Background/Purpose: Interstitial lung disease (ILD) is the major cause of mortality among scleroderma (systemic sclerosis, SSc) patients. Extracellular matrix (ECM) deposition is a hallmark of this and other fibrotic lung diseases. TGFβ plays a crucial role in ECM gene expression via regulation of Ca2+/calmodulin-dependent protein kinases and matrix metalloproteinases (MMPs). We recently demonstrated that M10 peptide, naturally derived from the cytosolic fragment of the hepatocyte growth factor receptor, blocks the TGFβ-mediated canonical pathway via interaction with SMAD2 and reduces fibrosis in vivo. In this study, we investigate the efficacy of M10 in the regulation of intracellular Ca²⁺ levels, ECM proteins, and in the bleomycin-induced therapeutic mouse model of SSc-ILD.

Methods: Lung fibroblasts were derived from lung tissues of SSc patients and from matched normal lungs obtained at autopsy. Antifibrotic in vivo effects of M10 (10 mg/kg, intraperitoneal, every 48h) were studied in the bleomycin-induced therapeutic mouse model of SSc-ILD. Ca²⁺ was measured by FLIPR Tetra cellular screening system equipped with Molecular Devices ScreenWorks® software. Expression levels of collagen, connective tissue growth factor (CTGF, CCN2), fibronectin and tenascin were measured by immunoblotting and real-time PCR. Expression levels of matrix metalloproteinases (MMPs) were determined by human MMP antibody array. Statistical analysis was performed using GraphPad Prism 7 software.

Results: In both normal lung fibroblasts and SSc-ILD lung fibroblasts, an acute increase of intracellular Ca²⁺ was observed 15sec following TGFβ administration, with a second peak of delayed Ca²⁺ efflux at 60sec. A high level of Ca²⁺ was maintained throughout the 10 min of the cellular screening process. In the presence of M10 peptide, TGFβ-mediated Ca²⁺ was significantly (p < 0.001) reduced in both acute and delayed states maintaining overall lower amplitude. M10 peptide suppressed TGFβ-mediated mRNA and protein expression of collagen I, CCN2, fibronectin, and tenascin. M10 peptide significantly induced the expression of MMP-10 in normal lung fibroblasts, whereas M10 peptide inhibited MMP-3 in SSc-ILD lung fibroblasts. In the bleomycin-induced therapeutic mouse model of SSc-ILD, M10 noticeably reduced collagen in lung parenchyma. A semi-quantitative evaluation of histopathology using the Ashcroft scale demonstrated a significant decrease in bleomycin-induced fibrosis in M10-treated mice as compared to mice treated with control (scrambled) peptide.

Conclusion: M10 peptide reduces ECM proteins, inhibits TGFβ-mediated Ca²⁺ efflux and regulates MMPs demonstrating strong antifibrotic effects in vitro and in vivo in an animal model of lung fibrosis and should be considered as a potential therapeutic agent for systemic sclerosis and other fibrosing diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/m10-a-small-fragment-of-the-hepatocyte-growth-factor-receptor-attenuates-fibrotic-changes-in-a-murine-model-of-scleroderma-lung-disease-and-in-human-lung-fibroblasts/