RESUMO
Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Our study is the first to assess the role of Ephb6 in blood pressure (BP) regulation. We observed that EphB6 and all three of its Efnb ligands were expressed on vascular smooth muscle cells (VSMC) in mice. We discovered that small arteries from castrated Ephb6 gene KO males showed increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo compared with their WT counterparts. Consistent with this finding, castrated Ephb6 KO mice presented heightened BP compared with castrated WT controls. In vitro experiments in VSMC revealed that cross-linking Efnbs but not Ephb6 resulted in reduced VSMC contractions, suggesting that reverse signaling through Efnbs was responsible for the observed BP phenotype. The reverse signaling was mediated by an adaptor protein Grip1. Additional experiments demonstrated decreased 24-h urine catecholamines in male Ephb6 KO mice, probably as a compensatory feedback mechanism to keep their BP in the normal range. After castration, however, such compensation was abolished in Ephb6 KO mice and was likely the reason why BP increased overtly in these animals. It suggests that Ephb6 has a target in the nervous/endocrine system in addition to VSMC, regulating a testosterone-dependent catecholamine compensatory mechanism. Our study discloses that Ephs and Efns, in concert with testosterone, play a critical role in regulating small artery contractility and BP.
Assuntos
Pressão Sanguínea/fisiologia , Hipertensão/metabolismo , Músculo Liso Vascular/enzimologia , Receptor EphB6/metabolismo , Testosterona/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Feminino , Humanos , Masculino , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Orquiectomia , RNA Interferente Pequeno/farmacologia , Receptor EphB6/genética , Transdução de Sinais/fisiologia , Vasoconstrição/fisiologiaRESUMO
While an increasing amount of evidence demonstrates the homeostatic functions of the cardiac oxytocin (OT) system, less is known about the role of this hormone in the injured heart. The current study examined the effect of OT infusion on cell apoptosis, expression of proliferating cell nuclear antigen (PCNA) and inflammation in the acute and subacute phases of myocardial infarction (MI). Prior MI male Sprague-Dawley rats were infused subcutaneously with OT 25 or 125 ng/(kg h) for 3 or 7 days. Saline-treated MI and sham-operated rats served as controls. Echocardiography and analysis of cardiac sections were used to disclose OT actions. Left ventricular fractional shortening, estimated to be 46.0 +/- 1.8% in sham controls, declined to 21.1 +/- 3.3% in vehicle-treated MI rats and was improved to 34.2 +/- 2.1 and to 30.9 +/- 2.5% after treatment with OT 25 and 125 ng/(kg h), respectively. OT infusion resulted in: (1) increase of cells expressing PCNA in the infarct zone, diminished cell apoptosis and fibrotic deposits in the remote myocardium; (2) suppression of inflammation by reduction of neutrophils, macrophages and T lymphocytes; (3) depression of the expression of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 with promotion of transforming growth factor-beta. OT treatment reduced expression of atrial and brain natriuretic peptides in the infarcted ventricle, as well as the concentration of both peptides in the circulation. These results indicate that continuous OT delivery reduces inflammation and apoptosis in infarcted and remote myocardium, thus improving function in the injured heart.
Assuntos
Infarto do Miocárdio/tratamento farmacológico , Miocardite/prevenção & controle , Ocitócicos/uso terapêutico , Ocitocina/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Ecocardiografia , Testes de Função Cardíaca , Leucócitos/patologia , Masculino , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/metabolismo , Miocardite/etiologia , Miocardite/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Peptídeos Natriuréticos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ocitócicos/farmacologia , Ocitocina/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley , Troponina T/metabolismoRESUMO
Beta-thalassemia is an inherited hematological disease caused by a decrease or absence of production of beta-globin that requires chronic therapeutic interventions. This condition leads to important arterial and venous thromboembolic events, transitory ischemic attacks, and microcirculatory obstructions, indicative of circulatory disturbances. To investigate the presence of microcirculatory disorders without the confounding effect of treatments, we used beta-thalassemic mice with typical clinical characteristics of human beta-thalassemia major. One impediment to the understanding of microcirculatory physiology, in particular for beta-thalassemic mice, has been the lack of an appropriate noninvasive imaging approach. We thus developed a novel noninvasive high-frequency ultrasound imaging method to evaluate murine vascular hemodynamic properties. In our beta-thalassemic mice, total peripheral vascular resistance was significantly increased (P < 0.01) compared with wildtype littermates, whereas mean blood pressure, heart rate, and cardiac output were similar (P = nonsignificant). Importantly, the vascular hemodynamics in beta-thalassemic mice were significantly affected according to the Pourcelot indexes measured in the common carotid artery and abdominal aorta (P < 0.01 and P < 0.05, respectively). Hence, our beta-thalassemia characterization of vascular hemodynamics by noninvasive ultrasonic approaches proves the existence and provides unique quantitative assessment of microcirculatory flow disturbances in those mice.
Assuntos
Microcirculação/patologia , Microscopia Acústica/métodos , Talassemia beta/patologia , Animais , Pressão Sanguínea/fisiologia , Débito Cardíaco/fisiologia , Frequência Cardíaca/fisiologia , Transplante de Fígado , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação/diagnóstico por imagem , Resistência Vascular/fisiologia , Talassemia beta/diagnóstico por imagemRESUMO
Several erythropoietin-producing hepatocellular receptor B family (EPHB) and their ligands, ephrinBs (EFNBs), are involved in blood pressure regulation in animal models. We selected 528 single nucleotide polymorphisms (SNPs) within the genes of EPHB6, EFNB2, EFNB3 and GRIP1 in the EPH/EFN signalling system to query the International Blood Pressure Consortium dataset. A SNP within the glutamate receptor interacting protein 1 (GRIP1) gene presented a p-value of 0.000389, approaching the critical p-value of 0.000302, for association with diastolic blood pressure of 60,396 individuals. According to echocardiography, we found that Efnb3 gene knockout mice showed enhanced constriction in the carotid arteries. In vitro studies revealed that in mouse vascular smooth muscle cells, siRNA knockdown of GRIP1, which is in the EFNB3 reverse signalling pathway, resulted in increased contractility of these cells. These data suggest that molecules in the EPHB/EFNB signalling pathways, specifically EFNB3 and GRIP1, are involved blood pressure regulation.
Assuntos
Pressão Sanguínea , Proteínas de Transporte/metabolismo , Efrina-B3/metabolismo , Contração Muscular , Músculo Liso Vascular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Proteínas de Transporte/genética , Efrina-B3/genética , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Polimorfismo de Nucleotídeo Único , Transdução de SinaisRESUMO
Non-invasive vascular elastography (NIVE) was recently introduced to characterize mechanical properties of superficial arteries. In this paper, the feasibility of NIVE and its applicability in the context of high-frequency ultrasound imaging is investigated. First, experiments were performed in vitro on vessel-mimicking phantoms. Polyvinyl alcohol cryogel was used to create two double-layer vessels with different mechanical properties. In both cases, the stiffness of the inner layer was made softer. Radial stress was applied within the lumen of the phantoms by applying incremental static pressure steps with a column of a flowing mixture of water-glycerol. The vessel phantoms were insonified at 32 MHz with an ultrasound biomicroscope to provide cross-section sequences of radio-frequency (RF) ultrasound data. The Lagrangian speckle model estimator (LSME) was used to assess the two-dimensional-strain tensors, and the composite Von Mises elastograms were computed. A new implementation of the LSME based on the optical flow equations was introduced. Deformation parameters were estimated using an inversion algorithm. For each in vitro experiment, both layers of approximately 1 mm were distinguished. Second, the use of the method for the purpose of studying small vessels (MicroNIVE) in genetically engineered rodents was introduced. Longitudinal scans of the carotid artery were performed at 40 MHz. The in vivo results give confidence in the feasibility of MicroNIVE as a potential tool to non-invasively study the impact of targeted genes on vascular remodelling in rodents.
Assuntos
Algoritmos , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/fisiopatologia , Hipertensão/diagnóstico por imagem , Hipertensão/fisiopatologia , Interpretação de Imagem Assistida por Computador/métodos , Modelos Biológicos , Ultrassonografia/métodos , Animais , Simulação por Computador , Elasticidade , Aumento da Imagem/métodos , Imageamento Tridimensional/métodos , Masculino , Imagens de Fantasmas , Ratos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Human ß-thalassemia major is one of the most prevalent genetic diseases characterized by decrease/absence of ß-globin chain production with reduction of erythrocyte number. The main cause of death of treated ß-thalassemia major patients with chronic blood transfusion is early cardiac complications that have been attributed to secondary iron overload despite optimal chelation. Herein, we investigated pathophysiological mechanisms of cardiovascular dysfunction in a severe murine model of ß-thalassemia from 6 to 15-months of age in the absence of confounding effects related to transfusion. Our longitudinal echocardiography analysis showed that ß-thalassemic mice first display a significant increase of cardiac output in response to limited oxygen-carrying erythrocytes that progressed rapidly to left ventricular hypertrophy and structural remodeling. Following this compensated hypertrophy, ß-thalassemic mice developed age-dependent deterioration of left ventricular contractility and dysfunction that led toward decompensated heart failure. Consistently, murine ß-thalassemic hearts histopathology revealed cardiac remodeling with increased interstitial fibrosis but virtual absence of myocardial iron deposits. Importantly, development of thalassemic cardiac hypertrophy and dysfunction independently of iron overload has uncoupled these cardiopathogenic processes. Altogether our study on ß-thalassemia major hemoglobinopathy points to two successive phases resulting from severe chronic anemia and from secondarily induced mechanisms as pathophysiologic contributors to thalassemic cardiopathy.
Assuntos
Cardiopatias/etiologia , Ferro/metabolismo , Miocárdio/metabolismo , Talassemia beta/complicações , Animais , Modelos Animais de Doenças , Progressão da Doença , Cardiopatias/metabolismo , Cardiopatias/fisiopatologia , Ventrículos do Coração/patologia , Homozigoto , Humanos , Masculino , Camundongos , Miocárdio/patologia , Fenótipo , Talassemia beta/genética , Talassemia beta/metabolismoRESUMO
AIMS: The hereditary ß-thalassemia major condition requires regular lifelong blood transfusions. Transfusion-related iron overloading has been associated with the onset of cardiovascular complications, including cardiac dysfunction and vascular anomalies. By using an untransfused murine model of ß-thalassemia major, we tested the hypothesis that vascular endothelial dysfunction, alterations of arterial structure and of its mechanical properties would occur despite the absence of treatments. METHODS AND RESULTS: Vascular function and structure were evaluated ex vivo. Compared to the controls, endothelium-dependent vasodilation with acetylcholine was blunted in mesenteric resistance arteries of ß-thalassemic mice while the endothelium-independent vasodilator (sodium nitroprusside) produced comparable vessel dilation, indicating endothelial cell impairment with preserved smooth muscle cell reactivity to nitric oxide (NO). While these findings suggest a decrease in NO bioavailability, Western blotting showed heightened expression of aortic endothelial NO synthase (eNOS) in ß-thalassemia. Vascular remodeling of the common carotid arteries revealed increased medial elastin content. Under isobaric conditions, the carotid arteries of ß-thalassemic mice exhibited decreased wall stress and softening due to structural changes of the vessel wall. CONCLUSIONS: A complex vasculopathy was identified in untransfused ß-thalassemic mice characterized by altered carotid artery structure and endothelial dysfunction of resistance arterioles, likely attributable to reduced NO bioavailability despite enhanced vascular eNOS expression.
Assuntos
Endotélio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Talassemia beta/metabolismo , Acetilcolina/farmacologia , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/genética , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/genética , Vasodilatadores/farmacologia , Sistema Vasomotor/efeitos dos fármacos , Sistema Vasomotor/metabolismo , Talassemia beta/genéticaRESUMO
Preeclampsia is the major cause of maternal and fetal mortality/morbidity. Because hypertension is an important risk factor for preeclampsia, we investigated whether hypertensive mice that overexpress human renin and angiotensinogen develop superimposed preeclampsia. Given that the mechanisms underlying this disease are still poorly understood, animal models are of great use for elucidation. Blood pressure and proteinuria were measured by telemetry and ELISA, respectively. Heart function was evaluated by echocardiography, whereas pathological cardiac hypertrophy-related genes were assessed by real-time PCR. Soluble fms-like tyrosine kinase 1 plasma concentrations were quantitated by ELISA and placental expression by real-time PCR. Transgenic mice develop de novo proteinuria during gestation and marked blood pressure elevation, which are hallmarks of superimposed preeclampsia on chronic hypertension. Abnormal placentation present in these mothers produced a significant decrease in pup and placental weight and was associated with an increased placental expression of soluble fms-like tyrosine kinase 1. We also found heightened circulating levels of this receptor, when adjusted for placental mass, as has been observed in women who suffer from preeclampsia. Cardiac hypertrophy could be observed in the transgenic mice and was exacerbated by gestation. As a result, heart function was significantly decreased, and markers of pathological hypertrophy were increased. Our data, thus, confirm the characterization of a new model of superimposed preeclampsia on chronic hypertension. Because chronically hypertensive women are at risk of developing the pathology, our model reflects a clinical reality and is, thus, an excellent tool to elucidate the molecular mechanisms triggering this disease.