RESUMO
BACKGROUND: Geometrical 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. METHODS: Patients 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. RESULTS: 46 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. CONCLUSION: Our 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.
Assuntos
Aneurisma Intracraniano , Humanos , Estudos Retrospectivos , Artéria Cerebral Média/patologia , Angiografia Cerebral/métodos , Imageamento TridimensionalAssuntos
Fator A de Crescimento do Endotélio Vascular , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Indutores da Angiogênese , Transdução de Sinais , Células Endoteliais/metabolismoRESUMO
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.
Assuntos
Estresse Oxidativo , Proteínas rac1 de Ligação ao GTP , Movimento Celular , Humanos , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Hypoxia and inflammation play a major role in revascularization following ischemia. Sildenafil inhibits phosphodiesterase-5, increases intracellular cGMP and induces revascularization through a pathway which remains incompletely understood. Thus, we investigated the effect of sildenafil on post-ischemic revascularization. The left femoral artery was ligated in control and sildenafil-treated (25 mg/kg per day) rats. Vascular density was evaluated and expressed as the left/right leg (L/R) ratio. In control rats, L/R ratio was 33 ± 2% and 54 ± 9%, at 7- and 21-days post-ligation, respectively, and was significantly increased in sildenafil-treated rats to 47 ± 4% and 128 ± 11%, respectively. A neutralizing anti-VEGF antibody significantly decreased vascular density (by 0.48-fold) in control without effect in sildenafil-treated animals. Blood flow and arteriolar density followed the same pattern. In the ischemic leg, HIF-1α and VEGF expression levels increased in control, but not in sildenafil-treated rats, suggesting that sildenafil did not induce angiogenesis. PI3-kinase, Akt and eNOS increased after 7 days, with down-regulation after 21 days. Sildenafil induced outward remodeling or arteriogenesis in mesenteric resistance arteries in association with eNOS protein activation. We conclude that sildenafil treatment increased tissue blood flow and arteriogenesis independently of VEGF, but in association with PI3-kinase, Akt and eNOS activation.
Assuntos
Membro Posterior , Isquemia , Óxido Nítrico Sintase Tipo III , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Citrato de Sildenafila , Animais , Membro Posterior/irrigação sanguínea , Membro Posterior/efeitos dos fármacos , Membro Posterior/metabolismo , Isquemia/tratamento farmacológico , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais , Citrato de Sildenafila/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Remodelação das Vias Aéreas , Asma/metabolismo , Miócitos de Músculo Liso/metabolismo , Hipersensibilidade Respiratória , Proteínas rac1 de Ligação ao GTP/metabolismo , Corticosteroides/farmacologia , Aminoquinolinas/administração & dosagem , Aminoquinolinas/farmacologia , Animais , Biópsia , Líquido da Lavagem Broncoalveolar/citologia , Estudos de Casos e Controles , Proliferação de Células , Modelos Animais de Doenças , Eosinófilos/metabolismo , Células Caliciformes/metabolismo , Humanos , Camundongos , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
BACKGROUND AND PURPOSE: The 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. METHODS: We 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. RESULTS: Among 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. CONCLUSIONS: The 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.
Assuntos
Aneurisma Roto/etiologia , Aneurisma Intracraniano/complicações , Fatores Etários , Idoso , Algoritmos , Aneurisma Roto/prevenção & controle , Feminino , Humanos , Aneurisma Intracraniano/diagnóstico por imagem , Aneurisma Intracraniano/patologia , Aprendizado de Máquina , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Neuroimagem , Fatores de Risco , Tomografia Computadorizada por Raios XRESUMO
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.
Assuntos
Envelhecimento/fisiologia , Aorta/patologia , Doenças Cardiovasculares/terapia , Endotélio/fisiologia , Moringa oleifera , Animais , Células Cultivadas , Suplementos Nutricionais , Humanos , Masculino , Contração Miocárdica , Óxido Nítrico Sintase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Sementes , Transdução de Sinais , VasodilataçãoRESUMO
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.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Piridazinas/síntese química , Piridazinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , HumanosRESUMO
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.
Assuntos
Quinases Associadas a rho/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Animais Geneticamente Modificados , Asma/fisiopatologia , Doenças Autoimunes/fisiopatologia , Biomarcadores , Doenças Cardiovasculares/fisiopatologia , Movimento Celular , Proliferação de Células , Endotélio/fisiologia , Glaucoma/fisiopatologia , Glucose/metabolismo , Humanos , Inflamação/fisiopatologia , Nefropatias/fisiopatologia , Leucócitos/metabolismo , Músculo Liso/fisiologia , Neoplasias/fisiopatologia , Doenças do Sistema Nervoso/fisiopatologia , Polimorfismo de Nucleotídeo Único , Transdução de Sinais , Quinases Associadas a rho/genéticaAssuntos
Anticorpos Neutralizantes/farmacologia , Antígenos de Dermatophagoides/administração & dosagem , Asma/tratamento farmacológico , Interleucina-17/imunologia , Contração Muscular/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Animais , Asma/induzido quimicamente , Asma/genética , Asma/imunologia , Movimento Celular/efeitos dos fármacos , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Quimiocina CXCL5/genética , Quimiocina CXCL5/imunologia , Modelos Animais de Doenças , Expressão Gênica , Humanos , Interleucina-17/antagonistas & inibidores , Interleucina-17/genética , Camundongos , Contração Muscular/imunologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/imunologia , Neutrófilos/imunologia , Neutrófilos/patologia , Pyroglyphidae/química , Pyroglyphidae/imunologia , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/patologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/patologiaRESUMO
Interventional strategies to treat atherosclerosis, such as transluminal angioplasty and stent implantation, often cause vascular injury. This leads to intimal hyperplasia (IH) formation that induces inflammatory and fibroproliferative processes and ultimately restenosis. We show that phosphoinositide 3-kinase γ (PI3Kγ) is a key player in IH formation and is a valid therapeutic target in its prevention/treatment. PI3Kγ-deficient mice and mice expressing catalytically inactive PI3Kγ (PI3Kγ KD) showed reduced arterial occlusion and accumulation of monocytes and T cells around sites of vascular lesion. The transfer of PI3Kγ KD CD4(+) T cells into Rag2-deficient mice greatly reduced vascular occlusion compared with WT cells, clearly demonstrating the involvement of PI3Kγ in CD4(+) T cells during IH formation. In addition we found that IH is associated with increased levels of Th1 and Th17 cytokines. A specific decrease in the Th1 response was observed in the absence of PI3Kγ activity, leading to decreased CXCL10 and RANTES production by smooth muscle cells. Finally, we show that treatment with the PI3Kγ inhibitor AS-605240 is sufficient to decrease IH in both mouse and rat models, reinforcing the therapeutic potential of PI3Kγ inhibition. Altogether, these findings demonstrate a new role for PI3Kγ activity in Th1-controlled IH development.
Assuntos
Neointima/enzimologia , Neointima/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Animais , Lesões das Artérias Carótidas/tratamento farmacológico , Lesões das Artérias Carótidas/enzimologia , Lesões das Artérias Carótidas/imunologia , Classe Ib de Fosfatidilinositol 3-Quinase/deficiência , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Inibidores Enzimáticos/farmacologia , Artéria Femoral/enzimologia , Artéria Femoral/imunologia , Artéria Femoral/lesões , Marcação de Genes , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neointima/tratamento farmacológico , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Tiazolidinedionas/farmacologiaRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Biflavonoides/farmacologia , Catequina/farmacologia , Cumarínicos/farmacologia , Produtos Finais de Glicação Avançada/efeitos dos fármacos , Mammea/química , Extratos Vegetais/farmacologia , Proantocianidinas/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Biflavonoides/química , Biflavonoides/isolamento & purificação , Catequina/química , Catequina/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/química , Cumarínicos/isolamento & purificação , Células Endoteliais , Frutas/química , Masculino , Estrutura Molecular , Triterpenos Pentacíclicos , Casca de Planta/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Proantocianidinas/química , Proantocianidinas/isolamento & purificação , Ratos , Ratos Wistar , Triterpenos/química , Triterpenos/isolamento & purificação , Triterpenos/farmacologia , Xantonas/química , Xantonas/isolamento & purificação , Xantonas/farmacologia , Ácido BetulínicoRESUMO
Glucose intolerance and dyslipidaemia are independent risk factors for endothelium dysfunction and CVD. The aim of the present study was to analyse the preventive effect of n-3 PUFA (EPA and DHA) on lipid and carbohydrate disturbances and endothelial dysfunction. Three groups of adult hamsters were studied for 20 weeks: (1) control diet (Control); (2) high-fat diet (HF); (3) high-fat diet enriched with n-3 PUFA (HFn-3) groups. The increase in body weight and fat mass in the HF compared to the Control group (P < 0.05) was not found in the HFn-3 group. Muscle TAG content was similar in the Control and HF groups, but significantly lower in the HFn-3 group (P = 0.008). Glucose tolerance was impaired in the HF compared to the Control group, but this impairment was prevented by n-3 PUFA in the HFn-3 group (P < 0.001). Plasma TAG and cholesterol were higher in the HF group compared to the Control group (P < 0.001), but lower in the HFn-3 group compared to the HF group (P < 0.001). HDL-cholesterol was lower in the HFn-3 group compared to the Control and HF groups (P < 0.0005). Hepatic secretion of TAG was lower in the HFn-3 group compared to the HF group (P < 0.005), but did not differ from the Control group. Hepatic gene expression of sterol regulatory element-binding protein-1c, diacylglycerol O-acyltransferase 2 and stearyl CoA desaturase 1 was lower in the HFn-3 group, whereas carnitine palmitoyl transferase 1 and scavenger receptor class B type 1 expression was higher (P < 0.05). In adipocytes and adipose macrophages, PPARγ and TNFα expression was higher in the HF and HFn-3 groups compared to the Control group. Endothelium relaxation was higher in the HFn-3 (P < 0.001) than in the HF and Control groups, and was correlated with glucose intolerance (P = 0.03) and cholesterol (P = 0.0003). In conclusion, n-3 PUFA prevent some metabolic disturbances induced by high-fat diet and improve endothelial function in hamsters.
Assuntos
Dieta Hiperlipídica , Endotélio Vascular/efeitos dos fármacos , Ácidos Graxos Ômega-3/metabolismo , Obesidade/metabolismo , Adipócitos/citologia , Tecido Adiposo/metabolismo , Ração Animal , Animais , Aorta/patologia , Composição Corporal , Peso Corporal , Antígenos CD36/metabolismo , Metabolismo dos Carboidratos , Cricetinae , Dislipidemias/metabolismo , Endotélio Vascular/metabolismo , Glucose/metabolismo , Intolerância à Glucose , Metabolismo dos Lipídeos , Lipídeos/sangue , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Macrófagos/citologia , Masculino , Mesocricetus , Músculos/metabolismo , Obesidade/fisiopatologiaRESUMO
OBJECTIVE: Estradiol (E2) mediates numerous beneficial effects assigned to estrogens, but whereas mechanisms have been described at the endothelial level, direct effects on vascular smooth muscle cells (VSMC) are poorly documented. As evidence accumulates regarding the role of RhoA in vascular pathophysiology and the benefit of RhoA-Rho associated protein kinase (Rock) pathway inhibition, we analyzed if E2 could inhibit it in VSMC. METHODS AND RESULTS: We show that in VSMC, E2 inhibits the RhoA-Rock pathway in a time- and concentration-dependent manner. The inhibition of RhoA-Rock pathway results from E2-induced phosphorylation of the Ser188 of RhoA. Using pharmacological, transfection, and in vitro phosphorylation experiments, we demonstrate that AMP-activated protein kinase subunit alpha 1 (AMPKα1) is activated by estrogen receptor stimulation and catalyzes RhoA phosphorylation induced by E2. Ex vivo, ovariectomy leads to an increase in the amplitude of phenylephrine- or serotonine-induced contractions of aortic rings in wild-type mice but not in AMPKα1-knock-out mice or E2-supplemented animals. These functional effects were correlated with a reduced level of RhoA phosphorylation in the aorta of ovariectomized female, male, and AMPKα1 knock-out mice. CONCLUSION: Our work thus defines AMPKα1 as (1) a new kinase for RhoA and (2) a new mediator of the vasoprotective effects of estrogen.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Estradiol/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Músculo Liso Vascular/citologia , Ovariectomia , Fosforilação/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Vasoconstrição/fisiologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Although overactivation of RhoA is recognized as a common component of vascular disorders, the molecular mechanisms regulating RhoA activity in vascular smooth muscle cells (VSMC) are still unclear. We have previously shown that in VSMC, RhoA is phosphorylated on Ser188 by nitric oxide (NO)-stimulated cGMP-dependent kinase (PKG), which leads to RhoA-Rho kinase pathway inhibition. In this study, we showed that expression of phosphoresistant RhoA mutants prevented the stimulation of VSMC migration and adhesion induced by NO-PKG pathway activation. In contrast, under basal conditions, phosphomimetic RhoA mutants stimulated VSMC adhesion and migration through a signaling pathway requiring Rac1 and the Rho exchange factor Vav3. RhoA phosphorylation or phosphomimetic RhoA mutants induced Rac1 activation but did not activate Vav3. Indeed, phosphorylated RhoA or phosphomimetic mutants trapped guanine dissociation inhibitor α (GDIα), leading to the release of Rac1 and its translocation to the membrane, where it was then activated by the basal Vav3 nucleotide exchange activity. In vivo, RhoA phosphorylation induced by PKG activation in the aortas of rats treated with sildenafil induced dissociation of Rac1 from GDIα and activation of the Rac1 signaling pathway. These results suggest that the phosphorylation of RhoA represents a novel potent and physiological GDIα displacement factor that leads to Rac1 activation and regulation of Rac1-dependent VSMC functions.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Miócitos de Músculo Liso/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Sequência de Bases , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Óxido Nítrico/metabolismo , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-vav/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais , Tionucleotídeos/farmacologia , Cicatrização/fisiologia , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Hypertension is one of the most frequent pathologies in the industrialized world. Although recognized to be dependent on a combination of genetic and environmental factors, its molecular basis remains elusive. Increased activity of the monomeric G protein RhoA in arteries is a common feature of hypertension. However, how RhoA is activated and whether it has a causative role in hypertension remains unclear. Here we provide evidence that Arhgef1 is the RhoA guanine exchange factor specifically responsible for angiotensin II-induced activation of RhoA signaling in arterial smooth muscle cells. We found that angiotensin II activates Arhgef1 through a previously undescribed mechanism in which Jak2 phosphorylates Tyr738 of Arhgef1. Arhgef1 inactivation in smooth muscle induced resistance to angiotensin II-dependent hypertension in mice, but did not affect normal blood pressure regulation. Our results show that control of RhoA signaling through Arhgef1 is central to the development of angiotensin II-dependent hypertension and identify Arhgef1 as a potential target for the treatment of hypertension.
Assuntos
Angiotensina II/fisiologia , Pressão Sanguínea/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipertensão/etiologia , Hipertensão/fisiopatologia , Janus Quinase 2/metabolismo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Tirosina/metabolismoRESUMO
AIMS: Rho guanine nucleotide exchange factors (Rho GEFs) are responsible for Rho protein activation by catalyzing the exchange of GDP for GTP. Although overactivation of Rho proteins is a common component of the pathogenesis of vascular disorders, the molecular mechanisms and the Rho GEFs regulating Rho protein activity and Rho-dependent functions in vascular smooth muscle cells (VSMC) are still unknown. The aim of this study was thus to identify Rho GEFs involved in the regulation of VSMC functions. METHODS AND RESULTS: By a functional screening based on small interfering RNA (siRNA)-mediated silencing of 27 Rho GEFs, we found that only silencing of the Rho GEF Vav3 inhibited rat VSMC proliferation. Conversely, overexpression of Vav3 potentiated VSMC proliferation, whereas the catalytically inactive Vav3 mutant had no effect. The stimulatory effect of Vav3 on VSMC proliferation was inhibited by the Src tyrosine kinase inhibitor SU6656 and by co-expression of the dominant-negative Rac1-N17 mutant. In agreement with this observation, expression of Vav3 induced enrichment of Rac1 to the membrane, activation of its effector PAK, and stimulated VSMC migration. Increased levels of Vav3 transcripts were found in stented arteries and arteries from hypertensive rats. Furthermore, siRNA targeting Vav3 decreased arterial wall cell proliferation. CONCLUSION: The Rho GEF Vav3 controls VSMC proliferation and migration through activation of Rac1/PAK signalling. Vav3 is a convergent point mediating Rac1 activation in response to different upstream mediators that promote VSMC proliferation and migration and thus appears to be a new potential therapeutic target that could be used to limit vascular proliferative diseases.
Assuntos
Movimento Celular/fisiologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Aorta/citologia , Aorta/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Proteínas Proto-Oncogênicas c-vav/genética , RNA Interferente Pequeno , Ratos , Ratos Endogâmicos WKY , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The small G protein Rho signaling pathways are recognized as major regulators of cardiovascular functions, and activation of Rho proteins appears to be a common component for the pathogenesis of hypertension and vascular proliferative disorders. Recent evidence suggests that modulation of Rho protein signaling by phosphorylation of Rho proteins provides an additional simple mechanism for coordinating Rho protein functions. Phosphorylation of RhoA by cAMP- or cGMP-activated kinase on Ser188 induces cytosolic sequestration of RhoA through increased interaction with guanine dissociation inhibitor, thereby resulting in inhibition of RhoA-dependent functions. Here we show that stimulation of angiotensin II (Ang II) type 2 receptor (AT(2)R) in vascular smooth muscle cells induces Ser188 phosphorylation of RhoA independently of cAMP- or cGMP-activated kinase. We identify the Ser/Thr kinase Ste20-related kinase SLK as a new kinase phosphorylating RhoA on Ser188. Activation of the signaling cascade involving Src homology 2 domain-containing protein-tyrosine phosphatase 1, casein kinase II and SLK is responsible for RhoA phosphorylation and inhibition of RhoA-mediated arterial contraction induced by AT(2)R activation. These results thus identify the molecular mechanism linking AT(2)R to RhoA inhibition and vasodilation.
Assuntos
Hipertensão/metabolismo , Músculo Liso Vascular/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Vasodilatação/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Angiotensina II/farmacologia , Animais , Aorta Torácica/citologia , Aorta Torácica/enzimologia , Caseína Quinase II/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Etinilestradiol/metabolismo , Masculino , Acetato de Megestrol/metabolismo , Músculo Liso Vascular/citologia , Óxido Nítrico Sintase/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , Vasoconstritores/farmacologiaRESUMO
OBJECTIVE: The use of sirolimus eluting stent (SES) has strongly limited the incidence of in-stent restenosis that still remains a problem at the stent edge. The aim of this study was to analyze the neointimal thickening after implantation of SES and to assess the influence of the stent implantation procedure on the neointimal thickening in the in-stent segment and at the edge of the stent in an ex-vivo model of stented human artery. METHODS: Both balloon expandable SES and the corresponding bare metal stent (BMS) were used in a model of human mammary artery culture. Stents were implanted either directly or after predilatation (10 atm, 60 seconds) and analysis of arterial segments were performed at 28 days poststenting. Cell proliferation and neointimal thickening were assessed by immunohistochemistry, western blotting, and histomorphometry, both in the in-stent segment and at the edge of the stent. Neointimal thickening was expressed as the ratio ([neointimal area/neointimal area + media area]). RESULTS: The in-stent neointimal thickening was dramatically inhibited in the SES group compared with the BMS group whatever the stenting technique was (predilatation: 0.22 +/- 0.05 vs 0.30 +/- 0.10; P < .04; direct stenting 0.16 +/- 0.04 vs 0.30 +/- 0.13; P <.01). This effect of SES was associated with a smallest expression of the small G protein RhoA and an increase of p27kip expression. In the BMS group, predilatation and direct stenting gave similar in-stent neointimal thickening. In contrast, in the SES group, in-stent neointimal thickening was significantly reduced when direct stenting was performed (0.16 +/- 0.04 [direct stenting] vs 0.22 +/- 0.05 [predilatation], P < .03). At the stent edge, a similar neointimal thickening was observed with both type of stent when predilatation was performed on the entire segment of the artery. Direct stenting significantly reduced the neointimal thickness at the stent edge when SES where used (0.06 +/- 0.01 [direct stenting] vs 0.19 +/- 0.06 [predilatation]; P < .001) but not in the BMS group. CONCLUSION: These results confirm the efficiency of sirolimus released form SES to inhibit RhoA expression and to increase p27kip level in the arterial wall and show the benefit of direct stenting to limit the edge effect with SES.
Assuntos
Implante de Prótese Vascular/instrumentação , Fármacos Cardiovasculares/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Artéria Torácica Interna/efeitos dos fármacos , Metais , Sirolimo/administração & dosagem , Stents , Túnica Íntima/efeitos dos fármacos , Cateterismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Artéria Torácica Interna/metabolismo , Artéria Torácica Interna/cirurgia , Artéria Torácica Interna/ultraestrutura , Microscopia Eletrônica de Varredura , Técnicas de Cultura de Órgãos , Desenho de Prótese , Fatores de Tempo , Túnica Íntima/metabolismo , Túnica Íntima/cirurgia , Túnica Íntima/ultraestrutura , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
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.