Proteomic studies of VEGFR2 in human placentas reveal protein associations with preeclampsia, diabetes, gravidity, and labor.

VEGFR2 (Vascular endothelial growth factor receptor 2) is a central regulator of placental angiogenesis. The study of the VEGFR2 proteome of chorionic villi at term revealed its partners MDMX (Double minute 4 protein) and PICALM (Phosphatidylinositol-binding clathrin assembly protein). Subsequently, the oxytocin receptor (OT-R) and vasopressin V1aR receptor were detected in MDMX and PICALM immunoprecipitations. Immunogold electron microscopy showed VEGFR2 on endothelial cell (EC) nuclei, mitochondria, and Hofbauer cells (HC), tissue-resident macrophages of the placenta. MDMX, PICALM, and V1aR were located on EC plasma membranes, nuclei, and HC nuclei. Unexpectedly, PICALM and OT-R were detected on EC projections into the fetal lumen and OT-R on 20-150 nm clusters therein, prompting the hypothesis that placental exosomes transport OT-R to the fetus and across the blood-brain barrier. Insights on gestational complications were gained by univariable and multivariable regression analyses associating preeclampsia with lower MDMX protein levels in membrane extracts of chorionic villi, and lower MDMX, PICALM, OT-R, and V1aR with spontaneous vaginal deliveries compared to cesarean deliveries before the onset of labor. We found select associations between higher MDMX, PICALM, OT-R protein levels and either gravidity, diabetes, BMI, maternal age, or neonatal weight, and correlations only between PICALM-OT-R (p < 2.7 × 10-8), PICALM-V1aR (p < 0.006), and OT-R-V1aR (p < 0.001). These results offer for exploration new partnerships in metabolic networks, tissue-resident immunity, and labor, notably for HC that predominantly express MDMX.

Chromosome 19 microRNA cluster enhances cell reprogramming by inhibiting epithelial-to-mesenchymal transition.

During implantation, cytotrophoblasts undergo epithelial-to-mesenchymal transition (EMT) as they differentiate into invasive extravillous trophoblasts (EVTs). The primate-specific microRNA cluster on chromosome 19 (C19MC) is exclusively expressed in the placenta, embryonic stem cells and certain cancers however, its role in EMT gene regulation is unknown. In situ hybridization for miR-517a/c, a C19MC cistron microRNA, in first trimester human placentas displayed strong expression in villous trophoblasts and a gradual decrease from proximal to distal cell columns as cytotrophoblasts differentiate into invasive EVTs. To investigate the role of C19MC in the regulation of EMT genes, we employed the CRISPR/dCas9 Synergistic Activation Mediator (SAM) system, which induced robust transcriptional activation of the entire C19MC cistron and resulted in suppression of EMT associated genes. Exposure of human iPSCs to hypoxia or differentiation of iPSCs into either cytotrophoblast-stem-like cells or EVT-like cells under hypoxia reduced C19MC expression and increased EMT genes. Furthermore, transcriptional activation of the C19MC cistron induced the expression of OCT4 and FGF4 and accelerated cellular reprogramming. This study establishes the CRISPR/dCas9 SAM as a powerful tool that enables activation of the entire C19MC cistron and uncovers its novel role in suppressing EMT genes critical for maintaining the epithelial cytotrophoblasts stem cell phenotype.

Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration.

Preeclampsia (PE) is a common cause of maternal morbidity, characterized by impaired trophoblast invasion and spiral artery transformation resulting in progressive uteroplacental hypoxia. Given the primary role of LIN28A and LIN28B in modulating cell metabolism, differentiation, and invasion, we hypothesized that LIN28A and/or LIN28B regulates trophoblast differentiation and invasion, and that its dysregulation may contribute to PE. Here we show that LIN28B is expressed ∼1300-fold higher than LIN28A in human term placenta and is the predominant paralog expressed in primary human trophoblast cultures. The expression of LIN28B mRNA and protein levels are significantly reduced in gestational age-matched preeclamptic vs. normal placentas, whereas LIN28A expression is not different. First trimester human placental sections displayed stronger LIN28B immunoreactivity in extravillous (invasive) cytotrophoblasts and syncytial sprouts vs. villous trophoblasts. LIN28B overexpression increased HTR8 cell proliferation, migration, and invasion, whereas LIN28B knockdown in JEG3 cells reduced cell proliferation. Moreover, LIN28B knockdown in JEG3 cells suppressed syncytin 1 (SYN-1), apelin receptor early endogenous ligand (ELABELA), and the chromosome 19 microRNA cluster, and increased mRNA expression of ITGß4 and TNF-α. Incubation of BeWo and JEG3 cells under hypoxia significantly decreased expression of LIN28B and LIN28A, SYN-1, and ELABELA, whereas TNF-α is increased. These results provide the first evidence that LIN28B is the predominant paralog in human placenta and that decreased LIN28B may play a role in PE by reducing trophoblast invasion and syncytialization, and by promoting inflammation.-Canfield, J., Arlier, S., Mong, E. F., Lockhart, J., VanWye, J., Guzeloglu-Kayisli, O., Schatz, F., Magness, R. R., Lockwood, C. J., Tsibris, J. C. M., Kayisli, U. A., Totary-Jain, H. Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration.

Modulation of LIN28B/Let-7 Signaling by Propranolol Contributes to Infantile Hemangioma Involution.

OBJECTIVE: Infantile hemangiomas (IHs) are the most common benign vascular neoplasms of infancy, characterized by a rapid growth phase followed by a spontaneous involution, or triggered by propranolol treatment by poorly understood mechanisms. LIN28/let-7 axis plays a central role in the regulation of stem cell self-renewal and tumorigenesis. However, the role of LIN28B/let-7 signaling in IH pathogenesis has not yet been elucidated. APPROACH AND RESULTS: LIN28B is highly expressed in proliferative IH and is less expressed in involuted and in propranolol-treated IH samples as measured by immunofluorescence staining and quantitative RT-PCR. Small RNA sequencing analysis of IH samples revealed a decrease in microRNAs that target LIN28B, including let-7, and an increase in microRNAs in the mir-498(46) cistron. Overexpression of LIN28B in HEK293 cells induced the expression of miR-516b in the mir-498(46) cistron. Propranolol treatment of induced pluripotent stem cells, which express mir-498(46) endogenously, reduced the expression of both LIN28B and mir-498(46) and increased the expression of let-7. Furthermore, propranolol treatment reduced the proliferation of induced pluripotent stem cells and induced epithelial-mesenchymal transition. CONCLUSIONS: This work uncovers the role of the LIN28B/let-7 switch in IH pathogenesis and provides a novel mechanism by which propranolol induces IH involution. Furthermore, it provides therapeutic implications for cancers in which the LIN28/let-7 pathway is imbalanced.

Telomere shortening and DNA damage of embryonic stem cells induced by cigarette smoke.

Embryonic stem cells (ESCs) provide a valuable in vitro model for testing toxicity of chemicals and environmental contaminants including cigarette smoke. Mouse ESCs were acutely or chronically exposed to smoke components, cigarette smoke condensate (CSC), or cadmium, an abundant component of CSC, and then evaluated for their self-renewal, apoptosis, DNA damage and telomere function. Acute exposure of ESCs to high dose of CSC or cadmium increased DNA damage and apoptosis. Yet, ESCs exhibited a remarkable capacity to recover following absence of exposure. Chronic exposure of ESCs to low dose of CSC or cadmium resulted in shorter telomeres and DNA damage. Together, acute exposure of ESCs to CSC or cadmium causes immediate cell death and reduces pluripotency, while chronic exposure of ESCs to CSC or cadmium leads to DNA damage and telomere shortening. Notably, a sub-proportion of ESCs during passages is selected to resist to smoke-induced oxidative damage to telomeres.

Fetal umbilical cord blood erythropoietin, interleukin-6, pH, pO(2), pCO(2), and base excess levels in histologic and/or clinical chorioamnionitis: is the response only inflammatory?

OBJECTIVE: To determine the correlation of histological chorioamnionitis (CA) with and without clinical CA with umbilical cord blood gases, erythropoietin (EPO), and interleukin-6 (IL-6) levels. METHODS: Umbilical artery blood gas analysis (pH, pO(2), pCO(2), BE) and umbilical vein EPO and IL-6 levels were measured in 202 infants from normal, histological, and no clinical CA and histological plus clinical CA pregnancies. RESULTS: Umbilical artery blood gas analyses were not different between normal controls and histological and clinical CA groups. Blanc Stage 1 histological CA had no abnormal EPO or IL-6 umbilical blood results. EPO in umbilical venous blood was elevated only in those infants with both histological and clinical CA. Umbilical vein IL-6 levels were elevated in all advanced microscopic and clinical CA. High and low EPO groups also have corresponding high and low IL-6 levels suggesting a common stimulus for these substances. CONCLUSIONS: Blanc stage I histological CA is probably clinically insignificant. CA is infrequently associated with abnormal umbilical artery blood gas levels. Advanced histological and clinical CA can elevate both EPO and IL-6 in umbilical blood and these may be key elements of mechanisms that effect fetal brain function.

Are placental chorionic capillary nucleated red blood cell counts useful compared to umbilical cord blood tests?

OBJECTIVE: To compare measurement of fetal nucleated red blood cell (NRBC) counts in paired histologic samples of the placenta and umbilical cord bloods. METHOD: Forty-five randomly selected pregnancies had two determinations of the NRBC count. A sample of umbilical venous blood had a NRBC count measured and sections of the placenta were examined for their villous capillary NRBC counts. RESULTS: Umbilical venous blood had NRBC/100 white blood cell counts ranging from 0 to 67. Paired evaluation of placental tissue had NRBC counts of 0-5 with 60% being zero compared to 8% zero counts in cord blood. There was no correlation between the paired counts (R(2) = 0.04). CONCLUSION: Umbilical cord blood provides different information on fetal NRBC count than does histologic study of the placenta.

Prediction of ABOG written examination performance from the third-year CREOG in-training examination results.

OBJECTIVE: To study the ability of the Council on Resident Education in Obstetrics and Gynecology (CREOG) in-training examination score to predict American Board of Obstetrics and Gynecology (ABOG) written examination performance. STUDY DESIGN: Twenty-six physicians took the CREOG examination during the third year of their residency and the ABOG written examination during their fourth year. These 2 test scores were compared. RESULTS: There was a statistically significant correlation between the 2 examination scores (r = 0.69, p < 0.001). CONCLUSION: The CREOG in-training examination at the third year of the residency correlates well with the ABOG written examination score and can be used to identify residents who need remedial study before finishing the program.

New potential regulators of uterine leiomyomata from DNA arrays: the ionotropic glutamate receptor GluR2.

In the post-Genome era, new concepts emerge about the growth regulation of uterine leiomyomata. Screening of leiomyoma and myometrial tissues with DNA arrays revealed numerous genes up-regulated in leiomyomata that were not known to be expressed in the human uterus. GluR2, a subunit of a ligand-gated cation channel, is up-regulated in leiomyomata relative to myometrium by 15- to 30-fold at the protein and mRNA level and is localized in endothelial cells. GluR2 pre-mRNA in leiomyoma and myometrial tissues is nearly 100% edited at the Q/R site, indicative of low Ca(2+) permeability of the ion channels. In spontaneous leiomyomata in women or leiomyomata induced in the guinea pig model, there is a likely synergism linking increased production of estradiol and all-trans retinoic acid with up-regulation of nuclear receptor PPARgamma and RXRalpha proteins to support tumor growth. GluR2 might be coupled to this synergism directly or via interleukin-17B, kinesin KIF5 or related genes also up-regulated in leiomyomata. GluR antagonists should be tested as inhibitors of leiomyoma growth.

Strategy for elucidating differentially expressed genes in leiomyomata identified by microarray technology.

OBJECTIVE: cDNA microarray technology identifies genes that are differentially expressed between tissues. Our previous study identified several genes that might contribute to the fibroid phenotype. We therefore sought to confirm genes involved in three distinct signal transduction pathways. DESIGN: Evaluation of differential mRNA and protein expression of Dlk, Frizzled-2, and CD-24 in fibroids compared with adjacent myometrium. University hospital. PATIENT(S): Five women undergoing medically indicated hysterectomy for symptomatic fibroids. INTERVENTION(S): Microarray analysis of up to 33000 genes, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time RT-PCR, Western blot, and immunohistochemistry. MAIN OUTCOME MEASURE(S): Expression of mRNA transcripts and protein in fibroid compared with myometrium.A more extensive microarray confirmed differential expression of Frizzled-2 and CD-24 but did not confirm Dlk overexpression. RT-PCR and real-time PCR demonstrated equivalent Dlk mRNA expression between fibroid and myometrium (ratio, 1.02), a slight Frizzled-2 overexpression (ratio, 2.09), and robust CD-24 overexpression in fibroids (ratio, 12.35). Western blot and immunohistochemistry confirmed Frizzled-2 overexpression, but did not confirm Dlk overexpression. CONCLUSION(S): Microarray technology is the first phase of tissue evaluation, but changes in gene expression must be confirmed. Confirmed genes can then be used to generate hypotheses testing their involvement in fibroid development.

Dose variation that is associated with approximated one-quarter tablet doses of misoprostol.

OBJECTIVE: The purpose of this study was to evaluate dose variation in approximated one-quarter tablet misoprostol fragments. STUDY DESIGN: Misoprostol 100 microg tablets were weighed, separated into two lots, and quartered with a razor blade or a pill cutter. Fragments were reweighed, and the misoprostol content was determined by high-performance liquid chromatography mass spectroscopy. RESULTS: Fragment weights varied more when a pill cutter was used (P <.0001). Fewer pill-cutter fragments than razor-cut fragments weighed within 10% of expected (24% vs 65%, P <.0001). Misoprostol content among the fragments that were determined by high-performance liquid chromatography mass spectroscopy was 103% +/- 12% of expected (range, 73%-124%). Tablet fragments that weighed >or=27.5 mg contained misoprostol in excess of 110% of expected in seven of eight fragments, although none from fragments that weighed

Insights from gene arrays on the development and growth regulation of uterine leiomyomata.

OBJECTIVE: To use microarray analysis as an unbiased approach to identify genes involved in the induction and growth of uterine leiomyomata. DESIGN: Screen by arrays for up to 12,000 genes in leiomyoma (L) and control myometrium (M) from nine patients. SETTING: University research laboratories. PATIENT(S): Nine patients in the follicular and luteal phases of the menstrual cycle. INTERVENTION(S): mRNA from L and M was converted to biotin-labeled cRNA and hybridized to cDNA oligonucleotide sequences on the arrays. MAIN OUTCOME MEASURE(S): Greater than two-fold change in gene expression between leiomyoma and matched myometrium. RESULT(S): Prominent among the 67 genes overexpressed in L relative to M were dlk or Pref-1, doublecortin, JM27, ionotropic glutamate receptor subunit 2, apolipoprotein E3, IGF2, semaphorin F, myelin proteolipid protein, MEST, frizzled, CRABP II, stromelysin-3, and TGFbeta3. The genes dlk, IGF2, and MEST are paternally expressed imprinted genes, and the others are involved in tissue differentiation and growth. Prominent among the 78 genes down-regulated in L relative to M were alcohol dehydrogenases 1alpha-gamma, tryptase, dermatopontin, thrombospondin, coxsackievirus receptor, nur77, and c-kit. CONCLUSION(S): Arrays offer large-scale screening of mRNA expression, which will help us differentiate between the genes and metabolic pathways necessary for leiomyoma growth and those regulating myometrial contractions.