Date of Award

1-1-2018

Embargo Period

2-22-2021

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Medicine

College

College of Graduate Studies

First Advisor

Galina S. Bogatkevich

Second Advisor

Richard M. Silver

Third Advisor

Alexander Awgulewitsch

Fourth Advisor

Gerard T. Hardiman

Abstract

Background: SSc-ILD is a complicated autoimmune disease and a major cause of mortality among scleroderma patients; TGFβ is a crucial profibrotic regulator. We previously demonstrated that a 10 amino acid peptide, M10, reduced collagen production and attenuated fibrosis in a preventive mouse model. In this study, we investigated therapeutic potentials of M10 in a bleomycin-induced fibrosis model in mice and its antifibrotic mechanisms in primary lung fibroblasts. Methods: Efficacy and pharmacokinetics (PK) of M10 were studied in C57BL/6J mice. Severity of fibrosis was evaluated by analyzing the lung mechanics, histology, and soluble collagen content. Concentration of M10 in plasma was estimated by ELISA. Expressions of extracellular matrix (ECM) proteins (e.g. collagen, fibronectin, and tenascin) were measured by immunoblotting and real-time PCR. Cytosolic Ca2+ was measured by the FLIPR Tetra cellular screening system. Expression and activity of matrix metalloproteinases (MMPs) were determined by human MMP antibody array, ELISA and fluorometric assays. Statistical analysis was performed using GraphPad Prism 7 software. Results: M10 significantly reduced lung fibrosis as measured by the Ashcroft score (p < 0.003) and soluble collagen content (p < 0.02) in the therapeutic mouse model. The maximum concentration of M10, 4.8 μg/ml, was achieved within 6 hours of M10-administration, and declined to a minimal level of 0.2 μg/ml by 48 hours. The experimentally obtained half-life of M10 (6.8 ± 0.7 hour) was comparable to the theoretically predicted half-life of M10 (7.2 hour) by ProtLifePred software. In lung fibroblasts, M10 slightly lowered ECM proteins on the mRNA level, significantly reduced collagen on the protein level, and regulated expressions of MMPs in a cell type-dependent manner. In normal lung fibroblasts, in the presence of TGFβ, M10 iv significantly induced expression of MMP-10 in a Ca2+ -independent way. In SSc-ILD lung fibroblasts, M10 significantly reduced expression of MMP-3 in Ca 2+ -dependent way. In both, normal and SSc-ILD lung fibroblasts, in the presence of M10, TGFβ-mediated Ca 2+ induction was significantly (p < 0.001) reduced. Conclusion: M10 exerts convincing stability and antifibrotic effects in in vivo models. M10 reduces ECM proteins, inhibits TGFβ-mediated Ca2+ efflux, and regulates MMP expression in lung fibroblasts. These data suggest that M10 possesses promising potential to reduce TGFβ-mediated outcomes in SSc-ILD.

Rights

All rights reserved. Copyright is held by the author.

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