Iñaki González-Foruria1,2,3, Pietro Santulli1,2, Sandrine Chouzenoux1, Francisco Carmona4, Charles Chapron1,2, Frédéric Batteux1,5. 1. Département Développement, Reproduction et Cancer, Institut Cochin, INSERM U1016, Equipe Pr. Batteux, Paris, France. 2. Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Universitaire Paris Centre, Centre Hospitalier Universitaire (CHU) Cochin, Department of Gynaecology Obstetrics II and Reproductive Medicine, 75679 Paris Cedex 14, France. 3. Institut Clínic of Gynecology, Obstetrics and Neonatology, Hospital Clínic. Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Faculty of Medicine - University of Barcelona, Barcelona, Spain. 4. Institut Clínic of Gynecology, Obstetrics and Neonatology, Hospital Clínic. Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Faculty of Medicine-University of Barcelona, Barcelona, Spain. 5. Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Universitaire Paris Centre, Centre Hospitalier Universitaire (CHU) Cochin, Service d'immunologie biologique, 75679 Paris Cedex 14, France.
Abstract
STUDY QUESTION: Is oxidative stress associated with the A disintegrin and metalloproteases (ADAM) metallopeptidase domain 17 (ADAM17)/Notch signalling pathway and fibrosis in the development of endometriosis? SUMMARY ANSWER: Oxidative stress is correlated with hyperactivation of the ADAM17/Notch signalling pathway and a consequent increase in fibrosis in patients with endometriosis. WHAT IS KNOWN ALREADY: It is nowadays accepted that oxidative stress plays an important role in the onset and progression of endometriosis. Oxidative stress is able to induce the synthesis of some members of the 'ADAM' family, such as ADAM17. ADAM17/Notch signalling is dysregulated in other profibrotic and inflammatory diseases. STUDY DESIGN, SIZE, DURATION: This was a prospective laboratory study conducted in a tertiary-care university hospital between January 2011 and April 2013. We investigated non-pregnant, younger than 42-year-old patients (n = 202) during surgery for a benign gynaecological condition. PARTICIPANTS/MATERIALS, SETTING, METHODS: After complete surgical exploration of the abdominopelvic cavity, 121 women with histologically proven endometriosis and 81 endometriosis-free control women were enrolled. Peritoneal fluid (PF) samples were obtained from all the study participants during surgery in order to detect advanced oxidation protein products (AOPPs) and metalloproteinase activity of ADAM17. Stromal cells from endometrial specimens (n = 8) were obtained from endometrium of control patients (Cs), and from eutopic (Es) and ectopic (Ps) endometrium of patients with deep infiltrating endometriosis (DIE) (n = 8). ADAM17, Notch and the fibrosis markers α-smooth muscle actin (α-SMA) and type-I collagen were assessed using immunoblotting in all the endometrial samples obtained. Additionally, fibrosis was assessed after using Notch cleavage inhibitors (DAPT and FLI-06). Notch and fibrosis were also evaluated after stimulation of stromal endometrial cells with ADAM17 purified protein, increasing concentrations of H2O2 and primary cell culture supernatants. MAIN RESULTS AND THE ROLE OF CHANCE: Patients with DIE presented higher PF AOPP and ADAM17 protein levels than controls (P < 0.01 and P < 0.05, respectively). In addition, these two markers were positively correlated (r = 0.614; P < 0.001). At the cellular level, ADAM17 activity was increased in Es and Ps compared to Cs (P < 0.001 and P < 0.01, respectively). Furthermore, Ps presented hyperactivation of Notch signalling (P < 0.05) and augmentation of fibrosis markers (P = 0.009 for α-SMA and P = 0.015 for type-I collagen) compared to controls. The use of DAPT and FLI-06 reduced both fibrosis markers in Ps but not in Cs. Stimulation with ADAM17, H2O2 and Ps supernatant culture significantly increased Notch and fibrosis in both Ps and Cs. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The control group consisted of women who underwent surgery for benign gynaecological conditions, which could lead to biases because some of these conditions may cause alterations in oxidative stress and the ADAM17/Notch pathways. The small sample size of endometrial biopsies used for each group of patients (n = 8) is a limitation of the study, and results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: We propose a novel pathway in endometriosis pathogenesis that correlates oxidative stress, hyperactivation of ADAM17/Notch signalling and a consequent increase in fibrosis. This study suggests that Notch signalling plays a key role in the fibrotic processes that take place in ectopic lesions of patients with DIE, as already observed in other pro-fibrotic diseases. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by grants from University Paris Descartes, INSERM and Fundación Alfonso Martín Escudero. The authors have no competing interests to declare.
STUDY QUESTION: Is oxidative stress associated with the A disintegrin and metalloproteases (ADAM) metallopeptidase domain 17 (ADAM17)/Notch signalling pathway and fibrosis in the development of endometriosis? SUMMARY ANSWER: Oxidative stress is correlated with hyperactivation of the ADAM17/Notch signalling pathway and a consequent increase in fibrosis in patients with endometriosis. WHAT IS KNOWN ALREADY: It is nowadays accepted that oxidative stress plays an important role in the onset and progression of endometriosis. Oxidative stress is able to induce the synthesis of some members of the 'ADAM' family, such as ADAM17. ADAM17/Notch signalling is dysregulated in other profibrotic and inflammatory diseases. STUDY DESIGN, SIZE, DURATION: This was a prospective laboratory study conducted in a tertiary-care university hospital between January 2011 and April 2013. We investigated non-pregnant, younger than 42-year-old patients (n = 202) during surgery for a benign gynaecological condition. PARTICIPANTS/MATERIALS, SETTING, METHODS: After complete surgical exploration of the abdominopelvic cavity, 121 women with histologically proven endometriosis and 81 endometriosis-free control women were enrolled. Peritoneal fluid (PF) samples were obtained from all the study participants during surgery in order to detect advanced oxidation protein products (AOPPs) and metalloproteinase activity of ADAM17. Stromal cells from endometrial specimens (n = 8) were obtained from endometrium of control patients (Cs), and from eutopic (Es) and ectopic (Ps) endometrium of patients with deep infiltrating endometriosis (DIE) (n = 8). ADAM17, Notch and the fibrosis markers α-smooth muscle actin (α-SMA) and type-I collagen were assessed using immunoblotting in all the endometrial samples obtained. Additionally, fibrosis was assessed after using Notch cleavage inhibitors (DAPT and FLI-06). Notch and fibrosis were also evaluated after stimulation of stromal endometrial cells with ADAM17 purified protein, increasing concentrations of H2O2 and primary cell culture supernatants. MAIN RESULTS AND THE ROLE OF CHANCE: Patients with DIE presented higher PF AOPP and ADAM17 protein levels than controls (P < 0.01 and P < 0.05, respectively). In addition, these two markers were positively correlated (r = 0.614; P < 0.001). At the cellular level, ADAM17 activity was increased in Es and Ps compared to Cs (P < 0.001 and P < 0.01, respectively). Furthermore, Ps presented hyperactivation of Notch signalling (P < 0.05) and augmentation of fibrosis markers (P = 0.009 for α-SMA and P = 0.015 for type-I collagen) compared to controls. The use of DAPT and FLI-06 reduced both fibrosis markers in Ps but not in Cs. Stimulation with ADAM17, H2O2 and Ps supernatant culture significantly increased Notch and fibrosis in both Ps and Cs. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The control group consisted of women who underwent surgery for benign gynaecological conditions, which could lead to biases because some of these conditions may cause alterations in oxidative stress and the ADAM17/Notch pathways. The small sample size of endometrial biopsies used for each group of patients (n = 8) is a limitation of the study, and results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: We propose a novel pathway in endometriosis pathogenesis that correlates oxidative stress, hyperactivation of ADAM17/Notch signalling and a consequent increase in fibrosis. This study suggests that Notch signalling plays a key role in the fibrotic processes that take place in ectopic lesions of patients with DIE, as already observed in other pro-fibrotic diseases. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by grants from University Paris Descartes, INSERM and Fundación Alfonso Martín Escudero. The authors have no competing interests to declare.