RESUMO
Endometriosis is an estrogen dependent, chronic inflammatory disease characterized by the growth of endometrial lining outside of the uterus. Mast cells have emerged as key players in regulating not only allergic responses but also other mechanisms such as angiogenesis, fibrosis, and pain. The influence of estrogen on mast cell function has also been recognized as a potential factor driving disease pathophysiology in number of allergic and chronic inflammatory conditions. However, precise information is lacking on the cross talk between endocrine and immune factors within the endometriotic lesions and whether that contributes to the involvement of mast cells with disease pathophysiology. In this study, we observed a significant increase in mast cell numbers within endometriotic lesions compared to matched eutopic endometrium from the same patients. Compared to eutopic endometrium, endometriotic lesions had significantly higher levels of stem cell factor (SCF), a potent growth factor critical for mast cell expansion, differentiation, and survival for tissue resident mast cells. Targeted mRNA Q-PCR array revealed that the endometriotic lesions harbour microenvironment (upregulation of CPA3, VCAM1, CCL2, CMA1, CCR1, and KITLG) that is conducive to mast cells recruitment and subsequent differentiation. To examine cross-talk of mast cells within the endometriotic lesion microenvironment, endometriotic epithelial cells (12Z) and endometrial stromal cells (hESC) incubated with mast cell-conditioned media showed significantly increased production of pro-inflammatory and chemokinetic cytokines. To further understand the impact of estrogen on mast cells in endometriosis, we induced endometriosis in C57BL/6 mice. Mature mast cells were significantly higher in peritoneal fluid of estrogen-treated mice compared to untreated mice within the sham operated groups. Mouse endometriotic lesion tissue revealed several genes (qRT-PCR) relevant in mast cell biology significantly upregulated in the estrogen treated, endometriosis-induced group compared to control endometrium. The endometriotic lesions from estrogen treated mice also had significantly higher density of Alcian blue stained mast cells compared to untreated lesions or control endometrium. Collectively, these findings suggest that endometriotic lesions provide a microenvironment necessary for recruitment and differentiation of mast cells. In turn, mast cells potentially release pro-inflammatory mediators that contribute to chronic pelvic pain and endometriosis disease progression.
Assuntos
Endometriose , Animais , Contagem de Células , Endometriose/patologia , Estrogênios , Feminino , Humanos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Humans and mice have cyclical regeneration of the endometrial epithelium. It is expected that such regeneration is ensured by tissue stem cells, but their location and hierarchy remain debatable. A number of recent studies have suggested the presence of stem cells in the mouse endometrial epithelium. At the same time, it has been reported that this tissue can be regenerated by stem cells of stromal/mesenchymal or bone marrow cell origin. Here, we describe a single-cell transcriptomic atlas of the main cell types of the mouse uterus and epithelial subset transcriptome and evaluate the contribution of epithelial cells expressing the transcription factor PAX8 to the homeostatic regeneration and malignant transformation of adult endometrial epithelium. According to lineage tracing, PAX8+ epithelial cells are responsible for long-term maintenance of both luminal and glandular epithelium. Furthermore, multicolor tracing shows that individual glands and contiguous areas of luminal epithelium are formed by clonal cell expansion. Inactivation of the tumor suppressor genes Trp53 and Rb1 in PAX8+ cells, but not in FOXJ1+ cells, leads to the formation of neoplasms with features of serous endometrial carcinoma, one of the most aggressive types of human endometrial malignancies. Taken together, our results show that the progeny of single PAX8+ cells represents the main source of regeneration of the adult endometrial epithelium. They also provide direct experimental genetic evidence for the key roles of the P53 and RB pathways in the pathogenesis of serous endometrial carcinoma and suggest that PAX8+ cells represent the cell of origin of this neoplasm.
Assuntos
Neoplasias do Endométrio/patologia , Endométrio/patologia , Epitélio/patologia , Homeostase , Neoplasias Císticas, Mucinosas e Serosas/patologia , Fator de Transcrição PAX8/metabolismo , Regeneração , Envelhecimento , Animais , Proliferação de Células , Modelos Animais de Doenças , Neoplasias do Endométrio/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Imunofenotipagem , Integrases/metabolismo , Camundongos Transgênicos , Neoplasias Císticas, Mucinosas e Serosas/genética , Fator de Transcrição PAX8/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Útero/metabolismoRESUMO
Many high-grade serous carcinomas (HGSCs) likely originate in the distal region of the Fallopian tube's epithelium (TE) before metastasizing to the ovary. Unfortunately, molecular mechanisms promoting malignancy in the distal TE are obfuscated, largely due to limited primary human TE gene expression data. Here we report an in depth bioinformatic characterization of 34 primary TE mRNA-seq samples. These samples were prepared from proximal and distal TE regions of 12 normal Fallopian tubes. Samples were segregated based on their aldehyde dehydrogenase (ALDH) activity. Distal cells form organoids with higher frequency and larger size during serial organoid formation assays when compared to proximal cells. Consistent with enrichment for stem/progenitor cells, ALDH+ cells have greater WNT signaling. Comparative evaluation of proximal and distal TE cell population's shows heightened inflammatory signaling in distal differentiated (ALDH-) TE. Furthermore, comparisons of proximal and distal TE cell populations finds that the distal ALDH+ TE cells exhibit pronounced expression of gene sets characteristic of HGSC sub-types. Overall, our study indicates increased organoid forming capacity, WNT/inflammatory signaling, and HGSC signatures underlie differences between distal and proximal regions of the human TE. These findings provide the basis for further mechanistic studies of distal TE susceptibility to the malignant transformation.
Assuntos
Biologia Computacional , Células Epiteliais/citologia , Tubas Uterinas/citologia , Via de Sinalização Wnt , Aldeído Desidrogenase/metabolismo , Diferenciação Celular , Células Epiteliais/patologia , Tubas Uterinas/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Inflamação/patologiaRESUMO
Endometriosis, a major reproductive pathology affecting 8-10% of women is characterized by chronic inflammation and immune dysfunction. Human antigen R (HuR) and Tristetraprolin (TTP) are RNA binding proteins that competitively bind to cytokines involved in inflammation including: tumor necrosis factor alpha (TNF-α), granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6) among others, and stabilize and destabilize them, respectively. The aim of this study was to examine RNA binding protein (RNABP) HuR/TTP axis in endometriosis patients compared to menstrual stage matched healthy fertile controls in hopes of better understanding their contribution to the pathogenesis of endometriosis. Additionally, using a targeted in vitro siRNA approach, we examined whether knock-down of TTP can play a functional role on other RNABPs that competitively bind to inflammatory targets of TTP in both endometriotic and endometrial epithelial cell lines. Our results suggest that RNABPs TTP and HuR are dysregulated in endometriotic lesions compared to matched eutopic patient samples as well endometrium from healthy controls. Silencing of TTP in endometriotic and endometrial epithelial cells revealed differential response to inflammatory cytokines and other RNABPs. Our results suggest potential involvement of HuR/TTP RNA binding protein axis in regulation of inflammation in endometriosis.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Endometriose/metabolismo , Transdução de Sinais , Tristetraprolina/metabolismo , Animais , Biópsia , Estudos de Casos e Controles , Coriocarcinoma/genética , Coriocarcinoma/patologia , Coristoma/patologia , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Proteína Semelhante a ELAV 1/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Endometriose/genética , Endometriose/patologia , Células Epiteliais/metabolismo , Feminino , Inativação Gênica , Humanos , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos BALB C , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tristetraprolina/genética , Regulação para Cima/genéticaRESUMO
Exosomes and microvesicles are extracellular vesicles released from cells and can contain lipids, miRNAs and proteins that affect cells at distant sites. Recently, microvesicles containing miRNA have been implicated in uterine microenvironment of pigs, a species with unique epitheliochorial (non-invasive) placentation. Here we report a novel role of conceptus-derived exosomes/microvesicles (hereafter referred to as extracellular vesicles; EVs) in embryo-endometrial cross-talk. We also demonstrate the stimulatory effects of EVs (PTr2-Exo) derived from porcine trophectoderm-cells on various biological processes including the proliferation of maternal endothelial cells (PAOEC), potentially promoting angiogenesis. Transmission immuno-electron microscopy confirmed the presence of EVs in tissue biopsies, PTr2-Exo and PAOEC-derived EVs (PAOEC-Exo). RT-PCR detected 14 select miRNAs in CD63 positive EVs in which miR-126-5P, miR-296-5P, miR-16, and miR-17-5P were the most abundant angiogenic miRNAs. Proteomic analysis revealed EV proteins that play a role in angiogenesis. In-vitro experiments, using two representative cell lines of maternal-fetal interface, demonstrated bidirectional EVs shuttling between PTr2 and PAOEC cells. Importantly, these studies support the idea that PTr2-Exo and PAOEC-Exo containing select miRNAs and proteins can be successfully delivered to recipient cells and that they may have a biological role in conceptus-endometrial cross-talk crucial for the pregnancy success.
Assuntos
Comunicação Celular , Endométrio/citologia , Vesículas Extracelulares/metabolismo , Feto/citologia , Troca Materno-Fetal , Animais , Biomarcadores/metabolismo , Proliferação de Células , Membrana Corioalantoide/metabolismo , Membrana Corioalantoide/ultraestrutura , Endométrio/metabolismo , Células Endoteliais/citologia , Exossomos/metabolismo , Vesículas Extracelulares/ultraestrutura , Feminino , MicroRNAs/metabolismo , Modelos Biológicos , Gravidez , Proteômica , Sus scrofa , Tetraspanina 30/metabolismo , Trofoblastos/citologiaRESUMO
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.
Assuntos
Quimiocinas/genética , Perda do Embrião/genética , Regulação da Expressão Gênica no Desenvolvimento , Troca Materno-Fetal/genética , Receptores de Quimiocinas/genética , Sus scrofa/embriologia , Sus scrofa/genética , Animais , Biópsia , Quimiocinas/metabolismo , Membrana Corioalantoide , Endométrio/metabolismo , Endométrio/patologia , Feminino , Perfilação da Expressão Gênica , Linfócitos/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Quimiocinas/metabolismoRESUMO
STUDY HYPOTHESIS: Placental growth factor (PGF) is expressed in the developing mouse brain and contributes to vascularization and vessel patterning. STUDY FINDING: PGF is dynamically expressed in fetal mouse brain, particularly forebrain, and is essential for normal cerebrovascular development. WHAT IS KNOWN ALREADY: PGF rises in maternal plasma over normal human and mouse pregnancy but is low in many women with the acute onset hypertensive syndrome, pre-eclampsia (PE). Little is known about the expression of PGF in the fetus during PE. Pgfââ(-/-) mice appear normal but recently cerebral vascular defects were documented in adult Pgfââ(-/-) mice. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Here, temporal-spatial expression of PGF is mapped in normal fetal mouse brains and cerebral vasculature development is compared between normal and congenic Pgfââ(-/-) fetuses to assess the actions of PGF during cerebrovascular development. Pgf/PGF, Vegfa/VEGF, Vegf receptor (Vegfr)1 and Vegfr2 expression were examined in the brains of embryonic day (E)12.5, 14.5, 16.5 and 18.5 C57BL/6 (B6) mice using quantitative PCR and immunohistochemistry. The cerebral vasculature was compared between Pgfââ(-/-) and B6 embryonic and adult brains using whole mount techniques. Vulnerability to cerebral ischemia was investigated using a left common carotid ligation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Pgf/PGF and Vegfr1 are highly expressed in E12.5-14.5 forebrain relative to VEGF and Vegfr2. Vegfa/VEGF is relatively more abundant in hindbrain (HB). PGF and VEGF expression were similar in midbrain. Delayed HB vascularization was seen at E10.5 and 11.5 in Pgfââ(-/-) brains. At E14.5, Pgfââ(-/-) circle of Willis showed unilateral hypoplasia and fewer collateral vessels, defects that persisted post-natally. Functionally, adult Pgfââ(-/-) mice experienced cerebral ischemia after left common carotid arterial occlusion while B6 mice did not. LIMITATIONS, REASONS FOR CAUTION: Since Pgfââ(-/-) mice were used, consequences of complete absence of maternal and fetal PGF were defined. Therefore, the effects of maternal versus fetal PGF deficiency on cerebrovascular development cannot be separated. However, as PGF was strongly expressed in the developing brain at all timepoints, we suggest that local PGF has a more important role than distant maternal or placental sources. Full PGF loss is not expected in PE pregnancies, predicting that the effects of PGF deficiency identified in this model will be more severe than any effects in PE-offspring. WIDER IMPLICATIONS OF THE FINDINGS: These studies provoke the question of whether PGF expression is decreased and cerebral vascular maldevelopment occurs in fetuses who experience a preeclamptic gestation. These individuals have already been reported to have elevated risk for stroke and cognitive impairments. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by awards from the Natural Sciences and Engineering Research Council, the Canada Research Chairs Program and the Canadian Foundation for Innovation to B.A.C. and by training awards from the Universidade Federal de Pernambuco and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil to R.L.L.; Queen's University to V.R.K. and the Canadian Institutes of Health Research to M.T.R. The work of P.C. is supported by the Belgian Science Policy BELSPO-IUAP7/03, Structural funding by the Flemish Government-Methusalem funding, and the Flemish Science Fund-FWO grants. There were no competing interests.
Assuntos
Isquemia Encefálica/genética , Encéfalo/metabolismo , Estenose Coronária/genética , Neovascularização Patológica/genética , Proteínas da Gravidez/deficiência , Animais , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Estenose Coronária/metabolismo , Estenose Coronária/patologia , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Feto , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fator de Crescimento Placentário , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Proteínas da Gravidez/genética , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Endometrial lymphocytes are recruited to the porcine maternal-fetal interface by conceptus-derived signals. The transiently recruited lymphocytes adopt a specialized phenotype in the endometrium that regulates various placental physiological processes, including angiogenesis. Small non-coding RNAs, microRNAs (miRNAs) are emerging as principal bio-molecules regulating the development of lymphocytes and their angiogenic functions. However, no information is available in the context of endometrial lymphocytes in pregnancy. We hypothesize that miRNAs are involved in the development of endometrial lymphocytes and their angiogenic functions at the porcine maternal-fetal interface. Using a targeted Q-PCR approach for selected miRNAs involved in immune cell development, angiogenesis, and anti-angiogenesis, we conducted a study to screen endometrial lymphocytes associated with healthy and spontaneously arresting conceptus attachment sites (CAS) at two well-defined periods of fetal loss. Comparisons were made with endometrium and trophoblasts associated with healthy and arresting CAS. In addition, levels of putative mRNA targets and subsequent functional clustering of genes were studied in order to predict the biological mechanisms affected. We found several significant differences for miRNAs involved in immune cell development and angiogenesis (miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19a) between endometrial lymphocytes associated with healthy and arresting CAS. Significant differences were also found in endometrium and trophoblasts for some miRNAs (miR-20b, miR-17-5P, miR-18a, miR-15b-5P, and miR-222). Finally, selected mRNA targets showed differential expression in all groups. Our data, although associative, are the first to unravel the selected miRNAs involved in immune cell development and provide insights into their possible regulation in abortive pregnancy.
Assuntos
Aborto Animal/imunologia , Endométrio/imunologia , Regulação da Expressão Gênica/imunologia , Linfócitos/imunologia , MicroRNAs/imunologia , Doenças dos Suínos/imunologia , Trofoblastos/imunologia , Aborto Animal/prevenção & controle , Animais , Endométrio/patologia , Feminino , Linfócitos/patologia , Gravidez , Suínos , Doenças dos Suínos/patologia , Trofoblastos/patologiaRESUMO
PROBLEM: To evaluate the expression of the tristetraprolin family and their selected targets during porcine pregnancy. METHOD OF STUDY: Using qPCR and Western blot, mRNA and protein levels were compared between endometrium and chorioallantoic membrane (CAM) associated with healthy and impaired conceptuses at gestation day (gd) 20 and gd50, respectively. Immunohistochemistry was performed to determine localization of TIS11 family members at gd20 and 50. RESULTS: Multiple significant differences (P < 0.05) in TIS11 family transcripts were observed in the aforementioned comparisons. GM-CSF was significantly higher in healthy endometrium and CAM from impaired conceptus attachment sites. TNF-α was elevated in CAM as compared to endometrium at gd50, regardless of conceptus health status. Immunohistochemical staining shows TIS11 family expressed in the glandular and luminal epithelium, as well as stromal cells in the uterus. CONCLUSIONS: The shift in the expression of tristetraprolin (TTP) and TIS11D points to a potential role of these genes in regulating spontaneous fetal loss.
Assuntos
Aborto Animal/metabolismo , Embrião de Mamíferos/metabolismo , Endométrio/metabolismo , Regulação da Expressão Gênica , Tristetraprolina/biossíntese , Aborto Animal/patologia , Animais , Embrião de Mamíferos/patologia , Endométrio/patologia , Feminino , Gravidez , SuínosRESUMO
MicroRNAs (miRNAs) are a recently discovered class of non-coding RNAs that are expressed in many cell types, where they regulate the expression of complementary RNAs, thus modulating the stability and translation of mRNAs. miRNAs are predicted to regulate the expression of â¼50% of all protein coding genes in mammals. Therefore, they participate in virtually all cellular processes investigated so far. Altered miRNAs expressions are associated with both physiological (pregnancy) and pathological processes (cancer). As the dynamic maternal-fetal interface plays a critical role in the maintenance of successful pregnancy, it is not surprising that the miRNAs that are unique to reproductive tissues are abundantly expressed. Research in this field has demonstrated the presence and dysregulation of a distinct set of pregnancy-associated miRNAs; however, most studies have centered on localizing various miRNAs in reproductive microdomains associated with normal or complicated pregnancies. Although several independent miRNA regulatory mechanisms associated with endometrial receptivity, immune cells, angiogenesis and placental development have been studied, miRNA-mediated regulation of pregnancy remains poorly understood. This review provides a summary of the current data on miRNA regulation as well as functional profiles of miRNAs that are found in the uterus, in immune cells associated with maternal tolerance to the fetus, and those involved in angiogenesis and placental development.