• Login
• Admin

Philp, Christopher J. (2016) The potential therapeutic effect of manipulating the extracellular matrix in idiopathic pulmonary fibrosis. PhD thesis, University of Nottingham.

PDF (Thesis for reader access - any sensitive & copyright infringing material removed) - Repository staff only - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader Download (6MB) Request Thesis

Abstract

Background: Idiopathic Pulmonary Fibrosis (IPF) is a physiologically devastating disease. The debilitating nature and high mortality rates make this one of the most lethal conditions, usually associated with median time to mortality of around 3 years. Increased deposition of extracellular matrix (ECM) and fibroblast accumulation are hallmarks of idiopathic pulmonary fibrosis (IPF). We hypothesise that the ECM in IPF is structurally abnormal by virtue of aberrant cross linking and promotes fibroblast accumulation. This study examined the structure and biological activity of IPF derived ECM and how this related to the expression of ECM cross linking enzymes as well as how inhibiting Transglutaminase 2 affects active fibrosis in the murine Bleomycin model.

Methods: Primary fibroblasts from 3 patients with IPF and 3 controls were isolated from biopsy samples and characterised by immunocytochemistry. ECM from these cells was deposited onto tissue culture plastic, cells removed using ammonium hydroxide and confirmed by electron microscopy (SEM). IPF and control cells were then grown on their own ECM or ECM derived from other cells. ECM was labelled with 3H-proline and digested with recombinant proteases and tritium liberation counted by scintillation as a measure of collagen proteolysis. A pilot study was carried out where C57BL/5J mice received a single intratracheal instillation of Bleomycin (2mg/kg) and administered cystamine dihydrochloride by intraperitoneal injection (IP), once a day for ten consecutive days at 40mg/kg or 100mg/kg, at 3 different time points.

Results: IPF derived fibroblasts had more distinct organisation of fibrous matrix filaments on the cell surface and between adjacent cells by SEM. Both control and IPF lung fibroblasts expressed transcripts for lysyl oxidase (LOX), LOXL1, LOXL2, LOXL3, LOXL4 and transglutaminase (TG) 2. IPF derived matrix increased expression of LOXL3 and TG2 transcripts, LOXL3 protein and TGase activity. Other cross linking enzymes were unchanged.

To assess if IPF matrix affected fibroblast accumulation, I measured fibroblast adhesion, proliferation by MTT and EDU assays, and apoptosis by cleaved caspase 3, cleaved PARP and TUNEL assay on the different matrices. IPF matrix enhanced proliferation over control matrix in response to PDGF-BB. To determine if this pro-proliferative effect was dependent upon aberrant cross-linking we generated ECM from normal and IPF fibroblasts treated with cystamine dihydrochloride (TG2 inhibitor) or β-amino-proprionitrile (LOX family inhibitor). The enhanced fibroblast proliferation seen on IPF matrix was reduced close to levels of normal matrix by each cross link inhibitor. There was no effect on apoptosis induced by either FAS ligand or staurosporine when cells were seeded onto IPF or control matrix suggesting IPF ECM does not protect seeded fibroblasts from apoptosis.

Bleomycin showed a trend towards increasing total lung hydroxyproline at day 24, 34 and 44 post administration however this was not statistically significant. Administration of cystamine at 40mg/kg/day showed no effect on total lung hydroxyproline. At day 34 post Bleomycin, cystamine administration showed a trend towards decreasing total lung hydroxyproline however again this was not statistically significant.

Conclusions: The data supports the hypothesis that IPF derived matrix is structurally and functionally different from normal matrix. This results in enhanced fibroblast proliferation, adhesion and increased cross linking activity by effects on gene transcription. Inhibition of matrix cross-linking reduced this enhanced fibroblast adhesion and proliferation. Administration of cystamine dihydrochloride via IP injection for ten consecutive days at 100mg/kg/day in the Bleomycin model showed a trend towards decreasing total lung hydroxyproline.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Johnson, S.R. Jenkins, R.G.
Keywords:	IPF, Fibrosis, Matrix, Transglutaminase, Lysyl oxidase
Subjects:	W Medicine and related subjects (NLM Classification) > WF Respiratory system
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	35802
Depositing User:	Philp, Christopher
Date Deposited:	10 Feb 2017 09:39
Last Modified:	08 Feb 2019 08:01
URI:	https://eprints.nottingham.ac.uk/id/eprint/35802

Actions (Archive Staff Only)

Edit View