Human Preterm Labor Research Articles

OR20-05 Regulation of Mouse Uterine Contractility by Regulator of G-Protein Signalling 2

INTRODUCTION: Uterine contractions in labour are produced by activation of receptors such as FP (PGF2α) and OXTR (oxytocin). Such receptors signal via Gαq proteins that can be selectively turned off by the regulator of G-protein signaling 2 (RGS2). RGS2 expression can be upregulated by Gαs-coupled receptor signaling. PGE2 elicits various uterine effects, some via activation of Gαs-coupled receptors. In primary human myometrial cells, PGE2 enhances RGS2 expression that attenuates subsequent oxytocin-stimulated calcium responses associated with contraction. RGS2 expression is higher in preterm non-labour versus labour human myometrium, suggesting that RGS2 promotes quiescence in the pregnant uterus. To directly study whether RGS2 dampens uterine contraction, we analyzed the ex vivo contractility of pregnant mouse uteri. We hypothesize that loss of RGS2 expression enhances oxytocin and PGF2α-stimulated uterine contractility. METHODS: Wildtype (WT) and RGS2 knockout (KO) female mice (8-14 weeks) were time-mated and euthanized on days 15-20 of pregnancy or during active labour (n=5-11 per group). Uteri were snap frozen for RNA/protein expression analysis by qPCR and western blot. Uterine sections from d18-19 were dissected into longitudinal strips for ex vivo contractility measurement. Tissues were submerged in Krebs-Henseleit buffer and tied to force transducers to measure isometric force development. Tissues were treated with or without 1μM PGE2 for 2 hours. In parallel, pretreated and non-pretreated tissues were exposed to increasing concentrations of oxytocin or PGF2α (0.1-10uM). All tissues were treated with 100mM KCl to determine the maximum contraction at the end of the experiment. Data were analyzed by ANOVA (Bonferroni post-hoc), or Student’s t-test, as appropriate. RESULTS: RGS2 expression in WT uteri sharply decreases at day 19 (p<0.05), remaining low during labour. OXTR and FP receptor expression increase at day 19 (p<0.01 and p<0.05, respectively) and are sustained through labour. Compared to WT, uteri from KO mice produced larger oxytocin-stimulated contractile amplitudes (p<0.05), and more frequent PGF2α-stimulated contractions (p<0.05). PGE2 pretreatment enhanced RGS2 expression in WT uteri, which reduced the peak amplitudes and integrals elicited by subsequent oxytocin treatment, compared to non-pretreated uteri (p<0.05). CONCLUSION: RGS2 may play a pro-quiescent role in pregnancy, since RGS2 loss enhances uterine contractility. Concomitant upregulation of pro-contractile receptors and RGS2 downregulation may facilitate labour activation. This mechanisms may be involved in the etiology of spontaneous preterm labour; loss of RGS2 expression observed in human preterm labour may allow for early contractile activation. Understanding RGS2’s role in quiescence is important to developing more effective strategies for managing preterm labour.

Differential Expression g‐H2AX and RAGE in the Placenta of Gestational Diabetes Mellitus (GDM), Preterm Labor (PTL) and Preeclampsia Patients

The integrity of the genome is fundamental for human health. Defective DNA repair response (DRR) is associated with several diseases, including pulmonary fibrosis, neurodegeneration, and in diseased placenta. The most destructive form of genomic damage is known to be DNA double strand breaks (DNA‐DSBs), in which both strands of DNA are ruptured. g‐H2AX is a component of the histone octomer and is phosphorylated in response to DNA‐DSBs. The receptor for advanced glycation end‐products (RAGE) is a multi‐ligand receptor primarily expressed on cell membranes, where it functions as a progression factor in inflammatory signaling; however, the nuclear isoform of RAGE (nRAGE) has been described as having a crucial role in DNA‐DSBs repair. The purpose of this study was to identify placental nRAGE and g‐H2AXexpression during normal gestation (Control), gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR), preterm delivery (PTL) and preeclampsia (PE). Immunohistochemistry was used to determine localization of both molecules present in human placenta. Immunoblot was further used to quantify nRAGE andg‐H2AX production. Assessing DNA degradation was used to confirm placental DNA damage during pregnancy complications. Relative to controls we observed: 1) Increased staining for of RAGE and g‐H2AX in PTL and PE placentas; 2) increased placental nRAGE protein in the PTL and PE placentas; 3) increased placental g‐H2AX protein in both PTL and PE conditions; and 4) increased DNA damage in the diseased placenta. These research suggest a potential role for nRAGE during DNA‐DSB detection and repair in human PTL and PE placentas. These results may provide insight into the physiological relevance of these molecules, and if so, their modification during gestation may help alleviate placental disease.Support or Funding InformationThis work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pro-inflammatory cytokine-induced microRNA-212-3p expression promotes myocyte contraction via methyl-CpG-binding protein 2: a novel mechanism for infection-related preterm parturition.

Preterm labour is a common pregnancy complication contributing to major maternal and fetal morbidity and mortality. We have found microRNA (miR)-212-3p, a potential infection-associated molecule, was significantly over-expressed during human preterm labour. However, the mechanism remains unknown. In this study, we have adopted a lipopolysaccharide (LPS)-induced Institute of Cancer Research murine preterm model to examine the role of miR-212-3p in the infection-induced preterm labour. Myometrial miR-212-3p expression was increased by nearly 4-fold in the term labour group (P = 0.10) and 12-fold (P = 0.03) in the LPS-induced preterm labour group compared with the non-labour group. In vitro cellular experiments confirmed that a series of pro-inflammatory cytokines, including interleukin (IL)1B (P = 0.02) and IL-6 (P = 0.01), rather than LPS (P = 0.08) itself could significantly upregulate miR-212-3p expression in human myometrial smooth muscle cells. Methyl-CpG-binding protein 2 (MeCP2), as a target gene of miR-212-3p confirmed by our dual luciferase assay, influenced myocyte contractility and connexin 43 expression which is an important contraction-associated protein. Therefore, we conclude that miR-212-3p may be involved in infection-induced preterm labour through MeCP2 and it is a promoting molecule and novel target for the diagnosis and treatment of preterm labour in the future.

Interleukin-33-induced expression of PIBF1 by decidual B cells protects against preterm labor.

Preterm birth (PTB) is a leading cause of neonatal death worldwide. Intrauterine and systemic infection and inflammation cause 30-40% of spontaneous preterm labor (PTL), which precedes PTB. Although antibody production is a major immune defense mechanism against infection, and B cell dysfunction has been implicated in pregnancy complications associated with PTL, the functions of B cells in pregnancy are not well known. We found that choriodecidua of women undergoing spontaneous PTL harbored functionally altered B cell populations. B cell-deficient mice were markedly more susceptible than wild-type (WT) mice to PTL after inflammation, but B cells conferred interleukin (IL)-10-independent protection against PTL. B cell deficiency in mice resulted in a lower uterine level of active progesterone-induced blocking factor 1 (PIBF1), and therapeutic administration of PIBF1 mitigated PTL and uterine inflammation in B cell-deficient mice. B cells are a significant producer of PIBF1 in human choriodecidua and mouse uterus in late gestation. PIBF1 expression by B cells is induced by the mucosal alarmin IL-33 (ref. 9). Human PTL was associated with diminished expression of the α-chain of IL-33 receptor on choriodecidual B cells and a lower level of active PIBF1 in late gestation choriodecidua. These results define a vital regulatory cascade involving IL-33, decidual B cells and PIBF1 in safeguarding term pregnancy and suggest new therapeutic approaches based on IL-33 and PIBF1 to prevent human PTL.

AB281. SPR-08 Altered S-nitrosation of contractile proteins underlies dysfunctional quiescence in human preterm labor

ObjectivePreterm labor is defined as labor prior to 37 weeks of gestation. There are no FDA approved drugs available to treat spontaneous preterm labor. Uterine smooth muscle is unique in that it relaxes in a cGMP-independent manner in the presence of nitric oxide (NO). However, NO-mediated relaxation in tissues from mothers who deliver preterm is blunted. S-nitrosation of cysteines via NO acts as an important mediator in a number of disease states. Our analysis of the myometrial S-nitrosoproteome has revealed that several smooth muscle contractile proteins are differentially S-nitrosated based upon the state of labor in women. An important regulator of protein S-nitrosation is the availability of S-nitrosoglutathione (GSNO), an endogenously expressed NO donor. The enzyme GSNO reductase (GSNOR) regulates GSNO levels in smooth muscle. We performed experiments to test the hypotheses that GSNOR activity is altered in the myometrium from women in preterm labor and that inhibition of GSNOR attenuates myometrial contraction and restores quiescence in spontaneous preterm labor.MethodsExperiments were performed in the Myometrial Research Laboratory at UNR Med. Uterine biopsies were obtained with IRB approval and written informed consent from mothers undergoing Cesarean section and transported to the laboratory in physiological buffer for immediate processing. GSNOR expression was measured by western blot and normalized to GAPDH expression. GSNOR activity was measured by the decrease in absorbance at 340 nm via conversion of NADH to NAD+ by GSNOR in the presence of GSNO. Eight (8) nM N6022, a potent and specific inhibitor of GSNOR, was used to verify GSNOR specific activity. Human myometrial tissue was dissected and six uterine strips from each patient were mounted in tissue baths and tested for their ability to contract in response to KCl prior to addition of test agents.ResultsHuman preterm myometrium fails to relax to NO addition whether in the presence or absence of oxytocin stimulation. GSNOR expression is increased in myometrial tissue from women in spontaneous preterm labor compared to term laboring controls. GSNOR enzymatic activity was also increased in protein lysates from myometrial tissue from patients undergoing spontaneous preterm labor than those in labor at term. A potent and selective inhibitor of GSNOR (N6022) decreases the peak force of contraction in guinea pig myometrium and restores sensitivity to NO.ConclusionsGSNOR is more highly expressed in preterm laboring myometrium. Preterm laboring myometrium exhibits a blunted response to NO and the GSNOR inhibitor N6022 restores sensitivity to relaxation by NO. Our data suggests that increased GSNOR activity in spontaneous preterm labor contributes to a preterm contractile phenotype through the enzymatic degradation of endogenous GSNO.Funding Source(s)NIH 1U54GM 104944

InlP, a New Virulence Factor with Strong Placental Tropism.

Intrauterine infection is a major detriment for maternal-child health and occurs despite local mechanisms that protect the maternal-fetal interface from a wide variety of pathogens. The bacterial pathogen Listeria monocytogenes causes spontaneous abortion, stillbirth, and preterm labor in humans and serves as a model for placental pathogenesis. Given the unique immunological environment of the maternal-fetal interface, we hypothesized that virulence determinants with placental tropism are required for infection of this tissue. We performed a genomic screen in pregnant guinea pigs that led to the identification of 201 listerial genes important for infection of the placenta but not maternal liver. Among these genes was lmrg1778 (lmo2470), here named inlP, predicted to encode a secreted protein that belongs to the internalin family. InlP is conserved in virulent L. monocytogenes strains but absent in Listeria species that are nonpathogenic for humans. The intracellular life cycle of L. monocytogenes deficient in inlP (ΔinlP) was not impaired. In guinea pigs and mice, InlP increased the placental bacterial burden by a factor of 3 log10 while having only a minor role in other maternal organs. Furthermore, the ΔinlP strain was attenuated in intracellular growth in primary human placental organ cultures and trophoblasts. InlP is a novel virulence factor for listeriosis with a strong tropism for the placenta. This virulence factor represents a tool for the development of new modalities to prevent and treat infection-related pregnancy complications.

Uterine natural killer cells dysregulation in idiopathic human preterm birth: a pilot study

Objective: To compare between uterine natural killer (uNK) cells in the placental samples of preterm birth and term labor.Study design: Two-arm case–control study. This study included 60 participants divided into two groups. The first group included 30 cases of idiopathic spontaneous preterm labor and the other group included 30 women who delivered by a spontaneous term vaginal delivery and with no history of previous preterm labor.Result(s): There were no CD16− CD56bright uNK cells in either groups; CD16+ CD56dim uNK cells were present in only 1 case out of 30 term delivery placentae (3.3%), whereas they were found in 21 cases out of 30 (70%) preterm placental samples with a significant statistical difference (p < 0.0001) and OR 67.667, 95% CI (7.95–575.69). CD16+CD56dim uNK cells were found to be invading both the villi and the decidua in 11 cases (70%), whereas those cells were found invading only the villi in 10 cases (33.3%).Conclusion: CD16+CD56dim cells are expressed in both the decidua and the villi of patients with idiopathic preterm labor suggesting an association between uNK cells dysregulation and idiopathic human preterm labor.

A HEXIM1-AIRE transcriptional counter-balance in trophoblasts regulates maternal-fetal mucosal immunity in pregnancy (MUC7P.755)

Abstract Trophoblasts are gatekeepers at maternal-fetal mucosal interface, but trophoblast-derived molecules that initiate and maintain maternal-fetal mucosal immunity are not fully understood. We found that HEXIM1, an inhibitor of P-TEFb essential for eukaryotic RNA Pol II-mediated transcription, is highly expressed in human villous and extravillous trophoblasts. Hexim1-/- mouse placentas exhibit abnormal placental immunity characterized by increased maternal leukocyte and T cell infiltration, a paucity of spiral arteries and reduced placental size. HEXIM1 interacts in trophoblasts with AIRE, a protein critical for central and peripheral immune tolerance, via 7SK snRNA. Such interaction is further dependent on the nuclear localization domains of HEXIM1 and AIRE. Extravillous trophoblasts in human preterm labor with placental inflammation have reduced HEXIM1 and increased AIRE expression. Silencing HEXIM1 in trophoblasts led to increased Treg induction by trophoblasts. Our findings identify trophoblasts as a new type of extrathymic AIRE-expressing immunoregulatory cell, reveal a novel function of epithelial cells in mucosal immune regulation via AIRE-mediated Treg induction, suggest HEXIM1 and AIRE establish a transcriptional counterbalance in trophoblasts to maintain maternal-fetal mucosal immunity in pregnancy by regulating P-TEFb and Pol II, and promote a better understanding of how certain pathogens, such as HIV-1, perturb mucosal homeostasis after hijacking the HEXIM1 machinery.

Statins prevent cervical remodeling, myometrial contractions and preterm labor through a mechanism that involves hemoxygenase-1 and complement inhibition.

Preterm birth (PTB) is a major public health problem, with a global prevalence of 9.6% and over a million annual neonatal deaths. In a mouse model of preterm labor (PTL) induced by intravaginal administration of a subclinical dose of lipopolysaccharide (LPS), we previously demonstrated that LPS ascends to the cervix, inducing complement activation, cervical remodeling and PTL. Here we show that complement activation also plays a role in myometrial contractions during PTL in this model. Increased levels of C5a were detected in the myometrium of LPS-treated mice but not in age-matched control or term myometrium. Human and mouse myometrium incubated with C5a showed increased frequency of contractions and expression of connexin 43, suggesting that C5a is an uterotonic molecule. Statins, which showed beneficial effects in preventing complement-mediated pregnancy complications, prevented cervical remodeling, myometrial contractions and PTL in the LPS model. The protective effects of statins in PTL were associated with increased synthesis, expression and activity of heme oxygenase (HO-1) in myometrium and cervix. Coadministration of HO-1 inhibitor tin-protoporphyrin-IX with pravastatin abrogated the protective effects of pravastatin on cervical remodeling and myometrial contractions leading to PTB. In addition, pravastatin inhibited complement activation in the cervix by increasing the synthesis and expression of complement inhibitor decay-accelerating factor. This study in mice suggests that statins might be useful to prevent PTL in humans. Clinical trials in humans are needed and if these results are confirmed, they may form the basis for a new clinical approach to prevent PTB.

The impact of undernutrition on the activation of premature birth and prostaglandin pathway

Human preterm labor is a syndrome that may arise from different causes. Before 30 weeks of gestation, infection appears to predominate amongst these. In later gestation, premature activation of the fetal HPA axis in response to an adverse intrauterine environment, e.g. hypoxemia, undernutrition (UN) or even infection, may underlie the stimulus to prematurity. A third pathway clearly involves vascular thrombotic processes. Tests on sheep have been shown to there is a premature birth which is a significant change was associated with precocious increases in adrenocorticotrophic hormone (ACTH) and cortisol in the fetal circulation, and increased levels of mRNA encoding proopiomelanocortin (POMC) and prohormone convertase-1 (PC-1) in the pars intermedia of the fetal pituitary. It would seem that these different pathways may occur separately, or be activated to different degrees in the same patient.

SIRT6 Is Decreased with Preterm Labor and Regulates Key Terminal Effector Pathways of Human Labor in Fetal Membranes1

Preterm birth is a major determinant of neonatal mortality and morbidity, affecting approximately one-third of preterm births as a result of prelabor rupturing of membranes. Infection and inflammation have strong causal links to preterm delivery, resulting in the activation of nuclear factor-kappaB (NFKB) and its downstream targets. Human sirtuin (SIRT) 6, which has ADP-ribosyl transferase and deacetylase activity, exhibits anti-inflammatory actions. The aims of this study were to determine the effect of 1) human preterm labor on SIRT6 expression in human gestational tissue and 2) the effect in primary amnion cells of SIRT6 inhibition, using small interfering RNA (siRNA) on prolabor mediators. To determine the effect of human preterm labor on SIRT6 expression, human fetal membranes were collected from women at preterm at the time of Cesarean section (no labor; n = 9) and from women after spontaneous labor and delivery (n = 9). SIRT6 mRNA and protein expression were significantly lower in fetal membranes after spontaneous preterm labor. Transfection of primary amnion cells with SIRT6 siRNA was associated with an increase in IL-1beta-induced proinflammatory cytokine gene expression and release (IL6, IL8, TNF [TNF-alpha]), cyclooxygenase ([COX]-2; official symbol PTGS2) expression and subsequent prostaglandin (PGE(2) and PGF(2alpha)) release, and MMP9 gene expression and release of pro-MMP9. To determine whether SIRT6 affects NFKB transcriptional activity, primary amnion cells were transfected with NFKB tagged with luciferase and stimulated with IL1B. As expected, IL1B induced NFKB transcriptional activity. However, when cells were also cotransfected with a vector expressing SIRT6, there was a decrease in NFKB transcriptional activity. In conclusion, SIRT6 plays a role in regulating the terminal effector pathways of human labor and delivery via the NFKB pathway.

Electrical pacemaker as a safe and feasible method for decreasing the uterine contractions of human preterm labor

The aim of the study was to test the safety and feasibility of a system designed to decrease the uterine contractions of human preterm labor using a weak electrical current. Patients in preterm labor had an electrode catheter placed in the posterior vaginal fornix and attached to an electrical pacemaker. Contraction intervals were determined during the 60-min study, in which minutes 0-20 was the preintervention control period (C1); 21-40 was the electrical intervention (EI), with a 10-s burst of current administered just before each expected contraction; and 41-60 was the postintervention control (C2). Mean intervals were calculated and compared mixed-model ANOVA. Five patients were studied. No maternal or fetal vital sign irregularities were seen. All the babies had a normal neonatal intensive care unit stay for gestational age. The respective mean contraction intervals for C1, EI, and C2 and their within- and between-subject standard deviations were 4.64 (±0.81, ±2.45), 5.71 (±3.03, ±1.62), and 3.83 (±0.83, ±1.31) min. There was a significant difference between EI and C2 (P=0.0078) and no difference between C1 and C2 or between EI and C1 (P=0.1373). EI appears to be a safe and feasible method for decreasing preterm contractions. To confirm and test effectiveness, longer interventions and additional cases are needed.

Epigenetic Regulation of Cytokine Production in Human Amnion and Villous Placenta

The mechanisms of human preterm labour appear inextricably linked to cytokine biosynthesis by gestational tissues. In turn, cytokine production by gestational tissues has been shown to be regulated by epigenetic mechanisms. In this paper, we demonstrate that cytokine production in gestational tissues is regulated epigenetically in a tissue-specific manner. Furthermore, we show that treatment with a histone deacetylation inhibitor can partially abrogate LPS-stimulated TNFα production in villous placenta but not amnion. LPS treatment significantly (P < 0.05) increased the production of IL-1β (∼10–34-fold), TNFα (∼23–>100-fold) and IL10 (∼6–10-fold) after 24 h of treatment in villous explants, as expected. There were no significant LPS effects on IL1Ra production. AZA treatment did not have any significant effect on any cytokines' production tested either alone or in combination with LPS. Interestingly, however, the stimulatory effects of LPS on TNFα production were partially mitigated (P < 0.05) by TSA treatment in villous explants. We suggest caution in the consideration of histone deacetylation inhibitors in pregnancy due to the different responses in gestational tissues.

Stretch and Inflammatory Cytokines Drive Myometrial Chemokine Expression Via NF-κB Activation

Both human preterm labor (PTL) and term labor are consistently associated with a chemokine-induced inflammatory infiltration of the myometrium. However, what regulates myometrial chemokine expression and whether the increase in expression precedes the onset of labor, and so may have a role in its causation, or occurs after, and is simply a consequence of labor, is uncertain. Therefore, we assessed 1) chemokine expression in nonlaboring and laboring myometrial samples obtained at and before term and 2) the factors that regulate myometrial chemokine expression. We found that term labor was characterized by an increase in CXCL8 and CCL2 in both upper and lower segments, whereas PTL was associated with a distinct pattern of chemokine expression, with increases in CCL5, CXCL5, and CCL20 in the lower segment myometrium only. Further, we found that chemokine expression in myometrial cell cultures was increased by stretch and inflammatory cytokines and reduced by prostglandins and oxytocin and that the primary mediator of stretch and cytokine effects was nuclear factor κB (NF-κB) and to a lesser extent MAPK. These data show that PTL appears to be associated with a distinct pattern of chemokine expression, that stretch and cytokines both drive myometrial chemokine expression primarily via activation of NF-κB. These data suggest that the modulation of NF-κB activity may be of potential benefit in the management of PTL.

Minireview: Fetal-Maternal Hormonal Signaling in Pregnancy and Labor

Mechanisms underlying the initiation of parturition remain unclear. Throughout most of pregnancy, uterine quiescence is maintained by elevated progesterone acting through progesterone receptor (PR). Although in most mammals, parturition is associated with a marked decline in maternal progesterone, in humans, circulating progesterone and uterine PR remain elevated throughout pregnancy, suggesting a critical role for functional PR inactivation in the initiation of labor. Both term and preterm labor in humans and rodents are associated with an inflammatory response. In preterm labor, intraamniotic infection likely provides the stimulus for increased amniotic fluid interleukins and migration of inflammatory cells into the uterus and cervix. However, at term, the stimulus for this inflammatory response is unknown. Increasing evidence suggests that the developing fetus may produce physical and hormonal signals that stimulate macrophage migration to the uterus, with release of cytokines and activation of inflammatory transcription factors, such as nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1), which also is activated by myometrial stretch. We postulate that the increased inflammatory response and NF-kappaB activation promote uterine contractility via 1) direct activation of contractile genes (e.g. COX-2, oxytocin receptor, and connexin 43) and 2) impairment of the capacity of PR to mediate uterine quiescence. PR function near term may be compromised by direct interaction with NF-kappaB, altered expression of PR coregulators, increased metabolism of progesterone within the cervix and myometrium, and increased expression of inhibitory PR isoforms. Alternatively, we propose that uterine quiescence during pregnancy is regulated, in part, by PR antagonism of the inflammatory response.

Monocyte Chemoattractant Protein-1 (CCL-2) Integrates Mechanical and Endocrine Signals That Mediate Term and Preterm Labor

Recent evidence suggests that leukocytes infiltrate uterine tissues at or around the time of parturition, implicating inflammation as a key mechanism of human labor. MCP-1 (also known as C-C chemokine motif ligand 2, CCL-2) is a proinflammatory cytokine that is up-regulated in human myometrium during labor. Myometrium was collected from pregnant rats across gestation and at labor. Total RNA and proteins were subjected to real-time PCR and ELISA, respectively. Ccl-2 gene and protein expression was significantly up-regulated in the gravid rat myometrium before and during labor, which might suggest that it is regulated positively by mechanical stretch of the uterus imposed by the growing fetus and negatively by physiological withdrawal of progesterone (P4). We confirmed in vivo that: 1) administration of P4 receptor antagonist RU486 induced an increase in Ccl-2 mRNA and preterm labor, whereas 2) artificial maintenance of elevated P4 levels at late gestation caused a significant decrease in gene expression and blocked labor; 3) Ccl-2 was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium; 4) in vitro static mechanical stretch of primary rat myometrial smooth muscle cells (25% elongation) induced a release of Ccl-2 protein, which was repressed by pretreatment with P4 (1 microM); and 5) stretch enhanced their monocyte chemoattractant activity. These data indicate that Ccl-2 protein serves to integrate mechanical and endocrine signals contributing to uterine inflammation and the induction of labor and thus may represent a novel target for therapeutic prevention of preterm labor in humans.

Enabling Coupled Quantitative Genomics and Proteomics Analyses from Rat Spinal Cord Samples

Translational research is progressing toward combined genomics and proteomics analyses of small and precious samples. In our analyses of spinal cord material, we systematically evaluated disruption and extraction techniques to determine an optimum process for the coupled analysis of RNA and protein from a single 5-mm segment of tissue. Analyses of these distinct molecular species were performed using microarrays and high resolution two-dimensional gels, respectively. Comparison of standard homogenization with automated frozen disruption (AFD) identified negligible differences in the relative abundance of genes (44) with all genes identified by either process. Analysis on either the Affymetrix or Applied Biosystems Inc. gene array platforms provided good correlations between the extraction techniques. In contrast, the AFD technique enabled identification of more unique proteins from spinal cord tissue than did standard homogenization. Furthermore use of an optimized CHAPS/urea extraction provided better protein recovery, as shown by quantitative two-dimensional gel analyses, than did solvent precipitation during TRIzol-based RNA extraction. Thus, AFD of tissue samples followed by protein and RNA isolation from separate aliquots of the frozen powdered sample is the most effective route to ensure full, quantitative analyses of both molecular entities.

Role of atypical protein kinase C isozymes and NF‐κB in IL‐1β‐induced expression of cyclooxygenase‐2 in human myometrial smooth muscle cells

Increased myometrial expression of cyclooxygenase-2 (Cox-2) at term results from elevated local levels of inflammatory cytokines, and its inhibition provides a potential route for intervention in human pre-term labor. We have identified a role for atypical protein kinase C (PKC) isozymes in IL-1beta-induced Cox-2 expression in human myometrial smooth muscle cells (HMSMC). The PKC inhibitor GF109203X (10 microM) inhibited IL-1beta-induced Cox-2 protein and RNA expression, which were also reduced by MAPK and nuclear factor kappaB (NF-kappaB) inhibitors. GF109203X did not affect MAPK activities, and neither did it replicate the effect of p38 MAPK inhibition on Cox-2 mRNA stability, suggesting that PKC operates through an independent mechanism. The effect of GF109203X remained intact after depletion of conventional and novel PKC isozymes by phorbol ester pre-treatment. In contrast LY379196 (10 microM), which at micromolar concentrations inhibits all but atypical PKCs, did not affect Cox-2 expression. A peptide corresponding to the pseudosubstrate sequence of atypical PKCs blocked Cox-2 protein expression, whereas the sequence from conventional PKCs was ineffective. GF109203X did not affect NF-kappaB binding to nuclear proteins, but strongly reduced NF-kappaB-dependent transcription in luciferase reporter assays. Our findings indicate that IL-1beta-induced Cox-2 expression in HMSMC in culture requires p38-MAPK-mediated mRNA stabilization and an independent activation of Cox-2 transcription which is dependent on the action of atypical PKCs, probably through direct stimulation of the transactivating activity of NF-kappaB.

An Initial Proteomic Analysis of Human Preterm Labor: Placental Membranes

Human preterm labor (PL) is the single most significant problem in modern Obstetrics and Gynecology, affecting approximately 10% of pregnancies worldwide, constituting the leading cause of perinatal mortality and morbidity, and contributing significantly to chronic childhood disease. Currently, our molecular understanding of PL remains staggeringly inadequate to reliably diagnose or rationally intervene in PL events; several molecular alterations have been implicated in PL, but these have proven of limited value as diagnostic/prognostic markers. The majority of PL events remain spontaneous and unpredictable: critical care emergencies. Here, we apply functional proteomics to dissect molecular mechanisms of human PL. Human placental tissue was collected in clearly differentiated cases of preterm and term labor. Highly refined two-dimensional gel electrophoresis (2DE) was used for protein separation, coupled with automated differential gel image analysis to compare the resulting proteomic maps. For this initial study, only the most important protein differences were selected for further analysis, that is, proteins that were unique to one sample, and absent from the other, with 100% reproducibility across the sample population. In total, 11 such proteins were identified by tandem mass spectrometry, falling into three distinct functional classes: structural/cytoskeletal components, ER lumenal proteins with enzymatic or chaperone functions, and proteins with anticoagulant properties. These expression changes form the groundwork for further molecular investigation of this devastating medical condition. This approach therefore holds the potential not only to define the underlying molecular components, but also to identify novel diagnostic tools and targets for rational drug intervention.

Adrenal and Thyroid Axis Function in Preterm Ventilated Baboons

Cortisol and thyroid hormones are critical to normal fetal development and neonatal transition, and baseline values and stimulation tests are abnormal after preterm birth. To evaluate cortisol and thyroxine (T₄) responses that are not influenced by uncontrolled antenatal events associated with human preterm labor, we measured cortisol and T₄ after standard-dose adrenocorticotropin (ACTH) and corticotropin-releasing hormone (CRH) stimulation tests, as well as high-dose CRH and thyrotropin-releasing hormone stimulation tests in baboons that were delivered for 3 separate protocols at 125 days of gestation (term is 186 days). The animals were surfactant treated and ventilated for up to 14 days. Some fetuses were exposed to fetal or maternal betamethasone, and some newborns were treated with 10 µg/kg T₄ for 9 days after birth. Baseline cortisol levels were in a stress range of 30–60 µg/dl by day 5. Cortisol did not increase consistently until day 11 in response to a high CRH dose or ACTH. T₄ treatment for 9 days after birth suppressed the cortisol responses and subsequent baseline T₄ levels. The hypothalamic-pituitary-adrenal (HPA) axis was unresponsive to standard dose stimulation tests until 11 days of age in preterm baboons, indicating HPA immaturity.