Blockade of cannabinoid CB1 receptors improves renal function, metabolic profile, and increased survival of obese Zucker rats.

Obesity is a major risk factor in the development of chronic renal failure. Rimonabant, a cannabinoid CB1 receptor antagonist, improves body weight and metabolic disorders; however, its effect on mortality and chronic renal failure associated with obesity is unknown. Obese Zucker rats received either rimonabant or vehicle for 12 months and were compared to a pair-fed but untreated group of obese rats. Mortality in the obese rats was significantly reduced by rimonabant along with a sustained decrease in body weight, transient reduction in food intake, and an increase in plasma adiponectin. This was associated with significant reduction in plasma total cholesterol, low-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio, triglycerides, glucose, norepinephrine, plasminogen activator inhibitor 1, and preservation of pancreatic weight and beta-cell mass index. The cannabinoid antagonist attenuated the increase in proteinuria, urinary N-acetylglucosaminidase excretion, plasma creatinine, and urea nitrogen levels while improving creatinine clearance. Renal hypertrophy along with glomerular and tubulointerstitial lesions were reduced by rimonabant. Although the drug did not modify hemodynamics, it normalized the pressor response to angiotensin II. Our study suggests that in a rat model of chronic renal failure due to obesity, rimonabant preserves renal function and increases survival.

The anti-obesity effect of rimonabant is associated with an improved serum lipid profile.

We investigated the effects of chronic treatment with the CB1 receptor antagonist rimonabant (10 mg/kg/day p.o. for 10 weeks) in mice with established obesity (5-month high-fat diet). Untreated obese mice showed a weight gain of 46% (45.0 +/- 0.6 g vs. 30.8 +/- 0.5 g) compared with age-matched animals fed a standard diet. Rimonabant treatment, commencing after 5-month high-fat diet, produced a marked and sustained decrease in body weight (34.5 +/- 0.8 g vs. 47.2 +/- 0.5 g in the high-fat vehicle group, p < 0.001). The anti-obesity effect of rimonabant was similar to that obtained by switching obese mice from high-fat diet to standard laboratory diet during 10 weeks (final weight 33.7 +/- 0.6 g) and was associated with only transient (14 days) reduction in energy intake. Serum leptin, insulin and glucose levels were markedly elevated in obese animals. Rimonabant treatment significantly reduced these elevations (leptin -81%, insulin -78%, glucose -67%, p < 0.001 in all cases vs. high-fat vehicle group). In addition, rimonabant treatment modestly but significantly increased serum adiponectin levels (+18%, p < 0.05 vs. high-fat vehicle group). Obese mice demonstrated abnormal serum lipid profiles. Although rimonabant did not modify high-density lipoprotein cholesterol (HDLc) and had modest effects on total cholesterol, it significantly reduced triglycerides and low-density lipoprotein cholesterol (LDLc) and, notably, increased the HDLc/LDLc ratio (12.4 +/- 0.8 vs. 7.9 +/- 0.2 in high-fat vehicle group, p < 0.001). Therefore, in a model of established obesity, chronic rimonabant treatment produces a marked and sustained decrease in body weight (equivalent to that achieved by dietary change) which is associated with favourable modifications in serum biochemical and lipid profiles.

Angiotensin AT1 receptor antagonist irbesartan decreases lesion size, chemokine expression, and macrophage accumulation in apolipoprotein E-deficient mice.

Recent data suggest that angiotensin II AT1 receptor antagonists may be beneficial in the treatment of atherosclerosis. To clarify how AT1 receptor antagonists reduce atherosclerosis, the effect of irbesartan on atherosclerotic lesion development was determined in low-fat, chow-fed apolipoprotein (Apo) E-deficient mice. Irbesartan (50 mg/kg per day) strongly decreased lesion development after a 12-week treatment period (lesion size: irbesartan treated, 20,524 +/- 4,200 microm(2) vs. control, 99,600 +/- 14,500; 79.4% inhibition, p < 0.001). This effect was not due to an effect of irbesartan on lipoprotein levels because irbesartan slightly increased total cholesterol levels and decreased the ratio of Apo A-I relative to Apo B levels. Immunochemical analysis of the atherosclerotic lesions using the mac3 monoclonal antibody showed the presence of macrophages in the lesions of control mice, whereas sections from irbesartan-treated animals only showed occasional labeling in the lesion area. These data suggest that irbesartan inhibits monocyte/macrophage influx into the vessel wall. Therefore, expression levels of monocyte chemoattractant protein-1 (MCP-1), as well as other chemokines involved in macrophage infiltration into the lesion area, were measured in the aortic sinus of control and irbesartan-treated animals. Irbesartan treatment strongly decreased MCP-1 mRNA levels as well as MCP-1 immunostaining in the lesion area. This effect of irbesartan on MCP-1 occurred without an effect on CCR2, the receptor of MCP-1. Expression of macrophage inflammatory protein (MIP)-1alpha, another CC chemokine expressed in atherosclerotic lesions, was also reduced after irbesartan treatment, without effect on CCR3 and CCR5, the receptors of MIP-1alpha. Concomitantly, the expression of the angiogenic chemokines KC and MIP-2, which are functionally related to interleukin-8, were downregulated, whereas their shared receptor CXCR2 was upregulated. These data suggest that inhibition of the inflammatory component of lesion progression plays an important role in the inhibitory effect of AT1 receptor antagonists on atherosclerotic lesion formation.

Cardiovascular effects of SL65.0472, a 5-HT receptor antagonist.

In this study, we describe the cardiovascular effects of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno[3,2-c] pyridin-4-yl) piperazin-l-yl] ethyl]-1, 2-dihydroquinoline-1-acetamide), a novel 5-hydroxytryptamine (5-HT) receptor antagonist developed for the treatment of cardiovascular disease, in several in vivo models. The haemodynamic profile of SL65.0472 was evaluated in anaesthetised dogs. Following i.v. bolus doses of 0.03 mg/kg i.v. and 0.3 mg/kg, no significant changes in cardiac output, contractility or rate, systemic and pulmonary pressures, regional blood flows and vascular resistances or electrocardiogram were noted. After 1 mg/kg i.v. SL65.0472 significantly reduced arterial blood pressure. In conscious spontaneously hypertensive rats administration of SL65.0472 0.5 mg/kg p.o. had no effect on mean arterial blood pressure or heart rate. Vasoconstriction produced by 5-HT results primarily from the stimulation of two receptor subtypes, 5-HT(1B) and 5-HT(2A) receptors. In anaesthetised dogs SL65.0472 antagonised sumatriptan-induced decreases in saphenous vein diameter (5-HT(1B)-receptor mediated) with an ID(50) of 10.1 microg/kg i.v. (95% c.l. 8.3-12.4). In anaesthetised pithed rats SL65.0472 inhibited 5-HT pressor responses (5HT(2A)-receptor mediated) with ID(50) values of 1.38 microg/kg i.v. (95% c.l. 1.15-1.64) and 31.1 microg/kg p.o. (95% c.l. 22.6-42.6). The duration of the 5-HT(2A)-receptor antagonist effect of SL65.0472 following oral administration was evaluated in conscious rats. SL65.0472 (0.1 mg/kg p.o.) markedly inhibited 5-HT pressor responses 1 and 6 h after administration. Therefore, in vivo, SL65.0472 potently antagonises vasoconstriction mediated by 5-HT(1B) and 5-HT(2A) receptors but has minimal direct haemodynamic effects.

Phenotypic and functional changes in regenerated porcine coronary endothelial cells : increased uptake of modified LDL and reduced production of NO.

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.

Hypercholesterolemia increases coronary endothelial dysfunction, lipid content, and accelerated atherosclerosis after heart transplantation.

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.

Impairment of G-protein-mediated signal transduction in the porcine coronary endothelium during rejection after heart transplantation.

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.

Alteration of endothelium-dependent hyperpolarizations in porcine coronary arteries with regenerated endothelium.

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.

Morphological heterogeneity with normal expression but altered function of G proteins in porcine cultured regenerated coronary endothelial cells.

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.

Time course of coronary endothelial dysfunction in acute untreated rejection after heterotopic heart transplantation.

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.

Snaring of the target vessel in less invasive bypass operations does not cause endothelial dysfunction.

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.

Stereospecific in vitro and in vivo effects of the new sinus node inhibitor (+)-S 16257.

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.

Endothelial effects of hemostatic devices for continuous cardioplegia or minimally invasive operations.

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.