Date of Award

2020

Embargo Period

12-10-2022

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Medicine

College

College of Graduate Studies

First Advisor

Carol Feghali-Bostwick

Second Advisor

Betty Tsao

Third Advisor

Amy Bradshaw

Fourth Advisor

Lynn Schnapp

Fifth Advisor

Amanda LaRue

Abstract

Pulmonary fibrosis is a hallmark of diseases such as systemic sclerosis (SSc, scleroderma) and idiopathic pulmonary fibrosis (IPF). SSc is a complex autoimmune disease that is characterized by progressive fibrosis of the skin and multiple visceral organs. Despites multiple studies, the etiology of this connective tissue disease is still unknown, causing high morbidity and mortality in the patients. To date, therapeutic options are limited, and organ transplantation remains the only viable option for SSc­associated lung disease. Therefore, identifying novel therapeutic targets is critical for advancing the treatment of SSc. Insulin-like growth factor-binding protein-5 (IGFBP-5) is the most conserved member of the IGFBP family of proteins that is overexpressed in SSc and IPF. Some of the mechanisms mediating the effects of IGFBP-5 and downstream signaling pathways have been identified, the question to whether the promotion of fibrosis was directly mediated by IGFBP-5 or via other pro-fibrotic factors that may be downstream of IGFBP-5 had remained unanswered. Therefore, we investigated the role of IGFBP-5 in the pathophysiology of SSc using in vitro, ex vivo and in vivo models. Our data showed that both exogenous and adenovirally expressed IGFBP-5 promote fibrosis by increasing the production of extracellular matrix (ECM) genes and the expression of pro-fibrotic genes in primary human lung fibroblasts and tissues. IGFBP-5 also increased expression of the pro-fibrotic growth factor CTGF and levels of the matrix crosslinking enzyme lysyl oxidase (LOX). IGFBP-5 promotes its own expression, generating a positive feedback loop. This suggests that IGFBP-5 likely acts in concert with other growth factors to drive fibrosis and tissue remodeling. Further, our findings demonstrated that LOX expression and activity correlated with fibrosis in vitro, ex vivo, and in vivo. LOX induced ECM production via upregulation of IL-6 and nuclear localization of c-Fos. Thus, LOX has a direct pathogenic role in SSc-associated fibrosis that is independent of its crosslinking function. The pro-fibrotic effects of IGFBP-5 was further examined in transgenic mice overexpressing human IGFBP-5. Our data show that the heterozygous and homozygous mice are viable and express human IGFBP-5 (hIGFBP-5). Transgenic mice had increased expression of extracellular matrix (ECM) genes, especially Col3a1, Fn and Lox in lung and skin tissues of mice expressing higher transgene levels. Histologic analysis of the skin tissues showed increased dermal thickness, and the lung histology showed subtle changes in the heterozygous and homozygous mice compared to the wild-type mice. These changes were more pronounced in animals expressing higher levels of hIGFBP-5. Bleomycin increased ECM gene expression in wild-type and accentuated the increase in ECM gene expression in transgenic mice, suggesting that transgene expression exacerbates bleomycin-induced pulmonary fibrosis. Primary lung fibroblasts cultured from lung tissues of homozygous transgenic mice showed significant increases in ECM gene expression and protein levels, further supporting the observation that IGFBP-5 results in a fibrotic phenotype in fibroblasts. Primary lung fibroblasts cultured from lung tissues of transgenic mice showed increased ECM production, suggesting that the effects of IGFBP-5 are cell-type and tissue-specific. In conclusion, these studies capitalize on knowledge gained about the impact of IGFBP-5 and LOX playing a direct pathogenic role in SSc. Our findings from the study provide a new platform for developing therapies targeting IGFBP-5 and/or LOX function to treat SSc and other fibrotic disorders.

Rights

All rights reserved. Copyright is held by the author.

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