P066 Fibrostenotic phenotype of fibroblasts in Crohn's disease is dependent on tissue stiffness and reversed by LOX inhibition

Abstract

Background: In Crohn's disease, intestinal inflammation often leads to fibrosis, characterized by excess extracellular matrix (ECM) deposition, increased tissue stiffness and stricture formation. Modulation of ECM is mainly mediated through activity of fibroblasts, which both deposit and degrade ECM components. To evaluate the role of fibroblasts in intestinal fibrosis in Crohn's disease, we compared phenotype and function of fibroblasts obtained from normal, inflamed and stenotic regions of the intestine. Methods: Fibroblasts were isolated from resection specimens of normal, inflamed and stenotic ileum within the same Crohn's disease patients and analyzed for gene expression profile. Fibroblasts were cultured in matrigel/collagen mix to measure ECM contraction in vitro. Matrix metalloproteinase (MMP) activity was measured upon culture in both soft and stiff matrices, mimicking normal and stenotic tissue conditions. Results: Transcriptional analysis showed that fibroblasts from stenotic ileum were distinct from both inflamed and normal fibroblasts with respect to genes involved in ECM organization and collagen production. In accordance with transcriptional data, stenotic fibroblasts showed an unexpected high activity of MMPs compared to normal and inflamed fibroblasts when cultured in the absence of ECM. This was counterintuitive, since MMP activity would be expected to be decreased in stenosis. However, when cultured in ECM with the compliance of their native stiff environment, stenotic fibroblasts displayed decreased MMP3 activity. This activity increased when cultured in soft environment. In sharp contrast, fibroblasts isolated from normal ileal regions had increased MMP3 activity upon stiffening of the ECM, suggesting a regulatory function to maintain tissue homeostasis. Functionally, stenotic fibroblasts induced significantly more ECM contraction than both normal and inflamed fibroblasts, consistent with tissue contraction in vivo. In addition, stenotic fibroblasts expressed increased levels of the collagen crosslinking enzyme lysyl oxidase (LOX), further contributing to tissue stiffness. Inhibition of LOX restored MMP3 activity of stenotic fibroblasts in a stiff ECM, resembling MMP3 activity level of fibroblasts cultured in soft matrix. Consequently, LOX inhibition prevented ECM contraction induced by stenotic fibroblasts. In normal fibroblasts, LOX inhibition did not affect ECM contraction. Conclusions: Stenotic fibroblasts display inherent alterations in gene expression and exhibit an aberrant response to tissue stiffness, contributing to ECM deposition and fibrosis. Altering the microenvironment by LOX inhibition corrects this phenotype, suggesting this as a potential anti-fibrotic agent in Crohn's disease.