Papel de la ruta CN/NFAT en el desarrollo de hipertrofia cardiaca patológica

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 20-02-2015La hipertrofia cardiaca patológica es un mecanismo por el cual el corazón reduce el estrés que se produce en su pared a consecuencia de patologías como hipertensión arterial, infarto de miocardio o estenosis aórtica. Este mecanismo se manifiesta con un aumento del tamaño del corazón y del área de los cardiomiocitos, la aparición de fibrosis en el miocardio y de disfunción cardiaca. La relación entre la hipertrofia y la activación de la ruta CN/NFAT es conocida desde hace años a través de trabajos con animales transgénicos o knock-out para CN o miembros de NFAT y con el fármaco inmunosupresor CsA, un inhibidor de CN; sin embargo, no se conoce en detalle la cinética de activación de la ruta en esta patología, ni el papel exacto de la CN en ella en la edad adulta. A la vez, existe mucha variabilidad en los resultados obtenidos con CsA, y se ha reportado que CsA podría tener efectos secundarios en animales y humanos derivados de su acción en dianas que no son la CN. Para circunvalar estos problemas, en este trabajo estudiamos esta patología en animales de genotipo silvestre y con deleción condicional y cardioespecífica de CN, mediante un modelo de infusión de AngII. Los resultados indican que la inhibición con CsA de CN consigue reducir todos los parámetros de la hipertrofia cardiaca excepto la fibrosis; en cambio, los animales en los que inducimos deleción genética de CN son capaces de inhibir también la fibrosis. Esto nos indica la importancia central de la CN en el desarrollo de la patología. Todos los animales tratados con AngII y co-tratados con CsA+AngII presentaban un aumento de la presión arterial. Esto sugiere que la hipertensión no es esencial en el desarrollo de la hipertrofia. Hemos comprobado que la activación de la ruta CN/NFAT es muy rápida tras la infusión de AngII, y que el fenotipo hipertrófico se produce tras 3 días de estimulación. También hemos observado que la deleción genética de CN o su inhibición con CsA impactan diferencialmente en el perfil génico que AngII induce. En el caso de colágenos, y a pesar de que la deleción de CN inhibía la fibrosis, sigue existiendo un aumento en su expresión, lo que sugiere que la presencia de CN en el cardiomiocito es necesaria en la deposición del colágeno producido en el tejido. Además, estudiamos una manera novedosa de inhibir el desarrollo de hipertrofia, usando el péptido inhibidor de CN, LxVP, por medio de vectores virales AAV9. Si bien la transducción que obtuvimos en el corazón fue alta, no conseguimos inhibir la activación de la ruta en su totalidad.The pathological cardiac hypertrophy is a mechanism by which the heart reduces the stress in its wall, as a consequence of diseases as arterial hypertension, myocardial infarction o aortic stenosis. This mechanism occurs with an increase in the heart size and cardiomyocyte area, and the appearance of cardiac fibrosis and dysfunction. The relationship between hypertrophy and CN/NFAT pathway activation is known for years through several studies where the authors used transgenic or knock-out animals for CN or different NFAT members and immunosuppressive drugs as CsA, which inhibits CN; however, neither the kinetics of activation of the pathway in this pathology, nor the exact role of CN in it in the adult age are known. Also, there is high variability in the results obtained with CsA as inhibitor, and has been reported that CsA could give secondary effects in both animals and humans derived of interference with other targets than CN. Therefore, we studied this pathology in wild type and conditional cardiospecific CnB knock-out mice, using an AngII-infusion model. The results indicate that CsA-mediated CN inhibition is able to reduce all features of cardiac hypertrophy except fibrosis; on the other hand, CnB deleted mice are able to inhibit fibrosis as well. This indicates the pivotal role of CN in the development of the pathology. All AngII- and CsA+AngII-treated animals presented an increase of blood pressure. This suggests that hypertension is not essential for the development of cardiac hypertrophy. Also, we have seen that the activation of CN/NFAT pathway is fast after the infusion of AngII, and that the hypertrophic phenotype is produced as soon as 3 days after the beginning of stimulation. Also, we have observed CnB deletion and CsA-mediated inhibition impact differentially in the AngII-induced gene expression profile. The case of collagens expression show us that, despite of fibrosis being inhibited by CnB deletion, there is still an increase in their expression, suggesting that the presence of CN in the cardiomyocyte is needed for the deposition of this collagen. Also, we studied a new approach to inhibit cardiac hypertrophy, using the CN inhibitory peptide, LxVP, delivered by viral vectors AAV9. We observed that, even with a high transduction achieved in the heart with this method, we couldn’t inhibit completely the pathway activation

Cardiomyocyte calcineurin is required for the onset and progression of cardiac hypertrophy and fibrosis in adult mice

Previous studies have demonstrated that activation of calcineurin induces pathological cardiac hypertrophy. In these studies, loss-of-function was mostly achieved by systemic administration of the calcineurin inhibitor cyclosporin A. The lack of conditional knockout models for calcineurin function has impeded progress toward defining the role of this protein during the onset and the development of cardiac hypertrophy in adults. Here, we exploited a mouse model of cardiac hypertrophy based on the infusion of a hypertensive dose of angiotensin II (AngII) to model the role of calcineurin in cardiac hypertrophy in adulthood. AngII-induced cardiac hypertrophy in adult mice was reduced by treatment with cyclosporin A, without affecting the associated increase in blood pressure, and also by induction of calcineurin deletion in adult mouse cardiomyocytes, indicating that cardiomyocyte calcineurin is required for AngII-induced cardiac hypertrophy. Surprisingly, cardiac-specific delection of calcineurin, but not treatment of mice with cyclosporin A, significantly reduced AngII-induced cardiac fibrosis and apoptosis. Analysis of pro-fibrotic genes revealed that AngII-induced expression of Tgfβ-family members and Lox was not inhibited by cyclosporin A but was markedly reduced by cardiac-specific calcineurin deletion. These results show that AngII induces a direct, calcineurin-dependent pro-hypertrophic effect in cardiomyocytes, as well as a systemic hypertensive effect that is independent of calcineurin activity

Long non-coding RNA LASSIE regulates shear stress sensing and endothelial barrier function

Blood vessels are constantly exposed to shear stress, a biomechanical force generated by blood flow. Normal shear stress sensing and barrier function are crucial for vascular homeostasis and are controlled by adherens junctions (AJs). Here we show that AJs are stabilized by the shear stress-induced long non-coding RNA LASSIE (linc00520). Silencing of LASSIE in endothelial cells impairs cell survival, cell-cell contacts and cell alignment in the direction of flow. LASSIE associates with junction proteins (e.g. PECAM-1) and the intermediate filament protein nestin, as identified by RNA affinity purification. The AJs component VE-cadherin showed decreased stabilization, due to reduced interaction with nestin and the microtubule cytoskeleton in the absence of LASSIE. This study identifies LASSIE as link between nestin and VE-cadherin, and describes nestin as crucial component in the endothelial response to shear stress. Furthermore, this study indicates that LASSIE regulates barrier function by connecting AJs to the cytoskeleton

4to. Congreso Internacional de Ciencia, Tecnología e Innovación para la Sociedad. Memoria académica

Este volumen acoge la memoria académica de la Cuarta edición del Congreso Internacional de Ciencia, Tecnología e Innovación para la Sociedad, CITIS 2017, desarrollado entre el 29 de noviembre y el 1 de diciembre de 2017 y organizado por la Universidad Politécnica Salesiana (UPS) en su sede de Guayaquil. El Congreso ofreció un espacio para la presentación, difusión e intercambio de importantes investigaciones nacionales e internacionales ante la comunidad universitaria que se dio cita en el encuentro. El uso de herramientas tecnológicas para la gestión de los trabajos de investigación como la plataforma Open Conference Systems y la web de presentación del Congreso http://citis.blog.ups.edu.ec/, hicieron de CITIS 2017 un verdadero referente entre los congresos que se desarrollaron en el país. La preocupación de nuestra Universidad, de presentar espacios que ayuden a generar nuevos y mejores cambios en la dimensión humana y social de nuestro entorno, hace que se persiga en cada edición del evento la presentación de trabajos con calidad creciente en cuanto a su producción científica. Quienes estuvimos al frente de la organización, dejamos plasmado en estas memorias académicas el intenso y prolífico trabajo de los días de realización del Congreso Internacional de Ciencia, Tecnología e Innovación para la Sociedad al alcance de todos y todas

Long non-coding RNA in vascular disease and aging

Cardiovascular diseases are the most prominent cause of death in Western society, especially in the elderly. With the increasing life expectancy, the number of patients with cardiovascular diseases will rise in the near future, leading to an increased healthcare burden. There is a need for new therapies to treat this growing number of patients. The discovery of long non-coding RNAs has led to a novel group of molecules that could be considered for their potential as therapeutic targets. This review presents an overview of long non-coding RNAs that are regulated in vascular disease and aging and which might therefore give insight into new pathways that could be targeted to diagnose, prevent, and/or treat vascular diseases

Long Non-Coding RNA in Vascular Disease and Aging

Cardiovascular diseases are the most prominent cause of death in Western society, especially in the elderly. With the increasing life expectancy, the number of patients with cardiovascular diseases will rise in the near future, leading to an increased healthcare burden. There is a need for new therapies to treat this growing number of patients. The discovery of long non-coding RNAs has led to a novel group of molecules that could be considered for their potential as therapeutic targets. This review presents an overview of long non-coding RNAs that are regulated in vascular disease and aging and which might therefore give insight into new pathways that could be targeted to diagnose, prevent, and/or treat vascular diseases

Long noncoding RNA in cardiac aging and disease

Cardiovascular diseases (CVDs) are the main cause of morbidity and mortality in Western society and present an important age-related risk. With the constant rise in life expectancy, prevalence of CVD in the population will likely increase further. New therapies, especially in the elderly, are needed to combat CVD. This review is focused on the role of long noncoding RNA (lncRNA) in CVD. RNA sequencing experiments in the past decade showed that most RNA does not code for protein, but many RNAs function as ncRNA. Here, we summarize the recent findings of lncRNA regulation in the diseased heart. The potential use of these RNAs as biomarkers of cardiac disease prediction is also discussed

Cardiomyocyte calcineurin is required for the onset and progression of cardiac hypertrophy and fibrosis in adult mice

Previous studies have demonstrated that activation of calcineurin induces pathological cardiac hypertrophy (CH). In these studies, loss-of-function was mostly achieved by systemic administration of the calcineurin inhibitor cyclosporin A. The lack of conditional knockout models for calcineurin function has impeded progress toward defining the role of this protein during the onset and the development of CH in adults. Here, we exploited a mouse model of CH based on the infusion of a hypertensive dose of angiotensin II (AngII) to model the role of calcineurin in CH in adulthood. AngII-induced CH in adult mice was reduced by treatment with cyclosporin A, without affecting the associated increase in blood pressure, and also by induction of calcineurin deletion in adult mouse cardiomyocytes, indicating that cardiomyocyte calcineurin is required for AngII-induced CH. Surprisingly, cardiac-specific deletion of calcineurin, but not treatment of mice with cyclosporin A, significantly reduced AngII-induced cardiac fibrosis and apoptosis. Analysis of profibrotic genes revealed that AngII-induced expression of Tgfβ family members and Lox was not inhibited by cyclosporin A but was markedly reduced by cardiac-specific calcineurin deletion. These results show that AngII induces a direct, calcineurin-dependent prohypertrophic effect in cardiomyocytes, as well as a systemic hypertensive effect that is independent of calcineurin activity.Spanish Ministerio de Economia, Industria y Competitividad (MEIC); Pro-CNIC Foundation; Severo Ochoa Center of Excellence (MEIC award) [SEV-2015-0505]; MEIC [SAF2012-34296, SAF2015-636333R]; Fundacio La Marato TV3 [CNIC is supported by the Spanish Ministerio de Economia, Industria y Competitividad (MEIC) and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505). Support was also provided by grants from MEIC (SAF2012-34296 and SAF2015-636333R to JMR), Fundacio La Marato TV3 (20151330 to JMR), Ministerio de Sanidad CIBERCV (CB16/11/00264 to JMR), Comunidad de Madrid (AORTASANA-CM; B2017/BMD-3676 to JMR), and Red de Investigacion Cardiovascular (RIC) (RD12/0042/0022 to JMR and RD12/0042/0056 to LJJ-B), and an FPU fellowship (AP 2009-1713 to NL-V). We thank Dr S. Bartlett for English language editing, Dr Fatima Sanchez Cabo for statistical advice, Dr M. R. Campanero, Dr J. F. Nistal, and Dr B. Ibanez for critical reading of the manuscript. We also thank Dr G. R. Crabtree for providing Cnb1Delta/flox mice, A. Peral for technical assistance, and the ultrasonographers A.V. Alonso and L. Flores for technical support.]; Ministerio de Sanidad CIBERCV [CB16/11/00264]; Comunidad de Madrid (AORTASANA-CM) [B2017/BMD-3676]; Red de Investigacion Cardiovascular (RIC) [RD12/0042/0022, RD12/0042/0056]; FPU fellowship [AP 2009-1713]S

Role of Calcineurin in the development of cardiac hypertrophy

CNIC Scientific Retreat. Madrid, 23-24 Nov. 2012Previous studies have shown that the Calcineurin (CN)/NFAT signalling pathway is an established important regulator of cardiac hypertrophy. However, most studies are based on gain- and loss-of-function initiated at or before birth, and there are no detailed studies about the role of this pathway in the progression of pathological cardiac remodelling during adulthood. To investigate the role of CN in the progression of disease we have analyzed the effect of cyclosporin A (CsA), a pharmacological CN inhibitor, on angiotensin II (AngII)-induced hypertension and cardiac hypertrophy in mice. Our results show that although CsA treatment does not alter the AngII-induced hypertension, it blocks the increase of cardiac mass, ventricular Wall thickness and cardiomyocyte size when hypertrophy is established (21 days after AngII infusion). Unexpectedly, AngII infusion induces hypertrophy as early as three days and this effect is also inhibited by CsA. Interestingly, CsA impairs cardiac function and has no effect on AngII-induced fibrosis. A complementary analysis based on the inducible genetic deletion of CN in heart just before the induction of hypertrophy indicates that CN deficiency results in a clear reduction of hypertrophy accompanied by a blockade of AngII-induced fibrosis. Furthermore, CN deficient mice, but not CsA-treated mice, do not develop systolic dysfunctionN

Adamts1 in vascular homeostasis and remodelling

IMPRS Max Planck - Annual Retreat. Bad Nauheim, junio 2015Aneurysms involving the aortic root and the ascending aorta leading to dissections are the major diseases affecting the aorta and a common cause of premature deaths ranking as high as the XXth cause of death in developed countries. The major constituent of the vessel wall is the extracellular matrix (ECM). It forms part of the basic structure of blood vessels and provides structural and mechanical support through elasticity, stiffness, and intercellular communication. Changes in ECM proteins expression, assembly, cross-linking, and degradation can trigger physiological pathological conditions in the vascular wall, including atherosclerosis, aneurysms, stenosis and hypertension (Hellenthal et al. 2009). Mutations in genes which encode ECM proteins which affects mechanical properties of tissues are present in some inherited connective tissue disorders such as Marfan syndrome (MS), Loeys-Dietz syndrome (LDS), vascular type of Ehlers-Danlos syndrome (EDS-IV), and familial forms of non-syndromic thoracic aneurysm and dissection (FTAAD) (Hoffjan 2012, Van Laer et al. 2014). TGFβ signaling pathway is overactivated in both syndromic and non-syndromic aortic diseases, TGFβ signaling pathway suggesting that it plays a pivotal role in these diseases. The ADAMTS family of of extracellular metalloproteinases degrade proteoglycans and therefore have the potential to modify tissue architecture and function (Stanton 2011). Recently, different works have involved the families, ADAMTS and ADAMTSL (Adamts-like) in fibril microfiber formation thus suggesting a role of these genes in the regulation of TGFβ signalling (Hoffjan et al 2012). Different mutations in ADAMTS/ADAMTSL superfamily members has been described as causative of connective tissue disorders without aortic phenotype (Le Goff et al. 2011). Adamts1 is widely expressed in aortic endothelial and VSMCs during development and in adulthood (Thai et al. 2002; Luque et. al. 2003) and under pathological vascular remodeling in (Jönsson-Rylanderand et al. 2005) and thoracic aneurysm (Pen et. al. 2013). However the role of this metalloproteinase in the vascular wall is poorly understood. Here, we show the potential role of Adamts1 in vascular wall homeostasis using two different approaches, a genetic model of Adamts1 deficient mice and a knocking-down model in aorta using short-interference RNA (siRNA) expressing lentiviruses. Both models, Adamts1 deficient mice and knocking-down present some vascular features that resembles aortic disorders, such as aortic ectasia, fibrosis, proteoglycan accumulation, elastin breaks, TGFb hyperactivation. These phenotype was exacerbated by AngII infusion. These data supports that Adamts1 is essential for vascular integrity in homeostasis and remodelingN