Bioactive lipid mediators in plasma are predictors of preeclampsia irrespective of aspirin therapy.

There are few early biomarkers to identify pregnancies at risk of preeclampsia (PE) and abnormal placental function. In this cross-sectional study, we utilized targeted ultra-performance liquid chromatography-ESI MS/MS and a linear regression model to identify specific bioactive lipids that serve as early predictors of PE. Plasma samples were collected from 57 pregnant women prior to 24-weeks of gestation with outcomes of either PE (n = 26) or uncomplicated term pregnancies (n = 31), and the profiles of eicosanoids and sphingolipids were evaluated. Significant differences were revealed in the eicosanoid, (±)11,12 DHET, as well as multiple classes of sphingolipids; ceramides, ceramide-1-phosphate, sphingomyelin, and monohexosylceramides; all of which were associated with the subsequent development of PE regardless of aspirin therapy. Profiles of these bioactive lipids were found to vary based on self-designated race. Additional analyses demonstrated that PE patients can be stratified based on the lipid profile as to PE with a preterm birth linked to significant differences in the levels of 12-HETE, 15-HETE, and resolvin D1. Furthermore, subjects referred to a high-risk OB/GYN clinic had higher levels of 20-HETE, arachidonic acid, and Resolvin D1 versus subjects recruited from a routine, general OB/GYN clinic. Overall, this study shows that quantitative changes in plasma bioactive lipids detected by ultra-performance liquid chromatography-ESI-MS/MS can serve as an early predictor of PE and stratify pregnant people for PE type and risk.

Aspirin Inhibits the Inflammatory Response of Protease-Activated Receptor 1 in Pregnancy Neutrophils: Implications for Treating Women with Preeclampsia.

Neutrophils expressing cyclooxygenase-2 (COX-2) extensively infiltrate maternal blood vessels in preeclampsia, associated with vascular inflammation. Because pregnancy neutrophils also express protease-activated receptor 1 (PAR-1, F2R thrombin receptor), which they do not in non-pregnant subjects, they can be activated by proteases. We tested the hypothesis that aspirin at a dose sufficient to inhibit COX-2 would reduce inflammatory responses in preeclampsia neutrophils. Neutrophils were isolated from normal pregnant and preeclamptic women at approximately 30 weeks' gestation. Normal pregnancy neutrophils were treated with elastase, a protease elevated in preeclampsia, or elastase plus aspirin to inhibit COX-2, or elastase plus pinane thromboxane, a biologically active structural analog of thromboxane and a thromboxane synthase inhibitor. Preeclamptic pregnancy neutrophils were treated with the same doses of aspirin or pinane thromboxane. Confocal microscopy with immunofluorescence staining was used to determine the cellular localization of the p65 subunit of nuclear factor-kappa B (NF-κB) and media concentrations of thromboxane were measured to evaluate the inflammatory response. In untreated neutrophils of normal pregnant women, p65 was localized to the cytosol. Upon stimulation with elastase, p65 translocated from the cytosol to the nucleus coincident with increased thromboxane production. When neutrophils were co-treated with aspirin or pinane thromboxane, elastase was not able to cause nuclear translocation of p65 or increase thromboxane. In untreated neutrophils of preeclamptic women, the p65 subunit was present in the nucleus and thromboxane production was elevated, but when preeclamptic neutrophils were treated with aspirin or pinane thromboxane, p65 was cleared from the nucleus and returned to the cytosol along with decreased thromboxane production. These findings suggest that COX-2 is a downstream mediator of PAR-1 and demonstrate that PAR-1- mediated inflammation can be inhibited by aspirin. Given the extensive and ubiquitous expression of PAR-1 and COX-2 in preeclamptic women, consideration should be given to treating women with preeclampsia using a dose of aspirin sufficient to inhibit COX-2.

Gene Expression of Pregnancy Neutrophils Differs for Protease versus Lipopolysaccharide Stimulation.

Neutrophils, which extensively infiltrate maternal systemic blood vessels in preeclampsia, express protease-activated receptor 1 (PAR-1) but only during pregnancy. Neutrophils are generally considered to be non-specific in their response, but the pregnancy-specific expression of PAR-1 could result in a gene expression profile unique to pregnancy, which could help explain why the maternal inflammatory response in preeclampsia is systemic rather than localized. We sought to determine if gene expression of pregnancy neutrophils would differ if stimulated by a protease versus bacterial lipopolysaccharide (LPS). We isolated neutrophils from normal pregnant women at 30 weeks' gestation and cultured them with elastase or LPS. We used elastase because it is a protease elevated in women with preeclampsia, and it activates pregnancy neutrophils via PAR-1. RNA was isolated from the neutrophils for sequencing of the transcriptomes. We discovered many differences in the gene expression profiles. For example, exposure to elastase resulted in three times more uniquely expressed genes than LPS, and the number of significantly differentially upregulated and downregulated genes was greater for elastase. Analysis of canonical pathways revealed similarities for innate immunity but also differences. LPS treatment enriched more pathways, but elastase activated more genes in each pathway. Elastase treatment enriched the MAPK signaling pathway, whereas LPS did not. This is significant because MAPK is a key mediator of transcriptional responses. These findings indicate that protease stimulation of pregnancy neutrophils results in a different profile than stimulation with LPS, which may help explain why the sterile inflammatory response of preeclampsia is systemic and unique to pregnancy.

Pregnancy-specific expression of protease-activated receptor 1: a therapeutic target for prevention and treatment of preeclampsia?

Neutrophils extensively infiltrate maternal blood vessels in preeclampsia. This could explain why multiple organs are affected in this enigmatic disorder. Lipid peroxides produced by the placenta are probably the first factors that activate neutrophils as they circulate through the intervillous space, but then a second factor specific to pregnancy comes into play, protease-activated receptor 1. The only time neutrophils express protease-activated receptor 1 is during pregnancy. This means that neutrophils can be activated by a mechanism specific to pregnancy, that is, by proteases. Two proteases that are elevated in preeclampsia and activate protease-activated receptor 1 are matrix metalloproteinase-1 and neutrophil elastase. There is an 8-fold increase in vascular protease-activated receptor 1 expression in women with preeclampsia, and protease-activated receptor 1 is also expressed on the placenta, a pregnancy-specific tissue. The question arises if the pregnancy-specific expression of protease-activated receptor 1 is essential to the pathophysiology of preeclampsia. Protease activation of protease-activated receptor 1 in neutrophils of women with normal pregnancies causes activation of RhoA kinase. RhoA kinase phosphorylates nuclear factor-kappa B causing its translocation from the cytosol into the nucleus, increasing the expression of inflammatory genes. This signaling pathway is blocked by inhibition of either protease-activated receptor 1 or RhoA kinase activity. In contrast, neutrophils obtained from preeclamptic women are already activated, with nuclear factor-kappa B localized in the nucleus. Surprisingly, inhibition of either protease-activated receptor 1 or RhoA kinase results in an efflux of nuclear factor-kappa B from the nucleus back into the cytoplasm. Cyclooxygenase-2 seems to be a downstream mediator between protease-activated receptor 1 and RhoA kinase because aspirin inhibits the nuclear translocation of nuclear factor-kappa B and inhibits neutrophil production of superoxide, thromboxane, and tumor necrosis factor alpha. Currently, low-dose aspirin is the standard of care to prevent preeclampsia in high-risk women. Generally, the actions of low-dose aspirin are attributed to selective inhibition of maternal platelet thromboxane production. However, a recent study showed that beneficial effects extend to the placenta, where aspirin corrected the imbalance of increased thromboxane and reduced prostacyclin and oxidative stress. Selective inhibition of placental thromboxane is possible because thromboxane and prostacyclin are compartmentalized. Thromboxane is produced by trophoblast cells and prostacyclin by endothelial cells, so as aspirin crosses the placenta, its levels decline, sparing prostacyclin. Placental oxidative stress is attenuated because cyclooxygenase-2 inhibition decreases the generation of reactive oxygen species to decrease the formation of isoprostanes. The clinical manifestations of preeclampsia can be explained by protease activation of protease-activated receptor 1 in different tissues. In neutrophils, it can account for their activation and inflammatory response. In vascular tissue, protease-activated receptor 1 activation leads to enhanced vascular reactivity to angiotensin II to cause hypertension. In the placenta, it leads to oxidative stress, increased soluble fms-like tyrosine kinase, and thromboxane production. Activation of protease-activated receptor 1 on endothelial cells causes contraction, leading to edema and proteinuria, and activation on platelets leads to coagulation abnormalities. As proteases that activate protease-activated receptor 1 are elevated in the circulation of women with preeclampsia, consideration should be given to the inhibition of protease-activated receptor 1 as a treatment. Recently, The Food and Drug Administration (FDA) approved a protease-activated receptor 1 inhibitor, creating an opportunity to test whether protease-activated receptor 1 inhibition can prevent and/or treat preeclampsia, but a standard dose of aspirin might be just as effective by blocking its downstream actions.

Epigenetic Regulation of Interleukin-17-Related Genes and Their Potential Roles in Neutrophil Vascular Infiltration in Preeclampsia.

DNA methylation is an epigenetic mechanism controlling gene expression, and reduced methylation is associated with increased gene expression. We hypothesized that IL-17 cytokines are regulated by DNA methylation, are elevated in the circulation of preeclamptic women, and stimulate vascular neutrophil chemokine expression, which could account for vascular infiltration of neutrophils in preeclampsia. We found significantly reduced DNA methylation of IL17A, IL17E, and IL17F genes in omental arteries of preeclamptic women, significantly reduced methylation of IL2, which regulates IL-17-producing T-lymphocytes, and significantly reduced methylation of genes encoding neutrophil chemokines and TNFα receptors related to lymphocyte function. Maternal plasma levels of IL-17A were significantly elevated in the second trimester of preeclamptic pregnancy as compared to normal pregnancy. To test if methylation regulates IL-17 cytokines, a lymphocyte cell line (Jurkat) was cultured with a hypomethylating agent. Hypomethylation increased expression of IL17E (aka IL25), IL17F, and IL2. IL17A was not expressed by Jurkat cells. To test the potential role of IL-17 cytokines in vascular neutrophil infiltration associated with preeclampsia, human vascular smooth muscle cells were cultured with IL-17 cytokines. IL-17A, but not IL-17E or IL-17F, increased gene expression of neutrophil chemokines (IL-8, CXCL5, and CXCL6) that are increased in vascular smooth muscle of preeclamptic women. The monocyte chemokine, CCL-2, was not increased. TNFα also increased neutrophil chemokines. IL-17 cytokines are regulated by DNA methylation; IL-17A is elevated in preeclampsia and stimulates expression of neutrophil chemokines in vascular smooth muscle. IL-17A could be responsible for vascular infiltration of neutrophils in preeclampsia.

Patterns of Maternal Neutrophil Gene Expression at 30 Weeks of Gestation, but Not DNA Methylation, Distinguish Mild from Severe Preeclampsia.

Neutrophils are activated and extensively infiltrate blood vessels in preeclamptic women. To identify genes that contribute to neutrophil activation and infiltration, we analyzed the transcriptomes of circulating neutrophils from normal pregnant and preeclamptic women. Neutrophils were collected at 30 weeks' gestation and RNA and DNA were isolated for RNA sequencing and 5-hydroxy-methylcytosine (5-hmC) sequencing as an index of dynamic changes in neutrophil DNA methylation. Women with normal pregnancy who went on to develop mild preeclampsia at term had the most uniquely expressed genes (697) with 325 gene ontology pathways upregulated, many related to neutrophil activation and function. Women with severe preeclampsia who delivered prematurely had few pathways up- or downregulated. Cluster analysis revealed that gene expression in women with severe preeclampsia was an inverse mirror image of gene expression in normal pregnancy, while gene expression in women who developed mild preeclampsia was remarkably different from both. DNA methylation marks, key regulators of gene expression, are removed by the action of ten-eleven translocation (TET) enzymes, which oxidize 5-methylcytosines (5mCs), resulting in locus-specific reversal of DNA methylation. DNA sequencing for 5-hmC revealed no differences among the three groups. Genome-wide DNA methylation revealed extremely low levels in circulating neutrophils suggesting they are de-methylated. Collectively, these data demonstrate that neutrophil gene expression profiles can distinguish different preeclampsia phenotypes, and in the case of mild preeclampsia, alterations in gene expression occur well before clinical symptoms emerge. These findings serve as a foundation for further evaluation of neutrophil transcriptomes as biomarkers of preeclampsia phenotypes. Changes in DNA methylation in circulating neutrophils do not appear to mediate differential patterns of gene expression in either mild or severe preeclampsia.

The Road to Low-Dose Aspirin Therapy for the Prevention of Preeclampsia Began with the Placenta.

The road to low-dose aspirin therapy for the prevention of preeclampsia began in the 1980s with the discovery that there was increased thromboxane and decreased prostacyclin production in placentas of preeclamptic women. At the time, low-dose aspirin therapy was being used to prevent recurrent myocardial infarction and other thrombotic events based on its ability to selectively inhibit thromboxane synthesis without affecting prostacyclin synthesis. With the discovery that thromboxane was increased in preeclamptic women, it was reasonable to evaluate whether low-dose aspirin would be effective for preeclampsia prevention. The first clinical trials were very promising, but then two large multi-center trials dampened enthusiasm until meta-analysis studies showed aspirin was effective, but with caveats. Low-dose aspirin was most effective when started <16 weeks of gestation and at doses >100 mg/day. It was effective in reducing preterm preeclampsia, but not term preeclampsia, and patient compliance and patient weight were important variables. Despite the effectiveness of low-dose aspirin therapy in correcting the placental imbalance between thromboxane and prostacyclin and reducing oxidative stress, some aspirin-treated women still develop preeclampsia. Alterations in placental sphingolipids and hydroxyeicosatetraenoic acids not affected by aspirin, but with biologic actions that could cause preeclampsia, may explain treatment failures. Consideration should be given to aspirin's effect on neutrophils and pregnancy-specific expression of protease-activated receptor 1, as well as additional mechanisms of action to prevent preeclampsia.

Placental Production of Eicosanoids and Sphingolipids in Women Who Developed Preeclampsia on Low-Dose Aspirin.

Low-dose aspirin, which selectively inhibits thromboxane synthesis, is now standard of care for the prevention of preeclampsia in at risk women, but some women still develop preeclampsia despite an aspirin regimen. To explore the "aspirin failures," we undertook a comprehensive evaluation of placental lipids to determine if abnormalities in non-aspirin sensitive lipids might help explain why some women on low-dose aspirin develop preeclampsia. We studied placentas from women with normal pregnancies and women with preeclampsia. Placental villous explants were cultured and media analyzed by mass spectrometry for aspirin-sensitive and non-aspirin-sensitive lipids. In women who developed severe preeclampsia and delivered preterm, there were significant elevations in non-aspirin-sensitive lipids with biologic actions that could cause preeclampsia. There were significant increases in 15- and 20-hydroxyeicosatetraenoic acids and sphingolipids: D-e-C18:0 ceramide, D-e-C18:0 sphingomyelin, D-e-sphingosine-1-phosphate, and D-e-sphinganine-1-phosphate. With regard to lipids sensitive to aspirin, there was no difference in placental production of thromboxane or prostacyclin, but prostaglandins were lower. There was no difference for isoprostanes, but surprisingly, anti-inflammatory omega 3 and 6 PUFAs were increased. In total, 10 of 30 eicosanoids and 5 of 42 sphingolipids were abnormal in women with severe early onset preeclampsia. Lipid changes in women with mild preeclampsia who delivered at term were of lesser magnitude with few significant differences. The placenta produces many aspirin-sensitive and non-aspirin-sensitive lipids. Abnormalities in eicosanoids and sphingolipids not sensitive to aspirin might explain why some aspirin-treated women develop preeclampsia.

Proteases Activate Pregnancy Neutrophils by a Protease-Activated Receptor 1 Pathway: Epigenetic Implications for Preeclampsia.

We tested a novel hypothesis that elevated levels of proteases in the maternal circulation of preeclamptic women activate neutrophils due to their pregnancy-specific expression of protease-activated receptor 1 (PAR-1). Plasma was collected longitudinally from normal pregnant and preeclamptic women and analyzed for MMP-1 and neutrophil elastase. Neutrophils were isolated for culture and confocal microscopy. Omental fat was collected for immunohistochemistry. Circulating proteases were significantly elevated in preeclampsia. Confocal microscopy revealed that tet methylcytosine dioxygenase 2 (TET2), a DNA de-methylase, and p65 subunit of NF-κB were strongly localized to the nucleus of untreated neutrophils of preeclamptic women, but in untreated neutrophils of normal pregnant women they were restricted to the cytosol. Treatment of normal pregnancy neutrophils with proteases activated PAR-1, leading to activation of RhoA kinase (ROCK), which triggered translocation of TET2 and p65 from the cytosol into the nucleus, mimicking the nuclear localization in neutrophils of preeclamptic women. IL-8, an NF-κB-regulated gene, increased in association with TET2 and p65 nuclear localization. Co-treatment with inhibitors of PAR-1 or ROCK prevented nuclear translocation and IL-8 did not increase. Treatment of preeclamptic pregnancy neutrophils with inhibitors emptied the nucleus of TET2 and p65, mimicking the cytosolic localization of normal pregnancy neutrophils. Expression of PAR-1 and TET2 were markedly increased in omental fat vessels and neutrophils of preeclamptic women. We conclude that elevated levels of circulating proteases in preeclamptic women activate neutrophils due to their pregnancy-specific expression of PAR-1 and speculate that TET2 DNA de-methylation plays a role in the inflammatory response.

Identification of a Cytopathogenic Toxin from Sneathia amnii.

Sneathia amnii is a poorly characterized emerging pathogen that has been implicated in amnionitis and urethritis. We found that S. amnii damages fetal membranes, and we identified and purified a cytotoxic exotoxin that lyses human red blood cells and damages cells from fetal membranes. The gene appears to be cotranscribed with a second gene that encodes a protein with identity to two-partner system transporters, suggesting that it is the "A," or secreted component of a type Vb system. The toxin is 1,881 amino acids with a molecular weight of approximately 200 kDa. It binds to red blood cell membranes and forms pores with a diameter of 2.0 to 3.0 nm, resulting in osmolysis. Because it appears to be the "A" or passenger component of a two-partner system, we propose to name this novel cytotoxin/hemolysin CptA for cytopathogenic toxin component A.IMPORTANCESneathia amnii is a very poorly characterized emerging pathogen that can affect pregnancy outcome and cause urethritis and other infections. To date, nothing is known about its virulence factors or pathogenesis. We have identified and isolated a cytotoxin, named CptA for cytopathogenic toxin, component A, that is produced by S. amnii CptA is capable of permeabilizing chorionic trophoblasts and lysing human red blood cells and, thus, may play a role in virulence. Except for small domains conserved among two-partner secretion system passenger proteins, the cytotoxin exhibits little amino acid sequence homology to known toxins. In this study, we demonstrate the pore-forming activity of this novel toxin.

Protease Amplification of the Inflammatory Response Induced by Commensal Bacteria: Implications for Racial Disparity in Term and Preterm Birth.

Decidual macrophages secrete proteases that activate protease-activated receptor 1 (PAR-1). We hypothesized that activation of the inflammatory response by bacteria is amplified by proteases, initiating labor. In addition, we hypothesized that commensal bacteria trigger an inflammatory response by activating NF-κB and TET methylcytosine dioxygenase 2 (TET2), a DNA de-methylase, via a protease amplified PAR-1, RhoA kinase (ROCK) pathway. To evaluate these hypotheses, we compared responses of mononuclear cells with Lactobacillus crispatus, prevalent in the vaginal microbiome of women of European ancestry, with L. iners and Fusobacterium nucleatum, which are more prevalent in vaginal samples collected from African-American women. Decidual tissue was collected at term not-in-labor (TNL), term labor (TL), spontaneous preterm labor (sPTL), and infected preterm labor (iPTL) and immunostained for PAR-1, TET2, and CD14. Mononuclear cells and THP-1 macrophage cells were treated with bacteria and elastase, a known activator of PAR-1. The inflammatory response was monitored by confocal microscopy of TET2 and the p65 subunit of NF-κB, as well as IL-8 production. Decidual staining for PAR-1, TET2, and CD14 increased TNL < TL < sPTL < iPTL. All treatments stimulated translocation of TET2 and p65 from the cytosol to the nucleus and increased IL-8, but L. iners and F. nucleatum caused more robust responses than L. crispatus. Inhibition of PAR-1 or ROCK prevented TET2 and p65 nuclear translocalization and increases in IL-8. Our findings demonstrate that proteases amplify the inflammatory response to commensal bacteria. The more robust response to bacteria prevalent in African-American women may contribute to racial disparities in preterm birth.

A20 Orchestrates Inflammatory Response in the Oral Mucosa through Restraining NF-κB Activity.

Deregulated immune response to a dysbiotic resident microflora within the oral cavity leads to chronic periodontal disease, local tissue destruction, and various systemic complications. To preserve tissue homeostasis, inflammatory signaling pathways involved in the progression of periodontitis must be tightly regulated. A20 (TNFAIP3), a ubiquitin-editing enzyme, has emerged as one of the key regulators of inflammation. Yet, the function of A20 in the oral mucosa and the biological pathways in which A20 mitigates periodontal inflammation remain elusive. Using a combination of in vivo and ex vivo disease models, we report in this study that A20 regulates inflammatory responses to a keystone oral bacterium, Porphyromonas gingivalis, and restrains periodontal inflammation through its effect on NF-κB signaling and cytokine production. Depletion of A20 using gene editing in human macrophage-like cells (THP-1) significantly increased cytokine secretion, whereas A20 overexpression using lentivirus infection dampened the cytokine production following bacterial challenge through modulating NF-κB activity. Similar to human cells, bone marrow-derived macrophages from A20-deficient mice infected with P. gingivalis displayed increased NF-κB activity and cytokine production compared with the cells isolated from A20-competent mice. Subsequent experiments using a murine ligature-induced periodontitis model showed that even a partial loss of A20 promotes an increased inflammatory phenotype and more severe bone loss, further verifying the critical function of A20 in the oral mucosa. Collectively, to our knowledge, these findings reveal the first systematic evidence of a physiological role for A20 in the maintenance of oral tissue homeostasis as a negative regulator of inflammation.

Matrix Metalloprotease-1 and Elastase Are Novel Uterotonic Agents Acting Through Protease-Activated Receptor 1.

Matrix metalloproteinase-1 (MMP-1) and neutrophil elastase are proteolytic enzymes involved in tissue remodeling, but a role for them as uterotonic agents has not been considered. However, both these proteases activate protease-activated receptor 1 (PAR-1) that mediates thrombin-induced contractions. Matrix metalloproteinase-1 and elastase are products of neutrophils that infiltrate intrauterine tissues at the time of labor, so we tested the hypothesis that these proteases might be novel uterotonic agents acting via PAR-1. Decidual tissue was collected from fetal membranes of term not-in-labor (TNL), term labor (TL), and preterm labor (PTL) women and analyzed for gene and protein expression of MMP-1 and neutrophil elastase. Contractile effects of MMP-1 and elastase were tested with uterine strips of day 19 and 20 gestation rats. Expression of MMP-1 and neutrophil elastase was increased in TL and PTL as compared to TNL. Expression of both the pro- and active enzymes of MMP-1 increased progressively from TNL to TL to PTL. Tumor necrosis factor-α, a neutrophil product, increased MMP-1 in decidual and myometrial cells. Both MMP-1 and elastase stimulated strong contractions of myometrial strips, which were prevented by inhibition of PAR-1 and inhibition of inositol trisphosphate receptor or calcium channel blockade. Indomethacin did not prevent protease-induced contractions. These data suggest that MMP-1 and neutrophil elastase may be important but heretofore unrecognized players in stimulating uterine contractions at the time of labor, and they may explain why indomethacin delays, but does not prevent, PTL because indomethacin inhibits the prostaglandin component but not the protease component of labor.

Increased expression of toll-like receptors 2 and 9 is associated with reduced DNA methylation in spontaneous preterm labor.

The cause of spontaneous preterm labor (sPTL) is not known, but it could be due to epigenetic alterations that increase the sensitivity of decidual tissue to inflammatory stimuli. We collected decidual tissue from women at term not in labor (TNL), women at term in labor (TL), and women with sPTL. Illumina Infinium HumanMethylation450 BeadChip analysis revealed significantly reduced DNA methylation for TLR-2 and TLR-9 in sPTL as compared to TL. Immunohistochemical staining documented significantly increased expression of TLR-2 and TLR-9 in decidual tissue of women with sPTL as compared to TL or TNL. TLR expression was not present in decidual cells, but localized to tissue leukocytes as revealed by staining for CD14, a macrophage antigen, and neutrophil elastase. Microarray analysis of inflammatory genes to assess innate immune response demonstrated marked increases in expression of inflammatory cytokines and chemokines in women with TL as compared to TNL. However, when sPTL was compared to TL, there was a further increase in inflammatory cytokines, and a remarkable increase in neutrophil chemokines. These results suggest that epigenetic mechanisms could play a role in increasing leukocyte infiltration, and increasing the sensitivity of decidual tissue to inflammatory stimuli that could precipitate labor prematurely.

Expression patterns of the chromosome 21 MicroRNA cluster (miR-99a, miR-125b and let-7c) in chorioamniotic membranes.

Trisomy 21 (T21) is the most common chromosome abnormality in humans and is associated with a spectrum of phenotypes, including cognitive impairment, congenital heart defects and immune system defects. In addition, T21 is also associated with abnormalities of fetal membranes including chorioamniotic separation, delayed fusion of the chorioamniotic membranes, defects in syncytiotrophoblast formation, as well as amniocyte senescence. There is evidence indicating miRNAs encoded by sequences on chromosome 21 (Chr-21) are involved in several of the cognitive and neurological phenotypes of T21, but the role of Chr-21 derived miRNAs in fetal membrane abnormalities associated with T21 has not been investigated. In the current study, we determined the expression patterns of three miRNAs derived from a cluster on Chr-21 - hsa-miR-99a, hsa-miR-125b and hsa-let-7c in chorioamniotic membranes obtained from term pregnancies with spontaneous rupture (n = 20). Tissue and location specific expression patterns within the chorioamniotic membranes were identified. The rupture zone in the choriodecidua had distinct expression patterns compared to other fetal membrane locations. Despite the increased gene dosage associated with T21, the expression of all three miRNAs was reduced in cultured T21 amniocytes as compared to cultured euploid amniocytes. In silico analysis of experimentally validated targets of the three miRNAs suggest these Chr-21 derived miRNAs play a potential role in fetal membrane rupture and the fetal membrane defects associated with T21.

Matrix Metalloproteinase 1 Causes Vasoconstriction and Enhances Vessel Reactivity to Angiotensin II via Protease-Activated Receptor 1.

Matrix metalloproteinase 1 (MMP-1) is an activator of protease-activated receptor 1 (PAR-1), which is known to mediate the release of endothelin 1 (ET-1) in endothelial cells and activate the RhoA kinase (ROCK) pathway. Recently, we reported increased serum and vascular MMP-1 in women with preeclampsia and hypothesized that the action of MMP-1 on PAR-1 might have vasoconstrictive effects. Resistance-sized omental arteries obtained from normal pregnant women were mounted on a myograph system and perfused with MMP-1 in a dose range of 0.025 to 25 ng/mL or with angiotensin II (Ang II) in a dose range of 0.001 to 10 µmol/L in the presence of intraluminal MMP-1 (2.5 ng/mL) perfusion. Angiotensin II dose response was also performed with omental arteries from women with preeclampsia. Matrix metalloproteinase 1 caused dose-dependent vasoconstriction in endothelium-intact, but not in endothelium-denuded, vessels from normal pregnant women, which was blocked by inhibitors of PAR-1 and ET-1 type A receptor blocker. Intraluminal perfusion with a constant amount of MMP-1 enhanced vessel reactivity to Ang II, which was blocked by inhibitors of PAR-1, ROCK, and ET-1. Enhanced vascular reactivity to Ang II was observed in endothelium-intact, but not in endothelium-denuded, arteries of women with preeclampsia. Inhibitors of PAR-1, ROCK, and ET-1 blocked enhanced vascular reactivity to Ang II in endothelium-intact preeclamptic arteries. These data demonstrate that MMP-1 has potent vasoconstrictor effects and the ability to enhance vascular reactivity to vasoconstrictor hormones, which are mediated by an endothelial PAR-1, ROCK, and ET-1 pathway. Increased circulating levels of MMP-1 and its increased expression in systemic vessels of women with preeclampsia may contribute to the development of maternal hypertension.

Neutrophil release of myeloperoxidase in systemic vasculature of obese women may put them at risk for preeclampsia.

Obesity is a risk factor for preeclampsia, but the reason for this risk is unknown. Neutrophils infiltrate into systemic blood vessels of both obese and preeclamptic women. Neutrophils are a major source of myeloperoxidase (MPO), which is associated with hypertension. We tested the hypothesis that systemic vasculature of both obese and preeclamptic women will have a significant presence of MPO as a result of neutrophil infiltration. We found that immunohistochemical staining of MPO was significantly greater in subcutaneous fat blood vessels of obese women than overweight women, which was significantly greater than normal weight women. Expression of MPO was significantly greater in maternal blood vessels of preeclamptic women than normal pregnant or normal nonpregnant women. In general, when vessels of overweight or normal pregnant women were stained it was primarily for leukocytes in the lumen and not infiltrated into the vessel. In contrast, in obese and preeclamptic women staining was present for leukocytes in the lumen, flattened, and adhered to the endothelium and infiltrated into the vessel wall. There was also extensive diffuse staining for MPO in vessels of obese and preeclamptic women. In conclusion, both obese and preeclamptic women have increased presence of MPO in systemic vasculature as a result of neutrophil infiltration. We speculate that obese women may be at risk of preeclampsia because their vasculature is already prone to hypertension.

Intracellular two-phase Ca2+ release and apoptosis controlled by TRP-ML1 channel activity in coronary arterial myocytes.

Activation of the death receptor Fas has been reported to produce a two-phase intracellular Ca(2+) release response in coronary arterial myocytes (CAMs), which consists of local Ca(2+) bursts via lysosomal transient potential receptor-mucolipin 1 (TRP-ML1) channels and consequent Ca(2+) release from the sarcoplasmic reticulum (SR). The present study was designed to explore the molecular mechanism by which lysosomal Ca(2+) bursts are coupled with SR Ca(2+) release in mouse CAMs and to determine the functional relevance of this lysosome-associated two-phase Ca(2+) release to apoptosis, a common action of Fas activation with Fas ligand (FasL). By confocal microscopy, we found that transfection of CAMs with TRP-ML1 small interfering (si)RNA substantially inhibited FasL (10 ng/ml)-induced lysosome Ca(2+) bursts and consequent SR Ca(2+) release. In contrast, transfection of CAMs with plasmids containing a full-length TRP-ML1 gene enhanced FasL-induced two-phase Ca(2+) release. We further demonstrated that FasL significantly increased the colocalization of the lysosomal marker Lamp1 with ryanodine receptor 3 and enhanced a dynamic trafficking of lysosomes to the SR. When CAMs were treated with TRP-ML1 siRNA, FasL-induced interactions between the lysosomes and SR were substantially blocked. Functionally, FasL-induced apoptosis and activation of calpain and calcineurin, the Ca(2+) sensitive proteins that mediate apoptosis, were significantly attenuated by silencing TRP-ML1 gene but enhanced by overexpression of TRP-ML1 gene. These results suggest that TRP-ML1 channel-mediated lysosomal Ca(2+) bursts upon FasL stimulation promote lysosome trafficking and interactions with the SR, leading to apoptosis of CAMs via a Ca(2+)-dependent mechanism.

DNA methylation is altered in maternal blood vessels of women with preeclampsia.

We analyzed 27,578 CpG sites that map to 14,495 genes in omental arteries of normal pregnant and preeclamptic women for DNA methylation status using the Illumina platform. We found 1685 genes with a significant difference in DNA methylation at a false discovery rate of <10% with many inflammatory genes having reduced methylation. Unsupervised hierarchical clustering revealed natural clustering by diagnosis and methylation status. Of the genes with significant methylation differences, 236 were significant at a false discovery rate of <5%. When data were analyzed more stringently to a false discovery rate of <5% and difference in methylation of >0.10, 65 genes were identified, all of which showed reduced methylation in preeclampsia. When these genes were mapped to gene ontology for molecular functions and biological processes, 75 molecular functions and 149 biological processes were overrepresented in the preeclamptic vessels. These included smooth muscle contraction, thrombosis, inflammation, redox homeostasis, sugar metabolism, and amino acid metabolism. We speculate that reduced methylation may contribute to the pathogenesis of preeclampsia and that alterations in DNA methylation resulting from preeclampsia may increase maternal risk of cardiovascular disease later in life.

Preeclampsia is associated with alterations in DNA methylation of genes involved in collagen metabolism.

Maternal vascular dysfunction is a hallmark of preeclampsia. A recently described vascular phenotype of preeclampsia involves increased expression of matrix metalloproteinase-1 (MMP-1) in endothelial cells, vascular smooth muscle, and infiltrating neutrophils. In contrast, the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) and collagen type Iα 1 is either reduced or not changed in the vessels, suggesting an imbalance in vessel collagen degradation and synthesis in preeclampsia. In the present study, we explored the possible contribution of DNA methylation to the altered expression of genes involved in collagen metabolism. We assayed the differences in DNA methylation in omental arteries from normal pregnant and preeclamptic women, and determined whether reduced DNA methylation increases the expression of MMP-1 in cultured vascular smooth muscle cells and a neutrophil-like cell line, HL-60. Several MMP genes, including MMP1 and MMP8, were significantly less methylated in preeclamptic omental arteries, whereas TIMP and COL genes either were significantly more methylated or had no significant change in their DNA methylation status compared with normal pregnancy. Experimentally induced DNA hypomethylation increased MMP-1 expression in cultured vascular smooth muscle cells and MMP-1 cells. Our findings suggest that epigenetic regulation contributes to the imbalance in genes involved in collagen metabolism in blood vessels of preeclamptic women.