I-28: Role of Mevalonate-Ras Homology (Rho)/Rho-Associated Coiled-Coil-Forming Protein Ki nase-Mediated Signaling Pathway in The Pathogenesis of Endometriosis-Associated Fibrosis

Background: Endometriosis, a disease affecting 3-10% of women of reproductive age, is characterized by the ectopic growth of endometrial glands and stroma surrounded by dense fibrous tissue. Whereas, normal eutopic endometrium shows scarless tissue repair during menstrual cycles, which suggests that the endometriotic tissues have distinct mechanisms of fibrogenesis. During the development of endometriotic lesions, excess fibrosis may lead to scarring and to alteration of tissue function. It has been suggested that type I collagen is a major contributor to endometriosis-associated fibrosis. Alpha-smooth muscle actin (SMA)-positive myofibroblastic cells were frequently detected in the fibrotic areas of endometriosis lesions. We have previously demonstrated that endometriotic stromal cells can differentiate to alpha-SMA-positive myofibroblasts. One approach to understanding the pathogenesis of endometriosis is to investigate the mechanisms underlying the fibrogenesis associated with this disease. Using 3-dimensional collagen gel culture model, we have evaluated the extracellular matrix contractility and myofibroblastic differentiation of endometriotic stromal cells. Endometriotic stromal cells showed enhanced extracellular matrix contractility in comparison with normal endometrial stromal cells. Activation of the mevalonate-Ras homology (Rho)/Rho-associated coiled-coil-forming protein kinase (ROCK)-mediated signaling pathway with simultaneously enhanced myofibroblastic differentiation is involved in this mechanism. In the present study, we investigated the effect of various agents that target mevalonate-Rho/ROCK mediated signaling pathway for the treatment of endometriosis- associated fibrosis using the 3-dimensional collagen gel culture system.Materials and Methods: Primary cultures of endometriotic cyst stromal cells were utilized for the experiments. The effects of simvastatin, Y-27632, fasudil, heparin and Decidualization on the contractile profile, morphology, cell density, and contraction-related molecule expression of these cells in the 3-dimensional collagen gel culture were investigated using laser scanning microscopy, collagen gel contraction assay, and Western blot analysis. The effects of these mevalonate-Rho/ROCK pathway-targeting agents on the cell proliferation, apoptosis, and cell cycle of endometriotic cyst stromal cells in 2-dimensional culture were also evaluated by methylthiazoletetrazolium (MTT) assay, 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: Mevalonate - Rho/ ROCK pathway - targeting agents examined in this study attenuated the contractility of endometriotic stromal cells by inhibiting mevalonate- Rho/ROCK pathway activation, cell proliferation, attachment to surrounding extracellular matrices, and the differentiation into the alpha-smooth muscle actin-positive myofibroblastic phenotype. These agents also induced the apoptosis and cell cycle arrest of cultured endometriotic stromal cells. Conclusion: Research on endometriotic stromal cell biology using 3-dimensional collagen matrices offers new opportunities to understand the reciprocal and adaptive interactions that occur between cells and surrounding matrix in a tissue-like environment. Such interactions are integrated with the regulation of endometriotic tissue morphogenesis and dynamics that characterizes endometriosis- associated fibrosis. It is suggested that the enhanced extracellular matrix contractility of endometriotic stromal cells in the 3-dimensional collagen gel culture is associated with myofibroblastic differentiation and the activation of mevalonate-Rho/ROCK-mediated signaling pathways, and that modulation of mevalonate- Rho/ROCK pathways seems to be a novel therapeutic target for the treatment and prevention of endometriosisassociated fibrosis. Keywords: Endometriosis, Contractility, Mevalonate-Rho/ ROCK Pathway, Fibrosis, Myofibroblast