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Homozygous mutation in arteriovenous malformation (AVM) causative genes in a fraction of endothelial cells (ECs) causes brain AVMs (bAVMs). Heterozygous mutation of an AVM causative gene, endoglin, in bone marrow (BM) caused capillary dysplasia in mouse brain. We hypothesize that homozygous mutation of activin receptor-like kinase 1 (Alk1, another AVM causative gene) in BM derived ECs (BMDECs) is sufficient to cause bAVM in brain angiogenic region, Alk1(−) ECs can clonally expand in bAVM, and the burden of Alk1(−) ECs correlates with bAVM severity. The BMs of PdgfbiCreER;Alk1(2f/2f);Ai14 mice with EC-specific tamoxifeninducible Cre and Alk1 floxed alleles were transplanted into wild-type mice to analyze the role of BMDECs in bAVM development. PdgfbiCreER;Alk1(2f/2f);confetti(+/−) mice with Cre-regulated confetti transgene were used to study EC clonal expansion. BAVMs were induced by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor followed with intra-peritoneal injection of tamoxifen. Wild-type mice transplanted with PdgfbiCreER;Alk1(2f/2f);Ai14 BM developed bAVMs after bAVM induction. Recombined BMDECs were detected in bAVMs. The presence of clusters of ECs expressing same confetti color in bAVMs suggests that Alk1(−) ECs have been clonally expanded. Increasing tamoxifen dose increased the number of Alk1(−) ECs and abnormal vessels in bAVMs of PdgfbiCreER;Alk1(2f/2f) mice. Our data indicated that homozygous mutation of Alk1 in BMDECs is sufficient to cause bAVM development in brain angiogenic region; clonal expansion of Alk1(−) ECs provides a likely mechanism for how a fraction of mutant ECs causes bAVM; and the burden of Alk1(−) ECs correlates with AVM severity.

Endothelial cells in the brain interact with other cell types, forming the blood-brain barrier. This barrier controls the movement of solutes into and out of the brain, regulating pathophysiological processes and drug delivery to the brain. Common isolation methods used to study these cells during embryonic development involve enzymatic treatment and cell sorting using specific markers. This process modifies the cell state and produces minute amounts of sample. Here, we describe a protocol for the enrichment of vascular cells from embryonic brains based on dextran separation. In this method, the brain is lightly disrupted with a pestle and then resuspended in a dextran solution. Low-speed centrifugation permits the separation of the parenchymal and vascular fractions. Further centrifugation steps improve fractionation. This method is simple and fast and produces enough sample for biochemical assays. Graphic abstract: [Image: see text] Purification of vascular fragments from an embryonic brain

As the resident macrophages of the brain and spinal cord, microglia are crucial for the phagocytosis of infectious agents, apoptotic cells and synapses. During brain injury or infection, bone-marrow derived macrophages invade neural tissue, making it difficult to distinguish between invading macrophages and resident microglia. In addition to circulation-derived monocytes, other non-microglial central nervous system (CNS) macrophage subtypes include border-associated meningeal, perivascular and choroid plexus macrophages. Using immunofluorescent labeling, flow cytometry and Cre-dependent ribosomal immunoprecipitations, we describe P2ry12-CreER, a new tool for the genetic targeting of microglia. We use this new tool to track microglia during embryonic development and in the context of ischemic injury and neuroinflammation. Because of the specificity and robustness of microglial recombination with P2ry12-CreER, we believe that this new mouse line will be particularly useful for future studies of microglial function in development and disease.

Fowler syndrome is a rare autosomal recessive brain vascular disorder caused by mutation in FLVCR2 in humans. The disease occurs during a critical period of brain vascular development, is characterized by glomeruloid vasculopathy and hydrocephalus, and is almost invariably prenatally fatal. Here, we sought to gain insights into the process of brain vascularization and the pathogenesis of Fowler syndrome by inactivating Flvcr2 in mice. We showed that Flvcr2 was necessary for angiogenic sprouting in the brain, but surprisingly dispensable for maintaining the blood-brain barrier. Endothelial cells lacking Flvcr2 had altered expression of angiogenic factors, failed to adopt tip cell properties, and displayed reduced sprouting, leading to vascular malformations similar to those seen in humans with Fowler syndrome. Brain hypovascularization was associated with hypoxia and tissue infarction, ultimately causing hydrocephalus and death of mutant animals. Strikingly, despite severe vascular anomalies and brain tissue infarction, the blood-brain barrier was maintained in Flvcr2 mutant mice. Our Fowler syndrome model therefore defined the pathobiology of this disease and provided new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.

Development encompasses the formation of a complex organism starting from a unicellular zygote. This process requires finely tuned changes in gene expression across several cells and tissues, which may be regulated by a combination of stable and dynamic epigenetic modifications that make the DNA more or less accessible to transcriptional regulators. Diverse interdependent epigenetic mechanisms have been described, including DNA and histone methylation, histone acetylation, and silencing by micro RNAs. In the developing embryo or fetus, the epigenetic status is influenced by diverse environmental conditions, including maternal nutrition. Depending on the stage of pregnancy, gestational nutritional inadequacies may lead to congenital embryonic defects, suboptimal fetal growth, and/or reprogramming of tissues, which may determine different phenotypes or predispose individuals to chronic diseases later in life. During early pregnancy, abnormal methylation of certain genes resulting from nutritional inadequacies has been shown to increase the risk for congenital malformations that lead to perinatal death or postnatal disabilities. During mid to late pregnancy, maternal malnutrition, both undernutrition and overnutrition, has been shown to promote adaptive epigenetic changes in different organs that allow the embryo or fetus to cope with the intrauterine environment but that persist throughout life and may increase susceptibility to chronic diseases. Preconceptional nutrition in the mother, and paternal nutrition, also influence the epigenetic marks in the embryo. Nutrition seems to use epigenetics to tailor diversity and susceptibility to disease in animals and humans. Understanding nutrigenomics may be useful to understand evolution and promote health in future generations.

The actions of insulin on intestinal cholesterol absorption and lipoprotein secretion are not well understood. Herein, we determined the effects of insulin on the levels of cholesterol transporter scavenger receptor, class B, type I (SR-BI), cellular cholesterol uptake, intracellular lipid accumulation, and lipoprotein secretion in a cellular model of human intestinal epithelium. Methods CaCo-2 cells were cultured to postconfluency in Transwell filters and stimulated with glucose (25 mM) in the presence or absence of insulin (100 nM) at their basolateral surface. SR-BI mRNA and protein levels were quantified by quantitative reverse transcription-PCR and immunoblot, respectively. Polarized localization of SR-BI was determined by cell surface proteins biotinylation and streptavidin precipitation. Activities of PI3K, AKT, mTOR, and SR-BI were pharmacologically antagonized. Cholesterol uptake was assessed by NBD-cholesterol incorporation. Apolipoprotein (apo) B concentration was quantified by ELISA. Subcellular localization of neutral lipids (BODIPY) and SR-BI (immunofluorescence) was determined by confocal microscopy. Results In polarized CaCo-2 cells, insulin increased SR-BI at the mRNA and protein levels. SR-BI was exclusively present at apical cell surface, as indicated by biotinylation and confocal microscopy analysis. Glucose did not modify SR-BI abundance or subcellular localization. Effects of insulin on SR-BI levels were abrogated by PI3K, AKT, or mTOR pharmacological antagonism. Cholesterol uptake, neutral lipid abundance, and apo B secretion were increased by insulin in CaCo-2 cells, and these effects were prevented by SR-BI pharmacological antagonism with block lipid transport-1. Conclusions insulin promotes cholesterol uptake, intracellular lipid store, and apo B-containing lipoproteins secretion by SR-BI-dependent mechanisms in a model of human intestinal epithelium.

espanolEl presente articulo tiene por objeto denotar el rol de los denominados “hallazgos casuales” en las diligencias investigativas de incautacion y registro de las comunicaciones digitales en Chile. Para tales efectos, luego de efectuar un breve analisis del principio de proporcionalidad y su relevancia en el tratamiento argumentativo de tal instituto, el trabajo aborda los hallazgos casuales desde una triple consideracion: en primer termino, realizando un analisis conceptual y metodologico de los hallazgos casuales en consideracion a sus principales manifestaciones en el Derecho comparado, particularmente en paises como Alemania, Italia y Espana; en segundo termino, examinando los hallazgos casuales en Chile y sus manifestaciones jurisprudenciales mas significativas, a partir de los articulos 215 (referido a la diligencia de entrada y registro) y 223 (relativo a la interceptacion de comunicaciones telefonicas) del Codigo Procesal Penal; y, por ultimo, abordando los hallazgos casuales de las comunicaciones digitales en nuestro pais, sus problemas practicos mas relevantes, asi como sus posibles formas de solucion. EnglishThe purpose of this article is to analyze the role of the so-called casual discoveries in the investigative proceedings of seizure and registration of digital communications in Chile. To achieve that goal, after making a brief analysis of the principle of proportionality and its relevance in the argumentative treatment, the work approaches the casual discoveries from a triple consideration: firstly, doing a conceptual and methodological analysis of the casual discoveries in consideration of its main manifestations in Comparative Law, particularly in countries such as Germany, Italy and Spain; secondly, examining casual discoveries in Chile and its most significant jurisprudential manifestations, based on articles 215 (referring to the entry and search procedure) and 223 (regarding the interception of telephone communications) of the Criminal Procedure Code; and, finally, tackling the casual discoveries of digital communications in Chile, its most relevant practical problems, as well as its possible ways of solution.

Scavenger receptor class B type 1 (SR-B1) is a membrane lipoprotein receptor/lipid transporter involved in the pathogenesis of atherosclerosis, but its role in obesity and fatty liver development is unclear. Here, we determined the effects of SR-B1 deficiency on plasma metabolic and inflammatory parameters as well as fat deposition in adipose tissue and liver during obesity. To induce obesity, we performed high-fat diet (HFD) exposure for 12 weeks in male SR-B1 knock-out (SR-B1−/−, n = 14) and wild-type (WT, n = 12) mice. Compared to HFD-fed WT mice, plasma from HFD-fed SR-B1−/− animals exhibited increased total cholesterol, triglycerides (TG) and tumor necrosis factor-α (TNF-α) levels. In addition, hypertrophied adipocytes and macrophage-containing crown-like structures (CLS) were observed in adipose tissue from HFD-fed SR-B1 deficient mice. Remarkably, liver from obese SR-B1−/− mice showed attenuated TG content, dysregulation in hepatic peroxisome proliferator-activated receptors (PPARs) expression, increased hepatic TG secretion, and altered hepatic fatty acid (FA) composition. In conclusion, we show that SR-B1 deficiency alters the metabolic environment of obese mice through modulation of liver and adipose tissue lipid accumulation. Our findings provide the basis for further elucidation of SR-B1's role in obesity and fatty liver, two major public health issues that increase the risk of advanced chronic diseases and overall mortality.

SR-BI is the main receptor for high density lipoproteins (HDL) and mediates the bidirectional transport of lipids, such as cholesterol and vitamin E, between these particles and cells. During early development, SR-BI is expressed in extraembryonic tissue, specifically in trophoblast giant cells in the parietal yolk sac. We previously showed that approximately 50% of SR-BI−/− embryos fail to close the anterior neural tube and develop exencephaly, a perinatal lethal condition. Here, we evaluated the role of SR-BI in embryonic vitamin E uptake during murine neural tube closure. Our results showed that SR-BI−/− embryos had a very low vitamin E content in comparison to SR-BI+/+ embryos. Whereas SR-BI−/− embryos with closed neural tubes (nSR-BI−/−) had high levels of reactive oxygen species (ROS), intermediate ROS levels between SR-BI+/+ and nSR-BI−/− embryos were detected in SR-BI−/− with NTD (NTD SR-BI−/−). Reduced expression of Pax3, Alx1 and Alx3 genes was found in NTD SR-BI−/− embryos. Maternal α-tocopherol dietary supplementation prevented NTD almost completely (from 54% to 2%, p −/− embryos and normalized ROS and gene expression levels. In sum, our results suggest the involvement of SR-BI in the maternal provision of embryonic vitamin E to the mouse embryo during neural tube closure.

High density lipoproteins (HDL) take up cholesterol from peripheral tissues via ABC transporters and deliver it to the liver via scavenger receptor class B type I (SR-B1). HDL are the main lipoproteins present in follicular fluid (FF). They are thought to derive from plasma, but their origin is still controversial. SR-B1 knock-out (KO) mice have provided important evidence linking HDL metabolism and female fertility. These mice have cholesterol-rich circulating HDL and female infertility that can be restored by treating mice with the cholesterol-lowering drug probucol. Ovulated oocytes from SR-B1 KO females are dysfunctional and show excess cholesterol. The mechanisms explaining the contribution of FF HDL to oocyte cholesterol homeostasis are unknown. Here, using quantitation of filipin fluorescence we show that in SR-B1 KO ovaries, cholesterol excess is first observed in immature oocytes in antral follicles. By performing cross-transplant experiments between WT and apolipoprotein A-I deficient (ApoA-I KO) mice, which lack the main protein component of HDL, we provide evidence supporting the plasmatic origin of FF HDL. Also, we demonstrate that probucol treatment in SR-B1 KO females results in lowering of cholesterol content in their oocytes. Incubation of oocytes from SR-B1 KO mice with purified WT HDL reduces their cholesterol content, suggesting that HDL promote efflux of excess cholesterol from oocytes. In agreement with this hypothesis, we identified ABC transporters in oocytes and observed that ABCA1 KO oocytes have excess cholesterol and lower viability than WT oocytes.

Background The high-density lipoprotein receptor SR-B1 mediates cellular uptake of several lipid species, including cholesterol and vitamin E. During early mouse development, SR-B1 is located in the maternal-fetal interface, where it facilitates vitamin E transport towards the embryo. Consequently, mouse embryos lacking SR-B1 are vitamin E-deficient, and around half of them fail to close the neural tube and show cephalic neural tube defects (NTD). Here, we used transcriptomic profiling to identify the molecular determinants of this phenotypic difference between SR-B1 deficient embryos with normal morphology or with NTD. Results We used RNA-Seq to compare the transcriptomic profile of three groups of embryos retrieved from SR-B1 heterozygous intercrosses: wild-type E9.5 embryos (WT), embryos lacking SR-B1 that are morphologically normal, without NTD (KO-N) and SR-B1 deficient embryos with this defect (KO-NTD). We identified over 1000 differentially expressed genes: down-regulated genes in KO-NTD embryos were enriched for functions associated to neural development, while up-regulated genes in KO-NTD embryos were enriched for functions related to lipid metabolism. Feeding pregnant dams a vitamin E-enriched diet, which prevents NTD in SR-B1 KO embryos, resulted in mRNA levels for those differentially expressed genes that were more similar to KO-N than to KO-NTD embryos. We used gene regulatory network analysis to identify putative transcriptional regulators driving the different embryonic expression profiles, and identified a regulatory circuit controlled by the androgen receptor that may contribute to this dichotomous expression profile in SR-B1 embryos. Supporting this possibility, the expression level of the androgen receptor correlated strongly with the expression of several genes involved in neural development and lipid metabolism. Conclusions Our analysis shows that normal and defective embryos lacking SR-B1 have divergent expression profiles, explained by a defined set of transcription factors that may explain their divergent phenotype. We propose that distinct expression profiles may be relevant during early development to support embryonic nutrition and neural tube closure. Electronic supplementary material The online version of this article (10.1186/s12864-018-5110-2) contains supplementary material, which is available to authorized users.

Scavenger Receptor Class B Type I (SR-BI) mediates bidirectional lipid uptake between HDL and cells. During murine early embryogenesis, SR-BI is expressed in trophoblast giant cells. SR-BI-/- embryos from SR-BI+/- intercrosses exhibit cephalic neural tube

The susceptibility to develop atherosclerosis is increased by intrauterine growth restriction and prenatal exposure to maternal hypercholesterolemia. Here, we studied whether mouse gestational hypercholesterolemia and atherosclerosis affected fetal development and growth at different stages of gestation. Female LDLR KO mice fed a proatherogenic, high cholesterol (HC) diet for 3 weeks before conception and during pregnancy exhibited a significant increase in non-HDL cholesterol and developed atherosclerosis. At embryonic days 12.5 (E12.5), E15.5, and E18.5, maternal gestational hypercholesterolemia and atherosclerosis were associated to a 22–24% reduction in male and female fetal weight without alterations in fetal number/litter or morphology nor placental weight or structure. Feeding the HC diet exclusively at the periconceptional period did not alter fetal growth, suggesting that maternal hypercholesterolemia affected fetal weight only after implantation. Vitamin E supplementation (1,000 UI of α-tocopherol/kg) of HC-fed females did not change the mean weight of E18.5 fetuses but reduced the percentage of fetuses exhibiting body weights below the 10th percentile of weight (HC: 90% vs. HC/VitE: 68%). In conclusion, our results showed that maternal gestational hypercholesterolemia and atherosclerosis in mice were associated to early onset fetal growth restriction and that dietary vitamin E supplementation had a beneficial impact on this condition.

The srbi gene encodes a lipoprotein receptor with high affinity for high density lipoprotein that is mainly expressed in the liver and in steroidogenic tissues. Disruption of this gene in mice and mutations in humans lead to alterations in lipoprotein metabolism and/or fertility. During murine development, scavenger receptor class B member I (SR-BI) is present in the yolk sac and the placenta and is only expressed in the embryo itself late in gestation. In humans, it has been detected in trophoblast cells and placenta. Although the proportion of mice carrying a null mutation in SR-BI obtained from heterozygous intercrosses is lower than the expected by the Mendelian ratio, suggesting the involvement of this receptor in intrauterine development, the cause of this demise has remained unknown. In this work, we show that embryos lacking SR-BI exhibit a high prevalence of exencephaly with a sex bias toward females. Immunolocalization studies confirmed that SR-BI is not expressed in the embryo at early stages of development and allowed a more detailed description of its localization in the cells that mediate maternal–fetal transport of nutrients. SR-BI-null embryos contain less cholesterol than their wild-type littermates, suggesting the involvement of SR-BI in materno-fetal cholesterol transport. Newborn SR-BI-deficient pups exhibit intrauterine growth restriction, suggesting that this receptor is also important for fetal growth. Altogether, the results of our work suggest that the presence of SR-BI in extraembryonic tissues is involved in the maternal–fetal transport of cholesterol and/or other lipids with a role during neural tube closure and fetal growth.

s / Placenta 36 (2015) 469e521 498 Conclusions: These results support a role for VIP during mouse early pregnancy pointing to TGC among the major local sources of the neuropeptide. VIP deficiency is reflected by TGC expressing an altered functional and invasive phenotype with increased VPAC2 expression. PA.32. INVOLVEMENT OF HDL RECEPTOR SR-BI-MEDIATED VITAMIN E UPTAKE IN MURINE NEURAL TUBE CLOSURE N. Santander , D. P erez , G. Echeverria , C. Mendoza , K. Cautivo , A. Rigotti , D. Busso . Department of Nutrition, Diabetes and Metabolism, Chile; Center for Molecular Nutrition and Chronic Diseases, Medical School, Pontifical University of Chile, Santiago, Chile SR-BI, the main HDL receptor, mediates the cellular uptake of cholesterol and vitamin E. SR-BI is present in trophoblast giants cells (TGC) during early pregnancy and 50% SR-BI-deficient (SR-BI / ) embryos die due to defective neural tube closure (NTD). Objectives: a) to compare vitamin E content and oxidative stress markers in SR-BI+/+ and SR-BI / TGC and embryos with and without NTD, b) to evaluate whether maternal vitamin E supplementation prevents NTD in SR-BI / embryos and compare vitamin E content in SR-BI+/+ and / embryos and TGC from chowand vitamin E-supplemented dams. Methods: Pregnant dams from SR-BI +/ intercrosses were fed a standard chow diet alone or supplementedwith 2000 UI vitamin E/kg. Embryos and TGC were recovered at E9.5 and snap frozen. Tissue homogenates were used to determine vitamin E by HPLC, reactive oxygen species (ROS) using dihydrodichlorofluorescein diacetate, and oxidative stress-related genes by qPCR. Yolk sacs were used for PCR genotyping. Results: Vitamin E content in SR-BI-/embryos was 5-fold lower than in SR-BI+/+ embryos (p

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification, and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain has eluded the field for over fifty years. The MFS transporter FLVCR1 was recently determined to be a choline transporter, and while this protein is not highly expressed at the blood-brain barrier (BBB), its relative FLVCR2 is. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus, and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here, we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in the inward- and outward-facing states using cryo-electron microscopy to 2.49 and 2.77 Å resolution, respectively. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of neurotherapeutics into the brain.