Inhibition of IAP (inhibitor of apoptosis) proteins represses inflammatory status via nuclear factor-kappa B pathway in murine endometriosis lesions.

PROBLEM: How is the role of inhibitor of apoptosis proteins (IAPs) in the development of murine endometriosis lesions? METHOD OF STUDY: BALB/c female mice (n = 36) were used for the murine endometriosis model. Endometriotic lesions were surgically induced in mice by transplanting mouse uterine tissue. After 4 weeks of IAP antagonist (BV6) treatment, the expression of inflammatory factors in the implants was evaluated using real-time RT-PCR. Inflammatory state, angiogenic activity, and nuclear factor-kappa B (NF-κB) activation were assessed by immunohistochemical staining. RESULTS: The number, size, and level of inflammatory cytokines (Vegf, Il-6, Ccl-2, Lif) gene expression in the murine endometriosis-like lesions were reduced by BV6 treatment. BV6 repressed the intensity and rate of positive cells of CD3, F4/80, and PECAM immunostaining; in addition, the expression of NF-κB p65 and phospho-NF-κB p65 was also attenuated. CONCLUSION: Inhibitor of apoptosis proteins antagonist represses the inflammation status of murine endometriosis-like lesions viaNF-κB pathway. IAPs may be a novel therapeutic target for endometriosis.

Lipopolysaccharide promotes the development of murine endometriosis-like lesions via the nuclear factor-kappa B pathway.

PROBLEM: Is lipopolysaccharide (LPS) involved in the development of endometriosis? METHOD OF STUDY: BALB/c mice (n=69) were used for the murine endometriosis model. Mice with surgically induced endometriosis were injected with LPS intraperitoneally. After 4 weeks of LPS injections with or without the nuclear factor-kappa B (NF-κB) inhibitor, the extent of endometriosis-like lesions was evaluated. Expression of inflammatory factors in the implants was evaluated using real-time RT-PCR. Cell proliferation, angiogenic activity, inflammation, and NF-κB phosphorylation were assessed by immunohistochemical staining. RESULTS: Lipopolysaccharide increased total number, size, and mRNA expression of Ptgs-2, Vegf, Ccl-2, and Il-6 in endometriosis-like lesions. LPS also increased the percentage of Ki67-positive cells and enhanced the intensity and rate of positive cells of CD3, F4/80, and PECAM. Intense expression of phospho-NF-κB p65 after LPS administration was observed. Treatment with the NF-kB inhibitor negated these LPS-induced effects. CONCLUSION: LPS-induced pelvic inflammation status enhanced the development of murine endometriosis-like lesions via NF-κB pathway.

Schizencephaly and Porencephaly Due to Fetal Intracranial Hemorrhage: A Report of Two Cases.

Schizencephaly and porencephaly are extremely rare types of cortical dysplasia. Case 1: Prenatal magnetic resonance imaging (MRI) showed wide clefts in the frontal and parietal lobes bilaterally. On postnatal day 3, MRI T2-weighted images showed multiple hypointensities in the clefts and ventricular walls, suggestive of hemosiderosis secondary to intracranial hemorrhage. Case 2: Prenatal MRI showed bilateral cleft and cyst formation in the fetal cerebrum, as well as calcification and hemosiderosis indicative of past hemorrhage. T2-weighted images showed hypointensities in the same regions as the calcification, corresponding with hemosiderosis due to intracranial hemorrhage on postnatal day 10. Thus, prenatal MRI was useful for diagnosing schizencephaly and porencephaly. Schizencephaly and porencephaly were thought to be due to fetal intracranial hemorrhage, which, in the porencephaly case, may have been related to a mutation of COL4A1.

Analysis of pregnancy outcome and decline of anti-Müllerian hormone after laparoscopic cystectomy for ovarian endometriomas.

AIM: Excision of ovarian endometrioma (OE) may induce the reduction of ovarian reserve. We evaluated pregnancy outcomes after laparoscopic cystectomy (LC), and the pre- and postoperative levels of anti-Müllerian hormone (AMH) to consider the ovarian reserve. METHODS: We enrolled 40 women with OE and 16 women with benign ovarian tumors who hoped to have a child and who underwent LC. To evaluate the ovarian reserve of 40 patients (OE group, n = 24; non-OE group, n = 16), we measured serum AMH levels before and after the surgery. RESULTS: In the 40 women who underwent LC for OE, the cumulative pregnancy rate was 50%. Prior to the cystectomy, serum AMH levels in the OE group, especially in patients over the age of 35, were significantly lower than those in the non-OE group. Rate of decline in serum AMH in the OE group was significant compared with that in the non-OE group 6 months after surgery. In patients over the age of 35 in the OE group, AMH levels 1 year after surgery decreased noticeably. CONCLUSION: LC for OE could be a preferred surgical approach, but effective therapeutic strategies will have to be developed to prevent damage to the ovarian reserve, especially for older patients.

Inhibitor of apoptosis proteins (IAPs) may be effective therapeutic targets for treating endometriosis.

STUDY QUESTION: What is the role of the inhibitor of apoptosis proteins (IAPs) in human endometriotic tissues and a mouse model of endometriosis? SUMMARY ANSWER: Four IAP proteins were expressed in endometriotic tissue indicating IAPs may be a key factor in the pathogenesis and progression of endometriosis. WHAT IS KNOWN ALREADY: Overexpression of IAPs protects against a number of proapoptotic stimuli. IAPs (c-IAP1, c-IAP2, XIAP and Survivin) are expressed in human ectopic endometrial stromal cells (ESCs) from ovarian endometriomas. STUDY DESIGN, SIZE, DURATION: Forty-eight women with or without ovarian endometrioma are included in this study. BALB/c mice (n = 24) were used for the mouse endometriosis model. Mice with surgically induced endometriosis were treated with an IAP antagonist (BV6) for 4 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human ectopic endometrial tissues from chocolate cysts and eutopic endometrial tissue were collected. ESCs were enzymatically isolated from these tissues. ESC proliferation was examined by 5-bromo-2'-deoxyuridine-enzyme-linked immunosorbent assay. IAPs expression in tissue derived from eutopic endometria and chocolate cysts was evaluated using real-time RT-PCR and immunohistochemistry. A homologous mouse endometriosis model was established by transplanting donor mouse uterine tissue into the abdominal cavities of recipient mice. After treating the mice with BV6 (i.p. 10 mg/ml), the extent of endometriosis-like lesions in mice was measured and proliferative activity assessed by Ki67 staining. All experiments were repeated a minimum of three times. MAIN RESULTS AND THE ROLE OF CHANCE: IAP (c-IAP1, c-IAP2, XIAP and Survivin) mRNA and protein in human ectopic endometrial tissues were expressed at higher levels than in eutopic endometrial tissues (P < 0.05). All four IAPs proteins were expressed in mouse endometriosis-like implants. BV6 inhibited BrdU incorporation of human ESCs (P < 0.05 versus control). BV6 also decreased the total number, weight, surface area and Ki67 positive cells in the endometriosis-like lesions in the mice (P < 0.05 versus control). LIMITATIONS, REASONS FOR CAUTION: Endometriotic lesions were surgically induced in mice by transplanting mouse uterine tissue only, not human pathological endometriotic tissue. Furthermore, the effects of BV6 on human ESCs and mouse endometriosis-like lesions may differ between the species. WIDER IMPLICATIONS OF THE FINDINGS: Our data support the hypothesis that IAPs are involved in the development of endometriosis, and therefore an inhibitor of IAPs has potential as a novel treatment for endometriosis. STUDY FUNDING/COMPETING INTERESTS: This work was supported by KAKENHI (Japan Society for the Promotion of Science, Grant-in-Aid: to F.T.; 21592098 and to T.H.; 24659731) and Yamaguchi Endocrine Research Foundation. The authors have no conflicts of interest to disclose.

Parthenolide reduces cell proliferation and prostaglandin E2 [corrected] in human endometriotic stromal cells and inhibits development of endometriosis in the murine model.

OBJECTIVE: To evaluate the effects of parthenolide on human endometriotic cells and murine endometriotic lesions. DESIGN: Experimental study. SETTING: University hospital and laboratory of animal science. PATIENT(S) AND ANIMAL(S): Twenty women with ovarian endometrioma and 30 mice. INTERVENTION(S): Ectopic endometrial tissue from the endometrioma was collected. MAIN OUTCOME MEASURE(S): Human endometriotic stromal cells (ESCs) were pretreated with parthenolide and exposed to tumor necrosis factor (TNF)-α. Interleukin 8 (IL-8) and COX-2 gene expressions were evaluated by real-time reverse transcription-polymerase chain reaction. Interleukin-8 protein, prostaglandin E2 (PGE2) level, and intranuclear p65 protein concentration were determined by ELISA. Cell proliferation was assessed by 5-bromo-2'-deoxyuridine-ELISA. Phosphorylation of signaling pathways in ESCs was evaluated by Western blotting. Gene expression and proliferative activity in murine endometriosis-like lesions were assessed by real-time reverse transcription-polymerase chain reaction and Ki67 staining, respectively. RESULT(S): With parthenolide pretreatment, TNF-α-induced IL-8 gene and protein expression in ESCs were diminished. Tumor necrosis factor α-induced COX-2 expression and PGE2 synthesis were also inhibited. Adding parthenolide repressed TNF-α-induced 5-bromo-2'-deoxyuridine incorporation and IκB phosphorylation in ESCs. As in vivo experiments, administering parthenolide reduced the number, surface area, and weight, the level of Vegf, Il-6, Mcp-1, and Lif gene expression, and the percentage of Ki67-positive cells in murine endometriosis-like lesions. CONCLUSION(S): Parthenolide repressed the development of endometriosis by suppressing the inflammatory peritoneal environment through the nuclear factor κB pathway.

Demethylation of a nonpromoter cytosine-phosphate-guanine island in the aromatase gene may cause the aberrant up-regulation in endometriotic tissues.

OBJECTIVE: To search for the demethylated cytosine-phosphate-guanine (CpG) islands within the aromatase gene in stromal cells derived from endometriotic chocolate cysts. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology and Department of Biosignaling, Tottori University, Yonago, Japan. PATIENT(S): Twenty-eight women who underwent laparoscopy (n=14) and laparotomy (n=14). INTERVENTION(S): Endometrial and endometriotic stromal cells were obtained from the uterus and chocolate cyst lining of the ovary. MAIN OUTCOME MEASURE(S): We searched for the CpG island and examined methylation profile and the association of methyl-binding proteins with the CpG island. RESULT(S): Up-regulation of aromatase messenger RNA (mRNA) expression was demonstrated in endometriotic cells. Three proximal promoters drove the mRNA expression. In endometrial cells, a marginal level of aromatase mRNA expression was observed. Treating endometrial cells with the demethylating agent 5-aza-2'-deoxycytidine markedly enhanced aromatase mRNA expression. The same promoters as in the endometriotic cells were used. To identify the unmethylated CpGs in endometriotic cells, we searched for CpG islands within the aromatase gene and subsequently examined the methylation profiles. Sequence analysis of bisulfite-treated genomic DNA demonstrated a stretch of CpG demethylation within a nonpromoter CpG island of the aromatase gene in endometriotic cells. In endometrial cells, the CpG sequences were heavily methylated and associated with methyl-CpG-binding proteins. CONCLUSION(S): The up-regulation of the aromatase gene in endometriosis may be ascribed to the epigenetic disorder associated with aberrant DNA demethylation in a nonpromoter CpG island.

The role of survivin in the resistance of endometriotic stromal cells to drug-induced apoptosis.

BACKGROUND: Decreased susceptibility of endometrial tissue to apoptosis may contribute to the pathogenesis of endometriosis. We investigate the role of survivin in the pathophysiology of endometriosis through the ability of ectopic and eutopic endometrial stromal cells (ESCs) to resist apoptosis. METHODS: Ectopic ESCs were obtained from ovarian chocolate cysts in patients undergoing laparoscopic surgery (n = 22). Eutopic ESCs were isolated from endometrial tissue of cyclic premenopausal women undergoing hysterectomy for fibroids (n = 22). Purified stromal cells were studied in vitro. The number of surviving cells and activation of caspases were assessed by WST-8 assay and immunoblotting. Expression of inhibitor of apoptosis proteins (IAP) family members: cIAP1 (birc2), cIAP2 (birc3), XIAP (birc4), survivin (birc5) were examined using cDNA array and real-time RT-PCR. Effects of gene silencing by small inhibitor RNAs (siRNA) were examined by WST-8-assay, Annexin-V staining and immunoblotting. RESULTS: After staurosporine (SS) treatment, 55% of eutopic ESCs survived versus 70% of ectopic ESCs. Procaspase-3 or -7 was more intensely activated by SS treatment in eutopic than in ectopic ESCs (P < 0.01). mRNAs for IAP-family genes, such as cIAP-1, XIAP and survivin, were highly expressed in ectopic ESCs before SS treatment. The fold induction of survivin expression after SS treatment was higher in ectopic than eutopic ESCs (2.8 +/- 0.27 versus 0.69 +/- 0.07, respectively). Survivin gene silencing in SS-treated ectopic ESCs led to an increase of apoptotic cells (P < 0.05, versus control siRNA). CONCLUSIONS: We demonstrated that survivin plays a critical role in susceptibility of ESCs to apoptosis. Our results indicate that a survivin inhibitor may be effective as a novel treatment for endometriosis.

TAK1 activation for cytokine synthesis and proliferation of endometriotic cells.

Endometriosis causes pelvic pain and infertility in women of reproductive age. We explored TNFalpha-induced specific signaling pathways and gene expressions in endometriotic stromal cells (ESCs). Based on the data of the pathway specific cDNA array, we analyzed the role of TAK1, which is believed to work as a common mediator for NF-kappaB and MAPK pathways. Using the NF-kappaB pathway array, we found that TNFalpha upregulated ICAM-3, IL-6, IL-8, TAK1, JNK2, RelA, and TLR4 expressions. TNFalpha augmented the phosphorylation of TAK1. By transfection of TAK1 siRNA, TNFalpha-induced phosphorylation of IkappaBalpha, JNK1/2, and p38MAPK, as well as IL-6 or IL-8 expression, were repressed. TAK1 silencing in TNFalpha-pretreated ESCs caused a decrease in the proportion of cells in S-phase, and reduced TNFalpha-promoted BrdU incorporation. We provide the first evidence that TNFalpha and its downstream TAK1, which are key mediators for NF-kappaB and MAPK pathways, may be involved in the pathogenesis of endometriosis.