Juan M. Gonzalez

Complement Activation Triggers Metalloproteinases Release Inducing Cervical Remodeling and Preterm Birth in Mice

Inflammation is frequently linked to preterm delivery (PTD). Here, we tested the hypothesis that complement activation plays a role in cervical remodeling and PTD. We studied two mouse models of inflammation-induced PTD. The first model was induced by vaginal administration of lipopolysaccharide (LPS) and the second one by administration of progesterone antagonist RU486. Increased cervical C3 deposition and macrophages infiltration and increased serum C3adesArg and C5adesArg levels were observed in both models when compared to gestational age matched controls. A significant increase in collagen degradation, matrix metalloproteinase 9 (MMP-9) activity and tissue distensibility was observed in the cervix in both models. Mice deficient in complement receptor C5a did not show increased MMP-9 activity and cervical remodeling and did not deliver preterm in response to LPS or RU486, suggesting a role for C5aR in the cervical changes that precede PTD. In vitro studies show that macrophages release MMP-9 in response to C5a. Progesterone diminished the amount of C5aR on the macrophages surface, inhibited the release of MMP-9 and prevented PTD. In addition, macrophages depletion also prevented cervical remodeling and PTD in LPS-treated mice. Our studies show that C5a-C5aR interaction is required for MMP-9 release from macrophages, and the cervical remodeling that leads to PTD. Complement inhibition and supplementation with progesterone may be good therapeutic options to prevent this serious pregnancy complication.

Cervical Remodeling/Ripening at Term and Preterm Delivery: The Same Mechanism Initiated by Different Mediators and Different Effector Cells

Background Premature cervical remodeling/ripening is believed to contribute to preterm delivery (PTD), the leading cause of perinatal morbidity and mortality. Despite considerable research, the causes of term and PTD remain unclear, and there is no effective treatment for PTD. We previously demonstrated that complement activation plays a causative role in cervical remodeling that leads to PTD in mice. Methodology/Principal Findings Here we found that complement activation is not required for the physiological process that leads to term delivery in mice. Neither increased C3 cervical deposition nor increased C3a and C5a serum levels were observed at term. In addition, macrophages infiltration was found in PTD in contrast to term delivery were no leukocytes were found. Despite the different role of complement and different cellular effector cells, PTD and term delivery share a common dowsntream pathway characterized by increased metalloproteinases (MMPs) release and increased collagen degradation. However, different sources of MMPs were identified. Macrophages are the source of MMPs in PTD while cervical fibroblasts and columnar epithelial cells synthesize MMPs at term delivery. A dramatic diminution in serum progesterone levels precedes parturition at term but not in PTD, suggesting that progesterone withdrawal initiates cervical remodeling at term. On the other hand, MMPs release in PTD is triggered by C5a. Conclusion and Significance In conclusion, preterm and term cervical remodeling occur through the same mechanism but they are initiated by different mediators and effector cells. That complement activation is required for PTD but not for the physiological process that leads to term delivery, suggests that complement is a potential specific biomarker and selective target to prevent PTD and thus avert neonatal mortality and morbidity.

Comparison of the mechanisms responsible for cervical remodeling in preterm and term labor

Understanding the mechanisms of term and preterm cervical remodeling is essential to prevent prematurity. Is preterm cervical remodeling caused by the same mechanisms that cause cervical remodeling at term, and are these changes accelerated in time? This question has been pondered by obstetricians seeking strategies to prevent preterm labor for many years. Mice represent an informative model of preterm birth. Thus, in this review we discuss the recent findings from mouse models that identify and characterize the initiators and cellular effectors of cervical remodeling at term and preterm labor/delivery. These studies suggest that similarities and differences exist between term and preterm cervical remodeling. Complement is an initiator or mediator in preterm labor/delivery, but is not involved in the physiological process that leads to term delivery. Therefore, complememt constitutes a specific and selective target for potentially preventing preterm delivery, thus improving neonatal health.

Maternal and neonatal circulating visfatin concentrations in patients with pre-eclampsia and a small-for-gestational age neonate.

Objective.u2003Maternal circulating visfatin concentrations are higher in patients with a small-for-gestational-age (SGA) neonate than in those who delivered an appropriate-for-gestational age (AGA) neonate or in those with pre-eclampsia. It has been proposed that enhanced transfer of visfatin from the foetal to maternal circulation may account for the high concentrations of maternal visfatin observed in patients with an SGA neonate. The aims of this study were: (1) to determine whether cord blood visfatin concentrations differ between normal neonates, SGA neonates and newborns of pre-eclamptic mothers; and (2) to assess the relationship between maternal and foetal circulating visfatin concentrations in patients with an SGA neonate and those with pre-eclampsia. Study design.u2003This cross-sectional study included 88 pregnant women and their neonates, as well as 22 preterm neonates in the following groups: (1) 44 normal pregnant women at term and their AGA neonates; (2) 22 normotensive pregnant women and their SGA neonates; (3) 22 women with pre-eclampsia and their neonates; and (4) 22 preterm neonates delivered following spontaneous preterm labour without funisitis or histologic chorioamnionitis, matched for gestational age with infants of pre-eclamptic mothers. Maternal plasma and cord blood visfatin concentrations were determined by ELISA. Non-parametric statistics were used for analyses. Results.u2003(1) The median visfatin concentration was lower in umbilical cord blood than in maternal circulation, in normal pregnancy, SGA and pre-eclampsia groups (pu2009<u20090.001 for all comparisons); (2) the median cord blood visfatin concentrations did not differ significantly between term AGA or SGA neonates, infants of mothers with pre-eclampsia and their gestational-age-matched preterm AGA neonates; (3) maternal and cord blood visfatin concentrations correlated only in the normal term group (ru2009=u20090.48, pu2009=u20090.04). Conclusion.u2003Circulating visfatin concentrations are lower in the foetal than in the maternal circulation and did not significantly differ between the study groups. Thus, it is unlikely that the foetal circulation is the source of the high maternal visfatin concentrations reported in patients with an SGA neonate.

Complement inhibition and statins prevent fetal brain cortical abnormalities in a mouse model of preterm birth

Premature babies are particularly vulnerable to brain injury. In this study we focus on cortical brain damage associated with long-term cognitive, behavioral, attentional or socialization deficits in children born preterm. Using a mouse model of preterm birth (PTB), we demonstrated that complement component C5a contributes to fetal cortical brain injury. Disruption of cortical dendritic and axonal cytoarchitecture was observed in PTB-mice. Fetuses deficient in C5aR (-/-) did not show cortical brain damage. Treatment with antibody anti-C5, that prevents generation of C5a, also prevented cortical fetal brain injury in PTB-mice. C5a also showed a detrimental effect on fetal cortical neuron development and survival in vitro. Increased glutamate release was observed in cortical neurons in culture exposed to C5a. Blockade of C5aR prevented glutamate increase and restored neurons dendritic and axonal growth and survival. Similarly, increased glutamate levels - measured by (1)HMRS - were observed in vivo in PTB-fetuses compared to age-matched controls. The blockade of glutamate receptors prevented C5a-induced abnormal growth and increased cell death in isolated fetal cortical neurons. Simvastatin and pravastatin prevented cortical fetal brain developmental and metabolic abnormalities -in vivo and in vitro. Neuroprotective effects of statins were mediated by Akt/PKB signaling pathways. This study shows that complement activation plays a crucial role in cortical fetal brain injury in PTL and suggests that complement inhibitors and statins might be good therapeutic options to improve neonatal outcomes in preterm birth.

The molecular basis for sonographic cervical shortening at term: identification of differentially expressed genes and the epithelial-mesenchymal transition as a function of cervical length

OBJECTIVEnThe purpose of this study was to determine whether cervical shortening of a ripe cervix at term is associated with changes in the cervical transcriptome.nnnSTUDY DESIGNnSonographically measured cervical lengths and biopsy specimens were obtained from 19 women at term who were not in labor with a ripe cervix. Affymetrix HG-U133 Plus 2.0 arrays (Affymetrix Inc, Santa Clara, CA) were used. Gene expression was analyzed as a function of cervical length. Gene Ontology, pathway analyses, quantitative real-time reverse transcription-polymerase chain reaction, and immunohistochemistry were performed.nnnRESULTSnCervical length shortening was associated with differential expression of 687 genes. Fifty-four biologic processes, 22 molecular functions, and 9 pathways were enriched. Quantitative real-time reverse transcription-polymerase chain reaction analysis confirmed differential expression of 13 genes. Bone morphogenetic protein-7, claudin-1, integrin beta-6, and endometrial progesterone-induced protein messenger RNA, and protein expressions were down-regulated with cervical shortening.nnnCONCLUSIONnSonographic cervical shortening in patients at term who are not in labor with a ripe cervix is associated with changes in the uterine cervix transcriptome. The epithelial-mesenchymal transition may participate in the mechanism of cervical shortening at term.

Elephant transcriptome provides insights into the evolution of eutherian placentation.

The chorioallantoic placenta connects mother and fetus in eutherian pregnancies. In order to understand the evolution of the placenta and provide further understanding of placenta biology, we sequenced the transcriptome of a term placenta of an African elephant (Loxodonta africana) and compared these data with RNA sequence and microarray data from other eutherian placentas including human, mouse, and cow. We characterized the composition of 55,910 expressed sequence tag (i.e., cDNA) contigs using our custom annotation pipeline. A Markov algorithm was used to cluster orthologs of human, mouse, cow, and elephant placenta transcripts. We found 2,963 genes are commonly expressed in the placentas of these eutherian mammals. Gene ontology categories previously suggested to be important for placenta function (e.g., estrogen receptor signaling pathway, cell motion and migration, and adherens junctions) were significantly enriched in these eutherian placenta–expressed genes. Genes duplicated in different lineages and also specifically expressed in the placenta contribute to the great diversity observed in mammalian placenta anatomy. We identified 1,365 human lineage–specific, 1,235 mouse lineage–specific, 436 cow lineage–specific, and 904 elephant-specific placenta-expressed (PE) genes. The most enriched clusters of human-specific PE genes are signal/glycoprotein and immunoglobulin, and humans possess a deeply invasive human hemochorial placenta that comes into direct contact with maternal immune cells. Inference of phylogenetically conserved and derived transcripts demonstrates the power of comparative transcriptomics to trace placenta evolution and variation across mammals and identified candidate genes that may be important in the normal function of the human placenta, and their dysfunction may be related to human pregnancy complications.

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.

Serum and plasma determination of angiogenic and anti-angiogenic factors yield different results: the need for standardization in clinical practice

Objective.u2003The importance of an anti-angiogenic state as a mechanism of disease in preeclampsia is now recognized. Assays for the determination of concentrations of soluble vascular endothelial growth factor receptor (sVEGFR)-1, sVEGFR-2, placental growth factor (PlGF) and soluble endoglin (sEng) have been developed for research and clinical laboratories. A key question is whether these factors should be measured in plasma or serum. The purpose of this study was to determine if there are differences in the concentrations of these analytes between plasma and serum in normal pregnancy and in preeclampsia. Methods.u2003Samples of maternal blood were obtained by venipuncture and collected in EDTA (lavender top) and serum collection tubes (red top). A standard laboratory procedure was implemented for the centrifugation, aliquoting and storage of samples. Plasma and serum from 70 women with normal pregnancies and 34 patients with preeclampsia were assayed for sVEGFR-1, sVEGFR-2, PlGF and sEng by ELISA. Nonparametric paired tests were used for analyses. Results.u2003A significant difference between plasma and serum concentration was observed for sVEGFR-1 and sVEGFR-2 in normal pregnancy, and for sVEGFR-1, sVEGFR-2, PlGF and sEng in women with preeclampsia. Conclusion.u2003The concentrations of sVEGFR-1, sVEGFR-2, PlGF and sEng when measured in maternal plasma and in serum are different. Therefore, the matrix used for the assay (serum versus plasma) needs to be considered when selecting thresholds for predictive studies and interpreting the growing body of literature on this subject.

Simple targeted arterial rendering (STAR) technique: a novel and simple method to visualize the fetal cardiac outflow tracts

To describe a novel and simple technique—simple targeted arterial rendering (STAR)—to visualize the fetal cardiac outflow tracts from dataset volumes obtained with spatiotemporal image correlation (STIC) and applying a new display technology (OmniView).