Cells Of Endometriotic Lesions Research Articles

ASPN Is a Potential Biomarker and Associated with Immune Infiltration in Endometriosis.

Objective: Endometriosis is a benign gynecological disease characterized by distant metastasis. Previous studies have discovered abnormal numbers and function of immune cells in endometriotic lesions. We aimed to find potential biomarkers of endometriosis and to explore the relationship between ASPN and the immune microenvironment of endometriosis. Methods: We obtained the GSE141549 and GSE7305 datasets containing endometriosis and normal endometrial samples from the Gene Expression Omnibus database (GEO). In the GSE141549 dataset, differentially expressed genes (DEGs) were found. The Least Absolute Shrinkage and Selection Operator (Lasso) regression and generalized linear models (GLMs) were used to screen new biomarkers. The expression levels and diagnostic utility of biomarkers were assessed in GSE7305, and biomarker expression levels were further validated using qRT-PCR and western blot. We identified DEGs between high and low expression groups of key biomarkers. Enrichment analysis was carried out to discover the target gene’s biological function. We analyzed the relationship between key biomarker expression and patient clinical features. Finally, the immune cells that infiltrate endometriosis were assessed using the Microenvironment Cell Population-Counter (MCP-counter), and the correlation of biomarker expression with immune cell infiltration and immune checkpoints genes was studied. Results: There were a total of 38 DEGs discovered. Two machine learning techniques were used to identify 10 genes. Six biomarkers (SCG2, ASPN, SLIT2, GEM, EGR1, and FOS) had good diagnostic efficiency (AUC > 0.7) by internal and external validation. We excluded previously reported related genes (SLIT2, EGR1, and FOS). ASPN was the most significantly differentially expressed biomarker between normal and ectopic endometrial tissues, as verified by qPCR. The western blot assay revealed a significant upregulation of ASPN expression in endometriotic tissues. The investigation for DEGs in the ASPN high- and low-expression groups revealed that the DEGs were particularly enriched in extracellular matrix tissue, vascular smooth muscle contraction, cytokine interactions, the calcium signaling pathway, and the chemokine signaling pathway. High ASPN expression was related to r-AFS stage (p = 0.006), age (p = 0.03), and lesion location (p < 0.001). Univariate and multivariate logistic regression analysis showed that ASPN expression was an independent influencing factor in patients with endometriosis. Immune cell infiltration analysis revealed a significant increase in T-cell, B-cell, and fibroblast infiltration in endometriosis lesions; cytotoxic lymphocyte, NK-cell, and endothelial cell infiltration were reduced. Additionally, the percentage of T cells, B cells, fibroblasts, and endothelial cells was favorably connected with ASPN expression, while the percentage of cytotoxic lymphocytes and NK cells was negatively correlated. Immune checkpoint gene (CTLA4, LAG3, CD27, CD40, and ICOS) expression and ASPN expression were positively associated. Conclusions: Increased expression of ASPN is associated with immune infiltration in endometriosis, and ASPN can be used as a diagnostic biomarker as well as a potential immunotherapeutic target in endometriosis.

Detection of Cannabinoid Receptor Expression by Endometriotic Lesions in Women with Endometriosis as an Alternative to Opioid-Based Pain Medication.

Emerging information suggests a potential role of medicinal cannabis in pain medication in addition to enhancing immune functions. Endometriosis is a disease of women of reproductive age associated with infertility and reproductive failure as well as chronic pain of varying degrees depending on the stage of the disease. Currently, opioids are being preferred over nonsteroidal anti-inflammatory drugs (NSAID) due to the latter's side effects. However, as the opioids are becoming a source of addiction, additional pain medication is urgently needed. Cannabis offers an alternative therapy for treating the pain associated with endometriosis. Information on the use and effectiveness of cannabis against endometriotic pain is lacking. Moreover, expression of receptors for endocannabinoids by the ovarian endometriotic lesions is not known. The goal of this study was to examine whether cannabinoid receptors 1 and 2 (CB1 and CB2) are expressed by ovarian endometriotic lesions. Archived normal ovarian tissues, ovaries with endometriotic lesions, and normal endometrial tissues were examined for the presence of endometrial stromal cells using CD10 (a marker of endometrial stromal cells). Expression of CB1 and CB2 were determined by immunohistochemistry, immunoblotting, and gene expression studies. Intense expression for CB1 and CB2 was detected in the epithelial cells in ovarian endometriotic lesions. Compared with stroma in ovaries with endometriotic lesions, the expression of CB1 and CB2 was significantly higher in the epithelial cells in endometriotic lesions in the ovary (P < 0.0001 and P < 0.05, respectively). Immunoblotting and gene expression assays showed similar patterns for CB1 and CB2 protein and CNR1 (gene encoding CB1) and CNR2 (gene encoding CB2) gene expression. These results suggest that ovarian endometriotic lesions express CB1 and CB2 receptors, and these lesions may respond to cannabinoids as pain medication. These results will form a foundation for a clinical study with larger cohorts.

Enhanced local and systemic inflammatory cytokine mRNA expression in women with endometriosis evokes compensatory adaptive regulatory mRNA response that mediates immune suppression and impairs cytotoxicity.

Endometriosis is a disease characterized by ectopic implantation of endometrium and impaired immune responses. To explore its pathogenic mechanisms, we studied the local and systemic cytokine mRNA profiles and their role in the immunity of patients with endometriosis and healthy controls. mRNA for eleven cytokines defining cytotoxic Th1, humoral Th2, regulatory Tr1/Th3, and inflammatory cytokine profiles was characterized locally in endometriotic tissue and endometrium, and systemically in PBMCs from women with endometriosis and healthy controls, using real-time qRT-PCR. In addition, immunohistochemical stainings with monoclonal antibodies were performed looking for T regulatory cells in endometriotic lesions. We found a downregulation of mRNA for cytokines mediating cytotoxicity and antibody response and an upregulation of inflammatory and T-regulatory cytokines in the endometriotic tissues and endometrium from the patients with endometriosis, suggesting enhanced local inflammation and priming of an adaptive regulatory response. Consistent with those findings, there was an abundancy of T regulatory cells in the endometriotic lesions. The ectopic implantation seen in endometriosis could be possible as a consequence of increased inflammation and priming of adaptive T regulatory cells, resulting in impaired cytotoxicity and enhanced immune suppression.

Interleukin-15 and Interleukin-7 are the Major Cytokines to Maintain Endometriosis

Objective: The objective of this study was to evaluate cytokines related to natural killer and T-regulatory cells in endometriotic lesions, peritoneal fluid (PF) and the peripheral blood (PB) of patients with deep infiltrative endometriosis. Study Design: A case-control study was conducted in a tertiary referral hospital. Sixty-four consecutive patients after laparoscopy were divided into 2 groups: with endometriosis (Group A – n = 32) and without endometriosis (Group B – n = 32). Main Outcome Measures: Interleukin (IL)-2, IL-4, IL-7, IL-10, IL-12, IL-15, transforming growth factor β1, and IFNγ concentration was measured using a Luminex^TM multiplex suspension bead array. Tissues from endometriotic lesions of patients with endometriosis and from eutopic endometrium were evaluated, as well as PF and PB of all patients. Results: Compared to the other analyzed groups, IL-15 concentration was significantly higher in the ectopic endometrium and IL-7 in the eutopic endometrium of the endometriosis group (p < 0.05). Compared to endometriosis group, IFNγ, IL-7, and IL-15 were observed to be significantly higher in the PF of the control group, and IL-10 was lower in the control group (p < 0.05). In PB, compared to endometriosis group, IL-4, IL-10, IL-12, IL-15, and IFNγ concentrations were significantly higher in the control group (p < 0.05). Conclusions: Our hypothesis is that deep endometriosis is a disease out of control. This disease’s nature is of progression and invasion of adjacent structures, and proof of this disease state is the disorganized secretion of cytokine regulation and inflammation, which seem to be among the factors responsible for the maintenance of the disease.

The expression and the role of bradykinin (BK) receptor in endometriosis

Objective: Endometriosis is a gynecological condition of which the primary symptoms are infertility and pelvic pain. Especially for infertility women, a new therapy which possesses reproductive expectation is needed. Hence, it is essential to explore inflammatory substances which have strong impact on the disease. Bradykinin (BK) is one of the most algogenic substances, also known as an inflammatory mediator in various diseases. Therefore, BK attracts much attention as a therapeutic target. As a first step toward understanding a possible role of bradykinin in endometriosis, we investigated the presence and the regulation of BK receptors in human endometriotic tissues. Material and method: This study protocol was approved by the institutional review boards. The expression of BK receptor (type I and II) in human endometriotic lesions was investigated by immunohistochemistory. Endometriotic stromal cells (ESC) were cultured with IL-1 (10ng/ml), followed by quantitative-PCR for BK receptors. In some experiments, after priming with IL-1 for 24h, ESC were stimulated with BK (1 M) for 8h, followed by quantitative-PCR for IL-6, COX-2 and endothelin-1. Result: Immunohistochemisty showed that BK receptors were present in epithelial and stromal cells in endometriotic lesions. IL-1 induced 10-fold increase in BK receptors mRNA in ESC (p<0.01). IL-1 induced IL-6 and COX-2 significantly (p<0.01) but not endothelin-1 mRNA. On addition of BK, IL-6 and COX-2 mRNA expressions were further enhanced to the levels of approximately 2-fold compared to IL-1 only (p<0.01). Endothelin-1 was increased 2-fold in ESC treated with IL-1 and BK, compared with control. These increases of mRNA expressions by BK were completely abrogated by 1 M HOE-140, an antagonist of BK receptor (p<0.01). Conclusion: In endometriotic lesions, BK receptors were present and served as chemical mediator. We showed that a strategy for blockingBK receptorsmight be anew therapy for endometriosis from the point of suppressing inflammation and pelvic pain.

Crucial role of hypoxia inducible factor in embryo implantation

Objective: Endometriosis is a gynecological condition of which the primary symptoms are infertility and pelvic pain. Especially for infertility women, a new therapy which possesses reproductive expectation is needed. Hence, it is essential to explore inflammatory substances which have strong impact on the disease. Bradykinin (BK) is one of the most algogenic substances, also known as an inflammatory mediator in various diseases. Therefore, BK attracts much attention as a therapeutic target. As a first step toward understanding a possible role of bradykinin in endometriosis, we investigated the presence and the regulation of BK receptors in human endometriotic tissues. Material and method: This study protocol was approved by the institutional review boards. The expression of BK receptor (type I and II) in human endometriotic lesions was investigated by immunohistochemistory. Endometriotic stromal cells (ESC) were cultured with IL-1 (10ng/ml), followed by quantitative-PCR for BK receptors. In some experiments, after priming with IL-1 for 24h, ESC were stimulated with BK (1 M) for 8h, followed by quantitative-PCR for IL-6, COX-2 and endothelin-1. Result: Immunohistochemisty showed that BK receptors were present in epithelial and stromal cells in endometriotic lesions. IL-1 induced 10-fold increase in BK receptors mRNA in ESC (p<0.01). IL-1 induced IL-6 and COX-2 significantly (p<0.01) but not endothelin-1 mRNA. On addition of BK, IL-6 and COX-2 mRNA expressions were further enhanced to the levels of approximately 2-fold compared to IL-1 only (p<0.01). Endothelin-1 was increased 2-fold in ESC treated with IL-1 and BK, compared with control. These increases of mRNA expressions by BK were completely abrogated by 1 M HOE-140, an antagonist of BK receptor (p<0.01). Conclusion: In endometriotic lesions, BK receptors were present and served as chemical mediator. We showed that a strategy for blockingBK receptorsmight be anew therapy for endometriosis from the point of suppressing inflammation and pelvic pain.

Aberrant expression of the pluripotency marker SOX-2 in endometriosis

Expression of the pluripotency factors SOX-2, OCT-4, KLF-4, and NANOG was analyzed by quantitative real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence microscopy in the endometrium, myometrium, and endometriotic tissue of 36 patients. Aberrant expression of SOX-2 may indicate a stem cell origin of endometriosis, whereas the presence of all progenitor markers in endometrial tissue marks the endometrium as a potential source for induced pluripotent stem cell generation.

The Immunoconjugate “Icon” Targets Aberrantly Expressed Endothelial Tissue Factor Causing Regression of Endometriosis

Endometriosis is a major cause of chronic pain, infertility, medical and surgical interventions, and health care expenditures. Tissue factor (TF), the primary initiator of coagulation and a modulator of angiogenesis, is not normally expressed by the endothelium; however, prior studies have demonstrated that both blood vessels in solid tumors and choroidal tissue in macular degeneration express endothelial TF. The present study describes the anomalous expression of TF by endothelial cells in endometriotic lesions. The immunoconjugate molecule (Icon), which binds with high affinity and specificity to this aberrant endothelial TF, has been shown to induce a cytolytic immune response that eradicates tumor and choroidal blood vessels. Using an athymic mouse model of endometriosis, we now report that Icon largely destroys endometriotic implants by vascular disruption without apparent toxicity, reduced fertility, or subsequent teratogenic effects. Unlike antiangiogenic treatments that can only target developing angiogenesis, Icon eliminates pre-existing pathological vessels. Thus, Icon could serve as a novel, nontoxic, fertility-preserving, and effective treatment for endometriosis.

Endometriotic stromal cells lose the ability to regulate cell-survival signaling in endometrial epithelial cells in vitro

In normal endometrium, stromal factors regulate the growth of epithelial cells. However, epithelial cells in endometriotic lesions display increased proliferation and decreased apoptosis. This work tested the hypothesis that in endometriosis stromal cells lose the ability to regulate survival signaling and cell growth in epithelial cells. Primary normal, endometriotic eutopic and ectopic epithelial cells were cultured in the presence of medium conditioned by normal, eutopic and ectopic endometriotic endometrial stromal cells. Endometriotic epithelial cells showed higher Survivin expression than normal epithelial cells. Conditioned medium (CM) from normal or eutopic endometriotic stromal cells significantly inhibited the Survivin expression and AKt phosphorylation in normal or eutopic endometriotic epithelial cells. However, CM from ectopic endometriotic stromal cells did not have an inhibitory effect on normal or ectopic endometriotic epithelial cells. Inhibition of AKt phosphorylation and Survivin expression in normal or eutopic endometriotic epithelial cells in the presence of stromal factors from normal or eutopic endometriotic stromal cells was enhanced by progesterone, whereas progesterone had little effect in the presence of stromal factors from ectopic endometriotic stromal cells. The inability of ectopic endometriotic stromal cells to regulated PI3K/AKt/Survivin signaling and mediate the progesterone response in endometriotic epithelial cells may facilitate epithelial cell proliferation in endometriosis and promote the survival of endometriotic lesions.

Macrophages and nerve fibres in peritoneal endometriosis

Endometriosis is considered to be an inflammatory disease, and macrophages are the most numerous immune cells in endometriotic lesions. However, the mechanisms underlying the elevation of macrophages and their role in the pathogenesis and manifestations of endometriosis still remain unclear. The number of macrophages stained for CD68 in endometriotic lesions (n = 24) and in peritoneum distant from the lesions (n = 14) from women with endometriosis was compared with the number of macrophages in normal peritoneum from women without endometriosis (n = 18). Peritoneal lesions were also double-stained for CD68 and protein gene product 9.5 to study the relationship between macrophages and nerve fibres. The densities of macrophages in peritoneal endometriotic lesions and unaffected peritoneum from women with endometriosis were both significantly higher than that in normal peritoneum from women without endometriosis (P < 0.001). More nerve fibres were also found in the areas where increased numbers of macrophages were identified. There was a significant elevation of macrophages in both normal peritoneum and peritoneal lesions from women with endometriosis compared with normal peritoneum from women without endometriosis. These cells may well play roles in the growth and development of endometriotic lesions and in the generation of pain through interaction with nerve fibres.

Basic and Translational Research on Proteinase-Activated Receptors: Proteinase-Activated Receptors in Female Reproductive Tissues and Endometriosis

During the menstrual cycle, dynamic morphological changes are observed in the ovarian follicle and the endometrium. These changes are associated with the onset of the inflammatory response in which many proteinases play various roles. Thrombin-induced activation of PAR1 (proteinase-activated receptor 1) stimulates the production of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in human granulosa cells, suggesting a possible role for PAR1 in the ovulatory process. In the endometrium, PAR2 expression increases during the menstrual period. PAR2 activation induces IL-8 production and cell proliferation in human endometrial stromal cells. PAR1 also stimulates proinflammatory cytokine production in human endometrial stromal cells. Thus, the PARs may be important in directing the dynamic changes of the endometrium. PARs also appear to play a role in endometriosis, a common gynecological disease, since activation of PAR1 and PAR2 induces the secretion of inflammatory cytokines and the proliferation of stromal cells in endometriotic lesions. Taken together, PARs appear to play diverse roles in the human reproductive organs.

Immunohistochemical analysis of CA125, CA19-9, and Ki-67 in stage III or IV endometriosis:

Background. Serum levels of CA125 and CA19-9 are often elevated in patients with endometriosis, but the clinical or biological significance of this is not well established. The aim of the present study was to compare serum and tissue levels of CA125 and CA19–9, and to examine the correlation between these levels and cell proliferation using immunohistochemical analysis in stage III or IV endometriosis. Methods. Forty-five cases diagnosed histologically as endometriosis were analyzed (26 cases were stage III and 19 were stage IV using the revised American Fertility Society classification). The preoperative serum levels of CA125 and CA19-9 were measured by immunoradiometric assay. Immunohistochemical analysis was performed using antibodies against CA125, CA19-9, and Ki-67 (a representative marker of cell proliferation). Results. The serum levels of CA125 and CA19-9 were elevated (over the cutoff values) in 25 cases and 21 cases, respectively. There was no significant correlation between serum CA125 and serum CA19-9 levels (correlation coefficient [ρ]=0.19). The serum CA19-9 level correlated well with the degree of immunostaining for CA19-9 (ρ=0.57), but not with the Ki-67 labeling index. The serum CA125 level did not show a strong correlation with CA125 staining (ρ=0.41), but it correlated well with the Ki-67 labeling index (ρ=0.53). Conclusions. The present study indicates that the serum CA125 level may correlate with the proliferative activity of epithelial cells in endometriotic lesions.

Immunohistochemical analysis of CA125, CA19–9, and Ki-67 in stage III or IV endometriosis: positive correlation between serum CA125 level and endometriotic epithelial cell proliferation

Serum levels of CA125 and CA 19-9 are often elevated in patients with endometriosis, but the clinical or biological significance of this is not well established. The aim of the present study was to compare serum and tissue levels of CA125 and CA19-9, and to examine the correlation between these levels and cell proliferation using immunohistochemical analysis in stage III or IV endometriosis. Forty-five cases diagnosed histologically as endometriosis were analyzed (26 cases were stage III and 19 were stage IV using the revised American Fertility Society classification). The preoperative serum levels of CA125 and CA19-9 were measured by immunoradiometric assay. Immunohistochemical analysis was performed using antibodies against CA125, CA199, and Ki-67 (a representative marker of cell proliferation). The serum levels of CA125 and CA19-9 were elevated (over the cutoff values) in 25 cases and 21 cases, respectively. There was no significant correlation between serum CA125 and serum CA19-9 levels (correlation coefficient [q]=0.19). The serum CA19-9 level correlated well with the degree of immunostaining for CA19-9 (q=0.57), but not with the Ki-67 labeling index. The serum CA125 level did not show a strong correlation with CA125 staining (q=0.41), but it correlated well with the Ki-67 labeling index (q=0.53). The present study indicates that the serum CA125 level may correlate with the proliferative activity of epithelial cells in endometriotic lesions.