Bernardo Krause Leyton ● Director / Profesor Titular

Frailty is increasingly becoming an important public health challenge worldwide because it is associated with older age, and with adverse outcomes such as reduced quality of life, increased mortality rates, hospitalizations, falls, depression, and dementia. Frailty is defined as dynamic state affecting an individual who experiences losses in one or more domains of human functioning (physical, psychological, social) that are caused by the influence of a range of variables, and which increases the risk of adverse outcomes. This more integral conceptual definition promotes the collaboration of scientists, social and behavioral professionals as well as clinicians from diverse specialties. In this proposal an interdisciplinary group (Biochemistry, Geriatric, Occupational Therapist, Kinesiologist, social worker, bioengineer, statistician among others) aims to evaluate frailty in Chile with a biopsychosocial approach with the final purpose to identify and manage frailty while taking into consideration all the dimensions. Additionally, we aim to design a multidomain personalized person-base intervention for a healthy aging that can uncover a circulating microRNA biomarker panel that can allow an early-detection of frailty, leading to a new multidimensional geriatric assessment. We propose the following hypothesis: A personalized multidimensional training program reduces the frailty prevalence, increasing adherence and participation in the program among community-living older adults. This intervention will be paralleled by a distinctive miRNA profile reflecting the multiple domains of frailty, as well as improvements in diverse psychosocial traits.

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.

El desarrollo de herramientas para el an ́alisis de funciones vitales a trav ́es de im ́agenes representa un campo de creciente inter ́es, especialmente para el estudio de marcadores tempranos de diversas patolog ́ıas, as ́ı como el desarrollo de aplicaciones diagn ́osticas a mediano plazo. En este contexto, el an ́alisis de la funci ́on vascular, a lo largo del ciclo vital, continua siendo un ́area con un alta demanda de nuevas tecnolog ́ıas, debido a su gran impacto a nivel de salud en la poblaci ́on. Esta propuesta busca generar una l ́ınea de investigaci ́on actualmente casi inexistente en Chile, las im ́agenes ultras ́onicas m ́edicas. En particular, nos gustar ́ıa introducir en Chile una t ́ecnica recientemente propuesta, denominada microscop ́ıa de localizaci ́on por ultrasonido (ULM), tambi ́en conocida como im ́agenes de su ́per- resoluci ́on. Esta t ́ecnica puede crear im ́agenes del sistema circulatoria con una resoluci ́on nunca antes vista, lo que permite visualizar vasos sangu ́ıneos de hasta 5μm. As ́ı, nuestra propuesta posee dos grandes objetivos. El primero es Optimizar y robusteces los procesos involucrados en el desarrollo de las im ́agenes de su ́per-resoluci ́on ultras ́onicas, esto a trav ́es de Optimizar los par ́ametros de adquisici ́on de datos y de procesamiento con el fin de robustecer la generaci ́on de este tipo de im ́agenes. Nuestro segundo objetivo es generar im ́agenes de su ́per- resoluci ́on de la red vascular interna de placenta humana ex-vivo y a buscar candidatos a marcadores a partir de estas im ́agenes. La ecograf ́ıa convencional es ampliamente usada en Chile y el mundo. Es preferida entre otras modalidades de im ́agenes (MRI, PET, TC) debido a su portabilidad, bajo costo, naturaleza no invasiva y a que utiliza radiaci ́on no ionizante, especialmente en condiciones como el embarazo, o en pacientes con manejo farmacol ́ogico complejo, entre otras. Recientemente, se han desarrollado nuevas modalidades de im ́agenes ultras ́onicas, propiciadas por la mejora en la industria de los semiconductores, lo que ha permitido un incremento en la capacidad de computo de los esc ́aneres ultraso ́nicos y en el nu ́mero de elementos piezoel ́ectrico de los transductores ultras ́onicos, generando un aumento significativo en la versatilidad y calidad de estas tecnolog ́ıas. Dentro de estas nuevas t ́ecnicas encontramos la microscop ́ıa de localizaci ́on por ultrasonido (ULM) o su ́per-resoluci ́on ultras ́onica. Esta revolucionaria t ́ecnica es capaz de superar el l ́ımite de difracci ́on y producir una resoluci ́on diez veces mayor en comparaci ́on con la ecograf ́ıa convencional. Puede ser usada para producir ima ́genes vasculares con una resoluci ́on sin precedentes de hasta 5 μm permitiendo as ́ı la visualizaci ́on de vasos sangu ́ıneos microsc ́opicos que hasta ahora no pueden ser vistos por ninguna t ́ecnica disponible cl ́ınicamente. ULM utiliza microburbujas (MBs) de gas (1 μm de di ́ametro) que actu ́an como fuentes acu ́sticas estoc ́asticas. Las MBs se inyectan en el torrente sangu ́ıneo y fluyen dentro del sistema circulatorio, donde aparecen y desaparecen de la regi ́on de inter ́es, lo que permite su localizaci ́on. Luego, la imagen de su ́per-resoluci ́on se construye a partir de la acumulaci ́on de cientos de miles de MBs localizadas. Actualmente, la mayor parte de la investigaci ́on en ULM se realiza desde el punto de vista de las ciencias de la ingenier ́ıa, que deja a veces a la ciencia fundamental como un aspecto secundario. Nuestro equipo, debido al car ́acter transdisciplinario de esta propuesta, esta constituido por investigadores de el ́area de la ingenier ́ıa, f ́ısica, biomedicina y matem ́aticas. Basados en las fortalezas de este equipo, proponemos estudiar esta tecnolog ́ıa desde la perspectiva de la ciencia fundamental, buscando limitaciones en ella, y estableciendo los mecanismos fisiol ́ogicos que se manifiestan a nivel de las im ́agenes de su ́per-resoluci ́on, permitiendo superar sus actuales limitaciones y potenciando su posible aplicaci ́on en el ́area m ́edica. En este contexto buscaremos como segundo objetivo el visualizar la microvasculatura de muestras de placenta ex-vivo, las que ser ́an donadas voluntariamente por pacientes del hospital regional de Rancagua, y complementar estas observaciones con par ́ametros funcionales y moleculares, con el fin de modelar desde distintas perspectivas nuevos marcadores de funci ́on vascular. Usualmente para lograr el avance en le desarrollo de este tipo de t ́ecnicas, se requieren condiciones experimentales altamente controlables, las que se logran utilizando sistemas que imitan el tejido en cuesti ́on, el que en nuestro caso es el sistema vascular. Para esto, se utilizan mayormente experimentos in-vitro fabricados a partir de microtubos de 50−150 μm de di ́ametro interno. Sin embargo, existe una gran diferencia entre las propiedades de este tipo de sistemas y las propiedades acu ́sticas de tejido in-vivo humano. Lo que requiere una gran cantidad de iteraciones experimentales retazando el desarrollo. As ́ı, al utilizar tejido humano ex-vivo pretendemos aumentar significativamente la velocidad de la curva de aprendizaje y por consiguiente lograr im ́agenes de su ́per-resoluci ́on humano compatibles dentro de la duraci ́on de esta propuesta. As ́ı mismo, el desarrollo de esta propuesta en un modelo vascular como la placenta humana representa un clara oportunidad para aportar en un ́area actualmente limitada en su capacidad diagn ́ostica, lo que restringe la aplicaci ́on de intervenciones efectivas durante el embarazo, con consecuencias en la salud de la madre y su progenie. Como proyecciones de este esquema colaborativo esperamos tener acceso a par ́ametros biol ́ogicos con los cuales generar nuevas formas de diagn ́ostico de alta precisi ́on, en especial a nivel de las estructuras involucradas, en primera instancia, con el desarrollo de alteraciones vasculares (i.e. vasos de pequen ̃o calibre). Con ello buscamos desarrollar un tecnolog ́ıa con base cient ́ıfica a trav ́es de la cual ser ́a posible obtener indicadores de mayor sensibilidad y especificidad para variadas condiciones, enfermedades o s ́ındromes, relacionados con la funci ́on vascular.

Las enfermedades cardiovasculares (ECV), incluidas la hipertensión, el accidente cerebrovascular y la enfermedad de las arterias coronarias, son las principales patologías a nivel mundial con un impacto significativo en la morbilidad y la mortalidad. El riesgo de enfermedades cardiovasculares se explica parcialmente por factores genéticos, pero en mayor medida por estímulos que tienen lugar durante el desarrollo temprano y el envejecimiento. La función vascular durante la vida está determinada principalmente por tensiones mecánicas resultantes de la presión sanguínea y el flujo, así como por vías de señalización activadas por la disponibilidad de oxígeno. En este contexto, alteraciones en las fuerzas de estiramiento (i.e. tensiones residuales circunferenciales y longitudinales de las arterias), la tensión de corte anormal (shear stress i.e: la fricción del flujo sanguíneo en la superficie arterial luminal) y la hipoxia (i.e. la disminución relativa de las presiones de oxígeno) son factores críticos en el desarrollo de ECV. Los miembros de este proyecto se han centrado en áreas particulares de estos procesos, sin embargo, se requieren esfuerzos adicionales interdisciplinarios para entender de manera integral los mecanismos mecánicos y moleculares involucrados en los orígenes de la disfunción cardiovascular. Recientemente se ha descrito a PIEZO1, un canal de cationes no selectivo, como uno de los principales activadores de la relajación dependiente de endotelio en respuesta a la tensión de corte. Sin embargo, la regulación de éste, por parte de los estímulos descritos anteriormente, no ha sido aún explorada. La presente propuesta busca caracterizar, a través de un estudio con enfoques en biomecánica, fisiología y biología molecular, los cambios en la función del mecano-sensor PIEZO1, en respuesta a estímulos físicos y biológicos en células endoteliales de arteria umbilical humana, con el fin de identificar potenciales blancos de modulación de la función vascular en condiciones que promueven las ECV.

There are more than 41 million deaths per year worldwide, 71% of them are due to non-communicable diseases (NCDs), with cardiovascular (CV) causes taking the first place. Adverse intrauterine conditions increase the risk of developing NCDs during the life course, a phenomenon known as Developmental Origins of Health and Disease (DOHaD). The most frequent and clinically relevant adverse condition during fetal life is intrauterine hypoxia (IH), which is associated with a high perinatal morbi-mortality. The molecular mechanisms that could be involve in IH causes and consequences are an important aspect in most of the pregnancies at high altitudes (> 2500m), and in 3-4% of the pregnancies with utero-placental complications at lowlands. Nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) are endogenous gasotransmitters with vasodilator properties that can regulate cardiovascular functions. These gasotransmitters may be affected by intrauterine chronic hypoxia- induced oxidative stress, eliciting vascular programming and deriving in cardiovascular dysfunction along life course. Although these gasotransmitters have shown to determine endothelial, smooth muscle and cardiac function, the role of them in the fetal programming CV dysfunction due to developmental hypoxia and their relationship with oxidative stress is still unknown. Our hypothesis is that the intrauterine development in chronic hypoxia programms the gasotransmitters pathways (NO, CO and H2S) in the heart and blood vessels through oxidative stress, which impacts in the short, medium and long term cardiovascular function. Therefore, an oral treatment with antenatal melatonin will prevent the hypoxic-induced cardiovascular impairment. In this proposal, we aim to study, in a well-characterized guinea pig model, the origins and outcomes of cardiovascular dysfunction resulting from IH, by characterizing the functional, structural and molecular aspects of vasoactive mechanisms dependent on endogenous gasotransmitters, in the heart and blood vessels. Moreover, as fetal growth restriction and cardiovascular programming are related with hypoxia induced-oxidative stress, we will implement an antenatal treatment with melatonin, a proved antioxidant, to test the prevention of IH-induced cardiovascular risks in the short-, mediate- and long-term. To address these aims, we will study the effects of intrauterine chronic hypoxia and oxidative stress on the NO, CO and H2S related pathways of the cardiovascular system in guinea pigs. We will describe the NO, CO and H2S related pathways, its epigenetic changes and their roles as important regulators of function, structure and biomechanical properties of heart and vessels, under hypoxia induced-oxidative stress. The aforementioned will be done in animals gestated under IH, with or without antenatal melatonin treatment, and follow the cardiovascular function from fetus to adulthood. The methodology is an in vivo non-invasive evaluation of the function and structure of the cardiovascular system (ultrasound); an ex vivo function and biomechanical characterization of heart and arteries (Langendorff, wire myography, biomechanical tests); and in vitro determinations of genetic, protein and epigenetic expression in heart and several vascular beds; at different stages of life. These approaches will permit us to correlate and integrate the short-, mediate- and long-term cardiovascular effects related to the gasotransmitters pathways, in the offspring gestated under chronic hypoxia. In addition, we will evaluate a supported treatment with melatonin, to prevent intrauterine growth restriction and cardiovascular impairment after IH exposure, to test the relationship of gasotransmitters pathways with intrauterine oxidative stress. We expect to unravel the mechanisms underlying IH- induced cardiovascular reprogramming focusing on oxidative stress and gasotransmitters, in order to potentiate melatonin treatment as a possible therapy in hypoxia related complicated pregnancies, either at high altitude or lowlands. This grant will effectively provide a valuable resource for the scientific and clinical community to pursue the understanding of cardiovascular NCDs programmed by intrauterine hypoxia, a worldwide burden still without effective therapeutic approach. Furthermore, this will add knowledge to the foundations for the development of novel therapies for intrauterine hypoxia, and the prevention of CV risk in fetuses, neonates and adults.

En la actualidad existe evidencia convincente del limitado impacto que tienen las estrategias destinadas a la prevención y tratamiento de la hipertensión y otras enfermedades cardiovasculares, una vez que éstas se manifiestan en el adulto. En este sentido, la identificación de los mecanismos que determinan a lo largo de la vida, especialmente en etapas tempranas del desarrollo, una mayor susceptibilidad a la aparición de estas enfermedades, representan un desafío para implementar estrategias efectivas de prevención o, alternativas terapéuticas adecuadas al grado de compromiso de la salud. Considerando la gran influencia que muestra la trayectoria de crecimiento y el estilo de vida sobre el desarrollo de enfermedades vasculares e hipertensión, en los últimos años ha existido un creciente interés en definir la participación de mecanismos que modifican la expresión de genes sin alterar la secuencia de estos (i.e. mecanismos epigenéticos) sobre el origen y progreso de éstas. El presente capítulo abordará qué es la epigenética, los mecanismos que median dichos efectos y el rol que cumple en definir a nivel molecular la programación de la(s) respuestas fisiológicas que condicionan el riesgo de desarrollar obesidad en las diferentes etapas del ciclo vital. La visión de los factores genéticos como causa de las enfermedades crónicas, incluyendo las cardiovasculares, ha sido el motor de la investigación biomédica en el último siglo. Sin embargo, el padre de la biología moderna, Charles Darwin, planteó fuertemente en su obra “El origen de las Especies mediante la Selección Natural” que la “ley superior” del origen de las especies es la adaptación a “las condiciones de existencia” (i.e. medioambiente, estilo de vida), la que estaría por sobre la “selección natural” de características heredables (i.e. genes). Posteriormente, sería Conrad Waddington en sus estudios de embriología quien propone la importancia de la interacción entre los genes y el ambiente. Lo que se manifiesta en la capacidad que tiene el organismo de generar, a partir un conjunto de genes (genoma) único, los distintos tipos y funciones celulares que lo constituyen, mediante respuestas a estímulos que se suceden en un tiempo y lugar específico. Dicha propiedad del medioambiente de moldear el fenotipo fue definida por Waddington como “epigenética”. Actualmente se denomina como epigenética al conjunto de “mecanismos modificadores del DNA que regulan la plasticidad fenotípica de una célula u organismo”. A través de estos mecanismos, nuestros genes pueden expresarse de manera adecuada en respuesta a cambios en el ambiente; pero a la vez definen respuestas fisiológicas a mediano y largo plazo basados en estas señales entregadas por el ambiente. En este contexto, el establecimiento de dichas respuestas limitaría, a través del ciclo vital, la plasticidad fenotípica del individuo. La presente línea de investigación se ha propuesto estudiar los cambios estructurales del sistema vascular y la activación de mecanismos epigenéticos desde etapas tempranas de la vida, que contribuirían al desarrollo de enfermedades cardiovasculares en el adulto. En este sentido plantemos que “el desarrollo de hipertensión y enfermedades cardiovasculares en el adulto resultaría de un programación estructural y epigenética a nivel vascular, mediada por niveles deficitarios de oxígeno (i.e. hipoxia) y el estrés oxidativo intrauterino”. Esta programación fetal establecería una fisiología vascular pro-hipertensiva que devendría posteriormente en una disfunción cardiovascular a medida que el individuo envejece.

Adverse intrauterine conditions, such as fetal growth restriction (FGR), 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 leads 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 and chronic hypoxia are common features in FGR. Several cellular processes require the participation of pro-oxidant molecules which are normally neutralized by antioxidant defenses. However, under determined conditions, such as chronic hypoxia, 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). The higher cardiovascular risk in adults born with FGR can be traced back to a reduced arterial compliance in pre-pubertal subjects and a decreased peripheral endothelial-dependent vascular relaxation at birth. This endothelial dysfunction (ED) is also observed in FGR placental vessels, suggesting an early onset of ED that could be evidenced in systemic and placental arteries. Our studies in human placentae have demonstrated that ED in FGR can be modulated by oxidative stress and is associated with changes in proteome profile as well as an epigenetic-mediated regulation of eNOS expression. Similarly, in guinea pigs, we have found that ED in FGR is also occurring in the fetal arteries and is prevented by maternal treatment with antioxidants. Analysis of eNOS expression and Nos3 promoter DNA methylation profile in endothelial cells from the aorta and umbilical arteries of FGR guinea pigs shows common molecular markers of ED in FGR systemic and umbilical vessels which are reverted by a maternal antioxidant treatment. Altogether, these data support the role of oxidative stress in the epigenetic programming of ED in FGR and the potential predictive value of studying human umbilical arteries endothelial cells (HUAEC). However, there are no studies addressing the time course and origins of the ED in FGR and the participation of additional epigenetic mechanisms, such as miRNAs, in this process. Here, we put forward two inter-related hypotheses. First, that arterial endothelium from babies with FGR show a genomic DNA methylation signature along with an altered expression of miRNAs miR-21, miR-126 & miR-155, which contributes to changes in the expression of enzymes related to NO synthesis, endothelial dysfunction, and vascular remodeling. Second, that FGR has, during gestation, dynamic changes in the expression of miRNAs miR-21, miR-126 & miR-155 as an early response to hypoxia and oxidative stress in the fetus leading, in the long term, to endothelial dysfunction and vascular remodeling. These hypothesis will be tested in primary cultures of HUAEC from FGR neonates and validated using guinea pig and chick embryo models of FGR according to the following general aims (GA): GA-1 To demonstrate, in human umbilical artery endothelial cells, whether FGR is associated with an altered epigenetic regulation (i.e. increased levels of miR-21, miR-126 & miR-155 and a differential genomic DNA methylation) of NO-related enzymes (i.e. eNOS, DDAH1, Nrf2, Arg2, and HO-1), which modifies the response to hypoxia and impairs angiogenic capacity.GA-2 To demonstrate, in FGR guinea pigs, whether circulating levels of miR-21, miR-126 & miR-155 are dynamically regulated during the development of fetal vascular dysfunction and if the prevention of oxidative stress by an antioxidant administration to mothers, contributes to this regulation. GA-3 To demonstrate, in FGR chicken embryos, whether the silencing of miR-21, miR-126 and miR-155 expression modifies the endothelial and vascular dysfunction induced by chronic hypoxia and oxidative stress. Our expected outcome is to demonstrate that miRNAs miR-21, miR-126 & miR-155 participates in the early defense to hypoxia and oxidative stress in the FGR, but leading at long-term to ED. Further, we propose that regulation of these miRNAs during gestation could prevent these effects. 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 treatments in complicated pregnancies in humans. Combined, our program of work offers insights into mechanisms underlying the association between intrauterine hypoxia, oxidative stress and the increased risk of developing cardiovascular disease in later life, and possible interventions considering administration of therapeutic agents at critical stages of intrauterine development.

As a scientist in Chile, I have been focused on understanding the mechanisms underlying fetal vascular dysfunction associated with altered fetal growth with a special interest in impaired fetal growth and epigenetic regulation. Fetal growth restriction (FGR), commonly defined by a weight below the 10th percentile is associated with increased perinatal morbidity and mortality with an incidence of 3 to 10% of all live births1, 2. Therefore, FGR remains at the forefront of basic science and clinical investigation, as it poses a significant problem on every nation’s wealth and health. Adverse conditions in complicated pregnancy known to induce FGR include reductions in fetal oxygenation or chronic fetal hypoxia3. Several studies in humans have established that high altitude pregnancy reduces fetal growth 4-6. Further, several studies in mammalian animal models of hypoxic pregnancy have also reported a significant effect in slowing fetal growth7. However, since most high altitude populations are also impoverished with a high prevalence of maternal undernutrition and since hypoxic exposure of mammalian animals during pregnancy can reduce maternal food intake7, the partial contributions of fetal under-nutrition versus fetal under-oxygenation in promoting FGR remain uncertain. The Giussani group at the University of Cambridge have combined the chick embryo model with hypoxic incubation to isolate the direct effects of chronic fetal hypoxia in promoting FGR. This group has shown that incubation at high altitude of fertilized eggs laid by sea level hens leads to FGR. Conversely, incubation at sea level of fertilized eggs laid by high altitude hens that normally show FGR completely recovered fetal growth. Importantly, incubation at high altitude of fertilized eggs laid by sea level with oxygen supplementation also prevented FGR8. Recent studies by the Cambridge group have also reported that isobaric rather than hypobaric chronic fetal hypoxia also leads to FGR in the chick embryo9. Further, they have reported that FGR as a result of isolated chronic fetal hypoxia is associated with significant oxidative stress and endothelial dysfunction in fetal peripheral circulations. Treatment of chick embryos during hypoxic incubation with the antioxidant melatonin rescued the endothelial dysfunction in peripheral circulations by the end of the incubation period. The mechanisms involved included reduced oxidative stress, enhanced antioxidant capacity, restored vascular endothelial growth factor expression and increased NO bioavailability9. Combined, therefore, studies by the Cambridge group have isolated a direct effect of hypoxia in promoting FGR and fetal endothelial dysfunction independent of effects at the level of the placenta or the mother and suggest that antioxidant therapy may also have a direct protective effect on the fetus. These findings are of particular interest to me because my research group in Chile has recently established a guinea pig experimental model of FGR. This is by progressive bilateral occlusion of the uterine arteries during the second half of gestation that gradually increases placental vascular resistance10, 11. Using this guinea pig model, we have recently shown that FGR induces epigenetic programming of eNOS expression in the fetal endothelium, which is prevented by a maternal treatment with N-acetylcysteine11. However, whether the effects of increased placental vascular resistance on the fetal epigenetic programming of eNOS expression is due to fetal under-nutrition versus fetal under-oxygenation in this model of FGR again remains uncertain. Therefore, the aim of my research proposal is to use the Cambridge chick embryo model of FGR as a result of hypoxic incubation to isolate the direct effects of chronic fetal hypoxia on epigenetic regulation promoting fetal vascular dysfunction. This would be the first step to enable better identification of potential targets for clinical intervention designed to protect the developing fetal circulation in pregnancy complicated by FGR and chronic fetal hypoxia.

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.

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.

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 5 to 25% depending on the nutritional and health conditions of 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, 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 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. epigenetic changes) of endothelial dysfunction in human umbilical and chorionic vessels, findings that have been further confirmed by comparing systemic (aorta and femoral arteries) and umbilical arteries in animal models of FGR. These traits suggest that umbilical artery endothelial cells (HUAEC) 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 (i.e. fetal hypoxia) and altered blood flow patterns (i.e. 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 stimuli related to FGR (i.e. hypoxia and altered 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. OG3 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 and enhance the characterization of in vivo shear stress patterns and mechanisms induced by chronic fetal hypoxia. This project is not only relevant to uncover the developmental approaches for diagnosing and treatments in complicated pregnancies.

Hay pruebas sólidas de que tanto la restricción del crecimiento intrauterino (RCIU) y la macrosomía fetal (peso al nacer > 4000 g) están fuertemente asociados con el aumento del riesgo de enfermedades crónicas en la vida adulta (i.e. hipertensión arterial y diabetes tipo 2). La asociación entre el desarrollo peri-concepcional y fetal con la salud en la vida adulta, llamada “Programación Fetal” se basa en la activación de mecanismos de detección y señalización de una diversidad de estímulos durante el desarrollo temprano. Los mecanismos que han sido ampliamente sugerido que participan en estos procesos son las modificaciones epigenéticas que podrían registrar los estímulos perinatales. Como testigo de los cambios perinatales a los que se ve expuesto el embrión y luego el feto en su desarrollo y crecimiento está la placenta, que junto con estar constituida por tejido que regula el intercambio de nutrientes y la modulación inmune está formada por una importante red vascular que permite obtener desde la sangre materna los nutrientes, el oxígeno, las señales hormonales y del sistema inmune. El sistema vascular de la placenta se proyecta igualmente en la circulación sistémica del feto, donde se sugiere que ocurren adaptaciones en las condiciones patológicas antes mencionadas (RCIU y macrosomía fetal secundaria a obesidad materna), condicionando así a estos niños a presentar un elevado riesgo de enfermedades cardiometabólicas en la vida adulta. Este proyecto propuso estudiar la presencia de marcas epigenéticas (cambios en el patrón de metilación de citosinas en la región promotora de genes y cambios en la modificación de histonas que participan en el estado de compactación/relajación de la cromatina) en las células vasculares de la arteria umbilical (HUAEC), cambios en la función vascular y expresión de genes, proteínas y actividad de enzimas claves en la función vascular en tres grupos de gestantes: 1) gestantes normopeso con fetos de crecimiento normal denominados “Adecuados para la Edad Gestacional” (AEG); 2) gestantes normopeso con fetos que presentaron Restricción del Crecimiento IntraUterino (RCIU) y 3) gestantes con obesidad pregestacional que tuvieron hijos que fueron Grandes para la Edad Gestacional (FMOM). Los objetivos y los resultados principales de este proyecto fueron: 1) Estudiar el tono vascular de las arterias umbilicales y coriónicas de la placenta en estas tres condiciones clínicas con acento en la participación de las vías dependiente de óxido nítrico, un vasodilatador dependiente del endotelio, clave en el tono vascular placentario. Se determinó que las arterias coriónicas y placentarias de los fetos RCIU y FMOM presentan disfunción vascular dependiente de óxido nítrico y que existe un desbalance entre la actividad de arginasa-2/eNOS que favorece esta disfunción. (ver Krause et al. Placenta 2012; 2013; Schneider et al., Placenta 2015). 2) Determinar si estos cambios en el tono vascular placentario y umbilical se correlacionan a cambios en la expresión de genes y proteínas claves en la función vascular (eNOS, Arg2, CAT-1) y de respuesta estrés oxidativo (NOX4, GPx-1, SOD-1 y HO-1) en los cultivos primarios de HUAEC de las tres condiciones clínicas, así como en los tejidos (cordones umbilicales y arterias coriónicas) (ver Schneider et al., Placenta 2015). Se determinó que estos vasos están marcados por una respuesta crónica a estrés oxidativo, presentando una limitada reserva vascular al estrés oxidativo, comparado con los tejidos provenientes de embarazos AEG (ver Schneider et al., Placenta 2015). 3) Estudiar la presencia de modificaciones epigenéticas presentes en la región promotora de los genes relacionados con la vía de síntesis del óxido nítrico (NO) y determinar si la respuesta al estrés oxidativo y/o hipoxia están programadas epigeneticamente. Se determinó que de todos los genes estudiados sólo eNOS y Arg2 presentan cambios en el patrón de metilación en la región promotora (ver Krause et al., Epigenetics 2013) que se correlacionan con las modificaciones de histonas encargadas de regular el estado de compactación de la cromatina (Caniuguir et al., en preparación). 4) Ahondar en la participación de los mecanismos epigenéticos (HDAC y DNMT) en la expresión basal de estos genes en las tres condiciones clínicas. Se determinó muy elegantemente como NO es quien regula a nivel epigenético la apertura de la cromatina de arginasa 2 y su actividad transcripcional (ver Krause et al., Biochem Pharmacol 2016). El desarrollo de este proyecto tuvo como fruto 18 artículos en revistas indexadas, la mitad de ellos directamente relacionados con resultados de este proyecto y los otros de colaboraciones nacidas durante estos años, que apuntan directamente a comprender los mecanismos que subyacen la programación fetal de enfermedades cardiometabólicas en tejido placentario, en niños pre-adolescentes, modelos animales de RCIU y obesidad materna, proyectos clínicos de seguimiento de niños, etc. Este proyecto aportó en forma sustantiva a la generación de nuevo conocimiento en relación a los mecanismos moleculares que subyacen a la programación vascular alterada en fetos con RCIU y FMOM, que están en mayor riesgo de desarrollar enfermedades vasculares en la vida postnatal, con la relevancia clínica de la potencial aplicación en la detección temprana de factores de riesgo cardiovascular en el nacimiento.