C. Olivares

Effects of a selective cyclooxygenase-2 inhibitor on endometrial epithelial cells from patients with endometriosis

BACKGROUND Celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, also has anti-proliferative properties and pro-apoptotic effects on different in vivo and in vitro models, two actions that may be efficacious in therapy for endometriosis. We evaluated the effects of celecoxib on apoptosis and proliferation, and vascular endothelial growth factor (VEGF) production and COX-2 expression and activity in endometrial epithelial cells (EECs). METHODS AND RESULTS Thirty-two endometriosis and 13 control women were included in the study. EECs from eutopic endometrium and control biopsies were cultured with different doses of celecoxib. Celecoxib at 50, 75 and 100 microM (versus vehicle control) inhibited EEC proliferation in cultures from controls (P < 0.05, P < 0.01 and P < 0.01, respectively) and patients with endometriosis (P < 0.05, P < 0.01 and P < 0.01), as assessed by (3)H-thymidine uptake. Celecoxib at 50, 75 and 100 microM induced apoptosis in EEC from controls (P < 0.05, P < 0.001 and P < 0.001) and patients with endometriosis (P < 0.001, P < 0.001 and P < 0.01), as revealed by the Acridine Orange-Ethidium Bromide technique. Western blot analysis showed that celecoxib was effective at increasing COX-2 protein at 100 microM in EEC from endometriosis patients (P < 0.05). In EEC from endometriosis patients, celecoxib at 25, 50 and 100 microM was also effective in reducing COX-2 activity, reflected in the reduction of prostaglandin E(2) (PGE(2)) synthesis (P < 0.001), and VEGF secretion (P < 0.001; P < 0.05 and P < 0.001), assessed by enzyme-linked immunosorbent assay. Exogenous PGE(2) did not reverse celecoxib-induced growth inhibition. CONCLUSIONS This study suggests a direct effect of celecoxib on reduction of endometrial growth and supports further research on selective COX-2 inhibition as a novel therapeutic modality in endometriosis.

The inhibitory effect of celecoxib and rosiglitazone on experimental endometriosis

OBJECTIVE To evaluate the effects of celecoxib and rosiglitazone on the implantation and growth of endometriotic-like lesions in a murine model of endometriosis. DESIGN Prospective experimental study. SETTING Animal research and laboratory facility. ANIMAL(S) Two-month-old female BALB/c mice. INTERVENTION(S) Surgically induced endometriosis in female BALB/C mice; 28 days of treatment with celecoxib, rosiglitazone, or their combination; counting, measuring, excising, and fixing lesions. MAIN OUTCOME MEASURE(S) Immunohistochemical examination for proliferating cell nuclear antigen (PCNA), CD31, and CD34 to assess cell proliferation and vascularization, with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique for apoptosis evaluation. RESULT(S) Celecoxib and the combined treatment (celecoxib and rosiglitazone) statistically significantly reduced the mean number of lesions established per mouse, and all treatments diminished the implant volume. In addition, cell proliferation within the implants was statistically significantly reduced, and apoptosis was statistically significantly enhanced by all treatments. Also, we found that all treatments diminished the vascularized area in the lesion. CONCLUSION(S) These results are promising and reveal that celecoxib and rosiglitazone, combined or separately, have a beneficial effect on overall endometriotic growth.

Natural therapies assessment for the treatment of endometriosis

STUDY QUESTION Can resveratrol and epigallocatechin-3-gallate (EGCG) inhibit the growth and survival of endometriotic-like lesions in vivo in a BALB/c model of endometriosis, and in vitro in primary cultures of human endometrial epithelial cells (EECs)? SUMMARY ANSWER Resveratrol and EGCG exerted a potent inhibitory effect on the development of endometriosis in a BALB/c murine model and on the survival of EECs. WHAT IS KNOWN ALREADY Endometriosis is a common condition associated with infertility and pelvic pain in women of reproductive age. Resveratrol and EGCG are two polyphenols with anticarcinogenic and antioxidant properties that have been proposed as natural therapies to treat endometriosis. STUDY DESIGN, SIZE, DURATION Fifty-six 2-month-old female BALB/c mice underwent surgical induction of endometriosis. Treatments with resveratrol or EGCG started 15 days post-surgery and continued for 4 weeks. Human biopsies were taken with a metal Novak curette from the posterior uterine wall from 16 patients with untreated endometriosis and 15 controls who underwent diagnostic laparoscopy for infertility. MATERIALS, SETTING, METHODS After the treatments, animals were sacrificed and lesions were counted, measured, excised and fixed. Immunohistochemistry for proliferating cell nuclear antigen and CD34 was performed for cell proliferation and vascularization assessment in the lesions. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) technique was performed for apoptosis evaluation. Peritoneal fluid was collected to analyze vascular endothelial growth factor levels. Human EECs were purified from proliferative-phase endometrial biopsies and cultured. The effect of both polyphenols on cell proliferation was determined by a colorimetric assay using the CellTiter 96®AQueous One Solution Cell Proliferation Assay kit and on apoptosis by the TUNEL technique, using an In Situ Cell Death Detection Kit with Fluorescein. MAIN RESULTS In the mouse model, both treatments significantly reduced the mean number (P < 0.05 versus control) and the volume of established lesions (P < 0.05 versus control). Treatments consistently statistically significantly diminished cell proliferation (resveratrol P < 0.01 and EGCG P < 0.05, versus control), reduced vascular density (resveratrol P < 0.01 and EGCG P < 0.001, versus control) and increased apoptosis within the lesions (resveratrol P < 0.01 and EGCG P < 0.05, versus control). Both compounds induced reduction in human EEC proliferation (P < 0.05 versus basal) and increased apoptosis (P < 0.05 versus basal) in primary cultures. LIMITATIONS In vitro studies were only carried out in epithelial cells from human eutopic endometrium. WIDER IMPLICATIONS OF THE FINDINGS The present findings are promising and will assist the development of novel natural treatments for endometriosis. STUDY FUNDING This study was supported by ANPCYT (PICT 6384 BID 1201 OC-AR) and CONICET (PIP 5471), Argentina. None of the authors has any conflict of interest to declare.

Targeting galectin‐1‐induced angiogenesis mitigates the severity of endometriosis

Endometriosis is characterized by the presence of endometrial tissue outside the uterus that causes severe pelvic pain and infertility in women of reproductive age. Although not completely understood, the pathophysiology of the disease involves chronic dysregulation of inflammatory and vascular signalling. In the quest for novel therapeutic targets, we investigated the involvement of galectin‐1 (Gal‐1), an endogenous glycan‐binding protein endowed with both immunosuppressive and pro‐angiogenic activities, in the pathophysiology of endometriotic lesions. Here we show that Gal‐1 is selectively expressed in stromal and endothelial cells of human endometriotic lesions. Using an experimental endometriosis model induced in wild‐type and Gal‐1‐deficient (Lgals1−/−) mice, we showed that this lectin orchestrates the formation of vascular networks in endometriotic lesions in vivo, facilitating their ectopic growth independently of vascular endothelial growth factor (VEGF) and the keratinocyte‐derived CXC‐motif (CXC‐KC) chemokine. Targeting Gal‐1 using a specific neutralizing mAb reduced the size and vascularized area of endometriotic lesions within the peritoneal compartment. These results underline the essential role of Gal‐1 during endometriosis and validate this lectin as a possible target for the treatment of disease. Copyright

Anastrozole and celecoxib for endometriosis treatment, good to keep them apart?

Endometriosis is a benign gynecological disease. Cyclooxygenase-2 (COX-2) and aromatase proteins have been shown to be overexpressed in eutopic endometrium from women suffering from this disease compared to disease-free women. Furthermore, inhibition of these molecules individually was demonstrated to have antiproliferative and proapoptotic effects both in vitro and in vivo in several models. In this study, the effect of combining celecoxib, a selective COX-2 inhibitor, and anastrozole, an aromatase inhibitor, on the implantation and growth of endometriotic like lesions in a murine model of endometriosis was evaluated. Endometriosis was surgically induced in female BALB/c mice. After 28 days of treatment with celecoxib, anastrozole, or their combination, animals were killed and lesions were counted, measured, excised, and fixed. Immunohistochemistry for proliferating cell nuclear antigen and CD34 was performed for assessment of cell proliferation and vascularization. TUNEL technique was performed for apoptosis evaluation. Celecoxib was the only treatment to significantly reduce the number of lesions established per mouse, their size and vascularized area. In addition, cell proliferation was significantly diminished and apoptosis was significantly enhanced by both individual treatments. When the therapies were combined, they reversed their effects. These results confirm that celecoxib and anastrozole separately decrease endometriotic growth, but when combined they might have antagonizing effects.

Inhibition of Hyaluronic Acid Synthesis Suppresses Angiogenesis in Developing Endometriotic Lesions

Background The development and long-term survival of endometriotic lesions is crucially dependent on an adequate vascularization. Hyaluronic acid (HA) through its receptor CD44 has been described to be involved in the process of angiogenesis. Objective To study the effect of HA synthesis inhibition using non-toxic doses of 4-methylumbelliferone (4-MU) on endometriosis-related angiogenesis. Materials and Methods The cytotoxicity of different in vitro doses of 4-MU on endothelial cells was firstly tested by means of a lactate dehydrogenase assay. The anti-angiogenic action of non-cytotoxic doses of 4-MU was then assessed by a rat aortic ring assay. In addition, endometriotic lesions were induced in dorsal skinfold chambers of female BALB/c mice, which were daily treated with an intraperitoneal injection of 0.9% NaCl (vehicle group; n = 6), 20mg/kg 4-MU (n = 8) or 80mg/kg 4-MU (n = 7) throughout an observation period of 14 days. The effect of 4-MU on their vascularization, survival and growth were studied by intravital fluorescence microscopy, histology and immunohistochemistry. Main Results Non-cytotoxic doses of 4-MU effectively inhibited vascular sprout formation in the rat aortic ring assay. Endometriotic lesions in dorsal skinfold chambers of 4-MU-treated mice dose-dependently exhibited a significantly smaller vascularized area and lower functional microvessel density when compared to vehicle-treated controls. Histological analyses revealed a downregulation of HA expression in 4-MU-treated lesions. This was associated with a reduced density of CD31-positive microvessels within the lesions. In contrast, numbers of PCNA-positive proliferating and cleaved caspase-3-positive apoptotic cells did not differ between 4-MU-treated and control lesions. Conclusions The present study demonstrates for the first time that targeting the synthesis of HA suppresses angiogenesis in developing endometriotic lesions. Further studies have to clarify now whether in the future this anti-angiogenic effect can be used beneficially for the treatment of endometriosis.

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.

Active compounds present in Rosmarinus officinalis leaves and Scutellaria baicalensis root evaluated as new therapeutic agents for endometriosis

RESEARCH QUESTION Can carnosic acid, (CA) rosmarinic acid (RA) and wogonin (WG) inhibit the growth of cultured human endometrial stromal cells and endometriotic-like lesions induced in a BALB/c model of endometriosis? DESIGN Primary stromal cell cultures were established from endometrial biopsies from women with endometriosis and controls. The human endometrial stromal cell line T-HESC was also used for in-vitro experiments. Endometriosis was surgically induced in BALB/c mice, which were randomly assigned to CA 2 mg/kg/day (n = 11); CA 20 mg/kg/day (n = 10); RA 1 mg/kg/day (n = 11); RA 3 mg/kg/day (n = 10); WG 20 mg/kg/day (n = 12); intraperitoneal vehicle control (n = 8) or oral vehicle control (n = 11). After surgery, CA and RA were administered intraperitoneally on days 14-28. WG was administered orally by intragastric gavage on days 14-26. RESULTS CA, RA and WG significantly inhibited in-vitro cell proliferation in primary and T-HESC cell cultures (P < 0.05). CA and WG induced cell cycle arrest of T-HESC at the G2/M phase (P < 0.01). RA reduced intracellular ROS accumulation (P < 0.001), whereas WG increased it (P < 0.05). WG significantly inhibited oestrogen receptor alpha expression in T-HESC (P < 0.01). In-vivo, CA, RA and WG significantly reduced lesions size (P < 0.05). All compounds significantly decreased the percentage of cells in proliferation (P < 0.05) whereas RA and WG further increased the percentage of apoptotic cells (P < 0.05) in endometriotic-like lesions. CONCLUSIONS The results are promising; further investigation of these compounds as new therapeutics is needed.