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Les antagonistes des récepteurs aux minéralocorticoïdes (MR) appartiennent à l'arsenal thérapeutique pour le traitement de diverses maladies cardiovasculaires, mais les mécanismes conférant leurs effets bénéfiques sont encore mal compris. Une partie de ces effets peuvent être liée à la régulation de l'expression du canal Ca2+ de type L Cav1.2, largement impliqué dans l'insuffisance cardiaque et l'hypertension. Nous montrons que MR fonctionne comme un facteur de transcription transformant le signal de l'aldostérone dans l'utilisation du 'cardiaque' promoteur alternatif P1, dirigeant l'expression du long N-terminal transcrit (Cav1.2-LNT. L'aldostérone augmente de façon concentration- et de temps dépendente l'expression de Cav1.2-LNT dans les cardiomyocytes en raison de l'activation du promoteur P1, par interactions des MR avec des séquences spécifiques de l'ADN sur le promoter P1. Ce mécanisme de cis-régulation induit l'activation de promoteur P1 dans les cellules vasculaires conduisant à une nouvelle signature moléculaire de Cav1.2-LNT associé à une sensibilité réduite aux bloqueurs des canaux Ca2+. Ces résultats révèlent Cav1.2-LNT comme une cible minéralocorticoïde spécifique qui pourrait influencer sur l'éfficacité thérapeutique dans les maladies cardiovasculaires. / The mineralocorticoid receptor (MR) antagonists belong to the current therapeutic armamentarium for the management of cardiovascular diseases, but the mechanisms conferring their beneficial effects are still poorly understood. Part of these MR effects might be related to the L-type Cav1.2 Ca2+ channel expression regulation, critically involved in heart failure and hypertension. Here, we show that MR acts as a transcription factor triggering aldosterone signal into specific alternative 'cardiac' P1-promoter usage, given rise to long (Cav1.2-LNT) N-terminal transcripts. Aldosterone increases Cav1.2-LNT expression in cardiomyocytes in a time- and dose-dependent manner due to MR-dependent P1-promoter activity, through specific DNA sequence-MR interactions. This cis-regulatory mechanism induced a MR-dependent P1-promoter switch in vascular cells leading to a new Cav1.2-LNT molecular signature with reduced Ca2+ channel blocker sensitivity. These findings uncover Cav1.2-LNT as a specific mineralocorticoid target that might influence the therapeutic outcome of cardiovascular diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / Factors released from coronary perivascular adipose tissue (PVAT), which surrounds large coronary arteries, have been implicated in the development of coronary disease. However, the precise contribution of coronary PVAT-derived factors to the initiation and progression of coronary vascular dysfunction remains ill defined. Accordingly, this investigation was designed to delineate the mechanisms by which PVAT-derived factors influence obesity-induced coronary smooth muscle dysfunction. Isometric tension studies of coronary arteries from lean and obese swine demonstrated that both lean and obese coronary PVAT attenuate vasodilation via inhibitory effects on smooth muscle K+ channels. Specifically, lean coronary PVAT attenuated KCa and KV7 channel-mediated dilation, whereas obese coronary PVAT impaired KATP channel-mediated dilation. Importantly, these effects were independent of alterations in underlying smooth muscle function in obese arteries. The PVAT-derived factor calpastatin impaired adenosine dilation in lean but not obese arteries, suggesting that alterations in specific factors may contribute to the development of smooth muscle dysfunction. Further studies tested the hypothesis that leptin, which is expressed in coronary PVAT and is upregulated in obesity, acts as an upstream mediator of coronary smooth muscle dysfunction. Long-term administration (3 day culture) of obese concentrations of leptin markedly altered the coronary artery proteome, favoring pathways associated with calcium signaling and cellular proliferation. Isometric tension studies demonstrated that short-term (30 min) exposure to leptin potentiated depolarization-induced contraction of coronary arteries and that this effect was augmented following longer-term leptin administration (3 days). Inhibition of Rho kinase reduced leptin-mediated increases in coronary artery contractions. Acute treatment was associated with increased Rho kinase activity, whereas longer-term exposure was associated with increases in Rho kinase protein abundance. Alterations in Rho kinase signaling were also associated with leptin-mediated increases in coronary vascular smooth muscle proliferation. These findings provide novel mechanistic evidence linking coronary PVAT with vascular dysfunction and further support a role for coronary PVAT in the pathogenesis of coronary disease.

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Spiral-artery (SA) remodeling is a fundamental process during pregnancy that involves the action of cells of the initial vessel, such as vascular smooth-muscle cells (VSMCs) and endothelial cells, but also maternal immune cells and fetal extravillous trophoblast cells (EVTs). Mast cells (MCs), and specifically chymase-expressing cells, have been identified as key to a sufficient SA remodeling process in vivo. However, the mechanisms are still unclear. The purpose of this study is to evaluate the effects of the MC line HMC-1 and recombinant human chymase (rhuCMA1) on human primary uterine vascular smooth-muscle cells (HUtSMCs), a human trophoblast cell line (HTR8/SV-neo), and human umbilical-vein endothelial cells (HUVEC) in vitro. Both HMC-1 and rhuCMA1 stimulated migration, proliferation, and changed protein expression in HUtSMCs. HMC-1 increased proliferation, migration, and changed gene expression of HTR8/SVneo cells, while rhuCMA treatment led to increased migration and decreased expression of tissue inhibitors of matrix metalloproteinases. Additionally, rhuCMA1 enhanced endothelial-cell-tube formation. Collectively, we identified possible mechanisms by which MCs/rhuCMA1 promote SA remodeling. Our findings are relevant to the understanding of this crucial step in pregnancy and thus of the dysregulated pathways that can lead to pregnancy complications such as fetal growth restriction and preeclampsia.

Atherosclerosis is a macrophage-dominated nonresolving inflammatory disease of the arterial wall. Macrophage processes, including apoptosis, influence lesion development in atherosclerosis. Cannabinoids, compounds structurally related to Δ9-tetrahydrocannabinol (THC), the active ingredient in marijuana, exert their effects through cannabinoid receptors, CB1 and CB2. Cannabinoid treatment, THC or Win55,212-2, reduces atherosclerosis in ApoE-null mice by a mechanism thought to involve CB2. However, the exact role of CB2 in atherosclerosis remains unclear. We found that CB2-null macrophages are resistant to oxysterol/oxLDL-induced apoptosis leading us to hypothesize that CB2 may modulate macrophage apoptosis in atherosclerosis. To determine the functions of CB2 in atherosclerosis, we fed low density lipoprotein receptor-null (Ldlr-/-) and Ldlr-/- mice genetically deficient in CB2, an atherogenic diet for 8 and 12 weeks. CB2 deficiency did not significantly affect aortic root lesion area after 8 or 12 weeks; however, after 12 weeks, CB2-deficient lesions displayed increased lesional macrophage and smooth muscle cell (SMC) content and a ~2-fold reduction in lesional apoptosis. CB2-deficienct lesions also displayed reduced collagen content and elevated elastin fiber fragmentation that was associated with elevated levels of the extracellular matrix degrading enzyme, matrix metalloproteinase 9 (MMP9). These results demonstrate that although CB2 signaling does not affect atherosclerotic lesion size it does modulate lesional apoptosis, cellularity and ECM composition. Ldlr-/- and CB2-deficient Ldlr-/- mice were also subjected to daily treatments with Win55,212-2, a synthetic cannabinoid, over the last 2 weeks of an 8 week atherogenic diet to identify CB2-dependent and CB2-independent effects of cannabinoid receptor stimulation on atherosclerosis. Win55,212-2 did not affect hypercholesterolemia, aortic root lesion area, lesional macrophage infiltration, or ECM composition in either genotype but did significantly reduce total plasma triglyceride levels and lesional SMC content, independent of CB2. Surprisingly, lesional apoptosis was dose-dependently repressed by Win55,212-2 in Ldlr-/- mice by a CB2-dependent mechanism. All together, these results support the suggestion that CB2 may be a target for novel therapies aimed at modulating lesional apoptosis and cellularity to increase lesion stability and reduce the vulnerability to rupture.

Peripheral Artery Disease is a very serious disease characterized by an arterial occlusion due to atherosclerotic plaques. In response to an arterial occlusion, arteriogenesis occurs, causing smooth muscle cells to transition from a contractile to synthetic state. Also following an arterial occlusion, functional impairment was seen in the collateral circuit. An immunofluorescence protocol was developed in order to assess the impact of collateral enlargement (arteriogenesis) on smooth muscle phenotype at various time points. Smooth muscle α-actin was used to mark all smooth muscle cells, Ki-67 was used to label proliferating smooth muscle cells, and a fluorescent nuclear stain was used to quantify the number of cells present. Samples of the profunda femoris and gracilis were dissected from each mouse hind limb (one ligated, one sham) at three different time points: 3 days, 7 days, and 14 days after a femoral artery ligation surgery. Smooth muscle cell phenotype and luminal cross-sectional area were assessed in the profunda femoris and the midzone of the gracilis collaterals. Smooth muscle cells were proliferating at 3 and 7 days following the occlusion in the gracilis collaterals and significant collateral vessel growth was observed over the two week period. No proliferation was observed in the profunda femoris and although there was an increasing trend in vessel size over the two week period, the averages were not significantly different. The phenotypic transition of the smooth muscle cells was not the cause of vascular impairment in the collateral circuit. This shows that further research is needed to characterize impairment in the collateral circuit.

Objectives: The mucosa of human detrusor strips impairs catecholamine-induced relaxation. In order to elucidate which signal transduction pathways are involved in this cross talk between the mucosa and detrusor, we have studied the effects of several pharmacological agonists and antagonists on noradrenaline-mediated relaxation in intact and mucosa-denuded detrusor strips. Patients and Methods: Strips of detrusor tissue were obtained from patients who had undergone cystectomy for bladder cancer and were set up for force measurement. KCl- or carbachol-precontracted strips were relaxed with increasing concentrations of noradrenaline in the absence and in the presence of nitric oxide synthase inhibitor, L-NAME; P2X-receptor antagonist, PPADS; ET A -receptor antagonist, BQ-123; ET B -receptor antagonist, BQ-788; cyclooxygenase inhibitor, diclofenac; AT 1 -receptor antagonist, candesartan; and NK 1 -receptor antagonist, L-703,606. Results: In intact strips, KCl-stimulated force was enhanced by all blockers; carbachol-stimulated force increased with L-703,606. In denuded strips, only L-NAME augmented the KCl-stimulated contraction. Noradrenaline relaxed the precontracted detrusor strips to a significantly larger extent and at lower concentrations in denuded than in intact strips. L-NAME, PPADS and BQ-123/BQ-788 had little effect on noradrenaline-induced relaxation, whereas diclofenac, candesartan and L-703,606 sensitized intact carbachol-stimulated detrusor strips to noradrenaline-induced relaxation. Conclusion: Inhibition of the noradrenaline-induced relaxation of precontracted human detrusor strips by the mucosa is attenuated by diclofenac, candesartan and L-703,606 suggesting the involvement of prostanoids, angiotensin and neurokinin pathways. Further experiments are required to unravel the exact mechanisms.

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No description available.