The Dysregulated IL-23/TH17 Axis in Endometriosis Pathophysiology.

Endometriosis is a chronic inflammatory disease in which endometrial-like tissue grows ectopically, resulting in pelvic pain and infertility. IL-23 is a key contributor in the development and differentiation of TH17 cells, driving TH17 cells toward a pathogenic profile. In a variety of inflammatory and autoimmune disorders, TH17 cells secrete proinflammatory cytokines, including IL-17, contributing to disease pathophysiology. Our studies and others have implicated IL-17 and TH17 cell dysregulation in endometriosis, which is associated with disease severity. In this article, we address whether IL-23-driven TH17 cells contribute to cardinal features of lesion proliferation, vascularization, and inflammation in endometriosis using patient samples, representative cell lines, and our established mouse model of endometriosis. The results indicated dysregulated expression of key genes in the IL-23/TH17 axis in patient ectopic and eutopic endometrial samples and increased IL-23 protein in patient plasma compared with controls. In vitro studies using primary human TH cells determined that rIL-23 mixture treatment increased pathogenic TH17 cell frequency. Similarly, rIL-23 treatment of cell lines (12Z cells, EECCs, HUVECs, and hESCs) representative of the endometriotic lesion microenvironment increased cytokines and growth factors, which play a role in lesion establishment and maintenance. In a syngeneic mouse model of endometriosis, rIL-23 treatment altered numbers of myeloid and T cell subsets in peritoneal fluid and increased giant cells within the lesion. Lesions from rIL-23-treated mice did not reveal significant alterations in proliferation/vascularization, although trends of increased proliferation and vascularization were observed. Collectively, these findings provide insights into the impact of the IL-23/TH17 axis on local immune dysfunction and broadly on endometriosis pathophysiology.

Neutrophil recruitment and function in endometriosis patients and a syngeneic murine model.

Endometriosis is a chronic inflammatory, gynecological disease characterized by the presence of endometrial-like tissue lesions outside of the uterus. Neutrophils are elevated in the systemic circulation and peritoneal fluid of endometriosis patients; however, whether and how neutrophils contribute to endometriosis pathophysiology remain poorly understood. With emerging roles for neutrophils in chronic and sterile inflammatory conditions, we sought to provide in-depth characterization of neutrophil involvement in endometriosis. We demonstrate that neutrophils reside within patient endometriotic lesions and that patient lesions possess a microenvironment that may influence neutrophil recruitment and function. We also provide the first evidence that systemic circulating neutrophils from endometriosis patients display distinct transcriptomic differences compared neutrophils from healthy control subjects. Time course characterization of our syngeneic, immunocompetent mouse model of endometriosis revealed that neutrophils are rapidly recruited to the peritoneal environment early after endometriotic lesion establishment and remain present in murine lesions long term. In vivo neutrophil depletion altered the systemic and peritoneal immune microenvironment of mice with endometriosis as demonstrated by changes in pro-inflammatory and angiogenic mediators. Taken together, these findings highlight a novel role for neutrophils in early events such as angiogenesis and modulation of the local inflammatory environment associated with endometriosis pathogenesis.

An epigenetic increase in mitochondrial fission by MiD49 and MiD51 regulates the cell cycle in cancer: Diagnostic and therapeutic implications.

Excessive proliferation and apoptosis-resistance are hallmarks of cancer. Increased dynamin-related protein 1 (Drp1)-mediated mitochondrial fission is one of the mediators of this phenotype. Mitochondrial fission that accompanies the nuclear division is called mitotic fission and occurs when activated Drp1 binds partner proteins on the outer mitochondrial membrane. We examine the role of Drp1-binding partners, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51), as drivers of cell proliferation and apoptosis-resistance in non-small cell lung cancer (NSCLC) and invasive breast carcinoma (IBC). We also evaluate whether inhibiting MiDs can be therapeutically exploited to regress cancer. We show that MiD levels are pathologically elevated in NSCLC and IBC by an epigenetic mechanism (decreased microRNA-34a-3p expression). MiDs silencing causes cell cycle arrest through (a) increased expression of cell cycle inhibitors, p27Kip1 and p21Waf1 , (b) inhibition of Drp1, and (c) inhibition of the Akt-mTOR-p70S6K pathway. Silencing MiDs leads to mitochondrial fusion, cell cycle arrest, increased apoptosis, and tumor regression in a xenotransplant NSCLC model. There are positive correlations between MiD expression and tumor size and grade in breast cancer patients and inverse correlations with survival in NSCLC patients. The microRNA-34a-3p-MiDs axis is important to cancer pathogenesis and constitutes a new therapeutic target.

Carbon monoxide increases utero-placental angiogenesis without impacting pregnancy specific adaptations in mice.

BACKGROUND: Cigarette smokers have a reduced risk of developing preeclampsia, possibly attributed to an increase in carbon monoxide (CO) levels. Carbon monoxide is a gasotransmitter that has been implicated in maintaining vascular tone, increasing angiogenesis, and reducing inflammation and apoptosis at physiological concentrations. Moderately increasing CO concentrations may have therapeutic potential to prevent or treat preeclampsia; however, the effects of CO on pregnancy are under studied. Our objective was to investigate the effect of CO on major angiogenic and inflammatory markers in pregnancy, and to evaluate the effect of CO on indicators of placental health. FINDINGS: Pregnant CD-1 mice were constantly exposed to either ambient air or 250 ppm CO from conception until gestation day (GD)10.5 or GD16.5. Using a qRT-PCR array, we identified that CO increased expression of major angiogenic genes at the implantation site on GD10.5, but not GD16.5. Pro-inflammatory cytokines in the plasma and tissue lysates from implantation sites in treated mice were not significantly different compared to controls. Additionally, CO did not alter the implantation site phenotype, in terms of proliferative capacity, invasiveness of trophoblasts, or abundance of uterine natural killer cells. CONCLUSIONS: This study suggests that CO exposure is pro-angiogenic at the maternal-fetal interface, and is not associated with demonstrable concerns during murine pregnancy. Future studies are required to validate safety and efficacy of CO as a potential therapeutic for vascular insufficiency diseases such as preeclampsia and intrauterine growth restriction.

Effects of placental growth factor deficiency on behavior, neuroanatomy, and cerebrovasculature of mice.

Preeclampsia, a hypertensive syndrome occurring in 3-5% of human pregnancies, has lifelong health consequences for fetuses. Cognitive ability throughout life is altered, and adult stroke risk is increased. One potential etiological factor for altered brain development is low concentrations of proangiogenic placental growth factor (PGF). Impaired PGF production may promote an antiangiogenic fetal environment during neural and cerebrovascular development. We previously reported delayed vascularization of the hindbrain, altered retinal vascular organization, and less connectivity in the circle of Willis in Pgf-/- mice. We hypothesized Pgf-/- mice would have impaired cognition and altered brain neuroanatomy in addition to compromised cerebrovasculature. Cognitive behavior was assessed in adult Pgf-/- and Pgf+/+ mice by four paradigms followed by postmortem high-resolution MRI of neuroanatomy. X-ray microcomputed tomography imaging investigated the three-dimensional cerebrovascular geometry in another cohort. Pgf-/- mice exhibited poorer spatial memory, less depressive-like behavior, and superior recognition of novel objects. Significantly smaller volumes of 10 structures were detected in the Pgf-/- compared with Pgf+/+ brain. Pgf-/- brain had more total blood vessel segments in the small-diameter range. Lack of PGF altered cognitive functions, brain neuroanatomy, and cerebrovasculature in mice. Pgf-/- mice may be a preclinical model for the offspring effects of low-PGF preeclampsia gestation.

RNA-Binding Proteins in Female Reproductive Pathologies.

RNA-binding proteins are key regulatory molecules involved primarily in post-transcriptional gene regulation of RNAs. Post-transcriptional gene regulation is critical for adequate cellular growth and survival. Recent reports have shown key interactions between these RNA-binding proteins and other regulatory elements, such as miRNAs and long noncoding RNAs, either enhancing or diminishing their response to RNA stabilization. Many RNA-binding proteins have been reported to play a functional role in mediation of cytokines involved in inflammation and immune dysfunction, and some have been classified as global post-transcriptional regulators of inflammation. The ubiquitous expression of RNA-binding proteins in a wide variety of cell types and their unique mechanisms of degradative action provide evidence that they are involved in reproductive tract pathologies. Aberrant inflammation and immune dysfunction are major contributors to the pathogenesis and disease pathophysiology of many reproductive pathologies, including ovarian and endometrial cancers in the female reproductive tract. Herein, we discuss various RNA-binding proteins and their unique contributions to female reproductive pathologies with a focus on those mediated by aberrant inflammation and immune dysfunction.

Developmental origins of pregnancy-induced cardiac changes: establishment of a novel model using the atrial natriuretic peptide gene-disrupted mice.

Pregnancy evokes many challenges on the maternal cardiovascular system that may unmask predispositions for future disease. This is particularly evident for women who develop pregnancy-related disorders, for example, pre-eclampsia and gestational diabetes or hypertension. Such pregnancy-related syndromes increase the risk for cardiovascular disease (CVD) postpartum. As a result, pregnancy has been termed as a cardiovascular stress test and an indicator or marker to predict the development of CVD later in life. In addition, pregnancy-related disorders impact the development of offspring also placing them at a higher risk for disease. Utilizing pregnancy as a physiological stressor, the current investigation sought to determine whether the cardiovascular system of offspring exposed to gestational hypertension in utero would respond adversely to the stress of pregnancy. Heterozygous atrial natriuretic peptide gene-disrupted (ANP+/-) offspring were generated by either crossing male wildtype ANP+/+ with female knockout ANP-/- to produce ANP+/-KO mice or crossing female wildtype ANP+/+ with male knockout ANP-/- to produce ANP+/-WT mice. To study the cardiovascular stress induced by pregnancy, female ANP+/-WT and ANP+/-KO mice were mated with male wildtype ANP+/+ mice to initiate pregnancy. Cardiac size and molecular expression of the renin-angiotensin (RAS) and natriuretic peptide systems (NPS) were compared between offspring groups. Our data demonstrate that gestational hypertension and lack of maternal ANP did not significantly impact the progression and regression of pregnancy-induced cardiac hypertrophy over gestation and postpartum in ANP+/- offspring. Additionally, the molecular cardiac expression of the RAS and NPS did not differ between offspring groups. Future investigation should assess potential differences in cardiac function and the impact of fetal-programming on offspring cardiovascular adaptations during pregnancy in more severe models of pregnancy-related hypertensive syndrome such as angiotensin II or isoproterenol infusion.

Influences of placental growth factor on mouse retinal vascular development.

BACKGROUND: Placental growth factor (PGF) is important for wound-healing and vascular collaterogenesis. PGF deficiency is associated with preeclampsia, a hypertensive disease of human pregnancy. Offspring born to preeclamptic mothers display cognitive impairments and brain vascular and neurostructural deviations. Low PGF production during development may contribute to alterations in offspring cerebrovascular beds. Retina is a readily accessible part of the central nervous system with a well-described pattern of vascular development in mice. Impacts of PGF deficiency were addressed during mouse retinal vascularization. RESULTS: Retinal vessels were compared between Pgf-/- and congenic C57BL/6 (B6) mice. PGF deficiency altered neonatal retinal vascularization patterns. Some anatomic alterations persisted into adulthood, particularly in males. Greater arterial wall collagen IV expression was found in adult Pgf-/- females. Pregnancy (studied in adult females at gestational days 11.5 or 18.5) induced subtle changes upon the mother's retinal vasculature but these pregnancy-induced changes did not differ between genotypes. Significant sex-related differences occurred between adult male and female B6 although sexually dimorphic retinal vascular differences were absent in B6 neonates. CONCLUSIONS: Overall, PGF has a role in retinal vascular angiogenesis and vessel organization during development but does not affect retinal vessel adaptations in adult females during pregnancy. Developmental Dynamics 246:700-712, 2017. © 2017 Wiley Periodicals, Inc.

Pregnancy and spontaneous fetal loss: A pig perspective.

Pigs have a unique, non-invasive epitheliochorial placenta where maternal and fetal layers lay in apposition. Indentation of fetal capillaries into the trophoblasts and maternal capillaries into the uterine epithelium reduce the distance between the fetal and maternal blood, ensuring nutrient transfer for proper conceptus development. Another unique feature of pig pregnancy is conceptus-mediated immune cell enrichment during the early stages of conceptus attachment (around gestation Day 15). This period coincides with the development of vasculature networks at the maternal-fetal interface, which is critical for successful conceptus growth. Specific chemokines, their receptors, and chemokine decoy receptor networks coordinate this immune cell enrichment and the positioning at the maternal-fetal interface. The recruited immune cells, in turn, adopt a specialized phenotype to support key processes of maternal-fetal adaptations, including tolerance to the semi-allogeneic fetus and supporting vascularization. Disturbance in coordinated cross talk between the conceptus and maternal endometrium is an important mechanism associated with spontaneous fetal loss. The exact mechanism of fetal loss is still not yet identified, although research in the last two decades point to various factors including genetics, nutrition, uterine capacity, placental efficiency, and imbalanced immune factors at the maternal-fetal interface. In this review, we summarize some of the recent advances in endometrial immune cell functions and their regulation. We also provide insights into endometrial/placental transcriptome, microRNA biology, and extravesicular transport across the maternal-fetal interface, as well as their potential implications in porcine pregnancy success or failure.

Cervical cerclage placement decreases local levels of proinflammatory cytokines in patients with cervical insufficiency.

BACKGROUND: Cervical insufficiency is characterized by premature, progressive dilation and shortening of the cervix during pregnancy. If left unattended, this can lead to the prolapse and rupture of the amniotic membrane, which usually results in midtrimester pregnancy loss or preterm birth. Previous studies have shown that proinflammatory cytokines such as interleukin-1ß, interleukin-6, interleukin-8, and tumor necrosis factor alpha are up-regulated in normal parturition but are also associated with preterm birth. Studies evaluating such markers in patients with cervical insufficiency have evaluated only their diagnostic potential. Even fewer studies have studied them within the context of cerclage surgery. OBJECTIVES(S): The objective of the study was to evaluate the impact of local and systemic inflammatory markers on the pathogenesis of cervical insufficiency and the effect of cerclage surgery on the local immune microenvironment of women with cervical insufficiency. STUDY DESIGN: We recruited 28 pregnant women (12-20 weeks' gestation) diagnosed with insufficiency and referred for cerclage surgery and 19 gestational age-matched normal pregnant women as controls. Serum and cervicovaginal fluid samples were collected before and after cerclage surgery and during a routine checkup for normal women and analyzed using a targeted 13-plex proinflammatory cytokine assay. RESULTS: Before surgery, patients with cervical insufficiency had higher levels of interleukin-1ß, interleukin-6, interleukin-12, monocyte chemoattractant protein-1 and tumor necrosis factor alpha in cervicovaginal fluid compared to controls, but after surgery, these differences disappeared. No differences were found in serum of insufficiency versus control women. In patients with insufficiency, the levels of interleukin-1ß, interleukin-6, interleukin-8, monocyte chemoattractant protein-1, and interferon gamma in cervicovaginal fluid declined significantly after cerclage compared with before intervention, but these changes were not detected in serum. CONCLUSION: Compared with normal women, patients with cervical insufficiency have elevated levels of proinflammatory cytokines in cervicovaginal fluid but not in serum, suggesting a dysregulation of the local immune environment. Cerclage intervention led to a significant decline in these proinflammatory cytokines, suggesting that cerclage may help reduce local inflammation in cervical insufficiency.

IL-17A Contributes to the Pathogenesis of Endometriosis by Triggering Proinflammatory Cytokines and Angiogenic Growth Factors.

Endometriosis is a chronic, inflammatory disease characterized by the growth of endometrial tissue in aberrant locations outside the uterus. Neoangiogenesis or establishment of new blood supply is one of the fundamental requirements of endometriotic lesion survival in the peritoneal cavity. IL-17A is emerging as a potent angiogenic and proinflammatory cytokine involved in the pathophysiology of several chronic inflammatory diseases such as rheumatoid arthritis and psoriasis. However, sparse information is available in the context of endometriosis. In this study, we demonstrate the potential importance of IL-17A in the pathogenesis and pathophysiology of endometriosis. The data show a differential expression of IL-17A in human ectopic endometriotic lesions and matched eutopic endometrium from women with endometriosis. Importantly, surgical removal of lesions resulted in significantly reduced plasma IL-17A concentrations. Immunohistochemistry revealed localization of IL-17A primarily in the stroma of matched ectopic and eutopic tissue samples. In vitro stimulation of endometrial epithelial carcinoma cells, Ishikawa cells, and HUVECs with IL-17A revealed significant increase in angiogenic (vascular endothelial growth factor and IL-8), proinflammatory (IL-6 and IL-1ß), and chemotactic cytokines (G-CSF, CXCL12, CXCL1, and CX3CL1). Furthermore, IL-17A promoted tubulogenesis of HUVECs plated on Matrigel in a dose-dependent manner. Thus, we provide the first evidence, to our knowledge, that endometriotic lesions produce IL-17A and that the removal of the lesion via laparoscopic surgery leads to the significant reduction in the systemic levels of IL-17A. Taken together, our data show a likely important role of IL-17A in promoting angiogenesis and proinflammatory environment in the peritoneal cavity for the establishment and maintenance of endometriosis lesions.

Protein Inhibitor of Activated STAT3 (PIAS3) Is Down-Regulated in Eutopic Endometrium of Women with Endometriosis.

Endometriosis is a major cause of chronic pelvic pain and infertility. Activation of STAT3 appears central to the inflammatory phenotype of eutopic endometrium in women with endometriosis. However, the molecular mechanism by which this occurs remains unknown. Our objective is to determine how STAT3 activity is regulated in endometriosis. Protein inhibitor of activated STAT3 (PIAS3) is a negative regulator of STAT3 activity. We examined the levels of PIAS3 in endometrium from women with and without endometriosis using Western blot analysis and immunohistochemistry. Levels of PIAS3 are significantly lower, in contrast with phosphorylation of STAT3, in women with endometriosis compared to women without endometriosis. Furthermore, induction of endometriosis in the baboon showed a significant reduction of PIAS3 expression during the progression of the disease. Interferon-γ (INFγ) reduces PIAS3 protein levels and increases phospho-STAT3 levels through CXCL10 in endometrial cells, Ishikawa, and 12Z cells. These results suggest that attenuation of PIAS3 causes aberrant activation of STAT3 in endometriosis, leading to inflammatory changes that may impair fertility or cause pain.

Altered expression of chemokines and their receptors at porcine maternal-fetal interface during early and mid-gestational fetal loss.

Chemokines play a significant role in pregnancy, especially during embryonic attachment and placental development. During early pregnancy, immune cells are recruited extensively to the endometrium in several species including pigs. However, this recruitment is solely mediated by the presence of the conceptus in pigs making it a unique feature compared with other species (humans, primates and mice). To understand the biological significance of chemokine expression and immune cell recruitment in the context of fetal loss, we investigate a well-characterized porcine fetal loss model during the window of early pregnancy at gestational day (gd) 20 and mid-pregnancy (gd50). These periods coincide with 25-40 % of conceptus loss. Using targeted quantitative polymerase chain reaction and Western blot approaches, we screened a specific set of chemokines. Comparisons were made with endometrial lymphocytes (ENDO LY), endometrium and chorioallantoic membranes (CAM) associated with spontaneously arresting and healthy conceptus attachment sites (CAS). mRNA expression studies revealed an increased expression of CXCR3 and CCR5 in ENDO LY and of CXCL10, CXCR3, CCL5 and CCR5 in the endometrium associated with arresting CAS at gd20. DARC was decreased in the endometrium at gd50. CCL1 was increased in CAM associated with arresting CAS at gd50. Some of these differences were also noted at the protein level (CXCL10, CXCR3, CCL5 and CCR5) in the endometrium and CAM. CD45+ immunohistochemistry demonstrated a significantly higher localization in ENDO LY in the endometrium associated with healthy versus arresting counterparts. Most of these differences were observed in early pregnancy and might contribute towards a shift in immune cell functions.

Chronic effects of an anti-angiogenic thrombospondin-1 mimetic peptide, ABT-898, on female mouse reproductive outcomes.

BACKGROUND: Angiogenesis is an essential process in endometriosis disease progression. Earlier, we demonstrated that anti-angiogenic peptide, ABT-898 prevents neoangiogenesis of human endometriotic lesions in a xenograft mouse model. Since angiogenesis is essential for normal ovarian and uterine function, we evaluated effects of ABT-898 on normal female reproductive processes in mice. METHODS: Cycling female C57BL/6N mice were dosed with ABT-898 (100 mg/kg) or 5 % dextrose control for 21 consecutive days to cover multiple estrous cycles (average estrous cycle 4 to 5 days in mice). Pregnant female mice were dosed with ABT-898 (100 mg/kg) or control on alternate days over the course of gestation, beginning at gestation day 7.5 to 17.5 (gestation length 21 days). Histological analysis along with CD31 and Vimentin immunohistochemistry were performed on ovaries and uteri obtained from treated and control mice. To understand the influence of ABT-898 on systemic angiogenic factors, a Pro Mouse Cytokine 9-plex assay was performed on plasma samples obtained from mice prior to treatment, during the second week of ABAT-898 or control treatment and on the last day of treatment. RESULTS: ABT-898 did not affect the number of estrous cycles over the 21 day treatment compared to control. Histological analysis of ovaries found no difference in the number of primordial, primary, secondary, and antral follicles between ABT-898 treated and control groups. Similarly, no difference was observed in the microvessel density between ABT-898 treated and control uteri, ovarian follicles or corpus luteum when assessed using CD31 or vimentin immunohistochemistry. Electron microscopy revealed similar capillary structure and appearance in both ABT-898 treated and control uteri. Although peripheral blood angiogenic cytokine profiles (IL-15, IL-18, M-CSF, b-FGF, PDGF-bb, MIG, MIP-2, LIF and VEGF) changed over the course of the intervention, there was no significant difference between ABT-898 and control groups at any of the studied time points. Treatment with ABT-898 during pregnancy had no effect on litter size at birth, pup weight at birth or pup weight at weaning. CONCLUSION: Our findings suggest that ABT-898 may not alter angiogenesis dependent reproductive processes in female mice. However, an extensive reproductive toxicology screening is required to substantiate use of ABT-898 in future.

Onset and Regression of Pregnancy-Induced Cardiac Alterations in Gestationally Hypertensive Mice: The Role of the Natriuretic Peptide System.

Pregnancy induces cardiovascular adaptations in response to increased volume overload. Aside from the hemodynamic changes that occur during pregnancy, the maternal heart also undergoes structural changes. However, cardiac modulation in pregnancies complicated by gestational hypertension is incompletely understood. The objectives of the current investigation were to determine the role of the natriuretic peptide (NP) system in pregnancy and to assess alterations in pregnancy-induced cardiac hypertrophy between gestationally hypertensive and normotensive dams. Previously we have shown that mice lacking the expression of atrial NP (ANP; ANP(-/-)) exhibit a gestational hypertensive phenotype. In the current study, female ANP(+/+) and ANP(-/-) mice were mated with ANP(+/+) males. Changes in cardiac size and weight were evaluated across pregnancy at Gestational Days 15.5 and 17.5 and Postnatal Days 7, 14, and 28. Nonpregnant mice were used as controls. Physical measurement recordings and histological analyses demonstrated peak cardiac hypertrophy occurring at 14 days postpartum in both ANP(+/+) and ANP(-/-) dams with little to no change during pregnancy. Additionally, left ventricular expression of the renin-angiotensin system (RAS) and NP system was quantified by real-time quantitative polymerase chain reaction. Up-regulation of Agt and AT(1a) genes was observed late in pregnancy, while Nppa and Nppb genes were significantly up-regulated postpartum. Our data suggest that pregnancy-induced cardiac hypertrophy may be influenced by the RAS throughout gestation and by the NP system postpartum. Further investigations are required to gain a complete understanding of the mechanistic aspects of pregnancy-induced cardiac hypertrophy.

Differential expression of plasma microRNA in preeclamptic patients at delivery and 1 year postpartum.

OBJECTIVE: Preeclampsia (PE) is a hypertensive disorder of pregnancy characterized by widespread maternal endothelial dysfunction. Although clinical signs subside following delivery, long-term risks associated with PE include hypertension, stroke, and cardiovascular disease. MicroRNAs (miRNAs) are emerging as critical regulators of biological function, and while alterations to the miRNAome have been described in the context of pregnancy and PE, the postpartum implications of PE on miRNA expression is unknown. The goal of this study was to characterize circulating miRNA profiles at the time of delivery and at 1 year postpartum for women who did and did not develop PE. STUDY DESIGN: Using a targeted reverse transcription polymerase chain reaction approach, selected miRNAs putatively involved in the pathophysiology of PE were examined in 17 normotensive control and 13 PE maternal plasma samples at the time of delivery and 1 year postpartum. Ingenuity Pathway Analysis was used to map putative messenger RNA targets of differentially expressed miRNA to global molecular networks based on gene function. RESULTS: Significant increases (P < .05) in 7 miRNAs with antiangiogenic, inflammatory, and apoptotic functions (miR-98-5p, miR-222-3p, miR-210-3p, miR-155-5p, miR-296-3p, miR-181a-5p, miR-29b-3p) were evident in maternal plasma at the time of severe PE compared to time-matched controls. Plasma samples from individuals who developed mild PE exhibited no changes compared to control samples for the subset of miRNAs analyzed here. Differential expression of plasma miRNA at the time of delivery for women with PE were largely resolved at 1 year postpartum, and reduced expression of only miR-221-3p (P < .05) was evident. Network analysis of putative targets of differentially regulated miRNA identified 11 interacting networks with enrichment for proteins involved in cardiovascular disease, organ system development and function, and cell signaling and interaction. CONCLUSION: The systemic effect of PE on maternal systems is evident in the circulating miRNAome with substantial alterations in miRNA expression in women who develop severe PE. In addition we provide novel evidence of disruption to miR-221 expression 1 year postpartum following a pregnancy complicated by PE compared to normotensive time-matched controls, which may allude to persistent inflammation in these women after delivery.

A peptide inhibitor of synuclein-γ reduces neovascularization of human endometriotic lesions.

Endometriosis is a chronic painful gynecological condition characterized by adherence and growth of endometrium outside of the uterine cavity. Neovascularization is essential to the developing endometriosis lesion to support its growth. Synuclein-γ (SNCG), a protein implicated in cellular proliferation, is associated with a broad range of malignancies as well as endometriosis. We hypothesized that SNCG plays an important role in the neovascularization and growth of endometriosis and blocking of SNCG will interfere with survival of endometriotic lesions in a mouse model. We developed SP012, a novel 12 amino acid peptide inhibitor of SNCG. SP012 inhibited three-dimensional endothelial cell tube formation in a dose-dependent manner. Using intravital microscopy, SP012 was shown to be successfully delivered to human endometriotic lesions in a xenograft mouse model in vivo. Alymphoid (BALB/c-Rag2-/-Il2rγ-/- lacking T, B and NK cells) mice were surgically induced with human endometriotic lesions and treated with SP012 or phosphate-buffered saline control. SP012 treated endometriotic lesions had decreased growth, development and vascularization at the time of necroscopy. Endometriotic lesions treated with SP012 also had fewer isolectin (+) microvessels. These results, using a mouse model, indicate that SNCG plays a role in the neovascularization and subsequent growth of human endometriotic lesions. Targeting SNCG function using peptide inhibitor might provide a potential therapeutic option for the treatment of endometriosis in the future.

Expression of leptin and its long form receptor at the porcine maternal-fetal interface: contrasting healthy and arresting conceptus attachment sites during early and mid-pregnancy.

BACKGROUND: It is well established that spontaneous conceptus loss in swine is associated with an imbalance of both angiogenic and immunological factors. Leptin (LEP), a metabolic hormone, has also been implicated in the promotion of angiogenesis. In this study, LEP and its long form receptor (OB-Rb) were evaluated during porcine pregnancy to assess their basal level of expression and their potential role in conceptus development. METHODS: Expression and secretion of LEP and OB-Rb were quantified in the endometrium of non-pregnant (n = 5), and in the endometrium and chorioallantoic membrane (CAM) of pregnant sows (parity 2 to 5) at gestational days (gd) 20 (n = 8) and 50 (n = 8). Data were analyzed by a 3-way ANOVA testing the effects of conceptus health, tissue type and gestational day. RESULTS: Leptin and OB-Rb transcripts were significantly higher (P < 0.05) in pregnant than in non-pregnant sows. Significantly greater LEP (P < 0.001) was detected in the endometrial tissue at gd20 compared with gd50. At the protein level, the lowest LEP expression (P < 0.01) was detected in the CAM at gd50, while OB-Rb protein was significantly lower (P < 0.01) at gd50 in the CAM than in the endometrium collected from gd20 and gd50 conceptus attachment sites. Immunofluorescence staining confirmed the expression of these proteins at both gestational days and in both tissue types. CONCLUSIONS: Changes in the expression patterns of LEP and OB-Rb between gd20 and gd50 suggest a role for the LEP/OB-R complex at the early stages of porcine pregnancy, possibly affecting the attachment process. Further mechanistic studies are warranted to understand the specific role of leptin in porcine pregnancy.

Thrombospondin-1 mimetic peptide ABT-898 affects neovascularization and survival of human endometriotic lesions in a mouse model.

Endometriosis is a common cause of pelvic pain and infertility in women, and a common indication for hysterectomy, yet the disease remains poorly diagnosed and ineffectively treated. Because endometriotic lesions require new blood supply for survival, inhibiting angiogenesis could provide a novel therapeutic strategy. ABT-898 mimics the antiangiogenic properties of thrombospondin-1, so we hypothesized that ABT-898 will prevent neovascularization of human endometriotic lesions and that ABT-898 treatment will not affect reproductive outcomes in a mouse model. Endometriosis was induced in BALB/c-Rag2(-/-)Il2rg(-/-) mice by surgical implantation of human endometrial fragments in the peritoneal cavity. Mice received daily injections of ABT-898 for 21 days. Flow cytometry was performed to measure circulating endothelial progenitor cells in peripheral blood. Cytokines were measured in plasma samples. Half of the ABT-898-treated and control mice were euthanized to assess neovascularization of endometriotic lesions, using CD31(+) immunofluorescence. The remaining mice were mated and euthanized at gestation day 12. Endometriotic lesions increased circulating endothelial progenitor cells 13 days after engraftment, relative to baseline. Endometriotic lesions from ABT-898-treated mice exhibited reduced neovascularization, compared with controls, and lesions had fewer CD31(+) microvessels. Chronic treatment with ABT-898 did not lead to any fetal anomalies or affect litter size at gestation day 12, compared with controls. Our results suggest that ABT-898 inhibits neovascularization of human endometriotic lesions without affecting mouse fecundity.

The dysregulation of leukemia inhibitory factor and its implications for endometriosis pathophysiology.

Endometriosis is an estrogen dominant, chronic inflammatory disease characterized by the growth of endometrial-like tissue outside of the uterus. The most common symptoms experienced by patients include manifestations of chronic pelvic pain- such as pain with urination, menstruation, or defecation, and infertility. Alterations to Leukemia Inhibitory Factor (LIF), a cytokine produced by the luminal and glandular epithelium of the endometrium that is imperative for successful pregnancy, have been postulated to contribute to infertility. Conditions such as recurrent implantation failure, unexplained infertility, and infertility associated diseases such as adenomyosis and endometriosis, have demonstrated reduced LIF production in the endometrium of infertile patients compared to fertile counterparts. While this highlights the potential involvement of LIF in infertility, LIF is a multifaceted cytokine which plays additional roles in the maintenance of cell stemness and immunomodulation. Thus, we sought to explore the implications of LIF production within ectopic lesions on endometriosis pathophysiology. Through immunohistochemistry of an endometrioma tissue microarray and ELISA of tissue protein extract and peritoneal fluid samples, we identify LIF protein expression in the ectopic lesion microenvironment. Targeted RT qPCR for LIF and associated signaling transcripts, identify LIF to be significantly downregulated in the ectopic tissue compared to eutopic and control while its receptor, LIFR, is upregulated, highlighting a discordance in ectopic protein and mRNA LIF expression. In vitro treatment of endometriosis representative cell lines (12Z and hESC) with LIF increased production of immune-recruiting cytokines (MCP-1, MCP-3) and the angiogenic factor, VEGF, as well as stimulated tube formation in human umbilical vein endothelial cells (HUVECs). Finally, LIF treatment in a syngeneic mouse model of endometriosis induced both local and peripheral alterations to immune cell phenotypes, ultimately reducing immunoregulatory CD206+ small peritoneal macrophages and T regulatory cells. These findings suggest that LIF is present in the ectopic lesions of endometriosis patients and could be contributing to lesion vascularization and immunomodulation.