Thu0333 2-Carba Cyclic Phosphatidic Acid (2ccpa) Suppresses Profibrotic Activity in Systemic Sclerosis Skin Fibroblasts and Bleomycin-Induced Skin Fibrosis in Mice

Abstract

BackgroundSystemic sclerosis (SSc) is a connective tissue disorder with progressive fibrosis in multiple organs including skin, lung and the gastrointestinal tract. Fibrosis is thought to be driven by activated fibroblasts. Therefore, inhibition of the profibrotic activity of activated fibroblasts may be a promising therapeutic approach in skin fibrosis in SSc. The autotaxin (ATX)/lysophosphatidic acid (LPA) axis is reportedly involved in fibrotic pathogenesis in SSc1. 2-carba cyclic phosphatidic acid (2ccPA) is a naturally occurring lipid mediator and one of its pleiotropic properties is to inhibit the ATX/LPA axis.ObjectivesWe investigated the anti-fibrotic effect of 2ccPA on human SSc skin fibroblasts and bleomycin-induced skin fibrosis in mice. Furthermore, we searched signaling pathways related to the anti-fibrotic effects of 2ccPA.MethodsThis study was approved by the ethics committee and the ethical review committee of animal experiments of Tokyo Women’s Medical University. We informed all participants of the content of this study, and written consent was obtained. Skin fibroblasts were taken from SSc patients and adult healthy individuals were purchased. The cells were incubated with 1-10 μM 2ccPA in the presence or absence of 10 ng/ml transforming growth factor-β1 (TGF-β1). Messenger RNA (mRNA) and protein expression for type I collagen, connective tissue growth factor (CTGF), α smooth muscle actin (αSMA), fibronectin (FN) and TGF-β1 were assessed by qRT-PCR or Western blotting. Procollagen type I C-peptide, prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF) levels in the supernatant were assessed by ELISA. Intracellular cyclic adenosine monophosphate (cAMP) levels were calculated using a commercially available EIA kit. Forskolin was used to increase intracellular cAMP levels in cultured SSc skin fibroblasts. An inhibitor of adenylate cyclase (AC), 2’-deoxyadenosine, was used to investigate whether the anti-fibrotic effect of 2ccPA was mediated via the AC/cAMP pathway. Furthermore, we used a mouse model of bleomycin-induced skin fibrosis to investigate the safety and anti-fibrotic effects of 2ccPA.ResultsTen μM 2ccPA significantly reduced mRNA and protein expression for type I collagen, CTGF and αSMA in SSc skin fibroblasts and adult healthy skin fibroblasts treated with TGF-β1. PGE2 and HGF levels in the supernatant of SSc skin fibroblasts were also reduced by 2ccPA treatment. 2ccPA increased intracellular cAMP levels as well as the AC stimulator, forskolin. In addition, forskolin decreased the mRNA expression of profibrotic markers. Reduction of COL1A1 mRNA expression by 2ccPA was blocked by treatment with 2’-deoxyadenosine in cultured SSc skin fibroblasts, suggesting that the anti-fibrotic activity of 2ccPA was partially mediated via AC stimulation. In mouse experiments, intraperitoneal injection of 10 mg/kg 2ccPA significantly reduced the development of skin thickness, collagen content and αSMA-positive cell counts.Conclusion2ccPA suppressed the profibrotic activity of SSc skin fibroblasts and...