Effects of linalool on postoperative peritoneal adhesions in rats.

The current study examines the effects of linalool in preventing postoperative abdominal adhesions. Twenty male Wistar rats were randomly divided into four groups. (1) Sham: in this group, the abdomen was approached, and without any manipulations, it was sutured. (2) Control: rats in this group underwent a surgical procedure to induce adhesions. This involved making three incisions on the right abdominal side and removing a 1×1-cm piece of the peritoneum on the left abdominal side. (3) Treatment groups: these groups underwent the same surgical procedure as the control group to induce adhesions. Animals in these groups received linalool orally with doses of 50 and 100 mg/kg, respectively, for a period of 14 days. Moreover, rats in the sham and control groups received normal saline via gavage for 14 days. The evaluation of TNF-α, TGF-ß, VEGF, and caspase 3 was performed using western blot and IHC methods. Furthermore, oxidative stress biomarkers such as MDA, TAC, GSH, and NO were assessed in the peritoneal adhesion tissue. The findings revealed that linalool significantly reduced peritoneal adhesions by reducing TNF-α, TGF-ß, VEGF, and caspase 3 levels. Moreover, MDA concentration was significantly decreased, while NO, TAC, and GSH levels were notably increased. Overall, linalool was effective in preventing adhesion formation and reduced inflammation, angiogenesis, apoptosis, and oxidative stress. Therefore, linalool as a potent antioxidant is suggested for reducing postoperative adhesions in rats.

Gene expression profiles separate endometriosis lesion subtypes and indicate a sensitivity of endometrioma to estrogen suppressive treatments through elevated ESR2 expression.

BACKGROUND: Endometriosis is a common, gynaecological disease characterised by the presence of endometrial-like cells growing outside the uterus. Lesions appear at multiple locations, present with variation in appearance, size and depth of invasion. Despite hormones being the recommended first-line treatment, their efficacy, success and side effects vary widely amongst study populations. Current, hormonal medication for endometriosis is designed to suppress systemic oestrogen. Whether these hormones can influence the lesions themselves is not yet clear. Evidence of hormone receptor expression in endometriotic lesions and their ability to respond is conflicting. A variation in their expression, activation of transcriptional co-regulators and the potential to respond may contribute to their variation in patient outcomes. Identifying patients who would benefit from hormonal treatments remain an important goal in endometriosis research. METHODS: Using gene expression data from endometriosis lesions including endometrioma (OMA, n = 28), superficial peritoneal lesions (SUP, n = 72) and deeply infiltrating lesions (DIE, n = 78), we performed principal component analysis, differential gene expression and gene correlation analyses to assess the impact of menstrual stage, lesion subtype and hormonal treatment on the gene expression. RESULTS: The gene expression profiles did not vary based on menstrual stage, but could distinguish lesion subtypes with OMA significantly differentiating from both SUP and DIE. Additionally, the effect of oestrogen suppression medication altered the gene expression profile in OMA, while such effect was not observed in SUP or DIE. Analysis of the target receptors for hormonal medication indicated ESR2 was differentially expressed in OMA and that genes that correlated with ESR2 varied significantly between medicated and non-medicated OMA samples. CONCLUSIONS: Our results demonstrate of the different lesion types OMA present with strongest response to hormonal treatment directly through ESR2. The data suggests that there may be the potential to target treatment options to individual patients based on pre-surgical diagnoses.

Endoplasmic reticulum stress and the unfolded protein response in skeletal muscle of subjects suffering from peritoneal sepsis.

We provide a descriptive characterization of the unfolded protein response (UPR) in skeletal muscle of human patients with peritoneal sepsis and a sepsis model of C57BL/6J mice. Patients undergoing open surgery were included in a cross-sectional study and blood and skeletal muscle samples were taken. Key markers of the UPR and cluster of differentiation 68 (CD68) as surrogate of inflammatory injury were evaluated by real-time PCR and histochemical staining. CD68 mRNA increased with sepsis in skeletal muscle of patients and animals (p < 0.05). Mainly the inositol-requiring enzyme 1α branch of the UPR was upregulated as shown by elevated X-box binding-protein 1 (XBP1u) and its spliced isoform (XBP1s) mRNA (p < 0.05, respectively). Increased expression of Gadd34 indicated activation of PRKR-Like Endoplasmic Reticulum Kinase (PERK) branch of the UPR, and was only observed in mice (p < 0.001) but not human study subjects. Selected cell death signals were upregulated in human and murine muscle, demonstrated by increased bcl-2 associated X protein mRNA and TUNEL staining (p < 0.05). In conclusion we provide a first characterization of the UPR in skeletal muscle in human sepsis.

Immunoregulation by type I interferons in the peritoneal cavity.

The peritoneal cavity, a fluid-containing potential space surrounding the abdominal and pelvic organs, is home to a rich network of immune cells that maintain tissue homeostasis and provide protection against infection. However, under pathological conditions such as peritonitis, endometriosis, and peritoneal carcinomatosis, the peritoneal immune system can become dysregulated, resulting in nonresolving inflammation and disease progression. An enhanced understanding of the factors that regulate peritoneal immune cells under both homeostatic conditions and in disease contexts is therefore required to identify new treatment strategies for these often life-limiting peritoneal pathologies. Type I interferons (T1IFNs) are a family of cytokines with broad immunoregulatory functions, which provide defense against viruses, bacteria, and cancer. There have been numerous reports of immunoregulation by T1IFNs within the peritoneal cavity, which can contribute to both the resolution or propagation of peritoneal disease states, depending on the specifics of the disease setting and local environment. In this review, we provide an overview of the major immune cell populations that reside in the peritoneal cavity (or infiltrate it under inflammatory conditions) and highlight their contribution to the initiation, progression, or resolution of peritoneal diseases. Additionally, we will discuss the role of T1IFNs in the regulation of peritoneal immune cells, and summarize the results of laboratory studies and clinical trials which have investigated T1IFNs in peritonitis/sepsis, endometriosis, and peritoneal carcinomatosis.

Keratin Granulomas in the Peritoneum on Frozen Section: A Case Report with Multiple Suspects and the Search for the Culprit.

Keratin granulomas in the peritoneum are a rare finding with multiple etiologies and can be especially challenging for both the pathologist and the surgeon when these lesions are grossly visible. We report a case of a unique frozen section diagnostic scenario of evaluation of keratin granulomas in the peritoneum of a 47-year-old woman in the setting of multiple potential culprits: endometrial endometrioid adenocarcinoma following fertility sparing treatment, and a concurrent dermoid cyst. We discuss the various etiologies of keratin granulomas in the peritoneum, mechanism of their formation, diagnostic significance, as well as implications of fertility sparing treatments. To the best of our knowledge, this is the only case of keratin granulomas in the peritoneum with multiple distinct potential pathologic culprits as well the only case following fertility sparing treatment.

The Inflammatory Feed-Forward Loop Triggered by the Complement Component C3 as a Potential Target in Endometriosis.

The complement system is a major component of humoral innate immunity, acting as a first line of defense against microbes via opsonization and lysis of pathogens. However, novel roles of the complement system in inflammatory and immunological processes, including in cancer, are emerging. Endometriosis (EM), a benign disease characterized by ectopic endometrial implants, shows certain unique features of cancer, such as the capacity to invade surrounding tissues, and in severe cases, metastatic properties. A defective immune surveillance against autologous tissue deposited in the peritoneal cavity allows immune escape for endometriotic lesions. There is evidence that the glandular epithelial cells found in endometriotic implants produce and secrete the complement component C3. Here, we show, using immunofluorescence and RT-qPCR, the presence of locally synthesized C3 in the ectopic endometriotic tissue, but not in the eutopic tissue. We generated a murine model of EM via injection of minced uterine tissue from a donor mouse into the peritoneum of recipient mice. The wild type mice showed greater amount of cyst formation in the peritoneum compared to C3 knock-out mice. Peritoneal washings from the wild type mice with EM showed more degranulated mast cells compared to C3 knock-out mice, consistent with higher C3a levels in the peritoneal fluid of EM patients. We provide evidence that C3a participates in an auto-amplifying loop leading to mast cell infiltration and activation, which is pathogenic in EM. Thus, C3 can be considered a marker of EM and its local synthesis can promote the engraftment of the endometriotic cysts.

Exosomes and their cargo are important regulators of cell function in endometriosis.

Endometriosis is a chronic oestrogen-dependent gynaecological disorder characterized by non-menstrual pelvic pain, infertility and the extrauterine growth of endometrial-like glands and stroma. It has been noted that the eutopic endometrium of women with endometriosis is functionally distinct from that of women without endometriosis. Moreover, ectopic endometrial implants are functionally different from the eutopic endometrium of women with endometriosis. However, the mechanisms directing these differences are ill-defined. It is proposed here that small membrane-bound extracellular vesicles called exosomes are important vehicles in the protection and transport of signalling molecules central to the dysregulation of endometrial function in women with endometriosis. Therefore, a critical review of the literature linking exosomes and their cargo to the pathobiology of endometriosis was conducted. Circulating peritoneal fluid and endometrial cell exosomes contained long non-coding RNA, miRNA and proteins involved in histone modification, angiogenesis and immune modulation that differed significantly in women with endometriosis compared with controls. Moreover, experimental evidence supports a role for exosomes and their cargo in angiogenesis, neurogenesis, immune modulation and endometrial stromal cell invasion. It is therefore suggested that exosomes play an important role in the pathophysiology of endometriosis.

Functional changes of immune cells: signal of immune tolerance of the ectopic lesions in endometriosis?

RESEARCH QUESTION: What is the potential role of immune cells and their inflammatory cytokines in the pathogenesis, development and establishment of endometriosis? DESIGN: Peritoneal fluid from 59 women (43 with endometriosis and 16 controls) who had undergone laparoscopic surgery was analysed. Changes in the population of innate and adaptive immune cells, cytokines, chemokines and growth factor expression were measured by flow cytometry, Luminex Technology and enzyme-linked immunosorbent assay. RESULTS: No differences were found in the frequencies of the innate and adaptive immune cells between women with and without endometriosis. In the peritoneal fluid of women with endometriosis, IL-1ß, IL-1RN, IL-2, IL-4, IL-8, IL-10, IL-12 (p70), IL-17α, FGF2, G-CSF, MCP-1, MIP-1α and TNF-α were significantly increased compared with controls. A correlation between IL-2, MCP-1, MIP-1α, TNF-α and the severity of endometriosis was observed. The concentration of neopterin, a possible biomarker for this disease, was increased in women with endometriosis compared with controls. CONCLUSIONS: The functional activity of immune cells seemed to be reduced despite their numbers remaining unchanged. The data indicate that a shift of TH cytokine profile occurs, which increases the TH1-TH2 ratio. This is driven by the increased levels of the cytokines (TNF-α and IL-2) in women with severe endometriosis.

Characterization and Proteomic Analysis of Endometrial Stromal Cell-Derived Small Extracellular Vesicles.

CONTEXT: Small extracellular vesicles (sEVs) have emerged as modulators of the disease microenvironment, thereby supporting disease progression. However, the potential role of EVs and their content to the pathophysiology of endometriosis remain unclear. OBJECTIVE: This work aimed to investigate whether the EVs from eutopic (Eu) and ectopic (Ec) endometrial stromal cells (ESCs) differ with respect to protein composition and role in endometriosis. METHODS: Human Eu and Ec endometrium-derived ESCs were isolated from samples of the same patients (n = 3). sEVs were isolated from ESCs via ultracentrifugation; these sEVs were characterized by Western blotting, transmission electron microscopy, and nanoparticle tracking analysis and analyzed using mass spectrometry. The potential role of EcESCs-derived sEVs (EcESCs-sEVs) in endometriosis was explored by assaying their effects on cell viability/proliferation, migration, and angiogenesis. RESULTS: In total, 105 ESCs-sEV-associated proteins were identified from EcESCs-sEVs and EuESCs-sEVs by mass spectrometry analysis. The protein content differed between EcESCs-sEVs and EuESCs-sEVs, with annexin A2 (ANXA2) being the most prominent difference-present in EcESCs-sEVs but not EuESCs-sEVs. We also found that sEVs-ANXA2 regulates the motility, proliferation, and angiogenesis of ESCs via the extracellularly regulated kinase (ERK)/STAT3 pathway. Notably, treatment of ESCs with sEVs-ANXA2 resulted in increased proliferation and motility, suggesting that sEVs-ANXA2 may be involved in regulating endometriosis. Our data suggest that EcESCs-sEVs-ANXA2 regulates the motility and the angiogenic potential of ESCs, implying a role for sEVs-ANXA2 in the pathogenesis of endometriosis. CONCLUSION: The study of sEVs-ANXA2 from Ec endometriotic cells uncovers a new mechanism of endometriosis progression and will inform the development of novel therapeutic strategies.

Risk of Bowel Obstruction in Patients with Mesenteric or Peritoneal Disease Receiving Peptide Receptor Radionuclide Therapy.

Although radiation-induced mesenteritis or peritonitis can potentially exacerbate the risk of bowel obstruction, there are no data in the literature on the incidence of intestinal obstruction related to peptide receptor radionuclide therapy. Methods: The records of all patients treated with 177Lu-DOTATATE at Moffitt Cancer Center between April 2018 and October 2019 were evaluated. The number of patients who developed bowel obstruction within 3 mo of a 177Lu-DOTATATE treatment was divided by the total number of patients with preexisting peritoneal or mesenteric disease. Management strategies and outcomes were evaluated. Results: Of a total of 159 patients treated, 81 had baseline mesenteric or peritoneal disease, among whom 5 (6%) experienced at least 1 episode of bowel obstruction within 3 mo of treatment. Two of the patients underwent surgical exploration during obstruction describing a "frozen abdomen." All 5 responded at least temporarily to high-dose corticosteroid treatment and regained bowel function, but 2 patients eventually succumbed to progressive peritoneal disease. Conclusion: Peptide receptor radionuclide therapy can lead to bowel obstruction in patients with mesenteric or peritoneal disease, likely by inducing inflammation. Corticosteroids can potentially play a role in treatment and prophylaxis.

Dopamine agonists as genital endometriosis target therapy.

OBJECTIVE: The present study was to find a pathogenic evidence for dopamine agonist application in patients with endometriosis associated pain syndrome. PATIENTS AND TECHNIQUE: The study involved 227 patients of reproductive age with histologically confirmed genital endometriosis (GE) of I-III degree according to ASRM classification. The control group included 12 women with no laparoscope detected gynecologic pathology. The levels of prolactin (PRL), peripheral blood (PB), and peritoneal fluid (PF) were evaluated by chemiluminescence immune assay. The pain syndrome was measured by McGill visual analogue scale. Statistica10 program (StatSoft, Inc., Tulsa, OK) was applied for obtained data processing. RESULTS: A correlation was established between GE rate and levels of PRL and PB (Rs = 0.28, p < .05) as well as a correlation of PRL in PB and PF (Rs = 0.29, p < .05). Patients receiving cabergoline combined with hormone therapy standard schemes manifested considerable pain syndrome relief. CONCLUSIONS: PRL involvement in GE pathogenesis and more intense therapeutic impact on pain syndrome in case of combined administration of dopamine and standard hormone therapy prove cabergoline application in clinical practice.

Mesothelial Cells Participate in Endometriosis Fibrogenesis Through Platelet-Induced Mesothelial-Mesenchymal Transition.

CONTEXT: While fibrosis in endometriosis has recently loomed prominently, the sources of myofibroblasts, the principal effector cell in fibrotic diseases, remain largely obscure. Mesothelial cells (MCs) can be converted into myofibroblasts through mesothelial-mesenchymal transition (MMT) in many fibrotic diseases and adhesion. OBJECTIVE: To evaluate whether MCs contribute to the progression and fibrogenesis in endometriosis through MMT. SETTING, DESIGN, PATIENTS, INTERVENTION, AND MAIN OUTCOME MEASURES: Dual immunofluorescence staining and immunohistochemistry using antibodies against calretinin, Wilms' tumor-1 (WT-1), and α-smooth muscle actin (α-SMA) were performed on lesion samples from 30 patients each with ovarian endometrioma (OE) and deep endometriosis (DE), and 30 normal endometrial (NE) tissue samples. Human pleural and peritoneal MCs were co-cultured with activated platelets or control medium with and without neutralization of transforming growth factor ß1 (TGF-ß1) and/or platelet-derived growth factor receptor (PDGFR) and their morphology, proliferation, and expression levels of genes and proteins known to be involved in MMT were evaluated, along with their migratory and invasive propensity, contractility, and collagen production. RESULTS: The number of calretinin/WT-1 and α-SMA dual-positive fibroblasts in OE/DE lesions was significantly higher than NE samples. The extent of lesional fibrosis correlated positively with the lesional α-SMA staining levels. Human MCs co-cultured with activated platelets acquire a morphology suggestive of MMT, concomitant with increased proliferation, loss of calretinin expression, and marked increase in expression of mesenchymal markers. These changes coincided with functional differentiation as reflected by increased migratory and invasive capacity, contractility, and collagen production. Neutralization of TGF-ß1 and PDGFR signaling abolished platelet-induced MMT in MCs. CONCLUSIONS: MCs contribute to lesional progression and fibrosis through platelet-induced MMT.

Reprogramming of Mesothelial-Mesenchymal Transition in Chronic Peritoneal Diseases by Estrogen Receptor Modulation and TGF-ß1 Inhibition.

In chronic peritoneal diseases, mesothelial-mesenchymal transition is determined by cues from the extracellular environment rather than just the cellular genome. The transformation of peritoneal mesothelial cells and other host cells into myofibroblasts is mediated by cell membrane receptors, Transforming Growth Factor ß1 (TGF-ß1), Src and Hypoxia-inducible factor (HIF). This article provides a narrative review of the reprogramming of mesothelial mesenchymal transition in chronic peritoneal diseases, drawing on the similarities in pathophysiology between encapsulating peritoneal sclerosis and peritoneal metastasis, with a particular focus on TGF-ß1 signaling and estrogen receptor modulators. Estrogen receptors act at the cell membrane/cytosol as tyrosine kinases that can phosphorylate Src, in a similar way to other receptor tyrosine kinases; or can activate the estrogen response element via nuclear translocation. Tamoxifen can modulate estrogen membrane receptors, and has been shown to be a potent inhibitor of mesothelial-mesenchymal transition (MMT), peritoneal mesothelial cell migration, stromal fibrosis, and neoangiogenesis in the treatment of encapsulating peritoneal sclerosis, with a known side effect and safety profile. The ability of tamoxifen to inhibit the transduction pathways of TGF-ß1 and HIF and achieve a quiescent peritoneal stroma makes it a potential candidate for use in cancer treatments. This is relevant to tumors that spread to the peritoneum, particularly those with mesenchymal phenotypes, such as colorectal CMS4 and MSS/EMT gastric cancers, and pancreatic cancer with its desmoplastic stroma. Morphological changes observed during mesothelial mesenchymal transition can be treated with estrogen receptor modulation and TGF-ß1 inhibition, which may enable the regression of encapsulating peritoneal sclerosis and peritoneal metastasis.

Progesterone receptor ligands for the treatment of endometriosis: the mechanisms behind therapeutic success and failure.

BACKGROUND: Despite intense research, it remains intriguing why hormonal therapies in general and progestins in particular sometimes fail in endometriosis. OBJECTIVE AND RATIONALE: We review here the action mechanisms of progesterone receptor ligands in endometriosis, identify critical differences between the effects of progestins on normal endometrium and endometriosis and envisage pathways to escape drug resistance and improve the therapeutic response of endometriotic lesions to such treatments. SEARCH METHODS: We performed a systematic Pubmed search covering articles published since 1958 about the use of progestins, estro-progestins and selective progesterone receptor modulators, to treat endometriosis and its related symptoms. Two reviewers screened the titles and abstracts to select articles for full-text assessment. OUTCOMES: Progesterone receptor signalling leads to down-regulation of estrogen receptors and restrains local estradiol production through interference with aromatase and 17 beta-hydroxysteroid dehydrogenase type 1. Progestins inhibit cell proliferation, inflammation, neovascularisation and neurogenesis in endometriosis. However, progesterone receptor expression is reduced and disrupted in endometriotic lesions, with predominance of the less active isoform (PRA) over the full-length, active isoform (PRB), due to epigenetic abnormalities affecting the PGR gene transcription. Oxidative stress is another mechanism involved in progesterone resistance in endometriosis. Among the molecular targets of progesterone in the normal endometrium that resist progestin action in endometriotic cells are the nuclear transcription factor FOXO1, matrix metalloproteinases, the transmembrane gap junction protein connexin 43 and paracrine regulators of estradiol metabolism. Compared to other phenotypes, deep endometriosis appears to be more resistant to size regression upon medical treatments. Individual genetic characteristics can affect the bioavailability and pharmacodynamics of hormonal drugs used to treat endometriosis and, hence, explain part of the variability in the therapeutic response. WIDER IMPLICATIONS: Medical treatment of endometriosis needs urgent innovation, which should start by deeper understanding of the disease core features and diverse phenotypes and idiosyncrasies, while moving from pure hormonal treatments to drug combinations or novel molecules capable of restoring the various homeostatic mechanisms disrupted by endometriotic lesions.

Oestrogen receptors and hypoxia inducible factor 1 alpha expression in abdominal wall endometriosis.

RESEARCH QUESTION: What are the protein levels and localization of oestrogen receptors (including ERa, ERb and G protein-coupled oestrogen receptor [GPER]) and hypoxia-inducible factor-1alpha (HIF-1a) in normal control endometrium (COEM) and ectopic endometrium from abdominal wall endometriosis (AWE). DESIGN: AWE (n = 20) were obtained during surgery; COEM (n = 40) were collected by curettage. All tissues were obtained during the proliferative or secretory phase. Formalin-fixed paraffin-embedded tissues were used for immunohistochemical study for oestrogen receptors and HIF-1a proteins. RESULT(S): The expression of oestrogen receptors and HIF-1a in AWE differed from that in the corresponding menstrual cycle phase of COEM. Compared with COEM, ERa and HIF-1a were decreased whereas ERb and GPER were increased in AWE. The greatest difference was in GPER, with increased protein expression in both the cytoplasm and nucleus of endometrial epithelial and stromal cells, as well as a distinct change in localization from cytoplasmic expression to nuclear and cytoplasmic expression, compared with COEM. CONCLUSIONS: Our data suggest that the expression changes of oestrogen receptors and HIF-1a, especially GPER, are associated with AWE, which may provide new clues to understanding the cause of endometriosis.

LncRNA MEG3-210 regulates endometrial stromal cells migration, invasion and apoptosis through p38 MAPK and PKA/SERCA2 signalling via interaction with Galectin-1 in endometriosis.

BACKGROUND: Endometriosis is a benign gynaecological disease with malignant characteristics that severely affects women's quality of life. Long noncoding RNA maternally expressed gene 3 (LncRNA MEG3) is a tumour suppressor that is downregulated in various cancer cells and tissues, and regulates multiple biological processes. Emerging studies have revealed that the interactions between MEG3 and proteins are involved in disease progression. Galectin-1 affects cell motility, signal transduction and vascularization, and is overexpressed in endometriosis. Our study is the first to explore the role of MEG3-210 transcript in endometriosis and to reveal the regulatory mechanism mediated by the interaction between MEG3-210 and Galectin-1. MATERIALS AND METHODS: Endometrial tissues and sera from patients with endometriosis and controls were collected. qRT-PCR was performed to detect the expression of MEG3-210 in the endometrium and endometrial stromal cells (ESCs). The CCK-8 assay, the Transwell assay, flow cytometry and animal models were conducted to evaluate the functions of MEG3-210 in vitro and in vivo. Bioinformatic analysis, Western blot assays, RNA-pull down assays and RNA immunoprecipitation were used to explore the potential mechanism of MEG3-210 in endometriosis. RESULTS: Our results showed that MEG3-210 expression was lower in the eutopic endometrium of women with endometriosis. MEG3-210 downregulation promoted ESCs migration, invasion, anti-apoptosis in vitro and growth of endometriotic lesions in vivo. Furthermore, MEG3-210 downregulation could activate p38 mitogen-activated protein kinase (p38 MAPK) and inhibit cAMP-dependent protein kinase A/sarcoplasmic reticulum Ca2+ ATPase 2 (PKA/SERCA2) signalling, which was mediated by Galectin-1. The protein levels of Galectin-1 in patients with endometriosis were elevated, and Galectin-1 siRNA could reduce the size of lesions. CONCLUSION: MEG3-210 regulates ESCs through p38 MAPK and PKA/SERCA signalling via interaction with Galectin-1. The novel regulatory mechanism may provide new insights into drug therapy and the diagnosis of endometriosis.

Interleukin-6 (IL-6) Activates the NOTCH1 Signaling Pathway Through E-Proteins in Endometriotic Lesions.

CONTEXT: NOTCH signaling is activated in endometriotic lesions, but the exact mechanisms remains unclear. IL-6, which is increased in the peritoneal fluid of women with endometriosis, induces NOTCH1 through E-proteins including E2A and HEB in cancer. OBJECTIVE: To study the role of E-proteins in inducing NOTCH1 expression under the regulation of IL-6 in endometriosis. SETTING AND DESIGN: The expression of E-proteins and NOTCH1 was first investigated in endometrium of women with endometriosis and the baboon model of endometriosis. Regulation of E-proteins and NOTCH1 expression was examined after IL-6 stimulation and siRNA mediated inhibition of E2A or/and HEB in human endometriotic epithelial cells (12Z) in vitro, and subsequently following IL-6 treatment in the mouse model of endometriosis in vivo. RESULTS: E2A, HEB, and NOTCH1 were significantly upregulated in glandular epithelium (GE) of ectopic endometrium compared to eutopic endometrium in both women and the baboon model. IL-6 treatment upregulated the expression of NOTCH1 together with E2A and HEB in 12Z cells. Small interfering RNA inhibition of E2A and HEB or HEB alone decreased NOTCH1 expression. Binding efficiency of both E2A and HEB was significantly higher at the binding sites on the human NOTCH1 promoter after IL-6 treatment. Finally, IL-6 treatment resulted in a significantly increased number of endometriotic lesions along with increased expression of E2A, HEB, and NOTCH1 in GE of the lesions compared with the vehicle group in an endometriosis mouse model. CONCLUSIONS: IL-6 induced NOTCH1 expression is mediated by E-proteins in the ectopic GE cells, which may promote endometriotic lesion development.

Cytokine and growth factor profile in endometriosis: a multiplex analysis of peritoneal fluid to assess diagnostic utility.

We aimed to assay cytokines and growth factors in peritoneal fluid samples from women with and without endometriosis to understand the inflammatory milieu, and assess their potential diagnostic utility. This cross-sectional study conducted at a tertiary care hospital included 54 women, aged 20-45 years, with regular menstrual history and undergoing diagnostic/therapeutic laparoscopy for infertility and/or pain. Peritoneal fluid samples were collected after insertion of trocar & laparoscope but prior to other surgical intervention. A multiplex immunoassay of 27 cytokines and growth factors was performed. The concentration of FGF2 and CSF3 were significantly lower in women with endometriosis than without endometriosis (p = .043 and .003, respectively). The levels of CCL2 and IL1RN were significantly higher in moderate-severe than in minimal-mild endometriosis (p = .038 and .043, respectively). Phase-specific comparison revealed that in proliferative phase, the levels of CSF2 and CSF3 were lower in women with endometriosis than without the disease (p = .047 and .013, respectively). The ROC curve analysis provided a cutoff value 0.78 and 0.76 for FGF2 and CSF3, respectively. Cytokines and growth factors such as FGF2, CSF3, CSF2, CCL2 and IL1RN seem to contribute to the pathogenesis of endometriosis and may have a potential utility for the diagnosis of endometriosis.

Interleukin-1ß inhibits estrogen receptor-α, progesterone receptors A and B and biomarkers of human endometrial stromal cell differentiation: implications for endometriosis.

Human blastocyst nidation in the uterus and successful pregnancy require coordinated endometrial expression of estrogen receptor (ER)-α, progesterone receptors (PR)-A and -B and the gap junction protein, connexin (Cx)43. Our prior work established that inflammation associated with conditions of reduced fecundity, particularly endometriosis, can perturb eutopic decidual function. In the current studies, we have modeled endometrial decidualization in primary human endometrial stromal cell cultures derived from normal controls (NESC) and from the eutopic endometria of women with endometriosis (EESC) to test the hypothesis that a proinflammatory cytokine, interleukin (IL)-1ß, can disrupt stromal cell differentiation. The cells were grown under a standard protocol with hormones (10 nM 17ß-estradiol, 100 nM progesterone and 0.5 mM dibutyryl cAMP) for up to 7 days in the absence or presence of IL-1ß. Time-course experiments showed that IL-1ß compromised decidual function in both NESC and EESC, which was accompanied by rapid phosphorylation of ER-α, PR and Cx43 and their cellular depletion. Inhibition of the extracellular signal-regulated kinase (ERK)1/2 pathway by a selective pharmacological blocker (PD98059) or siRNA interference, or the addition of hormones themselves, blocked the phosphorylation of ERK mediators; increased the production of steroid receptors, Cx43, prolactin, insulin-like growth factor binding protein-1 (IGFBP)-1 and vascular endothelial growth factor (VEGF) and accelerated the differentiation. The results indicate that inhibition of IL-1ß can enhance decidualization in NESC and EESC in vitro. Strategies to interfere with this pathway might be implemented as an in vivo approach to enhance fertility in women with endometriosis and, potentially, other inflammatory pathologies.

20(S)-Ginsenoside Rg3-loaded electrospun membranes to prevent postoperative peritoneal adhesion.

Postoperative peritoneal adhesions are one of the most common surgical complications. In this study, we developed a 20(S)-ginsenoside Rg3-loaded methoxy poly (ethylene glycol)-block-poly(L-lactide-co-glycolide) (mPEG-b-PLGA) electrospun membrane (PEM/Rg3) that could not only serve as a physical barrier, but also as a drug delivery system that releases 20(S)-ginsenoside Rg3 constantly to prevent postoperative peritoneal adhesions. The characteristics of PEM/Rg3, including scanning electron microscopy, water contact angle, and mechanical analyses, were assessed. Degradation and drug release assays of PEM/Rg3 were performed. The anti-adhesion efficacy of PEM/Rg3 was evaluated in an abdomen-cecum mouse model. The adhesion scores, adhesion areas, hematoxylin and eosin (H&E) staining, immunofluorescence, and western blotting were assessed. The 20(S)-ginsenoside Rg3 loaded mPEG-b-PLGA electrospun fibers were successfully fabricated. The fibers were smooth, with no obvious drug crystals. PEM/Rg3 membranes were biodegradable and could be degraded gradually to release 20(S)-Ginsenoside Rg3 constantly from the membranes. The animal study showed that PEM/Rg3 exhibited an excellent adhesion prevention ability when compared with the control group, the PEM group, and polylactic acid (PLA) commercial membrane (Surgiwrap™) group. Immunofluorescence and western blotting studies showed that PEM/Rg3 inhibited the expressions of interleukin 1 (IL-1), interleukin 6 (IL-6), and reactive oxygen species modulator-1 (ROMO1). The 20(S)-ginsenoside Rg3-loaded mPEG-b-PLGA electrospun membranes exhibited satisfactory anti-adhesion efficacy by inhibiting inflammatory responses and oxidative stress. This composite represents a promising strategy to prevent postoperative peritoneal adhesions.