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Parmar, Nimesh (2019) The role of galectin-3 in the development of idiopathic pulmonary fibrosis. PhD thesis, University of Nottingham.

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Abstract

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with a poor prognosis.Currently there is no cure for IPF but there are two licenced therapeutic drugs which can only slow down the progression of fibrosis.Transforming growth factor b(TGFβ) plays a crucial role in the pathogenesis of IPF.The b-galactoside binding lectingalectin-3 promotesfibrosis through modifying the activity of TGFβ.However, the precise mechanismof galectin-3 promoting fibrosiscurrently unknown.A galectin-3 inhibitor (TD139) is currently in clinical trials for the treatment of IPF. Additionally, galectin-3 is thought to play an important role in TGFβsignalling, where loss of galectin-3 in cell prevents TGFβinduced effects. The mechanism of this is also unclear.

Methods

Immunohistochemistry (IHC) was used to stain for galectin-3 in lung sections from patients with IPF and mice treated with bleomycin. Human lung fibroblasts (HLFs) isolated from healthy patients and IPF patients, and immortalised human bronchial epithelial cells (IHBECs) were used in the study. They were treated with galectin-3 inhibitor TD139 and then stimulated with recombinant human galectin-3 (rhGal-3). To delineate the possible mechanism of galectin-3 promoting TGFb activity HFLs were stimulated with LPA and the PAR1 agonist SFLLRN and treated with avb5 antibody, b1 inhibitor or TD139. HLFs were treated with b1 inhibitor oravb5 antibody and stimulated with rhGal-3. Solid-phase binding assays were used to investigate whether galectin-3 binds to components of the latent TGFβcomplex. Co-immunoprecipitation studies were undertaken to investigate galectin-3 and av integrin subunit interaction. HLFs and IHBECs were treated with TD139 and stimulated with recombinant TGFβ. Protein expression of phosphorylated Smad2 (pSmad2) was used as a measurement of TGFb signalling and was assessed by western blot. qPCR was used to measure galectin-3 at the mRNA level in HLFs treated with TGFb.2 antibody, b1 inhibitor or TD139. HLFs were treated with b1 inhibitor oravb5 antibody and stimulated with rhGal-3. Solid-phase binding assays were used to investigate whether galectin-3 binds to components of the latent TGFβcomplex. Co-immunoprecipitation studies were undertaken to investigate galectin-3 and av integrin subunit interaction. HLFs and IHBECs were treated with TD139 and stimulated with recombinant TGFβ. Protein expression of phosphorylated Smad2 (pSmad2) was used as a measurement of TGFb signalling and was assessed by western blot. qPCR was used to measure galectin-3 at the mRNA level in HLFs treated with TGFb.

Results

IHC showed an increase in galectin-3 expression in patients with IPF and in the mice treated with bleomycin; where expression was increased in airway epithelial cells, fibroblasts, and macrophages.RhGal-3 stimulation increased pSmad2 levels in HLFs but not in IHBECs. This was inhibited by TD139treatment. B1 and avb5 antibody blocked LPA induced pSmad2 increases. Galectin-3 inhibition with TD139 blocked both LPA and SFLLRN induced smad2 phosphorylation. B1 inhibition and avb5 antibody treatments had no effect on rhGal-3 induced pSmad2 increases. Solid-phase binding assay showed that galectin-3 binds to LTBP1 of the TGFβ latent complex. Co-immunoprecipitation studies revealed that galectin-3 associates with av integrin subunit. Treatment withTD139 inhibited recombinant TGFb induced smad2 phosphorylation in HLFs but not in IHBECs.TGFβ stimulation significantly increased galectin-3 expression at the mRNA level in IPF HLFs but not control HLFs.

Conclusion

Results show that galectin-3 promotes TGFβ signalling in fibroblasts but not in epithelial cells. It is thought there are 3 possible mechanisms by which galectin-3 promotes TGFβ activity.(1) galectin-3 forms a lattice complex with integrins to enhance integrin-LAP binding. (2) galectin-3 binding to the LTBP1 region of the large latent TGFβ complex and tightly anchoring the latent complex to the ECM resulting in little traction force needed for TGFβ to be released. (3) galectin-3 forming lattices between av integrins and the latent TGFβcomplex to increase accessibility of the integrin to bind to LAP leading to TGFβ release. Thus, the galectin-3-integrin site could be used as a therapeutic target for IPF treatment.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Jenkins, Gisli
Keywords:	Idiopathic pulmonary fibrosis; Transforming growth factor b; Galectin-3 promoting fibrosis; TGFβ induced effects
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:	59440
Depositing User:	Parmar, Nimesh
Date Deposited:	08 Apr 2020 14:03
Last Modified:	06 May 2020 09:02
URI:	https://eprints.nottingham.ac.uk/id/eprint/59440

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