LAUSR

Background Pulmonary fibrosis is a major cause of mortality in scleroderma (SSc) and Idiopathic Pulmonary Fibrosis (IPF). Fibrosis is driven by Inappropriate myofibroblast differentiation and persistence. Understanding this process, is vital for developing an effective treatment. Angiotensin II, is implicated in fibroblast activation in the heart and kidney, through interactions with growth factors (e.g. EGF and TGFβ). Objectives We examined the role of Angiotensin II in myofibroblast activation in the lung. Methods Lung fibroblasts were isolated from SSc, IPF, or control patient lungs (6 each). Fibroblasts were also cultured from PTEN null and wild-type mice. Protein expression after angiotensin II treatment (AngII) was investigated by western blotting. Myofibroblast differentiation and function was assayed through the contraction of 3D collagen gels and scratch migration assays. The signalling pathways involved were dissected using specific inhibitors: PI3-kinase/AKT (wortmannin, LY294002), TGFβ (1d11 neutralising antibody, SB431542 ALK5 inhibitor) Ataxia-Telangiectasia Mutated (ATM – Ku55933), AngII (Losartan). Results SSc and IPF lung fibroblasts showed increased AKT phosphorylation and suppressed PTEN expression (p<0.05). Their phenotype was more myofibroblast-like, with higher αSMA expression (p<0.05), increased collagen gel contraction (control; 207±14 vs SSc; 93±15 vs IPF 91±21, p<0.05), and enhanced migratory capacity (p<0.05). PTEN-null fibroblasts showed a similar phenotype. AngII treatment activated AKT, suppressed PTEN and induced myofibroblast differentiation in normal lung fibroblasts. In both AngII-treated and PTEN lung fibroblasts AKT activation required the ATM kinase. Inhibition of AKT either with PI3K or ATM inhibitor abrogated these effects. The increased expression of Myofibroblast-related genes after AngII treatment, was also blocked by inhibition of TGFβ with a neutralising antibody or an ALK5 inhibitor. AKT phosphorylation on the other hand was only partially blocked was partially blocked by TGFβ inhibition. Conclusions Our data demonstrate for the first time that AngII signals via the ATM kinase, which together with PTEN suppression are essential for the activation of AKT by AngII. AngII promotes myofibroblast differentiation, by stimulating the fibroblast TGFβ autocrine loop through AKT. Our data shows that activation of AKT through ATM and PTEN, may serve as the molecular link between pulmonary hypertension and lung fibrosis in fibrotic diseases. Acknowledgements Arthritis Research UK, Royal Free Hospital Charity and Scleroderma Research UK. Disclosure of Interest None declared