Endometriosis pathogenesis: the relationship between oxidative stress, fibrosis and immunological dysfunction

Abstract

This thesis includes two projects that evaluate the relationship between oxidative stress, the ADAM17/Notch signaling pathway, fibrosis and immunological dysfunction in the pathogenesis of endometriosis. The aim of the first project was to evaluate the role of oxidative stress and the ADAM17/Notch pathway on the fibrosis that is generated in the disease. For such purpose, 202 non-­‐pregnant women younger than 42 years old who were operated for a benign gynaecological condition were recruited. Peritoneal fluid (PF) was collected from all participants, as well as biopsies from eutopic and ectopic endometrium from rectal lesions of deep infiltrating endometriosis (DIE). Stromal endometrial cells from the biopsies were filtered and cultured. Oxidative stress was assessed in PF of the study participants by means of Advanced Oxidation Protein Products (AOPP). Besides, ADAM17 levels were also determined in all PF samples. At cellular levels, ADAM17, Notch intracellular domain (NICD) and fibrosis surrogates (alpha-­‐SMA and type-­‐I collagen) were evaluated. Among the 202 patients recruited, 121 presented endometriosis (either superficial endometriosis, ovarian endometriomas or DIE). Eighty-­‐one patients were considered as controls (uterine fibroids, non-­‐endometriotic benign ovarian cysts, tubal infertility, pelvic pain…). The results of the study showed that AOPP and ADAM17 were increased in PF of patients with DIE, compared to controls. In addition, both markers presented a significant positive correlation. At cellular levels, ADAM17 and NICD were increased in both eutopic and ectopic cells from patients with DIE compared to eutopic cells from controls. When fibrosis was assessed, we found that fibrosis markers were increased in ectopic cells from DIE compared to eutopic cells from controls. When gamma-­‐secretase inhibitors were added to inhibit the release of NICD, only those ectopic cells from deep lesions presented a decrease of fibrosis markers, showing a hyperactivation of the ADAM17/Notch pathway in endometriosis. Besides, and to further reinforce this

relationship, incubation of stromal endometrial cells with H2O2, ADAM17 and cell culture supernatants from DIE increased fibrosis markers independently from the origin of the cells. The second project was designed to investigate if natural killer (NK) NKG2D ligands shedding played a role in the pathogenesis of endometriosis. It has been shown that NK cells cytotoxicity is decreased in patients with endometriosis. Other studies have demonstrated that NKG2D ligands (MICA, MICB and ULBP family proteins) are released from cell surfaces after cleavage from metalloproteinases as ADAM17. After having established that the ADAM17/Notch pathway is hyperactivated in endometriosis, the next step was to determine if NKG2D ligands shedding was a phenomenon that occurred in endometriosis and decreased the cytotoxicity of NK cells. For this purpose, we evaluated 202 patients with the same characteristics as in the first study and we assessed the levels of NKG2D soluble ligands in PF of the study participants. The results of the second study demonstrated that PF levels of NKG2D ligands were increased in those patients with the most severe forms of endometriosis (DIE) compared to controls. In addition, positive clinical correlations between MICA and dysmenorrhea score and total rAFS score were found, suggesting that the NKG2D ligands shedding phenomenon plays a role in the pathogenesis of the disease.

The overall conclusions of this thesis work suggest that oxidative stress is increased in patients with endometriosis, leading to hyperactivation of the ADAM17/Notch signaling and to excessive fibrosis derived from this pathway. Besides, elevated NKG2D ligands found in PF of DIE patients suggest that NKG2D ligands shedding phenomenon plays a role in the pathogenesis of the disease.

Els treballs de la present tesi doctoral investiguen la relació que presenten l’estrés oxidatiu, la fibrosi i la disfunció immunològica en la fisiopatologia de l’endometriosi. Mitjançant una mostra de més de 202 pacients que es someteren a una intervenció quirúrgica per via abdominal per una patologia ginecològica benigna, es varen obtenir dades d’interés clínic de totes les pacients, a més de mostres biològiques de líquid peritoneal i teixit endometrial (eutòpic o ectòpic en aquelles pacients afectes d’endometriosi profunda). En les mostres obtingudes es varen realitzar uns estudis per quantificar els nivells d’estrés oxidatiu, la via de senyalització d’ADAM17/Notch, els marcadors de fibrosi com α-­‐SMA i els nivells de lligands solubles pel receptor NKG2D de les cèl.lules natural killers. Els resultats obtinguts demostraren que en la fisiopatologia de l’endometriosi, l’estrés oxidatiu es relaciona amb una sobreactivació de la via de senyalització d’ADAM17/Notch, i que aquesta a la vegada provoca un excés dels marcadors de fibrosi que s’expresen a nivell cel.lular. Paral.lelament també es demostrà que el fenomen de shedding dels lligands solubles pel receptor NKG2D pot representar una causa de la inhibició en la citotoxicitat de les cèl.lules natural killer, observada en aquesta malaltia.