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HYPOTHESIS/AIMS OF STUDY: Until recently, polypropylene meshes were often used as a surgical treatment option for Pelvic Organ Prolapse (POP). However, they have been associated with serious complications such as infammation, pain and erosion. As a result, transvaginal meshes were banned in Australia and New Zealand in 2017. At present, there is no optimal therapy for treatment of chronic POP leaving millions of women in despair. To overcome adverse events associated with trans-vaginal surgery using non degradable synthetic mesh for treating pelvic organ prolapse (POP), we are developing a tissue engineering construct comprising degradable nano-fbrous mesh and endometrial mesenchymal stem/stromal cells (eMSC). The aim of this study was to investigate in vivo foreign body response, angiogenesis and extracellular matrix gene expression to a nano-topographically controlled mesh made of poly (L-lactic acid)-co-poly(-caprolactone) and gelatin incorporating eMSC (PLCL/G/eMSC). Our team discovered a rare population of Mesenchymal stem cells in the endometrium (eMSCs) and has identifed a single marker to isolate these rare perivascular cells. In a phase IV clinical trial, our team has also showed that eMSCs can be collected with ease even from post-menopausal women and kept in an undiferentiated state for clinical use making them an ideal choice for autologous urogynecological application. We further more compared the diferences between the treatment capacity of eMSCs delivered using degradable and non-degradable. STUDY DESIGN, MATERIALS AND METHODS: PLCL polymer and gelatin (1:1, 10%w/w) was electrospun to form uniform nanofbers and assessed for fbre diameter, pore size, hydrophilicity and biomechanical properties. SUSD2+ eMSC were purifed from single cell suspensions obtained from endometrial biopsies from cycling women and by magnetic bead sorting and transfected with mCherry len-tivirus for cell tracking. SEM was used to characterise eMSC incorporation into the nanofber

Introduction: To overcome adverse events associated with transvaginal surgery using non degradable synthetic mesh for treating pelvic organ prolapse (POP), we are developing a tissue engineering construct comprising degradable nanofibrous mesh and endometrial mesenchymal stem/stromal cells (eMSC). The aim of this study was to investigate the in vitro cell-material properties and in vivo foreign body response to a nano-topographically controlled mesh made of poly (L-lactic acid)-copoly( -caprolactone) and gelatin incorporating eMSC (PLCL/G/eMSC). Method(s): PLCL alone and a 1:1 blend with gelatin (w/w) were electrospun from a 10% solution at 1 ml/hr and 18kV over 12.5 cm to form uniform nanofibers and assessed for fibre diameter, pore size, hydrophilicity and biomechanical properties. SUSD2+ eMSC were purified from single cell suspensions obtained from endometrial biopsies from cycling women by magnetic bead sorting and transfected with mCherry lentivirus for cell tracking. SEM was used to characterise eMSC incorporation into the PLCL and PLCL/G nanofiber meshes in vitro and their degradation in NSG mouse skin wound repair model of vaginal repair (2, 6 wks, 4 mice/gp). IF and confocal microscopy was used to assess total (F4/80+), M1 (F4/80+CCR7+) and M2 (F4/80+/CD206+) macrophages. Result(s): Addition of G to PLCL increased fiber size 40% (P<0.05) and pore size 25% (P<0.05) rendering meshes hydrophilic without change in tensile properties. In vitro, eMSC rapidly adhered, penetrated, incorporated and proliferated (P<0.01) in PLCL/G mesh with a 2.5 fold greater coverage than PLCL alone (P<0.05). In vivo, mCherry+ eMSC survived 1 wk in PLCL and PLCL/G implanted mesh, but only a few were retained in PLCL/G and none in PLCL at 6 wk. eMSC prevented PLCL/G nanofiber degradation 2 fold (P <0.0001) and increased cellular infiltration into the mesh 1.5 fold (P<0.05) at 6 wks. eMSC seeded on PLCL/G promoted macrophage switching from M1 to M2 phenotype to a greater extent than s

STUDY QUESTION: Do obese levels of advanced glycation end products (AGEs) within the uterine cavity detrimentally alter tissue function in embryo implantation and placental development? SUMMARY ANSWER: Obese levels of AGEs activate inflammatory signaling (p65 NF?B) within endometrial epithelial cells and alter their function, cause endoplasmic reticulum (ER) stress in endometrial stromal cells and impair decidualization, compromise implantation of blastocyst mimics and inhibit trophoblast invasion. WHAT IS KNOWN ALREADY: Obese women experience a higher incidence of infertility, recurrent miscarriage and pregnancy complications compared with lean women. Oocyte donation cycles suggest a detrimental uterine environment plays a role in these outcomes. STUDY DESIGN, SIZE, DURATION: Uterine lavage and tissues from lean (BMI 19.524.9, n = 17) and obese (BMI > 30, n = 16) women examined. Cell culture experiments utilizing human endometrial epithelial, trophectoderm and trophoblast cell lines and primary human stromal cells used to examine the functional impact of obese levels of AGEs. PARTICIPANTS/MATERIALS, SETTING, METHODS: Levels of AGEs examined within uterine lavage assessed by ELISA to determine differences between lean and obese women. Expression and localization of AGEs, receptor for AGEs (RAGE) and NF?B within endometrial tissues obtained from lean and obese women determined by immunohistochemistry. Endometrial epithelial cells (ECC-1), primary human stromal cells and trophoblast cells (HTR8-SVneo) treated with lean (2000 nmol/mol lysine) or obese (8000 nmol/mol lysine) uterine levels of AGEs and p65 NF?B (western immunoblot), real-time adhesion, proliferation migration and invasion (xCelligence real-time cell function analysis), decidualization (cell morphology and prolactin release), ER stress (western immunoblot for p-PERK) determined. Co-cultures of endometrial epithelial cells and blastocyst mimics (trophectoderm spheroids) similarly treated with lean or obese

The regenerative, proliferative phase of a woman's menstrual cycle is a critical period which lays the foundation for the subsequent, receptive secretory phase. Although endometrial glands and their secretions are essential for embryo implantation and survival, the proliferative phase, when these glands form, has been rarely examined. We hypothesized that alterations in the secreted proteome of the endometrium of idiopathic infertile women would reflect a disturbance in proliferative phase endometrial regeneration. Our aim was to compare the proteomic profile of proliferative phase uterine fluid from fertile (n = 9) and idiopathic infertile (n = 10) women. Proteins with >=2-fold change (P < 0.05) were considered significantly altered between fertile and infertile groups. Immunohistochemistry examined the endometrial localization of identified proteins. Western immunoblotting defined the forms of extracellular matrix protein 1 (ECM1) in uterine lavage fluid. Proteomic analysis identified four proteins significantly downregulated in infertile women compared to fertile women, including secreted frizzled-related protein 4 (SFRP4), CD44, and ECM1: two proteins were upregulated. Seven proteins were unique to the fertile group and six (including isoaspartyl peptidase/L-asparaginase [ASRGL1]) were unique to the infertile group. Identified proteins were classified into biological processes of tissue regeneration and regulatory processes. ASRGL1, SFRP4, and ECM1 localized to glandular epithelium and stroma, cluster of differentiation 44 (CD44) to stroma and immune cells. ECM1 was present in two main molecular weight forms in uterine fluid. Our results indicate a disturbance in endometrial development during the proliferative phase among infertile women, providing insights into human endometrial development and potential therapeutic targets for infertility.Copyright © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Background/Aims: Interleukin (IL)-17A and IL-17F are proinflammatory cytokines, which signal through a receptor complex consisting of IL-17RA and IL-17RC subunits. We sought to define the role of IL-17RA expression by leukocytes and stromal cells in nephritogenic immunity and injury in experimental glomerulonephritis. Method(s): Glomerulonephritis was induced in wild-type and IL-17RA-deficient (IL-17RA-/-) mice by sheep anti-mouse glomerular basement membrane globulin. Renal injury and immune responses were assessed at day 21. Glomerulonephritis was induced in bone marrow (BM) chimeric mice, with either BM or tissue cell (TC) deficiency of IL-17RA. To assess humoral responses, WT and IL-17RA-/- mice were sensitized to sheep globulin and euthanized 10 days later. Result(s): IL-17RA-/- mice had reduced glomerular crescent formation, neutrophils and macrophages compared to wild-type mice, while nephritic BM-TC+ mice developed less glomerular segmental necrosis. IL-17RA expression was required in both BM and TC for maximal systemic interferon-gamma expression. Antigen-specific humoral immune responses were impaired in the absence of IL-17RA. Compared to BM+TC+ mice, glomerular IgG and C3 deposition was reduced in BM+TC- and BM-TC+ mice, respectively. Humoral immunity was also impaired in BM- and TC-deficient chimeras. BM+TC- mice had fewer B cells expressing CXCR5, while IL-17RA-/- mice had abnormal germinal centre development after immunization, with reduced follicular B cell and follicular helper T-cell CXCR5 expression, explaining the impaired humoral immunity. Conclusion(s): IL-17RA contributes to experimental glomerulonephritis, with IL-17RA expression on both leukocytes and stromal cells being required for the full expression of nephritogenic humoral immunity.Copyright © 2016 S. Karger AG, Basel.

Mammographic density (MD) adjusted for age and body mass index is one of the strongest known risk factors for breast cancer. Given the high attributable risk of MD for breast cancer, chemoprevention with a safe and available agent that reduces MD and breast cancer risk would be beneficial. Cox-2 has been implicated in MD-related breast cancer risk, and was increased in stromal cells in high MD tissues in one study. Our study assessed differential Cox-2 expression in epithelial and stromal cells in paired samples of high and low MD human breast tissue, and in a validated xenograft biochamber model of MD. We also examined the effects of endocrine treatment upon Cox-2 expression in high and low MD tissues in the MD xenograft model. Paired high and low MD human breast tissue samples were immunostained for Cox-2, then assessed for differential expression and staining intensity in epithelial and stromal cells. High and low MD human breast tissues were separately maintained in biochambers in mice treated with Tamoxifen, oestrogen or placebo implants, then assessed for percentage Cox-2 staining in epithelial and stromal cells. Percentage Cox-2 staining was greater for both epithelial (p = 0.01) and stromal cells (p < 0.0001) of high compared with low MD breast tissues. In high MD biochamber tissues, percentage Cox-2 staining was greater in stromal cells of oestrogen-treated versus placebo-treated tissues (p = 0.05).

INTRODUCTION: Recently MSC have been identified in the regenerative endometrial lining of the uterus and specific markers (W5C5/ SUSD2) for their prospective isolation identified. Postmenopausal endometrium is atrophic but regenerates with systemic estrogen treatment and has even lead to a successful IVF pregnancy in a 64 year old woman.The aim of this study was to determine whether postmenopausal endometrium contains a population of endometrial MSC (eMSC) that might be responsible for its regenerative capacity which could be exploited for future clinical applications. We hypothesized that eMSC are present in postmenopausal endometrium at similar frequency as in premenopausal endometrium. METHOD(S): Single-cell suspensions of endometrial stromal cells were obtained from hysterectomy tissues of 17 premenopausal (pre-MP), 19 postmenopausal (post-MP) and 15 postmenopausal women enrolled into a phase IV clinical trial of estrogen replacement therapy (2mg oral estradiol valerate daily) (post-MP+E2) for 8 weeks. The proliferative potential and differentiation W5C5+ cells were assessed by colony forming unit CFU assays and differentiation-induction media culture. EMSC surface markers were determined using flow cytometry. RESULT(S): In post-MP+E2 Passage 1 (P1) stromal cultures, 3.7+/-1.8% (n=7) of the cells were W5C5+ cells and 7.7+/-6.3% (n=8) in post-MP cultures (p=0.69). The cloning efficiency for post-MP+E2 W5C5+ P1 cells was 3.41+/-2.71% (n=6), and comparable to post-MP W5C5+ cells (3.73+/-1.19%; n=8) but statistically lower than P1 Pre-MP cells (p>0.05) (n=6). W5C5, CD140b, CD146, CD29, CD44, CD73, CD105 surface markers were present in both PostMP groups. PostMP W5C5+ cells differentiated into chondrocytes, adipocytes, myocytes and osteocytes, but to a lesser degree than PreMP W5C5+ cells. CONCLUSION(S): Postmenopausal endometria possess eMSC at similar frequency and potency irrespective of whether or not it is regenerated with estrogen. Human endometrium is a promis

Background: Fibroblasts have essential roles in deposition of extracellular matrix and regulation of inflammation and are therefore central to wound healing, scar formation and fibrosis, all processes that are central to the pathology of endometriosis. Our aim was to characterise the activation status of stromal fibroblasts in eutopic versus ectopic endometrium from women with endometriosis. Method(s): Endometriotic lesions and eutopic endometrial samples were collected from women undergoing laparoscopy for the treatment of endometriosis (n=4 proliferative, n=7 secretory). Sections containing endometrial glands were immunostained for CD10 to identify endometrial stromal cells. Laser capture microscopy was then used on serial sections to collect glandular epithelium and CD10-postive stroma from ectopic lesions and eutopic endometrium, as well as CD10-negative tissue from around ectopic lesions. After confirming the quality of extracted RNA, quantitative PCR was used to examine the expression of selected genes involved in fibroblast activation (TGFbeta1, NFkappaB1, ACTA2, IFNgamma, SMAD2, 3 and 4). mRNA levels were expressed relative to an RNA spike or 18S rRNA. Result(s): Expression of TGFbeta1 and SMAD4 mRNA was significantly higher in eutopic versus ectopic glandular epithelium and SMAD3 was significantly higher in secretary-phase eutopic glandular epithelium relative to all other groups. However, there was no significant difference in the expression of these genes in eutopic stroma, ectopic CD10-positive stroma or ectopic CD10- negative stroma. There was no significant variation in ACTA2, NFkappaB1, or IFNgamma mRNA expression among any regions examined. Conclusion(s): Variations in TGFbeta1, SMAD3 and SMAD4 mRNA expression in eutopic relative to ectopic glandular epithelium are consistent with a specific function for the TGFbeta pathway in eutopic endometrium that is not replicated in ectopic lesions. However, our mRNA data do not support a difference in fibrobla

Local estrogen biosynthesis in breast adipose tissue, catalyzed by P450 aromatase, contributes to the growth of breast carcinomas. Aromatase expression is regulated by a number of alternative promoters, and in normal adipose tissue it is primarily regulated via the distal promoter I.4. However, in breast adipose containing a tumor, aromatase expression is regulated by the proximal promoter II in response to tumor-derived factors. Previously we have shown that peroxisomal proliferator-activated receptor gamma (PPARgamma) ligands inhibit aromatase expression in normal breast adipose tissue mediated by promoter I.4. In the present study, we investigated the effects of the PPARgamma ligand troglitazone and the retinoid X receptor (RXR) ligand LG101305 on aromatase expression mediated by promoter II. In cultured human breast adipose stromal cells, troglitazone or LG101305 alone inhibited aromatase activity and expression stimulated by inducers of promoter II, in a concentration-dependent manner, and this inhibition was greater in the presence of both ligands. Reporter gene assays showed that troglitazone and LG101305 inhibit transcription from promoter II of the CYP19 gene. However, EMSAs showed that PPARgamma and RXRalpha do not bind to promoter II of the CYP19 gene, indicating that PPARgamma- and RXR-mediated inhibition of aromatase expression via promoter II occurs through an indirect mechanism of action. Because ligands for PPARgamma and RXR inhibit aromatase expression in healthy breast adipose (via promoter I.4), as well as expression induced by tumor-derived factors (via promoter II), such compounds could find utility in the treatment of estrogen-dependent breast cancers.

Granulosa cell tumours of the ovary (GCT) exhibit high expression of estrogen receptor beta (ERbeta). A role for estrogen receptors in these tumours may depend on altered co-activator expression. This study examines the expression of the co-activators SRC-1a/e, SRC-2, SRC-3, SRA, and the corepressors NCoR and SMRT in GCT, epithelial ovarian tumours and normal ovary. No significant difference in the expression of SRC-1, SRC-2, SRC-3 or NCoR and SMRT was found. In particular, there was no correlation of co-activator expression with ERbeta expression. There was a significant upregulation in the expression of the novel RNA co-activator SRA in the serous tumours compared with the other tumour types and normal ovary. The findings suggest that ERbeta may require co-activators, other than members of the SRC family for the modulation of transcription in GCT. © 2004 Elsevier Ireland Ltd. All rights reserved.

The expression of aromatase is regulated in a tissue-specific fashion through alternative use of multiple promoter-specific first exons. To date, eight different first exons have been reported in human aromatase, namely I.1., I.2, I.3. I.4, I.5, PII, 2a, and 1f. Recently, we have found a new putative exon I in a RACE-generated library of THP-1 cells and have conducted studies to characterize this new exon I. We confirmed that the constructs containing -1552/+17 or less flanking sequence of this exon function as a promoter in THP-1 cells, JEG-3 cells and osteoblast-like cells obtained from a human fetus. Results of transfection assays using a series of deletion constructs and mutation constructs indicate that a 1-bp mismatch of the consensus TATA-like box (TTTAAT) and the consensus sequence of the initiator site, which is located 45 bp downstream of the putative TATA box, were functioning cooperatively as a core promoter. The putative transcription site was confirmed by the results of RT-PCR southern blot analysis. We examined the regulation and the expression of this exon, I.6, in several human cells and tissues by RT-PCR Southern blot analysis. THP-1 cells (mononuclear leukemic origin) and JEG-3 cells (choriocarcinoma origin) expressed exon I.6 in serum-free media. The level of expression was increased by serum and phorbol myristyl acetate (PMA) in both cell lines. Adipose stromal cells also expressed exon I.6 in the presence of PMA. In fetal osteoblasts, the expression of exon I.6 was increased most effectively by serum and less so by dexamethasone (DEX) + IL-1beta and DEX + IL-11, whereas induction by serum was suppressed by the addition of DEX. The level of expression was low in granulosa cells in culture and did not change with forskolin. On the other hand, dibutyryl cAMP suppressed PMA-stimulated expression of exon I.6 in THP- 1 cells and adipose stromal cells. This result supports the hypothesis that the expression of exon I.6 is regulated mainly via an AP-1

PURPOSE OF REVIEW: The human endometrium is a dynamic tissue, which undergoes cycles of growth and regression with each menstrual cycle. Endometrial regeneration also follows parturition and extensive resection and occurs in postmenopausal women taking estrogen replacement therapy. It is likely that adult stem/progenitor cells are responsible for this remarkable regenerative capacity. This review discusses the first published evidence for the existence of endometrial stem/progenitor cells in human and mouse endometrium. RECENT FINDINGS: Functional approaches have been used to identify candidate endometrial epithelial and stromal stem/progenitor cells, due to lack of known specific endometrial stem cell markers. Rare clonogenic cells and side population cells have been identified in human endometrial cell populations. In mouse endometrium, rare label-retaining cells have also been identified. The ability of transplanted human endometrial cells to grow endometrial tissue in animal hosts also suggests the presence of stem/progenitor cells. SUMMARY: These initial studies providing the first functional evidence for epithelial and stromal stem/progenitor cells in human and mouse endometrium lay the groundwork for further studies to characterize their stem cell properties. They also provide the impetus to discover specific markers that will enable their prospective isolation and allow their location in normal and pathological endometrium to be determined. © 2007 Lippincott Williams & Wilkins, Inc.

The clinical development of lenalidomide (RevlimidTM), then pomalidomide (ActimidTM) as members of immunomodulatory drugs (IMiDs) for the treatment of multiple myeloma (MM), exemplifies how insight into disease biology can lead to design of effective therapeutic agents. Increased experience and understanding of IMiD's diverse biological effects has lead to rational design of lenalidomide-based treatment-regimens over recent years. However, much about lenalidomide is yet to be understood and fully exploited. Here, we review what is known of lenalidomide's biological effects, clinical certainties and uncertainties in the treatment of MM, and explore its future potential with other synergistic therapeutic agents. © 2012 Wiley Periodicals, Inc.

The expression of an oestrogen receptor-related antigen has been measured by microspectrophotometry in the glandular epithelium and stroma of tissue sections from endometrial biopsies of women administered exogenous replacement of sex steroids because of premature ovarian failure. We have demonstrated that previously dormant endometrium is capable of expressing an antigen related to oestrogen receptors and that its distribution is influenced by the sex steroid environment. Immunohistochemical staining was more intense (P<0.001) in the glandular compared to the stromal cells in tissue exposed solely to oestradiol. With the administration of progesterone, the stromal staining intensity increased (P<0.0001) and that of the glandular staining decreased (P<0.0001). No assumption, however, has been made to infer that colour is stoichiometrically related to the concentration of antigen present. Further validation of immunohistochemical procedures is required because histological localization and measurement of immunohistochemical staining may be straightforward, but its relationship to antigen concentration remains unproven.

To better understand the transcriptional regulation of human endometrial remodeling, the localization of three members of the Ets family of transcription factors was examined at different stages of the menstrual cycle. Elf1 was found by immunohistochemistry to be predominantly localized to the glandular epithelium. In contrast, Ets1 and Ets2 were found at lower intensities in both glandular epithelial and stromal cells. Low expression during the menstrual phase of the cycle, and high expression and intensity of staining in decidualized stromal cells of the late secretory phase were common to Ets1, Ets2, and Elf1. These localization patterns were confirmed in cultured human endometrial stromal and epithelial cells by Western blotting, which also demonstrated different isoforms and phosphorylation products of Ets1 and Ets2 in the two cell types. This study has shown for the first time that members of the Ets family of transcription factors, previously found predominantly during development and in hematopoietic cells, are expressed in the human endometrium and display cell and cycle-stage specificity. Expression of Elf1 predominantly in the glandular epithelium may indicate that Elf1 plays a unique role in epithelium-specific gene regulation in the endometrium.

This review on normal and neoplastic growth of the prostate emphasizes the importance of epithelial-mesenchymal/stromal interactions. Accordingly, during prostatic development urogenital sinus mesenchyme (a) specifies prostatic epithelial identity, (b) induces epithelial bud formation, (c) elicits prostatic bud growth and regulates ductal branching, (d) promotes differentiation of a secretory epithelium, and (e) specifies the types of secretory proteins expressed. In reciprocal fashion, prostatic epithelium induces smooth muscle differentiation in the mesenchyme. Epithelial-mesenchymal interactions during development continue postnatally into adulthood as stromal-epithelial interactions which play a homeostatic role and in so doing reciprocally maintain epithelial and stromal differentiation and growth-quiescence. Prostatic carcinogenesis involves perturbation of these reciprocal homeostatic cell-cell interactions. The central role of mesenchyme in prostatic epithelial development has been firmly established through analysis of tissue recombinants composed of androgen-receptor-positive wild-type mesenchyme and androgen-receptor-negative epithelium. These studies revealed that at the very least ductal morphogenesis, epithelial cytodifferentiation, epithelial apoptosis and epithelial proliferation are regulated by stromal and not epithelial androgen receptors. Likewise, progression from non-tumorigenesis to tumorigenesis elicited by testosterone plus estradiol proceeds via paracrine mechanisms. Thus, stromal-epithelial interactions play critical roles in the hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development. © 2004 Elsevier Ltd. All rights reserved.

BACKGROUND. The actions of the extracellular-matrix degrading enzymes, matrix metalloproteinases (MMPs), are implicated in tumorigenesis. The cellular localization of MMP-2, MMP-9, membrane type 1 (MT1)-MMP, tissue inhibitors of metalloproteinases (TIMPs) 1-3, and the presence of active gelatinases were investigated in endometrial carcinoma. METHODS. Endometrial carcinomas were grouped according to histologic grade (Grades 1-3), depth of myometrial invasion (0, < 50%, > 50%) and the presence of vascular/lymphatic invasion. Twenty-nine endometrial carcinoma biopsies were investigated immunohistochemically to determine the tissue localization of MMP-2 (gelatinase A), MMP-9 (gelatinase B), MT1-MMP, and TIMPs 1-3. In situ hybridization was performed to localize MMP-2 and MMP-9 mRNA. The presence of active gelatinases was assessed using in situ zymography. RESULTS. Epithelial tumor cells were the main site of MMP-2, MMP-9, and MT1-MMP protein. Variable stromal cell localization was also observed, particularly in areas adjacent to tumor nests. Semiquantitative analysis revealed increases in MMP-9 and MMP-2 but not MT1-MMP staining scores in tumor epithelial cells in the transition from histologic Grade 1 to Grades 2 and 3. Matrix metalloproteinase-9 and MT1-MMP staining scores in tumor cells were significantly associated with the presence of myometrial invasion and vascular/lymphatic invasion, while MMP-2 did not correlate with these factors. In addition, MT1-MMP was colocalized with MMP-2, supporting its role in the activation of proMMP-2. Tumor cells from all histologic grades stained intensely for TIMP-2 and TIMP-3 proteins, while variable stromal staining was observed. In Grade 1 carcinomas TIMP-1 was predominantly immunolocalized to the stromal compartment with variable tumor cell localization being observed in Grades 2 and 3 carcinomas. Matrix metalloproteinase-9 and MMP-2 mRNAs were predominantly observed in tumor epithelial cells as well as in the stroma to varyin

The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single-cell suspensions of epithelial and stromal cells were cultured at cloning density (300-500/cm2) in serum medium or in serum-free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-alpha (TGFalpha), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF-I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGFalpha, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF-I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers e

Epidemiologic evidence supports a correlation between obesity and breast cancer in women. AMP-activated protein kinase plays an important role in energy homeostasis and inhibits the actions of cyclic AMP-responsive element binding protein-regulated transcription coactivator 2 (CRTC2). In postmenopausal women, the cyclic AMP-responsive element binding protein-dependent regulation of aromatase is a determinant of breast tumor formation through local production of estrogens. The present work aimed to examine the effect of adipokines on aromatase expression and identify additional mechanisms by which prostaglandin E2 causes increased aromatase expression in human breast adipose stromal cells. Treatment of human adipose stromal cells with forskolin and phorbol 12-myristate 13-acetate (PMA), to mimic prostaglandin E2, resulted in nuclear translocation of CRTC2. Aromatase promoter II (PII) activity assays showed that CRTC2 in addition to forskolin/PMA treatment significantly increased PII-induced activity. CRTC2 binding to PII was examined by chromatin immunoprecipitation, and forskolin/PMA treatment was associated with increased binding to PII. Treatment of human adipose stromal cells with leptin significantly up-regulated aromatase expression associated with nuclear translocation of CRTC2 and increased binding of CRTC2 to PII. Adiponectin treatment significantly decreased forskolin/PMA-stimulated aromatase expression, consistent with the decreased nuclear translocation of CRTC2 and the decreased binding of CRTC2 to PII. The expression and activity of the AMP-activated protein kinase LKB1 was examined and found to be significantly decreased following either forskolin/PMA or leptin treatment. In contrast, adiponectin significantly increased LKB1 expression and activity. In conclusion, the regulation of aromatase by CRTC2, in response to the altered hormonal milieu associated with menopause and obesity, provides a critical link between obesity and breast cancer. ©2009 Ameri

Effective osteoporosis therapy requires agents that increase the amount and/or quality of bone. Any modification of osteoclast-mediated bone resorption by disease or drug treatment, however, elicits a parallel change in osteoblast-mediated bone formation because the processes are tightly coupled. Anabolic approaches now focus on uncoupling osteoblast action from osteoclast formation, for example, by inhibiting sclerostin, an inhibitor of bone formation that does not influence osteoclast differentiation. Here, we report that oncostatin M (OSM) is produced by osteoblasts and osteocytes in mouse bone and that it has distinct effects when acting through 2 different receptors, OSM receptor (OSMR) and leukemia inhibitory factor receptor (LIFR). Specifically, mouse OSM (mOSM) inhibited sclerostin production in a stromal cell line and in primary murine osteoblast cultures by acting through LIFR. In contrast, when acting through OSMR, mOSM stimulated RANKL production and osteoclast formation. A key role for OSMR in bone turnover was confirmed by the osteopetrotic phenotype of mice lacking OSMR. Furthermore, in contrast to the accepted model, in which mOSM acts only through OSMR, mOSM inhibited sclerostin expression in Osmr-/- osteoblasts and enhanced bone formation in vivo. These data reveal what we believe to be a novel pathway by which bone formation can be stimulated independently of bone resorption and provide new insights into OSMR and LIFR signaling that are relevant to other medical conditions, including cardiovascular and neurodegenerative diseases and cancer.

Epithelial and stromal cells from endometria of ovariectomized estradiol-treated Corriedale ewes were separated and purified after collagenase digestion. The separation method utilized differences in the speed and ease of detachment of cultured epithelial and stromal cells attached to plastic in response to brief trypsin exposure. Cells were characterized according to morphological, growth, and histochemical criteria. Contamination of each cell type with the other was <1%. Separated cells were grown on plastic or on Matrigel-coated Millicell inserts with nitrocellulose membranes. Transmission and scanning electron microscope analyses demonstrated the existence of tight junctions and prominent microvilli in the epithelial cultures on inserts but not on plastic. Asymmetrical secretion of prostaglandin F(2alpha) (PGF(2alpha)) and prostaglandin E (PGE) by epithelial cells provided further evidence of polarization. Epithelial cell secretion of PGF(2alpha) was greater than that by stromal cells whereas PGE secretion by stromal cells was greater than that by epithelial cells. Epithelial secretion in the basal direction was approximately 4 and 3 times that of apical secretion for PGF(2alpha) and PGE, respectively. The separation protocol provides pure populations of ovine endometrial epithelial and stromal cells and the cultured epithelial cells exhibit characteristics of in vivo morphology and polarized function.

In situ hybridization histochemistry was used to detect mRNA coding for the plasma proteins alpha1-acid glycoprotein and alpha2-macroglobulin in the rat decidua during the period when the chorioallantoic placenta is established. It was found that alpha1-acid glycoprotein mRNA was localized to a subpopulation of decidual cells predominantly found in the decidua capsularis but extending into the decidua basalis at later times. The highest levels of alpha2-macroglobulin mRNA were found in the decidua basalis where there was some overlap with regions containing alpha1-acid glycoprotein mRNA. No alpha2-macroglobulin mRNA could be found in the inner part of the decidua capsularis where the highest levels of alpha1-acid glycoprotein mRNA were found. However, a thin outer layer of compressed stromal cells adjacent to the myometrium expressed the alpha2-macroglobulin gene which surrounded the cells containing alpha1-acid glycoprotein mRNA. This distribution of alpha2-macroglobulin mRNA is consistent with the hypothesis that the protein is produced locally to prevent non-specific proteolysis which may otherwise result from catabolic processes involved in tissue remodelling. The function of alpha1-acid glycoprotein is unknown but this protein is also likely to be involved in the maintenance of homeostasis during the period when contact between maternal and fetal systems is being established within the chorioallantoic placenta.

Interleukin-11 (IL-11) is crucial in the decidualization response of the uterine stroma to the implanting blastocyst in the mouse. This study examined the localization and expression of IL-11 in human endometrium throughout the menstrual cycle and of prolactin and leukaemia inhibitory factor (LIF) in secretory phase endometrium. The mRNA expression of IL-11 receptor alpha and the signalling component, gp130, in endometrial tissue were also determined. Immunoreactive IL-11 was highest in the secretory phase and present in decidualized stromal cells, glandular epithelial cells, endothelial and smooth muscle cells, and the mRNA expression was verified by in-situ hybridization. Decidual cells showed the most intense staining. IL-11 receptor alpha and gp130 mRNA were detected throughout the cycle with minimal variation. Expression of IL-11 mRNA and protein preceded that of prolactin. While immunoreactive prolactin was found in stromal, decidual and glandular epithelial cells, prolactin mRNA was confined to decidual cells. In contrast, endometrial LIF expression preceded IL-11 but was largely confined to the glandular epithelium. The sequence of appearance of LIF, IL-11 and prolactin suggests a synchronized role for each in the differentiation of the endometrium. The cyclical changes and cell type specific expression of IL-11 suggests a potential role in the decidualization of stromal cells.