RESUMO
Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.
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Colestase , Memória de Curto Prazo , Humanos , Camundongos , Animais , Colestase/tratamento farmacológico , Ácido Quenodesoxicólico/farmacologia , Ductos Biliares/cirurgia , Fígado , LigaduraRESUMO
RESEARCH QUESTION: What is the expression pattern of platelet-derived growth factor BB (PDGF-BB), and its receptors, across the menstrual cycle in healthy control women and those with abnormal uterine bleeding-endometrial disorder (AUB-E)? DESIGN: Immunohistochemical staining for PDGF-BB, platelet-derived growth factor receptor alpha (PDGFRα) and platelet-derived growth factor beta (PDGFRß) was performed in control and AUB-E endometrium from the proliferative, early, mid- and late secretory phases of the menstrual cycle (n = 5 each group). Control proliferative phase endometrium was cultured in PDGF-BB (0, 10 ng/ml) and vascular maturation assessed (n = 3). Endothelial cell to vascular smooth muscle cell (VSMC) association was assessed after treatment with PDGF-BB (0, 1, 10 ng/ml). Secretion of angiogenic growth factors by endothelial cells or VSMC was determined. RESULTS: Endothelial cell immunoreactivity for PDGF-BB was reduced in the mid and late secretory phases in AUB-E (P = 0.008). PDGFRα was also reduced in mid secretory phase endothelial cells, proliferative and early secretory phase glandular epithelium in AUB-E (P = 0.008). PDGFRß expression was not altered. Treatment of proliferative phase endometrium with PDGF-BB (10 ng/ml) reduced the percentage of vessels expressing contractile VSMC markers. PDGF-BB had no effect on angiogenic growth factor secretion by endothelial cells or VSMC in vitro and did not affect their association in an in-vitro endothelial cell-VSMC association assay. CONCLUSIONS: Reduced endothelial cell expression of PDGF-BB in the AUB-E endometrium may contribute to the reduced vascular maturation previously observed in these women.
Assuntos
Becaplermina , Células Endoteliais , Doenças Uterinas , Becaplermina/metabolismo , Células Cultivadas , Endométrio/metabolismo , Endométrio/fisiopatologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Hemorragia UterinaRESUMO
Appropriate growth and development of the endometrium across the menstrual cycle is key for a woman's quality of life and reproductive well-being. Recurrent pregnancy loss (RPL) and heavy menstrual bleeding (HMB) affect a significant proportion of the female population worldwide. These endometrial pathologies have a significant impact on a woman's quality of life as well as placing a high economic burden on a country's health service. An underlying cause for both conditions is unknown in approximately 50% of cases. Previous research has demonstrated that aberrant endometrial vascular maturation is associated with both RPL and HMB, where it is increased in RPL but reduced in HMB. TGFß1 is one of the key growth factors that regulate vascular maturation, by inducing phenotypic switching of vascular smooth muscle cells (VSMCs) from a synthetic phenotype to a more contractile one. Our previous data demonstrated an increase in TGFß1 in the endometrium of RPL, while others have shown a decrease in women with HMB. However, TGFß1 bioavailability is tightly controlled, and we therefore sought to perform an extensive immunohistochemical analysis of different components in the pathway in the endometrium of normal controls, women with HMB or RPL. In addition, two in vitro models were used to examine the role of TGFß1 in endometrial vascular maturation and endothelial cell (EC):VSMC association. Taken all together, the immunohistochemical data suggest a decrease in bioavailability, receptor binding capacity, and signaling in the endometrium of women with HMB compared with controls. In contrast, there is an increase in the bioavailability of active TGFß1 in the endometrium of women with RPL compared with controls. Endometrial explants cultured in TGFß1 had an increase in the number of vessels associated with contractile VSMC markers, although the total number of vessels did not increase. In addition, TGFß1 increased EC:VSMC association in an in vitro model. In conclusion, TGFß1 is a key regulator of endometrial vascular maturation and could be considered as a therapeutic target for women suffering from HMB and/or RPL.
RESUMO
Spiral artery (SpA) remodelling is essential for a successful pregnancy and is best described by its morphological features; vascular smooth muscle cell separation and loss, vessel dilatation, and invasion by extravillous trophoblast cells (EVT). Current opinion holds that EVT fully replace the endothelial cells (EC) of the SpA and take on EC-like characteristics. Placental bed biopsies (6-20 weeks gestation) were immunostained for EC and EVT, showing transient loss of EC. In vessels containing an endovascular EVT plug (n = 28) 77.6 ± 19.3% of the lumen was covered by EC while in vessels without endovascular EVT (n = 100) it was 100 ± 0%.
Assuntos
Células Endoteliais/fisiologia , Primeiro Trimestre da Gravidez/fisiologia , Trofoblastos/fisiologia , Útero/irrigação sanguínea , Remodelação Vascular , Feminino , Humanos , GravidezRESUMO
Excessive type I interferon (IFNα/ß) activity is implicated in a spectrum of human disease, yet its direct role remains to be conclusively proven. We investigated two siblings with severe early-onset autoinflammatory disease and an elevated IFN signature. Whole-exome sequencing revealed a shared homozygous missense Arg148Trp variant in STAT2, a transcription factor that functions exclusively downstream of innate IFNs. Cells bearing STAT2R148W in homozygosity (but not heterozygosity) were hypersensitive to IFNα/ß, which manifest as prolonged Janus kinase-signal transducers and activators of transcription (STAT) signaling and transcriptional activation. We show that this gain of IFN activity results from the failure of mutant STAT2R148W to interact with ubiquitin-specific protease 18, a key STAT2-dependent negative regulator of IFNα/ß signaling. These observations reveal an essential in vivo function of STAT2 in the regulation of human IFNα/ß signaling, providing concrete evidence of the serious pathological consequences of unrestrained IFNα/ß activity and supporting efforts to target this pathway therapeutically in IFN-associated disease.
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Doenças do Sistema Imunitário/genética , Interferon Tipo I/imunologia , Fator de Transcrição STAT2/genética , Mutação em Linhagem Germinativa , Humanos , Doenças do Sistema Imunitário/imunologia , Lactente , Masculino , Transdução de SinaisRESUMO
Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.
Assuntos
Feto/citologia , Hematopoese , Fígado/citologia , Fígado/embriologia , Células Sanguíneas/citologia , Microambiente Celular , Feminino , Feto/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Fígado/metabolismo , Tecido Linfoide/citologia , Análise de Célula Única , Células-Tronco/metabolismoRESUMO
Spontaneous preterm birth (sPTB, delivery <37 weeks gestation), accounts for approximately 10% of births worldwide; the aetiology is multifactorial with intra-amniotic infection being one contributing factor. This study aimed to determine whether asymptomatic women with a history of sPTB or cervical surgery have altered levels of inflammatory/antimicrobial mediators and/or microflora within cervical fluid at 22-24 weeks gestation. External cervical fluid was collected from women with history of previous sPTB and/or cervical surgery at 22-24 weeks gestation (n = 135). Cytokine and antimicrobial peptides were measured on a multiplex platform or by ELISA. qPCR was performed for detection of 7 potentially pathogenic bacterial species. IL-8 and IL-1ß levels were lower in women who delivered preterm compared to those who delivered at term (IL-8 P = 0.02; IL-1ß P = 0.04). There were no differences in elafin or human beta defensin-1 protein levels between the two groups. Multiple bacterial species were detected in a higher proportion of women who delivered preterm than in those who delivered at term (P = 0.005). Cervical fluid IL-8 and IL-1ß and microflora have the potential to be used as biomarkers to predict sPTB in high risk women.
Assuntos
Peptídeos Catiônicos Antimicrobianos/análise , Colo do Útero/imunologia , Citocinas/análise , Microbiota/imunologia , Nascimento Prematuro/diagnóstico , Adolescente , Adulto , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Biomarcadores/análise , Colo do Útero/microbiologia , Citocinas/imunologia , Citocinas/metabolismo , DNA Bacteriano/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Recém-Nascido , Microbiota/genética , Placenta/imunologia , Placenta/patologia , Valor Preditivo dos Testes , Gravidez , Segundo Trimestre da Gravidez/imunologia , Nascimento Prematuro/imunologia , Nascimento Prematuro/patologia , Prognóstico , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease. Approximately 30% of patients do not respond to therapy with ursodeoxycholic acid (UDCA). Previous studies have implicated increased senescence of cholangiocytes in patients who do not respond to UDCA. This may increase the release of cytokines which drive pathogenic T cell polarization. As FXR agonists are beneficial in treating UDCA non-responsive patients, the current study was designed to model the interactions between cholangiocytes and CD4+ T cells to investigate potential immunomodulatory mechanisms of bile acid receptor agonists. Human cholangiocytes were co-cultured with CD4+ T cells to model the biliary stress response. Senescent cholangiocytes were able to polarize T cells toward a Th17 phenotype and suppressed expression of FoxP3 (P = 0.0043). Whilst FXR and TGR5 receptor agonists were unable directly to alter cholangiocyte cytokine expression, FGF19 was capable of significantly reducing IL-6 release (P = 0.044). Bile acid receptor expression was assessed in PBC patients with well-characterized responsiveness to UDCA therapy. A reduction in FXR staining was observed in both cholangiocytes and hepatocytes in PBC patients without adequate response to UDCA. Increased IL-6 expression by senescent cholangiocytes represents a potential mechanism by which biliary damage in PBC could contribute to excessive inflammation.
RESUMO
During early human pregnancy the uterine mucosa transforms into the decidua, into which the fetal placenta implants and where placental trophoblast cells intermingle and communicate with maternal cells. Trophoblast-decidual interactions underlie common diseases of pregnancy, including pre-eclampsia and stillbirth. Here we profile the transcriptomes of about 70,000 single cells from first-trimester placentas with matched maternal blood and decidual cells. The cellular composition of human decidua reveals subsets of perivascular and stromal cells that are located in distinct decidual layers. There are three major subsets of decidual natural killer cells that have distinctive immunomodulatory and chemokine profiles. We develop a repository of ligand-receptor complexes and a statistical tool to predict the cell-type specificity of cell-cell communication via these molecular interactions. Our data identify many regulatory interactions that prevent harmful innate or adaptive immune responses in this environment. Our single-cell atlas of the maternal-fetal interface reveals the cellular organization of the decidua and placenta, and the interactions that are critical for placentation and reproductive success.
Assuntos
Comunicação Celular , Feto/citologia , Histocompatibilidade Materno-Fetal/imunologia , Placenta/citologia , Placenta/metabolismo , Gravidez/imunologia , Análise de Célula Única , Comunicação Celular/imunologia , Diferenciação Celular/genética , Decídua/citologia , Decídua/imunologia , Decídua/metabolismo , Feminino , Feto/imunologia , Feto/metabolismo , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Ligantes , Placenta/imunologia , RNA Citoplasmático Pequeno/genética , Análise de Sequência de RNA , Células Estromais/citologia , Células Estromais/metabolismo , Transcriptoma , Trofoblastos/citologia , Trofoblastos/imunologia , Trofoblastos/metabolismoRESUMO
STUDY QUESTION: Are there any phenotypic and structural/architectural changes in the vessels of endometrium and superficial myometrium during the normal menstrual cycle in healthy women and those with heavy menstrual bleeding (HMB)? SUMMARY ANSWER: Spatial and temporal differences in protein levels of endothelial cell (EC) markers and components of the extracellular matrix (ECM) were detected across the menstrual cycle in healthy women and these are altered in HMB. WHAT IS KNOWN ALREADY: HMB affects 30% of women of reproductive age with ~50% of cases being idiopathic. We have previously shown that the differentiation status of endometrial vascular smooth muscle cells (VSMCs) is altered in women with HMB, suggesting altered vessel maturation compared to controls. Endometrial arteriogenesis requires the co-ordinated maturation not only of the VSMCs but also the underlying ECs and surrounding ECM. We hypothesized that there are spatial and temporal patterns of protein expression of EC markers and vascular ECM components in the endometrium across the menstrual cycle, which are altered in women with HMB. STUDY DESIGN, SIZE, DURATION: Biopsies containing endometrium and superficial myometrium were taken from hysterectomy specimens from both healthy control women without endometrial pathology and women with subjective HMB in the proliferative (PP), early secretory (ESP), mid secretory (MSP) and late secretory (LSP) phases (N = 5 for each cycle phase and subject group). Samples were fixed in formalin and embedded in paraffin wax. PARTICIPANTS/MATERIALS, SETTING, METHODS: Serial sections (3µm thick) were immunostained for EC markers (factor VIII related antigen (F8RA), CD34, CD31 and ulex europaeus-agglutinin I (UEA-1) lectin), structural ECM markers (osteopontin, laminin, fibronectin and collagen IV) and for Ki67 to assess proliferation. Immunoreactivity of vessels in superficial myometrium, endometrial stratum basalis, stratum functionalis and luminal region was scored using either a modified Quickscore or by counting the number of positive vessels. MAIN RESULTS AND THE ROLE OF CHANCE: In control samples, all four EC markers showed a dynamic expression pattern according to the menstrual cycle phase, in both endometrial and myometrial vessels. EC protein marker expression was altered in women with HMB compared with controls, especially in the secretory phase in the endometrial luminal region and stratum functionalis. For example, in the LSP expression of UEA-1 and CD31 in the luminal region decreased in HMB (mean quickscore: 1 and 5, respectively) compared with controls (3.2 and 7.4, respectively) (both P = 0.008), while expression of F8RA and CD34 increased in HMB (1.4 and 8, respectively) compared with controls (0 and 5.8, respectively) (both P = 0.008). There was also a distinct pattern of expression of the vascular structural ECM protein components osteopontin, laminin, fibronectin and collagen IV in the superficial myometrium, stratum functionalis and stratum basalis during the menstrual cycle, which was altered in HMB. In particular, compared with controls, osteopontin expression in HMB was higher in stratum functionalis in the LSP (7.2 and 11.2, respectively P = 0.008), while collagen IV expression was reduced in stratum basalis in the MSP (4.6 and 2.8, respectively P = 0.002) and in stratum functionalis in the ESP (7 and 3.2, respectively P = 0.008). LIMITATIONS, REASONS FOR CAUTION: The protein expression of vascular EC markers and ECM components was assessed using a semi-quantitative approach in both straight and spiral arterioles. In our hospital, HMB is determined by subjective criteria and levels of blood loss were not assessed. WIDER IMPLICATIONS OF THE FINDINGS: Variation in the protein expression pattern between the four EC markers highlights the importance of choice of EC marker for investigation of endometrial vessels. Differences in expression of the different EC markers may reflect developmental stage dependent expression of EC markers in endometrial vessels, and their altered expression in HMB may reflect dysregulated vascular development. This hypothesis is supported by altered expression of ECM proteins within endometrial vessel walls, as well as our previous data showing a dysregulation in VSMC contractile protein expression in the endometrium of women with HMB. Taken together, these data support the suggestion that HMB symptoms are associated with weaker vascular structures, particularly in the LSP of the menstrual cycle, which may lead to increased and extended blood flow during menstruation. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.
Assuntos
Endométrio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Menorragia/metabolismo , Ciclo Menstrual/sangue , Miométrio/metabolismo , Adulto , Biomarcadores/metabolismo , Endométrio/irrigação sanguínea , Células Endoteliais/metabolismo , Feminino , Humanos , Menorragia/sangue , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Miométrio/irrigação sanguíneaRESUMO
High-risk primary biliary cholangitis (PBC), defined by inadequate response at one year to Ursodeoxycholic acid (UDCA), is associated with disease progression and liver transplantation. Stratifying high-risk patients early would facilitate improved approaches to care. Using long-term follow-up data to define risk at presentation, 6 high-risk PBC patients and 8 low-risk patients were identified from biopsy, transplant and biochemical archival records. Formalin-fixed paraffin-embedded (FFPE) liver biopsies taken at presentation were graded (Scheuer and Nakanuma scoring) and gene expression analysed using the NanoString® nCounter PanCancer Immunity 770-gene panel. Principle component analysis (PCA) demonstrated discrete gene expression clustering between controls and high- and low-risk PBC. High-risk PBC was characterised by up-regulation of genes linked to T-cell activation and apoptosis, INF-γ signalling and leukocyte migration and down-regulation of those linked to the complement pathway. CDKN1a, up-regulated in high-risk PBC, correlated with significantly increased expression of its gene product, the senescence marker p21WAF1/Cip, by biliary epithelial cells. Our findings suggest high- and low-risk PBC are biologically different from disease outset and senescence an early feature in high-risk disease. Identification of a high-risk 'signal' early from standard FFPE tissue sections has clear clinical utility allowing for patient stratification and second-line therapeutic intervention.
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Colangite/genética , Colangite/patologia , Perfilação da Expressão Gênica , Transcriptoma , Adulto , Idoso , Biomarcadores , Biópsia , Colangite/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Considerable work is being carried out on endometrial NK cells to determine whether they play a role in successful pregnancy outcome. In addition there is debate about whether measurements of uNK should be included in the clinical assessment for women with recurrent implantation failure or recurrent miscarriage. A hindrance to taking this forward is the fact that the density of uNK cells reported by different centres is very different. The aim of this study was to determine the reason for these differences and to develop a standardised method. Three centres participated in the study. Each centre exchanged five formalin fixed, wax embedded sections of endometrium from five women. Sections were immunostained for CD56. Images were taken of 10 random fields at ×400 magnification; total stromal and uNK cells were counted using Image J. Results were expressed as % positive uNK cells and the variation in counts obtained in each centre was compared. After initial analysis a standardised protocol was agreed and the process repeated. Significant variation was seen in the counts obtained after initial analysis (Centre A vs.B, mean difference=-0.72 P<0.001; A vs.C mean difference=-0.47 P<0.001; B vs.C, mean difference=0.25 P=0.085). Analysis suggested that differences may be due to duration of tissue fixation, the embedding and sectioning processes, selection of areas for assessment, definition of immunopositive cells and inclusion or exclusion of blood vessels. Adoption of a standardised protocol reduced the variation (Centre A vs.B mean difference=-0.105 P=0.744; A vs.C mean difference=0.219 P=0.150; B vs.C mean difference=0.32 P=0.031). Use of a standardised method is needed to establish a normal range for uNK cells and to develop a meaningful clinical test for uNK cell measurements.
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Aborto Habitual/imunologia , Endométrio/patologia , Células Matadoras Naturais/imunologia , Útero/patologia , Aborto Habitual/diagnóstico , Adulto , Antígeno CD56/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Contagem de Linfócitos , Inclusão em Parafina , Gravidez , Resultado da Gravidez , Padrões de ReferênciaRESUMO
Successful remodeling of the uterine spiral arteries is essential for a complication-free pregnancy and is best described in terms of its morphologic features. The molecular mediators and cellular sources of spiral artery remodeling are not known, although a role for uterine leukocytes has been proposed. Immunohistochemical assessment of placental bed biopsies demonstrated uterine NK cells, macrophages, and T lymphocytes in the wall and adventitia of spiral arteries at different stages of remodeling, regardless of the presence of extravillous trophoblast cells. Leukocytes were more prevalent in vessel adventitia than wall, and macrophages were the most abundant leukocyte population. Macrophages, separated from early pregnancy decidua, did not alter extravillous trophoblast cells invasion or vascular smooth muscle cell organization or differentiation status but did induce extracellular matrix breakdown (reduced immunostaining of laminin, P = 0.05; fibronectin, P = 0.02) and were able to phagocytose apoptotic vascular smooth muscle cells. Decidual macrophages were shown to secrete a wide range of cytokines (IL-1ß, -2, -4, -5, -6, -8, -10, and -13 and TNF-α), proteases (matrix metalloproteinase-1, -2, -7, -9, and -10), and angiogenic growth factors (angiogenin, keratinocyte growth factor, fibroblast growth factor B, vascular endothelial growth factor A, and angiopoietin-1 and -2). We conclude that spiral artery remodeling involves the coordinated activity of a range of cell types, including extravillous trophoblast cells, decidual uterine NK cells, and macrophages in a carefully, spatiotemporally regulated manner.
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Decídua/citologia , Células Matadoras Naturais/fisiologia , Macrófagos/citologia , Placenta/fisiologia , Útero/irrigação sanguínea , Remodelação Vascular/fisiologia , Células Cultivadas , Decídua/metabolismo , Feminino , Humanos , Células Matadoras Naturais/citologia , Macrófagos/metabolismo , Fagocitose , Gravidez , Trofoblastos/citologia , Trofoblastos/fisiologia , Útero/citologiaRESUMO
Heavy menstrual bleeding (HMB) affects 30% of women of reproductive age and significantly interferes with quality of life. Altered endometrial vascular maturation has been reported in HMB and recurrent miscarriage, the latter associated with increased uterine natural killer (uNK) cell numbers. This study compared endometrial leukocyte populations in controls and women with HMB. Formalin-fixed paraffin-embedded endometrial biopsies from controls (without endometrial pathology) and HMB were immunostained for CD14 (macrophages), CD56 (uNK cells), CD83 (dendritic cells), FOXP3 (regulatory T cells/Tregs), CD3 and CD8 (T cells). Leukocyte numbers were analysed as a percentage of total stromal cells in five randomly selected fields of view in the stratum functionalis of each sample. In control women across the menstrual cycle, 2-8% of total stromal cells were CD3(+) cells, 2-4% were CD8(+) T cells and 6-8% were CD14(+) macrophages. Compared with controls, CD3(+) cells were reduced during the mid-secretory phase (4%, P<0.01) and increased in the late secretory phase (12%, P=0.01) in HMB. CD83(+) dendritic cells and FOXP3(+) Tregs were scarce throughout the menstrual cycle in both groups. In controls, 2% of stromal cells in proliferative endometrium were CD56(+) uNK cells, increasing to 17% during the late secretory phase. In HMB, CD56(+) uNK cells were increased in the proliferative (5%, P<0.01) and early secretory (4%, P<0.02) phases, but reduced (10%, P<0.01) in the late secretory phase. This study demonstrates dysregulation of uNK cells in HMB, the functional consequence of which may have an impact on endometrial vascular development and/or endometrial preparation for menstruation.
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Endométrio/imunologia , Células Matadoras Naturais/imunologia , Menorragia/imunologia , Ciclo Menstrual/imunologia , Adulto , Antígenos CD/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Endométrio/patologia , Feminino , Humanos , Células Matadoras Naturais/patologia , Macrófagos/imunologia , Macrófagos/patologia , Menorragia/patologia , Pessoa de Meia-Idade , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
STUDY QUESTION: Are melanocortin receptors (MCR1-5) expressed in the endometrium? SUMMARY ANSWER: MCR1-5 are expressed in endometrium to varying degrees, with MC2R, MC3R and MC5R being the most abundant and the majority of expression being observed in glandular epithelium. WHAT IS KNOWN ALREADY: Women with Addison's disease who were being administered synthetic ACTH reported menstrual complications as a side effect. There is no previous literature on expression of the melanocortin receptors within the endometrium, and therefore whether ACTH may directly affect the endometrial vasculature. STUDY DESIGN, SIZE, DURATION: Endometrial biopsies were taken from hysterectomy specimens in control women without endometrial pathology (n = 4 for each of proliferative and late-secretory phases). Biopsies were formalin fixed and embedded in paraffin wax. Decidual samples (n = 7) were cultured in a range of concentrations of synthetic ACTH for 3 days before being formalin fixed and embedded in paraffin wax. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial paraffin embedded sections were immunostained for MCR1-5 and assessed using a modified quickscore with luminal epithelium, glandular epithelium, stromal cells, endothelial cells and vascular smooth muscle cells all being assessed separately. Cultured decidual biopsy paraffin embedded sections were immunostained for H-caldesmon and the number of layers of vascular smooth muscle cells surrounding the vessel assessed. MAIN RESULTS AND THE ROLE OF CHANCE: All five melanocortin receptors were shown to be immunolocalised to the endometrium, with MC5R, MC2R and MC3R being the most abundant and limited immunostaining being observed for MC1R and MC4R. Treatment of decidual biopsies with synthetic adrenocorticotropin (ACTH) resulted in loss of vascular integrity. LIMITATIONS, REASONS FOR CAUTION: This is an observational study and does not definitively demonstrate a link between synthetic ACTH administration and menstrual complications. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to demonstrate widespread expression of melanocortin receptors within the endometrium. Further study is required to determine the role of this hormone family in endometrial function. STUDY FUNDING/COMPETING INTERESTS: The work was part funded by MRC grant G09000001. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.
Assuntos
Endométrio/metabolismo , Regulação da Expressão Gênica , Receptor Tipo 2 de Melanocortina/metabolismo , Receptor Tipo 3 de Melanocortina/metabolismo , Receptores de Melanocortina/metabolismo , Hormônio Adrenocorticotrópico/química , Biópsia , Decídua/patologia , Células Endoteliais/citologia , Feminino , Humanos , Histerectomia , Músculo Liso Vascular/citologia , Inclusão em Parafina , Gravidez , Pré-Menopausa , Células Estromais/citologiaRESUMO
STUDY QUESTION: How does the smooth muscle content and differentiation stage of vascular smooth muscle cells (VSMCs) in endometrial blood vessels change according to the different phases of the menstrual cycle and is this altered in women with menorrhagia? SUMMARY ANSWER: The smooth muscle content (as a proportion of the vascular cross-sectional area) of endometrial blood vessels remained unchanged during the normal menstrual cycle and in menorrhagia; however, expression of the VSMC differentiation markers, smoothelin and calponin, was dysregulated in endometrial blood vessels in samples from women with menorrhagia compared with controls. WHAT IS KNOWN ALREADY: Menorrhagia affects 30% of women of reproductive age and is the leading indication for hysterectomy. Previous studies have suggested important structural and functional roles for endometrial blood vessels, including impaired vascular contractility. Differentiation of VSMC from a synthetic to contractile state is associated with altered cellular phenotype that contributes to normal blood flow and pressure. This vascular maturation process has been little studied in endometrium both across the normal menstrual cycle and in menorrhagia. STUDY DESIGN, SIZE, DURATION: Endometrial biopsies were taken from hysterectomy specimens or by pipelle biopsy prior to hysterectomy in controls without endometrial pathology and in women with menorrhagia (n = 7 for each of proliferative, early-secretory, mid-secretory and late-secretory phases for both groups). Biopsies were formalin fixed and embedded in paraffin wax. PARTICIPANTS/MATERIALS, SETTING, METHODS: Paraffin-embedded sections were immunostained for α smooth muscle actin (αSMA), myosin heavy chain (MyHC), H-caldesmon, desmin, smoothelin and calponin (h1 or basic). VSMC content was measured in 25 αSMA(+) vascular cross sections per sample and expressed as a ratio of the muscular area:gross vascular cross-sectional area. VSMC differentiation was analysed by the presence/absence of differentiation markers compared with αSMA expression. Smoothelin and calponin expression was also analysed in relation to total number of blood vessels by double immunostaining for endothelial cell markers. MAIN RESULTS AND THE ROLE OF CHANCE: Study of VSMC differentiation markers revealed decreased expression of calponin both in αSMA(+) vessels (P = 0.008) and in relation to total number of vessels (P = 0.001) in late secretory phase endometrium in menorrhagia compared with controls. Smoothelin expression in αSMA(+) vessels was increased (P = 0.03) in menorrhagia, although this was not significant in relation to the total number of vessels. In normal endometrium, the proportion of blood vessels expressing αSMA increased from 63% in proliferative endometrium to 81% in the late secretory phase (P = 0.002). The overall arterial muscle content did not differ between control and menorrhagia at any phase of the menstrual cycle, occupying 78-81% of gross vascular cross-sectional area during the different menstrual cycle phases. LIMITATIONS, REASONS FOR CAUTION: This study included both straight and spiral arterioles and analysed only stratum functionalis. The VSMC differentiation with respect to αSMA expression is an observational study and the data are presented as presence or absence of the differentiation markers in each field of view, corresponding with the vascular cross sections included in the study of vascular muscle content. WIDER IMPLICATIONS OF THE FINDINGS: Smoothelin and calponin have been widely implicated as important regulators of vascular tone, vascular contractility and rate of blood flow. Our results have uncovered a disparate pattern of calponin expression, potentially indicating a dysfunctional contraction mechanism in the endometrial blood vessels in menorrhagia, thus implicating calponin as a potential therapeutic target. STUDY FUNDING/COMPETING INTERESTS: This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.
Assuntos
Diferenciação Celular , Endométrio/irrigação sanguínea , Menorragia/patologia , Músculo Liso Vascular/patologia , Feminino , Humanos , VasoconstriçãoRESUMO
STUDY QUESTION: Are alterations in decidual expression of interleukin (IL)-6 and IL-8 associated with sporadic miscarriage? SUMMARY ANSWER: IL-6 and IL-8 secretion from decidual uterine natural killer (uNK) cells and macrophages isolated from women with spontaneous miscarriage was reduced compared with normal controls. WHAT IS KNOWN ALREADY: Miscarriage is a common gynaecological problem with huge financial and personal implications. Eleven to twenty per cent of all clinically recognized pregnancies are lost before the 20th week of gestation, with miscarriages often being divided into early (≤ 12 completed weeks from last menstrual period) and late (≥ 13 weeks). Spiral artery remodelling is a key feature of early pregnancy; failure of this process has been implicated in sporadic miscarriage. The molecular triggers that initiate spiral artery remodelling are not clear, although cytokines such as IL-6 and IL-8 may play a role. STUDY DESIGN, SIZE, DURATION: This was a laboratory-based study using decidual and placental bed biopsy samples from women with sporadic miscarriage (n = 30) and termination of pregnancy controls (n = 30). PARTICIPANTS/MATERIALS, SETTING, METHODS: Total adherent decidual cells, CD10(+) stromal cells, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from apparently normal pregnancies that were terminated at either 8-10 or 12-14 weeks' gestation. In addition, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from sporadic miscarriage at 8-10 weeks' gestation. Secreted IL-8 was measured in all isolated cell populations, while IL-6 was measured in CD14(+) macrophages and CD56(+) uNK cells from both sporadic miscarriage and normal controls. Placental bed biopsies were taken from women after sporadic miscarriage or termination of pregnancy at ≤ 12 completed weeks' or >13 weeks' gestational age, formalin-fixed, paraffin-embedded and immunostained for IL-6, IL-6Rα, GP130, IL-8, CXCR1, CXCR2 and CD13 (aminopeptidase N). Staining intensity for each factor was assessed in extravillous trophoblast cell populations, myometrial and decidual stroma, myometrial and decidual spiral arteries and decidual glandular epithelium. A CPA model was used to assess the potential role of IL-6 and IL-8 in spiral artery remodelling. MAIN RESULTS AND THE ROLE OF CHANCE: IL-8 was secreted by total adherent decidual cells, CD10(+) stromal cells and CD14(+) macrophages at both 8-10 and 12-14 weeks' gestation, with CD14(+) cells secreting the highest levels. Both CD14(+) and CD56(+) cells isolated from decidua of early sporadic miscarriage produced lower IL-6 (P = 0.04, P = 0.01, respectively) and IL-8 levels (P = 0.0007, P = 0.002, respectively) compared with normal cases. In addition, altered expression of IL-6, IL-8 and their receptors was observed in various cell types in placental bed (myometrial stroma, glandular epithelium, interstitial extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells) in sporadic miscarriage, particularly from later gestational ages. IL-6 and IL-8 disrupted vascular smooth muscle morphology and organization in an in vitro model of spiral artery remodelling. LIMITATIONS, REASONS FOR CAUTION: By the nature of sampling at the time of miscarriage, it was not possible to ascertain the cause or effect in the observed alterations of levels of IL-6 and IL-8 in sporadic miscarriage. WIDER IMPLICATIONS OF THE FINDINGS: Alterations in the expression of IL-6, IL-8 and their receptors may be associated with the aetiology of sporadic miscarriage, especially given the potential role of these cytokines in the regulation of trophoblast invasion and spiral artery remodelling. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by funding from Wellbeing of Women (RG1000). The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.
Assuntos
Aborto Habitual/metabolismo , Decídua/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Receptores de Interleucina-6/metabolismo , Receptores de Interleucina-8/metabolismo , Aborto Habitual/genética , Feminino , Idade Gestacional , Humanos , Interleucina-6/análise , Interleucina-6/genética , Interleucina-8/análise , Interleucina-8/genética , Neovascularização Fisiológica , Receptores de Interleucina-6/análise , Receptores de Interleucina-6/genética , Receptores de Interleucina-8/análise , Receptores de Interleucina-8/genéticaRESUMO
Uterine spiral artery remodeling is required for successful human pregnancy; impaired remodeling is associated with pregnancy complications, including late miscarriage, preeclampsia, and fetal growth restriction. The molecular triggers of remodeling are not known, but it is now clear that there are "trophoblast-independent" and "trophoblast-dependent" stages. Uterine natural killer (uNK) cells are abundant in decidualized endometrium in early pregnancy; they surround spiral arteries and secrete a range of angiogenic growth factors. We hypothesized that uNK cells mediate the initial stages of spiral artery remodeling. uNK cells and extravillous trophoblast (EVT) cells were isolated from early pregnancy decidua and placenta. Chorionic plate arteries from full-term placentas and spiral arteries from nonpregnant myometrium were cultured with angiogenic growth factors or conditioned medium (CM) from uNK cells or EVT or uNK cell/EVT cocultures. In both vessel models, uNK cell CM induced disruption of vascular smooth muscle cells (VSMCs) and breakdown of extracellular matrix components. Angiopoietin (Ang)-1, Ang-2, interferon-γ, and VEGF-C also disrupted VSMC integrity with an Ang-2 inhibitor abrogating the effect of uNK cell CM. These results provide compelling evidence that uNK cells contribute to the early stages of spiral artery remodeling; failure of this process could contribute to pregnancy pathology.
Assuntos
Células Matadoras Naturais/fisiologia , Trofoblastos/fisiologia , Artéria Uterina/fisiologia , Útero/irrigação sanguínea , Angiopoietina-1/metabolismo , Angiopoietina-1/farmacologia , Angiopoietina-2/metabolismo , Angiopoietina-2/farmacologia , Linhagem Celular , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Decídua/citologia , Decídua/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Interferon gama/metabolismo , Interferon gama/farmacologia , Células Matadoras Naturais/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miométrio/citologia , Miométrio/metabolismo , Placenta/citologia , Placenta/metabolismo , Gravidez , Técnicas de Cultura de Tecidos , Trofoblastos/citologia , Trofoblastos/metabolismo , Artéria Uterina/citologia , Artéria Uterina/efeitos dos fármacos , Útero/citologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/farmacologiaRESUMO
During human uterine spiral artery (SpA) remodeling, vascular smooth muscle cells (VSMCs) are lost and replaced by fibrinoid, incorporating extravillous trophoblast (EVT) cells. The aim of the current study was to determine the relative contributions of apoptosis and migration to VSMC loss during SpA remodeling. Immunohistochemistry (Apoptag, active caspase 3, lamin) of placental bed biopsies (8-20 wk gestation) demonstrated apoptotic cells in all samples; double immunolabeling identified these as trophoblasts, leukocytes, and endothelial cells. In total, 294 SpAs were studied, and only one apoptotic VSMC was identified. H-caldesmon-immunopositive VSMCs were observed surrounding and separate from SpA walls in partially remodeled vessels; the highest level of VSMC migration was observed in vessels with associated EVT cells (number of migrated cells 6.4 ± 1.2; distance migrated 3.5 ± 0.3 pixels) compared with those without (number of migrated cells 3.6 ± 0.5, P<0.001; distance migrated 2.8 ± 0.1 pixels, P<0.0001). VEGF-A, VEGF-C, TGF-ß1, and Ang-2 all stimulated human aorta VSMC invasion in vitro, although EVT cell culture supernatants did not. In summary, apoptosis is unlikely to play a major role in loss of VSMCs from SpAs during remodeling in normal pregnancy, but VSMCs appear to migrate away from the wall of the SpA, an effect enhanced by the presence of EVT cells.
Assuntos
Apoptose/fisiologia , Movimento Celular/fisiologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , Artéria Uterina/citologia , Artéria Uterina/fisiologia , Angiopoietina-2/farmacologia , Proteínas de Ligação a Calmodulina/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Laminas/metabolismo , Metaloproteinases da Matriz/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Gravidez , Fator de Crescimento Transformador beta1/farmacologia , Trofoblastos/citologia , Trofoblastos/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator C de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Invasion of uterine tissues by extravillous trophoblast cells (EVT) is essential for successful human pregnancy. EVT invasion is tightly regulated by a number of factors, including growth factors and cytokines, but the mechanisms that underlie their regulatory effect remain poorly understood. Interleukin (IL)-6 has been suggested to play a role in controlling EVT invasion. We hypothesized that IL-6 produced by cells in uterine decidua would regulate EVT invasiveness via IL-6Rα and gp130 receptors expressed by trophoblast cells. The effect of IL-6 on EVT signalling and cytokine production was also studied. Supernatants from disaggregated 'total' decidual cells, CD8(+) T cells, CD10(+) decidual stromal cells, CD14 macrophages, CD56(+) uterine natural killer cells, cytotrophoblast and EVT cells contained large quantities of IL-6 protein at both 8-10 and 12-14 weeks gestational age. IL-6Rα and gp130 were immunolocalized to EVT in placental bed biopsies from 8 to 20 weeks gestation and IL-6Rα expression was confirmed by western blotting. IL-6 had no effect on the invasive potential of EVT from chorionic villi or the immortalized EVT cell line HTR-8/SVneo in a Matrigel(®) invasion assay. IL-6 stimulated phosphorylation of several cell signalling proteins in EVT (8-14 weeks' gestation), although significance was lost after correction for multiple comparisons. Incubation with IL-6 decreased secretion of regulated upon activation, normal T-cell expressed and secreted (RANTES) by EVT cells. In conclusion, although IL-6 did not affect trophoblast cell invasion, it stimulated EVT cellular cascades and inhibited secretion of RANTES involved in a number of cellular processes.