Proyectos

Appropriate arterial function and structure are vital for a proper cardiovascular performance and therefore fundamental for a healthy life. Arterial function depends on cellular and molecular mechanisms but as well on the structural features. In fact, it has been shown that structural and biomechanical properties of vessels are very much related to several cardiovascular pathologies, such as systemic and pulmonary hypertension. Chronic lack of oxygen (hypoxia), may determine an impairment of the cardiovascular function, potentially deriving in pulmonary/systemic hypertension and cardiac failure. This is particularly relevant in human populations exposed to high altitude (above 2500m), either in chronic continuous (permanent inhabitants) or chronic intermittent (shifts of high altitude workers, such as miners, custom agents astronomers; and mountaineers) fashions. Most of the studies about vascular effects of chronic hypoxia have focused on function and molecular mechanisms involved in the pathophysiological responses. However, few is known about the biomechanical responses of systemic, pulmonary and cerebral arteries in these conditions, particularly in the chronic exposure to hypoxia as it happens in high-altitude population and in several diseases in lowlanders. The main goal of this project is to determine the vascular biomechanical characteristics of a representative rat paradigm of living under hypoxia and, in addition, establish the modelling, numerical simulation and experimental validation of the biomechanical responses of arterial vessels. Specifically, three different types of vessels (aorta, carotid and femoral), at different age (neonates, juveniles and adults) will be analyzed, either under normoxic, chronic permanent hypoxic or chronic intermittent hypoxic conditions. Small rodents have been extensively used as a paradigm of cardiovascular and vascular function, allowing massive steps in the knowledge of mechanisms involved in cardiovascular pathologies. Using a rat model, we will deeply analyze the biomechanical properties of the vasculature of animals exposed to chronic hypoxia (permanent or intermittent) in a hypobaric chamber, adding substantial data for the comprehension of the vascular biomechanical behaviour under these conditions. The need for a better understanding of the biomechanical response of arteries leads to the development of constitutive models that may define realistic and reliable stress-strain relationships. In this project, several constitutive models aimed at describing the biomechanical behaviour of soft tissues will be assessed. The specific aspects to be taken into account of the vessels biomechanical characterisation are: incompressibility of the tissue, presence of large deformations, isotropic and anisotropic material behaviours, residual stresses, rate dependent (i.e., viscous) effects, damage and active response. The material characterisation of the biomechanical behaviour of rat arteries will be performed via in-vitro biaxial and myograph tensile tests and ring opening test, considering standard, cyclic and relaxation loading conditions. From these experiments, the material characterisation will also involve the derivation of the material parameters. Specifically, we will address the development of algorithms for the treatment of non-linearities in the fitting procedure and sensitivity analysis to determine the consistency of the parameters found with this methodology. The analysis will also include comparison between the numerical predictions of the different constitutive models in their application to experimental data to be measured in this project. The analysis of the response of pressurised straight arterial vessels will be additionally carried out. Importantly, this test mimics the in-vivo physiological conditions of the arterial vessel. Aside from the internal pressure, axial stretching is usually considered. As a non-uniform biaxial stress state is commonly developed in this test, this fact extends the validity of the material characterisation. The material response will be described via the constitutive models previously characterised. Due to the complex stress and strain patterns developed in this problem, numerical simulations defined in the context of the finite element method will be performed. The obtained numerical results will be validated with the corresponding experimental measurements. Moreover, we expect to characterise the biomechanical properties of different vascular beds, representatives of systemic, pulmonary and cerebral circulations. We shall also describe the effects of chronic hypoxia on these properties. Furthermore, we will perform histological analyses to assess the ultrastructure and the wall components of the intima, media and adventitia layers and relate them with the biomechanical findings. The outcomes of this project will enhance the knowledge necessary to integrate the functional, structural and biomechanical properties of vascular tissues. Clearly, our data will provide useful information not only for vascular pathophysiology understanding, but also for optimization of medical diagnosis, prognosis and potential therapeutic approaches to the related pathologies

Overweight and Obesity are worldwide epidemic conditions defined as a body mass index (BMI) ≥ 25 and ≥30, respectively, characterized by an excessive accumulation of adipose tissue in the body that impairs both physical and psychosocial health and well-being [1]. Notably, according to the Chilean National Health Survey (ENS 2009-2010), 60% of woman in reproductive age (i.e. 15 and 44 years) are overweight or obese [2] with detrimental consequences on women as well as offspring health at long term. Epidemiological evidences have recognized pre-gestational obesity and, in a lesser degree, excessive gestational weight gain (GWG) as independent risk factors in the development of maternal complications and adverse perinatal outcomes [3]. This includes congenital malformations, perinatal death, large for gestational age babies (LGA, >p 90th), macrosomia (> 4 Kg at birth), birth dystocia, neonatal hypoglycemia, and others [4]. This evidences leaded the Institute of Medicine of the National Academy of Sciences of the United States (IOM) to provide guidelines for maternal weight gain in 1990, updated in 2009 [5] in order to improve maternal and perinatal outcomes. Conversely, a large number of evidence has shown that a deficient maternal nutrition during pregnancy affects the early in utero development, inducing adaptations/responses in the fetus and neonate which have been associated to increased risk of diseases in adulthood [6]. This concept, known as "Developmental Origins of Health and Disease” (DOHaD, referred also as intrauterine programming in this proposal) which was coined by Professor David Barker, who reported the association of low birth weight and the risk of T2DM and cardiovascular disease. Notably not only in utero under-nutrition (e.g. intrauterine growth restriction) but also over-nutrition (e.g. macrosomia, LGA) increase the cardiometabolic risk in the offspring [7-9]. Whilst the effect of maternal undernutrition on the risk of disease in the offspring has been extensively addressed, new efforts are required to clarify how increased maternal obesity and body fat previous and during pregnancy impinge an increased cardiometabolic risk in the progeny. In this context, in addition to perinatal complications associated with maternal obesity (MO), there is evidence of persistent adverse effects in the offspring throughout their lifespan. In fact, excessive maternal gestational weight gain is associated with a 2-fold increase in cardiometabolic risk in the offspring, which is evidenced since early infancy [10]. Notably, this effect is substantially higher (~5-fold increase) in offspring from women that initiate their pregnancy with obesity [11, 12]. This is relevant due to the high rate of childhood overweight/obesity in Chile (32.2% of the children ≤ 6 yo are overweight/obese, according to the ENS 2010), which is a strong predictor of later cardiometabolic complications in adulthood [13]. The effect of MO on susceptibility to obesity in the offspring appears to be independent of the presence of gestational diabetes (GDM) [14, 15]. This could have its origin during fetal development since it has been described that newborn whose mothers are obese, with normal glycaemia during gestation, present an increased adiposity versus lean mass compared to control pregnancies [16]. In this respect, it is noteworthy that the size of abdominal adipose tissue and hepatocellular lipid content in neonates strongly correlates with maternal BMI [17-19]. This has been also observed in offspring from pregnant obese non-human primates and mice, which exhibit increased accumulation of hepatic triglycerides and ceramides even before birth [17-19]. Furthermore, MO also relates with metabolic disturbances in the newborn, such as a reduced insulin sensitivity and altered inflammatory markers [20]. Altogether these data suggest that postnatal obesity could be programmed by MO during fetal development, however the mechanisms underlying the intrauterine programming of obesity are currently unknown. In this context inflammation, a mechanism exacerbated in pregnancies with MO, represents a potential pathway participating in this process.

Nuestro proyecto, espera dos grandes aportes. Un primer resultado de este proyecto corresponde al mejor conocimiento acerca de la interrelación que existe entre los problemas de salud mental y otras condiciones que afectan la salud de los trabajadores, como es el caso de los accidentes. Y lo mismo se puede señalar respecto del mejor conocimiento de los factores que inciden en el ausentismo, y por tanto en las consecuencias que este puede tener para los trabajadores y para las empresas. Un segundo resultado se refiere al desarrollo de instrumentos para la identificación de este tipo de problemas en los trabajadores. Uno de los primeros pasos para el desarrollo de intervenciones es contar con encuestas y/o escalas (instrumentos psicométricos) que sean eficientes en la identificación de estos síntomas mentales, lo más breve posibles, focalizada en aquellos síntomas más relevantes y adaptada para la realidad de nuestro país, y en especial para ser aplicada a trabajadores dentro de nuestras ambientes laborales.

Asthma is a chronic disease that affects young children starting mostly in the first years of life; with a high prevalence across countries globally. In Santiago, Chile, the prevalence of asthma among children 6-7 and 13-14 years of age is 11% and 15%, respectively; and for recurrent wheezing during the first year of life is as high as 22%. Asthma is the fourth most common cause of disability-adjusted life years for children aged 10–14 yrs and an important cause of reduced quality of life and exercise tolerance, higher rates of school absenteeism and hospitalization. A recent study done in Santiago reports that 30.4% of all infants visits to the emergency department presented with wheezing and wheezing exacerbations accounted for 8.4% hospital admission. Treatment of asthma currently focuses in reducing symptoms; however the morbidity remains high due to limited curative options and the unresolved etiology of asthma. There are no well-established methods or diagnostic tools to indentify the risk to develop asthma, and the current accepted practice base the diagnosis on parental or self-reported symptoms. One of the most accepted tools to predict asthma early in the infancy was developed by our team (the Asthma Predictive Index); and has been incorporated in most asthma early diagnosis guidelines globally. However, there is growing evidence supporting that beside genetic inheritance, maternal health during gestation represents an important factor conditioning the risk of asthma in the offspring. Thus a better understanding of pathogenesis of asthma in the neonatal period throughout uncovering the mechanisms that explain the associations between fetal life cues with asthma would contribute to an early prevention and treatment. Immune function plays a central role in the development of asthma, but the relative contribution of different immune cell types (i.e. mast cells, eosinophils, lymphocytes, neutrophils and monocytes/macrophages) to this disease is still under examination. Nonetheless, it is clear that asthma presents an altered expression of pro-inflammatory molecules (IL-12, TNF-α) and anti-inflammatory mediators (IL-10, IL-4). Notably, modulation of M1-M2 polarization of monocytes and macrophages seems to be crucial for the development of altered immune response in asthma, and this process would be tightly regulated by epigenetic mechanisms (i.e. DNA methylation, histone modifications). Numerous clinical and epidemiological studies have underlined the detrimental or beneficial role of nutritional factors in complex inflammation-related disorders such as allergy and asthma. It is now progressively better established that most of this risk is influenced in the very early stages of development, by a process of “early life programming” in which epigenetic mechanisms will actively participates. Remarkably, maternal obesity during gestation associates with increased plasma levels of TNF-α at birth, and a 4-fold increased risk of asthma in the offspring. Also growing data show that epigenetic mechanisms exert an important control on the altered immune function observed in autoimmune and inflammatory controlling the expression of key genes. However, whether maternal obesity during pregnancy influences the immune function and the risk of asthma in the offspring throughout epigenetic mechanisms has not been addressed. In this context we propose that “Maternal obesity during pregnancy increases the risk of developing asthma in the offspring by an epigenetic-mediated programming of the inflammatory response in monocytes. This programmed inflammatory response is characterized by a higher expression of pro-inflammatory molecules (IL-12, TNF-α) along with a lower expression of anti-inflammatory mediators (IL-10, IL-4Rα) which occurs due to changes in the methylation status of the promoter regions of these immune response- key genes”. Studying a cohort of 400 children (0–3 years old) born from mother with or without obesity during pregnancy this proposal will address whether: a) the increased risk of asthma in children born from obese mother can be observed at 3 years of life; b) the increased asthma risk in these children associates with an altered immune reactivity in monocytes at birth; and c) the altered immune reactivity in monocytes occurs along with changes in the DNA methylation status at the promoter regions of asthma-related genes. This study would reveal new molecular markers contributing to early diagnosis of asthma during childhood, as well as establish the real effects of epigenetic mechanisms modeling the immune function and responses at long term.

1. La hipertensión arterial esencial es el principal factor de riesgo cardiovascular, con una prevalencia del 30-40 % en la población adulta, que llega al 75% en mayores de 65 años. A pesar del desarrollo de fármacos, la efectividad de los tratamientos actuales es limitada. En cerca de 50% de los pacientes, el tratamiento farmacológico no logra controlar la presión arterial elevada. La hipertensión es de origen multifactorial, pero existe consenso que la disfunción endotelial juega un papel primordial en su desarrollo. La bio-disponibilidad del óxido nítrico (NO), que es el principal vasodilatador, está reducida en la mayoría de las enfermedades vasculares debido al estrés oxidativo y al aumento de la expresión y actividad de arginasa, una enzima que compite por el sustrato del NO. Este proyecto desarrollamos una nueva combinación farmacológica dirigida a proteger el NO, combinando las propiedades vasodilatadoras y anti-remodeladoras del inhibidor de la arginasa ácido 2(s)-Amino-6-Boronohexanoico (ABH) y de del antioxidante N-Acetilcisteína (NAC). Buscamos introducir una nueva forma de enfocar el tratamiento para la hipertensión arterial, atacando con esta formulación farmacológica los mecanismos moleculares que subyacen a la disfunción endotelial: baja bio-disponibilidad de NO y aumento del estrés oxidativo. En un modelo de hipertensión en ratas inducido por hipoxia intermitente crónica, cuya presión fue monitorizada por telemetría, evaluamos el efecto de la combinación sobre la hipertensión arterial y la disfunción vascular. La combinación ABH+NAC reduce la presión arterial elevada, revierte la reducción del diámetro interno de las arterias, regulariza la función endotelial y contráctil en arterias y disminuye el estrés oxidativo. No encontramos efectos adversos sobre la función renal y hepática. Estos resultados dieron origen a la presentación de dos solicitudes de patentes en Chile y de protección CTP en el extranjero para un nuevo método de síntesis de ABH y de los efectos benéficos de la combinación sobre la hipertensión y disfunción endotelial. En conjunto con el Laboratorio Andrómaco se estableció una estrategia de propiedad industrial ex post, así como las acciones científico-tecnológicas conducentes a las siguientes fases de desarrollo del medicamento: pruebas preclínicas en otros modelos de hipertensión, desarrollo galénico y/o fase clínica I. Dado que el resultado de producción de la formulación deberá pasar aún por pruebas clínicas antes de ser dispuesto a los beneficiarios, la patente de este nuevo fármaco podrá ser utilizada como activo principal para capturar capital de inversión que permita continuar con este proceso, o bien para licenciar a empresas con interés en el rubro. Junto a la Fundación del Adulto Mayor, desarrollamos actividades de extensión con la finalidad de informar a la comunidad y con ello contribuir a prevenir los efectos deletéreos de la hipertensión en la población de mayor riesgo cardiovascular. En esta propuesta se espera validar una nueva terapia antihipertensiva que ataque los mecanismos fisiopatológicos principales de la disfunción vascular que conduce a la hipertensión arterial esencial.

Compelling evidence shows that adverse intrauterine conditions increase the risk to develop cardiometabolic diseases in the adulthood. This concept has been called ‘Developmental Origins of Health and Disease’ (DOHaD) and relies on the activation of mechanisms sensing and signaling a diversity of stimuli during early development that later lead to higher risk of disease. The mechanisms that have been broadly suggested to be involved in these processes are epigenetic modifications in key gene promoters that could ‘record’ normal and abnormal perinatal stimuli. Intrauterine oxidative stress is a common feature in conditions with altered fetal growth (i.e. intrauterine growth restriction –IUGR-, or macrosomia). Several cellular processes require the participation of pro-oxidant molecules which are normally neutralized by antioxidant defenses. However, under determined conditions the pro-oxidants overcome these defenses inducing oxidative stress. The latter is an important stimulus that regulates vascular function and cardiovascular physiology, playing a key role in the development of cardiovascular diseases, regulating negatively the bioavailability of the main vasodilator nitric oxide (NO). In addition, the vascular system presents a high phenotypic plasticity during life, which is modulated and restricted by epigenetic mechanisms (including DNA methylation, histone post-translational modifications and micro RNAs). Interestingly, cultured placental endothelium derived from complicated pregnancies presents persistent abnormal phenotypes, characterized by altered expression of proteins involved in NO-dependent vasodilation (i.e. eNOS and arginase), suggesting an early onset of endothelial dysfunction. Preliminary data from in vitro experiments, show that this altered expression of eNOS in IUGR placenta-derived endothelial cells is accompanied by epigenetic alterations in the promoter of its gene. Moreover, these cells can be reprogrammed to a “normal type”, interfering with the molecular machinery that preserves the DNA methylation pattern. However, there are no studies addressing the role of the pro-oxidant status associated to IUGR on the epigenetic programming of placental vascular dysfunction, and whether these epigenetic changes reflect those present in other fetal vascular beds or in the adult cardiovascular system. Therefore, we hypothesized that the in utero oxidative stress, that characterizes IUGR, induces an epigenetic programming of the endothelial function, which is linked to abnormal umbilical and fetal vascular reactivity and higher risk of adult cardiovascular disease. These epigenetic changes lead to an altered expression of endothelial function-related proteins and to their response to superimposed oxidative stress in umbilical and systemic arteries in the fetus and adult guinea pig. If true, the development of IUGR in the presence of antioxidant treatment should prevent the vascular impairment in the fetus and the adult guinea pig. This hypothesis will be tested in an IUGR guinea pig model according to the following general aims (GA): GA-1. To determine whether the IUGR-associated oxidative stress induces endothelial dysfunction in umbilical and systemic arteries, altering the basal expression of proteins implicated in the NO-dependent vasodilation (NO-DV) pathway and their response to oxidative stress; GA-2. To determine in umbilical artery (UAEC) and aortic (AEC) endothelial cells whether endothelial dysfunction induced by fetal oxidative stress associates with epigenetic changes in the promoter of genes implicated in the NO-DV pathway, altering their response to oxidative stress in vitro; GA-3. To determine in adult life whether the IUGR-associated oxidative stress results in increased markers of endothelial dysfunction, oxidative stress and cardiovascular disease. GA-4. To determine in AEC of adults born with IUGR whether endothelial dysfunction induced by fetal oxidative stress is associated with epigenetic changes in the promoter of genes implicated in the NO-DV pathway and their response to oxidative stress in vitro, correlating them with those found at term of gestation. IUGR will be induced by uterine artery ligation in a pregnant sow, and the role of oxidative stress in the vascular programming will be analyzed treating IUGR pregnancies with the antioxidant N-acetylcysteine. The acute effect of pro- and anti-oxidants on vascular reactivity and NO-dependent vasodilation will be determined by wire-myography in IUGR near-term fetuses and adults guinea pig arteries. Further, the presence of proteins related with NO-dependent vasodilation (eNOS, arginase, HO-1, NQO1 and DHFR) in these vessels will be determined by immunohistochemistry. Endothelial epigenetic programming will be analyzed in primary cell cultures from UAEC and AEC in near-term fetuses and AEC in adult guinea pigs. Our expected outcome is to demonstrate that oxidative stress is one of the main sources of the IUGR-induced dysfunction in fetal and adult vascular beds. Further, we expect that the NAC treatment should be able to prevent partially or totally the programmed vascular impairment. This project is not only relevant to uncover the developmental mechanisms that determine short- and long-term vascular dysfunction, but also to propose eventual treatments in complicated pregnancies, that unfortunately present a very high rate in humans.