Tu'uhevaha J. Kaitu'u-Lino

Claudin-11 expression and localisation is regulated by androgens in rat Sertoli cells in vitro

Claudin-11 and occludin are protein components in tight junctions (TJs) between Sertoli cells which are important for the maintenance of the blood-testis barrier. Barrier formation occurs during puberty, with evidence suggesting hormonal regulation of both claudin-11 and occludin. This study aimed to investigate the regulation of claudin-11 and occludin mRNA expression by testosterone (T) and FSH and their immunolocalisation at rat Sertoli cell TJs in vitro, and to correlate any steroid regulation with the functional capacity of TJs. Sertoli cells formed functional TJs within 3 days as assessed by transepithelial electrical resistance (TER). Both T and dihydrotestosterone significantly (P < 0.01) increased TER twofold and claudin-11 mRNA two- to threefold within 3 days. FSH partially stimulated TER and claudin-11 mRNA, but estradiol had no effect. T also promoted claudin-11 localisation into extensive intercellular contacts. In contrast to claudin-11, Tand FSH did not change occludin mRNA expression, however, T promoted localisation of occludin at cell contacts in a similar manner to claudin-11. Addition of flutamide to T-stimulated cells caused a twofold decrease in both TER and claudin-11 mRNA expression, and resulted in the loss of both proteins from cell contacts. This effect was reversible following flutamide removal. It is concluded that androgens i) co-regulate claudin-11 mRNA expression and TER, implicating claudin-11 in TJ formation and ii) promote the localisation of claudin-11 and occludin at Sertoli cell contacts. Hence, the ability of androgens to maintain spermatogenesis in vivo is partly via their effects on TJ proteins and regulation of the blood-testis barrier.

MMP-14 Is Expressed in Preeclamptic Placentas and Mediates Release of Soluble Endoglin

Soluble endoglin is an anti-angiogenic protein that is released from the placenta and contributes to both maternal endothelial dysfunction and the clinical features of severe preeclampsia. The mechanism through which soluble endoglin is released from the placenta is currently unknown; however, recent work in colorectal cancer identified matrix metalloproteinase 14 (MMP-14) as the cleavage protease of endoglin. To determine whether this is also the mechanism responsible for soluble endoglin release in preeclampsia, we investigated the expression of MMP-14 within the placenta and the effects of its inhibition on soluble endoglin release. Placentas were obtained from severe, early onset preeclamptic pregnancies (n = 8) and gestationally matched preterm controls (n = 8). MMP-14 was predominately localized to the syncytiotrophoblast. Results from a proximity ligation assay showed protein interactions between endogenous MMP-14 and endoglin within the preeclamptic placenta. To demonstrate that this interaction produces soluble endoglin, we treated trophoblastic BeWo cells with either a broad-spectrum MMP inhibitor (GM6001) or MMP-14 siRNA. Both treatments produced a decrease in soluble endoglin (P ≤ 0.05). Treatment of mice bearing BeWo xenografts with GM6001 decreased circulating soluble endoglin levels in mouse serum (P ≤ 0.05). These findings indicate that MMP-14 is the likely cleavage protease of endoglin in the setting of preeclampsia. This approach provides a novel method for the development of potential therapeutics to reduce circulating soluble endoglin and ameliorate the clinical features of severe preeclampsia.

Reepithelialization of the Uterine Surface Arises from Endometrial Glands: Evidence from a Functional Mouse Model of Breakdown and Repair

The human endometrium is highly regenerative undergoing monthly cycles of growth and regression. Endometrial repair after menses is a critical component of the cycle; however, little is understood about the mechanisms behind this rapid process. Adult stem/progenitor cells identified in human and mouse endometrium may be responsible for its remarkable regenerative capacity; however, a functional role for stem/progenitor cells in menstruation is yet to be established. This study aimed to identify label retaining cells as candidate epithelial stem or progenitor cells involved in the rapid reepithelization of the uterine surface in our functional mouse model of endometrial breakdown and repair. Adult mice were pulse labeled with bromodeoxyuridine before endometrial breakdown and repair was induced. Throughout endometrial breakdown and repair, very rapid dilution of bromodeoxyuridine label was observed in the luminal epithelium, whereas label within the glandular epithelium remained constant. Importantly, glandular epithelial cells were shown to proliferate selectively in response to endometrial repair, and the majority strongly expressed estrogen receptor-alpha at this time. This is the first study to demonstrate a functionally diverse response during endometrial repair from the anatomically connected luminal and glandular epithelium and highlights the likelihood that the endometrial glands are the residence of epithelial progenitor cells contributing to reepithelialization of the uterine surface after menses.

Placental specific mRNA in the maternal circulation are globally dysregulated in pregnancies complicated by fetal growth restriction.

CONTEXT Fetal growth restriction (FGR) is a leading cause of perinatal mortality, yet no reliable screening test exists. Placental specific mRNA in the maternal circulation may reflect changes in the placental transcriptome in FGR and could be a novel biomarker for FGR. OBJECTIVE The aim of the study was to identify placental specific RNA detectable in the maternal circulation and examine whether they are differentially expressed in severe preterm FGR. DESIGN In silico screening was used to identify placental specific RNAs. Their expression in cases of severe FGR vs controls was examined in both maternal blood and placenta by microarray, RT-PCR, and in situ hybridization. RESULTS Via in silico analysis, we identified 137 genes very highly expressed in the placenta relative to other tissues. Using microarray, we found that they were detectable in the maternal blood and were globally dysregulated with preterm FGR; 75 genes (55%) had a ≥1.5-fold differential expression compared to controls. Eight genes (ERVWE-1, PSG1, PLAC4, TAC3, PLAC3, CRH, CSH1, and KISS1) were validated by RT-PCR to be significantly increased in both maternal blood and placenta in a larger cohort of severe FGR compared to controls. In situ hybridization confirmed PAPPA2 and ERVWE-1 localized to the syncytiotrophoblast. CONCLUSION There is global differential expression of placental specific mRNA in the maternal blood in pregnancies complicated by severe preterm FGR. Placental specific mRNA in maternal blood may represent a new class of biomarkers for preterm FGR.

Paternal obesity in a rodent model affects placental gene expression in a sex-specific manner

Fetal growth restriction (FGR) is a major obstetric complication stemming from poor placental development. We have previously demonstrated that paternal obesity in mice is associated with impaired embryo development and significantly reduced fetal and placental weights. We hypothesised that the FGR observed in our rodent model of paternal diet-induced obesity is associated with alterations in metabolic, cell signalling and stress pathways. Male C57BL/6 mice were fed either a normal or high-fat diet for 10 weeks before sperm collection for IVF and subsequent embryo transfer. On embryonic day 14, placentas were collected and RNA extracted from both male and female placentas to assess mRNA expression of 24 target genes using custom RT-qPCR arrays. Peroxisome proliferator-activated receptor alpha (Ppara) and caspase-12 (Casp12) expression were significantly altered in male placentas from obese fathers compared with normal (P<0.05), but not female placentas. PPARA and CASP12 proteins were localised within the placenta to trophoblast giant cells by immunohistochemistry, and relative protein abundance was determined by western blot analysis. DNA was also extracted from the same placentas to determine methylation status. Global DNA methylation was significantly increased in female placentas from obese fathers compared with normal (P<0.05), but not male placentas. In this study, we demonstrate for the first time that paternal obesity is associated with changes in gene expression and methylation status of extraembryonic tissue in a sex-specific manner. These findings reinforce the negative consequences of paternal obesity before conception, and emphasise the need for more lifestyle advice for prospective fathers.

Proton Pump Inhibitors Decrease Soluble fms-Like Tyrosine Kinase-1 and Soluble Endoglin Secretion, Decrease Hypertension, and Rescue Endothelial Dysfunction.

Preeclampsia is a severe complication of pregnancy. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. Oxidative stress and vascular inflammation exacerbate the endothelial injury. A drug that can block these pathophysiological steps would be an attractive treatment option. Proton pump inhibitors (PPIs) are safe in pregnancy where they are prescribed for gastric reflux. We performed functional studies on primary human tissues and animal models to examine the effects of PPIs on sFlt-1 and soluble endoglin secretion, vessel dilatation, blood pressure, and endothelial dysfunction. PPIs decreased sFlt-1 and soluble endoglin secretion from trophoblast, placental explants from preeclamptic pregnancies, and endothelial cells. They also mitigated tumor necrosis factor-&agr;–induced endothelial dysfunction: PPIs blocked endothelial vascular cell adhesion molecule-1 expression, leukocyte adhesion to endothelium, and disruption of endothelial tube formation. PPIs decreased endothelin-1 secretion and enhanced endothelial cell migration. Interestingly, the PPI esomeprazole vasodilated maternal blood vessels from normal pregnancies and cases of preterm preeclampsia, but its vasodilatory effects were lost when the vessels were denuded of their endothelium. Esomeprazole decreased blood pressure in a transgenic mouse model where human sFlt-1 was overexpressed in placenta. PPIs upregulated endogenous antioxidant defenses and decreased cytokine secretion from placental tissue and endothelial cells. We have found that PPIs decrease sFlt-1 and soluble endoglin secretion and endothelial dysfunction, dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved.

A New Role for Activin in Endometrial Repair after Menses

Abnormal uterine bleeding can severely affect the quality of life for women. After menstruation, the endometrium must adequately repair to limit and stop bleeding. Abnormal uterine bleeding may result from incorrect or inadequate endometrial repair after menstruation. Previous studies have shown an important contribution of activin to skin wound healing, with severely delayed wound repair observed in animals transgenically induced to overexpress activins natural inhibitor, follistatin. Activin subunits have also been identified within human endometrium; however, their role in endometrial repair is unknown. We assessed the contribution of activin to endometrial repair after menses using a human in vitro cell wounding method and our well-characterized mouse model of endometrial breakdown and repair applied to mice overexpressing follistatin. Endometrial repair after menses is initiated with reepithelialization of the uterine surface. To mimic this repair, we utilized a human endometrial epithelial cell line (ECC-1) and demonstrated significant stimulation of wound closure after activin A administration, and attenuation of this response by addition of follistatin. Immunolocalization of activin subunits, betaA and betaB, in control endometrium from the mouse model demonstrated specific epithelial and stromal localization and some leukocyte staining (betaA) around sites of endometrial repair, suggestive of a role for activin in this process. Follistatin-overexpressing animals had significantly higher circulating follistatin levels than wild-type littermates. There was a significant delay in endometrial repair after breakdown in follistatin transgenic animals compared with control animals. This study demonstrates for the first time a functional role for activin in endometrial repair after menses.

Targeted Nanoparticle Delivery of Doxorubicin Into Placental Tissues to Treat Ectopic Pregnancies

Abnormal trophoblast growth can cause life-threatening disorders such as ectopic pregnancy, choriocarcinoma, and placenta accreta. EnGeneIC Delivery Vehicles (EDVs) are nanocells that can promote tissue-specific delivery of drugs and may be useful to medically treat such disorders. The objective of this study was to determine whether EDVs loaded with the chemotherapeutic doxorubicin and targeting the epidermal growth factor receptor (EGFR, very highly expressed on the placental surface) can regress placental cells in vitro, ex vivo, and in vivo. In female SCID mice, EGFR-targeted EDVs induced greater inhibition of JEG-3 (choriocarcinoma cells) tumor xenografts, compared with EDVs targeting an irrelevant antigen (nontargeted EDVs) or naked doxorubicin. EGFR-targeted EDVs were more readily taken up by human placental explants ex vivo and induced increased apoptosis (M30 antibody) compared with nontargeted EDVs. In vitro, EGFR-targeted EDVs administered to JEG-3 cells resulted in a dose-dependent inhibition of cell viability, proliferation, and increased apoptosis, a finding confirmed by continuous monitoring by xCELLigence. In conclusion, EGFR-targeted EDVs loaded with doxorubicin significantly inhibited trophoblastic tumor cell growth in vivo and in vitro and induced significant cell death ex vivo, potentially mediated by increasing apoptosis and decreasing proliferation. EDVs may be a novel nanoparticle treatment for ectopic pregnancy and other disorders of trophoblast growth.

Identification of Label-Retaining Perivascular Cells in a Mouse Model of Endometrial Decidualization, Breakdown, and Repair

ABSTRACT The human endometrium is incredibly dynamic, undergoing monthly cycles of growth and regression during a womans reproductive life. Endometrial repair at the cessation of menstruation is critical for reestablishment of a functional endometrium receptive for embryo implantation; however, little is understood about the mechanisms behind this rapid and highly efficient process. This study utilized a functional mouse model of endometrial breakdown and repair to assess changes in endometrial vasculature that accompany these dynamic processes. Given that adult endometrial stem/progenitor cells identified in human and mouse endometrium are likely contributors to the remarkable regenerative capacity of endometrium, we also assessed label-retaining cells (LRC) as candidate stromal stem/progenitor cells and examined their relationship with endometrial vasculature. Newborn mouse pups were pulse-labeled with bromodeoxyuridine (BrdU) and chased for 5 wk before decidualization, endometrial breakdown, and repair were induced by hormonal manipulation. Mean vessel density did not change significantly throughout breakdown and repair; however, significantly elevated endothelial cell proliferation was observed in decidual tissue. Stromal LRC were identified throughout breakdown and repair, with significantly fewer observed during endometrial repair than before decidualization. A significantly higher percentage of LRC were associated with vasculature during repair than before decidualization, and a proportion were undergoing proliferation, indicative of their functional capacity. This study is the first to examine the endometrial vasculature and candidate stromal stem/progenitor cells in a functional mouse model of endometrial breakdown and repair and provides functional evidence suggesting that perivascular LRC may contribute to endometrial stromal expansion during the extensive remodeling associated with this process.

PAPPA2 is increased in severe early onset pre-eclampsia and upregulated with hypoxia

Severe early onset pre-eclampsia is a serious pregnancy complication, believed to arise as a result of persistent placental hypoxia due to impaired placentation. Pregnancy-associated plasma protein A2 (PAPPA2) is very highly expressed in the placenta relative to all other tissues. There is some evidence that PAPPA2 mRNA and protein are increased in association with pre-eclampsia. The aim of the present study was to characterise the mRNA and protein expression, as well as localisation, of PAPPA2 in an independent cohort of severe early onset pre-eclamptic placentas. We also examined whether exposing placental explants to hypoxia (1% oxygen) changed the expression of PAPPA2. Expression of PAPPA2 mRNA and protein was upregulated in severe early onset pre-eclamptic placentas compared with preterm controls and localised to the syncytiotrophoblast. Interestingly, protein localisation was markedly reduced in term placenta. Syncytialisation of BeWo cells did not change PAPPA2 expression. However, hypoxia upregulated PAPPA2 mRNA and protein expression in primary placental explants. Together, our data suggest that PAPPA2 may be upregulated in severe pre-eclampsia and, functionally, this may be mediated via increased placental hypoxia known to occur with this pregnancy disorder.