Idiopathic pulmonary fibrosis (IPF) is the leading cause of progressive fibrotic lung disease, accounting for 1:100 deaths in the UK. IPF is characterised by dysregulated alveolar epithelial injury and repair processes, resulting in fibrosis which is characterised by collagen-rich ECM deposition by myofibroblasts. SOX9 is a key candidate transcription factor because of its importance in healthy alveologenesis during lung development, and involvement in ECM secretion by myofibroblasts during fibrosis in liver and kidney. Therefore we hypothesise that SOX9 is critical in the response of alveoli to injury and to fibroblast deposition of ECM in pulmonary fibrosis. The work presented within this thesis demonstrates that SOX9 has an important and multi-faceted role in lung injury. A Wild type (WT) time course of lung injury demonstrates, using IHC protein expression, that SOX9 is upregulated from acute injury through to fibrosis in regions of alveolar damage and ECM deposition. Using a Sox9 Knockout (KO) bleomycin mouse model of acute lung injury damage to the alveolar compartment is evident in WT animals but reduced in Sox9 KO, demonstrated by immunohistochemistry (IHC). Bronchoalveolar lavage immune cell changes associated with acute lung injury are significantly reduced in Sox9 KO animals, quantified by flow cytometry. Using a bleomycin model where Sox9 KO is induced in the fibroproliferative phase of lung injury we demonstrate a significant reduction in fibrosis, measured by: hydroxyproline assay, Collagen 1 (COL1) protein expression, and Collagen 1 gene expression. The translatability of alveolar and stromal animal model findings is confirmed by IHC, using human healthy and IPF lung sections. SOX9 co-localises in IPF, but not healthy lung, to important ECM (COL1), stromal (PDGFR beta, alpha SMA) and alveolar proteins (KRT17, SFTPC). In vitro lung fibroblast Sox9 SiRNA knockdown demonstrates an important role for SOX9 in regulating lung fibroblast ECM secretion, with a significant reduction in COL1 expression. Further, fibroblast cell treatment with the pro-fibrotic cytokines transforming growth factor beta 1 and platelet derived growth factor DD upregulates SOX9 and COL1 expression. Exploring mechanisms of ECM regulation, SOX9 knockdown reduces expression of the mechanosensitive receptor Integrin beta 1. Culturing WT and Sox9 KO lung fibroblasts on hydrogel culture systems, that provide a normal (4Kpa) and fibrotic lung (12Kpa) stiffness environment, shows a reduction in COL1 expression only on stiff matrix and demonstrates mechanisms of SOX9 ECM regulation are mechanosensitive. Building on this preliminary evidence of SOX9's importance to Integrin beta1 we explore therapeutically targeting PAK1, a downstream effector of Integrin beta1. Pak1 null mice demonstrate reduced, compared to WT, experimental lung fibrosis. Taken together the data within this thesis suggests that SOX9 is critical in the development of acute lung injury and pulmonary fibrosis, with an important role in alveolar epithelium and fibroblast ECM secretion. Further, it suggests targeting relevant pathways may yield promising novel biomarkers and therapeutic targets.
- Pulmonary fibrosis
- Integrin beta1
- PAK1
- Integrins
- Extracellular matrix
- Acute lung injury
- Lung injury
- Mechanosensitive
- Idiopathic pulmonary fibrosis
- Transcription factor
- SOX9
Investigating the role of SOX9 in pulmonary fibrosis
Pearmain, L. (Author). 1 Aug 2022
Student thesis: Phd