New biologically active hydrogen sulfide donors.

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.

Cardiac FKBP12.6 overexpression protects against triggered ventricular tachycardia in pressure overloaded mouse hearts.

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.

Thromboxane A2/prostaglandin H2 receptor activation mediates angiotensin II-induced postischemic neovascularization.

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.

[The discovery of the selective If current inhibitor ivabradine (Procoralan): a new therapeutic approach to ischemic heart disease]. / La découverte de l'ivabradine (Procoralan), inhibiteur sélectif du courant pacemaker If: une nouvelle approche thérapeutique des cardiopathies ischémiques.

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.

SBP, DBP, and pulse blood pressure variability are temporally associated with the increase in pulse wave velocity in a model of aortic stiffness.

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.

Comparison of coronary endothelial dysfunction in the working and nonworking graft in porcine heterotopic heart transplantation.

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.

Consequences of reduced production of NO on vascular reactivity of porcine coronary arteries after angioplasty: importance of EDHF.

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.

Inhibiting the NO pathway with intracoronary L-NAME infusion increases endothelial dysfunction and intimal hyperplasia after heart transplantation.

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.

Surgical experience with retroperitoneal heterotopic heart transplantation in the large white domestic swine.

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.

[Selection and pharmacological characterisation of Procoralan, a selective inhibitor of the pacemaker If current]. / Sélection et caractérisation pharmacologique de Procoralan, un inhibiteur sélectif du courant pacemaker If.

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.

[Dysfunction of the nitric oxide pathway during coronary endothelium regeneration]. / Dysfonction de la voie du monoxyde d'azote au cours de la régénération de l'endothélium coronarien.

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.

Deamidated lipocalin-2 induces endothelial dysfunction and hypertension in dietary obese mice.

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.

Innovation in coronary artery disease and heart failure: clinical benefits of pure heart rate reduction with ivabradine.

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.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen--A novel biomarker of atherosclerotic plaque remodeling.

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.

I(f) inhibition in cardiovascular diseases.

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, ß-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 ß-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.

New insights in the contribution of voltage-gated Na(v) channels to rat aorta contraction.

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.

Effect of uncoupling endothelial nitric oxide synthase on calcium homeostasis in aged porcine endothelial cells.

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.

Status of the endothelium-derived hyperpolarizing factor pathway in coronary arteries after heterotopic heart transplantation.

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.

Chronic mild hyperhomocysteinemia induces aortic endothelial dysfunction but does not elevate arterial pressure in rats.

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.

Anti-ischemic effects of ivabradine, a selective heart rate-reducing agent, in exercise-induced myocardial ischemia in pigs.

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).