S. Matsuzaki

In Vitro Effects of a Small-Molecule Antagonist of the Tcf/ß-Catenin Complex on Endometrial and Endometriotic Cells of Patients with Endometriosis

Background Our previous studies suggested that aberrant activation of Wnt/ß-catenin signaling might be involved in the pathophysiology of endometriosis. We hypothesized that inhibition of Wnt/ß-catenin signaling might result in inhibition of cell proliferation, migration, and/or invasion of endometrial and endometriotic epithelial and stromal cells of patients with endometriosis. Objectives The aim of the present study was to evaluate the effects of a small-molecule antagonist of the Tcf/ß-catenin complex (PKF 115–584) on cell proliferation, migration, and invasion of endometrial and endometriotic epithelial and stromal cells. Methods One hundred twenty-six patients (78 with and 48 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of PKF 115–584 on cell proliferation, migration, and invasion and on the Tcf/ß-catenin target genes were evaluated in endometrial epithelial and stromal cells of patients with and without endometriosis, and in endometrial and endometriotic epithelial and stromal cells of the same patients. Results The inhibitory effects of PKF 115–584 on cell migration and invasion in endometrial epithelial and stromal cells of patients with endometriosis prepared from the menstrual phase were significantly higher than those of patients without endometriosis. Levels of total and active forms of MMP-9 were significantly higher in epithelial and stromal cells prepared from menstrual endometrium in patients with endometriosis compared to patients without endometriosis. Treatment with PKF 115–584 inhibited MMP-9 activity to undetectable levels in both menstrual endometrial epithelial and stromal cells of patients with endometriosis. The number of invasive cells was significantly higher in epithelial and stromal cells of endometriotic tissue compared with matched eutopic endometrium of the same patients. Treatment with PKF 115–584 decreased the number of invasive endometriotic epithelial cells by 73% and stromal cells by 75%. Conclusions The present findings demonstrated that cellular mechanisms known to be involved in endometriotic lesion development are inhibited by targeting the Wnt/β-catenin pathway.

Impact of intraperitoneal pressure of a CO2 pneumoperitoneum on the surgical peritoneal environment

BACKGROUNDnAnimal experiments have suggested that a high intraperitoneal pressure (IPP) might adversely affect the surgical peritoneal environment. The present experimental study investigates the impact of IPP of a CO(2) pneumoperitoneum on human peritoneum.nnnMETHODSnPatients undergoing laparoscopic surgery were subjected to either low (8 mmHg) or standard (12 mmHg) IPP. Normal peritoneum was collected from the parietal wall at the beginning of surgery and every 60 min thereafter. Expression levels of 168 genes that encode extracellular matrix proteins, adhesion molecules or inflammatory cytokine signaling molecules were measured in peritoneal tissues using real-time polymerase chain reaction (PCR)-based assay panels. Human peritoneal mesothelial cells (HPMCs) and human peritoneal fibroblasts (HPFBs) were incubated in a CO(2) insufflation chamber for 1 h at 12 or 8 mmHg. Hyaluronan (HA) synthesis and mRNA expression levels of hyaluronic acid synthases (HAS) and hyaluronidases (Hyal) in HPMCs and HPFBs were measured at 0, 4, 8, 12, 24 and 48 h after CO(2) gas exposure by ELISA and real-time PCR, respectively.nnnRESULTSnExpression levels of connective tissue growth factor (CTGF), matrix metalloproteinase-9, E-selectin, chemokine (C-X-C motif) ligand 2 (CXCL-2), Hyal-1 and Hyal-2 were significantly higher and those of HAS-1, HAS-3, thrombospondin-2 (TSP-2) and interleukin-10 were significantly lower in the 12 mmHg group compared with the 8 mmHg group. HA synthesis was significantly lower in the 12 mmHg group compared with the 8 mmHg group in HPMCs and HPFBs throughout the time course.nnnCONCLUSIONSnA low IPP (8 mmHg) may be better than the standard IPP (12 mmHg) to minimize the adverse impact on the surgical peritoneal environment during a CO(2) pneumoperitoneum.

Antifibrotic properties of epigallocatechin-3-gallate in endometriosis.

STUDY QUESTIONnIs epigallocatechin-3-gallate (EGCG) treatment effective in the treatment of fibrosis in endometriosis?nnnSUMMARY ANSWERnEGCG appears to have antifibrotic properties in endometriosis.nnnWHAT IS KNOWN ALREADYnHistologically, endometriosis is characterized by dense fibrous tissue surrounding the endometrial glands and stroma. However, only a few studies to date have evaluated candidate new therapies for endometriosis-associated fibrosis.nnnSTUDY DESIGN, SIZE, DURATIONnFor this laboratory study, samples from 55 patients (45 with and 10 without endometriosis) of reproductive age with normal menstrual cycles were analyzed. A total of 40 nude mice received single injection proliferative endometrial fragments from a total of 10 samples.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnThe in vitro effects of EGCG and N-acetyl-l-cysteine on fibrotic markers (alpha-smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin) with and without transforming growth factor (TGF)-β1 stimulation, as well as on cell proliferation, migration and invasion and collagen gel contraction of endometrial and endometriotic stromal cells were evaluated by real-time PCR, immunocytochemistry, cell proliferation assays, in vitro migration and invasion assays and/or collagen gel contraction assays. The in vitro effects of EGCG on mitogen-activated protein kinase (MAPK) and Smad signaling pathways in endometrial and endometriotic stromal cells were evaluated by western blotting. Additionally, the effects of EGCG treatment on endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice.nnnMAIN RESULTS AND THE ROLE OF CHANCEnTreatment with EGCG significantly inhibited cell proliferation, migration and invasion of endometrial and endometriotic stromal cells from patients with endometriosis. In addition, EGCG treatment significantly decreased the TGF-β1-dependent increase in the mRNA expression of fibrotic markers in both endometriotic and endometrial stromal cells. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels were significantly attenuated at 8, 12 and 24 h after treatment with EGCG. Epigallocatechin-3-gallate also significantly inhibited TGF-β1-stimulated activation of MAPK and Smad signaling pathways in endometrial and endometriotic stromal cells. Animal experiments showed that EGCG prevented the progression of fibrosis in endometriosis.nnnLIMITATIONS, REASONS FOR CAUTIONnThe attractiveness of epigallocatechin-3-gallate as a drug candidate has been diminished by its relatively low bioavailability. However, numerous alterations to the EGCG molecule have been patented, either to improve the integrity of the native compound or to generate a more stable yet similarly efficacious molecule. Therefore, EGCG and its derivatives, analogs and prodrugs could potentially be developed into agents for the future treatment and/or prevention of endometriosis.nnnWIDER IMPLICATIONS OF THE FINDINGSnEpigallocatechin-3-gallate is a potential drug candidate for the treatment and/or prevention of endometriosis.nnnSTUDY FUNDING/COMPETING INTERESTSnThis study was supported in part by Karl Storz Endoscopy & GmbH (Tuttlingen, Germany). No competing interests are declared.

Co-operation between the AKT and ERK signaling pathways may support growth of deep endometriosis in a fibrotic microenvironment in vitro

STUDY QUESTIONnHow can deep endometriotic stromal cells proliferate and persist in a fibrotic environment?nnnSUMMARY ANSWERnThe serine/threonine kinase AKT and extracellular regulated kinase (ERK) signaling pathways may co-operate to support growth of deep endometriotic lesions by enhancing endometriotic stromal cell proliferation and survival in a fibrotic microenvironment in vitro.nnnWHAT IS KNOWN ALREADYnEndometriosis, particularly deep infiltrating endometriosis, is characterized histologically by dense fibrous tissue that is primarily composed of type I collagen. This tissue may cause pelvic pain and infertility, which are major clinical issues associated with endometriosis. Proliferation of normal fibroblasts is tightly regulated, and fibrillar, polymerized type I collagen inhibits normal fibroblast proliferation. However, no studies to date have investigated how deep endometriotic stromal cells can proliferate and persist in a fibrotic environment.nnnSTUDY DESIGN, SIZE, DURATIONnEndometrial and/or endometriotic tissues from 104 patients (61 with and 43 without endometriosis) of reproductive age with normal menstrual cycles were analyzed. A total of 25 nude mice received a single injection of endometrial fragments from a total of five samples.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnWe evaluated cell proliferation, caspase 3/7 activity, and the AKT and ERK signaling pathways in endometrial and endometriotic stromal cells on three-dimensional (3D) polymerized collagen matrices in vitro. In addition, to determine whether aberrant activation of the AKT and ERK pathways is involved during progression of fibrosis in endometriosis in vivo, we evaluated the expression of phosphorylated AKT and ERK1/2 in endometriotic implants in a nude mouse model of endometriosis. Finally, we evaluated the effects of MK2206 (an AKT inhibitor) and U0126 (a MEK inhibitor) on cell proliferation, caspase 3/7 activity, and phosphorylation of AKT and ERK1/2 of endometriotic stromal cells on 3D polymerized collagen matrices.nnnMAIN RESULTS AND THE ROLE OF CHANCEnProliferation of endometriotic stromal cells was significantly less inhibited than that of endometrial stromal cells (P < 0.05) on 3D polymerized collagen. Levels of phosphorylated AKT, phosphorylated p70S6K and phosphorylated ERK1/2 were significantly higher in endometriotic stromal cells than in endometrial stromal cells at 24 h (P < 0.05) and at 72 h (P < 0.05) on 3D polymerized collagen. Phosphorylated AKT expression was significantly increased on Days 21 and 28 compared with those on Days 3 and 7 (all P < 0.05) in endometriotic implants during progression of fibrosis in a nude mouse model of endometriosis. Inhibition of AKT or ERK1/2 with MK2206 or U0126, respectively, did not significantly increase caspase 3/7 activity in endometriotic stromal cells on either two-dimensional or 3D collagen matrices. Western blot analysis showed that MK2206 alone decreased levels of phosphorylated AKT; however, it increased levels of phosphorylated ERK in endometriotic cells compared with vehicle-treated cells (both P < 0.05). In addition, U0126 treatment decreased levels of phosphorylated ERK; however, it resulted in increased levels of phosphorylated AKT in endometriotic stromal cells compared with vehicle-treated cells (both P < 0.05).nnnLIMITATIONS, REASONS FOR CAUTIONnEndometriosis involves a number of processes, such as invasion, metastasis, angiogenesis, and apoptosis resistance, and a variety of signaling pathways may be involved in promoting development and progression of the disease. In addition, further animal experiments are required to determine whether the AKT and ERK signaling pathways co-operate to support growth of endometriotic lesions in a fibrotic microenvironment in vivo.nnnWIDER IMPLICATIONS OF THE FINDINGSnCo-targeting the AKT and ERK pathways may be an effective therapeutic strategy for endometriosis treatment.nnnSTUDY FUNDING/COMPETING INTERESTSnThis study was supported in part by Karl Storz Endoscopy & GmbH (Tuttlingen, Germany). No competing interests are declared.

Soft matrices inhibit cell proliferation and inactivate the fibrotic phenotype of deep endometriotic stromal cells in vitro

STUDY QUESTIONnCan deep infiltrating endometriotic stromal cells (DES) sense changes in extracellular matrix (ECM) stiffness and respond to them?nnnSUMMARY ANSWERnSoft matrices inhibit cell proliferation and inactivate the fibrotic phenotype of DES in vitro.nnnWHAT IS KNOWN ALREADYnDeep infiltrating endometriosis (DIE) is characterized histologically by dense fibrous tissue. Tissue stiffening is a hallmark of fibrosis. Studies show that matrix stiffness is involved in the progression of numerous diseases, including cancer and fibrosis. However, no studies to date have investigated whether tissue stiffening could influence cell behavior in DIE. Previous in vitro studies typically analyzed cells grown on rigid plastic or glass substrates with stiffness in the gigapascal (gPa) range, which is much stiffer than that occurring in vivo. To investigate how changes in ECM stiffness affect the behavior of DES, it is critical to model in vivo tissue compliance conditions in vitro.nnnSTUDY DESIGN, SIZE, DURATIONnFor this laboratory study, paired endometrial and endometriotic samples from 40 patients who had histological evidence of DIE and endometrial samples from 23 patients without endometriosis were analyzed (uterine fibroma: n = 10, tubal infertility: n = 13).nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnAll participants were 20-37 years old and had regular menstrual cycles of 26-32 days. The abundance of F-actin, alpha smooth muscle actin (αSMA), Ki67, and procollagen type I in DES and endometrial stromal cells (EES) on polyacrylamide gel substrates of varying stiffness (2, 4, 8, 16 and/or 30 kPa) was determined by immunofluorescence confocal microscopy. mRNA level of type I collagen, matrix metalloproteinase-1 (MMP-1), MMP-14 and cyclin D1 was measured by real-time PCR. The cellular proliferation index (CPI), assessed as the percentage of Ki67-positive cells among the total number of nuclei stained by 4,6-diamidino-2-phenylindole (DAPI) was determined.nnnMAIN RESULTS AND THE ROLE OF CHANCEnIncreased matrix stiffness induced F-actin stress fiber formation in both EES and DES, whereas αSMA-containing stress fibers were induced only in DES. Furthermore, increased stiffness increased the CPI in both EES (16 or 30 kPa versus 2 kPa, P < 0.05) and DES (16 or 30 kPa versus 2, 4 or 8 kPa, P < 0.05). Increased stiffness increased the percentage of procollagen I-positive cells as well as mRNA levels of type I collagen in both EES and DES in a matrix stiffness-dependent manner (2, 8 and 30 kPa) (P < 0.05). Increased stiffness also increased MMP-14 mRNA levels in EES (30 versus 2 kPa, P < 0.05), but decreased MMP-1 mRNA levels in DES in a matrix stiffness-dependent manner (2, 8 and 30 kPa; P < 0.05). Treatment with transforming growth factor (TGF)-β1 further increased type I collagen mRNA levels in both EES and DES when compared with cells grown on a substrate of the same stiffness (2, 8 or 30 kPa, with versus without TGF-β1, P < 0.05). Treatment with TGF-β1 also increased MMP-1 (8 or 30 kPa, P < 0.05 versus no TGF-β1) and MMP-14 mRNA levels (2, 8 or 30 kPa, P < 0.05 versus no TGF-β1) in EES, but decreased MMP-1 mRNA levels (2, 8 or 30 kPa, P < 0.05 versus no TGF-β1) in DES. On a soft substrate (2 kPa), both EES and DES exhibited a small rounded morphology with diffuse labeling for F-actin. No F-actin-positive stress fibers were observed in either EES or DES grown on 2 kPa substrates. There were more Ki67-positive EES when grown on 2, 4 or 8 kPa compared with Ki67-positive DES (P < 0.05).nnnLIMITATIONS, REASONS FOR CAUTIONnA tremendous gap exists between the present in vitro model and in vivo deep endometriotic tissues. Cell culture systems that more closely mimic the cellular complexity typical of in vivo endometriotic tissues are required to develop novel strategies for treatment of DIE. A disadvantage of polyacrylamide is its cytotoxicity but in the two-dimensional culture models used here, where cells are seeded above the polyacrylamide gel, this should not have a major impact. Finally, the soft substrates we used in vitro (2 and 4 kPa) may represent the elasticity of the endometrium in vivo, however, currently there are no data regarding tissue stiffness in DIE in vivo.nnnWIDER IMPLICATIONS OF THE FINDINGSnHormonal suppressive therapy is not usually effective for treating DIE. Interrupting the mechanical interactions between endometriotic fibroblasts and aberrant ECM may be a novel strategy for treatment of DIE.nnnSTUDY FUNDING/COMPETING INTERESTSnThis study was supported in part by Karl Storz Endoscopy & GmbH (Tuttlingen, Germany). No competing interests are declared.

Adenosine triphosphate-binding cassette transporter G2 expression in endometriosis and in endometrium from patients with and without endometriosis.

OBJECTIVEnTo investigate adenosine triphosphate (ATP)-binding cassette transporter G2 (ABCG2) expression in endometriosis and in samples of endometrium from patients with and without endometriosis.nnnDESIGNnProspective study.nnnSETTINGnUniversity hospital.nnnPATIENT(S)nPatients with and without endometriosis.nnnINTERVENTION(S)nEndometrial and endometriotic tissues obtained throughout the menstrual cycle.nnnMAIN OUTCOME MEASURE(S)nDensity of ABCG2(+) microvessels, density of CD31(+) microvessels.nnnRESULT(S)nNo statistically significant differences in the density of ABCG2(+) microvessels were observed between endometrium of patients with and without endometriosis in the proliferative phase and early, middle, and late secretory phases. The density of ABCG2(+) microvessels was statistically significantly higher in the menstrual endometrium of patients with endometriosis compared with patients without endometriosis. The density of ABCG2(+) microvessels was reduced in the ectopic endometrium compared with matched eutopic endometrium except in cases of deep infiltrating endometriosis. The density of ABCG2(+) microvessels was statistically significantly higher in deep infiltrating endometriosis compared with ovarian endometriosis and red and black peritoneal lesions throughout the menstrual cycle.nnnCONCLUSION(S)nABCG2 is strongly expressed in the endothelial cells of microvessels of eutopic endometrium, and the density of ABCG2(+) microvessels is reduced in ectopic endometrium except in cases of deep infiltrating endometriosis. ABCG2(+) microvessels may represent an integral part of the pathophysiology of deep infiltrating endometriosis.

Targeting the Wnt/β-catenin pathway in endometriosis: a potentially effective approach for treatment and prevention

Endometriosis is a chronic, estrogen-dependent disease associated with infertility and pelvic pain. Endometriosis is defined by the presence of extra-uterine endometrial tissue. It affects approximately 10% of reproductive-aged women. However, the underlying etiology, pathogenesis and pathophysiology remain to be fully elucidated. Knowledge of these factors is indispensable for the development of targeted therapies for prevention and treatment of endometriosis. Several studies, including those from our laboratory, have suggested that aberrant activation of the Wnt/β-catenin pathway may be involved in the pathophysiology of endometriosis. This is a review of the literature focused on the aberrant activation of the Wnt/β-catenin pathway in patients with endometriosis, and on how targeting the Wnt/targeting pathway may be a potentially effective approach for treating and/or preventing endometriosis.

In vitro and in vivo effects of MK2206 and chloroquine combination therapy on endometriosis: Autophagy may be required for regrowth of endometriosis

A high recurrence rate after medical treatment is a major clinical problem for patients with endometriosis. Here, we have evaluated the in vitro effects of combined treatment with MK2206 (an AKT inhibitor) + chloroquine on cell growth and regrowth of endometriotic stromal cells and the in vivo effects on endometriotic implants in a mouse xenograft model of endometriosis.

Effects of low intraperitoneal pressure and a warmed, humidified carbon dioxide gas in laparoscopic surgery: a randomized clinical trial

Laparoscopic surgery technology continues to advance. However, much less attention has been focused on how alteration of the laparoscopic surgical environment might improve clinical outcomes. We conducted a randomized, 2u2009×u20092 factorial trial to evaluate whether low intraperitoneal pressure (IPP) (8u2009mmHg) and/or warmed, humidified CO2 (WH) gas are better for minimizing the adverse impact of a CO2 pneumoperitoneum on the peritoneal environment during laparoscopic surgery and for improving clinical outcomes compared to the standard IPP (12u2009mmHg) and/or cool and dry CO2 (CD) gas. Herein we show that low IPP and WH gas may decrease inflammation in the laparoscopic surgical environment, resulting in better clinical outcomes. Low IPP and/or WH gas significantly lowered expression of inflammation-related genes in peritoneal tissues compared to the standard IPP and/or CDxa0gas. The odds ratios of a visual analogue scale (VAS) pain score >30 in the ward was 0.18 (95% CI: 0.06, 0.52) at 12u2009hours and 0.06 (95% CI: 0.01, 0.26) at 24u2009hours in the low IPP group versus the standard IPP group, and 0.16 (95% CI: 0.05, 0.49) at 0u2009hours and 0.29 (95% CI: 0.10, 0.79) at 12u2009hours in the WH gas group versus the CD gas group.

Reply to the letter from Barra et al.

We thank the Editor-in Chief, Professor Amrita Ahluwalia, for her fair handling of the letter from Barra et al. (2018), as well as her openness. We acknowledge the interest of Barra et al. in our work, but we are obliged to respond to their letter. The major concern of Barra et al. (2018) is the future clinical use of MK2206, an Akt inhibitor, in patients with endometriosis. First, we would like to clarify that the results of our in vitro and animal experiments (Matsuzaki et al., 2018) did not support future clinical trials of MK2206 alone, chloroquine (CQ) alone, or combination MK2206 + CQ in patients with endometriosis. There are two main reasons why MK2206 + CQ treatment should not be evaluated in future clinical trials, as we discussed in the article (Matsuzaki et al., 2018). First, treatment with MK2206 + CQ or CQ alone affects growth of endometrial stromal cells (EES) rather than that of endometriotic stromal cells (DES). Endometriosis, a common gynaecological disorder that causes infertility and pelvic pain, affects approximately 10% of women of reproductive age. Therefore, the ideal drugs for patients with endometriosis should affect only diseased endometriotic lesions and should not affect ‘normal’ endometrium within the same patients. However, we showed that the IC50 of MK2206 + CQ treatment was significantly lower in EES compared to that in DES within the same patients. Furthermore, our clonogenic assay demonstrated that CQ alone affected survival of EES rather than that of DES, within the same patients. Second, DES have the potential to regrow after discontinuation of MK2206 alone, CQ alone, or MK2206 + CQ. A high recurrence rate after treatment, with or without surgery, is a major clinical problem for patients with endometriosis. Before validation of the effects of candidate molecules can be performed in animal experiments or clinical trials, it is important to evaluate whether candidate molecules can prevent regrowth of endometriotic cells in vitro. The results of the present mouse study are not consistent with those of previous animal studies with the mTOR inhibitors. Kacan et al. (2017) demonstrated that in an autologous rat model of endometriosis, treatment with everolimus alone disrupted the epithelial cells of endometriotic implants. Lee and Kim (2014) did not investigate the effect of either temsirolimus or everolimus in a rat model of endometriosis. In a study not cited by Barra et al., temsirolimus disrupted the lesion histopathology of deep endometriotic endometriosis in a mouse xenograft model of endometriosis (Leconte et al., 2011). However, our study clearly showed that MK2206 alone is not effective in reducing the size of implants or inducing apoptosis in a xenograft model of endometriosis (Matsuzaki et al., 2018). The rationale of our study was not based on the important role of the PI3K/Akt/mTOR pathway in the pathogenesis of endometriosis, but on our previous findings that MK2206 treatment may induce a cytoprotective autophagy in endometriosis (Matsuzaki et al., 2017). Our previous study showed that the Akt and ERK signalling pathways may compensate for each other, resulting in apoptosis resistance in DES (Matsuzaki and Darcha, 2015). Therefore, we evaluated MK2206 + U0126 [a MAPK/ERK kinase (MEK) inhibitor] in endometriosis in vitro (Matsuzaki et al., 2017). We showed that U0126 + MK2206 synergistically inhibits cell growth of DES (Matsuzaki et al., 2017). However, the rate of cell regrowth of DES after drug discontinuation was disappointingly high (Matsuzaki et al., 2017). Our further experiments suggested that MK2206 treatment may induce autophagy, which may inhibit cell death, resulting in cell survival and subsequent cell proliferation following U0126 + MK2206 treatment (Matsuzaki et al., 2017). These observations serve as the rationale for our present study. Our primary target in the present study was not the PI3K/ Akt/mTOR pathway, but rather the ‘autophagy’, which may play a cytoprotective role in endometriosis. Barra et al. (2018) incorrectly stated that our study (Matsuzaki et al., 2018) demonstrated the efficacy of MK2206 + CQ in inducing autophagy of endometriotic stromal and epithelial cells. Actually, our study showed that MK2206 + CQ inhibited, and did not induce, canonical autophagy in DES (Matsuzaki et al., 2018). Furthermore, we did not evaluate this in endometriotic epithelial cells. British Journal of Pharmacology British Journal of Pharmacology (2018) 175 3628–3629 3628