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P041 Differences in NOTCH signalling between stricturing and penetrating behaviour in Crohn’s disease

M. Rodriguez-Antequera1, J. Cosin-Roger2,3, D. Macias-Ceja3, P. Salvador4, L. Gisbert-Ferrándiz4, S. Coll4, J. Manyé5, R. Alós6, F. Navarro-Vicente7, S. Calatayud2,4, M. D. Barrachina2,4, D. Ortiz-Masia1,2

1Universidad de Valencia, Medicine, Valencia, Spain, 2CIBERehd, Valencia, Spain, 3Fisabio, Valencia, Spain, 4Universidad de Valencia, Pharmacology, Valencia, Spain, 5CIBERehd, Badalona, Spain, 6Hospital de Sagunto, Sagunto, Spain, 7Hospital de Manises, Manises, Spain

Background

Fibrosis and fistula development constitute the main complications associated to Crohn’s disease. Notch signalling has been implicated in lung, kidney, liver, and cardiac fibrosis and in various disease conditions such as scleroderma. We aim to analyse here the pattern of NOTCH ligands, receptors, and effectors expression in surgical resections from stenotic and fistulizing CD patients and to determine the potential role of these ligands in favouring fistula and fibrosis.

Methods

CD patients (n = 41) were categorised according to Montreal classification (age at diagnosis, location, and behaviour). mRNA was isolated from resections of patients presenting a stricturing (B2, n = 26) or a penetrating (B3, n = 15) behaviour or from unaffected mucosa of patients with colorectal cancer (control, n = 15). The expression of Notch ligands, receptors, and effectors (HES1 and MATH1) was determined by RT-PCR or WB. Correlations between data were analysed using Pearson’s correlation coefficient (*p < 0.05).

Results

A higher mRNA expression of NOTCH3 and NOTCH4 receptors was detected in CD patients compared with controls; in addition, the expression of these markers was higher in the fistulizing than in the stenotic behaviour (Table 1). The fistulizing group presented a generalised overexpression of NOTCH ligands (JAG2, DLL3, and DLL4) compared with controls and among them, only DLL3 expression was up-regulated in the stenotic group (Table 1). Similar levels of HES1 and MATH1 mRNA expression were detected between different groups while protein levels of HES1 were higher in the fistulising group than in control or stenotic groups (3.4 ± 0.1 A.U*#, 2.8 ± 0.2 A.U and 2.0 ± 0.1 A.U, respectively). The expression of DLL3 significantly correlated with FSP1 (r = 0.77, p = 0.04*), DESMIN (r = 0.80, p = 0.03*), and SNAIL1 (r = 0.59, p < 0.04*), only in intestinal tissue from the fistulizing CD group.

ΔCTNOTCH1NOTCH2NOTCH3NOTCH4JAG2DLL1DLL3DLL4HES1
Non-IBD14.2 ± 0.49.9 ± 0.217.4 ± 0.213.4 ± 0.316.02 ± 0.223.2 ± 0.320.4 ± 0.718.9 ± 0.29.5 ± 0.3
B214.5 ± 0.29.9 ± 0.216.0 ± 0.3*11.5 ± 0.3*15.0 ± 0.323.1 ± 0.515.7 ± 0.7**19.5 ± 0.49.8 ± 0.3
B313.6 ± 0.29.2 ± 0.414.8 ± 0.3**#9.6 ± 0.9**#14.1 ± 0.2**23.0 ± 0.416.4 ± 0.9*17.6 ± 0.3*#9.2 ± 0.3

Relative mRNA expression of NOTCH ligands and receptors vs. the housekeeping gene β-ACTIN in intestinal mucosa. Significant differences vs. the respective Non-IBD patients are shown by *p < 0.05 or **p < 0.05 and vs. B2 CD patients by #p < 0.05.

Conclusion

Activation of the Notch signalling pathway is detected in Crohn’s disease patients presenting a penetrating (B3) behaviour compared with those with a structuring (B2) phenotype and it may be involved in fistula development over fibrosis.