FGR-associated placental insufficiency and capillary angiogenesis involves disruptions in human placental miRNAs and mRNAs.

Fetal growth restriction (FGR) is one of the most common pregnancy complications culminating in adverse fetal outcome, including preterm birth, neonatal mortality and stillbirth. Compromised placental development and function, especially disruption in angiogenesis and inadequate nutrient supply are contributing factors. Fetal sex also influences placental function. Knowledge of gene expression changes and epigenetic factors contributing to placental dysfunction in FGR pregnancies will help identify biomarkers and help target interventions. This study tested the hypothesis that FGR pregnancies are associated with disruptions in miRNA - an epigenetic factor and mRNAs involving key mediators of angiogenesis and microvessel development. Changes in expression of key genes/proteins involved in placental dysfunction by RT-PCR and immunohistochemistry and miRNA changes by RNA sequencing were undertaken with term placenta from 12 control and 20 FGR pregnancies. Findings showed changes in expression of genes involved in steroidogenesis, steroid action, IGF family members, inflammatory cytokines and angiogenic factors in FGR pregnancies. In addition, upregulation of MIR451A and downregulation of MIR543 in placentas from FGR group with female newborns and upregulation of MIR520G in placentas from FGR group with male newborns were also noted. MIR451A and MIR543 have been implicated in angiogenesis. Consistent with gene changes, CD34, the microvessel angiogenesis marker, also showed reduced staining only in female FGR group. These findings provide evidence that epigentically regulated gene expression changes in angiogenesis and capillary development influence placental impairment in FGR pregnancies. Our preliminary observations also support for these changes to be driven in a sex-specific manner.

Developmental programming: Adipose depot-specific regulation of non-coding RNAs and their relation to coding RNA expression in prenatal testosterone and prenatal bisphenol-A -treated female sheep.

Inappropriate developmental exposure to steroids is linked to metabolic disorders. Prenatal testosterone excess or bisphenol A (BPA, an environmental estrogen mimic) leads to insulin resistance and adipocyte disruptions in female lambs. Adipocytes are key regulators of insulin sensitivity. Metabolic tissue-specific differences in insulin sensitivity coupled with adipose depot-specific changes in key mRNAs, were previously observed with prenatal steroid exposure. We hypothesized that depot-specific changes in the non-coding RNA (ncRNA) - regulators of gene expression would account for the direction of changes seen in mRNAs. Non-coding RNA (lncRNA, miRNA, snoRNA, snRNA) from various adipose depots of prenatal testosterone and BPA-treated animals were sequenced. Adipose depot-specific changes in the ncRNA that are consistent with the depot-specific mRNA expression in terms of directionality of changes and functional implications in insulin resistance, adipocyte differentiation and cardiac hypertrophy were found. Importantly, the adipose depot-specific ncRNA changes were model-specific and mutually exclusive, suggestive of different regulatory entry points in this regulation.

Developmental programming: Impact of prenatal bisphenol-A exposure on liver and muscle transcriptome of female sheep.

Gestational Bisphenol A (BPA) exposure leads to peripheral insulin resistance, and hepatic and skeletal muscle oxidative stress and lipotoxicity during adulthood in the female sheep offspring. To investigate transcriptional changes underlying the metabolic outcomes, coding and non-coding (nc) RNA in liver and muscle from 21-month-old control and prenatal BPA-treated (0.5 mg/kg/day from days 30 to 90 of gestation; Term: 147 days) female sheep were sequenced. Prenatal BPA-treatment dysregulated: expression of 194 genes (138 down, 56 up) in liver and 112 genes (32 down, 80 up) in muscle (FDR < 0.05 and abs log2FC > 0.5); 155 common gene pathways including mitochondrial-related genes in both tissues; 1415 gene pathways including oxidative stress and lipid biosynthetic process specifically in the liver (FDR < 0.01); 192 gene pathways including RNA biosynthetic processes in muscle (FDR < 0.01); 77 lncRNA (49 down, 28 up), 14 microRNAs (6 down, 8 up), 127 snoRNAs (63 down, 64 up) and 55 snRNAs (15 down, 40 up) in the liver while upregulating 6 lncRNA and dysregulating 65 snoRNAs (47 down, 18 up) in muscle (FDR < 0.1, abs log2FC > 0.5). Multiple ncRNA correlated with LCORL, MED17 and ZNF41 mRNA in liver but none of them in the muscle. Discriminant analysis identified (p < 0.05) PECAM, RDH11, ABCA6, MIR200B, and MIR30B in liver and CAST, NOS1, FASN, MIR26B, and MIR29A in muscle as gene signatures of gestational BPA exposure. These findings provide mechanistic clues into the development and/or maintenance of the oxidative stress and lipid accumulation and potential for development of mitochondrial and fibrotic defects contributing to the prenatal BPA-induced metabolic dysfunctions.

A Transcription Start Site Map in Human Pancreatic Islets Reveals Functional Regulatory Signatures.

Identifying the tissue-specific molecular signatures of active regulatory elements is critical to understand gene regulatory mechanisms. Here, we identify transcription start sites (TSS) using cap analysis of gene expression (CAGE) across 57 human pancreatic islet samples. We identify 9,954 reproducible CAGE tag clusters (TCs), ∼20% of which are islet specific and occur mostly distal to known gene TSS. We integrated islet CAGE data with histone modification and chromatin accessibility profiles to identify epigenomic signatures of transcription initiation. Using a massively parallel reporter assay, we validated the transcriptional enhancer activity for 2,279 of 3,378 (∼68%) tested islet CAGE elements (5% false discovery rate). TCs within accessible enhancers show higher enrichment to overlap type 2 diabetes genome-wide association study (GWAS) signals than existing islet annotations, which emphasizes the utility of mapping CAGE profiles in disease-relevant tissue. This work provides a high-resolution map of transcriptional initiation in human pancreatic islets with utility for dissecting active enhancers at GWAS loci.

Endotoxin- and mechanical stress-induced epigenetic changes in the regulation of the nicotinamide phosphoribosyltransferase promoter.

Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.

The NAMPT promoter is regulated by mechanical stress, signal transducer and activator of transcription 5, and acute respiratory distress syndrome-associated genetic variants.

Increased nicotinamide phosphoribosyltransferase (NAMPT) transcription is mechanistically linked to ventilator-induced inflammatory lung injury (VILI), with VILI severity attenuated by reduced NAMPT bioavailability. The molecular mechanisms of NAMPT promoter regulation in response to excessive mechanical stress remain poorly understood. The objective of this study was to define the contribution of specific transcription factors, acute respiratory distress syndrome (ARDS)-associated single nucleotide polymorphisms (SNPs), and promoter demethylation to NAMPT transcriptional regulation in response to mechanical stress. In vivo NAMPT protein expression levels were examined in mice exposed to high tidal volume mechanical ventilation. In vitro NAMPT expression levels were examined in human pulmonary artery endothelial cells exposed to 5 or 18% cyclic stretch (CS), with NAMPT promoter activity assessed using NAMPT promoter luciferase reporter constructs with a series of nested deletions. In vitro NAMPT transcriptional regulation was further characterized by measuring luciferase activity, DNA demethylation, and chromatin immunoprecipitation. VILI-challenged mice exhibited significantly increased NAMPT expression in bronchoalveolar lavage leukocytes and in lung endothelium. A mechanical stress-inducible region (MSIR) was identified in the NAMPT promoter from -2,428 to -2,128 bp. This MSIR regulates NAMPT promoter activity, mRNA expression, and signal transducer and activator of transcription 5 (STAT5) binding, which is significantly increased by 18% CS. In addition, NAMPT promoter activity was increased by pharmacologic promoter demethylation and inhibited by STAT5 silencing. ARDS-associated NAMPT promoter SNPs rs59744560 (-948G/T) and rs7789066 (-2,422A/G) each significantly elevated NAMPT promoter activity in response to 18% CS in a STAT5-dependent manner. Our results show that NAMPT is a key novel ARDS therapeutic target and candidate gene with genetic/epigenetic transcriptional regulation in response to excessive mechanical stress.

Mechanical stress induces pre-B-cell colony-enhancing factor/NAMPT expression via epigenetic regulation by miR-374a and miR-568 in human lung endothelium.

Increased lung vascular permeability and alveolar edema are cardinal features of inflammatory conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). We previously demonstrated that pre-B-cell colony-enhancing factor (PBEF)/NAMPT, the proinflammatory cytokine encoded by NAMPT, participates in ARDS and VILI inflammatory syndromes. The present study evaluated posttranscriptional regulation of PBEF/NAMPT gene expression in human lung endothelium via 3'-untranslated region (UTR) microRNA (miRNA) binding. In silico analysis identified hsa-miR-374a and hsa-miR-568 as potential miRNA candidates. Increased PBEF/NAMPT transcription (by RT-PCR) and expression (by Western blotting) induced by 18% cyclic stretch (CS) (2 h: 3.4 ± 0.06 mRNA fold increase (FI); 10 h: 1.5 ± 0.06 protein FI) and by LPS (4 h: 3.8 ± 0.2 mRNA FI; 48 h: 2.6 ± 0.2 protein FI) were significantly attenuated by transfection with mimics of hsa-miR-374a or hsa-miR-568 (40-60% reductions each). LPS and 18% CS increased the activity of a PBEF/NAMPT 3'-UTR luciferase reporter (2.4-3.25 FI) with induction reduced by mimics of each miRNA (44-60% reduction). Specific miRNA inhibitors (antagomirs) for each PBEF/NAMPT miRNA significantly increased the endogenous PBEF/NAMPT mRNA (1.4-3.4 ± 0.1 FI) and protein levels (1.2-1.4 ± 0.1 FI) and 3'-UTR luciferase activity (1.4-1.7 ± 0.1 FI) compared with negative antagomir controls. Collectively, these data demonstrate that increased PBEF/NAMPT expression induced by bioactive agonists (i.e., excessive mechanical stress, LPS) involves epigenetic regulation with hsa-miR-374a and hsa-miR-568, representing novel therapeutic strategies to reduce inflammatory lung injury.