observed in sham-operated Ctr mice in response to 50 nM isoproterenol was reduced in DT mice, and abolished in TAC-DT mice. NCX1 function was reduced in Sham-DT and TAC-DT compared with Sham-Ctr and TAC-Ctr mice, respectively (p < 0.05 for the 2 comparisons). In mice killed after isoproterenol injection and burst pacing, RyR2 S2814 phosphorylation was decreased by 50% in TAC-DT versus TAC-Ctr mice (p < 0.05), with no change in RyR2 S2808 and PLB S16 and T17 phosphorylation. Cardiac FKBP12.6 overexpression in the mouse blunts pressure overload-induced maladaptive LV remodelling and protects against catecholamine-promoted burst pacing-induced ventricular tachycardia by decreasing cardiac sensitivity to adrenergic stress and RyR2 S2814 phosphorylation, and decreasing NCX1 activity.

Objective: Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine., Design and Methods: A monoclonal antibody targeting a specific MMP-9 generated fragment of collagen III was used in a competitive ELISA. The assay was validated in urine and arterial tissue of Apolipoprotein-E knockout (ApoE-KO) mice., Results: The lower limit of detection was 0.5ng/mL, intra- and inter-assay coefficients of variation were below 10%. By the end of 20weeks of the study, urine levels of the novel CO3-610 biomarker in ApoE-KO mice increased by two-fold (p<0.0001) and were three-fold higher than in control mice. Western blots confirmed high expression of CO3-610 in arterial extracts of ApoE-KO mice., Conclusion: We have developed a novel competitive ELISA, capable of measuring a urine biomarker indicative of pathological extracellular matrix remodeling in a mouse model of atherosclerosis., (Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

The link between elevated heart rate and cardiovascular events is established in healthy individuals and in patients with cardiovascular disease. The new agent, ivabradine, specifically and selectively inhibits the I(f) current, with the sole action of heart rate reduction, with no impact on any other cardiac parameters. The benefits of "pure" heart rate reduction with ivabradine have been the focus of one of the largest clinical development programs ever performed, involving >20,000 individuals. Ivabradine has anti-ischemic and antianginal efficacy in monotherapy, as well as in combination with other antianginals, such as beta-blockers, and is safe and well tolerated. Two major morbidity-mortality trials, BEAUTIFUL and SHIFT, showed that heart rate reduction with ivabradine dramatically improves prognosis in patients with coronary artery disease and left ventricular dysfunction, symptomatic angina, or chronic heart failure. The development of ivabradine represents a clear innovation in the management of cardiovascular disease., (© 2011 New York Academy of Sciences.)

Heart rate (HR) is determined by the pacemaker activity of cells from the sinoatrial node (SAN), located in the right atria. Spontaneous electrical activity of SAN cells results from a diastolic depolarization (DD). Despite controversy in the exact contribution of funny current (I(f)) in pacemaking, it is a major contributor of DD. I(f) is an inward Na(+)/K(+) current, activated upon hyperpolarization and directly modulated by cyclic adenosine monophosphate. The f-proteins are hyperpolarization-activated cyclic nucleotide-gated channels, HCN4 being the main isoform of SAN. Ivabradine (IVA) decreases DD and inhibits I(f) in a use-dependent manner. Under normal conditions IVA selectively reduces HR and limits exercise-induced tachycardia, in animals and young volunteers. Reduction in HR with IVA both decreases myocardial oxygen consumption and increases its supply due to prolongation of diastolic perfusion time. In animal models and in human with coronary artery disease (CAD), IVA has anti-anginal and anti-ischemic efficacy, equipotent to classical treatments, β-blockers, or calcium channel blockers. As expected from its selectivity for I(f), the drug is safe and well tolerated with minor visual side effects. As a consequence, IVA is the first inhibitor of I(f) approved for the treatment of stable angina. Available clinical data indicate that IVA could improve the management of stable angina in all patients including those treated with β-blockers. As chronic elevation of resting HR is an independent predictor of mortality, pure HR reduction by inhibition of I(f) could, beyond the control of anti-anginal symptoms, improve the prognosis of CAD and heart failure; this therapeutic potential is currently under evaluation with IVA., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Despite increasing evidence for the presence of voltage-gated Na(+) channels (Na(v)) isoforms and measurements of Na(v) channel currents with the patch-clamp technique in arterial myocytes, no information is available to date as to whether or not Na(v) channels play a functional role in arteries. The aim of the present work was to look for a physiological role of Na(v) channels in the control of rat aortic contraction., Methodology/principal Findings: Na(v) channels were detected in the aortic media by Western blot analysis and double immunofluorescence labeling for Na(v) channels and smooth muscle alpha-actin using specific antibodies. In parallel, using real time RT-PCR, we identified three Na(v) transcripts: Na(v)1.2, Na(v)1.3, and Na(v)1.5. Only the Na(v)1.2 isoform was found in the intact media and in freshly isolated myocytes excluding contamination by other cell types. Using the specific Na(v) channel agonist veratridine and antagonist tetrodotoxin (TTX), we unmasked a contribution of these channels in the response to the depolarizing agent KCl on rat aortic isometric tension recorded from endothelium-denuded aortic rings. Experimental conditions excluded a contribution of Na(v) channels from the perivascular sympathetic nerve terminals. Addition of low concentrations of KCl (2-10 mM), which induced moderate membrane depolarization (e.g., from -55.9+/-1.4 mV to -45.9+/-1.2 mV at 10 mmol/L as measured with microelectrodes), triggered a contraction potentiated by veratridine (100 microM) and blocked by TTX (1 microM). KB-R7943, an inhibitor of the reverse mode of the Na(+)/Ca(2+) exchanger, mimicked the effect of TTX and had no additive effect in presence of TTX., Conclusions/significance: These results define a new role for Na(v) channels in arterial physiology, and suggest that the TTX-sensitive Na(v)1.2 isoform, together with the Na(+)/Ca(2+) exchanger, contributes to the contractile response of aortic myocytes at physiological range of membrane depolarization.

Aims: The requirement of endothelial NO synthase (NOS3) calcium to produce NO is well described, although the effect of NO on intracellular calcium levels [Ca(2+)](i) is still confusing. Therefore, NO and [Ca(2+)](i) cross-talk were studied in parallel in endothelial cells possessing a functional or a dysfunctional NO pathway., Methods and Results: Dysfunctional porcine endothelial cells were obtained either in vitro by successive passages or in vivo from regenerated endothelium 1 month after coronary angioplasty. Activity of NOS3 was characterized by conversion of arginine to citrulline, BH(4) intracellular availability, cGMP, and superoxide anion production. Imaging of the Ca(2+) indicator FURA 2-AM was recorded and sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) pump activity was analysed by (45)Ca(2+) uptake into cells. In endothelial cells with a functional NO pathway, NOS3 inhibition increased [Ca(2+)](i) and, conversely, an NO donor decreased it. In aged cells with an uncoupled NOS3 as shown by the reduced BH(4) level, the increase in superoxide anion and the lower production of cGMP and the decrease in NO bioavailability were linearly correlated with the increase in basal [Ca(2+)](i). Moreover, when stimulated by bradykinin, the calcium response was reduced while its decay was slowed down. These effects on the calcium signalling were abolished in calcium-free buffer and were similarly induced by SERCA inhibitors. In aged cells, NO improved the reduced SERCA activity and tended to normalize the agonist calcium response., Conclusion: In control endothelial cells, NO exerts a negative feedback on cytosolic Ca(2+) homeostasis. In aged cells, uncoupled NOS3 produced NO that was insufficient to control the [Ca(2+)](i). Consequently, under resting conditions, SERCA activity decreased and [Ca(2+)](i) increased. These alterations were reversible as exogenous NO, in a cGMP-independent way, refilled intracellular calcium stores, reduced calcium influx, and improved the agonist-evoked calcium response. Therefore, prevention of the decrease in NO in dysfunctional endothelium would normalize the calcium-dependent functions.

Background: After the first year of transplantation, the major limitation to long-term survival is the development of graft coronary vasculopathy, characterized by a pathologic activation of the endothelium with an attendant loss of its regulatory properties on homeostasis of the vascular wall. The present study was designed to evaluate the integrity of coronary vascular relaxations attributed to the endothelium-derived hyperpolarizing factor (EDHF) and to study hyperpolarization of smooth muscle cells after heterotopic heart transplantation., Methods: Six weeks after heart transplantation in a porcine model, vascular reactivity studies of control, native and allograft epicardial coronary artery rings were performed in standard organ chamber experiments. Moreover, membrane potential measurements were made with intracellular microelectrodes in rings of native and allograft coronary arteries., Results: There was a significant decrease in endothelium-dependent relaxations to 5-hydroxytryptamine (5-HT), high doses of bradykinin (BK) alone and BK plus N-omega-nitro-L-arginine (L-NNA) in rings from allograft compared to native, whereas the variation was significantly increased in response to cromakalim, a K(+)-ATP channel opener. Electrical and mechanical recordings showed no alteration in the resting membrane potential of smooth muscle cells, depolarization during contraction to prostaglandin F(2alpha) (PGF(2alpha)), or hyperpolarization in the presence of BK + L-NNA in rings of allograft vs native., Conclusions: In this swine model of heart transplantation, part of the reduction in endothelium-dependent relaxations to BK may be attributed to an alteration in the activity of EDHF. This impairment of EDHF-mediated relaxations may compound the endothelial dysfunction preceding the development of coronary graft vasculopathy.

Background and Purpose: Ivabradine, a specific and use-dependent I(f) inhibitor, exerts anti-ischaemic activity purely by reducing heart rate. The aim of this work was to characterize its effect on the predominant HCN channel isoform expressed in human sino-atrial nodes (hSAN), to determine its kinetics in HCN channels from multicellular preparations and rate-dependency of its action., Experimental Approach: RT-PCR analysis of the four HCN channel isoforms was carried out on RNAs from hSAN. Patch-clamp and intracellular recordings were obtained from CHO cells stably expressing hHCN4 and isolated SAN, respectively. Beating rate of rat isolated atria was followed using a transducer., Key Results: hHCN4 mRNAs were predominant in hSAN. Ivabradine induced a time-dependent inhibition of hHCN4 with an IC(50) of 0.5 microM. In rabbit SAN, ivabradine progressively reduced the frequency of action potentials: by 10% after 3 h at 0.1 microM, by 14% after 2 h at 0.3 microM and by 17% after 1.5 h at 1 microM. After 3h, ivabradine reduced the beating rate of rat right atria with an IC(30) of 0.2 microM. The onset of action of ivabradine was use-dependent rather than time-dependent with slower effects than caesium, an extracellular I (f) blocker. Ivabradine 3 microM decreased the frequency of action potentials in SAN from guinea-pig, rabbit and pig by 33%, 21% and 15% at 40 min, respectively., Conclusions and Implications: The use-dependent inhibition of hHCN4 current by ivabradine probably contributes to its slow developing effect in isolated SAN and right atria and to its increased effectiveness in species with rapid SAN activity.

Objective: We analyzed the involvement of thromboxane (TX) A2/prostaglandin (PG) H2 (TP) receptor in ischemia-induced neovascularization in mice., Methods and Results: Unilateral hindlimb ischemia was induced by right femoral artery ligature in male C57BL/6J mice (n=7 per group). Animals were then treated with or without TP receptor antagonist (S18886, 5 or 10 mg/kg per day; ramatroban, 10 mg/kg per day) or aspirin (30 mg/kg per day) in drinking water for 21 days. Hindlimb ischemia raised plasma level of TXB2, the stable metabolite of TXA2, by 4.7-fold. This increase was blocked by aspirin treatment whereas S18886 (5 or 10 mg/kg per day) had no effect. However, neither S 18886 nor aspirin affected postischemic neovascularization. We next assessed the putative involvement of TXA2 signaling in angiotensin II (Ang II) proangiogenic pathway. Ang II (0.3 mg/kg per day) enhanced TXB2 plasma levels by 2.6-fold over that of control (P<0.01). Ang II-induced TXB2 upregulation was reduced by cotreatment with Ang II type I receptor antagonist (candesartan, 20 mg/kg per day). Angiographic score, capillary number, and foot perfusion were improved by 1.7-, 1.7-, and 1.4-fold, respectively, in Ang II-treated mice compared with controls (P<0.05). Ang II proangiogenic effect was associated with a 1.6-fold increase in VEGF-A protein content (P<0.05) and a 1.4-fold increase in the number of Mac-3-positive cells (ie, macrophages) in ischemic areas (P<0.05). Interestingly, treatments with TP receptor antagonists or aspirin hampered the proangiogenic effects of Ang II., Conclusions: Endogenous activation of TXA2 receptor by eicosanoids did not modulate spontaneous neovascularization in the setting of ischemia. Conversely, TXA2 signaling is involved in Ang II-induced AT1-dependent vessel growth.

Coronary artery disease is still a major cause of morbidity and mortality in the industrialized countries and its prevalence is predicted to grow with the current aging of the population in these countries. In spite of the rapid pace of progress and increasing use of myocardial revascularization procedures, in particular percutaneous coronary interventions, the medical treatment of coronary artery disease has lost none of its relevance in the majority of patients, though conventional drugs have their limitations and the pharmacological approach to ischemic heart disease needs to be improved in terms of efficacy and tolerance to ensure better prevention of mortality and improvement in quality of life. Since increased heart rate plays a major role in coronary artery disease, not only as a trigger of most ischemic episodes, but also as an independent predictor of mortality, inhibition of the pacemaker If current in view of inducing a direct and selective decrease in heart rate represents an ideal conceptual target and an attractive therapeutic approach to coronary artery disease. The screening of original benzocycloalkane compounds at the Servier Research Institute resulted in the selection of ivabradine (Procoralan) for clinical development. Preclinical data showed that ivabradine inhibits the If current originating in the sinus node, induces a selective reduction in heart rate both at rest and during exercise, preserves myocardial contractility, atrioventricular conduction and ventricular repolarization and prevents exercise-induced myocardial ischemia as effectively as a b-blocker while offering better protection of regional myocardial contractility. These data were confirmed in humans, in particular the anti-ischemic efficacy of ivabradine, at least as effective as that of a b-blocker in patients with stable angina. Large ongoing clinical trials are seeking to assess the therapeutic value of ivabradine in ischemic heart failure and its potential for improving the prognosis of coronary artery disease by reducing mortality and the occurrence of major cardiovascular events.

Mild hyperhomocysteinemia is prevalent in the general population and has been linked to endothelial dysfunction and high arterial pressure (AP) in clinical studies. The present study was designed to determine whether a rise in AP was induced by mild hyperhomocysteinemia and whether the potential rise in AP is secondary or prior to endothelial dysfunction. Experiments were performed in a rat model of mild hyperhomocysteinemia induced by oral administration of homocysteine for 1-4 months. Aortic endothelial dysfunction was observed 2 months after homocysteine treatment while endothelium-independent vasodilation was normal. In parallel, homocysteine treatment increased phenylephrine-induced contraction in aortas with endothelium, but did not modify the contraction in aortas without endothelium, suggesting a decrease of basal NO production. In conscious unrestrained rats, AP was not significantly different 1, 2, 3 and 4 months after homocysteine treatment. In correlation, endothelial function of a resistance vessel (mesenteric artery), mainly non-NO nonprostanoid factor mediated, was preserved, indicating that homocysteine treatment only affected the NO pathway. In conclusion, mild hyperhomocysteinemia alone is not sufficient to elevate arterial blood pressure, at least in the rat model. Aortic endothelial dysfunction produced by mild hyperhomocysteinemia is independent of hemodynamic factors., (Copyright 2005 S. Karger AG, Basel.)

The screening of a series of benzocycloalkane derivatives led to the selection of Procoralan (ivabradine), the first selective inhibitor of the depolarizing If (funny) current of the sinus node, for the treament of myocardial ischaemia. In vitro, this compound reduces the spontaneous beating rate of isolated right rat atria and the firing rate of the action potential of rabbit sinus node preparations. This effect is explained by a reduction in the diastolic depolarisation slope of the action potential and underlies a selective inhibition of the pacemaker If current. In vivo, it induces a selective reduction in heart rate both at rest and during exercise. It preserves myocardial contractility, atrioventricular conduction and ventricular repolarisation duration. Ivabradine exerts a similar anti-ischaemic activity in exercise-induced myocardial ischaemia in pigs to that of a beta-blocker and, furthermore, it limits to a greater extent ischaemic myocardial contractile dysfunction.

Objective: Myocardial contractility is regulated by adrenergic stimulation, the strength-length relationship and the force-frequency relationship or Bowditch effect. The latter mechanism was clearly demonstrated in muscle strips, in the isolated heart as well as in in-vivo experiments. The aim of this study was to further investigate the role of the force-frequency effect on the contractile response to exercise or isoproterenol infusion in conditions of restricted increases in heart rate i.e., AV-block, sinus node block and beta-adrenoceptor block., Methods: Nineteen dogs were instrumented with a left ventricular miniature pressure gauge, catheters in the aorta, pulmonary artery and left atrium and pacing leads on the left atrium and left ventricle. In order to control the chronotropic response during sympathetic stimulation, permanent AV-block was induced in nine dogs, sinus node block using UL-FS 49 and beta-adrenoceptor block using propranolol was studied in ten dogs., Results: Adrenergic stimulation (isoproterenol 0.4 micro g/kg or exercise) after total AV-block failed to increase LVdP/dt. However, increasing LV pacing rate (from 50 up to 200 bpm) prior to adrenergic stimulation elicited a significant increase in LVdP/dt (4762 +/- 166 mmHg/s vs. 6485 +/- 381 mmHg/s, p < 0.05). In dogs in sinus rhythm, heart rate and LVdP/dt response to isoproterenol and exercise following pre-treatment with UL-FS 49 is significantly reduced, with heart rate increasing from 103 +/- 7 up to 154 +/- 5 bpm and LV dP/dt(max) from 2925 +/- 171 mmHg/s to 6249 +/- 400 mmHg/s compared to the response in control conditions (HR 220 +/- 3 bpm and LV dP/dt(max) 7473 +/- 616 mmHg/s) (p < 0.05). When heart rate is matched using atrial pacing, the LVdP/dt(max) response reached comparable values as observed in control conditions (7310 +/- 550 mmHg/s). Similar responses were obtained during exercise. Beta-adrenoceptor blockade attenuates considerably the heart rate and LVdP/dt response to sympathetic stimulation. Adjusting heart rate with atrial pacing restores only partially LVdP/dt(max)., Conclusion: During sympathetic stimulation, the chronotropic response plays a major role for the concomitant full expression of the inotropic response. In conditions where increases in heart rate are absent or severely restricted such as in permanent AV-block, sinus node block and beta-adrenoceptor block, the inotropic response will also be impaired.

The effects of ivabradine, a novel heart rate-reducing agent that inhibits the cardiac pacemaker current If, were compared with those of the beta-adrenergic blocker propranolol, in a model of exercise-induced regional myocardial ischemia in pigs. Five Yucatan micropigs were chronically instrumented to measure hemodynamics, regional myocardial contractility, and local electrograms, and a fixed stenosis of the left anterior descending coronary artery was induced using a clip. Each animal underwent three experiments on different days, each consisting of two treadmill exercise sessions, 4 hours apart. Ivabradine 5 mg/kg, propranolol 5 mg/kg, or vehicle was administered orally 3 hours before the second exercise session. Exercises before treatment and after vehicle produced reproducible hemodynamic changes and regional myocardial ischemia in the area perfused by the stenosed coronary artery, indicated by ST segment shift and regional contractile dysfunction. Ivabradine and propranolol were equipotent in reducing heart rate at rest and limiting tachycardia during exercise. Ivabradine, unlike propranolol, did not reduce left ventricular contractility at rest or during exercise, and did not increase atrio-ventricular conduction time. Both compounds reduced the exercise-induced ST segment shift in the ischemic region by approximately 80%, but ivabradine preserved systolic shortening to a significantly greater degree than propranolol (P < 0.05).

Increased accumulation of lipoproteins and cholesterol within cells from regenerated endothelium may be responsible for their reported dysfunction. This study compared the presence and uptake of oxidized forms of low-density lipoprotein (LDL) in cells derived from native and regenerated endothelium. Four weeks after balloon denudation, primary cultures of native and regenerated endothelial cells were prepared from porcine coronary arteries. Regenerated endothelium stained more strongly using an antibody against oxidized lipoproteins. The increase in oxidized forms of apolipoprotein-B-100 exhibited by cells from regenerated endothelium was not due to an increase in extracellular-induced oxidation of native LDL, measured as the production of thiobarbituric-acid-reactive substances, being identical in both cell types. Intracellular cholesterol and cholesterol ester content were unchanged in regenerated cells. Using flow cytometry, accumulation of oxidized LDL was investigated further by quantifying the uptake of a mildly oxidized preparation of 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate-labelled LDL. The parameters of uptake, EC(50) and E(max), were not different between cells from native and regenerated endothelium suggesting that the number of LOX-1 receptors was identical in the two cell types. Moreover, a negative correlation between the increased uptake of acetylated LDL and decreased cGMP production in response to bradykinin was observed in cells from regenerated endothelium. Thus, the increased incorporation of modified LDL and their intracellular oxidation could be responsible for the alteration in NO production. The presence of oxidized forms of LDL may be a marker of endothelium regeneration and could be involved in the endothelial dysfunction of pig coronary arteries 4 weeks after balloon denudation., (Copyright 2003 S. Karger AG, Basel)

Background: The endothelium protects the vascular wall through the nitric oxide (NO) release. Coronary endothelial dysfunction occurs early after heart transplantation and predicts the development of intimal thickening typical of graft coronary vasculopathy., Objective: We designed this study to examine the effect of endothelial NO synthase (eNOS) inhibition on the endothelial dysfunction caused by rejection and on the development of accelerated atherosclerosis after heart transplantation., Methods: To study the effect on these 2 end-points of inhibiting eNOS with intracoronary L-nitro arginine methyl ester (L-NAME; 1 mg/kg/day), infused with an osmotic pump for 30 days, we used a porcine model of heterotopic heart transplantation with pre-operative immunologic typing, to permit slow rejection without the need for immunosuppression. The endothelium-dependent relaxations of allografted coronary arteries, allografted arteries infused with L-NAME, allografted arteries mounted with the pump, and vehicle and native coronary arteries were compared 30 days after graft implantation using standard organ chamber experiments. We evaluated intimal thickening using a semi-quantitative scale (0-4+ grading)., Results: A significant decrease in relaxations to serotonin (5-HT) occurred in allografted arteries infused directly with L-NAME compared with allografted arteries from swine receiving 5-HT, and relaxations in the latter were decreased compared with those of swine receiving the vehicle and native coronary arteries (p < 0.05). We found no significant differences in endothelium-dependent relaxations to bradykinin among coronary rings from all groups. We observed a significant increase in the prevalence and severity of intimal thickening in allografted coronary arteries infused with L-NAME compared with allografts not infused (p < 0.05), which had significantly more intimal thickening compared with native coronary arteries (p < 0.05)., Conclusion: These results demonstrate that inhibiting the NO pathway worsens the endothelial dysfunction caused by rejection after heart transplantation and accelerates the intimal thickening process, leading to graft coronary vasculopathy. Strategies designed to preserve endothelial integrity and function of the endothelial NO pathway should be used to prevent graft coronary vasculopathy.

The markers of the bioavailability of NO (endothelium-dependent relaxation to acetylcholine and cyclic GMP content) in the thoracic aorta of apolipoprotein-E-deficient (ApoE KO) mice, 20 weeks old with enriched cholesterol diet or 35 weeks old on regular chow, are not decreased, in contrast with other models of atherosclerosis. However, severe hypercholesterolemia, the presence of atherosclerotic lesions and increased NADPH oxidase activity have been reported as early as at 20 weeks of age. The present experiments were designed to test if an increased capacity of NO production in these mice explains this paradox. The expressions of the 3 isoforms of NO synthase (NOS) were compared in ApoE KO and C57Bl/6J mice using Western blot and localized by immunohistochemistry. No adaptive increase in the expression of NOS was detected in ApoE KO mice. NO bioavailability could also be preserved by upregulation of enzymes involved in redox status such as CuZn or Mn superoxide dismutase and catalase. However, these enzymes were less expressed in ApoE KO mice than in control mice. These results highlight that ApoE KO mice represent an atypical model of atherosclerosis., (Copyright 2003 S. Karger AG, Basel)

Background: The nonworking heterotopic heart transplantation model has been used extensively for the study of rejection and coronary endothelial function in different species. The effect of left ventricular loading in a working heart transplantation model, which may be associated with different coronary flow patterns and local nitric oxide release, on the development of coronary endothelial dysfunction and intimal hyperplasia, is unknown., Methods: Porcine retroperitoneal "nonworking" heterotopic transplantations (n=10) and "working" heart (with left ventricular filling) transplantations (n=7) were performed. The left ventricular pressure was 0+/-0 mm Hg and 91+/-11 mm Hg in the nonworking and working groups, respectively. In the latter, the left ventricle to systemic arterial pressure ratio was 0.76+/-0.08., Results: Sixty days after transplantation, epicardial coronary arteries from working and nonworking allografts developed a comparable selective endothelial dysfunction of Gi-protein mediated relaxations. There were no statistically significant differences in the prevalence of intimal hyperplasia, but the severity of intimal hyperplasia was significantly greater in allograft coronary arteries from the working hearts., Conclusion: Working heterotopic allografts develop an endothelial dysfunction comparable with that of nonworking allografts, which validates the use of the simpler nonworking graft for the study of endothelial function. The similar prevalence of intimal hyperplasia with the development of more severe coronary lesions in working hearts may be due to differences in local nitric oxide release in these two models.

1 The consequences of the reduced production of nitric oxide (NO) by cells from regenerated endothelium were investigated by measuring membrane potential of smooth muscle cells (SMCs), isometric tension and cyclic nucleotides content in porcine coronary arteries with intimal thickening, four weeks following angioplasty. 2 Under basal conditions, SMCs of coronary arteries with regenerated endothelium were depolarized by 10 mV. This depolarization was associated with 82% decreased level of cGMP without alteration in cAMP. 3 Sodium nitroprusside (SNP, 1 micro M) repolarized SMCs of the previously denuded coronary arteries. This repolarization was abolished by 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 micro M) and not suppressed by glibenclamide (10 micro M), iberiotoxin (IbTX, 100 nM) and the combination of charybdotoxin (ChTX, 40 nM) plus apamin (100 nM). 4 Four-aminopyridine (4-AP, 1-5 mM) generated spontaneous rhythmic activities only in coronary arteries with regenerated endothelium which were abolished by SNP. Nevertheless, 4-AP did not suppress the repolarization induced by SNP. 5 In vascular segments with regenerated endothelium, contracted with prostaglandin F(2alpha) (PGF(2alpha)), relaxation to bradykinin (BK, 30 nM) was unaltered despite a reduced production of cGMP (-70%). Indomethacin (10 micro M) plus N(omega)-nitro-L-arginine (L-NA, 30 micro M) reduced relaxation (-12% and -35% for native and regenerated endothelium, respectively) but did not abolish it. 6 The hyperpolarizations induced by BK were not altered by the presence of indomethacin and L-NA and were unchanged in segments with regenerated endothelium. 7 These data are consistent with a contribution of impairment in NO production to the depolarization of SMCs. Nevertheless, EDHF responses to BK are sufficient to maintain a normal relaxation after angioplasty.

Accelerated coronary atherosclerosis following heart transplantation is the main limiting factor for long-term survival, aside from graft failure and complications due to immunosuppression. Graft coronary vasculopathy is due to chronic rejection of the vascular wall leading to intimal hyperplasia in coronary arteries. Numerous heterotopic heart transplantation models have been used in different species to study the immunology and pathophysiology following graft implantation. This study reports our experience with the retroperitoneal heterotopic heart transplantation in Large White domestic swine using immunological typing. This approach mimics the kinetics of slow low-grade rejection in clinical human heart transplantation. One hundred and fifty-four retroperitoneal (n = 154) heterotopic heart transplantations were performed using Large White swine sampled for the major histocompatibility class (MHC) class I antigen and blood type using the microlymphotoxicity technique. Acute rejection studies were performed by intentional mismatch of the swine lymphocyte alloantigen (SLA) and chronic rejection studies were done in allografts implanted in donor-recipients matched for blood type and class I antigen to assess the effects of rejection per se, hypercholesterolemia, intracoronary L-NAME infusion, and endothelial denudation on the development of graft coronary vasculopathy. Assessment of in vitro coronary arterial vascular reactivity in standard organ chamber experiments comprised the core of vascular biology studies in this large animal model. Eighty (52%) transplanted recipients survived until the elective date of sacrifice at 60 days, 14 (9.1%) died during the surgery, 21 (13.6%) died <24 h after the transplant, and 8 (5.2%) died of late deaths. The retroperitoneal heterotopic heart transplantation model with blood typing and determination of the SLA class I antigen is a useful model for the study of immunological and vascular events due to graft rejection after heart transplantation.

Experiments were designed to determine whether or not aging per se or cellular density affects endothelial NO-synthase (eNOS) activity in cultured coronary endothelial cells of the pig. A diminished activity could explain the reduced endothelium-dependent relaxation to bradykinin previously observed during regeneration after endothelial injury. The results demonstrate that cell cultures derived from eight-day old regenerated endothelium exhibit a normal basal production of cyclic GMP, but a reduced response to bradykinin or the Ca2+ ionophore A23187. With multiple cellular passages, used to mimick aging, the basal production of cyclic GMP remained stable during the first passage, to decrease moderately after one month (4th passage). By contrast, the response to bradykinin was reduced as of the second passage, to remain stable thereafter. In cultured aortic endothelial cells, an increase in cellular density was accompanied by a reduced number of active eNOS-site, as well as a reduction of NO production in the response to both bradykinin and A23187. These results suggest that both the increased cellular density and cell senescence explain the endothelial dysfunction during regeneration. They permit a better understanding of the changes in vascular reactivity in the course of endothelial regeneration, and of its pathological consequences.

Porcine coronary arteries with regenerated endothelium exhibit impaired endothelium-dependent relaxations. Experiments were designed to analyze the structural and functional changes occurring in regenerated endothelial cells. Primary cultures from regenerated endothelium contained giant endothelial cells, with an increased number of cells with diameter >14.5 microm, a reduced ability to proliferate, and signs of apoptosis. The uptake of fluorescent acetylated LDL was increased 2-fold in cultures from regenerated endothelium. The increased uptake of acetylated LDL was confirmed ex vivo in injured coronary arteries. In cultures from regenerated endothelium, cGMP production was decreased under basal conditions and during stimulation with serotonin, bradykinin, and A23187. Thus, during regeneration, there is accelerated senescence of endothelial cells accompanied by increased incorporation of modified LDL and reduction of NO production without decrease in endothelial NO synthase expression. These alterations help to explain the altered endothelium-dependent responses 28 days after balloon injury.

Hyperlipidemia may increase endothelial damage and promote accelerated atherogenesis in graft coronary vasculopathy. To study the effects of hypercholesterolemia on coronary endothelial dysfunction, intimal hyperplasia, and lipid content, a porcine model of heterotopic heart transplantation, allowing nonacute rejection without immunosuppressive drugs, was used. A high cholesterol diet was fed to donor and recipient swine 1 month before and after transplantation. The endothelial function of coronary arteries of native and transplanted hearts from cholesterol-fed animals was studied in organ chambers 30 days after implantation and compared with endothelial function in arteries from animals fed a normal diet. The total serum cholesterol increased 3-fold in donors and recipients. Endothelium-dependent relaxations to serotonin, to the alpha(2)-adrenergic agonist UK14,304, and to the direct G-protein activator sodium fluoride were decreased significantly in allografted hearts compared with native hearts from both groups. Relaxations to the calcium ionophore A23187 and bradykinin were decreased significantly in allografts from animals fed the high cholesterol diet. The prevalence of intimal hyperplasia was significantly increased in coronary arteries from hypercholesterolemic swine. There was a significant increase in the lipid content of allograft arteries of hypercholesterolemic recipients. Hypercholesterolemia causes a general coronary endothelial dysfunction, increases the prevalence of intimal hyperplasia, and augments the incorporation of lipids in the vascular wall after heart transplantation. Hyperlipidemia accelerates graft coronary atherosclerosis through its effects on the endothelium.

Background: Endothelial dysfunction is an early event leading to atherosclerosis. It also occurs after orthotopic heart transplantation and can be used to predict the development of intimal hyperplasia in the coronary artery wall. The present study was designed to assess the time course and specific alterations underlying endothelial dysfunction due to rejection after heart transplantation., Methods: A porcine model of heterotopic heart transplantation was used. Preoperative serum typing for the class I antigen of the swine lymphocyte alloantigen was performed to ensure compatibility for this antigen. This permitted survival of the graft with a low grade rejection without immunosuppression. Rings (with or without endothelium) of epicardial coronary arteries of native and transplanted hearts were studied in organ chambers filled with modified Krebs-Ringer bicarbonate solution and compared 1, 30 and 60 days after transplantation., Results: Myocardial contractility was normal in all grafts studied at 60 days after transplantation and all coronary arteries were patent. Myocardial biopsies showed the progression of rejection from day 1 to day 60 after implantation. All endothelium-dependent relaxations were normal one day after transplantation. Endothelium-dependent relaxations to serotonin and to the alpha 2-adrenergic agonist UK14304 (which both activate receptors coupled to Gi-proteins) and to sodium fluoride (a direct activator of G-proteins) were decreased 30 days after transplantation, while those to the calcium ionophore, A23187, and bradykinin were shifted to the right and those to ADP were normal. At 60 days, endothelium-dependent relaxations mediated by the Gi-protein pathway were decreased further while the concentration-relaxation curves to the other agonists were further shifted to the right. Endothelium-independent relaxations to the nitric oxide donor, Sin-1, were progressively reduced at 30 and 60 days, but maximal relaxations were maintained at 60 days. Histomorphometric studies showed a progressive increase in the percentage of coronary rings with intimal thickening from day 1 to day 60 after transplantation., Conclusions: The progressive endothelial dysfunction reported in this model of accelerated coronary atherosclerosis after transplantation without immunosuppression involves preferentially the pertussis-toxin-sensitive Gi-protein-mediated pathway. Endothelium-independent relaxations are decreased at 60 days, as are all other endothelium-dependent relaxations. Decreased endothelium-dependent vasodilatation may contribute to the development of coronary graft vasculopathy.

Experiments were designed to determine in two arteries (the guinea-pig carotid and the porcine coronary arteries) whether or not the endothelium-derived hyperpolarizing factor (EDHF) can be identified as potassium ions, and to determine whether or not the inwardly rectifying potassium current and the Na+/K+ pump are involved in the hyperpolarization mediated by EDHF. The membrane potential of vascular smooth muscle cells was recorded with intracellular microelectrodes in the presence of N(omega)-L-nitro-arginine (L-NA) and indomethacin. In vascular smooth muscle cells of guinea-pig carotid and porcine coronary arteries, acetylcholine and bradykinin induced endothelium-dependent hyperpolarizations (-18+/-1 mV, n = 39 and -19+/-1 mV, n = 7, respectively). The hyperpolarizations were not affected significantly by ouabain (1 microM), barium chloride (up to 100 microM) or the combination of ouabain plus barium. In both arteries, increasing extracellular potassium concentration by 5 or 10 mM induced either depolarization or in a very few cases small hyperpolarizations which never exceeded 2 mV. In isolated smooth muscle cells of the guinea-pig carotid artery, patch-clamp experiments shows that only 20% of the vascular smooth muscle cells expressed inwardly rectifying potassium channels. The current density recorded was low (0.5+/-0.1 pA pF(-1), n = 8). These results indicate that, in two different vascular preparations, barium sensitive-inwardly rectifying potassium conductance and the ouabain sensitive-Na+/K+ pump are not involved in the EDHF-mediated hyperpolarization. Furthermore, potassium did not mimic the effect of EDHF pointing out that potassium and EDHF are not the same entity in those arteries.

The present study was designed to test the ability of regenerated endothelium to evoke endothelium-dependent hyperpolarizations. Hyperpolarizations induced by serotonin and bradykinin were compared in isolated porcine coronary arteries with native or regenerated endothelium, 4 weeks after balloon endothelial denudation. The experiments were performed in the presence of inhibitors of nitric oxide synthase (Nomega-nitro-L-arginine) and cyclooxygenase (indomethacin). The transmembrane potential was measured using conventional glass microelectrodes. Smooth muscle cells from coronary arteries with regenerated endothelium were depolarized in comparison with control coronary arteries from the same hearts. Spontaneous membrane potential oscillations of small amplitude or spikes were observed in some of these arteries but never in arteries with native endothelium. In coronary arteries from control pigs, both serotonin and bradykinin induced concentration-dependent hyperpolarizations. In the presence of ketanserin, 10 micromol/L serotonin induced a transient hyperpolarization in control coronary arteries. Four weeks after balloon denudation, the response to serotonin was normal in arteries with native endothelium, but the hyperpolarization was significantly lower in coronary arteries with regenerated endothelium. In control arteries, the endothelium-dependent hyperpolarization obtained with bradykinin (30 nmol/L) was reproducible. Four weeks after balloon denudation, comparable hyperpolarizations were obtained in coronary arteries with native endothelium. By contrast, in arteries with regenerated endothelium, the hyperpolarization to bradykinin became voltage-dependent. In the most depolarized cells, the hyperpolarization to bradykinin was augmented. The changes in resting membrane potential and the alteration in endothelium-dependent hyperpolarizations observed in the coronary arteries with regenerated endothelium may contribute to the reduced response to serotonin and the unchanged relaxation to bradykinin described previously.

Background: The safe development of minimally invasive coronary artery bypass operations might require alternatives to conventional suture-based anastomotic techniques. In this setting, nonpenetrating stapling is an attractive option because of its simplicity of use and potential for improved endothelial preservation., Methods and Results: In the experimental part of this study, porcine internal mammary arteries were anastomosed to left anterior descending coronary arteries using either an 8-0 polypropylene running suture or nonpenetrating microclips (7 anastomoses in each group). The endothelium-dependent relaxations to bradykinin of the arterial rings bearing the anastomosis and of noninstrumented rings were compared in organ chamber experiments. There were no significant differences in maximal relaxations (mean +/- SEM) between the microclipped and sutured anastomoses (81%+/-7% versus 74%+/-10%), which were both significantly lower than those of control coronary rings (98%+/-2%, p = 0.001 versus the two anastomosed groups). Histologic examination showed a comparable preservation of the coronary and graft endothelium with both techniques. The clinical part of the study comprised 7 patients in whom the left internal mammary artery was conventionally sutured to the left anterior descending whereas 13 saphenous vein grafts were anastomosed to their target vessels by nonpenetrating staples. There were no clip-related complications. An angiographic assessment of the venous grafts was performed within 10 days postoperatively in all patients. One graft is presumably occluded. The 12 remaining conduits were patent with stapled anastomoses featuring a widely open "shark-mouth" configuration., Conclusions: These preliminary data suggest that non-penetrating stapling is an easy-to-use technique that competes well with conventional suturing, at least in terms of immediate results. Further studies are warranted to better define its potential place within the armamentarium of minimally invasive coronary artery bypass techniques.

In situ direct measurement of nitric oxide (NO) in biological media is now possible by means of electrochemical detection. In the literature, there are principally two amperometric approaches based on the direct oxidation of NO either on a sensor made from platinum/ iridium (Pt/Ir) alloy coated with a three-layered membrane or on a nickel porphyrin and Nafion-coated carbon fiber electrode. Nonetheless, the exact nature of the experimental amperometric signal obtained with the Pt/Ir system was never authenticated as being related to NO. This study compared responses of two sensors to the inhibition effect of Nomega-nitro-L-arginine (L-NA) as the amperometric signals produced by 5-hydroxytryptamine (5-HT) on isolated pig coronary preparations. These amperometric signals could be attributed to NO only for the nickel porphyrin and Nafion-coated carbon fiber electrode. Indeed, voltammetric characterization of the electrochemical response demonstrated only variations of the baseline current upon additions of either SNAP or NO on the Pt/Ir electrode instead of anodic peak current displayed at 0.63-0.75 V for the other system. Nitrites induced baseline current variations with the Pt/Ir electrode, similar to those obtained with S-nitroso-N-acetyl-dl-penicillamine (SNAP) or NO. This study highlights the potential hazards and pitfalls that may be associated with the use of a Pt/Ir sensor calibrated with SNAP solutions for the detection of NO production in various biological systems.

Background: The accurate performance of coronary anastomoses on the beating heart requires some form of myocardial immobilization that can be achieved pharmacologically. Different classes of drugs can be used to induce bradycardia, but the most effective in this setting of off-pump operation has not yet been determined., Methods: Fifty-six isolated buffer-perfused rabbit hearts were divided into seven equal groups. Control hearts were continuously perfused throughout the experimental time course. A second group of hearts underwent 60 minutes of potassium arrest (at 37 degrees C) followed by 1 hour of reperfusion. The following pharmacologic approaches were tested in the remaining five groups: short-acting beta-blockade (esmolol, 6 x 10(-3) mol/L and 3 x 10(-4) mol/L), opening of adenosine triphosphate-dependent potassium channels (nicorandil, 10(-3) mol/L and 10(-5) mol/L), and inhibition of the pacemaker current, which largely accounts for the diastolic depolarization of sinoatrial node cells (S 16257-2, 3 x 10(-6) mol/L). Each drug was infused at a constant rate for 60 minutes, after which hearts were perfused for 1 additional hour with drug-free buffer. Heart rate and isovolumic measurements of function and coronary flow were serially taken during and after drug infusion., Results: The worst recovery of systolic and, moreover, diastolic function was yielded by potassium arrest. Neither esmolol nor nicorandil was able to induce a significant bradycardia. However, nicorandil did not impair function which, conversely, was markedly depressed after esmolol therapy. Significant bradycardia (p < 0.0001 versus corresponding baseline values and versus all other groups) was only achieved with pacemaker current inhibition, which was otherwise associated with an excellent preservation of contractility, diastolic function, and coronary flow., Conclusions: Inhibition of the pacemaker current seems to be an effective approach for inducing intraoperative bradycardia without compromising left ventricular function or flow.

1. The purpose of these experiments was to determine whether or not the endothelium-dependent hyperpolarizations of the vascular smooth muscle cells (observed in the presence of inhibitors of nitric oxide synthase and cyclo-oxygenase) can be attributed to the production of an endogenous cannabinoid. 2. Membrane potential was recorded in the guinea-pig carotid, rat mesenteric and porcine coronary arteries by intracellular microelectrodes. 3. In the rat mesenteric artery, the cannabinoid receptor antagonist, SR 141716 (1 microM), did not modify either the resting membrane potential of smooth muscle cells or the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (17.3 +/- 1.8 mV, n = 4 and 17.8 +/- 2.6 mV, n = 4, in control and presence of SR 141716, respectively). Anandamide (30 microM) induced a hyperpolarization of the smooth muscle cells (12.6 +/- 1.4 mV, n = 13 and 2.0 +/- 3.0 mV, n = 6 in vessels with and without endothelium, respectively) which could not be repeated in the same tissue, whereas acetylcholine was still able to hyperpolarize the preparation. The hyperpolarization induced by anandamide was not significantly influenced by SR 141716 (1 microM). HU-210 (30 microM), a synthetic CB1 receptor agonist, and palmitoylethanolamide (30 microM), a CB2 receptor agonist, did not influence the membrane potential of the vascular smooth muscle cells. 4. In the rat mesenteric artery, the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (19.0 +/- 1.7 mV, n = 6) was not altered by glibenclamide (1 microM; 17.7 +/- 2.3 mV, n = 3). However, the combination of charybdotoxin (0.1 microM) plus apamin (0.5 microM) abolished the acetylcholine-induced hyperpolarization and under these conditions, acetylcholine evoked a depolarization (7.7 +/- 2.7 mV, n = 3). The hyperpolarization induced by anandamide (30 microM) (12.6 +/- 1.4 mV, n = 13) was significantly inhibited by glibenclamide (4.0 +/- 0.4 mV, n = 4) but not significantly affected by the combination of charybdotoxin plus apamin (17.3 +/- 2.3 mV, n = 4). 5. In the guinea-pig carotid artery, acetylcholine (1 microM) evoked endothelium-dependent hyperpolarization (18.8 +/- 0.7 mV, n = 15). SR 141716 (10 nM to 10 microM), caused a direct, concentration-dependent hyperpolarization (up to 10 mV at 10 microM) and a significant inhibition of the acetylcholine-induced hyperpolarization. Anandamide (0.1 to 3 microM) did not influence the membrane potential. At a concentration of 30 microM, the cannabinoid agonist induced a non-reproducible hyperpolarization (5.6 +/- 1.3 mV, n = 10) with a slow onset. SR 141716 (1 microM) did not affect the hyperpolarization induced by 30 microM anandamide (5.3 +/- 1.5 mV, n = 3). 6. In the porcine coronary artery, anandamide up to 30 microM did not hyperpolarize or relax the smooth muscle cells. The endothelium-dependent hyperpolarization and relaxation induced by bradykinin were not influenced by SR 141716 (1 microM). 7. These results indicate that the endothelium-dependent hyperpolarizations, observed in the guinea-pig carotid, rat mesenteric and porcine coronary arteries, are not related to the activation of cannabinoid CB1 receptors.

Two normotensive strains of rat, the Lou and Brown Norway (BN) strains, have contrasting levels of plasma angiotensin-converting enzyme (ACE). To investigate the degree of genetic determination of ACE expression, a polymorphic marker of the ACE gene was analyzed in inbred rats of the two strains. The two inbred strains were shown to bear different alleles for a polymorphic marker at the ACE gene. The segregation of the alleles of this marker and the plasma ACE levels were studied in a group of F2 rats issued from a cross between Lou and BN rats. The degree of genetic determination of plasma ACE activity was estimated to be 94% in the F2 cohort. The ACE locus accounts for 74% of total plasma ACE variance. ACE activity and mRNA expression in lungs were also genetically determined. The difference observed in ACE mRNA accumulation in the lungs between the two strains was due to a difference in the transcriptional rate of the ACE gene, as shown in nuclear run-on experiments. No differences were observed in arterial blood pressure of homozygous F2 progeny. In these animals, ACE genotype did not interfere with the pressor or the depressor responses to ACE-dependent vasoactive peptides. There was a significant effect of strain on constitutive or inducible membrane or soluble ACE activity in primary cultures of vascular cells. Neointima formation in the carotid artery 14 days after balloon injury was also influenced by the genotype in F2 homozygous progeny, whereas the medial area was not. These results demonstrate that there is a close relationship between the genetically determined ACE expression and the inducibility of the ACE gene. The degree of genetic determination of ACE expression in inbred rat strains offers a unique opportunity to study the interaction between genetic and environmental determinants of ACE expression and its involvement in response to experimental cardiovascular and renal injury.

The effects of the two isomers, (+)-S 16257 and (-)-S 16260, of a new bradycardic agent, (+/-)-S 15544 (7,8-dimethoxy 3-[3-[[(4.5-dimethoxybenzocyclobutan-1-yl)methyl] methylamino]propyl]1,3,4,5-tetrahydro-2H-3-benzazepin-2-one), were compared in vitro and in vivo on cardiac spontaneous rate and repolarization time. In the isolated rabbit sino-atrial node, the three compounds (3 microM) were equi-effective to reduce the action potential firing rate. In anesthetized pigs, both isomers (0.03, 0.1, 0.3 and 1 mg kg(-1) i.v.) were equipotent to reduce heart rate. For all compounds, the negative chronotropic effect resulted from a reduction in the slope of diastolic depolarization of pacemaker cells. In sino-atrial node cells, (-)-S 16260 (3 microM) increased action potential duration while (+)-S 16257 had a smaller effect. In driven guinea-pig papillary muscles exposed to increasing concentrations of compounds (0.1 to 10 microM) a small prolongation of action potential duration was observed. This prolongation was more marked in rabbit Purkinje fibers stimulated at a low rate. In all cardiac preparations the highest prolongation was observed with (-)-S 16260. In vivo, (-)-S 16260 prolonged QTc at the two highest doses tested while (+)-S 16257 had no effect. In conclusion, resolution of (+/-)-S 15544 into its two enantiomers yielded compounds with the same bradycardic effects. Of the isomers, (+)-S 16257 has an increased specificity with minimal direct effect on action potential repolarization.

1. Experiments were designed to investigate whether the pertussis toxin-dependent endothelial dysfunction following balloon injury is due to a reduced expression or an insufficient function of G-proteins. 2. Endothelium-dependent responses of porcine coronary arteries were examined in vitro by use of conventional organ chambers. Morphological analysis was performed by isolating and culturing the endothelial cells from these arteries. The expression of Gi-proteins in regenerated endothelial cells was measured by Western blots and immunolabelling. The function of G-proteins was assessed by measuring the GTPase activity of cultured endothelial cells. 3. Eight days following denudation, endothelial regrowth was confirmed by histological examination and by demonstrating the presence of endothelium-dependent relaxations to bradykinin and 5-hydroxytryptamine (5-HT). In primary culture, the regenerated endothelial cells displayed a 'cobblestone' pattern as seen with native endothelial cells. 4. Twenty eight days after denudation, the endothelium-dependent relaxations induced by 5-HT were impaired, but those to bradykinin were maintained. However, the latter were reduced when endothelium-dependent hyperpolarization was prevented. 5. Twenty eight days after denudation, multinucleated giant cells were present in the regenerated but not in the native cultured endothelial cell populations. These regenerated endothelial cells incorporated less tritiated thymidine than native endothelial cells. 6. The intensities of the bands on the immunoblot of the regenerated endothelial cells, when several antibodies against Gi alpha 1/alpha 2/alpha 3 were used, were the same as those obtained in native endothelial cells. The immunolabelling with the same antibodies was similar between the giant cells and the regenerated endothelial cells of normal size. The hydrolysis of GTP was lower in regenerated than in native endothelial cell membranes. 7. In conclusion, endothelium-dependent relaxations mediated by Gi-proteins are impaired in balloon denuded coronary arteries. This dysfunction following regeneration cannot be explained by a reduced expression of Gi proteins but rather reflects an abnormal function of the G-proteins in the regenerated endothelium.

Background: Endothelial dysfunction is one of the early events leading to atherosclerosis. It occurs early after orthotopic heart transplantation and precedes the appearance of accelerated graft coronary artery disease believed to stem from chronic rejection of the endothelium. Acute rejection may contribute to the development of graft vasculopathy., Methods: To assess the time course and specific mechanisms of coronary endothelial dysfunction in acute untreated rejection, a swine model of retroperitoneal heterotopic heart transplantation was used. Large white swine (age 10 +/- 2 weeks, weight 25 +/- 5 kg) were serum-typed for class I antigen of the swine leukocyte antigen system and selected to ensure a similar degree of incompatibility. Donor hearts were preserved with normothermic blood cardioplegia and regional hypothermia; the mean ischemic time was 64 +/- 15 minutes. Myocardial contractility decreased from day 5 (normal) to day 14 (weak), but electrical activity was preserved. All coronary arteries were patent, and International Society for Heart and Lung Transplantation grade 4 rejection was present in all hearts beyond 5 days. The endothelial function of epicardial coronary arterial rings of native and transplanted hearts was studied in organ chambers filled with modified Krebs-Ringer bicarbonate solution and compared 1, 5, 9, and 14 days after transplantation., Results: Maximal endothelium-independent relaxations were unaffected at all stages. Endothelium-dependent relaxations to serotonin and alpha 2-adrenergic agonist UK 14304 (which activate receptors coupled to Gi-proteins) and to sodium fluoride (a direct G-protein activator) deteriorated progressively over time. At 14 days maximal relaxations to the calcium ionophore A23187, adenosine diphosphate, and bradykinin were also reduced, but to a lesser degree than those to serotonin and sodium fluoride. Histomorphometric studies of the allograft coronary artery rings showed progressive intimal hyperplasia from day 5 to day 14, with an increase in the incidence from 29% +/- 8.3% to 61.5% +/- 12%., Conclusions: These studies show that endothelial dysfunction in untreated acute rejection after heart transplantation develops beyond 5 days and initially involves G-proteins; the dysfunction worsens over time to finally affect all endothelial mechanisms and vascular smooth muscle. The progression of the associated intimal hyperplasia parallels the alteration in endothelial function, suggesting a permissive role of the dysfunction in the development of this acute form of coronary graft vasculopathy.

Background: Minimally invasive coronary artery bypass grafting aims to achieve less patient discomfort and a more rapid return to active life. Most approaches have used maintenance of the beating heart and control of the target coronary vessel by different hemostatic devices. The purpose of this study was to assess the effects of commonly used coronary artery snares and of the occlusion of the coronary vessel necessary for minimally invasive coronary artery operations on coronary endothelial function., Methods: Coronary artery bypass grafting with an internal mammary artery to left anterior descending artery anastomosis was performed in a porcine model with a 30-minute period of ischemia and a subsequent 30-minute period of reperfusion, using snares on either side of the anastomotic site to achieve hemostasis of the operative field. Endothelium-dependent relaxation to serotonin was studied in conventional organ chamber experiments with rings taken from the left anterior descending artery at the proximal snare site, the anastomotic site in the segment that underwent the ischemia-reperfusion cycle, the distal snare site, and at a control segment. Responses to potassium chloride and bradykinin were also compared., Results: There were no significant differences in endothelium-dependent relaxation values among the four sites studied., Conclusions: These results confirm that snaring of the coronary artery for achieving hemostasis at the anastomotic site when performing coronary artery bypass grafting on the beating heart does not cause endothelial dysfunction.

Background: Improvements in myocardial protection may include the continuous delivery of normothermic blood cardioplegia. Technical aids are required for optimal visualization of the operative field during the performance of coronary anastomoses if cardioplegia is to be given continuously or during minimally invasive operations. However, the effects of the different hemostatic devices on coronary endothelial function are unknown., Methods: We compared the effects on endothelial function of two commonly used hemostatic techniques, coronary clamping and gas jet insufflation, with those of a technique using extravascular balloon occlusion to mimic systolic luminal closure by the surrounding myocardium. The three techniques were applied for 15 minutes on porcine epicardial coronary arteries from explanted hearts. For coronary clamping, standard bulldog clamps were used. Gas jet insufflation was applied by blowing oxygen (12 L/min) tangentially at a 45-degree angle 1 cm away from a 3-mm arteriotomy. Extravascular balloon occlusion was achieved with a needle-tipped silicone loop, the midportion of which, once positioned beneath the coronary artery, was inflated to push a myocardial "cushion" against the back of the vessel until its occlusion. Control rings were taken from the same coronary artery. The endothelial function of control and instrumented arterial rings was then studied in organ chambers filled with modified Krebs-Ringer bicarbonate solution., Results: Contractions to potassium chloride and prostaglandin F2 alpha and endothelium-independent relaxation to sin-1, a nitric oxide donor, were unaffected in all groups. Endothelium-dependent relaxation to serotonin was impaired after clamping and preserved after gas jet insufflation and extravascular balloon occlusion. Maximal endothelium-dependent relaxation to serotonin was as follows: for coronary clamping, 63% +/- 6% versus 87% +/- 3% in controls; for gas jet insufflation, 67% +/- 12% versus 88% +/- 7%; and for extraluminal balloon occlusion, 79% +/- 6% versus 85% +/- 5%., Conclusions: Whereas commonly used hemostatic devices may impair endothelial function, extravascular balloon occlusion appears to achieve effective hemostasis while preserving endothelial integrity.

Unsaturated fatty acids constitutive of cardiac membranal lipid matrix are one of the primary targets for reactive oxygen species generated during ischemia-reperfusion cycle. Lipid peroxidation is a cascade of intricate reactions involving the successive formations of fatty acids hydroperoxides and aldehydic compounds such as alkenals derived from the oxidative fragmentation of these hydroperoxides. The potential deleterious effects of different classes of lipid peroxidation products on cardiac cells were compared using three in vitro approaches: (i) cardiomyocyte integrity, (ii) electromechanical activity of papillary muscle, and (iii) atrial contractility. The following products of lipid peroxidation were tested: (i) photoperoxidized arachidonic acid pooling hydroperoxidized derivatives and aldehydic compounds, (ii) fatty acids hydroperoxides, and (iii) 4-hydroxynonenal, a characteristic alkenal derived from the oxidative fragmentation of hydroperoxidized n-6 fatty acids. Only fatty acids hydroperoxides induced drastic loss of cellular integrity and severe disturbances in electromechanical activity of cardiomyocytes. 4-hydroxynonenal induced only a slight leak of lactate dehydrogenase at high concentrations and did not modify the electromechanical behavior of cardiac preparations. Under our conditions, monohydroperoxidized fatty acids but not 4-hydroxynonenal induced acute cardiac cell damages. In conclusion, lipid hydroperoxides can be considered both as markers of oxidative injury and relay sources of oxidative stress.

The novel flavones 6-28, which display structural analogies with the two well-known lipid peroxidation inhibitors, probucol [1] and butylated hydroxytoluene [2], were synthesized and studied in vitro for their ability to inhibit the copper sulfate or endothelial cell-induced lipid peroxidation of human low-density lipoprotein (LDL). Most of the flavones were active in the range of 0.1-1 microM.

Objective: As a result of oxidative stress to membrane lipid matrix, the peroxidation of polyunsaturated fatty acids induced the transient formation of lipid hydroperoxides (ROOH). The aim of this study was to evaluate the damaging effects of ROOH on the cardiac cell and the link between the alterations observed and intracellular calcium overload., Methods: Necrosis of cultured rat cardiac cells was determined by measuring the release of lactate dehydrogenase (LDH). In guinea-pig papillary muscles, action potential (AP) and isometric tension were recorded with standard microelectrodes and a transducer, respectively. The reactive oxygen species (ROS) scavenging properties of tested compounds were determined using a cell-free model of lipid photoperoxidation., Results: 15(S)-HpETE (15(S)-hydroperoxyeicosatetraenoic acid), an arachidonic acid hydroperoxide, induced a concentration-dependent loss of cardiomyocytes membrane integrity. The release of LDH induced by 15(s)-HpETE (30 microM) was prevented by a ROS scavenger, BW755C (10 microM), but not by a sarcolemmal calcium channel blocker, Amlodipine (10 microM), or a calcium overload protective agent, R56865 (10 microM). Cardiomyocytes necrosis induced by calcium paradox was prevented by Amlodipine (10 microM) and R56865 (10 microM), but not by BW755C (10 microM). Superfusion of papillary muscles with 15(S)-HpETE (20 microM) induced a membrane depolarization and a marked reduction in the AP amplitude and duration. Concomitantly, a transient positive inotropic effect and a progressive rise in diastolic tension were observed. These alterations were maximal after 15 min and associated with delayed after-depolarizations (DADs) and after-contractions. Every alteration was inhibited by BW755C (10 microM) and R56865 (30 microM), but not by Amlodipine (1 microM). Ryanodine (1 microM), a blocker of sarcoplasmic reticulum calcium channel, only prevented the appearance of DADs and after-contractions. Only BW755C exhibited ROS scavenging properties., Conclusions: ROOH induced enzyme leakage and electromechanical alterations in cardiac cells. These effects of ROOH implicated oxidative mechanisms and resulted in an intracellular calcium overload.

Coexistence of hypertension and lipid disorders enhances the development of atherosclerosis. However it is still unclear whether this promoting effect of hypertension results only from hemodynamic changes or whether part of it is mediated by humoral or neurogenic factors independently of blood pressure alteration. The aim of this study is to determine whether mineralocorticoids, which are known to be involved in the pathogenesis of hypertension, can influence the atherosclerotic process in Watanabe heritable hyperlipidemic rabbits (WHHL) independently of pressure changes. For this purpose, DOCA (200 or 400 mg/kg) or vehicle were implanted subcutaneously for 4 weeks in 3 months old WHHL or New Zealand (NZ) rabbits, without nephrectomy and with a fluid intake solution of 1% NaCl +0.2% KCl. DOCA treatment, independently of hemodynamic changes, significantly increases the size of atherosclerotic lesions in parallel with the aortic cholesterol esters content in the arch and thoracic aorta of WHHL rabbits. Plasmatic and aortic cholesterol and triglyceride content remains unchanged by DOCA treatment. Alteration of endothelial function usually found in WHHL rabbits is accentuated only for the dose of 400 mg/kg. Aortic sensitivity to serotonin is not altered, but the maximal contraction to this agonist is decreased in both strains by mineralocorticoid treatment. These results indicate the importance of non-hemodynamic factors related to hypertension which are implicated also in atherogenesis and support the clinical observations that a reduction of arterial pressure in hypertensive atherosclerotic patients is not sufficient to reduce the progression of this vascular disease.

We investigated the role of the renin-angiotensin system in neointima formation in a species in which converting enzyme inhibitors have been so far ineffective in suppressing abnormal vascular repair. The effects of converting enzyme inhibition by perindopril and selective blockade of angiotensin subtype 1 receptor by DuP 753 were assessed on neointima formation after balloon injury of rabbit carotid artery. Myointimal growth was measured by histomorphometric analysis. In rabbits treated 6 days before and for 14 days after injury, perindopril (2 mg/kg per day PO, n = 7) significantly reduced neointima formation (-51%, P < .01). DuP 753 (1 mg/d, n = 8) infused perivascularly for 14 days in the vicinity of injured carotid artery also markedly suppressed myointimal thickening (-60%, P < .01). To determine whether angiotensin subtype 2 receptor was implicated in this vascular response, we infused CGP 42112A, a specific subtype 2 receptor ligand, continuously for 14 days according to the same protocol of DuP 753 administration. CGP 42112A (1 mg/d) did not change the neointima-media ratio, indicating that angiotensin subtype 2 receptors were not involved in myointimal hyperplasia in rabbits. Thus in rabbits, the renin-angiotensin system plays a major role in neointima formation, and the protective effect of perindopril appears to be mediated mainly by inhibition of angiotensin II production, because blockade of the subtype 1 receptor reduced myointimal growth in a manner similar to that of converting enzyme inhibition and because intracarotid infusion of angiotensin II (500 ng/min) at the site of injury enhanced the vascular response (+39%, P < .05). Bradykinin (500 ng/min) administered in the same conditions as angiotensin II did not modify neointima formation.

Epidemiological studies have indicated that hypertension enhances the development of atherosclerosis in patients with lipid disorders. However, it was still unclear whether this promoting effect resulted only from hemodynamic changes or whether part of it was mediated by humoral or neurogenic factors independent of blood pressure alteration. The aim of this study was to determine whether mineralocorticoids, which are known to be involved in the pathogenesis of hypertension, could be implicated in the atherosclerotic process independent of pressure changes. For this purpose, the effect of deoxycorticosterone (DOCA, 200 mg/kg s.c.) on aortic atherosclerosis was studied in Watanabe heritable hyperlipidemic (WHHL) rabbits in comparison with New Zealand rabbits. After 4 weeks of treatment, DOCA significantly increased the size of atherosclerotic lesions in the arch and thoracic aorta (+115%) in parallel with the aortic cholesterol ester content (+83%). The vascular free cholesterol and triglyceride content remained unchanged on DOCA treatment, as were arterial pressure and plasma cholesterol levels. None of these effects was observed in New Zealand rabbits. DOCA did not accentuate the alteration of endothelial function usually found in WHHL rabbits. The sensitivity to serotonin was not altered, but the maximal contraction to this agonist was decreased in both strains by mineralocorticoid treatment.

1. S 16257 is a new bradycardic agent. Its electropharmacological profile has been compared to that of the known bradycardic compound UL-FS 49 (Zatebradine). Intracellular recordings of action potentials (APs) were performed with conventional glass microelectrodes. 2. In the rabbit isolated sino-atrial node (SAN) tissue, S 16257 and UL-FS 49 (1 microM, 3 microM and 10 microM) were equipotent in slowing spontaneous APs firing predominantly by decreasing the rate of diastolic depolarization (at 3 microM, -23.8 +/- 3.9% and -27.9 +/- 2.6%, respectively). For the two compounds a maximal effect was obtained at 3 microM. In these preparations, action potential duration at 50% of total repolarization (APD50) was more affected by UL-FS 49 than S 16257 at any concentration tested (at 3 microM, +8.9 +/- 2.9% and +29.1 +/- 3.7% for S 16257 and UL-FS 49, respectively; P < or = 0.01). 3. To estimate the direct effects on AP duration, driven cardiac preparations were exposed to these agents. In guinea-pig papillary muscles, paced at a frequency of 1 Hz, increasing concentrations of S 16257 or UL-FS 49 (0.1 to 10 microM, 30 min exposure for each concentration) slightly prolonged AP repolarization. This prolongation was more marked for UL-FS 49 (at 1 microM, +6.1 +/- 0.6% and +11.2 +/- 1.3% elevation of APD50, for S 16257 and UL-FS 49, respectively). 4. Application of UL-FS 49 (3 microM) to rabbit Purkinje fibres, triggered at a frequency of 0.25 Hz, induced a marked prolongation of APD50 and APD90 (+149.4 +/- 51.2% and +86.0 +/- 15.4%, respectively). S 16257 (3 MicroM) induced only a weak prolongation of AP (+ 14.1 +/- 5.0% and + 14.8 +/- 3.3% for APD50 and APD90, respectively) significantly smaller than in the case of UL-FS 49.5. These results show that S 16257 slows the rate of spontaneous AP firing in isolated SAN mainly by a reduction of the diastolic depolarization of the cells, which suggests an inhibition of the pace-maker current (If). S 16257 and UL-FS 49 are equipotent in their bradycardic effect but S 16257 is more specific as it induces less increase in myocardial repolarization time.