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P073 An increased autophagy and decreased apoptosis is detected in intestinal fibroblasts from Crohn’s Disease patients

Gisbert-Ferrandiz, L.(1);Queralt, M.(1);Cosín-Roger, J.(2);Coll, S.(1);Bauset, C.(1);Ortiz-Masia, D.(3);Llau, J.(1);Navarro-Vicente, F.(4);Calatayud, S.(1);Barrachina, M.D.(1);

(1)Universitat de València, Pharmacology, Valencia, Spain;(2)Unidad Mixta Facultad Medicina - Hospital Dr Peset- FISABIO, Pharmacology, Valencia, Spain;(3)CIBERehd-Univ. de València, Medicine, Valencia, Spain;(4)Hospital Manises, Surgery, Valencia, Spain

Background

Fibrosis is a complication commonly present in Crohn’s disease (CD) patients with a structuring (B2) or penetrating (B3) phenotype, with no effective treatment. This process is characterized by a disequilibrium between the production and degradation of the extracellular matrix (ECM), mainly regulated by myofibroblasts. We aim to analyse here, the expression of markers of autophagy, apoptosis and proliferation in intestinal fibroblasts from CD patients.

Methods

Fibroblasts were isolated from the damaged intestinal mucosa of CD patients with a penetrating and stenotic behaviour. Control cells were obtained from the non-damaged intestine of patients with colorectal cancer. Protein levels of markers of autophagy and apoptosis were determined by Western Blot in isolated fibroblasts. The proliferation marker Ki67 was analysed by immunohistochemistry (IHC) in 5 µm slides of intestinal tissue from control or CD patients. Statistical significance was measured by t-test.

Results

In fibroblasts from CD patients, we detected a significant decrease in the ratio phospho-mTOR / mTOR (Fig. A) in parallel with a non-significant increment in the LC3 II / LC3 I protein ratio (174% ± 46.5), and a decrease in p62 protein levels (84.8% ± 5.5). When compared between CD behaviours, a significant decreased in the phospho-mTOR / mTOR protein ratio was detected in fibroblasts from B2- compared to that obtained in cells from B3-CD patients (Fig. B). The analysis of the expression of an apoptosis marker, Caspase 3, revealed a decreased of cleaved caspase 3 protein levels in CD fibroblasts compared to levels detected in control cells (Fig C). Finally, we observed in the lamina propria of the intestine from CD patients an increased number of Ki67 positive cells, compared to that detected in control tissue.

Conclusion

Our data show an increased autophagy and decreased apoptosis in isolated intestinal fibroblasts from CD patients; the high number of cells proliferating in the lamina propria of the intestinal tissue of these patients, strongly suggests a higher viability of these cells in the fibrotic context.

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