Search

International audience; Purpose. Asthma is a frequent chronic inflammatory bronchial disease affecting more than 300 million patients worldwide, 70% of whom are secondary to allergy. The diversity of asthmatic endotypes contributes to their complexity. The inter-relationship between allergen and other exposure and the airway microbiome adds to the phenotypic diversity and defines the natural course of asthma. Here, we compared the mouse models of house dust mite (HDM)-induced allergic asthma. Allergic sensitization was performed via various routes and associated with outcomes.Methods: Mice were sensitized with HDM via the oral, nasal or percutaneous routes. Lung function, barrier integrity, immune response and microbiota composition were analyzed.Results: Severe impairment of respiratory function was observed in the mice sensitized by the nasal and cutaneous paths. It was associated with epithelial dysfunction characterized by an increased permeability secondary to junction protein disruption. Such sensitization paths induced a mixed eosinophilic and neutrophilic inflammatory response with high interleukin (IL)-17 airway secretion. In contrast, orally sensitized mice showed a mild impairment of respiratory function. Epithelial dysfunction was mild with increased mucus production, but preserved epithelial junctions. Regarding lung microbiota, sensitization provoked a significant loss of diversity. At the genus level, Cutibacterium, Acinetobacter, Streptococcus and Lactobacillus were found to be modulated according to the sensitization pathway. An increase in theanti-inflammatory microbiota metabolites was observed in the oral-sensitization group.Conclusions: Our study highlights the strong impact of the sensitization route on the pathophysiology and the critical phenotypic diversity of allergic asthma in a mouse model.

How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here, we examined how extracellular matrix (ECM) rigidity affects cell division. We found that cells divide more rapidly when cultured on rigid substrates. While we observed no effect of ECM rigidity on rounding or postmitotic spreading duration, we found that changes in matrix stiffness impact mitosis progression. We noticed that ECM elasticity up-regulates the expression of the linker of nucleoskeleton and cytoskeleton (LINC) complex component SUN2, which in turn promotes metaphase-to-anaphase transition by acting on mitotic spindle formation, whereas when cells adhere to soft ECM, low levels of SUN2 expression perturb astral microtubule organization and delay the onset of anaphase.

Hemodynamic forces play an important role in vascular network development and homeostasis. In physiological condition, shear stress generated by laminar flow promotes endothelial cells (EC) health and induces their alignment in the direction of flow. In contrast, altered hemodynamic forces induce endothelial dysfunction and lead to the development of vascular disorders such as atherosclerosis and aneurysms. Following mechano-sensor activation, Rho protein-mediated cytoskeletal rearrangement is one of the first steps in transforming flow-induced forces into intracellular signals in EC via guanine nucleotide exchange factors (RhoGEFs) that mediate the spatio-temporal activation of these Rho proteins. Here we identified ARHGEF18 as a flow-sensitive RhoGEF specifically activating RhoA. Both ARHGEF18 expression and activity were controlled by shear stress level. ARHGEF18 promotes EC adhesion, focal adhesion formation and migration. ARHGEF18 localized to the tight junction by interacting with ZO-1 and participated to shear stress-induced EC elongation and alignment via its nucleotide exchange activity and the activation of p38 MAPK. Our study therefore characterized ARHGEF18 as the first flow-sensitive RhoA GEF in ECs, whose activity is essential for the maintenance of intercellular junctions and a properly organized endothelial monolayer under physiological flow conditions.

Asthma is a frequent respiratory condition whose pathophysiology relies on altered interactions between bronchial epithelium, smooth muscle cells (SMC) and immune responses. Those leads to classical hallmarks of asthma: airway hyper-responsiveness, bronchial remodelling and chronic inflammation. Airway smooth muscle biology and pathophysiological implication in asthma are now better understood. Precise deciphering of intracellular signalling pathways regulating smooth muscle contraction highlighted the critical roles played by small GTPases of Rho superfamily. Beyond contractile considerations, active involvement of airway smooth muscle in bronchial remodelling mechanisms is now established. Not only cytokines and growth factors, such as fibroblats growth factor or transforming growth factor-β, but also extracellular matrix composition have been demonstrated as potent phenotype modifiers for airway SMC. Although basic science knowledge has grown significantly, little of it has translated into improvement in asthma clinical practice. Evaluation of airway smooth muscle function is still limited to its contractile activity. Moreover, it relies on tools, such as spirometry, that give only an overall assessment and not a specific one. Interesting technics such as forced oscillometry or specific imagery (CT and MRI) give new perspectives to evaluate other aspects of airway muscle such as bronchial remodelling. Finally, except for the refinement of conventional bronchodilators, no new drug therapy directly targeting airway smooth muscle proved its efficacy. Bronchial thermoplasty is an innovative and efficient therapeutic strategy but is only restricted to a small proportion of severe asthmatic patients. New diagnostic and therapeutic strategies specifically oriented toward airway smooth muscle are needed to improve global asthma care.

International audience; Background and purpose: Pulmonary hypertension (PH) is a multifactorial chronic disease characterized by an increase in pulmonary artery (PA) resistance leading to right ventricle (RV) failure. Endothelial dysfunction and alteration of NO/cGMP signaling in PA play a major role in PH. We recently described the involvement of the Rho protein Rac1 in the control of systemic blood pressure through its involvement in NO-mediated relaxation of arterial smooth muscle cell (SMC). The aim of this study was thus to analyze the role of SMC Rac1 in PH.Experimental approach: PH is induced by exposure of control and SMC Rac1-deficient (SM-Rac1-KO) mice to chronic hypoxia (10% O2 , 4 weeks). PH is assessed by the measurement of RV systolic pressure and hypertrophy. PA reactivity is analyzed by isometric tension measurements. PA remodeling is quantified by immunofluorescence in lung sections and ROS are detected using the DHE probe and electronic paramagnetic resonance analysis. Rac1 activity is determined by immunofluorescence.Key results: Rac1 activation is observed in PA of hypoxic mice and patients with idiopathic PH. Hypoxia-induced rise in RV systolic pressure, RV hypertrophy and loss of endothelium-dependent relaxation were significantly decreased in SM-Rac1-KO mice compared to control mice. SMC Rac1 deletion also limited hypoxia-induced PA remodeling and ROS production in PASMCs.Conclusion and implications: Our results provide evidence for a protective effect of SM Rac1 deletion against hypoxic PH. Rac1 activity in PASMCs plays a causal role in PH by favoring ROS-dependent PA remodeling and endothelial dysfunction induced by chronic hypoxia.

BackgroundGeometrical parameters, including arterial bifurcation angle, tortuosity, and arterial diameters, have been associated with the pathophysiology of intracranial aneurysm (IA) formation. The aim of this study was to investigate whether these parameters were present before or if they resulted from IA formation and growth.MethodsPatients from nine academic centers were retrospectively identified if they presented with a de novo IA or a significant IA growth on subsequent imaging. For each patient, geometrical parameters were extracted using a semi-automated algorithm and compared between bifurcations with IA formation or growth (aneurysmal group), and their contralateral side without IA (control group). These parameters were compared at two different times using univariable models, multivariable models, and a sensitivity analysis with paired comparison.Results46 patients were included with 21 de novo IAs (46%) and 25 significant IA growths (54%). The initial angle was not different between the aneurysmal and control groups (129.7±42.1 vs 119.8±34.3; p=0.264) but was significantly wider at the final stage (140.4±40.9 vs 121.5±34.1; p=0.032), with a more important widening of the aneurysmal angle (10.8±15.8 vs 1.78±7.38; p=0.001). Variations in other parameters were not significant. These results were confirmed by paired comparisons.ConclusionOur study suggests that wider bifurcation angles that have long been deemed causal factors for IA formation or growth may be secondary to IA formation at pathologic bifurcation sites. This finding has implications for our understanding of IA formation pathophysiology.

Small molecule inhibitors of GTPases are increasingly considered for the treatment of multiple human pathologies. The GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1) plays major roles in vital cellular processes, notably in the control cell motility and dynamic, the regulation of oxidative stress, and in inflammatory and immune surveillance. As such, Rac1 is viewed as a potential target to combat cancers but also diverse inflammatory, metabolic, neurodegenerative, respiratory, cardiovascular, viral, and parasitic diseases. Potent and selective Rac1 inhibitors have been identified and designed, such as compounds GYS32661 and MBQ-167 both in preclinical development for the treatment of advanced solid tumors. The pleiotropic roles and ubiquitous expression of the protein can be viewed as limitations for anticancer approaches. However, the frequent overexpression and/or hyperactivation of the Rac1 in difficult-to-treat chemoresistant cancers, make Rac1 an attractive target in oncology. The key roles of Rac1 in multiple cellular pathways, together with its major implications in carcinogenesis, tumor proliferation and metastasis, support the development of small molecule inhibitors. The challenge is high and the difficulty shall not be underestimated, but the target is innovative and promising in combination with chemo- and/or immuno-therapy. Opportunities and challenges associated with the targeting of Rac1 are discussed.

International audience; Various 3-amino-, 3-aryloxy- and alkoxy-6-arylpyridazines have been synthesized by an electrochemical reductive cross-coupling between 3-amino-, 3-aryloxy- or 3-alkoxy-6-chloropyridazines and aryl or heteroaryl halides. In vitro antiproliferative activity of these products was evaluated against a representative panel of cancer cell lines (HuH7, CaCo-2, MDA-MB-231, HCT116, PC3, NCI-H727, HaCaT) and oncogenicity prevention of the more efficient derivatives was highlighted on human breast cancer cell line MDA-MB 468-Luc prior establishing their interaction with p44/42 and Akt-dependent signaling pathways.

International audience; Asthma is often associated with a Th2-type immune response with well-known cellular and molecular actors such as eosinophils, Th2 lymphocytes and associated cytokines such as interleukin-5 or IL-4. Nevertheless, some of the asthmatic patients show clinical manifestations and characteristics that do not correspond to the current pattern of the pathophysiology of asthma. Thus, recently new cellular and molecular actors in the development of asthma have been demonstrated in animal models and in humans. Among these are components of the innate immune system such as type 2 innate lymphoid cells or adaptive immune system such as Th9 lymphocytes. At the cellular level, the role of small G proteins in asthma is also highlighted as well as the role of major cytokines like IL-17 or those derived from the epithelium. A better knowledge of the physiopathology of asthma and the taking into account of these new actors allows the identification of new therapeutic targets for different endotypes of patients.

Background and purposeThe ever-growing availability of imaging led to increasing incidentally discovered unruptured intracranial aneurysms (UIAs). We leveraged machine-learning techniques and advanced statistical methods to provide new insights into rupture intracranial aneurysm (RIA) risks.MethodsWe analysed the characteristics of 2505 patients with intracranial aneurysms (IA) discovered between 2016 and 2019. Baseline characteristics, familial history of IA, tobacco and alcohol consumption, pharmacological treatments before the IA diagnosis, cardiovascular risk factors and comorbidities, headaches, allergy and atopy, IA location, absolute IA size and adjusted size ratio (aSR) were analysed with a multivariable logistic regression (MLR) model. A random forest (RF) method globally assessed the risk factors and evaluated the predictive capacity of a multivariate model.ResultsAmong 994 patients with RIA (39.7%) and 1511 patients with UIA (60.3 %), the MLR showed that IA location appeared to be the most significant factor associated with RIA (OR, 95% CI: internal carotid artery, reference; middle cerebral artery, 2.72, 2.02–3.58; anterior cerebral artery, 4.99, 3.61–6.92; posterior circulation arteries, 6.05, 4.41–8.33). Size and aSR were not significant factors associated with RIA in the MLR model and antiplatelet-treatment intake patients were less likely to have RIA (OR: 0.74; 95% CI: 0.55–0.98). IA location, age, following by aSR were the best predictors of RIA using the RF model.ConclusionsThe location of IA is the most consistent parameter associated with RIA. The use of ‘artificial intelligence’ RF helps to re-evaluate the contribution and selection of each risk factor in the multivariate model.

Background Severe asthma is a chronic lung disease characterised by inflammation, airway hyperresponsiveness (AHR) and airway remodelling. The molecular mechanisms underlying uncontrolled airway smooth muscle cell (aSMC) proliferation involved in pulmonary remodelling are still largely unknown. Small G proteins of the Rho family (RhoA, Rac1 and Cdc42) are key regulators of smooth muscle functions and we recently demonstrated that Rac1 is activated in aSMC from allergic mice. The objective of this study was to assess the role of Rac1 in severe asthma-associated airway remodelling. Methods and results Immunofluorescence analysis in human bronchial biopsies revealed an increased Rac1 activity in aSMC from patients with severe asthma compared with control subjects. Inhibition of Rac1 by EHT1864 showed that Rac1 signalling controlled human aSMC proliferation induced by mitogenic stimuli through the signal transducer and activator of transcription 3 (STAT3) signalling pathway. In vivo, specific deletion of Rac1 in SMC or pharmacological inhibition of Rac1 by nebulisation of NSC23766 prevented AHR and aSMC hyperplasia in a mouse model of severe asthma. Moreover, the Rac1 inhibitor prevented goblet cell hyperplasia and epithelial cell hypertrophy whereas treatment with corticosteroids had less effect. Nebulisation of NSC23766 also decreased eosinophil accumulation in the bronchoalveolar lavage of asthmatic mice. Conclusion This study demonstrates that Rac1 is overactive in the airways of patients with severe asthma and is essential for aSMC proliferation. It also provides evidence that Rac1 is causally involved in AHR and airway remodelling. Rac1 may represent as an interesting target for treating both AHR and airway remodelling of patients with severe asthma.

Introduction Pulmonary arterial hypertension (PAH) is a chronic disease characterized by an increase in pulmonary artery (PA) resistance leading to right ventricle (RV) failure. Endothelial dysfunction and alteration of NO/cGMP signaling play a major role in PAH. We previously described the major role of smooth muscle cell (SMC) Rac1 in NO-induced dilation of systemic arteries. Objective Here, our aim was to analyze the role of SMC Rac1 in pulmonary circulation and in PAH. Method Rac1 activity was assessed by immunofluorescence in lung biopsies. PAH has been induced by exposure of wild-type (WT) and SMC Rac1-deficient mice (SM-Rac1-KO mice) to chronic hypoxia (10% O2, 4 weeks). In vitro, the proliferation of PASMC in hypoxic condition was measured by EdU staining and contractility of PA rings was analyzed by myography. Oxidative stress was assessed using the DHE probe and by electron paramagnetic resonance (EPR). Results A 3-fold increase of Rac-GTP in PA was observed in PAH patients compared to control (n = 4-6). To determine whether Rac1 activation plays a causal role in PAH, WT and SM-Rac1-KO mice were exposed to hypoxia. As expected, exposure to chronic hypoxia induced an increase in systolic pressure in RV and RV hypertrophy which were reduced by 32.5% and 60% respectively, in SM-Rac1-KO mice compared to WT (n = 5–7). PA remodeling was also significantly reduced in hypoxic SM-Rac1-KO compared to WT mice, while PA contractility was not affected by SMC Rac1 deletion. In vitro, exposure of primary PASMC to hypoxia stimulated proliferation and ROS production that were decreased by 50% and 66%, respectively, by the Rac1 inhibitor, EHT1864. Conclusion Our results show that SMC Rac1 activity is increased in PA of PAH patients and plays a causal role in hypoxia-induced PAH in mice by stimulating ROS-dependent PA remodeling. These data suggest that pharmacological Rac1 inhibition might have a beneficial effect in the treatment of PAH.

Aims The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. Methods and results Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. Conclusion This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.

International audience; An aneurysm is a vascular disorder represented by a ballooning of a blood vessel. The blood vessel's wall is distorted by the blood flow, and a bulge forms there. When ruptured, the aneurysm may cause irreversible damage and could even lead to premature death. Intra-cranial aneurysms are the ones presenting the higher risks. In this work, thanks to a graph based approach, we detect the bifurcations located on the circle of Willis within brain mice cerebral vasculature. Once properly located in the 3D stack, we characterize the cerebral arteries bifurcations, i.e. we gather several properties of the bifurcation, such as their angles, or area cross section, in order to further estimate geometrical patterns that can favor the risk of occurrence of an intra-cranial aneurysm. Effectively, apart from genetic predisposition, and environmental risk factors (high blood pressure, smoking habits, ...) the anatomical disposition of the brain vasculature may influence the chances of an aneurysm to form. Our objectives in this paper is to obtain accurate measurements on the 3D bifurcations.

Vascular aging is characterized by functional and structural changes of the vessel wall, including endothelial dysfunction, with decreased endothelial NO⋅ bioavailability and elevated vasoconstrictor and inflammatory mediator production, vascular rigidity, and tone impairment. Moringa oleifera (MOI) is a little tree, and different parts of which are used in traditional medicine in tropical Africa, America, and Asia for therapeutic applications in several disorders including cardiovascular disease. The present study is aimed at assessing the effect of MOI on aging-associated alteration of the endothelial function in Wistar rats. Middle-aged Wistar rats (46-week-old males) have been fed with food containing or not 750 mg/kg/day of MOI seed powder for 4 weeks. A group of young Wistar rats (16-week-old) was used as control. Measurement of isometric contraction, western blot analysis, and immunostaining has then been performed in the aortas and mesenteric arteries to assess the endothelium function. MOI treatment improved carbachol-induced relaxation in both aortas and mesenteric arteries of middle-aged rats. In the aortas, this was associated with an increased Akt signalling and endothelial NO synthase activation and a downregulation of arginase-1. In the mesenteric arteries, the improvement of the endothelial-dependent relaxation was related to an EDHF-dependent mechanism. These results suggest a vascular protective effect of MOI seeds against the vascular dysfunction that develops during aging through different mechanisms in conductance and resistance arteries.

Asthma is often associated with a Th2-type immune response with well-known cellular and molecular actors such as eosinophils, Th2 lymphocytes and associated cytokines such as interleukin-5 or IL-4. Nevertheless, some of the asthmatic patients show clinical manifestations and characteristics that do not correspond to the current pattern of the pathophysiology of asthma. Thus, recently new cellular and molecular actors in the development of asthma have been demonstrated in animal models and in humans. Among these are components of the innate immune system such as type 2 innate lymphoid cells or adaptive immune system such as Th9 lymphocytes. At the cellular level, the role of small G proteins in asthma is also highlighted as well as the role of major cytokines like IL-17 or those derived from the epithelium. A better knowledge of the physiopathology of asthma and the taking into account of these new actors allows the identification of new therapeutic targets for different endotypes of patients.

Introductionn: Severe asthma is a chronic lung disease characterized by inflammation, airway hyperresponsiveness (AHR), airway remodelling and corticoid resistance. The molecular mechanisms underlying airway smooth muscle cells (aSMC) contraction and proliferation involved in AHR and remodelling are still largely unknown. Small GTPases of the Rho family (RhoA, Rac1 and Cdc42) are key regulators of SM functions. Recently, we demonstrated that Rac1 controls bronchoconstriction by modulating aSMC contraction (Andre et al., JACI, 2017). The objective of this study was to assess the role of SM Rac1 in severe asthma-associated airway remodelling. Methods and Results: We developed a new model of severe allergic asthma (house dust mite (HDM)-sensitized mice) characterized by AHR, strong airway remodelling with SMC hyperplasia and a reduced sensitivity to inhaled corticoids. In mice harbouring SMC specific Rac1 deletion, AHR and aSMC hyperplasia were not observed. A similar inhibition of AHR and aSMC hyperplasia was obtained by the pharmacological inhibition of Rac1 by nebulisation of NSC23766. Moreover, the Rac1 inhibitor prevented goblet cell hyperplasia and epithelial cell hypertrophy whereas corticoids treatments had no effect. Nebulisation of NSC23766 also decreased eosinophil and neutrophil populations in bronchoalveolar lavages from HDM sensitized asthmatic mice. Conclusion: In this study, we generated a new mouse model of severe allergic asthma that closely mimics the human pathology. We demonstrated that Rac1 plays an essential role in aSMC contraction and hyperplasia, and that its inhibition prevents AHR and airway remodelling associated with severe asthma.

International audience; Intracranial aneurysms (IAs) are acquired cerebrovascular abnormalities characterized by localized dilation and wall thinning in intracranial arteries, possibly leading to subarachnoid hemorrhage and severe outcome in case of rupture. Here, we identified one rare nonsense variant (c.1378A>T) in the last exon of ANGPTL6 (Angiopoietin-Like 6)—which encodes a circulating pro-angiogenic factor mainly secreted from the liver—shared by the four tested affected members of a large pedigree with multiple IA-affected case subjects. We showed a 50% reduction of ANGPTL6 serum concentration in individuals heterozygous for the c.1378A>T allele (p.Lys460Ter) compared to relatives homozygous for the normal allele, probably due to the non-secretion of the truncated protein produced by the c.1378A>T transcripts. Sequencing ANGPTL6 in a series of 94 additional index case subjects with familial IA identified three other rare coding variants in five case subjects. Overall, we detected a significant enrichment (p = 0.023) in rare coding variants within this gene among the 95 index case subjects with familial IA, compared to a reference population of 404 individuals with French ancestry. Among the 6 recruited families, 12 out of 13 (92%) individuals carrying IA also carry such variants in ANGPTL6, versus 15 out of 41 (37%) unaffected ones. We observed a higher rate of individuals with a history of high blood pressure among affected versus healthy individuals carrying ANGPTL6 variants, suggesting that ANGPTL6 could trigger cerebrovascular lesions when combined with other risk factors such as hypertension. Altogether, our results indicate that rare coding variants in ANGPTL6 are causally related to familial forms of IA.

Rho-associated kinases ROCK1 and ROCK2 are key regulators of actin cytoskeleton dynamics downstream of Rho GTPases that participate in the control of important physiologic functions, S including cell contraction, migration, proliferation, adhesion, and inflammation. Several excellent review articles dealing with ROCK function and regulation have been published over the past few years. Although a brief overview of general molecular, biochemical, and functional properties of ROCKs is included, an effort has been made to produce an original work by collecting and synthesizing recent studies aimed at translating basic discoveries from cell and experimental models into knowledge of human physiology, pathophysiological mechanisms, and medical therapeutics. This review points out the specificity and distinct roles of ROCK1 and ROCK2 isoforms highlighted in the last few years. Results obtained from genetically modified mice and genetic analysis in humans are discussed. This review also addresses the involvement of ROCKs in human diseases and the potential use of ROCK activity as a biomarker or a pharmacological target for specific inhibitors.

Background and purpose The early management of the cardiovascular dysfunction of septic shock is critical as it is associated with a poor outcome. Although the use of catecholamines is a common therapy in this syndrome, no data are available on the involvement of β-adrenoceptor (β-AR) subtypes and only few studies report an alteration of β-adrenergic-induced vasodilation in septic shock. The purpose of the study was to evaluate vascular β 1 , β 2 and β 3 -AR expression and function in an endotoxemic rat model. Experimental approach Endotoxemia was induced in rats by intravenous injection of lipopolysaccharide (LPS). β 1 , β 2 and β 3 -AR mRNA expression was evaluated by RT-PCR in aorta and vascular β 1 , β 2 and β 3 -AR responses were determined on conducting (aorta) and/or resistance (mesenteric and renal) arteries by constructing relaxation curves in response to different β-AR agonists. Results The maximal effect of isoproterenol decreased by 31 to 61% in the three vascular beds of LPS-treated rats compared to controls. In aortas from LPS-treated rats, β 1 and β 3 -AR mRNA expression was decreased and associated to a reduced β 1 and β 3 -induced vasodilation. Conversely, albeit β 2 -AR mRNA was unchanged, the maximal β 2 -AR-induced vasodilation increased by 49% in aortas from LPS-treated rats compared to controls. This increase was not affected by endothelium removal but was abolished in the presence of a β 2 -AR antagonist or an adenylate cyclase inhibitor. Conclusions In endotoxemia, β 2 -AR vasodilation was increased by a potential recruitment of β 2 -AR located on smooth muscle cells. This study suggests that vascular β 2 -AR should be a putative new therapeutic target in septic shock.

International audience; AKI is a frequent complication in hospitalized patients. Unfortunately, there is no effective pharmacologic approach for treating or preventing AKI. In rodents, mineralocorticoid receptor (MR) antagonism prevents AKI induced by ischemia-reperfusion (IR). We investigated the specific role of vascular MR in mediating AKI induced by IR. We also assessed the protective effect of MR antagonism in IR-induced AKI in the Large White pig, a model of human AKI. In mice, MR deficiency in smooth muscle cells (SMCs) protected against kidney IR injury. MR blockade by the novel nonsteroidal MR antagonist, finerenone, or genetic deletion of MR in SMCs associated with weaker oxidative stress production. Moreover, ischemic kidneys had higher levels of Rac1-GTP, required for NADPH oxidase activation, than sham control kidneys, and genetic deletion of Rac1 in SMCs protected against AKI. Furthermore, genetic deletion of MR in SMCs blunted the production of Rac1-GTP after IR. Pharmacologic inhibition of MR also prevented AKI induced by IR in the Large White pig. Altogether, we show that MR antagonism, or deletion of the MR gene in SMCs, limited the renal injury induced by IR through effects on Rac1-mediated MR signaling. The benefits of MR antagonism in the pig provide a rational basis for future clinical trials assessing the benefits of this approach in patients with IR-mediated AKI.

International audience; BACKGROUND: Understanding the pathophysiologic mechanism of intracranial aneurysm (IA) formation is a prerequisite to assess the potential risk of rupture. Nowadays, there are neither reliable biomarkers nor diagnostic tools to predict the formation or the evolution of IA. Increasing evidence suggests a genetic component of IA but genetics studies have failed to identify genetic variation causally related to IA.OBJECTIVE: To develop diagnostic and predictive tools for the risk of IA formation and rupture.METHODS: The French ICAN project is a noninterventional nationwide and multicentric research program. Each typical IA of bifurcation will be included. For familial forms, further IA screening will be applied among first-degree relatives. By accurate phenotype description with high-throughput genetic screening, we aim to identify new genes involved in IA. These potential genetic markers will be tested in large groups of patients. Any relevant pathway identified will be further explored in a large cohort of sporadic carriers of IA, which will be well documented with clinical, biological, and imaging data.EXPECTED OUTCOMES: Discovering genetic risk factors, better understanding the pathophysiology, and identifying molecular mechanisms responsible for IA formation will be essential bases for the development of biomarkers and identification of therapeutic targets.DISCUSSION: Our protocol has many assets. A nationwide recruitment allows for the inclusion of large pedigrees with familial forms of IA. It will combine accurate phenotyping and comprehensive imaging with high-throughput genetic screening. Last, it will enable exploiting metadata to explore new pathophysiological pathways of interest by crossing clinical, genetic, biological, and imaging information.

International audience; Advanced glycation end-products (AGEs) are associated with many pathogenic disorders such as pathogenesis of diabetes or endothelial dysfunction leading to cardiovascular events. Therefore, the identification of new anti-AGE molecules or extracts aims at preventing such pathologies. Many Clusiaceae and Calophyllaceae species are used in traditional medicines to treat arterial hypertension as well as diabetes. Focusing on these plant families, an anti-AGE plant screening allowed us to select Mammea neurophylla for further phytochemical and biological studies. Indeed, both DCM and MeOH stem bark extracts demonstrated in vitro their ability to prevent inflammation in endothelial cells and to reduce vasoconstriction. A bioguided fractionation of these extracts allowed us to point out 4-phenyl- and 4-(1-acetoxypropyl)coumarins and procyanidins as potent inhibitors of AGE formation, potentially preventing endothelial dysfunction. The fractionation steps also led to the isolation of two new compounds, namely neurophyllols A and B from the DCM bark extract together with thirteen known mammea A and E coumarins (mammea A/AA, mammea A/AB, mammea A/BA, mammea A/BB, mammea A/AA cycloD, mammea A/AB cycloD, disparinol B, mammea A/AB cycloE, ochrocarpin A, mammea A/AA cycloF, mammea A/AB cycloF, mammea E/BA, mammea E/BB) as well as δ-tocotrienol, xanthones (1-hydroxy-7-methoxyxanthone, 2-hydroxyxanthone) and triterpenes (friedelin and betulinic acid). During this study, R,S-asperphenamate, previously described from fungal origin was also purified.

Background The molecular mechanisms responsible for airway smooth muscle cells' (aSMCs) contraction and proliferation in airway hyperresponsiveness (AHR) associated with asthma are still largely unknown. The small GTPases of the Rho family (RhoA, Rac1, and Cdc42) play a central role in SMC functions including migration, proliferation, and contraction. Objective The objective of this study was to identify the role of Rac1 in aSMC contraction and to investigate its involvement in AHR associated with allergic asthma. Methods To define the role of Rac1 in aSMC, ex and in vitro analyses of bronchial reactivity were performed on bronchi from smooth muscle (SM)-specific Rac1 knockout mice and human individuals. In addition, this murine model was exposed to allergens (ovalbumin or house dust mite extract) to decipher in vivo the implication of Rac1 in AHR. Results The specific SMC deletion or pharmacological inhibition of Rac1 in mice prevented the bronchoconstrictor response to methacholine. In human bronchi, a similar role of Rac1 was observed during bronchoconstriction. We further demonstrated that Rac1 activation is responsible for bronchoconstrictor-induced increase in intracellular Ca 2+ concentration and contraction both in murine and in human bronchial aSMCs, through its association with phospholipase C β2 and the stimulation of inositol 1,4,5-trisphosphate production. In vivo , Rac1 deletion in SMCs or pharmacological Rac1 inhibition by nebulization of NSC23766 prevented AHR in murine models of allergic asthma. Moreover, nebulization of NSC23766 decreased eosinophil and neutrophil populations in bronchoalveolar lavages from mice with asthma. Conclusions Our data reveal an unexpected and essential role of Rac1 in the regulation of intracellular Ca 2+ and contraction of aSMCs, and the development of AHR. Rac1 thus appears as an attractive therapeutic target in asthma, with a combined beneficial action on both bronchoconstriction and pulmonary inflammation.

Platelet activation by thrombin is an auto-amplification loop of thrombin generation, a major factor in the formation of atherosclerotic plaques. The small G protein RhoA, under the direct control of the exchange factor Arhgef1, modulates several cellular functions in inflammation. The objective was to study the RhoA pathway and its control by Arhgef1 in platelet aggregation and thrombin generation due to PAR receptor activation by thrombin. We used a knockout mouse model for the exchange factor Arhgef1 (Arhgef1 −/−). In response to an agonist (collagen, ADP and thrombin), the expression of surface glycoproteins and the aggregation of washed platelets were not altered in the Arhgef1 −/− mice compared to Argef1 +/+ mice. In contrast, platelet activation studied by the secretion of granules a, exposure to phosphatidylserine and release of microparticles were decreased in the Arhgef1 −/− mice. Thrombin generation in whole platelet-rich blood was also reduced by 25%. These changes result in a lengthening of the time of occurrence of an occlusive thrombus in the carotid induced by FeCl3. In conclusion, the results confirm the involvement of the RhoA pathway in platelet activation and demonstrate an Arhgef1-dependent mechanism. The results in mice show a new auto-amplification mechanism of thrombin generation by platelets through PAR and membrane phospholipids. Redistribution of phospholipid linked rearrangements of the membrane complex induced by inflammation suggests that the RhoA pathway potentiates the deleterious effects of thrombin in atherothrombosis.

Drug-eluting stents have been developed to reduce the risk of restenosis after angioplasty. To facilitate the adhesion of a poly(lactic acid) (PLA) overlayer loaded with rapamycin (20 wt%), a biodegradable macromonomer based on poly(lactic acid) (HEMA-PLA) was grafted onto the metallic stent by electrografting in a one-step reaction involving the immobilization of aryl diazonium onto the metal followed by an in situ surface electro-polymerization. The HEMA-PLA coating was chemically characterized. Mechanical performance during stent expansion was tested. Morphology examinations showed a strong adhesion of PLA topcoat in the presence of the electrografted layer. Biocompatibility and degradation of the coating were studied in vitro and in vivo in rabbit iliac arteries. These 28 days implantations resulted in a minimal inflammatory process with a partial degradation of the coating. These results suggest that this kind of anchoring of a biodegradable layer shows great potential for drug-eluting stents.

International audience; BACKGROUND Hypertension is characterized by a maintained high blood pressure leading to cardiac complications such as left ventricular hypertrophy and fibrosis and an increased risk of heart failure and myocardial infarction. This study investigated the cardiac effects of oral administration of Moringa oleifera (MOI) seed powder in spontaneous hypertensive rats (SHR).METHODS SHR received food containing MOI seed powder (750mg/d, 8 weeks) or normal food. In vivo measurement of hemodynamic parameters by telemetry and cardiac structure and function analysis by echocardiography were performed. Histological studies were performed to determine fibrosis and protein expression.RESULTS MOI treatment did not modify blood pressure in SHR but reduced nocturnal heart rate and improved cardiac diastolic function (reduction of isovolumetric relaxation time and deceleration time of the E wave, increase of ejection volume and cardiac output compared to nontreated SHR). Left ventricular anterior wall thickness, interseptal thickness on diastole, and relative wall thickness were reduced after MOI treatment. Furthermore, we found a significant reduction of fibrosis in the left ventricle of MOI-treated SHR. This antihypertrophic and antifibrotic effect of MOI was associated with increased expression of peroxisome proliferator-activated receptor (PPAR)-α and δ, reduced cardiac triglyceride level, and enhanced plasmatic prostacyclins.CONCLUSIONS Our data show a beneficial effect of MOI on the cardiac structure and function in SHR associated with an upregulation of PPAR-α and δ signaling. This study thus provides scientific rational support for the empirical use of MOI in the traditional Malagasy medicine against cardiac diseases associated with blood pressure overload.

Sustained overactivation of RhoA is a common component for the pathogenesis of several cardiovascular disorders, including hypertension. Although activity of Rho proteins depends on Rho exchange factors (Rho-GEFs), the identity of Rho-GEFs expressed in vascular smooth muscle cells (VSMC) and participating in the control of Rho protein activity and Rho-dependent functions remains unknown. To address this question, we analyzed by quantitative RT-PCR the expression profile of 28 RhoA-GEFs in arteries of normotensive (saline-treated) and hypertensive (ANG II-treated) rats. Sixteen RhoA-GEFs were downregulated in mesenteric arteries of hypertensive rats, among which nine are also downregulated in cultured VSMC stimulated by ANG II (100 nM, 48 h), suggesting a direct effect of ANG II. Inhibition of type 1 ANG II receptors (losartan, 1 μM) or Rho kinase (fasudil, 10 μM) prevented ANG II-induced RhoA-GEF downregulation. Functionally, ANG II-induced downregulation of RhoA-GEFs is associated with decreased Rho kinase activation in response to endothelin-1, norepinephrine, and U-46619. This work thus identifies a group of RhoA-GEFs that controls RhoA and RhoA-dependent functions in VSMC, and a negative feedback of RhoA/Rho kinase activity on the expression of these RhoA-GEFs that may play an adaptative role to limit RhoA/Rho kinase activation.

Resume Dans ce projet concernant la mise au point de nouvelles endoprotheses vasculaires (stents) liberant de facon controlee des molecules pharmacologiques, nous avons prepare des macromonomeres de polyacide lactique biodegradables et biocompatibles, de masse molaire controlee (600 g/mol), ayant une double liaison permettant par la suite l’electrogreffage sur un stent metallique. Cette sous-couche biodegradable a pour role d’ameliorer l’interface entre le metal et la matrice polymere degradable qui sera deposee ulterieurement sur le stent pour assurer une bonne liberation du principe actif (DES). Nous avons teste la faisabilite de l’electrogreffage de cette sous-couche a base de PLA, sa degradation in vitro puis in vivo ainsi que la recolonisation du stent par des cellules. Les resultats positifs obtenus dans cette etude sont tout a fait encourageants pour la mise au point de nouveaux DES.

Introduction Severe asthma is a lung chronic disease characterized by inflammation, airway hyperresponsiveness (AHR), airway remodelling and corticoid resistance. The molecular mechanisms underlying airway smooth muscle cells (aSMC) contraction and proliferation involved in airway hyperresponsiveness (AHR) and airway remodelling are still largely unknown. Small GTPases of the Rho family (RhoA, Rac1 and Cdc42) play a central role in smooth muscle functions. Recently, we demonstrated that Rac1 play a major role in the control of bronchoconstriction by modulating airway SMC contraction (Andre-Gregoire et al., J Allergy Clin Immunol, 2017). The objective of this study was to assess the role of smooth muscle Rac1 in severe asthma. Methods We developed a new model of severe allergic asthma (house dust mite (HDM) sensitized mice) characterized by AHR and strong airway remodelling, including SMC hyperplasia. Interestingly, as in human severe asthma, this model is associated to an altered sensitivity to inhaled corticoids. To assess the role of Rac1 in aSMCs, SMC-Rac1-KO mice were sensitized to HDM. Results Our results demonstrated that deletion of SMC Rac1 totally prevents AHR and smooth muscle mass increase in this murine model. Furthermore, nebulisation of the Rac1 inhibitor NSC23766 prevents AHR and SMC hyperplasia, but also goblet cell hyperplasia and epithelial cell hypertrophy whereas corticoids treatments has no effect on these parameters. In addition, we observed that nebulisation of NSC23766 decreased eosinophil and neutrophil populations in bronchoalveolar lavages from HDM sensitized asthmatic mice. Discussion In this study, we generated a new model of severe allergic asthma that mimic closely the human pathology. We demonstrated that Rac1 plays an essential role in aSMC contraction and hyperplasia, and that its inhibition prevents AHR and airway remodelling associated to severe asthma. Compared to classical bronchodilators, Rac1 inhibition presents the additional advantage to decrease pulmonary inflammation. Conclusion Consistant with these datas, inhibition of Rac1 activity may represent a novel therapeutic approach for patients with severe asthma.

Activation of the thrombin receptors, called PAR, leads to a remodeling of the actin cytoskeleton depending on the small G proteins of the Rho family. The main one, RhoA, controls platelet contractility. The Arhgef1 nucleotide exchange factor, responsible for the activation of RhoA, is not well known. Our goal is to identify the role of Arhgef1 in platelet activity and thrombin generation in response to thrombin-activated PAR receptors. Thrombin generation, platelet activation and aggregation are measured by fluorimetry, flow cytometry, and turbidimetry in the blood of Arhgef1+/+ and Arhgef1−/− mice. The formation of a thrombus in the carotid artery in response to the local application of FeCl3, is followed by Echo-Doppler. In response to thrombin, aggregation of washed platelets and intracellular calcium are similar in Arhgef1−/− and Arhgef1+/+ mice. However, the secretion of alpha granules, exposure of phosphatidylserine and release of microparticles associated with platelet activation are decreased compared to Arhgef1+/+ mice. The generation of whole blood thrombin was significantly reduced (25%) in Arhgef1−/− mice compared to Arhgef1+/+. These changes result in an increase in the time of an occlusive thrombus occurrence induced by FeCl3 in the carotid artery. Our results show the involvement of the platelet RhoA pathway in the self-amplification of thrombin generation by a mechanism dependent on Arhgef1 and PAR receptors. Changes in the distribution of membrane phospholipids suggest that this pathway could participate in the deleterious effects of thrombin in atherothrombosis.

Angiotensin II (ANG II) is a major regulator of blood pressure that essentially acts through activation of ANG II type 1 receptor (AT1R) of vascular smooth muscle cells (VSMC). AT1R activates numerous intracellular signaling pathways, including the small G protein RhoA known to control several VSMC functions. Nevertheless, the mechanisms leading to RhoA activation by AT1R are unknown. RhoA activation can result from activation of RhoA exchange factor and/or inhibition of Rho GTPase-activating protein (GAP). Here we hypothesize that a RhoGAP could participate to RhoA activation induced by ANG II in rat aortic VSMC. The knockdown of the RhoGAP p190A by small interfering RNA (siRNA) abolishes the activation of RhoA-Rho kinase pathway induced after 5 min of ANG II (0.1 μM) stimulation in rat aortic VSMC. We then show that AT1R activation induces p190A dephosphorylation and inactivation. In addition, expression of catalytically inactive or phosphoresistant p190A mutants increases the basal activity of RhoA-Rho kinase pathway, whereas phosphomimetic mutant inhibits early RhoA activation by ANG II. Using siRNA and mutant overexpression, we then demonstrate that the tyrosine phosphatase SHP2 is necessary for 1) maintaining p190A basally phosphorylated and activated by the tyrosine kinase c-Abl, and 2) inducing p190A dephosphorylation and RhoA activation in response to AT1R activation. Our work then defines p190A as a new mediator of RhoA activation by ANG II in VSMC.

Background and purpose: Pulmonary arterial hypertension (PAH) is associated with increased contraction and proliferation of pulmonary vascular smooth muscle cells. The anti-diabetic drug metformin has been shown to have relaxant and anti-proliferation properties. We thus examined the effect of metformin in PAH. Experimental approach: Metformin effects were analysed in hypoxia- and monocrotaline-induced PAH in rats. Ex vivo and in vitro analyses were performed in lungs, pulmonary artery rings and cells. Key results: In hypoxia- and monocrotaline-induced PAH, the changes in mean pulmonary arterial pressure and right heart hypertrophy were nearly normalized by metformin treatment (100 mg·kg−1·day−1). Pulmonary arterial remodelling occurring in both experimental models of PAH was also inhibited by metformin treatment. In rats with monocrotaline-induced PAH, treatment with metformin significantly increased survival. Metformin increased endothelial nitric oxide synthase phosphorylation and decreased Rho kinase activity in pulmonary artery from rats with PAH. These effects are associated with an improvement of carbachol-induced relaxation and reduction of phenylephrine-induced contraction of pulmonary artery. In addition, metformin inhibited mitogen-activated protein kinase activation and strongly reduced pulmonary arterial cell proliferation during PAH. In vitro, metformin directly inhibited pulmonary artery smooth muscle cell growth. Conclusions and implications: Metformin protected against PAH, regardless of the initiating stimulus. This protective effect may be related to its anti-remodelling property involving improvement of endothelial function, vasodilatory and anti-proliferative actions. As metformin is currently prescribed to treat diabetic patients, assessment of its use as a therapy against PAH in humans should be easier.

The complex and multifactorial pathogenesis of pulmonary hypertension (PH) involves constriction, remodeling, and in situ thrombosis of pulmonary vessels. Both serotonin (5-HT) and Rho kinase signaling may contribute to these alterations.To investigate possible links between the 5-HT transporter (5-HTT) and RhoA/Rho kinase pathways, as well as their involvement in the progression of human and experimental PH.Biochemical and functional analyses of lungs, platelets, and pulmonary artery smooth muscle cells (PA-SMCs) from patients with idiopathic PH (iPH) and 5-HTT overexpressing mice.Lungs, platelets, and PA-SMCs from patients with iPH were characterized by marked elevation in RhoA and Rho kinase activities and a strong increase in 5-HT binding to RhoA indicating RhoA serotonylation. The 5-HTT inhibitor fluoxetine and the type 2 transglutaminase inhibitor monodansylcadaverin prevented 5-HT-induced RhoA serotonylation and RhoA/Rho kinase activation, as well as 5-HT-induced proliferation of PA-SMCs from iPH patients that was also inhibited by the Rho kinase inhibitor fasudil. Increased Rho kinase activity, RhoA activation, and RhoA serotonylation were also observed in lungs from SM22-5-HTT(+)mice, which overexpress 5-HTT in smooth muscle and spontaneously develop PH. Treatment of SM22-5-HTT(+) mice with either fasudil or fluoxetine limited PH progression and RhoA/Rho kinase activation.RhoA and Rho kinase activities are increased in iPH, in association with enhanced RhoA serotonylation. Direct involvement of the 5-HTT/RhoA/Rho kinase signaling pathway in 5-HT-mediated PA-SMC proliferation and platelet activation during PH progression identify RhoA/Rho kinase signaling as a promising target for new treatments against PH.

The molecular mechanism via which keratinocyte differentiation assembles multiple layers of cells (stratification) is poorly understood. We describe here a novel function of the Rho family member RhoE as a regulator of epidermal morphogenesis. RhoE protein levels are specifically and transiently up-regulated upon keratinocyte differentiation. RhoE up-regulation requires the activity of Rho kinase (ROCK) I, suggesting that both RhoE and ROCKI are important during keratinocyte differentiation. RhoE overexpression results in a striking enlargement of cell size and the number of stratified cells. In contrast, RhoE depletion induces hyperproliferation and delays initiation of keratinocyte differentiation. Interestingly, up-regulation of RhoE protein is seen primarily in basal, undifferentiated cells, in which commitment to differentiation and stratification takes place. RhoE activation in basal cells negatively modulates integrin adhesion, thereby facilitating detachment from the substratum and migration to form suprabasal layers. Thus, RhoE integrates two processes essential for keratinocyte differentiation and stratification: regulation of proliferative status and integrin adhesion.

Increasing evidence has accumulated to implicate overactivation of Rho protein as a common component for the pathogenesis of several cardiovascular disorders including hypertension, coronary and cerebral vasospasm, atherosclerosis, and diabetes. Recent advances in Rho protein signaling research indicate that the Rho exchange factors (Rho GEFs) which activate Rho proteins by catalyzing the exchange of GDP for GTP are major regulators of Rho protein activity. In addition, linkage analysis and association studies have recently identified Rho GEFs as susceptibility genes for cardiovascular diseases. All of these data are converging to suggest that as upstream activators of Rho proteins, Rho GEFs expressed in cardiovascular cells are good candidate targets for the treatment of cardiovascular disorders.

Urotensin II (U-II), a vasoactive cyclic neuropeptide which activates the G protein-coupled receptor UT receptor, exerts various cardiovascular effects and may play a role in the pathophysiology of atherosclerosis. In this study, we report that the UT receptor is expressed and functional on human PBMC and rat splenocytes. PBMC surface expression of the UT receptor was mainly found in monocytes and NK cells, also in a minority of B cells, but not in T cells. Stimulation of monocytes with LPS increased UT receptor mRNA and protein expression. Cloning and functional characterization of the human UT receptor gene promoter revealed the presence of NF-κB-binding sites involved in the stimulation of UT receptor gene expression by LPS. Activation of the UT receptor by U-II induced chemotaxis with maximal activity at 10 and 100 nM. This U-II effect was restricted to monocytes. Analysis of the signaling pathway involved indicated that U-II-mediated chemotaxis was related to RhoA and Rho kinase activation and actin cytoskeleton reorganization. The present results thus identify U-II as a chemoattractant for UT receptor-expressing monocytes and indicate a pivotal role of the RhoA-Rho kinase signaling cascade in the chemotaxis induced by U-II.

This study was designed to identify the global pattern of differentially expressed genes in human varicose veins. Using suppressive subtractive hybridization, we identified overexpression of genes known to be associated with extracellular matrix remodeling, including collagen III, tissue inhibitor of metalloproteinases I, dermatopontin, matrix Gla protein (MGP) and tenascin C. Real-time polymerase chain reaction analysis confirmed the differential expression of these genes. The overexpression of MGP transcript was associated with increased MGP level in varicose veins, in particular the undercarboxylated form of the protein. Smooth muscle cells from varicose veins showed increased proliferation rate and enhanced matrix mineralization. This observation correlated with the presence of ectopic mineralization areas in the varicose vein walls. The use of warfarin, to inhibit MGP activity, or siRNA targeting MGP transcript induced a reduction in the exacerbated proliferation of varicose vein smooth muscle cells. Our results suggest that high expression of MGP in varicose veins may contribute to venous wall remodeling by affecting proliferation and mineralization processes probably through impaired carboxylation of MGP. In addition, suppressive subtractive hybridization results also produce a profile of differentially expressed genes in varicose veins, in particular extracellular matrix components. Further study of these genes will provide insights into their specific roles in the etiology of venous disease.

OBJECTIVE: Myogenic tone, which has a major role in the regulation of local blood flow, refers to the ability of vascular smooth muscle to adapt its contractility to changes in transmural pressure. Although Rho-kinase is involved in myogenic tone, the pathway involved remains unclear, especially concerning translocation to the plasma membrane and activation of RhoA. As caveolae have a key role in the signal transduction of membrane-bound proteins, we tested the hypothesis that RhoA might be activated by pressure and that its activation might involve caveolin-1, which has been shown to be involved in vascular functions. METHODS: Myogenic tone was studied in isolated rat mesenteric resistance arteries (118+/-15 microm internal diameter with a pressure of 75 mmHg) submitted to pressure steps (25, 75, and 150 mmHg). Pharmacological blockade of caveolae or RhoA-Rho-kinase pathway was assessed by confocal microscopy in pressurized arteries to analyze protein co-localization and by co-immunoprecipitation in order to confirm protein interactions. Caveolin-1-deficient mice were used to confirm the role of the protein in myogenic tone. RESULTS: Pressure-induced myogenic tone was significantly reduced by RhoA inactivation with TAT-C3 (90.5% inhibition at 150 mmHg) and by the Rho-kinase inhibitor Y27632 (91.8% inhibition at 150 mmHg). In arteries pressurized at 150 mmHg, RhoA was localized to the plasma membrane (localization by confocal microscopy and increased quantity of RhoA in the membrane fraction after protein extraction). Thus, translocation of RhoA to the plasma membrane was associated with pressure-induced tone. In addition, caveolae disruption with methyl-beta-cyclodextrin reduced myogenic tone by 66% at 150 mmHg. Further, myogenic tone was significantly reduced to 24% of control in caveolin-1-deficient mice (active tone was 32.3+/-2.8 microm and 9.1+/-3.7 microm in +/+ and -/- mice, respectively, n = 5 per group), suggesting a key role of caveolin-1 in myogenic tone. Finally, RhoA and caveolin-1 co-immunoprecipitation and co-localization significantly increased when myogenic tone developed at 150 mmHg (co-localization showed 26+/-13% merging at 25 mmHg versus 97+/-21% at 150 mmHg, n = 5). Co-immunoprecipitation was prevented by TAT-C3 and by methyl beta-cyclodextrin. CONCLUSION: RhoA activation is critical for the development of myogenic tone in resistance arteries. This activation induced translocation of RhoA to the plasma membrane within caveolae, where the interaction of RhoA with caveolin-1 leads selectively to the activation of a Rho-kinase-dependent force development.