DOI: 10.6084/m9.figshare.28760102.v1
title: Supplementary Material for: In vitro effects of ABT-263, a BH3 mimetic, on fibrotic phenotype in endometriotic and endometrial stromal cells.

description: Objectives: A recent study has shown that myofibroblasts are primed for apoptosis when survival pathways are inhibited under fibrosis. This knowledge of apoptosis priming led to the development of methods to induce apoptosis specifically in myofibroblasts by blocking specific pro-survival proteins of the BCL-2 family with small molecules that mimic the function of the BH3-only proteins, termed BH3-mimetics. The current study aimed to investigate the in vitro effects of ABT-263 (Navitoclax), a BH3 mimetic, alone and in combination with oestrogen and/or progesterone, on fibrosis in endometriotic and endometrial stromal cells.
Design: Prospective study using immunocytochemistry with primary cultured human cells.
Participants/Materials: Primary culture of Stromal cells of endometrial and/or endometriotic tissues (deep infiltrating endometriosis [DIE] of 28 patients) and those of women who underwent tubal ligation or reversal as ‘true’ healthy controls (n=22)
Setting: Gynaecological research laboratory.

Methods: Double immunofluorescence staining for Ki-67 and α-smooth muscle actin (αSMA) or collagen Type I (Col I) and αSMA was performed to investigate the in vitro effects of ABT-263, alone and in combination with oestrogen and/or progesterone, on fibrosis in endometriotic and endometrial stromal cells. The total cellular proliferation index (T-PI) (percentage of Ki-67-positive cells among the total number of DAPI-stained nuclei), fibroblast proliferation index (Fb-PI) (percentage of Ki-67-positive and αSMA+ stress fibres negative cells among the total number of DAPI-stained nuclei), myofibroblast proliferation index (Myo-PI) (percentage of Ki-67-positive and αSMA+ stress fibres -positive cells among the total number of DAPI-stained nuclei), myofibroblast index (Myo-I) (percentage of cells with αSMA+ stress fibres among the total number of DAPI-stained nuclei), total collagen index (T-Col-I) (percentage of Col I+ cells among the total number of DAPI-stained nuclei), collagen in myofibroblasts index (Myo-Col-I) (percentage of Col I+ cells among the total number of myofibroblasts), and collagen in fibroblasts index (Fb-Col-I) (percentage of Col I+ cells among the total number of fibroblasts) were calculated from 10 random high-power (x400) fields through each section. 
Results: The present in vitro experiments showed that targeting myofibroblasts by ABT-263 (Navitoclax), a BH3 mimetic, in endometriosis resulted in decreased proliferation (Myo-PI) and numbers of collagen type I-expressing myofibroblasts (Myo-Col-I), and total number of collagen type I-expressing cells (T-Col-I), but increased proliferation (Fb-PI) and numbers of collagen type I-expressing fibroblasts (Fb-Col-I).
Limitations: First, we did not conduct any mechanistic studies. Second, we did not confirm the present findings in an animal model of endometriosis. Third, we did not include endometriotic cells derived from ovarian endometriosis.
Conclusions. These findings suggest that fibrosis (collagen type I) and/or myofibroblasts (key source of collagen type I) may control the proliferation of endometriotic fibroblasts. Thus, the present study does not support the current concept that fibrosis is a therapeutic target for endometriosis. Until more robust evidence is obtained to support the conclusion that regression of tissue fibrosis in endometriotic lesions is indeed beneficial to patients with endometriosis, we should be very cautious in considering fibrosis a therapeutic target for endometriosis.
Trial registration: Not applicable