P0214 Notch Pathway-Mediated epithelial-mesenchymal transition and Fibrosis in Complicated Crohn’s Disease

Abstract Background The dysregulation of epithelial-mesenchymal transition (EMT) is linked to numerous pathologies, including fibrotic diseases. The Notch pathway may selectively mediate fibrogenic processes, such as EMT. Previous studies by our group have demonstrated elevated expression of NOTCH3, NOTCH4 receptors, and the DLL4 ligand in the intestines of patients with complicated Crohn’s disease (CD)1. This study aims to explore the potential of the Notch pathway as a therapeutic target in intestinal fibrosis associated with CD. Methods We analyzed ileal samples from Crohn’s disease patients with complicated lesions to assess the localization of NOTCH3/4 receptors using immunohistochemistry (IHQ) and performed RT-qPCR and Western blot (WB) analysis. In vitro studies were conducted using DLL4-coated plates (1 µg/ml), with or without the γ-Secretase Inhibitor DAPT (10 μL/ml), to evaluate protein and mRNA expression of HES1 and EMT markers (VIMENTIN, SNAIL, and E-CADHERIN) in two primary epithelial cell lines: small intestine (HIEC6) and colon (CoEpi) cells over 48 hours. Data, presented as fold induction (mean ± SEM) relative to water-treated cells or control tissues, were analyzed using parametric or non-parametric ANOVA (Kruskal-Wallis) based on normality testing (*P < 0.05). Results RT-qPCR analysis revealed significantly increased mRNA levels of the Notch receptors NOTCH3 and NOTCH4. WB analysis showed elevated levels of NOTCH3 (235±87*), but not NOTCH4 (87±13), protein in affected versus control tissues (100±8 and 100±7, respectively). In terms of cellular localization, NOTCH3 was detected in endothelial and smooth muscle cells, lamina propria cells, and with lower intensity in the epithelium. NOTCH4 was primarily found in cells of the lamina propria. In vitro assays indicated that DLL4 significantly altered mRNA and protein expression of HES1 and certain EMT markers (VIMENTIN and SNAIL) in both epithelial cell lines. Co-treatment with DAPT significantly reversed some EMT markers induced by DLL4 (VIMENTIN and SNAIL) (Figure 1). Conclusion NOTCH3 may play a role in the fibrogenic processes of CD, with elevated expression observed. The activation of EMT markers by DLL4 suggests that Notch signaling has a critical function in driving fibrosis and tissue remodeling in CD. References M Rodriguez-Antequera, J Cosin-Roger, D Macias-Ceja, P Salvador, L Gisbert-Ferrándiz, S Coll, J Manyé, R Alós, F Navarro-Vicente, S Calatayud, M D Barrachina, D Ortiz-Masia, P041 Differences in NOTCH signalling between stricturing and penetrating behaviour in Crohn’s disease, Journal of Crohn’s and Colitis, Volume 13, Issue Supplement_1, March 2019, Page S107, https://doi.org/10.1093/ecco-jcc/jjy222.165