Alejandro González Candia ● Profesor Asociado

EPIGENOMIC PROGRAMMING IN THE EARLY FETAL BLOOD-BRAIN BARRIER BY GESTATIONAL HYPOXIA: CONSEQUENCES FOR THE NEURO-ENDOTHELIAL LIFESPAN.

Non-communicable diseases (NCDs) are responsible for 74% of worldwide human deaths, with cardiovascular causes in the first place (1). NCDs are determined by a combination of environmental, genetic and epigenetic factors. In fact, adverse intrauterine conditions, such as reduced oxygen availability (hypoxia) and oxidative stress, can increase the risk of developing diseases during life, a phenomenon known as Fetal Programming or Developmental Origins of Health and Disease (DOHaD). Intrauterine hypoxia (IUH) affects most of the pregnancies in high altitudes populations (> 2500m) (2-4) and 3-4% in lowlands, with uteroplacental and developmental complications (4,5). We, and a couple of others, have recently shown that IUH determines cardiovascular oxidative stress during lifespan affecting endothelial function and vasodilator capacity, similar to what is seen with aging. The hypoxia-induced responses during development are responsible for fetal survival, but also determine mechanisms that program postnatal cardiovascular function that may increase cardiovascular health risks and accelerate aging (6). This proposal aims to determine the mechanisms and trace the origins and outcomes of cardiovascular dysfunction resulting from intrauterine hypoxia and oxidative stress, and further identify the interrelated senescence mechanisms in the heart and blood vessels. To assess the aforementioned, we will study the effects of IUH on cardiovascular aging along lifespan, as important regulators of the function, structure and biomechanical properties of the cardiovascular system.

The quality of fetal and early post-natal environment influences lifelong health and predicts the risk for a range of non-communicable chronic diseases (NCDs). These observations form the basis of the "Developmental Origins of Health and Disease" (DOHaD hypothesis), which indicates that the intrauterine signals that compromise fetal growth also act to "program" tissue differentiation in a manner that predisposes later illnesses. Interestingly, the DOHaD hypothesis asserts that some aging-associated diseases that occur in adults are closely related to the development and conditions in the intrauterine environment. Thus, aging and aging-associated diseases can be viewed, at least in part, as the result of a developmental program activated early in embryogenesis and persists throughout the organism's lifespan. On the other hand, one of the main consequences of this programming is Fetal Growth Restriction (FGR) and which remains a leading cause of perinatal morbidity and mortality, affecting about 10% of pregnancies, but the incidence is reportedly sixfold greater in low-income countries depending on the region surveyed's nutrition and health access availability. FGR is clinically defined as a fetal weight below the 10th percentile of normal for gestational age, associated with some loss of fetal-placental blood flow diagnosed by ultrasound, and it is a condition in which the potential growth of the fetus is negatively influenced by environmental and maternal factor; the short-term consequences of FGR are low birth weight (LBW) and the corresponding phenotype, which is associated with increased perinatal morbidity and mortality. Besides, the long-term effects include a 2 to 3-fold increase in the risk of developing cerebrovascular disease in adulthood. Indeed, many neurodevelopmental dysfunctions originated in the antenatal period, but few studies have focused on how growth restriction interferes with normal brain development of the blood-brain barrier (BBB) in the FGR neonate. The BBB is a cellular network formed by a monolayer of neuro-endothelial and mural cells. The BBB regulates the transport of molecules into and out of the central nervous system (CNS) (selective permeability and integrity of the BBB). In cerebrovascular aging, BBB breakdown and dysfunction lead to leakages of components into the central nervous system (CNS), contributing to neurological deficits; growing evidence from genomic data shows that FGR vascular dysfunction is mediated by aging, with a series of prominent hallmarks, including genetic and epigenetic alterations. These aging-associated epigenetic changes include DNA methylation, histone modification, chromatin remodeling, and non-coding RNA (ncRNA) regulation; however, how this mechanism regulates the aging process and contributes to aging-related BBB dysfunction remains elusive. We hypothesize that the impaired fetal growth conditions associated with epigenetic programming of aging-related DNA methylation, chromatin remodeling, and miRNA-omic profile of complex junctional genes in the neuro-endothelium, which can alter BBB integrity and permeability, increasing cerebral damage which impacts the perinatal and adulthood neurocognitive function. This hypothesis will be addressed by the study of the effects of gestational chronic hypoxia on the aging epigenetic programming of gene expression of junctional complexes: Tight junction, adherens junction, and Gap junction family’s molecules, as important regulators of the permeability para and transcellular of the BBB. For this, we will use a well-established Guinea pig model of cerebrovascular programming (DOHaD model) to demonstrate DNA methylation shift, chromatin remodeling, miRNA-omic profile, and transcriptomic analyses in neuro-endothelial cells isolated from cortex and hippocampus from animals gestated under hypobaric hypoxia at two stages of life (juvenile period and adulthood). The methodology for this project is an in vivo assess locomotor, exploratory activity, and memory acquisition evaluation, and in vitro determinations of epigenetic regulation of aging in BBB from the cortex, hypothalamus, and neuro- endothelial cell culture primary at different stages of life in animals gestated under hypoxia. Our expected outcome is to improve the knowledge about neuro-endothelial epigenetic programming by aging induced by the FGR and enhance the characterization of those epigenomic patterns and mechanisms associated with BBB breakdown by intrauterine hypoxia. This project aims to demonstrate that the effect of gestational hypoxia can accelerate the permeability of the BBB by epigenetic mechanisms not yet studied and that these changes continue throughout life, producing further deterioration of brain function

Antecedentes: los sobrevivientes de cáncer son una población en rápido crecimiento con necesidades de salud específicas. En los EE. UU., aproximadamente 17 millones de adultos son sobrevivientes de cáncer, lo que representa el 5 % de la población adulta, y las proyecciones indican que este número aumentará a 20 millones para 2026, según el Instituto Nacional del Cáncer de Estados Unidos. Los eventos cardiovasculares son la principal causa (no maligna) de muerte entre los sobrevivientes de cáncer, con un riesgo de muerte aproximadamente siete veces mayor en relación con sus contrapartes de la misma edad. Por tanto, el estudio de los factores de riesgo CV en pacientes oncológicos pediátricos supervivientes es un tema en auge, con ensayos clínicos prospectivos en marcha y que actualmente carecen de una base mecanística. Cada año en Chile se presentan aproximadamente 400 casos nuevos de cáncer infantil, y sabiendo que la sobrevida global es cercana al 60%, llegamos a un número apreciable de niños (alrededor de 240) que logran superar su enfermedad año tras año. Sin embargo, desde el inicio del programa, la monitorización del riesgo CV ha sido insuficiente, a pesar de que el 60-70% de los protocolos del PINDA (Programa Infantil para Drogas Antineoplásicas) incluyen antraciclinas, que son agentes quimioterápicos relacionados con alto riesgo de cardiotoxicidad. En relación a las medidas de intervención disiponibles, es bien sabido que el entrenamiento aeróbico conduce a una mayor capacidad/aptitud cardiorrespiratoria, CCR (inglés CRF; cardiorrespiratoty fitness). Por otro lado, el tipo de entrenamiento, en el caso de resistencia aumenta significativamente la fuerza muscular y mejora la forma física, asociándose a mejoría de los outcomes cardiovasculares en adultos sobreviviente de cáncer. En esta población se ha visto que un CCR más baja se asocia con una mayor carga de síntomas y un mayor riesgo de morbilidad y mortalidad CV por afecciones no cancerosas; sin embargo, la relación entre la aptitud física expresada por la CCR y la ocurrencia de cardiotoxicidad no ha sido bien caracterizadas en la población infantil, o en la transición infantil-adulto, ya que cuentan con un vacío de seguimiento CV. Con base en estos antecedentes, planteamos la hipótesis de que mejorar la condición/aptitud física en una cohorte de sobrevivientes de cáncer infantil a través de la intervención con ejercicios se asocia con una disminución de los factores de riesgo cardiovascular y los marcadores ocurrencia de cardiotoxicidad.

During the last decades, compelling evidence shows how the context in which early life takes place impinges risk or protection for later development of non-communicable chronic diseases. In this regard, impaired fetal growth, as occur in the fetal growth restriction (FGR), leads to a higher risk for later cardiovascular diseases, an effect that would be mediated by accelerated aging at molecular, structural, and functional levels. FGR remains a leading cause of perinatal morbidity and mortality, affecting ~10% of pregnancies, but ranging from 5 to 25% depending on the population surveyed, with a higher prevalence among pregnant women of low socioeconomic status. In the clinic, FGR is normally defined by a fetal weight below the 10th percentile, however, new evidence shows that impaired intrauterine growth may affect several neonates born over the 10th percentile, especially late in pregnancy, which may be missed from the perinatal survey for preventing adverse outcomes. This points out the need for further studies to improve the understanding and identification of altered fetal growth trajectories and their consequences on vascular function. Studies in the placenta show that FGR vascular dysfunction is also found at birth in chorionic and umbilical arteries. We have demonstrated the presence of functional and molecular markers (e.g. vasodilator mediators and epigenetic changes) of endothelial dysfunction in human FGR umbilical and chorionic vessels, findings that have been further confirmed by comparing systemic and umbilical arteries in guinea pigs and chicken FGR models. These traits suggest that umbilical artery endothelial cells (HUAEC), in complement with approaches in animal models, can be used as a surrogate to explore the vascular programming within the fetus, however, their translation to clinical preventive applications for promoting healthy aging deserves further studies. It worth noting that fetal reduced oxygen supply (fetal hypoxia) and altered blood flow patterns (e.g. shear stress) are key clinical markers in the FGR, independently of the constraints leading to impaired growth, and both factors exert a tight control of vascular development and function across life. However, how these key stimuli interact and impose an epigenetic program on the endothelial function remains elusive. This proposal will focus on the crosstalk between hypoxia and shear stress that results in the endothelial programming related to impaired fetal growth, and the molecular mechanisms that mediate the vascular responses to these stimuli. Furthermore, we will address if these molecular markers may allow detecting early vascular aging in FGR subjects beyond the 10th centile cutoff. We hypothesize that “Impaired fetal growth conditions are associated with epigenetic programming of aging- and mechanosensing-related miRNAs and transcripts in the endothelium, which can be triggered by the confluence of altered flow patterns and hypoxia resulting in molecular and structural pro-hypertensive biomechanical vascular properties”. This hypothesis will be addressed by three General Objectives (GO) involving ex vivo, in vitro, and in vivo observational and mechanistic approaches: GO1 To demonstrate, in HUAEC, whether late FGR results in epigenetic changes related to the regulation of vascular aging and the expression of mechanosensing mechanisms involved in the endothelial-dependent relaxation, and their relationship with general prenatal parameters of vascular health. GO1 will be performed by recruiting HUAEC samples from late FGR and control pregnancies, to assess transcriptomic and DNA methylation analyses that will be crossed with prenatal clinical data. GO2 To study, in vivo, whether hypoxia and shear stress differentially regulate mechanosensing pathways involved in the endothelial-dependent relaxation and their relationship with the in vivo and ex vivo vascular properties (e.g. functional and biomechanical). GO2 will be performed in chicken embryos exposed to hypoxia and treated with agents targeting mechanosensing pathways, in which wall shear stress will be determined by Ultrasound Localization Microscopy, with complementary functional, structural, and molecular analyses. GO 3. To study, in cultured HUAEC, whether stimuli related to impaired fetal growth converge in the regulation of mechanosensing- and aging-related transcripts and miRNA, contributing to the cellular programming of endothelial dysfunction. GO3 will be performed in HUAEC exposed, in vitro, to sustained hypoxia and diverse flow patterns (shear stress), in which target DNA methylation, miRNA, transcripts, and proteins will be assessed. Our expected outcome is to improve the knowledge about the endothelial epigenetic programming after FGR in humans and enhance the characterization of the shear stress patterns and mechanisms associated with chronic fetal hypoxia. These effects will be isolated by studying, in vivo, hypoxic chicken embryos and, in vitro, cultured HUAEC exposed to FGR-like flow patterns. This project is not only relevant to uncover the developmental mechanisms that determine short- and long-term vascular dysfunction but also to open potential approaches for preventing, diagnosing, and treating FGR-pregnancies.

Proyecto que busca establecer los efectos de un suplemento en base a miel sobre la salud y el bienestar, así como la respuesta al tratamiento en personas con úlcera gástricas.

Goal: This grant aims to describe the role of epigenetics programming in the progression of BBB injury in growth-restricted neonates. Furthermore, we will identify the molecules and mechanisms of junctional complexes and adhesion molecules of the BBB that are affected by gestational hypoxia. The original contribution of this project is to trace the time course of origins and outcomes of the BBB permeability and integrity impairment related to epigenetic mechanisms, due to gestational chronic hypoxia.