Proyectos

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.

Objetivo de la investigación: i) Evaluar el rol protector del ácido protocatecuico sobre la alteración en la señalización de la insulina e inflamación en tejido adiposo visceral de sujetos normo peso y obesos. ii) Estudiar el rol protector del ácido protocatecuico sobre la alteración en la señalización de la insulina inducida por alta glucosa en tejido visceral de sujetos delgados.

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.