CD-associated fibrosis and the resulting clinically relevant stenosis and strictures represent a common and severe complication in CD patients. To date, conventional options for prevention or treatment of such fibrotic complications are insufficient and even surgery is often only a temporarily beneficial approach. The limitation in available treatment options is mainly due to the fact that the pathogenesis of CD-related fibrosis is only poorly understood. Here, we demonstrate the presence of EMT-associated events in fibrotic areas of colonic tissue specimens derived from CD patients.
A characteristic feature of EMT is the translocation of β-catenin from the cytosol, where it connects E-cadherin to the actin cytoskeleton, into the nucleus to regulate gene expression. During EMT, β-catenin translocates from the cell membrane into the cytoplasm indicative for the disintegration of the epithelial zonulae adherentes. Further, the cytoplasmic pool of β-catenin translocates into the nucleus and initiates the expression of EMT-associated genes, such as α-SMA, vimentin or TGFβ [18,19]. TGFβ has been shown to regulate expression and activity of the Snail transcription factor family member, SLUG, via β-catenin in epithelial cell systems and SLUG has also been implicated in the pathogenesis of EMT via down-regulation of E-cadherin [20]. Characteristically, activated fibroblasts express FAP and presence of FAP has been implicated in the pathology of cancers, chronic inflammatory disorders, fibrosis and other pathologies indicating possible roles for FAP in facilitating cell invasion and growth [21].
Though EMT has already been associated with the pathogenesis of fibrosis in many systems, such as kidney, liver or lung [11], the evidence for EMT being involved in the development of intestinal fibrosis in CD patients is lacking. A recent study by Flier et al. identified EMT as a source of fibroblasts in the TNBS model of inflammation-associated intestinal fibrosis. They demonstrated that intra-rectal administration of TNBS to mice induces inflammation and fibrosis that is histological and immunologically similar to human CD. The fibrotic areas in these animals featured a large amount of cells expressing both, epithelial and mesenchymal markers, indicative for the onset of EMT. In particular, fibrotic areas revealed significantly more fibroblasts being α-SMA-positive as well as being positive for both, the epithelial cell marker E-cadherin and the fibroblast marker, fibroblast-specific protein 1 (FSP1) [8]. However, the observation period of these animals was certainly limited when compared to year-long duration of fibrogenesis in humans. Additionally, these mice did not develop strictures that display one of the most severe complications of human intestinal fibrosis. In intestinal tissue samples derived from three patients suffering from either active UC or CD, they showed co-localisation of α-SMA and E-cadherin in colonic crypts [8]. Nevertheless, this patient number was certainly too small for demonstrating finally conclusive results and the patients were also not featuring strictures as a complication of intestinal fibrosis.
In our new animal model of intestinal fibrosis occurring after heterotopic transplantation of small bowel resections in rats we found rapid loss of crypt structures which was followed by lymphocyte infiltration and obliteration of the intestinal lumen by fibrous tissue. Loss of intestinal epithelium was demonstrated by lack of cytokeratines while collagen expression was increased with time after transplantation. Interestingly, lumen obliteration was connected with increased expression of factors associated with EMT such as β6 integrin, IL-13 and TGFβ. The myofibroblast phenotype in fibrotic areas was demonstrated by presence of the mesenchymal markers α-SMA smooth muscle actin and vimentin in the obliterated lumen. These observations demonstrate that a variety of histologic and molecular features of fibrosis associated EMT can be observed in the heterotopic intestinal grafts [22]. Therefore, the data obtained in our new animal model so far fully confirmed our data obtained in human samples suggesting that the observations made in the animal model might actually reflect real fibrosis. This of particular interest, since it not only underlines the relevance of our data presented in this manuscript, but also gives us the possibility to perform further research using a reproducible animal model. This might finally critically contribute to a better understanding of the complex pathogenesis of intestinal fibrosis and the development and validation of new therapeutic options.
Thus, the observations presented here are in good accordance with other findings from our laboratory, demonstrating that EMT also is present in human CD and contributes to the pathogenesis of CD-associated fistulae [16,23-25]. Of note, intestinal fistulae are commonly surrounded by areas featuring fibrotic lesions. However, neither the findings in the animal models nor our previous data provided conclusive evidence that EMT might be involved in the pathogenesis of intestinal fibrosis in CD patients.
Here, we demonstrated that areas of intestinal fibrosis in CD patients feature EMT-associated gene expression events using colonic tissue specimens from fibrotic areas of CD patients. We found significant staining of TGFβ as well as of the EMT-associated transcription factor SLUG in fibrotic areas. Additionally, we detected nuclear localisation of β-catenin as a prominent feature of fibrotic regions. All of these molecules are associated with the onset of EMT. In particular, in intestinal epithelial cells TGFβ can induce SLUG expression and activation (meaning nuclear localisation) via β-catenin. Further, activated SLUG as well as activated (meaning nuclear) β-catenin play an important role for the downregulation and disassembling of E-cadherin [12,15,18-20]. All of these events (expression of TGFβ and nuclear localisation of SLUG and β-catenin) are characteristic for EMT and were visible in human tissue samples featuring a CD-associated fibrosis.
Of interest, FAP staining was strongly detectable in fibroblasts adjacent to the fibrotic areas. Though FAP expression is absent in most adult tissues, it is expressed under chronic inflammatory and fibrotic conditions as well as in certain epithelial tumours. On a functional level, FAP features dipeptidyl- and endopeptidase activity and seems to be involved in tumour cell proliferation [21,26]. A recent study even suggested that FAP inhibition might be beneficial for treating epithelial-derived tumours, since pharmacologic targeting of FAP inhibited tumour stromagenesis by affecting integrin-mediated signalling and led to a decreased recruitment and overall number of myofibroblasts [27]. Therefore, one might speculate, whether FAP could be a potential target for the treatment of CD-associated intestinal fibrosis.