OP0239 Systemic Sclerosis: A Pathological Model Linking Oxidative Stress, DNA Damage and Fibrosis

Background Fibroblasts isolated from lesional areas of Systemic Sclerosis (SSc) patients overproduce reactive oxygen species (ROS), overexpress type I collagen and a-smooth muscle actin (a-SMA) and show chromosomal aberrations (1, 2). Recent studies have demonstrated a prominent role for ATM pathway in fibrosis (3). ATM (ataxia telangiectasia mutated) is regarded as the major regulator of the cellular response to DNA damage, in particular to DNA double strand breaks (DSBs). In response to DSBs, ATM undergoes catalytic activation and phosphorylates intermediary protein substrates, such as CHK2, p53 and H2AX, leading to cell cycle arrest and DNA repair or apoptosis. Moreover, ATM has also been implicated in the regulation of oxidative stress, and its effects in this pathway were recently shown to modulate cytoplasmatic pathways (4). Objectives The aim of this study was to demonstrate a possible correlation between ROS induced-DNA damage and fibrosis in SSc. Methods Fibroblasts were isolated from skin biopsies of SSc patients or healthy volunteers. AT fibroblasts were obtained from Coriell Institute. Cells were transiently transfected with specific siRNA against ATM with Lipofectamine (Invitrogen). Total RNA was isolated, reverse-transcribed, and quantitative real-time PCR reactions were performed using SYBR-Green Master Mix. To analyzed protein expression, cells were lysed and subjected to western blot with specific antibodies. For immunocytochemistry, cells were fixed in PFA, permeabilized and labelled with specific antibodies to be evaluated through fluorescence microscopy. Results SSc fibroblasts show higher levels of activated ATM compared to normal cells. Normal fibroblasts stimulated by bleomycin shift to a SSc like-phenotype with high levels of ROS, overexpression of type I collagen and phosphorylation of ATM. Incubation of SSc fibroblasts or activated normal fibroblasts with a synthetic inhibitor of ATM (KU55933) or trasfection with specific siRNA against ATM lead to downregulation of ROS production and type I collagen expression. AT fibroblasts, lacking of functional ATM, show no changes in type I collagen transcript abundance after bleomycin tretament. Conclusions DNA damage leads to changes in gene-profiles, and also in protein synthesis, degradation and trafficking. Our data suggest an important link between DNA damage induced by ROS and overexpression of type I collagen in SSc fibroblasts. Modulation of ROS levels and ATM activation may provide novel therapeutic targets to prevent fibrosis, key feature of Systemic Sclerosis. References Sambo P, Arhtritis Rheum 2001; 44: 2653-2664. Baroni S, N Engl J Med 2006; 354:2667-76. Daugherity EK, Cell Cycle 2012; 11: 1918-1928. Shiloh Y, Nature Reviews 2013; 14: 197-210. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2836