Aortic Carboxypeptidase-Like Protein Promotes The Fibroblast To Myofibroblast Transition

Introduction: Organ fibrosis is characterized by the overgrowth, hardening, and scarring of tissues and impacts both cardiovascular and pulmonary systems. Fibrosis is exacerbated by extracellular matrix (ECM) remodeling and accumulation of active transforming growth factor β (TGFβ) which promote the production of smooth muscle alpha actin (SMA)-positive myofibroblasts and collagen secretion. Aortic Carboxypeptidase-Like Protein (ACLP) is an ECM protein secreted by vascular smooth muscle cells and myofibroblasts. ACLP knockout mice are protected from bleomycin-induced fibrosis and have decreased accumulation of myofibroblasts. However, the mechanism of ACLP action is currently unknown. Hypothesis: Because of ACLP’s expression in the fibrotic ECM and the accumulation of TGFβ in organ fibrosis, we hypothesize that ACLP stimulates the fibroblast to myofibroblast transition by promoting SMA and collagen expression, in part, by activating TGFβ signaling. Methods: We used gain of function and loss of function studies in primary mouse lung fibroblasts and cultured human lung fibroblasts with recombinant protein, co-culture, and siRNA-mediated knockdown. Results and Conclusions: As primary lung fibroblasts differentiate into myofibroblasts, ACLP protein expression precedes SMA and collagen protein expression. Recombinant ACLP (rACLP) induces SMA and collagen protein and gene expression in both primary mouse lung fibroblasts and cultured human lung fibroblasts. Knockdown of ACLP slows and reverts the fibroblast to myofibroblast transition. Treatment of fibroblasts with rACLP induces phosphorylation of Smad3 and Smad1, and both this phosphorylation and induction of SMA expression is dependent on TGFβ receptor kinase activity. In conclusion, these data suggest that ACLP stimulates the fibroblast to myofibroblast transition via the TGFβ receptors. This project has unveiled both novel signaling events that occur in early stages of disease progression and molecules that could serve as targets of drugs designed to slow the progression of organ fibrosis.