Effects of pharmacological inhibition of hyaluronic acid synthesis on experimental endometriosis.

BACKGROUND: Dysregulated hyaluronic acid (HA) metabolism has been shown to be implicated in several pathologies including endometriosis. 4-Methylumbelliferone (4MU) is an HA synthesis inhibitor with proven antitumour activity. In this study, we aim to evaluate the effect of 4MU on endometriosis development both in vivo and in vitro. METHODS: Endometriosis was surgically induced by uterine tissue auto-transplantation in 32 two-month-old BALB/c mice. Animals were designated into the early or late starting treatment group, which initiated on day 2 or day 15 after surgery, respectively. Within each group, 4MU 200 mg/kg/day or vehicle (Control) were administered by oesophageal gavage for 28 days. After sacrifice, the percentage of developed lesions, lesion size, cell proliferation, vascularization and HA deposition within the endometriotic-like lesions were evaluated. Cell viability was assessed in endometrial epithelial cells (ECC-1) and in endometrial stromal cells (t-HESC); and migration was evaluated in t-HESC. RESULTS: There was a significant reduction in the percentage of developed lesions in mice that started the 4MU treatment on day 2 compared with its respective control group, and compared with those that started treatment on day 15. However, no significant changes were found when analysing endometriotic-like lesion's cell proliferation, vascularization and HA deposition. In vitro, both cell viability and migration were inhibited by 4MU treatment. CONCLUSIONS: The inhibition of HA synthesis could be a beneficial and alternative option to treat endometriosis at the early stage of the disease. Further research is necessary to elucidate 4MU's mechanism of action and better strategies for delivering this promising drug.

Resveratrol impairs cellular mechanisms associated with the pathogenesis of endometriosis.

RESEARCH QUESTION: Does resveratrol exert a potent inhibitory effect on the development of endometriosis by interfering with some pivotal processes? DESIGN: In-vitro cultures of primary endometriotic stromal cells, immortalized endometrial stromal (St-T1b) and endometriotic epithelial (12Z) cells were used to assess the effects of resveratrol on endometrial cell mechanisms. The effects of resveratrol on 12Z and St-T1b cell viability were assessed by MTT assay, apoptosis by FITC Annexin V assay and cleaved caspase-3 levels and cell migration by wound healing assay. The effect of resveratrol on the expression of genes related to cell migration, angiogenesis and cell stemness was evaluated by qRT-PCR. RESULTS: Resveratrol significantly decreased cell viability (P= 0.0065 to P = 0.0180), cell migration (P < 0.001 to P = 0.0225) and increased the number of apoptotic cells (P = 0.0031 to P = 0.0432) in both cell lines. In cell lines and primary culture, the treatment reduced MMP-2/TIMP-1 (P < 0.001 to P = 0.0180), VEGF (P = 0.0052 to P = 0.0243) and Ang-1 mRNA (P < 0.001 to P = 0.0382) expression. Among the stem cell phenotype markers, resveratrol 100 µM increased mRNA expression levels of Notch-1 (P < 0.001 to P = 0.0018), KLF-4 (P = 0.0011 to P = 0.0137), SOX-2 (P < 0.001 to P = 0.0070) and TERT (P < 0.001 to P = 0.0193) in both cell lines and primary cultures. The mRNA expression level of Snail-1 increased in the cell lines (P < 0.001 to P = 0.0087), whereas OCT-4 mRNA expression increased in St-T1b (P = 0.0396) and primary cultures (P = 0.0148). Vimentin mRNA expression showed a significant upregulation in primary cultures (P < 0.001). The expression of Msi-1 (P = 0.0145) and NANOG (P = 0.0080) decreased only in St-T1b cells. CONCLUSION: Resveratrol showed inhibitory effects on cell behaviour related to the development of endometriosis by differentially affecting growth, apoptosis, migration and stem cell phenotype of endometrial and endometriotic cells in vitro.

Effect of urolithins A and B on ectopic endometrial growth in a murine model of endometriosis.

Endometriosis is an often painful disease in reproductive-aged women, in which endometrial-like tissue grows outside the uterine cavity. Since the limited current therapeutic alternatives fail in alleviating the symptoms and based on our previous research in in vitro models using the same compounds as the ones used in the present study, we aimed to evaluate the effects of urolithins A (UA) and B (UB) on the growth and survival of endometriotic-like lesions in a murine model of endometriosis. Female BALB/C mice were surgically induced with endometriosis and treated with 2.5 mg kg-1 day-1 intraperitoneal UA or UB. The mice were monitored daily and weighed and the estrous stage was determined. After 28 days of treatment, lesions were counted, measured, excised, and fixed. Both urolithins proved not to affect the estrous cycle or body weight of the mice. UA completely prevented endometriotic-like lesions, while UB diminished the implant volume (p < 0.05). Treatment also reduced epithelial and stromal cell proliferation within the implants (p < 0.001 and p < 0.01, respectively) and apoptosis was enhanced (p < 0.05 and p < 0.01, respectively). These results are promising and reveal that urolithins A and B, separately, have a beneficial effect on the overall endometriotic growth without affecting the body weight or estrous cycle.

Interplay between Endometriosis and Pregnancy in a Mouse Model.

OBJECTIVES: To evaluate the effect of endometriosis on fertility and the levels of the IL-2 and IFN-γ in the peritoneal fluid in a mouse model; to evaluate the effect of pregnancy on endometriotic lesion growth, apoptosis and cell proliferation. STUDY DESIGN: Two month old C57BL/6 female mice underwent either a surgical procedure to induce endometriosis or a sham surgery. Four weeks after surgery mice were mated and sacrificed at day 18 of pregnancy. Number of implantation sites, fetuses and fetal weight were recorded. Endometriotic lesions were counted, measured, excised and fixed. Apoptosis and cell proliferation were evaluated in lesions by TUNEL and immunohistochemistry for PCNA respectively. Levels of IL-2 and IFN-γ were assessed by ELISA in the peritoneal fluid. RESULTS: Pregnancy rate (i.e. pregnant mice/N) decreased in mice with endometriosis. However there were no significant differences in resorption rate, litter size and pup weight between groups. IFN-γ augmented in endometriosis mice independently of pregnancy outcome. Additionally IFN-γ increased in pregnant endometriosis mice compared to pregnant sham animals. While IFN-γ increased in non pregnant versus pregnant mice in the sham group, IL-2 was increased in non pregnant mice in the endometriosis group. The size of endometriotic lesions increased in pregnant mice while apoptosis increased in the stroma and cell proliferation decreased in the epithelium of these lesions. Additionally, leukocyte infiltration, necrosis and decidualization were increased in the same lesions. CONCLUSIONS: Pregnancy rate is reduced in this mouse model of endometriosis. Levels of IL-2 are increased in the peritoneal fluid of mice with endometriosis suggesting a role of this cytokine in infertility related to this disease. The size of endometriotic lesions is increased in pregnant mice; however pregnancy has a beneficial effect on lesions by decreasing cell proliferation and by increasing apoptosis, decidualization and necrosis.

Effect of vascular endothelial growth factor inhibition on endometrial implant development in a murine model of endometriosis.

The main factor involved in neovascularization of ectopic endometrial tissue in endometriosis is the vascular endothelial growth factor (VEGF), which is produced both by the endometrial implant and by peritoneal macrophages. On the other hand, bevacizumab is an antiangiogenic agent used in the treatment of different tumors, like colorectal, pulmonary, and recently mammary. We evaluated the effect of the inhibition of VEGF activity with bevacizumab (Avastin) on ectopic endometrial growth in a murine model of endometriosis. Two months old female BALB/c mice had surgery performed to induce endometriotic-like lesions. Treatment with bevacizumab started on post-surgery day 15 and continued during 2 weeks. Then, animals were sacrificed, peritoneal fluid was collected, and endometriotic-like lesions were counted, measured, and removed. Cell proliferation, vascular density, and apoptosis were assessed by immunohistochemistry for proliferating cell nuclear antigen (PCNA), immunohistochemistry for CD34, and Terminal Deoxynucleotidil Transferase-Mediated dUTP Nick End Labeling (TUNEL), respectively. Vascular endothelial growth factor levels were evaluated in the peritoneal fluid by enzyme-linked immunoassay (ELISA). Treatment with bevacizumab significantly inhibited endometriotic lesion development (P < .05). Consistently, bevacizumab significantly inhibited cell proliferation in lesions (P < .01), reduced vascular density (P < .001), as well as increased the apoptotic cell percentage (P < .001). In addition, bevacizumab reduced VEGF levels in peritoneal fluid of endometriosis-induced animals (P < .05). In conclusion, this study suggests a direct effect of bevacizumab on the reduction of endometrial implant growth and supports further research on VEGF inhibition as a novel therapeutic modality in endometriosis.