Sophie Vieujean1,2, Shurong Hu1,3, Emeline Bequet1,4, Catherine Salee1, Charlotte Massot1, Noëlla Bletard5, Nicolas Pierre1, Florence Quesada Calvo1, Dominique Baiwir6, Gabriel Mazzucchelli7, Edwin De Pauw7, Carla Coimbra Marques8, Philippe Delvenne5, Florian Rieder9, Edouard Louis1,2, Marie-Alice Meuwis1. 1. Laboratory of Translational Gastroenterology, University of Liège, Liège, Belgium. 2. Hepato-Gastroenterology and Digestive Oncology, University Hospital CHU of Liège, Liège, Belgium. 3. Department of Gastroenterology, Center of Inflammatory Bowel Disease, a Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. 4. Division of Hepato-Gastroenterology, Department of Paediatrics, University Hospital of Liège, Liège, Belgium. 5. Pathological Anatomy and Cytology, University Hospital CHU of Liège, Liège, Belgium. 6. GIGA Proteomics Facility, University of Liège, Liège, Belgium. 7. MolSys Research Unit, Laboratory of Mass Spectrometry, University of Liège, Liège, Belgium. 8. Abdominal Surgery Department, University Hospital CHU of Liège, Liège, Belgium. 9. Gastroenterology, Hepatology & Nutrition, Cleveland Clinic, Cleveland, OH, USA.
Abstract
BACKGROUND AND AIMS: Intestinal fibrosis is a common complication of Crohn's disease [CD]. It is characterised by an accumulation of fibroblasts differentiating into myofibroblasts secreting excessive extracellular matrix. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. METHODS: We performed a pilot proteomic study comparing the proteome of surface epithelium, isolated by laser-capture microdissection, in normal and fibrotic zones of resected ileal CD strictures [13 zones collected in five patients]. Proteins of interests were validated by immunohistochemistry [IHC] in ileal and colonic samples of stricturing CD [n = 44], pure inflammatory CD [n = 29], and control [n = 40] subjects. The pro-fibrotic role of one selected epithelial protein was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. RESULTS: Proteomic study revealed an endoplasmic reticulum [ER] stress proteins increase in the epithelium of CD ileal fibrotic strictures, including anterior gradient protein 2 homologue [AGR2] and binding-immunoglobulin protein [BiP]. This was confirmed by IHC. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2 intracellular expression and its secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin, led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. By using recombinant protein and blocking agent for AGR2, we demonstrated that the secretion of this protein by epithelial cells can play a role in the myofibroblastic differentiation. CONCLUSIONS: The development of CD fibrotic strictures could involve epithelial ER stress and particularly the secretion of AGR2.
BACKGROUND AND AIMS: Intestinal fibrosis is a common complication of Crohn's disease [CD]. It is characterised by an accumulation of fibroblasts differentiating into myofibroblasts secreting excessive extracellular matrix. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. METHODS: We performed a pilot proteomic study comparing the proteome of surface epithelium, isolated by laser-capture microdissection, in normal and fibrotic zones of resected ileal CD strictures [13 zones collected in five patients]. Proteins of interests were validated by immunohistochemistry [IHC] in ileal and colonic samples of stricturing CD [n = 44], pure inflammatory CD [n = 29], and control [n = 40] subjects. The pro-fibrotic role of one selected epithelial protein was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. RESULTS: Proteomic study revealed an endoplasmic reticulum [ER] stress proteins increase in the epithelium of CD ileal fibrotic strictures, including anterior gradient protein 2 homologue [AGR2] and binding-immunoglobulin protein [BiP]. This was confirmed by IHC. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2 intracellular expression and its secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin, led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. By using recombinant protein and blocking agent for AGR2, we demonstrated that the secretion of this protein by epithelial cells can play a role in the myofibroblastic differentiation. CONCLUSIONS: The development of CD fibrotic strictures could involve epithelial ER stress and particularly the secretion of AGR2.
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