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1.  Tissue-specific Leptin promoter DNA methylation is associated with maternal and infant perinatal factors 
Leptin a regulator of body weight is involved in reproductive and developmental functions. Leptin promoter DNA methylation (LEP) regulates gene expression in a tissue-specific manner and has been linked to adverse pregnancy outcomes. In non-pathologic human pregnancies, we assessed LEP methylation, genotyped the single nucleotide polymorphism (SNP) rs2167270 in placental (n=81), maternal and cord blood samples (n=60), and examined the association between methylation, genotype, and perinatal factors. Maternal blood LEP methylation was lower in pre-pregnancy obese women (P=0.01). Cord blood LEP methylation was higher in small for gestational age (SGA) (P=4.6×10−3) and A/A genotype (P=1.6×10−4), lower (−1.47, P=0.03) in infants born to pre-pregnancy obese mothers and correlated (P=0.01) with maternal blood LEP. Gender was associated with placental LEP methylation (P=0.05). These results suggest that LEP epigenetic control may be influenced by perinatal factors including: maternal obesity, infant growth, genotype and gender in a tissue-specific manner and may have multigenerational implications.
doi:10.1016/j.mce.2013.07.024
PMCID: PMC3795868  PMID: 23911897
leptin; epigenetics; DNA methylation; rs2167270; pregnancy; maternal obesity; small for gestational age
2.  Genetic and Epigenetic Variation of the Glucocorticoid Receptor (NR3C1) in Placenta and Infant Neurobehavior 
Developmental psychobiology  2012;55(7):673-683.
The intrauterine environment can impact the developing infant by altering the function of the placenta through changes to the epigenetic regulatory features of this tissue. Genetic variation, too, may impact infant development or may modify the relationship between epigenetic alterations and infant outcomes. To examine the association of this variation with early life infant neurodevelopment, we examined the extent of DNA methylation of the glucocorticoid receptor gene (NR3C1) promoter and a common SNP in the promoter region in a series of 186 placentas from healthy newborn infants. We associated these molecular features with specific summary measures from the NICU Network Neurobehavioral Scales. After controlling for genotype and confounders, we identified significant associations of NR3C1 methylation with infant quality of movement (P=0.05) and with infant attention (P=0.05), and a potential interaction between methylation and genotype on infant attention score. These results suggest that epigenetic alteration of the NR3C1 gene in the placentas of genetically susceptible infants can have impacts on neurodevelopment which may have lifelong impact on neurobehavioral and mental health outcomes. Further research is needed to more precisely define these relationships and the interaction between epigenetic alterations and genetic variations on infant health.
doi:10.1002/dev.21061
PMCID: PMC3458180  PMID: 22714792
Epigenetic; glucocorticoid receptor; placenta; neurodevelopment; human; attention; quality of movement
3.  Metformin Protects Cardiomyocyte from Doxorubicin Induced Cytotoxicity through an AMP-Activated Protein Kinase Dependent Signaling Pathway: An In Vitro Study 
PLoS ONE  2014;9(8):e104888.
Doxorubicin (Dox) is one of the most widely used antitumor drugs, but its cumulative cardiotoxicity have been major concerns in cancer therapeutic practice for decades. Recent studies established that metformin (Met), an oral anti-diabetic drug, provides protective effects in Dox-induced cardiotoxicity. Met has been shown to increase fatty acid oxidation, an effect mediated by AMP activated protein kinase (AMPK). Here we delineate the intracellular signaling factors involved in Met mediated protection against Dox-induced cardiotoxicity in the H9c2 cardiomyoblast cell line. Treatment with low dose Met (0.1 mM) increased cell viabilities and Ki-67 expressions while decreasing LDH leakages, ROS generations and [Ca2+]i. The protective effect was reversed by a co-treatment with compound-C, an AMPK specific inhibitor, or by an over expression of a dominant-negative AMPKα cDNA. Inhibition of PKA with H89 or a suppression of Src kinase by a small hairpin siRNA also abrogated the protective effect of the low dose Met. Whereas, with a higher dose of Met (1.0 mM), the protective effects were abolished regardless of the enhanced AMPK, PKA/CREB1 and Src kinase activity. In high dose Met treated cells, expression of platelet-derived growth factor receptor (PDGFR) was significantly suppressed. Furthermore, the protective effect of low dose Met was totally reversed by co-treatment with AG1296, a PDGFR specific antagonist. These data provide in vitro evidence supporting a signaling cascade by which low dose Met exerts protective effects against Dox via sequential involvement of AMPK, PKA/CREB1, Src and PDGFR. Whereas high dose Met reverses the effect by suppressing PDGFR expression.
doi:10.1371/journal.pone.0104888
PMCID: PMC4134245  PMID: 25127116
4.  Neonatal sepsis 
Virulence  2013;5(1):170-178.
Neonatal sepsis continues to be a common and significant health care burden, especially in very-low-birth-weight infants (VLBW <1500 g). Though intrapartum antibiotic prophylaxis has decreased the incidence of early-onset group B streptococcal infection dramatically, it still remains a major cause of neonatal sepsis. Moreover, some studies among VLBW preterm infants have shown an increase in early-onset sepsis caused by Escherichia coli. As the signs and symptoms of neonatal sepsis are nonspecific, early diagnosis and prompt treatment remains a challenge. There have been a myriad of studies on various diagnostic markers like hematological indices, acute phase reactants, C-reactive protein, procalcitonin, cytokines, and cell surface markers among others. Nonetheless, further research is needed to identify a biomarker with high diagnostic accuracy and validity. Some of the newer markers like inter α inhibitor proteins have shown promising results thereby potentially aiding in early detection of neonates with sepsis. In order to decrease the widespread, prolonged use of unnecessary antibiotics and improve the outcome of the infants with sepsis, reliable identification of sepsis at an earlier stage is paramount.
doi:10.4161/viru.26906
PMCID: PMC3916371  PMID: 24185532
neonatal sepsis; epidemiology; microbiology; biomarkers; algorithms; newer tests; screening; group B streptococcus; antibiotic prophylaxis
5.  Neonatal Intensive Care Unit: Predictive Models for Length of Stay 
Purpose
Hospital length of stay (LOS) is important to administrators and families of neonates admitted to the intensive care unit (NICU). A prediction model for NICU LOS was developed using as predictors birth weight, gestational age, and two severity of illness tools, the Score for Neonatal Acute Physiology (SNAPPE) and the Morbidity Assessment Index for Newborns (MAIN).
Materials and Methods
Consecutive admissions (n=293) to a New England regional level III NICU were retrospectively collected in 1999. Multiple predictive models were compared for complexity and goodness-of-fit, coefficient of determination (R2) and predictive error. The optimal model was validated prospectively with consecutive admissions (n=615) in 2002. Observed and expected LOS was compared.
Results
LOS was longer in the 2002 cohort than the 1999 cohort, without differences in birth weight, gestational age, MAIN or SNAPPE. The MAIN models had best AIC, highest R2 (0.786) and lowest predictive error. The best SNAPPE model underestimated LOS, with substantial variability, yet was fairly well calibrated by birthweight category.
Conclusions
Length of stay prediction is improved by accounting for severity of illness in the first week of life beyond factors known at birth. Prospective validation of both MAIN and SNAPPE models is warranted.
doi:10.1038/jp.2012.62
PMCID: PMC4073289  PMID: 22678140
Neonatal Intensive Care Units; Length of Stay; Severity of Illness Index; Benchmarking; Projections and Predictions; Score for Neonatal Acute Physiology
6.  Pathway-Based Genetic Analysis of Preterm Birth 
Genomics  2013;101(3):163-170.
Preterm birth in the United States is now 12%. Multiple genes, gene networks, variants have been associated with this disease. Using a custom database for preterm birth (dbPTB) with a refined set of genes extensively curated from literature and biological databases, we analyzed a GWAS of preterm birth for complete genotype data on nearly 2000 preterm and term mothers. We used both the curated genes and a genome-wide approach to carry out a pathway-based analysis. There were 19 significant pathways, which withstood FDR correction for multiple testing that were identified using both the curated genes and the genome-wide approach. The analysis based on the curated genes was more significant than genome-wide in 15 out of 19 pathways. This approach demonstrates the use of a validated set of genes, in the analysis of otherwise unsuccessful GWAS data, to identify gene-gene interactions in a way that enhances statistical power and discovery.
doi:10.1016/j.ygeno.2012.12.005
PMCID: PMC3570639  PMID: 23298525
Preterm birth; Pathway analysis; GWAS
7.  Placental miRNA Expression Profiles Associated with Measures of Infant Neurobehavioral Outcomes 
Pediatric research  2013;74(3):272-278.
Background
A growing body of research suggests that the intrauterine environment influences fetal neurodevelopment by altering the functional placental epigenome. A number of miRNAs are expressed in the placenta, may be sensitive to dysregulation by environmental exposures, and are associated with adverse pregnancy outcomes. Our study aimed to identify relationships between placental miRNA expression and newborn neurobehavior.
Methods
We examined the association between the expression of miR-16, miR-21, miR-93, miR-135b, miR-146a, and miR-182 in total RNA from the placentas of 86 term infants as measured by quantitative real-time PCR and newborn neurobehavioral outcomes as assessed using the NICU Network Neurobehavioral Scales (NNNS).
Results
Bivariate analysis revealed that placental miR-16 expression is negatively associated with attention score (p=0.006), while expression of miR-146a and miR-182 are both positively associated with quality of movement score (p=0.016 and p=0.016, respectively). Controlling for potential confounders, high miR-16 expression is significantly associated with reduced attention score (p=0.04), and high miR-146a expression and high miR-182 expression are significantly associated with increased quality of movement score (p=0.04 and p=0.01, respectively).
Conclusions
These results suggest that placental miRNA expression is associated with early neurobehavioral outcomes and miRNAs in the placenta may contribute to the developmental origins of infant neurobehavior.
doi:10.1038/pr.2013.102
PMCID: PMC3766495  PMID: 23783433
8.  Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery 
Objective
The purpose of this study was to determine in a mouse model whether uterine natural killer (uNK) cell cytotoxic activation induces infection/inflammation-associated preterm labor and delivery.
Study design
Wild type or interleukin (IL)-10−/− mice were injected intraperitoneally with lipopolysaccharide on gestational day 14. Mice were either killed for collection of uteroplacental tissue, spleen, and serum or allowed to deliver. Uteroplacental tissue was used for histology and characterization of uNK cells.
Results
Low-dose lipopolysaccharide treatment triggered preterm labor and delivery in IL-10−/−, but not wild type mice, in a manner independent of progesterone levels. Preterm labor and delivery in IL-10−/− mice was associated with an increased number and placental infiltration of cytotoxic uNK cells and placental cell death. Depletion of NK cells or tumor necrosis factor (TNF)α neutralization in these mice restored term delivery. Furthermore, TNFα neutralization prevented uNK cell infiltration and placental cell apoptosis.
Conclusion
The uNK cell-TNFα-IL-10 axis plays an important role in the genesis of infection/inflammation-induced preterm labor/delivery.
doi:10.1016/j.ajog.2008.10.043
PMCID: PMC3893044  PMID: 19114277
cytokines; inflammation; preterm birth; uterine natural killer cells
9.  HE4 (WFDC2) gene overexpression promotes ovarian tumor growth 
Scientific Reports  2014;4:3574.
Selective overexpression of Human epididymal secretory protein E4 (HE4) points to a role in ovarian cancer tumorigenesis but little is known about the role the HE4 gene or the gene product plays. Here we show that elevated HE4 serum levels correlate with chemoresistance and decreased survival rates in EOC patients. HE4 overexpression promoted xenograft tumor growth and chemoresistance against cisplatin in an animal model resulting in reduced survival rates. HE4 displayed responses to tumor microenvironment constituents and presented increased expression as well as nuclear translocation upon EGF, VEGF and Insulin treatment and nucleolar localization with Insulin treatment. HE4 interacts with EGFR, IGF1R, and transcription factor HIF1α. Constructs of antisense phosphorothio-oligonucleotides targeting HE4 arrested tumor growth in nude mice. Collectively these findings implicate increased HE4 expression as a molecular factor in ovarian cancer tumorigenesis. Selective targeting directed towards the HE4 protein demonstrates therapeutic benefits for the treatment of ovarian cancer.
doi:10.1038/srep03574
PMCID: PMC3880958  PMID: 24389815
10.  PT19c, Another Nonhypercalcemic Vitamin D2 Derivative, Demonstrates Antitumor Efficacy in Epithelial Ovarian and Endometrial Cancer Models 
Genes & Cancer  2013;4(11-12):524-534.
Hypercalcemia remains a major impediment to the clinical use of vitamin D in cancer treatment. Approaches to remove hypercalcemia and development of nonhypercalcemic agents can lead to the development of vitamin D–based therapies for treatment of various cancers. In this report, in vitro and in vivo anticancer efficacy, safety, and details of vitamin D receptor (VDR) interactions of PT19c, a novel nonhypercalcemic vitamin D derived anticancer agent, are described. PT19c was synthesized by bromoacetylation of PTAD-ergocalciferol adduct. Broader growth inhibitory potential of PT19c was evaluated in a panel of chemoresistant breast, renal, ovarian, lung, colon, leukemia, prostate, melanoma, and central nervous system cancers cell line types of NCI60 cell line panel. Interactions of PT19c with VDR were determined by a VDR transactivation assay in a VDR overexpressing VDR-UAS-bla-HEK293 cells, in vitro VDR-coregulator binding, and molecular docking with VDR-ligand binding domain (VDR-LBD) in comparison with calcitriol. Acute toxicity of PT19c was determined in nontumored mice. In vivo antitumor efficacy of PT19c was determined via ovarian and endometrial cancer xenograft experiments. Effect of PT19c on actin filament organization and focal adhesion formation was examined by microscopy. PT19c treatment inhibited growth of chemoresistant NCI60 cell lines (log10GI50 ~ −4.05 to −6.73). PT19c (10 mg/kg, 35 days) reduced growth of ovarian and endometrial xenograft tumor without hypercalcemia. PT19c exerted no acute toxicity up to 400 mg/kg (QDx1) in animals. PT19c showed weak VDR antagonism, lack of VDR binding, and inverted spatial accommodation in VDR-LBD. PT19c caused actin filament dysfunction and inhibited focal adhesion in SKOV-3 cells. PT19c is a VDR independent nonhypercalcemic vitamin D–derived agent that showed noteworthy safety and efficacy in ovarian and endometrial cancer animal models and inhibited actin organization and focal adhesion in ovarian cancer cells.
doi:10.1177/1947601913507575
PMCID: PMC3877664  PMID: 24386512
vitamin D; hypercalcemia; antitumor efficacy; ovarian cancer; endometrial cancer
11.  Targeting human CD34+ hematopoietic stem cells with anti-CD45 × anti-myosin light-chain bispecific antibody preserves cardiac function in myocardial infarction 
We have previously shown that targeting human CD34+ hematopoietic stem cells (HSC) with a bispecific antibody (BiAb) directed against myosin light chain (MLC) increases delivery of cells to the injured hearts and improves cardiac performance in the nude rat. In this study, we have sought to validate our previous observations and to perform more detailed determination of ventricular function in immunocompetent mice with myocardial infarction (MI) that were treated with armed CD34+ HSC. We examined whether armed CD34+ HSC would target the injured heart following MI and restore ventricular function in vitro. MI was created by ligation of the left anterior descending artery. After 48 h, adult ICR mice received either 0.5 × 106 human CD34+ HSC armed with anti-CD45 × anti-MLC BiAb or an equal volume of medium through a single tail vein injection. Two weeks after stem cell administration, ventricular function of hearts from mice receiving armed CD34+ HSC was significantly greater compared with the same parameters from control mice. Immunohistochemistry confirmed the accumulation of CD34+ HSC in MI hearts infused with stem cells. Angiogenesis was significantly enhanced in CD34+ HSC-treated heart as determined by vascular density per area. Furthermore, histopathological examination revealed that the retained cardiac function observed in CD34+ HSC-treated mice was associated with decreased ventricular fibrosis. These results suggest that peripheral administration of armed CD34+ HSC results in localization of CD34+ HSC to injured myocardium and restores myocardial function.
doi:10.1152/japplphysiol.01109.2007
PMCID: PMC3792713  PMID: 18292296
CD34+ hematopoietic stem cell; bispecific antibody; mouse; myocardium
12.  Developmental genes targeted for epigenetic variation between twin-twin transfusion syndrome children 
Clinical Epigenetics  2013;5(1):18.
Background
Epigenetic mechanisms are thought to be critical in mediating the role of the intrauterine environment on lifelong health and disease. Twin-twin transfusion syndrome (TTTS) is a rare condition wherein fetuses share the placenta and develop vascular anastomoses, which allow blood to flow between the fetuses. The unequal flow results in reciprocal hypo- and hypervolemia in the affected twins, striking growth differences and physiologic adaptations in response to this significant stressor. The donor twin in the TTTS syndrome can be profoundly growth restricted and there is likely a nutritional imbalance between the twins. The consequences of TTTS on fetal programming are unknown. This condition can now be effectively treated through the use of fetal laparoscopic procedures, but the potential for lifelong morbidity related to this condition during development is apparent. As this condition and the resulting uteroplacental discordance can play a role in the epigenetic process, we sought to investigate the DNA methylation profiles of childhood survivors of TTTS (n = 14). We focused on differences in both global measures and genome-wide CpG specific DNA methylation between donor and recipient children in this pilot study in order to generate hypotheses for further research.
Results
We identified significant hypomethylation of the LINE1 repetitive element in the peripheral blood of donor children and subtle variation in the genome-wide profiles of CpG specific methylation most prominent at CpG sites which are targets for polycomb group repressive complexes.
Conclusions
These preliminary results suggest that coordinated epigenetic alterations result from the intrauterine environment experienced by infants with TTTS and may, at least in part, be responsible for downstream health conditions experienced by individuals surviving this condition.
doi:10.1186/1868-7083-5-18
PMCID: PMC4016001  PMID: 24090360
Twin-twin transfusion syndrome; Growth restriction; DNA methylation; Intrauterine environment; Fetal
13.  Inter-Alpha Inhibitor Protein (IAIP) Administration Improves Survival From Neonatal Sepsis In Mice 
Pediatric research  2010;68(3):242-247.
Inter-alpha Inhibitor proteins (IaIp) are serine proteases inhibitors which modulate endogenous protease activity and have been shown to improve survival in adult models of sepsis. We evaluated the effect of IaIp on survival and systemic responses to sepsis in neonatal mice. Sepsis was induced in 2-day-old mice with LPS, E. coli and Group B Streptococci. Sepsis was associated with 75% mortality. IaIp, given by intraperitoneal administration at doses between 15–45 mg/kg from 1–6 hours following the onset of sepsis improved survival to nearly 90% (p = 0.0159) in both LPS induced sepsis and with live bacterial infections. The greatest effect was on reversal of hemorrhagic pneumonitis. The effects were dose and time dependent. Systemic cytokine profile and tissue histology were examined. Survival was compared in interleukin 10 knock out animals. Systemic cytokine levels, including TNF-α and IL-10 were increased following induction of sepsis and modulated significantly following IaIp administration. Because the effect of IaIp was still demonstrable in IL-10 deficient mice, we conclude the beneficial effect(s) of IaIp is due to suppression of pro-inflammatory cytokines like TNF-α rather than augmentation of IL-10. IaIp may offer significant benefits as a therapeutic adjunct to treatment of sepsis in neonates and adults.
doi:10.1203/PDR.0b013e3181e9fdf0
PMCID: PMC2928396  PMID: 20520583
14.  Patterning in Placental 11-B Hydroxysteroid Dehydrogenase Methylation According to Prenatal Socioeconomic Adversity 
PLoS ONE  2013;8(9):e74691.
Background
Prenatal socioeconomic adversity as an intrauterine exposure is associated with a range of perinatal outcomes although the explanatory mechanisms are not well understood. The development of the fetus can be shaped by the intrauterine environment through alterations in the function of the placenta. In the placenta, the HSD11B2 gene encodes the 11-beta hydroxysteroid dehydrogenase enzyme, which is responsible for the inactivation of maternal cortisol thereby protecting the developing fetus from this exposure. This gene is regulated by DNA methylation, and this methylation and the expression it controls has been shown to be susceptible to a variety of stressors from the maternal environment. The association of prenatal socioeconomic adversity and placental HSD11B2 methylation has not been examined. Following a developmental origins of disease framework, prenatal socioeconomic adversity may alter fetal response to the postnatal environment through functional epigenetic alterations in the placenta. Therefore, we hypothesized that prenatal socioeconomic adversity would be associated with less HSD11B2 methylation.
Methods and Findings
We examined the association between DNA methylation of the HSD11B2 promoter region in the placenta of 444 healthy term newborn infants and several markers of prenatal socioeconomic adversity: maternal education, poverty, dwelling crowding, tobacco use and cumulative risk. We also examined whether such associations were sex-specific. We found that infants whose mothers experienced the greatest levels of socioeconomic adversity during pregnancy had the lowest extent of placental HSD11B2 methylation, particularly for males. Associations were maintained for maternal education when adjusting for confounders (p<0.05).
Conclusions
Patterns of HSD11B2 methylation suggest that environmental cues transmitted from the mother during gestation may program the developing fetus’s response to an adverse postnatal environment, potentially via less exposure to cortisol during development. Less methylation of placental HSD11B2 may therefore be adaptive and promote the effective management of stress associated with social adversity in a postnatal environment.
doi:10.1371/journal.pone.0074691
PMCID: PMC3764127  PMID: 24040322
15.  Fetal Effects of Psychoactive Drugs 
Clinics in perinatology  2009;36(3):595-619.
doi:10.1016/j.clp.2009.06.002
PMCID: PMC2767264  PMID: 19732616
cocaine; methamphetamine; SSRI; maternal depression; fetal behavior
16.  Placenta Imprinted Gene Expression Association of Infant Neurobehavior 
The Journal of Pediatrics  2011;160(5):854-860.e2.
Objective
To identify links between altered gene imprinting in the placenta and infant neurobehavioral profiles.
Study design
We used qRT-PCR to examine the expression of 22 imprinted candidate genes in a series of 106 term human primary placenta tissues and associated that expression with summary scores from the NICU Network Neurobehavioral Scales performed on the corresponding infants. Clustering of the expression data was used to define distinct classes of expression.
Results
Significant associations were identified between classes of expression and the NICU Network Neurobehavioral Scales quality of movement (P=0.02) and handling (P=0.006) scores. Multivariable regression demonstrated an independent effect of imprinted gene expression profile on these neurobehavioral scores after controlling for confounders.
Conclusion
These results suggest that alterations in imprinted gene expression in the placenta are associated with infant neurodevelopmental outcomes. Our results suggest a role for the placenta and genomic imprinting in the placenta beyond intrauterine growth regulation.
doi:10.1016/j.jpeds.2011.10.028
PMCID: PMC3311768  PMID: 22153677
neurodevelopment; epigenetics; fetal programming; trophoblast; mental health
17.  Haplotype-dependent Differential Activation of the Human IL-10 Gene Promoter in Macrophages and Trophoblasts: Implications for Placental IL-10 Deficiency and Pregnancy Complications 
Problem
Polymorphic changes in the IL-10 gene promoter have been identified that lead to altered IL-10 production. We hypothesized that because of these genotypic changes, the IL-10 promoter might be expressed in a cell type–specific manner and may respond differentially to inflammatory triggers.
Method of study
We created reporter gene promoter constructs containing GCC, ACC, and ATA haplotypes using DNA from patients harboring polymorphic changes at −1082 (G → A), −819 (C → T), and −592 (C → A) sites in the IL-10 promoter. These individual luciferase reporter constructs were transiently transfected into either primary term trophoblasts or THP1 monocytic cells. DNA-binding studies were performed to implicate the role of the Sp1 transcription factor in response to differential promoter activity.
Results
Our results suggest that the GCC promoter construct was activated in trophoblast cells in response to lipopolysaccharide (LPS), as demonstrated by reporter gene expression, but not in monocytic cells. The ACC construct showed weaker activation in both cell types. Importantly, while the ATA promoter was constitutively activated in both cell types, its expression was selectively repressed in response to LPS, but only in trophoblasts. DNA-nuclear protein binding assays with nuclear extracts from LPS treated or untreated cells suggested a functional relevance for Sp1 binding differences at the −592 position.
Conclusions
These results demonstrate cell type–specific effects of the genotypic changes in the IL-10 gene promoter. These responses may be further modulated by bacterial infections or other inflammatory conditions to suppress IL-10 production in human trophoblasts.
doi:10.1111/j.1600-0897.2010.00854.x
PMCID: PMC3634123  PMID: 20482524
Human trophoblasts; IL-10 gene promoter; polymorphism; promoter dysregulation
18.  LAT1 Expression in Pre and Post Implantation Embryos and Placenta 
Placenta  2009;30(3):270-276.
Objectives
LAT-1 (L-type amino acid transporter 1) is a system L, Na+-independent amino acid transporter responsible for transport of large neutral amino acids. Dysregulated expression of LAT-1 is characteristic of many primary human cancers and is related to tumor invasion. Primary rat hepatocytes in culture increase LAT-1 mRNA in response to amino acid depletion. Transformed hepatic cell lines demonstrate constitutive expression of LAT-1. These observations suggest that LAT-1 expression confers a growth and survival advantage under limited amino acid availability. LAT-1 is highly expressed in the placenta. It has been shown previously that amino acids are fundamental regulators of cell function and energy metabolism in pre-implantation embryos. Our objectives were to analyze qualitatively and quantitatively LAT-1 expression in pre-implantation stages of mouse embryo development and to identify cell types expressing LAT-1 in post implantation stages.
Methods
LAT-1 was quantified by real-time qPCR. Localization of expression was by laser capture microdissection, in situ hybridization and immunohistochemistry.
Results
Our results show increasing mRNA levels of LAT-1 as the embryo develops from zygote to blastocyst with highest levels at hatching blastocyst. Expression studies of LAT-1 on microdissected samples from developing mouse placenta show highest levels of LAT-1 mRNA in trophoblast giant cells (TGC’s) at the time of implantation (E7.5), followed by maternal decidua, ectoplacental cone and epiblast. At later stages of development (E9.5 and E11.5) no differential expression of LAT-1 was observed. In situ hybridization and immunohistochemistry also showed differential expression of LAT-1 mRNA and protein, respectively, with darkest staining in TGC’s at E7.5. By E9.5 and E11.5 mRNA expression was no longer preferentially localized to TGC’s, hybridization was equal across the different cell types and regions. LAT-1 protein expression, however, still showed highest intensity of staining in TGC’s at E9.5 and E11.5.
Conclusions
Since trophoblast giant cells are invasive cells that displace and phagocytose the uterine epithelial cells, these data suggest that LAT-1 may play a role in the invasive phenotype. The mechanism of LAT-1 regulation during placentation, therefore, might provide valuable clues to its role in tumor progression and invasion.
doi:10.1016/j.placenta.2008.12.015
PMCID: PMC2683020  PMID: 19193433
19.  Ex vivo expanded human cord blood-derived hematopoietic progenitor cells induce lung growth and alveolarization in injured newborn lungs 
Respiratory Research  2013;14(1):37.
Background
We investigated the capacity of expanded cord blood-derived CD34+ hematopoietic progenitor cells to undergo respiratory epithelial differentiation ex vivo, and to engraft and attenuate alveolar disruption in injured newborn murine lungs in vivo.
Methods
Respiratory epithelial differentiation was studied in CD34+ cells expanded in the presence of growth factors and cytokines (“basic” medium), in one group supplemented with dexamethasone (“DEX”). Expanded or freshly isolated CD34+ cells were inoculated intranasally in newborn mice with apoptosis-induced lung injury. Pulmonary engraftment, lung growth and alveolarization were studied at 8 weeks post-inoculation.
Results
SP-C mRNA expression was seen in 2/7 CD34+ cell isolates expanded in basic media and in 6/7 isolates expanded in DEX, associated with cytoplasmic SP-C immunoreactivity and ultrastructural features suggestive of type II cell-like differentiation. Administration of expanding CD34+ cells was associated with increased lung growth and, in animals treated with DEX-exposed cells, enhanced alveolar septation. Freshly isolated CD34+ cells had no effect of lung growth or remodeling. Lungs of animals treated with expanded CD34+ cells contained intraalveolar aggregates of replicating alu-FISH-positive mononuclear cells, whereas epithelial engraftment was extremely rare.
Conclusion
Expanded cord blood CD34+ cells can induce lung growth and alveolarization in injured newborn lungs. These growth-promoting effects may be linked to paracrine or immunomodulatory effects of persistent cord blood-derived mononuclear cells, as expanded cells showed limited respiratory epithelial transdifferentiation.
doi:10.1186/1465-9921-14-37
PMCID: PMC3610254  PMID: 23522153
Alveolar type II cell; Bronchopulmonary dysplasia; Dexamethasone; Stem cell; Regeneration
20.  Maternal Depression and Anxiety are Associated with Altered Gene Expression in the Human Placenta without Modification by Antidepressant Use: Implications for Fetal Programming 
Developmental psychobiology  2011;53(7):711-723.
Objective
We sought to determine if maternal depression, anxiety, and/or treatment with selective serotonin reuptake inhibitors (SSRIs) affect placental human serotonin transporter (SLC6A4), norepinephrine transporter (SLC6A2), and 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) gene expression.
Methods
Relative mRNA expression was compared among placental samples (n=164) from healthy women, women with untreated depression and/or anxiety symptoms during pregnancy, and women who used SSRIs.
Results
SLC6A4 expression was significantly increased in placentas from women with untreated mood disorders and from women treated with SSRIs, compared to controls. SLC6A2 and 11β-HSD2 expression was increased in non-control groups, though the differences were not significant. SLC6A4, SLC6A2, and 11β-HSD2 expression levels were positively correlated.
Conclusions
The finding that maternal depression/anxiety affects gene expression of placental SLC6A4 suggests a possible mechanism for the effect(s) of maternal mood on fetal neurodevelopmental programming. SSRI treatment does not further alter the elevated SLC6A4 expression levels observed with exposure to maternal depression or anxiety.
doi:10.1002/dev.20549
PMCID: PMC3155003  PMID: 21547899
serotonin transporter; norepinephrine transporter; 11beta-hydroxysteroid dehydrogenase; depression; anxiety; placenta; fetal programming
21.  In vitro silencing of the insulin receptor attenuates cellular accumulation of fibronectin in renal mesangial cells 
Background
Insulin receptor (InsR) and insulin signaling proteins are widely distributed throughout the kidney cortex. Insulin signaling can act in the kidney in multiple ways, some of which may be totally independent of its primary role of the maintenance of whole-body glucose homeostasis. However, descriptions of the insulin signaling in renal glomerular mesangial cells (MCs) are quite limited and the roles of insulin signaling in MC functions have not been sufficiently elucidated.
Results
InsR silencing induced a unique phenotype of reduced fibronectin (FN) accumulation in renal glomerular MCs. Transcription level of FN was not significantly changed in the InsR silenced cells, suggesting the phenotype switching was caused by post-transcriptional modification. The decreased expression of InsR was associated with enhanced activity of insulin-like growth factor-1 receptor (IGF-1R)/PI3K/Akt signaling pathway which contributed in part to the attenuation of cellular FN accumulation. Formation of IGF-1R homodimer was increased in the InsR silenced cells. The InsR silenced cells also showed increased sensitivity to exogenous IGF-1, and increased PI3K activity was reversed significantly by incubating cells with IGF-1R specific antagonist, AG538. PI3K/Akt dependent activation of cAMP responsive element-binding protein (CREB)-1 induced expression of matrix metalloproteinase (MMP)-9 and suppressing MMP activity by doxycycline partially reversed FN accumulation in the InsR silenced cells.
Conclusions
The effects of InsR silencing on cellular FN accumulation in vitro are, at least partially, mediated by increased degradation of FN by MMPs which is induced by enhanced signaling sequence of IGF-1R/PI3K/Akt/CREB-1.
doi:10.1186/1478-811X-10-29
PMCID: PMC3507851  PMID: 23061721
Insulin receptor; Fibronectin; Mesangial cells; CREB-1; MMP-9
22.  Infant growth restriction is associated with distinct patterns of DNA methylation in human placentas 
Epigenetics  2011;6(7):920-927.
The placenta acts not only as a conduit of nutrient and waste exchange between mother and developing fetus, but also functions as a regulator of the intrauterine environment. Recent work has identified changes in the expression of candidate genes, often through epigenetic alteration, which alter the placenta's function and impact fetal growth. In this study, we used the Illumina Infinium HumanMethylation27 BeadChip array to examine genome-wide DNA methylation patterns in 206 term human placentas. Semi-supervised recursively partitioned mixture modeling was implemented to identify specific patterns of placental DNA methylation that could differentially classify intrauterine growth restriction (IUGR) and small for gestational age (SGA) placentas from appropriate for gestational age (AGA) placentas, and these associations were validated in a masked testing series of samples. Our work demonstrates that patterns of DNA methylation in human placenta are reliably and significantly associated with infant growth and serve as a proof of principle that methylation status in the human term placenta can function as a marker for the intrauterine environment, and could potentially play a critical functional role in fetal development.
doi:10.4161/epi.6.7.16079
PMCID: PMC3154432  PMID: 21758004
epigenetics; DNA methylation; placenta; intrauterine growth restriction; small for gestational age; development; human
23.  Efficacy of a Non-Hypercalcemic Vitamin-D2 Derived Anti-Cancer Agent (MT19c) and Inhibition of Fatty Acid Synthesis in an Ovarian Cancer Xenograft Model 
PLoS ONE  2012;7(4):e34443.
Background
Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models.
Methodology/Principal Finding
Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c–VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein.
Significance
Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis.
doi:10.1371/journal.pone.0034443
PMCID: PMC3317945  PMID: 22509304
24.  Na+,K+-ATPase Activity and Subunit Protein Expression 
Reproductive Sciences  2011;18(4):359-373.
We examined the effects of development, exogenous, and endogenous glucocorticoids on Na+,K+-ATPase activity and subunit protein expression in ovine cerebral cortices and renal cortices. Ewes at 60%, 80%, and 90% gestation, newborns, and adults received 4 dexamethasone or placebo injections. Cerebral cortex Na+,K+-ATPase activity was higher (P < .05) in placebo-treated newborns than fetuses of placebo-treated ewes and adults, α1-expression was higher at 90% gestation than the other ages; α2-expression was higher in newborns than fetuses; α3-expression was higher in newborns than 60% gestation; β1-expression was higher in newborns than the other ages, and β2-expression higher at 60% than 80% and 90% gestation, and in adults. Renal cortex Na+,K+-ATPase activity was higher in placebo-treated adults and newborns than fetuses. Cerebral cortex Na+,K+-ATPase activity was higher in dexamethasone- than placebo-treated adults, and α1-expression higher in fetuses of dexamethasone- than placebo-treated ewes at 60% and 80% gestation. Renal cortex Na+,K+-ATPase activity and α1-expression were higher in fetuses of dexamethasone- than placebo-treated ewes at each gestational age, and β1-expression was higher in fetuses of dexamethasone- than placebo-treated ewes at 90% gestation and in dexamethasone- than placebo-treated adults. Cerebral cortex Na+,K+-ATPase activity, α1-expression, β1-expression, and renal cortex α1-expression correlated directly with increases in fetal cortisol. In conclusion, Na+,K+-ATPase activity and subunit expression exhibit specific developmental patterns in brain and kidney; exogenous glucocorticoids regulate activity and subunit expression in brain and kidney at some ages; endogenous increases in fetal cortisol regulate cerebral Na+,K+-ATPase, but exogenous glucocorticoids have a greater effect on renal than cerebral Na+,K+-ATPase.
doi:10.1177/1933719110385137
PMCID: PMC3343057  PMID: 20959645
brain; kidney; glucocorticoids; Na+; K+-ATPase
25.  Placental 11-Beta Hydroxysteroid Dehydrogenase Methylation Is Associated with Newborn Growth and a Measure of Neurobehavioral Outcome 
PLoS ONE  2012;7(3):e33794.
Background
There is growing evidence that the intrauterine environment can impact the neurodevelopment of the fetus through alterations in the functional epigenome of the placenta. In the placenta, the HSD11B2 gene encoding the 11-beta hydroxysteroid dehydrogenase enzyme, which is responsible for the inactivation of maternal cortisol, is regulated by DNA methylation, and has been shown to be susceptible to stressors from the maternal environment.
Methodology/Principal Findings
We examined the association between DNA methylation of the HSD11B2 promoter region in the placenta of 185 healthy newborn infants and infant and maternal characteristics, as well as the association between this epigenetic variability and newborn neurobehavioral outcome assessed with the NICU Network Neurobehavioral Scales. Controlling for confounders, HSD11B2 methylation extent is greatest in infants with the lowest birthweights (P = 0.04), and this increasing methylation was associated with reduced scores of quality of movement (P = 0.04).
Conclusions/Significance
These results suggest that factors in the intrauterine environment which contribute to birth outcome may be associated with placental methylation of the HSD11B2 gene and that this epigenetic alteration is in turn associated with a prospectively predictive early neurobehavioral outcome, suggesting in some part a mechanism for the developmental origins of infant neurological health.
doi:10.1371/journal.pone.0033794
PMCID: PMC3303854  PMID: 22432047

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