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A2917 - The Pathological Effects of Decellularized Fibrotic Extracellular Matrix from IPF Patients
Author Block: R. Nho1, J. Im2; 1Medicine, Univ of Minnesota, Minneapolis, MN, United States, 2Medicine, University of Minnesota, Minneapolis, MN, United States.
1. Rationale: Idiopathic pulmonary fibrosis (IPF) is a deadly fibrotic lung disease. IPF is characterized by the relentless production of a collagen rich matrix and the presence of apoptosis-resistant fibrotic fibroblasts. Forkhead box O3a (FoxO3a) is a direct regulator of cell death inducing and cell cycle inhibitor proteins, and FoxO3a deregulation is associated with various human diseases. When normal lung fibroblasts interact with collagen matrix, FoxO3a activity is increased, which subsequently promotes their cell death. In contrast, FoxO3a becomes abnormally suppressed when IPF fibroblasts attach to collagen, protecting them from matrix driven apoptosis. We found that FoxO3a suppression is due to aberrant miR-96 activation in IPF fibroblasts, which confers an apoptosis-resistant phenotype on collagen rich matrix. Since type I collagen is the major protein found in the fibroblastic foci in IPF patients, we conducted experiments with the extracellular matrix (ECM) derived from IPF and non-IPF patients to elucidate matrix-driven fibrotic process via miR-96/FoxO3a-dependent cellular signaling.
2. Methods: Decellularized ECM (n=3, each) was prepared using 1%SDS and 1% Triton-X-100. IPF (n=6) and non IPF cells (n=5) were attached to decellularized matrix from non- IPF and IPF patients. Real time PCR and Western analysis were performed to measure miR-96 and FoxO3a. antimiR-96 or non-functional miR were injected to C57/BL6 mice after bleomycin treatment, and miR-96 expression was measured in mouse lungs by hydroxyproline and sircol assay.
3. Results: miR-96 was highly increased while FoxO3a was reduced when non-IPF lung (control) fibroblasts (n=5) are cultured on a decellularized ECM derived from IPF patients (IPF-ECM). In contrast, when IPF lung fibroblasts (n=6) were cultured on a decellularized ECM from non-IPF patients (control-ECM), miR-96 expression remained high, FoxO3a remained low, and their expression was not significantly altered by IPF-ECM. These findings suggest that the fibrotic ECM from IPF patients has pathological properties that cause neighboring normal lung fibroblasts to acquire a fibrotic phenotype via miR-96/FoxO3a alteration. To support miR-96 regulation by fibrotic ECM, reduced FoxO3a and enhanced miR-96 expression were also found in the fibrotic lungs of bleomycin injured C57/BL6 mice (n=8). However, the injection of antimiR-96 reactivated FoxO3a via miR-96 suppression, which reduced collagen production in bleomycin injured mouse lungs.
4. Conclusion: Our findings highlight the crucial role of fibrotic ECM produced by miR-96 altered fibrotic fibroblasts on the development of lung fibrosis. We propose that targeting of miR-96 may be an effective strategy to limit the development of lung fibrosis via FoxO3a activation.