Vav3 proto-oncogene deficiency leads to sympathetic hyperactivity and cardiovascular dysfunction

El pdf del artículo es el manuscrito de autor.-- et al.Although much is known about environmental factors that predispose individuals to hypertension and cardiovascular disease, little information is available regarding the genetic and signaling events involved. Indeed, few genes associated with the progression of these pathologies have been discovered despite intensive research in animal models and human populations. Here we identify Vav3, a GDP-GTP exchange factor that stimulates Rho and Rac GTPases, as an essential factor regulating the homeostasis of the cardiovascular system.Vav3-deficient mice exhibited tachycardia, systemic arterial hypertension and extensive cardiovascular remodeling. These mice also showed hyperactivity of sympathetic neurons from the time of birth. The high catecholamine levels associated with this condition led to the activation of the renin-angiotensin system, increased levels of kidney-related hormones and the progressive loss of cardiovascular and renal homeostasis. Pharmacological studies with drugs targeting sympathetic and renin-angiotensin responses confirmed the causative role and hierarchy of these events in the development of theVav3-null mouse phenotype. These observations uncover the crucial role of Vav3 in the regulation of the sympathetic nervous system (SNS) and cardiovascular physiology, and reveal a signaling pathway that could be involved in the pathophysiology of human disease states involving tachycardia and sympathetic hyperactivity with unknown etiologies.This work was supported by grants from the US National Institutes of Health to X.R.B. and the Spanish Ministry of Education and Science to X.R.B. and J.M.L.-N. V.S. is supported by a European Molecular Biology Organization (EMBO) longterm postdoctoral fellowship.Peer reviewe

High Throughput Screen for Inhibitors of Rac1 GTPase by Flow Cytometry

High throughput (HT) screening is at the starting point for most drug discovery programs. As the range of targets being pursued widens new technologies have to be deployed to enable assays built to measure the activity of proteins previously deemed challenging. Flow cytometry is a technology providing multi-parametric analysis of single cells or other particles in suspension, such as beads. High throughput (HT) flow cytometry has become a very attractive screening platform for drug discovery. In this chapter we describe a 1536 well format high throughput screen of 500,000 compounds to find inhibitors of Rac1 GTPase to prevent allergic airway hyper-responsiveness in asthma. We discuss the assay development, miniaturization and validation carried out prior to the full screening campaign. We then describe how we have automated our iQue® HD screener instruments and how we proceed with the data analysis and explain why we chose to run this screen on a flow cytometer and how it enabled us to reduce cost and timelines for the project

0341: AMPK exerts an insulin-sensitizing effect on cardiac glucose uptake by multiple molecular mechanisms including cytoskeleton reorganization

BackgroundInsulin-resistant cardiomyocytes are characterized by a decreased ability of insulin to stimulate glucose uptake. We have previously shown that the activation of AMPK by metformin or phenformin restores insulin-sensitivity in insulin-resistant cardiomyocytes. The aim of our present work is to understand by which molecular mechanisms AMPK exerts its insulin sensitizing effect. In this study we focused on the mTOR/p70S6K pathway and on cytoskeleton reorganization. mTOR/p70S6K, which is known to be inhibited by AMPK, is able to reduce insulin signaling via a negative feedback loop involving serine phosphorylation of IRS-1. On the other hand, cytoskeleton reorganization, which is a known target of AMPK, is responsible for the translocation of the glucose transporter GLUT4 to the plasma membrane.MethodsAdult rat cardiomyocytes were primary cultured and treated with different agents including insulin, AMPK activator (phenformin), mTOR inhibitor rapamycin and/or actin cytoskeleton inhibitor latrunculin B. Glucose uptake was assessed by detritiation of 2-3H-glucose.ResultsFirst, we tested if rapamycin was able to mimic AMPK activators. Similarly to phenformin, rapamycin increased the insulin-dependent phosphorylation of Akt involved in the regulation of glucose uptake. Despite the ability of rapamycin to induce this Akt over-phosphorylation, rapamycin was not able to restore the insulin-dependent stimulation of glucose uptake like phenformin did. On the other hand, latrunculin B abolished the insulin-sensitizing action of phenformin on glucose uptake, in insulin-sensitive as well as in insulinresistant cells.Conclusionsactin cytoskeleton reorganization but not mTOR/p70S6K, is involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake. The role played by Small G proteins, known to be involved in the regulation of actin cytoskeleton is under investigation

Vav3 Is Involved in GABAergic Axon Guidance Events Important for the Proper Function of Brainstem Neurons Controlling Cardiovascular, Respiratory, and Renal Parameters

Vav3 is a phosphorylation GDP/GTP exchange factor for Rho/Rac GTPases. Recently, it has been described that Vav3 knockout mice develop hypertension and sympathoexcitation. In this work, we report the neurological cause of this phenotype

Mamífero no humano carente de la expresión del gen Vav3, procedimiento de obtención y sus aplicaciones

Mamífero no humano carente de la expresión del gen Vav3, procedimiento de obtención y sus aplicaciones. La invención se relaciona con un mamífero no humano knockout útil como modelo experimental cuyo genoma posee el gen Vav3 mutado y cuyos niveles de RNA mensajero o proteína para la proteína Vav3 endógena se encuentran alterados. Otro objeto de la invención lo constituye un procedimiento de obtención de dicho animal no humano, así como una célula eucariota mutada. Este modelo animal o línea celular pueden emplearse en un procedimiento para determinar el efecto de un fármaco útil para la prevención y/o el tratamiento de enfermedades o trastornos derivados de dicha deficiencia de la proteína Vav3, por ejemplo, problemas de coordinación motora, hipertensión esencial, hiperplasia cardiaca y arterial, taquicardia y esquizofrenia.Peer reviewedConsejo Superior de Investigaciones Científicas (España)B1 Patente con informe sobre el estado de la ténic

GTP-binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo

24 páginas, 5 figuras, 1 tabla.-- El pdf del artículo es el manuscrito de autor.Rho/Rac proteins constitute a subgroup of the Ras superfamily of GTP hydrolases. Although originally implicated in the control of cytoskeletal events, it is currently known that these GTPases coordinate diverse cellular functions, including cell polarity, vesicular trafficking, the cell cycle and transcriptomal dynamics. In this review, we will provide an overview on the recent advances in this field regarding the mechanism of regulation and signaling, and the roles in vivo of this important GTPase family.Funding agencies: XRB’s work is supported by grants from the US National Cancer Institute (5R01-CA73735-10), the Spanish Ministry of Education and Science (SAF2003-00028), the Castilla-León Autonomous Government (SA053A05), and the Red Temática de Investigación Cooperativa en Cáncer (RD06/0020/0001, Spanish Ministry of Health). VS was supported by an EMBO long term postdoctoral fellowship. IMB was partially supported by a Spanish Ministry of Education and Science FPU fellowship (FP2000-6489). The Centro de Investigación del Cáncer is supported by endowments from the CSIC, University of Salamanca, Castilla-León Autonomous Government, the Red Temática de Investigacián Cooperativa de Centros de Cáncer (C03/10, Spanish Ministry of Health), and the Foundation for Cancer Research of Salamanca.Peer reviewe

Utilización de mamíferos no humanos carentes de la expresión y/o actividad de la proteína Vav2 en campos cardiovascular, renal y neurológicos

Solicitud de patente (concesión en curso).-- Referencia OEPM: P200602969.-- Fecha de presentación: 21/11/2006.-- Solicitantes: Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca.La invención se relaciona con la utilización de mamíferos no humanos que han sido alterados genéticamente para eliminar el gen Vav2 y/o reducir la expresión de los niveles de su RNA mensajero o de la proteína que codifica, la molécula Vav2. Este modelo animal, o líneas celulares derivadas del mismo, pueden emplearse en procedimientos experimentales de diverso tipo para la identificación y caracterización de fármacos útiles para la prevención y/o el tratamiento de enfermedades o trastornos de alta incidencia en humanos como la hipertensión esencial, hiperplasia cardiaca y arterial, taquicardia, fallos renales y procesos fibróticos. Este modelo es también de utilidad para la identificación de marcadores genéticos, bioquímicos, fisiológicos y de otro tipo que tengan valor pronóstico sobre enfermedades humanas poco caracterizadas como las mencionadas más arriba.Peer reviewe

A mouse model for Costello syndrome reveals an Ang II-mediated hypertensive condition

11 páginas, 8 figuras, 2 tablas.-- et al.Germline activation of H-RAS oncogenes is the primary cause of Costello syndrome (CS), a neuro-cardio-facio-cutaneous developmental syndrome. Here we describe the generation of a mouse model of CS by introduction of an oncogenic Gly12Val mutation in the mouse H-Ras locus using homologous recombination in ES cells. Germline expression of the endogenous H-RasG12V oncogene, even in homozygosis, resulted in hyperplasia of the mammary gland. However, development of tumors in these mice was rare. H-RasG12V mutant mice closely phenocopied some of the abnormalities observed in patients with CS, including facial dysmorphia and cardiomyopathies. These mice also displayed alterations in the homeostasis of the cardiovascular system, including development of systemic hypertension, extensive vascular remodeling, and fibrosis in both the heart and the kidneys. This phenotype was age dependent and was a consequence of the abnormal upregulation of the renin-Ang II system. Treatment with captopril, an inhibitor of Ang II biosynthesis, prevented development of the hypertension condition, vascular remodeling, and heart and kidney fibrosis. In addition, it partially alleviated the observed cardiomyopathies. These mice should help in elucidating the etiology of CS symptoms, identifying additional defects, and evaluating potential therapeutic strategies.This work was supported by grants from the Spanish Ministry of Education and Science to M. Barbacid (SAF2003-05172 and SAF2004-20477-E) and X.R. Bustelo (SAF2006-01789); from the VI Framework Programme of the European Union to M. Barbacid (LSHC-CT-2004-503438, LSHG-CT-2007-037665, and LSHGCT-2006-037188); from the National Cancer Institute, NIH, to X.R. Bustelo (5R01-CA73735-10); from the Fondo de Investigación Sanitaria to C. Guerra (PI042124); from the Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, to X.R. Bustelo (RD06/0020/0001); from the Autonomous Community of Madrid to M. Barbacid (GR/SAL/0587/2004) and C. Guerra (GR/SAL/0349/2004); from the Autonomous Government of Castilla-León to X.R. Bustelo (SA053A05); and from the Fundación de la Mutua Madrileña de Automóviles to M. Barbacid. All Spanish funding to X.R. Bustelo is cosponsored by the European Union FEDER program. A.J. Schuhmacher is supported by a FPU fellowship from the Spanish Ministerio de Educación y Ciencia (MEC). V. Sauzeau is partially supported by a Juan de la Cierva postdoctoral contract from the MEC.Peer Reviewe

In Vitro and In Vivo Approaches to Assess Rho Kinase Activity

International audienceIncreased arterial tone and the resulting rise in peripheral vascular resistance are major determinants of the elevated arterial pressure in hypertension. The RhoA/Rho kinase signaling pathways are now recognized as a major regulator of vascular smooth muscle contraction and arterial tone. Here we describe methods to directly and indirectly assess Rho kinase activity in vitro and in cells and tissues

Régulation de la protéine G monomérique RhoA par la voie de signalisation NO/PKG dans le système vasculaire (implication dans l'hypertension artérielle pulmonaire chronique)

La protéine RhoA est impliquée dans de nombreuses fonctions et pathologies vasculaires. L'objectif de ma thèse a été d'identifier de nouveaux récepteurs qui activent RhoA et d'étudier son contrôle par les facteurs endothéliaux. Mes résultats mettent en évidence le couplage des récepteurs aux nucléotides extracellulaires à l'activation de RhoA/Rho-kinase responsable de la contraction et de la migration des cellules musculaires lisses. De même, les effets contractiles et mitogènes de l'urotensine II impliquent l'activation de cette voie. L'ensemble des pathologies artérielles dépendantes de RhoA est associé à une altération de l'endothélium. L'hypothèse est que RhoA pourrait être régulée par les facteurs endothéliaux. Mes travaux montrent que le NO, via la PKG, inhibe RhoA et ainsi la contraction des cellules musculaires lisses. D'autre part, nous montrons que la voie NO/PKG exerce un contrôle positif sur l'expression de RhoA qui est impliquée dans l'hypertension artérielle pulmonaire.NANTES-BU Sciences (441092104) / SudocSudocFranceF