Placental transcriptomic signatures of prenatal and preconceptional maternal stress.

Prenatal exposure to maternal psychological stress is associated with increased risk for adverse birth and child health outcomes. Accumulating evidence suggests that preconceptional maternal stress may also be transmitted intergenerationally to negatively impact offspring. However, understanding of mechanisms linking these exposures to offspring outcomes, particularly those related to placenta, is limited. Using RNA sequencing, we identified placental transcriptomic signatures associated with maternal prenatal stressful life events (SLEs) and childhood traumatic events (CTEs) in 1 029 mother-child pairs in two birth cohorts from Washington state and Memphis, Tennessee. We evaluated individual gene-SLE/CTE associations and performed an ensemble of gene set enrichment analyses combing across 11 popular enrichment methods. Higher number of prenatal SLEs was significantly (FDR < 0.05) associated with increased expression of ADGRG6, a placental tissue-specific gene critical in placental remodeling, and decreased expression of RAB11FIP3, an endocytosis and endocytic recycling gene, and SMYD5, a histone methyltransferase. Prenatal SLEs and maternal CTEs were associated with gene sets related to several biological pathways, including upregulation of protein processing in the endoplasmic reticulum, protein secretion, and ubiquitin mediated proteolysis, and down regulation of ribosome, epithelial mesenchymal transition, DNA repair, MYC targets, and amino acid-related pathways. The directional associations in these pathways corroborate prior non-transcriptomic mechanistic studies of psychological stress and mental health disorders, and have previously been implicated in pregnancy complications and adverse birth outcomes. Accordingly, our findings suggest that maternal exposure to psychosocial stressors during pregnancy as well as the mother's childhood may disrupt placental function, which may ultimately contribute to adverse pregnancy, birth, and child health outcomes.

Human Enteroid Monolayers: A Novel, Functionally-Stable Model for Investigating Oral Drug Disposition.

To further the development of an in vitro model which faithfully recapitulates drug disposition of orally administered drugs, we investigated the utility of human enteroid monolayers to simultaneously assess intestinal drug absorption and first-pass metabolism processes. We cultured human enteroid monolayers from three donors, derived via biopsies containing duodenal stem cells that were propagated and then differentiated atop permeable Transwell® inserts, and confirmed transformation into a largely enterocyte population via RNA-seq analysis and immunocytochemical (ICC) assays. Proper cell morphology was assessed and confirmed via bright field microscopy and ICC imaging of tight junction proteins and other apically and basolaterally localized proteins. Enteroid monolayer barrier integrity was demonstrated by elevated transepithelial electrical resistance (TEER) that stabilized after 10 days in culture and persisted for 42 days. These results were corroborated by low paracellular transport probe permeability at 7 and 21 days in culture. The activity of a prominent drug metabolizing enzyme, CYP3A, was confirmed at 7, 21, and 42 days culture under basal, 1α,25(OH)2 vitamin D3-induced, and 6',7'-dihydroxybergamottin-inhibited conditions. The duration of these experiments is particularly noteworthy, as this is the first study assessing drug metabolizing enzymes and transporters (DMET) expression/function for enteroids cultured for greater than 12 days. The sum of these results suggests enteroid monolayers are a promising ex vivo model to investigate and quantitatively predict an orally administered drug's intestinal absorption and/or metabolism. Significance Statement This study presents a novel ex vivo model of the human intestine, human intestinal organoid (enteroid) monolayers, that maintain barrier function and metabolic functionality for up to 42-days in culture. The incorporation of both barrier integrity and metabolic function over an extended period within the same model is an advancement over historically used in vitro systems, which either lack one or both of these attributes or have limited viability.

Mono(2-ethylhexyl) phthalate induces transcriptomic changes in placental cells based on concentration, fetal sex, and trophoblast cell type.

Phthalates are ubiquitous plasticizer chemicals found in consumer products. Exposure to phthalates during pregnancy has been associated with adverse pregnancy and birth outcomes and differences in placental gene expression in human studies. The objective of this research was to evaluate global changes in placental gene expression via RNA sequencing in two placental cell models following exposure to the phthalate metabolite mono(2-ethylhexyl) phthalate (MEHP). HTR-8/SVneo and primary syncytiotrophoblast cells were exposed to three concentrations (1, 90, 180 µM) of MEHP for 24 h with DMSO (0.1%) as a vehicle control. mRNA and lncRNAs were quantified using paired-end RNA sequencing, followed by identification of differentially expressed genes (DEGs), significant KEGG pathways, and enriched transcription factors (TFs). MEHP caused gene expression changes across all concentrations for HTR-8/SVneo and primary syncytiotrophoblast cells. Sex-stratified analysis of primary cells identified different patterns of sensitivity in response to MEHP dose by sex, with male placentas being more responsive to MEHP exposure. Pathway analysis identified 11 KEGG pathways significantly associated with at least one concentration in both cell types. Four ligand-inducible nuclear hormone TFs (PPARG, PPARD, ESR1, AR) were enriched in at least three treatment groups. Overall, we demonstrated that MEHP differentially affects placental gene expression based on concentration, fetal sex, and trophoblast cell type. This study confirms prior studies, as enrichment of nuclear hormone receptor TFs were concordant with previously published mechanisms of phthalate disruption, and generates new hypotheses, as we identified many pathways and genes not previously linked to phthalate exposure.

Temporal changes in the brain lipidome during neurodevelopment of Smith-Lemli-Opitz syndrome mice.

Neurodevelopment is an intricately orchestrated program of cellular events that occurs with tight temporal and spatial regulation. While it is known that the development and proper functioning of the brain, which is the second most lipid rich organ behind adipose tissue, greatly rely on lipid metabolism and signaling, the temporal lipidomic changes that occur throughout the course of neurodevelopment have not been investigated. Smith-Lemli-Opitz syndrome is a metabolic disorder caused by genetic mutations in the DHCR7 gene, leading to defective 3ß-hydroxysterol-Δ7-reductase (DHCR7), the enzyme that catalyzes the last step of the Kandutsch-Russell pathway of cholesterol synthesis. Due to the close regulatory relationship between sterol and lipid homeostasis, we hypothesize that altered or dysregulated lipid metabolism beyond the primary defect of cholesterol biosynthesis is present in the pathophysiology of SLOS. Herein, we applied our HILIC-IM-MS method and LiPydomics Python package to streamline an untargeted lipidomics analysis of developing mouse brains in both wild-type and Dhcr7-KO mice, identifying lipids at Level 3 (lipid species level: lipid class/subclass and fatty acid sum composition). We compared relative lipid abundances throughout development, from embryonic day 12.5 to postnatal day 0 and determined differentially expressed brain lipids between wild-type and Dhcr7-KO mice at specific developmental time points, revealing lipid metabolic pathways that are affected in SLOS beyond the cholesterol biosynthesis pathway, such as glycerolipid, glycerophospholipid, and sphingolipid metabolism. Implications of the altered lipid metabolic pathways in SLOS pathophysiology are discussed.

Genetic associations of breast and prostate cancer are enriched for regulatory elements identified in disease-related tissues.

Although genome-wide association studies (GWAS) have identified hundreds of risk loci for breast and prostate cancer, only a few studies have characterized the GWAS association signals across functional genomic annotations with a particular focus on single nucleotide polymorphisms (SNPs) located in DNA regulatory elements. In this study, we investigated the enrichment pattern of GWAS signals for breast and prostate cancer in genomic functional regions located in normal tissue and cancer cell lines. We quantified the overall enrichment of SNPs with breast and prostate cancer association p values < 1 × 10-8 across regulatory categories. We then obtained annotations for DNaseI hypersensitive sites (DHS), typical enhancers, and super enhancers across multiple tissue types, to assess if significant GWAS signals were selectively enriched in annotations found in disease-related tissue. Finally, we quantified the enrichment of breast and prostate cancer SNP heritability in regulatory regions, and compared the enrichment pattern of SNP heritability with GWAS signals. DHS, typical enhancers, and super enhancers identified in the breast cancer cell line MCF-7 were observed with the highest enrichment of genome-wide significant variants for breast cancer. For prostate cancer, GWAS signals were mostly enriched in DHS and typical enhancers identified in the prostate cancer cell line LNCaP. With progressively stringent GWAS p value thresholds, an increasing trend of enrichment was observed for both diseases in DHS, typical enhancers, and super enhancers located in disease-related tissue. Results from heritability enrichment analysis supported the selective enrichment pattern of functional genomic regions in disease-related cell lines for both breast and prostate cancer. Our results suggest the importance of studying functional annotations identified in disease-related tissues when characterizing GWAS results, and further demonstrate the role of germline DNA regulatory elements from disease-related tissue in breast and prostate carcinogenesis.

Elevated CO2 impairs olfactory-mediated neural and behavioral responses and gene expression in ocean-phase coho salmon (Oncorhynchus kisutch).

Elevated concentrations of CO2 in seawater can disrupt numerous sensory systems in marine fish. This is of particular concern for Pacific salmon because they rely on olfaction during all aspects of their life including during their homing migrations from the ocean back to their natal streams. We investigated the effects of elevated seawater CO2 on coho salmon (Oncorhynchus kisutch) olfactory-mediated behavior, neural signaling, and gene expression within the peripheral and central olfactory system. Ocean-phase coho salmon were exposed to three levels of CO2 , ranging from those currently found in ambient marine water to projected future levels. Juvenile coho salmon exposed to elevated CO2 levels for 2 weeks no longer avoided a skin extract odor that elicited avoidance responses in coho salmon maintained in ambient CO2 seawater. Exposure to these elevated CO2 levels did not alter odor signaling in the olfactory epithelium, but did induce significant changes in signaling within the olfactory bulb. RNA-Seq analysis of olfactory tissues revealed extensive disruption in expression of genes involved in neuronal signaling within the olfactory bulb of salmon exposed to elevated CO2 , with lesser impacts on gene expression in the olfactory rosettes. The disruption in olfactory bulb gene pathways included genes associated with GABA signaling and maintenance of ion balance within bulbar neurons. Our results indicate that ocean-phase coho salmon exposed to elevated CO2 can experience significant behavioral impairments likely driven by alteration in higher-order neural signal processing within the olfactory bulb. Our study demonstrates that anadromous fish such as salmon may share a sensitivity to rising CO2 levels with obligate marine species suggesting a more wide-scale ecological impact of ocean acidification.

Dried blood spot compared to plasma measurements of blood-based biomarkers of brain injury in neonatal encephalopathy.

BACKGROUND: Data correlating dried blood spots (DBS) and plasma concentrations for neonatal biomarkers of brain injury are lacking. We hypothesized that candidate biomarker levels determined from DBS can serve as a reliable surrogate for plasma levels. METHODS: In the context of a phase II multi-center trial evaluating erythropoietin for neuroprotection in neonatal encephalopathy (NE), DBS were collected at enrollment ( < 24 h), day 2, 4, and 5. Plasma was collected with the first and last DBS. The relationship between paired DBS-plasma determinations of brain-specific proteins and cytokines was assessed by correlation and Bland-Altman analyses. For analytes with consistent DBS-plasma associations, DBS-derived biomarker levels were related to brain injury by MRI and 1-year outcomes. RESULTS: We enrolled 50 newborns with NE. While S100B protein, tumor necrosis factor α, interleukin (IL)1 ß, IL-6, IL-8 demonstrated significant DBS-plasma correlations, Bland-Altman plots demonstrated that the methods are not interchangeable, with a 2 to 4-fold error between measurements. No significant relationships were found between DBS levels of TNFα, IL-6, and IL-8 and outcomes. CONCLUSION: Further work is needed to optimize elution and assay methods before using DBS specimens as a reliable surrogate for plasma levels of candidate brain injury biomarkers in NE.

In utero exposure to diesel exhaust is associated with alterations in neonatal cardiomyocyte transcription, DNA methylation and metabolic perturbation.

BACKGROUND: Developmental exposure to particulate matter air pollution is harmful to cardiovascular health, but the mechanisms by which this exposure mediates susceptibility to heart disease is poorly understood. We have previously shown, in a mouse model, that gestational exposure to diesel exhaust (DE) results in increased cardiac hypertrophy, fibrosis and susceptibility to heart failure in the adult offspring following transverse aortic constriction. RESULTS: In this study, we have analyzed gene expression in neonatal cardiomyocytes after gestational exposure by RNA-sequencing and have identified 300 genes that are dysregulated, including many involved in cardiac metabolism. We subsequently determined that these cardiomyocytes exhibit reduced metabolic activity as measured by Seahorse extracellular flux analysis. We also surveyed for modifications in DNA methylation at global regulatory regions using reduced representation bisulfite sequencing and found hypomethylation of DNA in neonatal cardiomyocytes isolated from in utero DE exposed neonates. CONCLUSION: We have demonstrated that in utero exposure to diesel exhaust alters the neonatal cardiomyocyte transcriptional and epigenetic landscapes, as well as the metabolic capability of these cells. Understanding how exposure alters the developing heart through dysregulation of gene expression, metabolism and DNA methylation is vital for identifying therapeutic interventions for air pollution-related heart failure.

Human Cervical Mucus Plugs Exhibit Insufficiencies in Antimicrobial Activity Towards Group B Streptococcus.

Preterm birth is a leading cause of neonatal mortality and lacks an effective therapy. Ascending microbial infections from the lower genital tract lead to infection of the placenta, amniotic fluid, and fetus causing preterm birth or stillbirth. Directly in the path of an ascending infection is the cervical mucus plug (CMP), a dense mucoid structure in the cervical canal with potential antimicrobial properties. In this study, we aimed to define the components of CMP responsible for antimicrobial activity against a common lower genital tract organism associated with preterm birth and stillbirths, namely, group B streptococcus (GBS). Using a quantitative proteomic approach, we identified antimicrobial factors in CMPs that were collected from healthy human pregnancies. However, we noted that the concentration of antimicrobial peptides present in the human CMPs were insufficient to directly kill GBS, and antimicrobial activity, when observed, was due to antibiotics retained in the CMPs. Despite this insufficiency, CMP proteins were able to activate leukocytes in whole blood resulting in increased rates of bacterial killing, suggesting a role for the CMP in enhancing complement-mediated killing or leukocyte activation. This study provides new insight into how the human CMP may limit ascending bacterial infection.

In utero exposure to diesel exhaust particulates is associated with an altered cardiac transcriptional response to transverse aortic constriction and altered DNA methylation.

In utero exposure to diesel exhaust air pollution has been associated with increased adult susceptibility to heart failure in mice, but the mechanisms by which this exposure promotes susceptibility to heart failure are poorly understood. To identify the potential transcriptional effects that mediate this susceptibility, we have performed RNA sequencing analysis on adult hearts from mice that were exposed to diesel exhaust in utero and that have subsequently undergone transverse aortic constriction. We identified 3 target genes, Mir133a-2, Ptprf, and Pamr1, which demonstrate dysregulation after exposure and aortic constriction. Examination of expression patterns in human heart tissues indicates a correlation between expression and heart failure. We subsequently assessed DNA methylation modifications at these candidate loci in neonatal cultured cardiac myocytes after in utero exposure to diesel exhaust and found that the promoter for Mir133a-2 is differentially methylated. These target genes in the heart are the first genes to be identified that likely play an important role in mediating adult sensitivity to heart failure. We have also shown a change in DNA methylation within cardiomyocytes as a result of in utero exposure to diesel exhaust.-Goodson, J. M., Weldy, C. S., MacDonald, J. W., Liu, Y., Bammler, T. K., Chien, W.-M., Chin, M. T. In utero exposure to diesel exhaust particulates is associated with an altered cardiac transcriptional response to transverse aortic constriction and altered DNA methylation.

Genetic determinants of susceptibility to silver nanoparticle-induced acute lung inflammation in mice.

Silver nanoparticles (AgNPs) are employed in a variety of consumer products; however, in vivo rodent studies indicate that AgNPs can cause lung inflammation and toxicity in a strain- and particle type-dependent manner, but mechanisms of susceptibility remain unclear. The aim of this study was to assess the variation in AgNP-induced lung inflammation and toxicity across multiple inbred mouse strains and to use genome-wide association (GWA) mapping to identify potential candidate susceptibility genes. Mice received doses of 0.25 mg/kg of either 20-nm citrate-coated AgNPs or citrate buffer using oropharyngeal aspiration. Neutrophils in bronchoalveolar lavage fluid (BALF) served as markers of inflammation. We found significant strain- and treatment-dependent variation in neutrophils in BALF. GWA mapping identified 10 significant single-nucleotide polymorphisms (false discovery rate, 15%) in 4 quantitative trait loci on mouse chromosomes 1, 4, 15, and 18, and Nedd4l (neural precursor cell expressed developmentally downregulated gene 4-like; chromosome 18), Ano6 (anocatmin 6; chromosome 15), and Rnf220 (Ring finger protein 220; chromosome 4) were considered candidate genes. Quantitative RT-PCR revealed significant inverse associations between mRNA levels of these genes and neutrophil influx. Nedd4l, Ano6, and Rnf220 are candidate susceptibility genes for AgNP-induced lung inflammation that warrant additional exploration in future studies.-Scoville, D. K., Botta, D., Galdanes, K., Schmuck, S. C., White, C. C., Stapleton, P. L., Bammler, T. K., MacDonald, J. W., Altemeier, W. A., Hernandez, M., Kleeberger, S. R., Chen, L.-C., Gordon, T., Kavanagh, T. J. Genetic determinants of susceptibility to silver nanoparticle-induced acute lung inflammation in mice.

Evidence of cardiac involvement in the fetal inflammatory response syndrome: disruption of gene networks programming cardiac development in nonhuman primates.

BACKGROUND: Most early preterm births are associated with intraamniotic infection and inflammation, which can lead to systemic inflammation in the fetus. The fetal inflammatory response syndrome describes elevations in the fetal interleukin-6 level, which is a marker for inflammation and fetal organ injury. An understanding of the effects of inflammation on fetal cardiac development may lead to insight into the fetal origins of adult cardiovascular disease. OBJECTIVE: The purpose of this study was to determine whether the fetal inflammatory response syndrome is associated with disruptions in gene networks that program fetal cardiac development. STUDY DESIGN: We obtained fetal cardiac tissue after necropsy from a well-described pregnant nonhuman primate model (pigtail macaque, Macaca nemestrina) of intrauterine infection (n=5) and controls (n=5). Cases with the fetal inflammatory response syndrome (fetal plasma interleukin-6 >11 pg/mL) were induced by either choriodecidual inoculation of a hypervirulent group B streptococcus strain (n=4) or intraamniotic inoculation of Escherichia coli (n=1). RNA and protein were extracted from fetal hearts and profiled by microarray and Luminex (Millipore, Billerica, MA) for cytokine analysis, respectively. Results were validated by quantitative reverse transcriptase polymerase chain reaction. Statistical and bioinformatics analyses included single gene analysis, gene set analysis, Ingenuity Pathway Analysis (Qiagen, Valencia, CA), and Wilcoxon rank sum. RESULTS: Severe fetal inflammation developed in the context of intraamniotic infection and a disseminated bacterial infection in the fetus. Interleukin-6 and -8 in fetal cardiac tissues were elevated significantly in fetal inflammatory response syndrome cases vs controls (P<.05). A total of 609 probe sets were expressed differentially (>1.5-fold change, P<.05) in the fetal heart (analysis of variance). Altered expression of select genes was validated by quantitative reverse transcriptase polymerase chain reaction that included several with known functions in cardiac injury, morphogenesis, angiogenesis, and tissue remodeling (eg, angiotensin I converting enzyme 2, STEAP family member 4, natriuretic peptide A, and secreted frizzled-related protein 4; all P<.05). Multiple gene sets and pathways that are involved in cardiac morphogenesis and vasculogenesis were downregulated significantly by gene set and Ingenuity Pathway Analysis (hallmark transforming growth factor beta signaling, cellular morphogenesis during differentiation, morphology of cardiovascular system; all P<.05). CONCLUSION: Disruption of gene networks for cardiac morphogenesis and vasculogenesis occurred in the preterm fetal heart of nonhuman primates with preterm labor, intraamniotic infection, and severe fetal inflammation. Inflammatory injury to the fetal heart in utero may contribute to the development of heart disease later in life. Development of preterm labor therapeutics must also target fetal inflammation to lessen organ injury and potential long-term effects on cardiac function.

Impact of processing methods on urinary biomarkers analysis in neonates.

BACKGROUND: In neonates, the validation of urinary biomarkers to diagnose acute kidney injury is a rapidly evolving field. The neonatal population poses unique challenges when assessing the collection, storage, and processing of urinary samples for biomarker analysis. Given this, establishing optimal and consistent sample processing in this population for meaningful use in ongoing clinical trials is important. METHODS: Urine from a cohort of 19 hospitalized neonatal intensive care unit patients enrolled in the Preterm Erythropoietin Neuroprotection Trial (Clinical Trial NCT01378273) was collected for biomarker analysis by indirect techniques using Fisher-brand cotton balls placed in the diapers. Fourteen urinary biomarkers were measured using commercially available kits via electrochemiluminescence on multiarray plates and compared between paired samples processed with centrifugation prior to storage versus prior to analysis. RESULTS: None of the biomarker concentrations differed between samples undergoing centrifugation prior to storage versus prior to analysis. The difference between samples was within 2% of the estimated concentration for the protein in 12 of 14 biomarkers (86%), and all paired biomarker concentrations were within 4%. The percentage error analysis did not show a difference between paired samples, with biomarker percentage errors smaller than the stated immunoassay coefficient of variance. CONCLUSIONS: The urinary concentrations of biomarkers were comparable between paired samples, demonstrating that indirectly collected neonatal urine samples do not require centrifugation after collection and before storage. The ability to use routine urine collection and storage methods to obtain samples for subsequent quantitative immunoassay analysis should facilitate studies of newborns and young children.

Time Course of Inflammatory Gene Expression Following Crush Injury in Murine Skeletal Muscle.

BACKGROUND: Early inflammation and secretion of proinflammatory cytokines such as IL-1ß, IL-6, and TNF-α act as the key drivers to regulate inflammation after muscle injury. However, the effects of these key proinflammatory drivers in a noninvasive crush injury model are not well known. Understanding these effects is important for treating crush injuries that occur during natural disasters and military conflicts. PURPOSE: We studied the timed mRNA expression of IL-1ß, IL-6, and TNF-α in a noninvasive murine crush injury model to further understand their impact on proinflammatory cytokine pathways that are activated within the first 48 hours after a crush muscle injury. METHODS: A total of 25 mice were anesthetized and placed on a crush injury apparatus platform with the apparatus piston situated in direct contact with intact skin overlying the right gastrocnemius muscle. Pressure at 45 psi was applied to the piston for 30 seconds for two applications. The mice recovered for either 4, 8, 24, or 48 hours postinjury, after which we harvested the gastrocnemius muscle of both legs. Microarray, confirmatory real-time polymerase chain reaction, and immunolabeling experiments were followed by a microarray time-course analysis. RESULTS: Muscle IL-1ß mRNA rose 270-fold within 4 hours and declined rapidly at 8 hours to 196-fold, 24 hours to 96-fold, and 48 hours to 10-fold. Muscle IL-6 followed the same pattern, with a 34-fold increase at 4 hours, 29-fold increase at 8 hours, 10-fold increase at 24 hours, and 5-fold increase at 48 hours. Ingenuity Pathway Analysis of IL-6 identified activation of two major downstream signaling pathways (IL-6/Stat3 and IL-1ß/Egr1) as key activators of inflammation, regeneration, and fibrosis. DISCUSSION: Closed crush muscle injury produced robust muscle cytokine expression levels, and the microarray findings allowed us to generate our most novel hypothesis: that high expression of IL-1ß, IL-6, and TNF-α may be related to the downregulation of mitochondrial genes early after injury and triggers activation of genes in the repair and fibrosis machinery. The significance of these findings and the identified expression pathways of IL1-ß, IL-6, and TNF-α and their downstream targets in skeletal muscle will allow us to further investigate targets for improved muscle recovery and limb-saving interventions.

A Novel Three-Dimensional Human Peritubular Microvascular System.

Human kidney peritubular capillaries are particularly susceptible to injury, resulting in dysregulated angiogenesis, capillary rarefaction and regression, and progressive loss of kidney function. However, little is known about the structure and function of human kidney microvasculature. Here, we isolated, purified, and characterized human kidney peritubular microvascular endothelial cells (HKMECs) and reconstituted a three-dimensional human kidney microvasculature in a flow-directed microphysiologic system. By combining epithelial cell depletion and cell culture in media with high concentrations of vascular endothelial growth factor, we obtained HKMECs of high purity in large quantity. Unlike other endothelial cells, isolated HKMECs depended on high vascular endothelial growth factor concentration for survival and growth and exhibited high tubulogenic but low angiogenic potential. Furthermore, HKMECs had a different transcriptional profile. Under flow, HKMECs formed a thin fenestrated endothelium with a functional permeability barrier. In conclusion, this three-dimensional HKMEC-specific microphysiologic system recapitulates human kidney microvascular structure and function and shows phenotypic characteristics different from those of other microvascular endothelial cells.

Long-term outdoor air pollution and DNA methylation in circulating monocytes: results from the Multi-Ethnic Study of Atherosclerosis (MESA).

BACKGROUND: DNA methylation may mediate effects of air pollution on cardiovascular disease. The association between long-term air pollution exposure and DNA methylation in monocytes, which are central to atherosclerosis, has not been studied. We investigated the association between long-term ambient air pollution exposure and DNA methylation (candidate sites and global) in monocytes of adults (aged ≥55). METHODS: One-year average ambient fine particulate matter (PM2.5) and oxides of nitrogen (NOX) concentrations were predicted at participants' (n = 1,207) addresses using spatiotemporal models. We assessed DNA methylation in circulating monocytes at 1) 2,713 CpG sites associated with mRNA expression of nearby genes and 2) probes mapping to Alu and LINE-1 repetitive elements (surrogates for global DNA methylation) using Illumina's Infinium HumanMethylation450 BeadChip. We used linear regression models adjusted for demographics, smoking, physical activity, socioeconomic status, methyl-nutrients, and technical variables. For significant air pollution-associated methylation sites, we also assessed the association between expression of gene transcripts previously associated with these CpG sites and air pollution. RESULTS: At a false discovery rate of 0.05, five candidate CpGs (cg20455854, cg07855639, cg07598385, cg17360854, and cg23599683) had methylation significantly associated with PM2.5 and none were associated with NOX. Cg20455854 had the smallest p-value for the association with PM2.5 (p = 2.77 × 10-5). mRNA expression profiles of genes near three of the PM2.5-associated CpGs (ANKHD1, LGALS2, and ANKRD11) were also significantly associated with PM2.5 exposure. Alu and LINE-1 methylation were not associated with long-term air pollution exposure. CONCLUSIONS: We observed novel associations between long-term ambient air pollution exposure and site-specific DNA methylation, but not global DNA methylation, in purified monocytes of a multi-ethnic adult population. Epigenetic markers may provide insights into mechanisms underlying environmental factors in complex diseases like atherosclerosis.

Choriodecidual Group B Streptococcal Infection Induces miR-155-5p in the Fetal Lung in Macaca nemestrina.

The mechanisms underlying fetal lung injury remain poorly defined. MicroRNAs (miRNAs) are small noncoding, endogenous RNAs that regulate gene expression and have been implicated in the pathogenesis of lung disease. Using a nonhuman primate model of choriodecidual infection, we sought to determine if differentially expressed miRNAs were associated with acute fetal lung injury. After inoculating 10 chronically catheterized pregnant monkeys (Macaca nemestrina) with either group B streptococcus (GBS) at 1 × 10(6) CFU (n = 5) or saline (n = 5) in the choriodecidual space, we extracted fetal lung mRNA and miRNA and profiled the changes in expression by microarray analysis. We identified 9 differentially expressed miRNAs in GBS-exposed fetal lungs, but of these, only miR-155-5p was validated by quantitative reverse transcription-PCR (P = 0.02). Significantly elevated miR-155-5p expression was also observed when immortalized human fetal airway epithelial (FeAE) cells were exposed to proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]). Overexpression of miR-155-5p in FeAE cells in turn increased the production of IL-6 and CXCL10/gamma interferon-induced protein 10, which are implicated in leukocyte recruitment but also in protection from lung injury. Interestingly, while miR-155-5p decreased fibroblast growth factor 9 (FGF9) expression in a luciferase reporter assay, FGF9 levels were actually increased in GBS-exposed fetal lungs in vivo. FGF9 overexpression is associated with abnormal lung development. Thus, upregulation of miR-155-5p may serve as a compensatory mechanism to lessen the increase in FGF9 and prevent aberrant lung development. Understanding the complicated networks regulating lung development in the setting of infection is a key step in identifying how to prevent fetal lung injury leading to bronchopulmonary dysplasia.

Network analysis of microRNA and mRNA seasonal dynamics in a highly plastic sensorimotor neural circuit.

BACKGROUND: Adult neurogenesis and the incorporation of adult-born neurons into functional circuits requires precise spatiotemporal coordination across molecular networks regulating a wide array of processes, including cell proliferation, apoptosis, neurotrophin signaling, and electrical activity. MicroRNAs (miRs) - short, non-coding RNA sequences that alter gene expression by post-transcriptional inhibition or degradation of mRNA sequences - may be involved in the global coordination of such diverse biological processes. To test the hypothesis that miRs related to adult neurogenesis and related cellular processes are functionally regulated in the nuclei of the avian song control circuit, we used microarray analyses to quantify changes in expression of miRs and predicted target mRNAs in the telencephalic nuclei HVC, the robust nucleus of arcopallium (RA), and the basal ganglia homologue Area X in breeding and nonbreeding Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelli). RESULTS: We identified 46 different miRs that were differentially expressed across seasons in the song nuclei. miR-132 and miR-210 showed the highest differential expression in HVC and Area X, respectively. Analyzing predicted mRNA targets of miR-132 identified 33 candidate target genes that regulate processes including cell cycle control, calcium signaling, and neuregulin signaling in HVC. Likewise, miR-210 was predicted to target 14 mRNAs differentially expressed across seasons that regulate serotonin, GABA, and dopamine receptor signaling and inflammation. CONCLUSIONS: Our results identify potential miR-mRNA regulatory networks related to adult neurogenesis and provide opportunities to discover novel genetic control of the diverse biological processes and factors related to the functional incorporation of new neurons to the adult brain.