Introduction
The Notch signaling pathway and senescence are both complex processes that play important roles in a wide range of biological processes, including tissue repair and regeneration, as well as the development of age-related diseases. Studies have suggested that the Notch pathway may be involved in the regulation of senescence and in the development of fibrosis. Senescence is a normal physiological process that plays a role in tissue repair and regeneration, however the accumulation of senescent cells has been implicated in the development of fibrosis. Fibrosis represent the main complications related to Crohn´s disease (CD). Previous studies carried out by our group have shown a high expression of NOTCH pathway in CD samples (Rodriguez-Antequera, et al., 2019).
Aims & Methods
The general aim of the present study is to determine the possible potential of Notch pathway as a therapeutic target in intestinal fibrosis associated with Crohn's disease. Specifically, we pretend: to analyze the localization of the receptors of NOTCH3/4 and the expression of NOTCH markers in the intestinal tissue of patients with complicated CD, and to study the relevance of the Notch pathway in the senescence of intestinal fibroblasts. We have analyzed in intestinal samples from CD patients with complicated lesions: the localization of NOTCH3/4 receptors by IH, the protein expression of senescence/apoptotic markers (BCL2, P16, MCL1 and P53) and NOTCH markers (NOTCH3/4, DLL3/4 and HES1) by WB. We correlated the expression of NOTCH pathway markers with senescence markers of each patient. We carry out in vitro studies and analyze: the protein expression of HES1 (effector Notch pathway) and the protein expression of senescence/apoptotic proteins in HSIF fibroblasts treated with DLL4 or DLL3. Results are expressed as fold induction (mean±SEM). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test or by unpaired Student’s t test.
Results
NOTCH4 is found more specifically in the crypts of the mucosa, as well as in cells of the lamina propria. NOTCH3 was located preferentially in muscular areas -muscularis mucosa, endothelium and muscularis externa. The expression of DLL4/3, NOTCH4 and HES1 were significantly higher in the affected tissue compared to the ileal samples of control patients. BCL2, MCL1, P16 and P53 showed significantly elevated levels in the affected CD tissue, compared to the ileal sample of control patients. P16, the main marker of senescence, correlates positively and significantly with DLL3/4, NOTCH3 and HES1 in intestinal tissue (Table). DLL3, and not DLL4, produced in intestinal fibroblasts a significant increase in the protein expression levels of BCL2 and P53, compared to the vehicle.
Table. Protein correlation of NOTCH markers with senescence/apoptotic markers in intestinal mucosa. The table show the Pearson r. The results of applying the Spearman’s correlation analysis were considered significant at P < 0.05*, P < 0.01** and P < 0.001***.
| DLL3 | DLL4 | NOTCH3 | NOTCH4 | HES1 |
| MCL1 | r=0,885***
| r=-0,242
| r=0,763***
| r=0,388
| r=-0,138
|
| BCL2 | r=-0,0183
| r=0,559**
| r=-0,101
| r=0,4383
| r=0,445*
|
| P53 | r=-0,099
| r=0,828***
| r=-0,236
| r=0,397
| r=0,668***
|
| P16 | r=0,473*
| r=0,428*
| r=0,428*
| r=0,311
| r=0,487*
|
Conclusion
NOTCH pathway is involved in the regulation of key cellular functions and processes essential for the pathogenesis of intestinal fibrosis in CD patients. DLL3 seems to have a relevant role in activation of senescence in fibroblasts.
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
Disclosure
Nothing to disclose