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.
CD34+ hematopoietic stem cell; bispecific antibody; mouse; myocardium
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).
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.
To identify links between altered gene imprinting in the placenta and infant neurobehavioral profiles.
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.
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.
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.
neurodevelopment; epigenetics; fetal programming; trophoblast; mental health
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.
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.
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.
Human trophoblasts; IL-10 gene promoter; polymorphism; promoter dysregulation
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.
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.
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.
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.
Alveolar type II cell; Bronchopulmonary dysplasia; Dexamethasone; Stem cell; Regeneration
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.
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.
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.
Insulin receptor; Fibronectin; Mesangial cells; CREB-1; MMP-9
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.
LAT-1 was quantified by real-time qPCR. Localization of expression was by laser capture microdissection, in situ hybridization and immunohistochemistry.
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.
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.
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.
epigenetics; DNA methylation; placenta; intrauterine growth restriction; small for gestational age; development; human
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.
brain; kidney; glucocorticoids; Na+; K+-ATPase
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.
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).
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.
Genome-wide association studies (GWAS) query the entire genome in a hypothesis-free, unbiased manner. Since they have the potential for identifying novel genetic variants, they have become a very popular approach to the investigation of complex diseases. Nonetheless, since the success of the GWAS approach varies widely, the identification of genetic variants for complex diseases remains a difficult problem. We developed a novel bioinformatics approach to identify the nominal genetic variants associated with complex diseases. To test the feasibility of our approach, we developed a web-based aggregation tool to organize the genes, genetic variations and pathways involved in preterm birth. We used semantic data mining to extract all published articles related to preterm birth. All articles were reviewed by a team of curators. Genes identified from public databases and archives of expression arrays were aggregated with genes curated from the literature. Pathway analysis was used to impute genes from pathways identified in the curations. The curated articles and collected genetic information form a unique resource for investigators interested in preterm birth. The Database for Preterm Birth exemplifies an approach that is generalizable to other disorders for which there is evidence of significant genetic contributions.
The trend toward single-room neonatal intensive care units (NICUs) is increasing; however scientific evidence is, at this point, mostly anecdotal. This is a critical time to assess the impact of the single-room NICU on improving medical and neurobehavioral outcomes of the preterm infant. We have developed a theoretical model that may be useful in studying how the change from an open-bay NICU to a single-room NICU could affect infant medical and neurobehavioral outcome. The model identifies mediating factors that are likely to accompany the change to a single-room NICU. These mediating factors include family centered care, developmental care, parenting and family factors, staff behavior and attitudes, and medical practices. Medical outcomes that plan to be measured are sepsis, length of stay, gestational age at discharge, weight gain, illness severity, gestational age at enteral feeding, and necrotizing enterocolitis (NEC). Neurobehavioral outcomes include the NICU Network Neurobehavioral Scale (NNNS) scores, sleep state organization and sleep physiology, infant mother feeding interaction scores, and pain scores. Preliminary findings on the sample of 150 patients in the open-bay NICU showed a “baseline” of effects of family centered care, developmental care, parent satisfaction, maternal depression, and parenting stress on the neurobehavioral outcomes of the newborn. The single-room NICU has the potential to improve the neurobehavioral status of the infant at discharge. Neurobehavioral assessment can assist with early detection and therefore preventative intervention to maximize developmental outcome. We also present an epigenetic model of the potential effects of maternal care on improving infant neurobehavioral status.
preterm; neurobehavior; NNNS; NICU; very-low-birthweight infants; single-room NICU design; epigenetics
Background: Fetal programming describes the theory linking environmental conditions during embryonic and fetal development with risk of diseases later in life. Environmental insults in utero may lead to changes in epigenetic mechanisms potentially affecting fetal development.
Objectives: We examined associations between in utero exposures, infant growth, and methylation of repetitive elements and gene-associated DNA in human term placenta tissue samples.
Methods: Placental tissues and associated demographic and clinical data were obtained from subjects delivering at Women and Infants Hospital in Providence, Rhode Island (USA). Methylation levels of long interspersed nuclear element-1 (LINE-1) and the Alu element AluYb8 were determined in 380 placental samples from term deliveries using bisulfite pyrosequencing. Genomewide DNA methylation profiles were obtained in a subset of 184 samples using the Illumina Infinium HumanMethylation27 BeadArray. Multiple linear regression, model-based clustering methods, and gene set enrichment analysis examined the association between birth weight percentile, demographic variables, and repetitive element methylation and gene-associated CpG locus methylation.
Results: LINE-1 and AluYb8 methylation levels were found to be significantly positively associated with birth weight percentile (p = 0.01 and p < 0.0001, respectively) and were found to differ significantly among infants exposed to tobacco smoke and alcohol. Increased placental AluYb8 methylation was positively associated with average methylation among CpG loci found in polycomb group target genes; developmentally related transcription factor binding sites were overrepresented for differentially methylated loci associated with both elements.
Conclusions: Our results suggest that repetitive element methylation markers, most notably AluYb8 methylation, may be susceptible to epigenetic alterations resulting from the intrauterine environment and play a critical role in mediating placenta function, and may ultimately inform on the developmental basis of health and disease.
birth weight; epigenetics; fetal programming; in utero exposures; placenta; retrotransposon
We compared Inter alpha Inhibitor proteins (LALP) levels in infants with proven necrotizing enterocolitis (NEC) and with infants who had other, non-specific abdominal disorders.
A prospective observational study of infants in the NICU. NEC was diagnosed according to Bell’s staging criteria. Infants in the control group had non-specific abdominal disorders but no radiographic evidence of NEC and no disease progression. All infants with radiographic NEC were included. Plasma IaIp was quantitated using ELISA.
Seventeen infants had confirmed NEC and 34 infants had non-specific disorders that improved rapidly. Gestational age, postnatal age, weight, sex, maternal obstetric variables, rupture of membranes and mode of delivery did not differ. Mean IaIp level in the NEC group was significantly lower (137±38 mg/L, 95% CI=118–157) than the control group (258±53 mg/L, 95%CI: 238–277), p < 0.0001.
The demonstration that IaIp are significantly reduced in neonates with NEC suggests LALP serve useful as a sensitive biomarker, allowing patients to be placed on appropriate therapy and reducing antibiotic overuse in infants with suspected but unproven NEC. Administration of LALP may significantly reduce the severity of systemic inflammation and associated tissue injury.
Necrotizing enterocolitis; inter alpha inhibitor; serine protease inhibitors; serpins
Maternal cigarette smoking during pregnancy is associated with poor fetal outcome and aberrant miRNA expression is associated with adverse pregnancy outcomes. In 25 human placentas, we analyzed the expression of four candidate miRNA previously implicated in growth and developmental processes: miR-16, miR-21, miR-146a and miR-182, and used three immortalized placental cell lines to identify if specific components of cigarette smoke were responsible for alterations to miRNA expression. miR-16, miR-21 and miR-146a were significantly downregulated in cigarette smoke-exposed placentas compared to controls. TCL-1 cells exposed to both nicotine and benzo(a)pyrene exhibited significant, dose-dependent downregulation of miR-146a. These results suggest that miR-146a is particularly responsive to exposures, and that smoking may elicit some of its downstream effects through alteration of miRNA expression.
miRNA; placenta; cigarette smoking; nicotine; benzo(a)pyrene; epigenetics
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.
We have demonstrated an important role of bone marrow-derived stem cells in preservation of myocardial function. We investigated whether Akt-1 of lin−c-kit+ stem cells preserves ventricular function following myocardial infarction (MI).
Methods and results
Isolated lin−c-kit+ cells were conjugated with anti-c-kit heteroconjugated to anti-vascular cell adhesion molecule to facilitate the attachment of stem cells into damaged tissues. Female severe combined immunodeficient mice were used as recipients. MI was created by ligation of the left descending artery. After 48 h, animals were divided into four groups: (i) sham (n = 5): animals underwent thoracotomy without MI; (ii) MI (n = 5): animals underwent MI and received medium; (iii) MI + wild-type (Wt) stem cells (n = 6): MI animals received 5 × 105 Wt lin−c-kit+ stem cells; (iv) MI + Akt-1−/− stem cells (n = 6): MI animals received 5 × 105 Akt-1−/− lin−c-kit+ stem cells. Two weeks later, left ventricular function was measured in the Langendorff mode. The peripheral administration of Wt armed stem cells into MI animals restored ventricular function, which was absent in animals receiving Akt-1−/− cells. Real-time PCR indicates a decrease in SRY3, a Y chromosome marker in hearts receiving Akt-1−/− cells. An increase in angiogenic response was demonstrated in hearts receiving Wt stem cells but not Akt-1−/− stem cells.
Our results demonstrate that the peripheral administration of Wt lin−c-kit+ stem cells restores ventricular function and promotes angiogenic response following MI. These benefits were abrogated in MI mice receiving Akt-1−/− stem cells, suggesting the pivotal role of Akt-1 in mediating stem cells to protect MI hearts.
Akt-1; Myocardial infarction; Stem cells; Heart; Mouse
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 it’s over expression is related to tumor invasion. LAT-1 is highly expressed in the trophoblast giant cells (TGCs) at the time of implantation. Since trophoblast giant cells are highly invasive during the process of endometrial implantation and placentation, LAT-1 may play a role in the invasive phenotype. Our objectives were to identify the effects of increased and decreased LAT-1 expression on mouse trophoblast invasion. We therefore examined the role of amino acid deprivation, pharmacologic blockade specific to leucine transport and gene silencing (siRNA) on LAT-1 expression and trophoblast cell invasion. We utilized mouse primary trophoblast stem (TS) cells. LAT-1 mRNA expression was quantified by real-time qPCR, protein by Western blotting and cell invasion was measured in Transwell plates through Matrigel. Amino acid transport using uptake of tritiated leucine. Under limited leucine availability and/or pharmacologic blockage, LAT-1 gene expression was significantly increased, p<0.05. This was associated with a 3-fold increase in cell invasion, p<0.05. In contrast, following siRNA-mediated gene silencing decreased LAT-1 expression (both mRNA and protein) was associated with decreased cell invasion and decreased leucine uptake, p<0.05. Upregulation of LAT-1 gene expression via limited amino acid availability or following pharmacologic blockade of transport leads to an increase in mouse trophoblast stem cell invasiveness. Downregulation of LAT-1 expression via genetic silencing leads to inhibition of invasiveness. These results demonstrate that LAT-1 plays an important role in trophoblast invasion.
trophoblast stem cell; L amino acid transporter; LAT-1; Slc7a5; invasion; implantation
The placenta and its myriad functions are central to successful reproductive outcomes. These functions can be influenced by the environment encountered throughout pregnancy. Such influences can alter the appropriate genetic programming needed to allow for sustained pregnancy and appropriate fetal development. This altered programming may result from epigenetic alterations related to environmental exposures. Epigenetic alterations are now being linked to several important reproductive outcomes, including early pregnancy loss, intrauterine growth restriction, congenital syndromes (eg. Beckwith-Weidemann syndrome), preterm birth and preeclampsia. The diversity of environmental exposures linked to adverse reproductive effects continues to grow. Much attention has focused on the role of endocrine disruptors and other xenobiotics in infertility, but recent work is demonstrating that these chemicals may have adverse effects in pregnancy and development as well. Environmental oxygen is also critical in early pregnancy success. There are clear links between altered oxygen levels and placentation amongst other effects. As research continues to increase our understanding of the molecular processes including epigenetic regulation that influence pregnancy, it will be critical to specifically examine how the environment, broadly defined, may play a role at altering these critical functions.
cocaine; methamphetamine; SSRI; maternal depression; fetal behavior
Results from our previous trial revealed that infants with delayed cord clamping (DCC) had significantly less intraventricular hemorrhage (IVH) and late onset sepsis (LOS) than infants with immediate cord clamping (ICC). A priori, we hypothesized that infants with DCC would have better motor function by 7 months CA.
Infants between 24 and 316 weeks were randomized to ICC or DCC and follow-up evaluation was completed at 7 months corrected age.
We found no differences in the Bayley Scales of Infant Development (BSID) scores between the DCC and ICC groups. However, a regression model of effects of DCC on motor scores controlling for gestational age, IVH, bronchopulmonary dysplasia, sepsis, and male gender suggested higher motor scores of male infants with DCC.
Delayed cord clamping at birth appears to be protective of very low birth weight male infants against motor disability at 7 months corrected age.
cord clamping; motor outcomes; very low birth weight infants; randomized controlled trial; gender
Cell-based therapy in adult lung injury models is associated with highly variable donor cell engraftment and epithelial reconstitution. The role of marrow-derived cell therapy in neonatal lung injury is largely unknown. In this study, we determined the fate and effects of adult bone marrow cells in a model of neonatal lung injury. Wild-type mice placed in a normoxic or hyperoxic (95% O2) environment received bone marrow cells from animals expressing green fluorescent protein (GFP) at Postnatal Day (P)5. Controls received vehicle buffer. Lungs were analyzed between Post-Transplantation (TPX) Day 2 and Week 8. The volume of GFP-immunoreactive donor cells, monitored by stereologic volumetry, remained constant between Post-TPX Weeks 1 and 8 and was similar in normoxic and hyperoxia-exposed recipients. Virtually all marrow-derived cells showed colocalization of GFP and the pan-macrophage marker, F4/80, by double immunofluorescence studies. Epithelial transdifferentiation was not seen. Marrow cell administration had adverse effects on somatic growth and alveolarization in normoxic mice, while no effects were discerned in hyperoxia-exposed recipients. Reexposure of marrow-treated animals to hyperoxia at P66 resulted in significant expansion of the donor-derived macrophage population. In conclusion, intranasal administration of unfractionated bone marrow cells to newborn mice does not achieve epithelial reconstitution, but establishes persistent alveolar macrophage chimerism. The predominantly adverse effects of marrow treatment in newborn lungs are likely due to macrophage-associated paracrine effects. While this model and route of cell therapy may not achieve epithelial reconstitution, the role of selected stem cell populations and/or alternate routes of administration for cell-based therapy in injured newborn lungs deserve further investigation.
stem cells; cell therapy; lung injury; newborns; BPD
Myocardial hypoxic-ischemic injury is the cause of significant morbidity and mortality worldwide. The cardiomyocyte response to hypoxic-ischemic injury is known to include changes in cell cycle regulators. The cyclin-dependent kinase inhibitor p57Kip2 is involved in cell cycle control, differentiation, stress signaling and apoptosis. In contrast to other cyclin-dependent kinase inhibitors, p57Kip2 expression diminishes during postnatal life and is reactivated in the adult heart under conditions of cardiac stress. Overexpression of p57Kip2 has been previously shown to prevent apoptotic cell death in vitro by inhibiting stress-activated kinases. Therefore, we hypothesized that p57Kip2 has a protective role in cardiomyocytes under hypoxic conditions. To investigate this hypothesis, we created a transgenic mouse (R26loxpTA-p57k/+) that expresses p57Kip2 specifically in cardiac tissue under the ventricular cardiomyocyte promoter Mlc2v.
Transgenic mice with cardiac specific overexpression of p57Kip2 are viable, fertile and normally active and their hearts are morphologically indistinguishable from the control hearts and have similar heart weight/body weight ratio. The baseline functional parameters, including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), LVdp/dtmax, heart rate (HR) and rate pressure product (RPR) were not significantly different between the different groups as assessed by the Langendorff perfused heart preparation. However, after subjecting the heart ex vivo to 30 minutes of ischemia-reperfusion injury, the p57Kip2 overexpressing hearts demonstrated preserved cardiac function compared to control mice with higher left ventricular developed pressure (63 ± 15 vs 30 ± 6 mmHg, p = 0.05), rate pressure product (22.8 ± 4.86 vs 10.4 ± 2.1 × 103bpm × mmHg, p < 0.05) and coronary flow (3.5 ± 0.5 vs 2.38 ± 0.24 ml/min, p <0.05).
These data suggest that forced cardiac expression of p57Kip2 does not affect myocardial growth, differentiation and baseline function but attenuates injury from ischemia-reperfusion in the adult mouse heart.
This study compared the effects of immediate (ICC) and delayed (DCC) cord clamping on very low birth weight (VLBW) infants on 2 primary variables: bronchopulmonary dysplasia (BPD) and suspected necrotizing enterocolitis (SNEC). Other outcome variables were late-onset sepsis (LOS) and intraventricular hemorrhage (IVH).
This was a randomized, controlled unmasked trial in which women in labor with singleton fetuses <32 weeks' gestation were randomly assigned to ICC (cord clamped at 5–10 seconds) or DCC (30–45 seconds) groups. Women were excluded for the following reasons: their obstetrician refused to participate, major congenital anomalies, multiple gestations, intent to withhold care, severe maternal illnesses, placenta abruption or previa, or rapid delivery after admission.
Seventy-two mother/infant pairs were randomized. Infants in the ICC and DCC groups weighed 1151 and 1175 g, and mean gestational ages were 28.2 and 28.3 weeks, respectively. Analyses revealed no difference in maternal and infant demographic, clinical, and safety variables. There were no differences in the incidence of our primary outcomes (BPD and suspected NEC). However, significant differences were found between the ICC and DCC groups in the rates of IVH and LOS. Two of the 23 male infants in the DCC group had IVH versus 8 of the 19 in the ICC group. No cases of sepsis occurred in the 23 boys in the DCC group, whereas 6 of the 19 boys in the ICC group had confirmed sepsis. There was a trend toward higher initial hematocrit in the infants in the DCC group.
Delayed cord clamping seems to protect VLBW infants from IVH and LOS, especially for male infants.
delayed cord clamping; intraventricular hemorrhage; IVH; late-onset sepsis; VLBW infants; randomized; controlled trial
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.
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.
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.
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.
serotonin transporter; norepinephrine transporter; 11beta-hydroxysteroid dehydrogenase; depression; anxiety; placenta; fetal programming