Shepherd, Thomas Charles; (2025) Exploring the Mechanisms of Fibrosis in Stenotic Phenotypes of Crohn’s Disease. Masters thesis (M.Phil), UCL (University College London).

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Abstract

Background: Up to 50% of patients with Crohn’s disease (CD) will develop intestinal strictures involving and evolving from a process of currently unpreventable and irreversible fibrosis. The pathogenesis of this selective fibrogenesis is unknown but involves complex pathophysiology. Once initiated, characteristic TGF-β-driven myofibroblast (MF) activation and excess extracellular matrix (ECM) deposition is self-perpetuating. Methods: Proliferation, activation, and migration, with and without exogenous TGF-β stimulation, was investigated in primary human MF isolated from strictured and adjacent non-strictured ileum. Limited experiments on MF isolated from a 6 second CD patient with inflamed colonic disease were also performed synchronously until the viability of this culture was lost. Culture of MF subtypes in isolation and in medium previously conditioned by prior incubation of another MF subtype (cross culture) was performed to assess paracrine effects. RNA analysis of genes known to exert fibrotic influence was performed on biopsies of one patient taken from strictured and adjacent non-strictured intestine. Results: Proliferation MF isolated from strictured ileum show a higher proliferative attitude. Activation However, MF activation, assessed by α-SMA expression, and collagen 1A expression, a significant ECM component, were greatest in MF isolated from non-strictured ileum compared to that of strictured ileum and colonic samples. Cross Culture and conditioned media Activation of MF isolated from strictured ileum was enhanced by culture in conditioned medium from incubated MF from non strictured ileum. TGF-β treatment also enhanced activation and COL1A expression. RNA gene expression Profibrotic gene expression of ACTA2, COL1A, COL3A and TIMP-1 was increased in strictured intestine. Non-strictured intestine had pronounced expression of other profibrotic genes, TGF-β, TNF-α, IL-6 and CCL2. Summary: These novel foundation experiments give new prominence to apparently macroscopically quiescent tissue adjacent to strictures. These areas could be considered biologically primed, and a reservoir of profibrotic mediators, involved in fibrosis initiation, development and progression.

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