The Association between Prenatal Nicotine Exposure and Offspring's Hearing Impairment.

OBJECTIVE: The objective of this study is to evaluate whether there is an association between in-utero exposure to nicotine and subsequent hearing dysfunction. PATIENTS AND METHODS: Secondary analysis of a multicenter randomized trial to prevent congenital cytomegalovirus (CMV) infection among gravidas with primary CMV infection was conducted. Monthly intravenous immunoglobulin hyperimmune globulin therapy did not influence the rate of congenital CMV. Dyads with missing urine, fetal or neonatal demise, infants diagnosed with a major congenital anomaly, congenital CMV infection, or with evidence of middle ear dysfunction were excluded. The primary outcome was neonatal hearing impairment in one or more ears defined as abnormal distortion product otoacoustic emissions (DPOAEs; 1 to 8 kHz) that were measured within 42 days of birth. DPOAEs were interpreted using optimized frequency-specific level criteria. Cotinine was measured via enzyme-linked immunosorbent assay kits in maternal urine collected at enrollment and in the third trimester (mean gestational age 16.0 and 36.7 weeks, respectively). Blinded personnel ran samples in duplicates. Maternal urine cotinine >5 ng/mL at either time point was defined as in-utero exposure to nicotine. Multivariable logistic regression included variables associated with the primary outcome and with the exposure (p < 0.05) in univariate analysis. RESULTS: Of 399 enrolled patients in the original trial, 150 were included in this analysis, of whom 46 (31%) were exposed to nicotine. The primary outcome occurred in 18 (12%) newborns and was higher in nicotine-exposed infants compared with those nonexposed (15.2 vs. 10.6%, odds ratio [OR] 1.52, 95% confidence interval [CI] 0.55-4.20), but the difference was not significantly different (adjusted odds ratio [aOR] = 1.0, 95% CI 0.30-3.31). This association was similar when exposure was stratified as heavy (>100 ng/mL, aOR 0.72, 95% CI 0.15-3.51) or mild (5-100 ng/mL, aOR 1.28, 95% CI 0.33-4.95). There was no association between nicotine exposure and frequency-specific DPOAE amplitude. CONCLUSION: In a cohort of parturients with primary CMV infection, nicotine exposure was not associated with offspring hearing dysfunction assessed with DPOAEs. KEY POINTS: · Nicotine exposure was quantified from maternal urine.. · Nicotine exposure was identified in 30% of the cohort.. · Exposure was not associated with offspring hearing dysfunction..

Cell Lines as Biological Models: Practical Steps for More Reliable Research.

Research in toxicology relies on in vitro models such as cell lines. These living models are prone to change and may be described in publications with insufficient information or quality control testing. This article sets out recommendations to improve the reliability of cell-based research.

Inflammatory gene networks in term human decidual cells define a potential signature for cytokine-mediated parturition.

BACKGROUND: Inflammation is a proximate mediator of preterm birth and fetal injury. During inflammation several microRNAs (22 nucleotide noncoding ribonucleic acid (RNA) molecules) are up-regulated in response to cytokines such as interleukin-1ß. MicroRNAs, in most cases, fine-tune gene expression, including both up-regulation and down-regulation of their target genes. However, the role of pro- and antiinflammatory microRNAs in this process is poorly understood. OBJECTIVE: The principal goal of the work was to examine the inflammatory genomic profile of human decidual cells challenged with a proinflammatory cytokine known to be present in the setting of preterm parturition. We determined the coding (messenger RNA) and noncoding (microRNA) sequences to construct a network of interacting genes during inflammation using an in vitro model of decidual stromal cells. STUDY DESIGN: The effects of interleukin-1ß exposure on mature microRNA expression were tested in human decidual cell cultures using the multiplexed NanoString platform, whereas the global inflammatory transcriptional response was measured using oligonucleotide microarrays. Differential expression of select transcripts was confirmed by quantitative real time-polymerase chain reaction. Bioinformatics tools were used to infer transcription factor activation and regulatory interactions. RESULTS: Interleukin-1ß elicited up- and down-regulation of 350 and 78 nonredundant transcripts (false discovery rate < 0.1), respectively, including induction of numerous cytokines, chemokines, and other inflammatory mediators. Whereas this transcriptional response included marked changes in several microRNA gene loci, the pool of fully processed, mature microRNA was comparatively stable following a cytokine challenge. Of a total of 6 mature microRNAs identified as being differentially expressed by NanoString profiling, 2 (miR-146a and miR-155) were validated by quantitative real time-polymerase chain reaction. Using complementary bioinformatics approaches, activation of several inflammatory transcription factors could be inferred downstream of interleukin-1ß based on the overall transcriptional response. Further analysis revealed that miR-146a and miR-155 both target genes involved in inflammatory signaling, including Toll-like receptor and mitogen-activated protein kinase pathways. CONCLUSION: Stimulation of decidual cells with interleukin-1ß alters the expression of microRNAs that function to temper proinflammatory signaling. In this setting, some microRNAs may be involved in tissue-level inflammation during the bulk of gestation and assist in pregnancy maintenance.

Mechanistic modeling of the effects of myoferlin on tumor cell invasion.

Myoferlin (MYOF) is a member of the evolutionarily conserved ferlin family of proteins, noted for their role in a variety of membrane processes, including endocytosis, repair, and vesicular transport. Notably, ferlins are implicated in Caenorhabditis elegans sperm motility (Fer-1), mammalian skeletal muscle development and repair (MYOF and dysferlin), and presynaptic transmission in the auditory system (otoferlin). In this paper, we demonstrate that MYOF plays a previously unrecognized role in cancer cell invasion, using a combination of mathematical modeling and in vitro experiments. Using a real-time impedance-based invasion assay (xCELLigence), we have shown that lentiviral-based knockdown of MYOF significantly reduced invasion of MDA-MB-231 breast cancer cells in Matrigel bioassays. Based on these experimental data, we developed a partial differential equation model of MYOF effects on cancer cell invasion, which we used to generate mechanistic hypotheses. The mathematical model predictions revealed that matrix metalloproteinases (MMPs) may play a key role in modulating this invasive property, which was supported by experimental data using quantitative RT-PCR screens. These results suggest that MYOF may be a promising target for biomarkers or drug target for metastatic cancer diagnosis and therapy, perhaps mediated through MMPs.

Maternal Body Mass Index, Myometrium Contractility and Uterotonic Receptor Expression in Pregnancy.

Pregnant individuals with obesity (body mass index, BMI ≥ 30 kg/m2) are more likely to experience prolonged labor and have double the risk of cesarean compared with individuals with normal weight (BMI < 25 kg/m2). The aim of this study was to evaluate whether obesity in pregnancy is associated with reduced spontaneous and oxytocin-stimulated myometrial contractile activity using ex vivo preparations. We also assessed the relationship between maternal BMI and the expression of oxytocin (OXTR) and prostaglandin (FP) receptors in the myometrial tissue. We enrolled 73 individuals with a singleton gestation undergoing scheduled cesarean delivery at term in a prospective cohort study. This included 49 individuals with a pre-pregnancy BMI ≥ 30 kg/m2 and 24 with BMI < 25.0 kg/m2. After delivery, a small strip of myometrium was excised from the upper edge of the hysterotomy. Baseline spontaneous and oxytocin stimulated myometrial contractile activity was measured using ex vivo preparations. Additionally, expression of oxytocin and prostaglandin receptors from myometrial samples were compared using qRT-PCR and western blot techniques. Spontaneous and oxytocin-stimulated contraction frequency, duration, and force were not significantly different in myometrial samples from the obese and normal-weight individuals. Myometrial OXTR gene and protein expression was also similar in the two groups. While FP gene expression was lower in the myometrial samples from the obese group, protein expression did not differ. These data help to address an important knowledge gap related to the biological mechanisms underlying the association between maternal obesity and dysfunctional labor.

Aspirin modulates production of pro-inflammatory and pro-resolving mediators in endothelial cells.

Endothelial cells synthesize biochemical signals to coordinate a response to insults, resolve inflammation and restore barrier integrity. Vascular cells release a variety of vasoactive bioactive lipid metabolites during the inflammatory response and produce pro-resolving mediators (e.g., Lipoxin A4, LXA4) in cooperation with leukocytes and platelets to bring a halt to inflammation. Aspirin, used in a variety of cardiovascular and pro-thrombotic disorders (e.g., atherosclerosis, angina, preeclampsia), potently inhibits proinflammatory eicosanoid formation. Moreover, aspirin stimulates the synthesis of pro-resolving lipid mediators (SPM), so-called Aspirin-Triggered Lipoxins (ATL). We demonstrate that cytokines stimulated a time- and dose-dependent increase in PGI2 (6-ketoPGF1α) and PGE2 formation that is blocked by aspirin. Eicosanoid production was caused by cytokine-induced expression of cyclooxygenase-2 (COX-2). We also detected increased production of pro-resolving LXA4 in cytokine-stimulated endothelial cells. The R-enantiomer of LXA4, 15-epi-LXA4, was enhanced by aspirin, but only in the presence of cytokine challenge, indicating dependence on COX-2 expression. In contrast to previous reports, we detected arachidonate 5-lipoxygenase (ALOX5) mRNA expression and its cognate protein (5-lipoxygenase, 5-LOX), suggesting that endothelial cells possess the enzymatic machinery necessary to synthesize both pro-inflammatory and pro-resolving lipid mediators independent of added leukocytes or platelets. Finally, we observed that, endothelial cells produced LTB4 in the absence of leukocytes. These results indicate that endothelial cells produce both pro-inflammatory and pro-resolving lipid mediators in the absence of other cell types and aspirin exerts pleiotropic actions influencing both COX and LOX pathways.

Low-dose aspirin increases 15-epi-lipoxins A4 in pregnancies at high-risk for developing preeclampsia.

BACKGROUND: LDA triggers biosynthesis of endogenous anti-inflammatory molecules, aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4), which may counteract inflammatory process of preeclampsia (PE), and play role in LDA's mechanism of action in PE prevention in high-risk patients. OBJECTIVE: Investigate the effects of daily LDA on levels of 15-epi-LXA4 in pregnancies at high-risk for developing PE. MATERIALS AND METHODS: Secondary analysis of multi-centered randomized controlled trial investigating effects of daily LDA (60 mg) in high-risk pregnancies. Maternal samples were drawn at three points: before LDA initiation (13-26 weeks' gestation), 24-28 weeks' gestation (at least two weeks after LDA) and 34-36 weeks' gestation. 15-epi-LXA4 levels were measured by ELISA. RESULTS: Analysis included 82 patients: 63 receiving daily LDA and 29 receiving daily placebo starting between 13 and 25 weeks gestation. Prior to randomization, baseline 15-epi-LXA4 levels were similar between both groups (75.9 pg/mL [IQR; 63.8-114.0] vs 136.2 pg/mL [52.4-476.2]; p = 0.10). Patients receiving daily LDA were noted to have significantly increased levels of 15-epi-LXA4 after LDA administration (136.2 pg/mL [IQR; 52.4-476.2] vs 1758.2 pg/mL [905.4-6638.5]; p < 0.001). They also had higher 15-epi-LXA4 levels compared to those receiving placebo at 24-28 weeks' (50.3 [38.1-94.2] vs 1758.2 [905.4-6638.5]; p < 0.001 and 34-38 weeks' gestation (57.9 [41.9-76.7] vs 2310.3 pg/mL [656.9-10609.4]; p < 0.001). After LDA administration in the second trimester, patients who developed PE had decrease in 15-epi-LXA4 levels compared to those without PE (942 pg/mL [348.3-1810.3] vs 1758.2 pg/mL [905.4-6638.5]; p = 0.129). CONCLUSION: Daily LDA administration increases 15-epi-LXA4 levels in high-risk pregnancies for PE. In LDA group, pregnancies complicated by PE have lower levels of 15-epi-LXA4 compared to pregnancies without PE.

Nuclear factor-kappa B localization and function within intrauterine tissues from term and preterm labor and cultured fetal membranes.

BACKGROUND: The objective of this study was to quantify the nuclear localization and DNA binding activity of p65, the major transactivating nuclear factor-kappa B (NF-kappaB) subunit, in full-thickness fetal membranes (FM) and myometrium in the absence or presence of term or preterm labor. METHODS: Paired full-thickness FM and myometrial samples were collected from women in the following cohorts: preterm no labor (PNL, N = 22), spontaneous preterm labor (PTL, N = 21), term no labor (TNL, N = 23), and spontaneous term labor (STL, N = 21). NF-kappaB p65 localization was assessed by immunohistochemistry, and DNA binding activity was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based method. RESULTS: Nuclear p65 labeling was rare in amnion and chorion, irrespective of clinical context. In decidua, nuclear p65 labeling was greater in the STL group relative to the TNL cohort, but there were no differences among the TNL, PTL, and PNL cohorts. In myometrium, diffuse p65 nuclear labeling was significantly associated with both term and preterm labor. There were no significant differences in ELISA-based p65 binding activity in amnion, choriodecidual, and myometrial specimens in the absence or presence of term labor. However, parallel experiments using cultured term fetal membranes demonstrated high levels of p65-like binding even the absence of cytokine stimulation, suggesting that this assay may be of limited value when applied to tissue specimens. CONCLUSIONS: These results suggest that the decidua is an important site of NF-kappaB regulation in fetal membranes, and that mechanisms other than cytoplasmic sequestration may limit NF-kappaB activation prior to term.

Mathematical modeling of preadipocyte fate determination.

White adipose tissue is the major energy storage depot for neutral lipids and is also a key endocrine regulator of a host of homeostatic activities, including metabolism, feeding behaviors, cardiovascular functions and reproduction. Abnormal fat accretion in the setting of obesity can lead to insulin resistance and type 2 diabetes, and has been linked to some cancers and arteriosclerosis. Thus, a thorough appreciation of the intricate signaling events that must take place as quiescent adipocyte precursors are recruited into the proliferating cell population that then must 'decide' to differentiate into fully functional fat cells is critical to our understanding of diseases related to excess adipogenesis. We are beginning to gain insights into the molecular regulators of adipocyte differentiation. A significant advance would be to construct mathematical modeling tools that would assist cell biologists in predicting how environmental and/or intrinsic inputs could influence preadipocyte fate decision making. We have developed a model of how preadipocytes may use the dynamic interplay of two transcription factors, nuclear factor-kappa B (NF-kappaB) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in early proliferation and differentiation events in vitro. Critical to the model is the feedback signaling between NF-kappaB and its inhibitor, I kappaB. The model is based on differential equations that describe the interactions of stimuli for NF-kappaB activation and mitogen concentrations and allows one to make predictions about how mouse 3T3-L1 preadipocytes choose between proliferation, differentiation or quiescence. Those predictions are supported by experiments on mouse 3T3-L1 cells.

Progesterone Receptor Signaling Selectively Modulates Cytokine-Induced Global Gene Expression in Human Cervical Stromal Cells.

Preterm birth (PTB) is the leading cause of morbidity and mortality in infants <1 year of age. Intrauterine inflammation is a hallmark of preterm and term parturition; however, this alone cannot fully explain the pathobiology of PTB. For example, the cervix undergoes a prolonged series of biochemical and biomechanical events, including extracellular matrix (ECM) remodeling and mechanochemical changes, culminating in ripening. Vaginal progesterone (P4) prophylaxis demonstrates great promise in preventing PTB in women with a short cervix (<25 mm). We used a primary culture model of human cervical stromal fibroblasts to investigate gene expression signatures in cells treated with interleukin-1ß (IL-1ß) in the presence or absence of P4 following 17ß-estradiol (17ß-E2) priming for 7-10 days. Microarrays were used to measure global gene expression in cells treated with cytokine or P4 alone or in combination, followed by validation of select transcripts by semiquantitative polymerase chain reactions (qRT-PCR). Primary/precursor (MIR) and mature microRNAs (miR) were quantified by microarray and NanoString® platforms, respectively, and validated by qRT-PCR. Differential gene expression was computed after data normalization followed by pathway analysis using Kyoto Encyclopedia Genes and Genomes (KEGG), Panther, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) upstream regulator algorithm tools. Treatment of fibroblasts with IL-1ß alone resulted in the differential expression of 1432 transcripts (protein coding and non-coding), while P4 alone led to the expression of only 43 transcripts compared to untreated controls. Cytokines, chemokines, and their cognate receptors and prostaglandin endoperoxide synthase-2 (PTGS-2) were among the most highly upregulated transcripts following either IL-1ß or IL-1ß + P4. Other prominent differentially expressed transcripts were those encoding ECM proteins, ECM-degrading enzymes, and enzymes involved in glycosaminoglycan (GAG) biosynthesis. We also detected differential expression of bradykinin receptor-1 and -2 transcripts, suggesting (prominent in tissue injury/remodeling) a role for the kallikrein-kinin system in cervical responses to cytokine and/or P4 challenge. Collectively, this global gene expression study provides a rich database to interrogate stromal fibroblasts in the setting of a proinflammatory and endocrine milieu that is relevant to cervical remodeling/ripening during preparation for parturition.

Isolation of highly enriched apical plasma membranes of the placental syncytiotrophoblast.

The human placenta is a complex organ whose proper function is crucial for the development of the fetus. The placenta contains within its structure elements of the maternal and fetal circulatory systems. The interface with maternal blood is the lining of the placenta, that is a unique compartment known as the syncytiotrophoblast. This large syncytial structure is a single cell layer in thickness, and the apical plasma membrane of the syncytiotrophoblast interacts directly with maternal blood. Relatively little is known about the proteins that reside in this unique plasma membrane or how they may change in various placental diseases. Our goal was to develop methods for isolating highly enriched preparations of this apical plasma membrane compatible with high-quality proteomics analysis and herein describe the properties of these isolated membranes.

Myoferlin regulates epithelial cancer cell plasticity and migration through autocrine TGF-ß1 signaling.

Epithelial cancer cells can undergo an epithelial-mesenchymal transition (EMT), a complex genetic program that enables cells to break free from the primary tumor, breach the basement membrane, invade through the stroma and metastasize to distant organs. Myoferlin (MYOF), a protein involved in plasma membrane function and repair, is overexpressed in several invasive cancer cell lines. Depletion of myoferlin in the human breast cancer cell line MDA-MB-231 (MDA-231MYOFKD) reduced migration and invasion and caused the cells to revert to an epithelial phenotype. To test if this mesenchymal-epithelial transition was durable, MDA-231MYOFKD cells were treated with TGF-ß1, a potent stimulus of EMT. After 48 hr with TGF-ß1, MDA-231MYOFKD cells underwent an EMT. TGF-ß1 treatment also decreased directional cell motility toward more random migration, similar to the highly invasive control cells. To probe the potential mechanism of MYOF function, we examined TGF-ß1 receptor signaling. MDA-MB-231 growth and survival has been previously shown to be regulated by autocrine TGF-ß1. We hypothesized that MYOF depletion may result in the dysregulation of TGF-ß1 signaling, thwarting EMT. To investigate this hypothesis, we examined production of endogenous TGF-ß1 and observed a decrease in TGF-ß1 protein secretion and mRNA transcription. To determine if TGF-ß1 was required to maintain the mesenchymal phenotype, TGF-ß receptor signaling was inhibited with a small molecule inhibitor, resulting in decreased expression of several mesenchymal markers. These results identify a novel pathway in the regulation of autocrine TGF-ß signaling and a mechanism by which MYOF regulates cellular phenotype and invasive capacity of human breast cancer cells.

Cellular Mechanics of Primary Human Cervical Fibroblasts: Influence of Progesterone and a Pro-inflammatory Cytokine.

The leading cause of neonatal mortality, pre-term birth, is often caused by pre-mature ripening/opening of the uterine cervix. Although cervical fibroblasts play an important role in modulating the cervix's extracellular matrix (ECM) and mechanical properties, it is not known how hormones, i.e., progesterone, and pro-inflammatory insults alter fibroblast mechanics, fibroblast-ECM interactions and the resulting changes in tissue mechanics. Here we investigate how progesterone and a pro-inflammatory cytokine, IL-1ß, alter the biomechanical properties of human cervical fibroblasts and the fibroblast-ECM interactions that govern tissue-scale mechanics. Primary human fibroblasts were isolated from non-pregnant cervix and treated with estrogen/progesterone, IL-1ß or both. The resulting changes in ECM gene expression, matrix remodeling, traction force generation, cell-ECM adhesion and tissue contractility were monitored. Results indicate that IL-1ß induces a significant reduction in traction force and ECM adhesion independent of pre-treatment with progesterone. These cell level effects altered tissue-scale mechanics where IL-1ß inhibited the contraction of a collagen gel over 6 days. Interestingly, progesterone treatment alone did not modulate traction forces or gel contraction but did result in a dramatic increase in cell-ECM adhesion. Therefore, the protective effect of progesterone may be due to altered adhesion dynamics as opposed to altered ECM remodeling.

Chemotherapeutic implants via subcritical CO2 modification.

Polymer-based biomaterials have a broad range of current applications in medicine. Many implants generate a favorable biomedical outcome solely by providing short-term mechanical stability that allows healing of the surrounding tissues. An example is polymeric reconstructive resorbable plates having initial strengths sufficient to stabilize bone segments while allowing the osteosynthesis needed to restore original function following tumor resection. Simultaneous, localized delivery of the widely employed chemotherapeutic paclitaxel following tumor removal presents a particularly desirable goal in this context. By using compressed/subcritical CO(2) at moderate pressures (as opposed to the more familiar supercritical pressures) to embed paclitaxel in clinically utilized reconstructive plating, the form of the implant can be preserved while adding an inherently localized chemotherapeutic function. In vitro tests demonstrate the efficacy of the embedded paclitaxel against adherent MCF-7 breast cancer cells within the immediate area of the polylactic acid (PLA). CO(2) can be utilized to add dual structural-chemotherapeutic function to polymeric surfaces without a change in form. The ability to 'piggyback' chemotherapeutic function into nearly any polymeric surface should find widespread utility.

Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds.

A mechanistic understanding of adipose tissue differentiation is critical for the treatment and prevention of obesity and type 2 diabetes. Conventional in vitro models of adipogenesis are preadipocytes or freshly isolated adipocytes grown in two-dimensional (2D) cultures. Optimal results using in vitro tissue culture models can be expected only when adipocyte models closely resemble adipose tissue in vivo. Thus the design of an in vitro three-dimensional (3D) model which faithfully mimics the in vivo environment is needed to effectively study adipogenesis. Pluripotent embryonic stem (ES) cells are a self-renewing cell type that can readily be differentiated into adipocytes. In this study, a 3D culture system was developed to mimic the geometry of adipose tissue in vivo. Murine ES cells were seeded into electrospun polycaprolactone scaffolds and differentiated into adipocytes in situ by hormone induction as demonstrated using a battery of gene and protein expression markers along with the accumulation of neutral lipid droplets. Insulin-responsive Akt phosphorylation, and beta-adrenergic stimulation of cyclic AMP synthesis were demonstrated in ES cell-derived adipocytes. Morphologically, ES cell-derived adipocytes resembled native fat cells by scanning electron and phase contrast microscopy. This tissue engineered ES cell-matrix model has potential uses in drug screening and other therapeutic developments.

Agonist-Dependent Downregulation of Progesterone Receptors in Human Cervical Stromal Fibroblasts.

Progesterone (P(4)) maintains uterine quiescence during the majority of pregnancy, whereas diminished progesterone receptor (PR) expression and/or activity (ie, functional P(4) withdrawal) promotes parturition. To investigate the regulation of PR expression in cervical stroma, fibroblasts from premenopausal hysterectomy specimens were prepared. Greater than 99% of the cultures were vimentin positive (mesenchymal cell marker) with only occasional cytokeratin-8 positivity (epithelial cell marker) and no evidence of CD31-positive (endothelial cell marker) cells. Cells were immunolabeled with antibodies directed against PRs (PR-A and PR-B), estrogen receptor α (ER-α), and glucocorticoid receptor-α/ß (GR-α/ß). All cells were uniformly immunopositive for ER-α and GR-α/ß but did not express PRs. Incubation of cells with 10(-8) mol/L 17ß-estradiol induced a time-dependent increase in PR-A and PR-B messenger RNAs (mRNAs) by quantitative real-time polymerase chain reactions and proteins by immunoblotting and immunofluorescence. Incubation of cervical fibroblasts with PR ligands (medroxyprogesterone acetate or Org-2058) downregulated PR-A and PR-B levels. Coincubation of cells with PR ligands plus RU-486, a PR antagonist, partially abrogated agonist-induced receptor downregulation. Dexamethasone, a pure glucocorticoid, had no inhibitory effect on PR expression. These results indicate that progestins and estrogens regulate PR expression in cervical fibroblasts. We postulate that hormonal regulation of PR expression in the cervical stroma may contribute to functional P(4) withdrawal in preparation for parturition.

Despite transcriptional and functional coordination, cyclooxygenase-2 and microsomal prostaglandin E synthase-1 largely reside in distinct lipid microdomains in WISH epithelial cells.

Cytokine-induced prostaglandin (PG)E(2) synthesis requires increased expression of cyclooxygenase-2 (COX-2) in human WISH epithelial cells. Recently, an inducible downstream PGE synthase (microsomal PGE synthase-1, mPGES-1) has been implicated in this inflammatory pathway. We evaluated cooperation between COX-2 and mPGES-1 as a potential mechanism for induced PGE(2) production in WISH cells. Cytokine stimulation led to increased expression of both enzymes. Selective pharmacological inhibition of these enzymes demonstrated that induced PGE(2) release occurred through a dominant COX-2/mPGES-1 pathway. Unexpectedly, immunofluorescent microscopy revealed that the expression of these enzymes was not tightly coordinated among cells after cytokine challenge. Within cells expressing high levels of both mPGES-1 and COX-2, immunolabeling of high-resolution semithin cryosections revealed that COX-2 and mPGES-1 were largely segregated to distinct regions within continuous intracellular membranes. Using biochemical means, it was further revealed that the majority of mPGES-1 resided within detergent-insoluble membrane fractions, whereas COX-2 was found only in detergent-soluble fractions. We conclude that although mPGES-1 and COX-2 show transcriptional and functional coordination in cytokine-induced PGE(2) synthesis, complementary morphological and biochemical data suggest that a majority of intracellular mPGES-1 and COX-2 are segregated to discrete lipid microdomains in WISH epithelial cells.

Adipose tissue model using three-dimensional cultivation of preadipocytes seeded onto fibrous polymer scaffolds.

A better understanding of the mechanism of adipose tissue differentiation is of paramount importance in the development of therapeutic strategies for the treatment and prevention of obesity and type 2 diabetes mellitus. Optimal results using tissue culture models can be expected only when the in vitro adipocyte resembles adipose tissue in vivo as closely as possible. In this study, we used tissue-engineering principles to develop a three-dimensional (3-D) culture system to mimic the geometry of adipose tissue in vivo. Mouse preadipocyte 3T3-L1 cells were seeded onto nonbiodegradable fibrous polyethylene terephthalate scaffolds and differentiated with a hormone cocktail consisting of insulin, dexamethasone, isobutylmethylxanthine, and fetal calf serum. Cell morphology, growth, differentiation, and function were studied by immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, enzyme-linked immunosorbent assay, and oil red O staining. Cells grown on 3-D fibrous scaffolds were differentiated in situ by hormone induction with high efficiency (approximately 90%) as shown by scanning electron microscopy. Immunocytochemistry, immunoblot analysis, and RT-PCR revealed that the 3-D constructs expressed adipocyte-specific genes, including peroxisome proliferator-activated receptor gamma, leptin, adipsin, aP2, adiponectin, GLUT4, and resistin. Adipocytes matured on 3-D constructs secreted leptin at levels even greater than that of fully differentiated adipocytes in 2-D conventional cell cultures. Finally, adipocyte-specific phenotypic function was demonstrated by accumulation of neutral lipids in larger fat droplets. In conclusion, preadipocytes grown on 3-D matrices acquire morphology and biological features of mature adipocytes. This new culture model should have significant utility for in vitro studies of adipocyte cell biology and development.