Scientific Abstract
Proposal No. IBD-0241R2
Principal Investigator: Gerhard Rogler, M.D., Ph.D.
Applicant Organization: University of Zürich (Switzerland)
Project Title: Snail blockage as a potential new approach to fistula therapy
Period of Award: October 1, 2009 – September 30, 2011
The transmural inflammation characterizing Crohn’s disease (CD) predisposes the formation of fistulae, which is a major problem for a large number of CD patients due to a significant morbidity. Around 35% of all patients with CD suffer from fistulae at least once during their disease course. The therapeutic options for treating fistulae in CD are limited so far. Antibiotics, immunosuppressants and anti-TNF antibodies are established treatments; however, definitive fistula closure rates upon conservative treatment are around 30-40%.
Only little is known about the pathogenesis of fistula formation. We recently showed that more than 2/3 of CD fistulae are covered with flat, mesenchymal-like cells (transitional cells; TC) forming a patchy basement membrane. Recent data from our group provided evidence that epithelial-mesenchymal transformation (EMT) is involved in the development of TC and subsequently in the pathogenesis of fistula formation. EMT is a process of re-programming epithelial cells allowing them to migrate more effectively and giving them an “invasive” potential. EMT has been suggested to be involved in fibrosis in different tissues and diseases. Transcription factors of the SNAIL family as well as Twist are known to be crucial regulators during EMT. The TC covering CD fistulae were strongly positive for cytokeratins 8 and 20 indicating their epithelial origin. ß6-integrin and tissue growth factor-beta (TGF-β had highest staining intensities in the transitional zone between the epithelium and the TC. In addition expression of junctional proteins such as E-cadherin was reduced in TC as compared to regular fistulae epithelium, and a translocation of β-catenin from the membrane to the cytoplasm was observed. In addition, myofibroblasts and mesenchymal-like cells isolated from fistulae had a higher cytokeratin 8 expression in culture compared to non-fistulae myofibroblasts.
Based on these findings we hypothesize that:
A. Snail and Twist are driving factors during EMT in the development of fistula in CD.
B. Inhibition or knock down of Snail or Twist can block EMT and contribute to fistula healing in IBD patients.
C. Investigating the role of Snail or Twist in fistula EMT may contribute to the development of new treatment options for CD patients with fistulae.
The specific aims and experimental design include:
A. Investigation of the expression of Snail family members and Twist as well as EGF and FGF-2 in surgical ex vivo fistula specimens, primary fistula fibroblast cultures and TC cultures.
B. Knock down of Snail and Twist expression in cell cultures by siRNA techniques. Re-differentiation into an epithelial phenotype of TC cells will be monitored by quantitative PCR for E-cadherin, β-catenin, β6-integrin and vimentin.
C. Knock down of Snail and Twist expression in fistula specimens. Morphological changes as well as expression of E-cadherin, β-catenin, β6-integrin and vimentin will be studied.
The transmural inflammation characterizing Crohn’s disease (CD) predisposes the formation of fistulae, which is a major problem for a large number of CD patients due to a significant morbidity. Around 35% of all patients with CD suffer from fistulae at least once during their disease course. The therapeutic options for treating fistulae in CD are limited so far. Antibiotics, immunosuppressants and anti-TNF antibodies are established treatments; however, definitive fistula closure rates upon conservative treatment are around 30-40%.
Only little is known about the pathogenesis of fistula formation. We recently showed that more than 2/3 of CD fistulae are covered with flat, mesenchymal-like cells (transitional cells; TC) forming a patchy basement membrane. Recent data from our group provided evidence that epithelial-mesenchymal transformation (EMT) is involved in the development of TC and subsequently in the pathogenesis of fistula formation. EMT is a process of re-programming epithelial cells allowing them to migrate more effectively and giving them an “invasive” potential. EMT has been suggested to be involved in fibrosis in different tissues and diseases. Transcription factors of the SNAIL family as well as Twist are known to be crucial regulators during EMT. The TC covering CD fistulae were strongly positive for cytokeratins 8 and 20 indicating their epithelial origin. ß6-integrin and tissue growth factor-beta (TGF-β had highest staining intensities in the transitional zone between the epithelium and the TC. In addition expression of junctional proteins such as E-cadherin was reduced in TC as compared to regular fistulae epithelium, and a translocation of β-catenin from the membrane to the cytoplasm was observed. In addition, myofibroblasts and mesenchymal-like cells isolated from fistulae had a higher cytokeratin 8 expression in culture compared to non-fistulae myofibroblasts.
Based on these findings we hypothesize that:
A. Snail and Twist are driving factors during EMT in the development of fistula in CD.
B. Inhibition or knock down of Snail or Twist can block EMT and contribute to fistula healing in IBD patients.
C. Investigating the role of Snail or Twist in fistula EMT may contribute to the development of new treatment options for CD patients with fistulae.
The specific aims and experimental design include:
A. Investigation of the expression of Snail family members and Twist as well as EGF and FGF-2 in surgical ex vivo fistula specimens, primary fistula fibroblast cultures and TC cultures.
B. Knock down of Snail and Twist expression in cell cultures by siRNA techniques. Re-differentiation into an epithelial phenotype of TC cells will be monitored by quantitative PCR for E-cadherin, β-catenin, β6-integrin and vimentin.
C. Knock down of Snail and Twist expression in fistula specimens. Morphological changes as well as expression of E-cadherin, β-catenin, β6-integrin and vimentin will be studied.