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.

Endocrine disruptor hexachlorobenzene induces cell migration and invasion, and enhances aromatase expression levels in human endometrial stromal cells.

Endometriosis is the presence and growth of endometrial tissue outside of the uterus. Previous studies have suggested that endocrine disrupting chemicals such as organochlorine pesticides could be a risk factor for endometriosis. Hexachlorobenzene (HCB) is a weak ligand of the aryl hydrocarbon receptor (AhR) and promotes metalloproteinase and cyclooxygenase-2 (COX-2) expression, as well as, c-Src activation in human endometrial stromal cells (T-HESC) and in rat endometriosis model. Our aim was to evaluate the effect of HCB exposure on oestrogen receptor (ER) ɑ and ß, progesterone receptor (PR) and aromatase expression, as well as, on cell migration and invasion in T-HESC and primary cultures of endometrial stromal cells from eutopic endometria of control subjects (ESC). Results show that HCB increases ERɑ and aromatase protein levels and reduces PR content in both T-HESC and ESC. However, the pesticide only increases ERß expression in ESC, without changes in T-HESC. Moreover, cell migration and invasion are promoted by pesticide exposure involving the AhR, c-Src, COX-2 and ER pathways in T-HESC. HCB also triggers ERɑ activation via phosphorylation in Y537 through AhR/c-Src pathway. Our results provide experimental evidence that HCB induces alterations associated with endometriosis, suggesting that these mechanisms could contribute to pesticide exposure-induced endometriosis development.

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.

Active compounds present inRosmarinus officinalis leaves andScutellaria 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.

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), 20 mg/kg 4-MU (n = 8) or 80 mg/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.

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.

Percepción del tiempo: resultados de una intervención grupal breve para el cambio del perfil temporal / Time perception: results of a brief group intervention to change time perspective profiles

La teoría de la perspectiva del tiempo de Zimbardo (1999) plantea una clasificación de las dimensiones de la orientación temporal, medidas con el Zimbardo Time Perspective Inventory (ZTPI), la cual propone, por una parte, que si se tiene una actitud positiva y moderada hacia el pasado, el presente y el futuro, se puede desarrollar mayor bienestar y buena salud tanto mental como física, mientras que actitudes negativas o extremas reflejarían pautas de vida poco saludables. El principal objetivo de esta investigación es evaluar el efecto de una intervención grupal breve para la modificación del perfil temporal. Para este fin, se realizó un estudio cuasiexperimental con grupo control en una muestra de 28 estudiantes universitarios; 14 de ellos formaron el grupo experimental y 14 formaron el grupo control. Los resultados muestran que la intervención realizada al grupo experimental modificó la orientación temporal de los participantes hacia un perfil equilibrado. Para terminar, se concluye que, con una intervención breve y grupal que potencie un pasado positivo y un futuro y un presente más saludables, es posible lograr un perfil equilibrado. Posteriores estudios podrían evaluar el impacto de la modificación del perfil temporal en población clínica.

Zimbardo's time perspective theory (1999) provides a classification of the dimensions of time perspective, which can be measured with the Zimbardo Time Perspective Inventory (ZTPI). This inventory proposes that if people have a positive and balanced attitude toward the past, present, and future, they can develop greater well-being and good mental and physical health. A negative attitude can reflect unhealthy life patterns. The main aim of this study was to assess the effect of a brief group intervention to modify time perspective profiles. Research was conducted under a quasi-experimental design with a control group. The sample included 28 university students, 14 in the experimental group and 14 in the control group. The results show that intervention in the experimental group modified the time perspective of each participant toward a balanced time perspective profile. In conclusion, a balanced time perspective profile can be achieved with a brief group intervention that seeks to favor a positive past and a healthier present and future. Further studies should assess the impact of modifying time perspective among the clinical population.

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.

Output capabilities of personal music players and assessment of preferred listening levels of test subjects: outlining recommendations for preventing music-induced hearing loss.

OBJECTIVES/HYPOTHESIS: Our goal was to assess the impact of personal music players, earphones, and music styles on output, the subject's preferred listening levels, and outline recommendations for the prevention of music-induced hearing loss. STUDY DESIGN: Experimental study. Personal music players' output capabilities and volunteers' preferred output levels were assessed in different settings. Based on current noise-induced hearing loss exposure limits, recommendations were outlined. METHODS: On three different devices and earphone types and 10 music styles, free field equivalent sound pressure output levels were assessed by applying a microphone probe inside the auditory canal. Forty-five hearing-healthy volunteers were asked to select preferred listening levels in different background noise scenarios. RESULTS: Sound pressure output reached 126 dB. No difference was found between device types, whereas earbud and supra-aural earphones showed significantly lower outputs than in-ear earphones (P < .001). Three distinct music style groups were identified with as much as 14.4 dB difference between them. In silence, 17.8% of volunteers spontaneously selected a listening level above 85 dB. With 90 dB background noise, 40% selected a level above 94 dB. Earphone attenuation capability was found to correlate significantly with preferred level reductions (r = 0.585, P < .001). In-ear and especially supra-aural earphones reduced preferred listening levels the most. CONCLUSIONS: Safe-use recommendations were outlined, whereas selecting the lowest volume setting comfortable remained the main suggestion. High background noise attenuating earphones may help in reducing comfortable listening levels and should be preferred. A risk table was elaborated, presenting time limits before reaching a risky exposure.

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.

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.

Apoptosis is increased and cell proliferation is decreased in out-of-phase endometria from infertile and recurrent abortion patients.

BACKGROUND: Various endometrial abnormalities have been associated with luteal phase deficiency: a significant dyssynchrony in the maturation of the glandular epithelium and the stroma and a prevalence of out-of-phase endometrial biopsy specimens. Out-of phase endometrium is a controversial disorder related to failed implantation, infertility and early pregnancy loss. Given that the regulation of the apoptotic process in endometrium of luteal phase deficiency is still unknown, the aim of this study was to evaluate cell proliferation, apoptosis and the levels of the main effector caspase, caspase-3 in the luteal in-phase and out-of-phase endometrium. METHODS: Thirty-seven endometrial samples from sterile or recurrent abortion patients were included in this study: 21 in-phase samples (controls) and 16 samples with out-of-phase endometrium. Biopsy specimens of eutopic endometrium were obtained from all subjects during days 21-25 of the menstrual cycle. The endometrium with endometrial maturity of cycle day 25 or less at the time of menstruation was considered out-of phase. Endometrial tissues were fixed in 10% buffered formaldehyde. For apoptosis quantification, sections were processed for in situ immunohistochemical localization of nuclei exhibiting DNA fragmentation, by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxygenin nick-end labeling (TUNEL) technique. Expressions of Proliferating Cell Nuclear Antigen (PCNA) as a marker of cell proliferation, and of cleaved caspase-3 as a marker of apoptosis, were assessed by immunohistochemistry in the luteal in-phase and out-of-phase endometrium from infertile and recurrent abortion patients. RESULTS: Luteal out-of-phase endometrium had increased apoptosis levels compared to in-phase endometrium (p < 0.05). Caspase-3 evaluation confirmed these results: the luteal out-of-phase endometrium showed augmented cleaved caspase-3 expression (p < 0.005). As well, our data demonstrated that the luteal out-of-phase endometrium expresses decreased PCNA levels (p < 0.05), showing that cell proliferation is diminished in this tissue. CONCLUSIONS: this study represents the first report describing variations at the cell proliferation and cell death levels in the out-of-phase endometrium in comparison with in-phase endometrium from infertile and recurrent abortion patients. Further studies are needed to elucidate a potential role of these alterations in the physiopathology of luteal phase deficiency.