Intestinal fibrosis

Molecular Aspects of Medicine xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Molecular Aspects of Medicine journal homepage: www.elsevier.com/locate/mam

Intestinal fibrosis Marco Vincenzo Lenti, Antonio Di Sabatino∗ First Department of Internal Medicine, San Matteo Hospital Foundation, University of Pavia, Pavia, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: Collagen Crohn's disease Extracellular matrix Myofibroblast Strictureplasty Ulcerative colitis

Extensive tissue fibrosis is the end-stage process of a number of chronic conditions affecting the gastrointestinal tract, including inflammatory bowel disease (Crohn's disease, ulcerative colitis), ulcerative jejunoileitis, and radiation enteritis. Fibrogenesis is a physiological, reparative process that may become harmful as a consequence of the persistence of a noxious agent, after an excessive duration of the healing process. In this case, after replacement of dead or injured cells, fibrogenesis continues to substitute normal parenchymal tissue with fibrous connective tissue, leading to uncontrolled scar formation and, ultimately, permanent organ damage, loss of function, and/or strictures. Several mechanisms have been implicated in sustaining the fibrogenic process. Despite their obvious etiological and clinical distinctions, most of the above-mentioned fibrotic disorders have in common a persistent inflammatory stimulus which sustains the production of growth factors, proteolytic enzymes, and pro-fibrogenic cytokines that activate both non-immune (i.e., myofibroblasts, fibroblasts) and immune (i.e., monocytes, macrophages, T-cells) cells, the interactions of which are crucial in the progressive tissue remodeling and destroy. Here we summarize the current status of knowledge regarding the mechanisms implicated in gut fibrosis with a clinical approach, also focusing on possible targets of antifibrogenic therapies.

1. Introduction Fibrosis is a pathophysiological mechanism of repair that leads to the deposition of connective tissue in the extracellular matrix (ECM) after an injury has occurred (Pakshir and Hinz, 2018). This is a fundamental step of the wound healing process (Fig. 1) that could be triggered in virtually any human tissue by several noxious agents, including infections, autoimmune reactions, physical, chemical, and mechanical injuries. Fibrogenesis may become a pathological process when it is uncontrolled and persistent, or else, when the acute injury is repeated or is followed by a chronic damage. The fibrogenic process can cause anatomical alterations and/or loss of function, with obvious different clinical implications depending on the organ involved. The gastrointestinal tract is a tubular organ system and therefore fibrosis is responsible for the narrowing of the lumen, thus causing strictures. Intestinal strictures are the end product of chronic transmural inflammation and dysregulated wound healing which lead to scar formation and, ultimately, tissue distortion. Aim of this review is to describe the molecular bases and the clinical implications of intestinal

diseases complicated by fibrosis, including inflammatory bowel disease (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC), and other less common fibrogenic disorders of the small bowel, such as ulcerative jejunoileitis (UJI) and radiation enteritis. IBD is an immune-mediated disorder of the gastrointestinal tract (Baumgart and Sandborn, 2012; Di Sabatino et al., 2015) affecting more than 3 million people in western countries, and with a rapidly growing incidence in developing countries (Kaplan, 2015). Fibrosis is one of the most threatening complications of CD, occurring in more than one-third of patients and causing intestinal obstruction due to recurrent stricture formation (Pariente et al., 2011). Fibrostenotic complications are burdened by substantial morbidity and mortality, and are responsible for a significant proportion of hospitalizations, surgical interventions, and health care costs (Silverstein et al., 1999). In the absence of a reliable, non-invasive biomarker, strictures are often diagnosed when the process is already irreversible, and surgery is inevitable. UC instead has always been considered a mucosal disease, but more recent evidence suggests that fibrosis occurs in both acute and chronic UC (de Bruyn et al., 2015) and may negatively affect response to therapies. However,

Abbreviations: ANCA, anti-neutrophil cytoplasmic antibody; ASCA, anti-saccharomyces cerevisiae antibody; bFGF, basic fibroblast growth factor; CD, Crohn's disease; CMUSE, cryptogenic multifocal ulcerous stenosing enteritis; CT, computer tomography; EMT, epithelial-mesenchymal transition; ECM, extracellular matrix; FAP, fibroblast activation protein; IBD, inflammatory bowel disease; IL, interleukin; miRNA, microRNA; MMP, matrix metalloproteinase; MR, magnetic resonance; PPAR, peroxisome proliferator activated receptor; PSC, primary sclerosing cholangitis; ROS, reactive oxygen species; TIMP, tissue inhibitor of metalloproteinase; TGF, transforming growth factor; TNF, tumor necrosis factor; UC, ulcerative colitis; UJI, ulcerative jejunoileitis; US, ultrasound; VEGF, vascular endothelial growth factor ∗ Corresponding author. Clinica Medica I, Fondazione IRCCS Policlinico San Matteo, Università di Pavia, Piazzale Golgi 19, 27100, Pavia, Italy. E-mail address: [email protected] (A. Di Sabatino). https://doi.org/10.1016/j.mam.2018.10.003 Received 22 September 2018; Received in revised form 19 October 2018; Accepted 28 October 2018 0098-2997/ © 2018 Elsevier Ltd. All rights reserved.

Please cite this article as: Lenti, M.V., Molecular Aspects of Medicine, https://doi.org/10.1016/j.mam.2018.10.003

Molecular Aspects of Medicine xxx (xxxx) xxx–xxx

M.V. Lenti, A. Di Sabatino

Fig. 1. Schematic representation of fundamental processes occurring after an injury. First (left-hand side), after an injury in the gut has occurred, the subsequent cell death triggers an inflammatory response that causes degradation of the extracellular matrix (ECM) and tissue damage. Thereafter, myofibroblast activation and proliferation leads to regeneration and repair of the damaged tissues. When the activation and proliferation of myofibroblasts is abnormal, this results in excessive myofibroblast survival, with consequent deposition of ECM and scar formation (right-hand side). Abbreviations: ECM, extracellular matrix.

3. Fibrogenic cellular and molecular mechanisms

given that UC only involves the colon that has a wider lumen compared to the small bowel, strictures are rather infrequent. Less common fibrogenic intestinal disorders are UJI, an immunemediated disorder characterized by chronic idiopathic ulcerations and scarring of the small bowel, mostly occurring as a complication of refractoriness in celiac disease (Bayless et al., 1967; Biagi et al., 2000; Di Sabatino and Corazza, 2009); radiation enteritis, caused by abdominal or pelvic radiotherapy; and “cryptogenic multifocal ulcerous stenosing enteritis” (CMUSE), a benign syndrome of unknown etiology characterized by recurrent episodes of small intestinal obstruction caused by benign multifocal ulcerated stenoses (Freeman, 2009), also known as “chronic nonspecific multiple ulcers of the small intestine” (Matsumoto et al., 2007). Despite the important clinical impact, the underlying mechanisms of fibrogenesis are yet to be completely understood, and no targeted therapies able to revert fibrosis are currently available.

3.1. Introduction to general mechanisms Several mediators and effectors proved to have a role in the fibrogenic process affecting the gut, including cytokines, chemokines, and many cellular players. The essential multi-step process of fibrogenesis includes, among others, cellular damage, production of transforming growth factor (TGF)-β1, recruitment of inflammatory cells, release of reactive oxygen species (ROS), and activation of myofibroblasts and collagen producing cells (Kisseleva and Brenner, 2008). The deposition of collagen causes chronic hypoxia that stimulates neoangiogenesis through the up-regulation of vascular endothelial growth factor (VEGF) (Taha et al., 2004), favoring in turn the deposition of further fibrotic tissue in a vicious circle. Inflammation is a powerful stimulus that triggers fibrosis. However, once fibrosis has established, the mere control of inflammation is not enough for reverting this process. In a mouse model of IBD infected by S. typhimurium, delayed eradication of the pathogen was able to repress inflammation, without preventing fibrosis, and early eradication significantly ameliorated, but did not completely prevent, fibrosis (Johnson et al., 2012). This suggests that fibrosis is regulated by mechanisms other than inflammation. Actually, tissue damage in IBD is the result of a dysregulation of the whole wound healing process, with defective chronic wound healing occurring in the gut mucosa and submucosa, determining inflammation and ulceration (Fig. 2). Instead, uncontrolled deposition of ECM occurring deeper in the intestinal wall, often with features of scar surrounding granulomatous inflammation, may have a significant role in confining inflammation and preventing tissue destruction with fistula formation (Pinzani, 2010). Finally, tissue tension also plays a central role in the biomechanical development and progression of fibrosis (Wells, 2013). Mechanical forces may activate myofibroblasts, thus favoring progressive ECM accumulation and determining tissue remodeling. The fibrogenic mechanisms affecting the gut are believed to be similar to those seen in other organs and tissues (Rieder et al., 2017). Most of our knowledge derives from IBD (both non-human and human studies), particularly CD, while little is known regarding the other less common disorders. We will therefore mainly focus on fibrogenic mechanisms underlying CD.

2. Literature search strategy In July 2018 we searched Medline (PubMed) by using the medical subject heading terms “fibrosis”, “fibrogenesis”, “strictures”, and “chronic inflammation” matched with “gut”, “small bowel”, “intestine”, and “gastrointestinal tract” for all articles published within the last thirty years, but we did not exclude a priori highly regarded older publications. More than one thousand papers were found with this search strategy, the majority of which were unrelated to the subject of this review (molecular and clinical aspects of intestinal fibrosis) and were not considered. In order to refine the search strategy, we also specifically searched Medline for “inflammatory bowel disease”, “Crohn's disease”, “ulcerative colitis”, “ulcerative jejunoileitis”, “ulcerous stenosing enteritis”, and “radiation enteritis” matched with “fibrosis”, “fibrosis markers”, and “treatment”. We therefore selected only studies (both non-human and human, mainly focusing on these latter) exploring molecular, cellular, and clinical characteristics of fibrosis in IBD (both CD and UC), UJI, CMUSE, and radiation enteritis. We also searched the reference lists of pivotal review articles for additional papers we judged to be relevant to this review.

2

Molecular Aspects of Medicine xxx (xxxx) xxx–xxx

M.V. Lenti, A. Di Sabatino

Fig. 2. Schematic representation of the molecular mechanisms underlying the fibrogenic process leading to stricture formation (left-hand side) and ulcer formation (right hand-side) in Crohn's disease (CD). In CD, the cross-talk between macrophage and T cells, sustained by Th1 pro-inflammatory cytokines, including interferon (IFN)-ɣ and interleukin (IL)-12, results in the production of tumor necrosis factor (TNF)-α, which promotes myofibroblast production of transforming growth factor (TGF)-β1. This latter inhibits the production of matrix metalloproteinases (MMPs) and favors the production of tissue inhibitors of metalloproteinases (TIMPs), which causes abnormal extracellular matrix (ECM) protein deposition, with consequent fibrosis and stricture formation. However, TNF-α also directly promotes MMP production by activated myofibroblasts, being responsible for ulcer formation. Chronic defective wound healing occurring in the mucosa and submucosa of patients with CD may also be responsible for fistula development. Theoretically, anti-fibrotic therapies, by interfering with the tissue remodeling process, might favor ulcer or fistula development. Abbreviations: CD, Crohn's disease; ECM, extracellular matrix; IFN, interferon; IL, interleukin; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase; TNF, tumor necrosis factor.

2002). Migration is likely to arise from any layer of the intestinal wall, depending on inflammation intensity being triggered by many inflammatory mediators in an autocrine or paracrine fashion (Rieder and Fiocchi, 2008). Besides fibronectin, which is one of the most powerful inducers of myofibroblast migration (Leeb et al., 2004), in CD strictures there is an overexpression of the adhesion molecule N-cadherin that further enhances the migration process (Burke et al., 2011). Myofibroblasts have been shown to be activated also by mechanical forces in different tissues, including heart, lung, liver, and kidney (Wells, 2013; Stempien-Otero et al., 2016), and this is likely to occur in the gut. Apart from cell proliferation and migration, another mechanism seems to dramatically increase the growth of mesenchymal cells in CD, namely the epithelial-mesenchymal transition (EMT) (Jiang et al., 2018). EMT is a dynamic process in which epithelial cells acquire a migratory function with fibroblast features (Jiang et al., 2018). This may be a physiological reaction that occurs to counteract an excessive damage or inflammatory process. In stricturing CD, EMT seems to be sustained by IL-13 and TGF-β up-regulation, with subsequent nuclear translocation of β-catenin and activation of EMT-related transcription factors (Jiang et al., 2018), and associated with the epithelial downregulation of the miRNA-200 family (Mehta et al., 2018). However, there is not enough convincing evidence to support the implication of EMT in the pathogenesis of intestinal stricture development in CD. The contribution of endothelial-to-mesenchymal transition in CD is even less clear (Rieder et al., 2011). Finally, it has been hypothesized that myofibroblasts could derive from mesenchymal progenitors of the bone

3.2. Cellular players Mesenchymal cells, after being stimulated by inflammation, promote the deposition of collagen in the ECM, particularly myofibroblasts (Regan et al., 2000; Andoh et al., 2007; Biancheri et al., 2013; Kurahara et al., 2015; Rieder et al., 2017; Pakshir and Hinz, 2018). Basically, all mesenchymal cell types may play a role in intestinal fibrosis, as they are in a persistent state of transdifferentiation among fibroblasts, myofibroblasts, and smooth muscle cells (Rieder and Fiocchi, 2008). The fine mechanisms underlying this transition are elusive and how to discriminate fibroblasts from myofibroblasts is still a matter of debate (Pakshir and Hinz, 2018). Nonetheless, fibroblasts and myofibroblasts are indeed major players in tissue fibrosis, and this is particularly true for CD (Andoh et al., 2007; Di Sabatino et al., 2009; Biancheri et al., 2013). Mesenchymal cells under specific stimuli can differentiate into myofibroblasts that deposit collagen and have mechanic properties thanks to the ability of contracting and making post-translational modifications (i.e., remodeling) (Pakshir and Hinz, 2018). The deposition of fibrotic tissue is subsequently favored by the inhibition of ECM degradation, due to an imbalance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) (Regan et al., 2000; Latella et al., 2014). As shown in Fig. 2, the result of this imbalance may lead to either ECM protein deposition or to ulcer formation. It is thought that the main source of myofibroblasts derives from cells proliferation and migration within the inflamed gut. In fact, as it was previously shown, myofibroblasts seem to proliferate at a faster pace in IBD (McKaig et al., 3

Molecular Aspects of Medicine xxx (xxxx) xxx–xxx

M.V. Lenti, A. Di Sabatino

normal adult tissues, but only on activated fibroblasts during wound healing and on the stroma of most human epithelial cancers. We have previously assessed FAP expression in surgical specimens of the colon of patients with fibrostenosing CD and of the inflamed colon of patients with UC undergoing colectomy (Rovedatti et al., 2011). FAP was strongly overexpressed in the submucosa and the muscle layer of CD strictures, with no expression in CD non-strictured areas, UC, and normal gut mucosa. Moreover, FAP expression was significantly higher in CD stricture myofibroblasts compared to the other groups. Finally, after stimulation with tumor necrosis factor (TNF)-α or TGF-β, FAP expression was significantly higher in strictured than non-strictured myofibroblasts. Similarly, in a more recent study, myofibroblast overexpression of FAP in stenotic CD compared to non-stenotic CD has been confirmed (Truffi et al., 2018). Many other cytokines have been implied in intestinal fibrosis, including interleukin (IL)-13, IL-17A, and IL-33. The role of IL-17A is well established, as it is increased in stricturing CD and induces the intestinal myofibroblast secretion of collagen, TIMP-1, MMP-3, and MMP-12 (Biancheri et al., 2013). On the contrary, IL-17E seems to be not implicated in the pathogenesis of fibrosis in CD. The role of IL-13 in CD fibrosis is still unclear (Bailey et al., 2012; Biancheri et al., 2014b), whereas some evidence supports a profibrogenic role for IL-33, which was found to be overexpressed in the strictured ileum of children affected by fibrostenosing CD (Masterson et al., 2015).

Fig. 3. Schematic representation of the interconnections among inflammation, neoangiogenesis, and fibrosis with regards to cytokines and main cellular players. Inflammation arising in the gut is largely a T-cell dependent process stimulated, among other cytokines, by tumor necrosis factor (TNF) α. An excessive inflammation up-regulates vascular endothelial growth factor (VEGF) that in turn mainly acts on endothelial cells, stimulating capillary formation. Parallelly, persistent inflammation also stimulates collagen deposition through transforming growth factor (TGF)-β1 up-regulation, acting on myofibroblasts. Macrophages (center of the triangle) represent the “core” cells of this scheme, being highly plastic and influenced by all the others. Abbreviations: M, macrophage; TNF, tumor necrosis factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.

3.4. Growth factors Growth factors have an established role in gut fibrogenesis. Both basic fibroblast growth factor (bFGF) and VEGF are up-regulated in both CD and UC (Bousvaros et al, 1997, 1999; Griga et al, 1998, 1999; Thörn et al., 2000; Kanazawa et al., 2001; Di Sabatino et al., 2004). bFGF is a signaling protein that promotes wound healing through the modulation of fibroblast proliferation, whereas VEGF binds to a surface receptor located on endothelial cells and increases vascular permeability and capillary angiogenesis (Neufeld et al., 1999). We have previously shown that bFGF is increased in patients with CD in comparison to controls, and it is higher in patients with strictures compared to other CD phenotypes (Di Sabatino et al., 2004). Furthermore, highest bFGF levels were found in CD patients with a marked increase of bowel wall thickness and undetectable intramural blood flow. These findings corroborate previous in vitro experiments showing that bFGF up-regulates collagen production in cultured fibroblasts of IBD patients compared to controls (Lawrance et al., 2001). Unlike bFGF, VEGF is overexpressed in active CD, and its level correlates with intramural bowel vascularity detected by Doppler imaging (Di Sabatino et al., 2004). Fig. 3 schematically depicts the relationship among inflammation, neoangiogenesis, and fibrosis in CD.

marrow, similarly to what is seen in other immune-mediated disorders (Rieder and Fiocchi, 2008). Fig. 2 schematically represents the essential steps leading to ECM protein deposition with consequent stricture development and ulcer formation in CD.

3.3. Cytokines TGF-β is a multifunctional profibrogenic cytokine involved in fibrosis in almost any organ and tissue, including the gastrointestinal tract (Kisseleva and Brenner, 2008). In physiological conditions, TGF-β promotes the immune homeostasis within the intestinal mucosa, by preventing detrimental activation of inflammatory cells against the normal constituents of gut microbiota (Konkel and Chen, 2011; Biancheri et al., 2014a). In fact, in Tgfb1 gene null murine models, a systemic inflammatory status was reported, also involving the intestinal mucosa (Shull et al., 1992; Kulkarni and Karlsson, 1993). The TGF-β1 isoform can promote ECM synthesis and fibroblast contraction (Leask and Abraham, 2004). It has been previously shown that myofibroblasts isolated from strictures of patients suffering from CD overproduce collagen, TGF-β1, TIMP-1, and have reduced migratory ability compared with those isolated from intestinal areas without strictures (Di Sabatino et al., 2009). The increase of TGF-β1 transcripts was also associated with the molecular signature (phosphorylated Smad2-3) of increased TGF-β1 signaling, that is decreased Smad7 levels, and fewer MMP-3 and MMP-12 proteins. However, TGF-β signaling pathway seems to be defective in IBD, as proved by the reduced levels of phosphorylated Smad3 and Smad3-bound Smad4 in inflamed IBD intestinal mucosa (Monteleone et al., 2001). TGF-β (β1, β2, and β3) and its receptors are up-regulated in the intestine of patients affected by CD (in the lamina propria cells, lymphocytes, epithelial cells, and in fibroblasts) (di Mola et al., 1999), and their binding induces the activation of the Smad transcriptional proteins that represent a possible therapeutic target. In addition to TGF-β, another pivotal protein implicated in fibrotic evolution of damaged tissue is fibroblast activation protein (FAP). FAP is a glycoprotein belonging to the post-prolyl dipeptidyl aminopeptidase enzyme family (Aertgeerts et al., 2005). FAP is not expressed in

3.5. MicroRNAs MicroRNAs (miRNAs) are small non-coding, endogenous RNAs of 21–25 nucleotides that influence cell signaling negatively regulating protein translation (Wahid et al., 2010). These small molecules are attracting interest in many different fields, spacing, among others, from oncological to cardiovascular and gastrointestinal disorders. In patients with CD, miRNA-29a, b, and c have been demonstrated to be reduced in the mucosa of strictured gut and miRNA-29b transfection is able to prevent TGF-β-induced collagen production (Nijhuis et al., 2014). miRNA-29 is also upregulated by NOD2 signaling, and this is proved by the fact that CD patients with NOD2 polymorphisms fail to induce miRNA-29 (Brain et al., 2013). 4. Clinical features In healthy adult individuals, the small bowel has a smaller diameter compared to the colon, spacing from a mean of approximately 25 mm of 4

Molecular Aspects of Medicine xxx (xxxx) xxx–xxx

M.V. Lenti, A. Di Sabatino

pattern) positivity (Di Sabatino et al., 2017). A rare fibrogenic disorder of the small bowel is UJI, a chronic condition that may complicate the course of celiac disease, in which idiopathic ulcerations cause scarring of the small bowel (Biagi et al., 2000). At early stages the pain is usually mild and recurrent, whereas when stenoses become severe, intestinal obstruction occurs. Scarring is a frequent finding, and is usually transverse, thus easily leading to intestinal obstruction. Ulcerations may have different extensions and may deepen in the mucosa, causing bleeding and perforation. Other two rare intestinal disorders complicated by fibrosis are CMUSE, usually presenting with abdominal pain, gastrointestinal bleeding, and anemia (Hwang et al., 2017), and radiation enteritis that should be borne in mind in patients with a history of abdominal or pelvic irradiation (Hauer-Jensen et al., 2014).

the duodenum, to 19 mm of the terminal ileum (Cronin et al., 2010). Therefore, strictures are more likely to become clinically symptomatic when the small bowel is involved. The typical clinical picture of small bowel strictures is characterized by abdominal pain and intestinal obstruction in more severe cases. We will briefly discuss the specific features of the single diseases. CD is a chronic, immune-mediated disorder characterized by periods of remission and relapse, with an unpredictable course. CD comprises heterogeneous phenotypes in terms of age of onset, disease location (terminal ileum and colon are most commonly affected, followed by other parts of the small bowel and upper gastrointestinal tract), and disease behavior (non-stenotic/non-penetrating, stenotic/stricturing, penetrating/fistulizing) (Silverberg et al., 2005; Gomollón et al., 2017). CD may onset in people of any age, and any part of the gastrointestinal tract may be involved, causing transmural inflammation and skip lesions. Apart from strictures, its complications include, among others, the development of fistulas, abscesses, small-bowel carcinoma, and colorectal cancer (Di Sabatino et al., 2013; Gomollón et al., 2017; Vanoli et al., 2017). The clinical picture of CD reflects the heterogeneity of this condition. Patients with CD may experience a broad range of manifestations, sometimes mild and unspecific, and sometimes requiring admission in an emergency department. This heterogeneity considerably increases the diagnostic delay of this condition, generally greater compared to patients with UC (7 months vs 2 months) (Cantoro et al., 2017). Abdominal pain is present in most of the patients with CD, the localization depending on disease location (Di Sabatino et al., 2013). Strictures are present in up to 40% of patients at the time of CD diagnosis (Fiorino et al., 2017). Abdominal pain may be mild at an early stage of the disease. Being terminal ileum involved in most cases, abdominal pain is usually experienced in the right lower quadrant, sometimes mimicking acute appendicitis. Diarrhea is another frequent symptom, though obstructive symptoms (abdominal pain, constipation, nausea, vomiting) are frequent in patients with stricturing disease. Bloody diarrhea or hematochezia are not usual presentations, unless the distal colon is involved (20–30% of the cases). Disease behavior also tends to change over time, thus changing the clinical picture accordingly. A more severe disease is characterized by malnutrition, anorexia, weight loss, and fever, eventually leading to septic shock, intestinal obstruction or perforation, electrolyte imbalance, and death. Patients with CD may also experience a broad range of extraintestinal manifestations, in some cases years before its diagnosis (Vavricka et al., 2015; Harbord et al., 2016). These include peripheral or axial arthritis, anemia, erythema nodosum and pyoderma gangrenosum, oral aphthous ulcers, episcleritis, and uveitis. As regard UC, a chronic, relapsing-remitting disease that affects the colonic mucosa, starting from the rectum until the cecum, in a continuous fashion (Di Sabatino et al., 2015), although it is not classically considered a fibrogenic condition, submucosal fibrosis has been reported in patients with longstanding and/or refractory UC (Gordon et al., 2018). Similarly to CD, UC may onset at virtually any age, with a severe course even in elderly patients (Fries et al., 2017). According to the Montreal classification, UC extension can be classified as follows: proctitis (only the rectum is involved), left-sided colitis (colon distal to the splenic flexure), and extensive colitis (proximal to the distal colon, until pancolitis) (Silverberg et al., 2005). The clinical picture of patients with UC is generally less variegate compared to those with CD, and this may explain, at least partially, the lower diagnostic delay found in UC (Cantoro et al., 2017). The main clinical feature is the presence of bloody diarrhea (hematochezia). Cramping abdominal pain may also be present, especially in more extensive disease. As for CD, the aforementioned extraintestinal manifestations may also occur in patients with UC. Notably, primary sclerosing cholangitis (PSC) is another extraintestinal manifestation that may occur in patients with UC (very rare in CD), characterized by inflammation and fibrosis of the biliary tree and anti-neutrophil cytoplasmic antibodies (ANCA; perinuclear

5. Diagnosis Regardless of etiology, intestinal fibrosis is usually diagnosed when it becomes clinically evident, i.e. when strictures have occurred. Regarding diagnostic imaging, different techniques may be used for diagnosing stenoses, including ultrasound (US), computer tomography (CT), and magnetic resonance (MR). Barium contrasted x-ray examinations are being progressively abandoned, but still they may be useful to assess the extension and location of a stricture. However, this examination fails to discriminate between an inflamed or a fibrotic stricture that is a valuable information for deciding treatment. Available radiological techniques have variable accuracy in detecting and characterizing stenoses, and most studies have been conducted in patients with stricturing CD. Abdominal US can discriminate inflamed and fibrotic stenoses with a good accuracy, but MR enterography is more sensitive in defining their extension in CD (Castiglione et al., 2013). The European Federation of Societies for Ultrasound in Medicine and Biology have drafted specific indications for the use of contrastenhanced US (which have the additional advantage of quantifying vascularization) in IBD, with various levels of evidence, including evaluation of disease activity, distinction between inflamed and fibrotic strictures, characterization of abscesses and fistulae (Piscaglia et al., 2012). US elasticity imaging is a non-invasive technique that was specifically developed to evaluate fibrosis. Bowel wall stiffness can be evaluated through the strain ratio between the mesenteric tissue and the bowel wall with this technique. Patients with CD showing a strain ratio ≥2 (i.e., with severe ileal fibrosis) at baseline are more likely to undergo surgery, thus predicting failure to anti-TNF-α therapy as reported in a recent study (Orlando et al., 2018). Both CT and MR enterography can discriminate with a good accuracy between inflamed and fibrotic strictures in CD patients (Rieder et al., 2018). In particular, mural hyperenhancement and bowel wall thickening are the most sensitive markers of inflammation, whereas the absence of mural hyperenhancement is compatible with fibrosis (Booya et al., 2006; Park and Lim, 2013; Bruining et al., 2018). Other signs of inflammation include mural stratification, engorged vasa recta (“comb sign”), and increased attenuation of the mesenteric fat (Park and Lim, 2013; Bruining et al., 2018). A focused panel, composed by renowned expert radiologists and gastroenterologists, have recently drafted a consensus paper for the evaluation, interpretation, and utilization of CT and MR enterography for patients with small bowel CD (Bruining et al., 2018), defining their role in both the diagnosis and follow-up. Finally, a very recent study explored the role of magnetization transfer MR compared to contrast-enhanced MR in diagnosing fibrosis in CD patients (Li et al., 2018). Magnetization transfer was more accurate in detecting variable degrees of fibrosis. US elasticity and magnetization transfer MR are promising techniques that are not yet widely available in clinical practice. Gastrointestinal endoscopy is the mainstay for the diagnosis of disorders characterized my mucosal alterations. A stenosis can be simply defined as a narrowing of the lumen that cannot be passed with 5

Molecular Aspects of Medicine xxx (xxxx) xxx–xxx

M.V. Lenti, A. Di Sabatino

contrast for fluoroscopic observation, constantly monitoring the hydraulic with a manometer. First reports on the efficacy and safety of endoscopic balloon dilation in CD were reported nearly 30 years ago, showing that this technique could lead to a rapid symptom relieve and could delay the need for surgery (Blomberg et al., 1991). According to a recent meta-analysis, endoscopic balloon dilation appears to be a safe alternative to surgery, especially in CD patients with strictures < 4 cm in length, with a low rate (4%) of major adverse events (including perforation and/or hemorrhage requiring interventions, blood transfusion, abscess or fistula formation, sepsis) (Navaneethan et al., 2016). A good efficacy and safety profile were also shown in upper gastrointestinal strictures complicating CD (Singh et al., 2017). According to an expert panel, the following items should be considered when performing balloon dilation: 18 mm as the maximal luminal diameter after dilation (in one or more sessions), an inflation time of at least 1 min, and 5 cm as the maximum stricture length that should be dilated (Rieder et al., 2018). Dilation is considered successful when a previously non-passable stricture can be passed with an adult colonoscope with a reasonable amount of pressure applied (Rieder et al., 2018). Surgical resection rate for CD has slightly decreased over the last years, being elective operations now more common than those performed on an urgent basis (Ma et al., 2018). Surgical resection is the preferred option in case of ileocecal CD without signs of inflammation, when endoscopic treatment is not feasible or unsuccessful (Bemelman et al., 2018). Laparoscopic treatment, rather than laparotomic, should be preferred whenever possible, due to the lower complication rate. In order to avoid complications and loss of function (i.e., short bowel syndrome in case of extensive small bowel resections), minimal bowel resection or strictureplasty are the best available options. Strictureplasty is a surgical procedure that has the aim of restoring the normal bowel transit without removing the fibrotic tract. Apart from the obvious advantage of preserving the anatomical continuity, strictureplasty may relieve tissue tension, with subsequent myofibroblast inactivation, thus possibly reducing progression of fibrosis. Different techniques for strictureplasty have been proposed so far, depending on the length and site of the stricture, including Heineke-Mikulicz (the most commonly used conventional technique), Michelassi (the most commonly used non-conventional technique), and the Finney technique (Bemelman et al., 2018). According to a meta-analysis including 1112 patients who underwent 3259 strictureplasties (most of which Heineke-Mikulicz), septic complications (leak, fistulae, or abscesses) occurred in 4% of the whole population, and the five-year recurrence was 28% (Yamamoto et al., 2007). As already mentioned, colonic fibrosis may also complicate the course of UC, but rarely becomes clinically evident, and no studies have ever evaluated the effects of available therapies in preventing this complication. However, given that most colonic stenoses are seen in patients with longstanding history of UC, it is reasonable to assume that controlling inflammation could prevent -or at least decrease-the occurrence of fibrosis in UC. As benign colonic stenoses in UC are rare and given that the risk of developing colorectal cancers increases with increasing disease duration, malignancy should always be ruled out (Fumery et al., 2015). In patients with UC and concurrent PSC, vedolizumab, adalimumab, or infliximab were not effective treatments for PSC (Tse et al., 2018). Therefore, the mere control of intestinal inflammation is not enough for blocking the progression of PSC that eventually leads to liver fibrosis.

the endoscope (Daperno et al., 2004). Therefore, its role in the characterization of the inflamed/fibrotic components and the extension of the stricture is limited; however, biopsy specimens can be taken for histopathological assessment and for ruling out malignancies. No histopathological grading of fibrosis is currently available. The results of an expert consensus survey regarding the definition and diagnosis of strictures in CD has just been published (Rieder et al., 2018). A small bowel stricture could either be diagnosed with crosssectional imaging or ileocolonoscopy alone, being MR enterography considered the best imaging technique. 6. Therapeutic approaches The therapeutic approach of intestinal strictures depends on the cause, localization, and extension of the stricture. Therapeutic options include medical treatment, targeting the inflammatory component of the stricture, endoscopic treatment, and surgery. No medical therapy targeting fibrosis is currently available in clinical practice. In the absence of new targeted therapies, the prevention and treatment of the underlying profibrogenic disorder represent the only possible way to decrease, but not abolish, fibrosis-related complications. While inflammatory mechanisms in IBD have been extensively investigated, knowledge on fibrogenic mechanisms leading to intestinal stricture development remains relatively limited. Despite the advances in the medical management of CD in the last 25 years, now including six different monoclonal antibodies targeting either anti-TNF-α (infliximab, adalimumab, certolizumab pegol), integrins (natalizumab, vedolizumab), or anti-IL12/23 (ustekinumab) (Fischer and Neurath, 2017), the incidence of intestinal strictures and the requirement for surgical interventions have not diminished since the introduction of infliximab in 1998 (de Buck van Overstraeten et al., 2012). We have now accumulated a 20-year experience with the use of biologic therapy for the treatment of IBD. Whether to indefinitely continue or not a biologic therapy is still a matter of debate. What we know is that antiTNF-α agent discontinuation and lack of adherence lead to a significantly higher risk of disease relapse, especially in stricturing CD (hazard ratio 1.5) (Casanova et al., 2017; Lenti and Selinger, 2017). The old assumption that the use of infliximab in stricturing CD could paradoxically worsen obstruction due to the rapid healing has been overcome by more recent evidence. Actually, the use of anti-TNF-α antibodies is generally safe in patients with stricturing CD and could decrease the need for surgery over time (Allocca et al., 2017). The use of adalimumab, a fully-human monoclonal antibody, in stricturing CD patients proved to be safe and able to maintain surgery-free remission in half of the included patients over a 4-year follow-up (Bouhnik et al., 2018). These results are encouraging, but surgery-free remission is still rather low. A possible explanation to this finding is that defective chronic wound healing occurring in the mucosa and submucosa of patients with CD is likely to be improved by anti-TNF-α agents, whereas deeper, transmural fibrosis cannot be resolved by these drugs (Fig. 2). Vedolizumab is a gut-selective monoclonal antibody that proved to be effective and safe in many real-world studies, even in stricturing CD and with better results in anti-TNF-α naïve patients (Engel et al., 2018; Kopylov et al., 2018). However, long-term outcomes regarding the occurrence of strictures are not known. Real-world data on ustekinumab are still scanty, but according to a recent multicentric study, patients with stricturing CD are less likely to achieve remission at six months (odds ratio 0.29) (Ma et al., 2017). Therapeutic gastrointestinal endoscopy can be used in virtually any type of fibrotic stricture, regardless of etiology, but most of the experience derives from the treatment of CD strictures. In particular, endoscopic balloon dilation is a minimal-invasive procedure that consists in placing a radial expanding balloon dilator, with or without wire guidance, in the stenotic tract and inflating the balloon as needed (Siddiqui et al., 2013). Some balloons can be sequentially inflated to different diameters. Balloons can also be expanded with a radiopaque

7. Perspectives Intestinal fibrosis represents a challenge for both basic scientists and physicians, in terms of pathogenic mechanisms, diagnosis, and clinical management, due to the lack of reliable and easily transferrable experimental models of fibrosis, the scarcity of predictive markers, and the lack of drugs targeting fibrosis. New methods for studying fibrosis, and early markers and new drugs are eagerly awaited, given that the 6

Molecular Aspects of Medicine xxx (xxxx) xxx–xxx

M.V. Lenti, A. Di Sabatino

Table 1 Non-invasive biomarkers and imaging techniques in Crohn's disease fibrosis.

Fig. 4. Window of opportunity to modulate fibrosis. Fibrosis is a multistep process that eventually depends on the balance between extracellular matrix (ECM) deposition, in which prevails the role of tissue inhibitor of metalloproteinases (TIMPs), and ECM degradation, in which prevails the role of matrix metalloproteinases (MMPs). Currently, once the pathologic process has become irreversible, we have no therapies able to counteract further ECM proteins deposition and consequent scar formation. Abbreviations: ECM, extracellular matrix; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase.

GENETIC

SEROLOGIC

IMAGING

- CARD15/NOD2 variants - TIMP/MMP variants

-Anti-glycan antibodies ASCA Anti-I2 Anti-CBir1 Anti-OmpC ACCA ALCA AMCA Anti-L -Growth factors bFGF VEGF YLK-40 -ECM proteins PIIINP Fibronectin Laminin Collagen IV Hyaluronic acid -ECM proteases TIMP1 MMP12 microRNA

-Contrast-enhanced US -CT enterography -MR enterography -US elasticity -Magnetization tranfer MR

Abbreviations: ACCA: anti-chitobioside carbohydrate antibody; ALCA, anti-laminaribioside carbohydrate antibodies; AMCA: anti-mannobioside carbohydrate antibody; anti-CBir1: anti-bacterial flagellin CBir1 antibody; anti-I2: antiPseudomonas-associated sequence I2 antibody; anti-L: antilaminarin carbohydrate antibody; anti-OmpC: anti-Escherichia coli outer membrane protein C antibody; ASCA: anti-Saccharomyces cerevisiae antibody; bFGF: basic fibroblast growth factor; CT, computer tomography; ECM: extracellular matrix; MMP, matrix metalloproteinase; MR, magnetic resonance; PDGF: platelet-derived growth factor; PIIINP: N-terminal propeptide of type III collagen; TIMP: tissue inhibitor of matrix metalloproteinases; US, ultrasound; VEGF, vascular endothelial growth factor; YLK-40, human chitinase 3-like 1.

window of opportunity to treat or modulate fibrosis is very small (Fig. 4). Murine models of fibrosis carry a number of flaws that limit their usefulness, as they lack of clinical transferability. Therefore, alternative methods have been proposed. Stem cell-derived human intestinal organoids turned out to be an interesting model for studying fibrosis in CD, as it also contains myofibroblasts (Rodansky et al., 2015). Decellularized scaffolds constitute a more complex model of increasing interest in different medical subspecialties, as they may become an alternative therapy to organ transplantation, especially heart and liver (Mazza et al., 2015; Yu et al., 2016). Intestinal differentiation has been studied in porous, synthetic 3D tissue scaffold with villous features, in order to reproduce the natural microenvironment as best as possible (Costello et al., 2014). More recently, human intestinal epithelium has been elegantly reproduced with a 3D porous, silk protein, scaffolding system with a geometrically-engineered hollow lumen (Chen et al., 2015). This model could also be used for reproducing pathological conditions, as in the case of profibrotic disorders of the gastrointestinal tract. Experimental models for the study of mechanical forces and gut tissue tension are lacking and should be developed. A number of markers of fibrosis could be potentially used for identifying fibrostenosing CD patients, even if the lack of solid evidence and the scarce availability have limited their use in clinical practice (Giuffrida et al., 2016). Table 1 shows all the possible biomarkers (genetic and serologic) and diagnostic imaging of fibrosis for CD. Patients with NOD2 gene variants are at higher risk for developing early ileal strictures and postoperative recurrence (odds ratio 3.29) (AlvarezLobos et al., 2005). We have already discussed about the role of TIMP-1 in favoring fibrosis; however, serum TIMP-1 level does not correlate with stricturing CD (Kapsoritakis et al., 2008). The vast and increasing number of antimicrobial antibodies found in patients with CD are another prove of the abnormal and altered immune response underlying this condition. The presence of these antibodies may be related to a more complex disease phenotype or more aggressive disease course, but the studies published so far failed to define a phenotype-specific antibody signature in CD (Giuffrida et al., 2016). In particular, anti-glycan

antibodies generally fail to discriminate different CD phenotypes, even if they may be associated with a more aggressive IBD (Paul et al., 2015). MiRNAs are among the most interesting candidate predictors of fibrosis in CD (Lewis et al., 2015). MiRNAs profiling of serum from CD patients was assessed to identify those associated with fibrostenosing phenotype. In particular, serum levels of miRNA-19a-3p and miRNA19b-3p were found to be lower in CD patients with a stricturing phenotype than in controls (Lewis et al., 2015). Moreover, this association with stricturing CD was independent of many potential confounding variables. More studies are needed to determine the prognostic value of this marker. The design of new trials exploring potential anti-fibrotic drugs is an impellent, though yet unmet, need. A group of renowned IBD experts have recently drafted a core set of endpoints for anti-fibrotic trials through a two-round Delphi-style process, defining 13 critical endpoints including, among others, complete clinical response, long-term efficacy, radiological remission, normal quality of life, deep remission, complete absence of occlusive symptoms, symptom-free survival, bowel damage progression, and no disability (Danese et al., 2018). This consensus will help harmonization of future trials. When considering new anti-fibrotic drugs, we cannot disregard that they would also inhibit the wound healing process, possibly favoring the occurrence of fistulas in case of extensive granulomatous inflammation in the intestinal wall, as in the case of CD (Fig. 2). TGF-β is the core cytokine of fibrosis. Rho kinases are pleiotropic proteins able to modulate TGF-β signaling in the intestine and other organs (Huang et al., 2015; Shimada et al., 2011). A very recent in vitro/ ex vivo study showed that a rho kinase inhibitor (AMA0825) is able to reverse established fibrosis in a chronic mouse dextran sulfate sodium model and prevented ex vivo profibrotic protein secretion from stricturing CD biopsies (Holvoet et al., 2017). The main limitation of rho 7

Molecular Aspects of Medicine xxx (xxxx) xxx–xxx

M.V. Lenti, A. Di Sabatino

intestinal fibrosis into effective anti-fibrotic therapies, as demonstrated by the unsuccessful studies published so far, and the use of untargeted strategies could even potentially aggravate mucosal damage (defective wound repair). Markers with bio-predictive power and non-invasive diagnostic tools able to assess fibrosis progression/regression are still awaited.

kinase inhibitors is that they have important cardiovascular effects (Bian et al., 2017), and therefore a locally-acting, non-absorbable, formulation should be developed. Other compounds have been shown to downregulate the TGF-β signaling. In an animal model of colitis-induced fibrogenesis, TGF-β was downregulated by cilengitide, an anti-integrin compound that targets smooth muscle cells (Li et al., 2013). Angiotensin converting enzyme inhibitors proved to have pleiotropic effects, including modulation of inflammation and fibrosis (Paul et al., 2006). The transanal delivery of enalaprilat in mice suffering from experimental colitis was able to reduce colonic inflammation and fibrosis (Koga et al., 2008). No trials exploring the efficacy of these compounds in CD are currently ongoing. Prolyl-specific aminodipeptidases include dipeptidyl-aminodipeptidase IV, that is currently used for the treatment of type II diabetes mellitus. Unfortunately, an inhibitor able to discriminate between dipeptidyl-aminodipeptidase and FAP is not yet available in clinical practice, even if FAP certainly represents a possible therapeutic target of fibrogenesis in CD and other fibrogenic disorders (Juillerat-Jeanneret and Gerber-Lemaire, 2009). It has been recently showed that the ex vivo treatment of stenotic tissue from ileal surgical specimens of patients with CD with an anti-FAP monoclonal antibody was able to induce a dose-dependent decrease of type I collagen and TIMP-1 production, without altering MMP-3 and MMP-12 secretion (Truffi et al., 2018). Treatment with an anti-FAP antibody could therefore restore ECM homeostasis in CD patients. MiRNAs are increasingly studied as potential targets of therapies, especially for cardiovascular diseases (Gangwar et al., 2018). However, no drugs targeting miRNAs are currently available in clinical practice, nor have been extensively studied. Peroxisome proliferator activated receptor (PPAR)-ɣ is a nuclear receptor that regulates inflammatory processes in the gut (Dubuquoy et al., 2006). PPAR-ɣ signaling regulates TGF-β-dependent fibrogenesis in systemic sclerosis, a rheumatologic condition characterized by progressive deposition of ECM proteins in the skin and other organs (Wei et al., 2010). Human primary intestinal myofibroblasts were isolated from normal colon tissue and treated with TGF-β1 with or without PPAR-ɣ agonists troglitazone or rosiglitazone (Koo et al., 2017). These agents were able to inhibit TGF-β1-induced synthesis of procollagen1A1, fibronectin, and α-smooth muscle actin. PPAR-ɣ agonists are currently being used for the treatment of type II diabetes and could represent an anti-fibrotic therapy. However, systematic studies in humans are still lacking. Blocking the vicious circle of neoangiogenesis has been supposed to be a therapeutic target of fibrosis. Bevacizumab is an anti-VEGF monoclonal antibody currently used for the treatment of different neoplastic conditions and macular degeneration. However, bevacizumab, at least in vitro, significantly increases the expression of fibrosis-related inflammatory cytokines (Chu et al., 2017). Moreover, due to the possible side effects, bevacizumab and VEGF tyrosine kinase inhibitors could even worsen IBD (Alkim et al., 2015). From this point of view, up-regulation of VEGF seems to be a physiological response to counteract hypoxia occurring with fibrosis, and therefore blocking its action could have a detrimental effect in late stages of the disease.

Sources of funding, grant support None. Financial disclosure None. Conflicts of interest None to declare. Author contributions All authors equally participated in the drafting of the manuscript or critical revision of the manuscript for important intellectual content and provided approval of the final submitted version. ADS is the guarantor of the article. Acknowledgments Dr. Marco Vincenzo Lenti is grateful to University of Pavia for supporting his research projects. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mam.2018.10.003. References Aertgeerts, K., et al., 2005. Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein alpha. J. Biol. Chem. 280 (20), 19441–19444. https://doi:10.1074/jbc.C500092200. Alkim, C., et al., 2015. Angiogenesis in inflammatory bowel disease. Int. J. Inflamm. 2015, 970890. https://doi:10.1155/2015/970890. Allocca, M., et al., 2017. Efficacy of tumour necrosis factor antagonists in stricturing Crohn's disease: a tertiary center real-life experience. Dig. Liver Dis. 49 (8), 872–877. https://doi:10.1016/j.dld.2017.03.012. Alvarez-Lobos, M., et al., 2005. Crohn's disease patients carrying Nod2/CARD15 gene variants have an increased and early need for first surgery due to stricturing disease and higher rate of surgical recurrence. Ann. Surg. 242 (5), 693–700. Andoh, A., et al., 2007. Role of intestinal subepithelial myofibroblasts in inflammation and regenerative response in the gut. Pharmacol. Ther. 114 (1), 94–106. https:// doi:10.1016/j.pharmthera.2006.12.004. Baumgart, D.C., Sandborn, W.J., 2012. Crohn's disease. Lancet 380 (9853), 1590–1605. https://doi:10.1016/S0140-6736(12)60026-9. Bailey, J.R., et al., 2012. IL-13 promotes collagen accumulation in Crohn's disease fibrosis by down-regulation of fibroblast MMP synthesis: a role for innate lymphoid cells? PloS One 7 (12), e52332. https://doi:10.1371/journal.pone.0052332. Bayless, T.M., et al., 1967. Intestinal ulceration–a complication of celiac disease. N. Engl. J. Med. 276 (18), 996–1002. https://10.1056/NEJM196705042761802. Bemelman, W.A., et al., 2018. ECCO-ESCP consensus on surgery for Crohn's disease. J. Crohns. Colitis. 12 (1), 1–16. https://doi:10.1093/ecco-jcc/jjx061. Biagi, F., Lorenzini, P., Corazza, G.R., 2000. Literature review on the clinical relationship between ulcerative jejunoileitis, coeliac disease, and enteropathy-associated T-cell. Scand. J. Gastroenterol. 35 (8), 785–790. Bian, H., et al., 2017. Rho-kinase signaling pathway promotes the expression of PARP to accelerate cardiomyocyte apoptosis in ischemia/reperfusion. Mol. Med. Rep. 16 (2), 2002–2008. https://doi:10.3892/mmr.2017.6826. Biancheri, P., et al., 2013. The role of interleukin 17 in Crohn's disease-associated intestinal fibrosis. Fibrogenesis Tissue Repair 6 (1), 13. https://doi:10.1186/17551536-6-13. Biancheri, P., et al., 2014a. The role of transforming growth factor (TGF)-β in modulating the immune response and fibrogenesis in the gut. Cytokine Growth Factor Rev. 25 (1), 45–55. https://doi:10.1016/j.cytogfr.2013.11.001. Biancheri, P., et al., 2014b. Absence of a role for interleukin-13 in inflammatory bowel

8. Conclusions The incidence of fibrosis-related complications has not markedly changed over the last ten years, implying that the progression of intestinal fibrosis may be at least in part independent from the control of the inflammatory process. This is particularly true for deep intestinal wall fibrosis, that cannot be considered as a part of the physiologic wound healing process. Origin and function of the cells involved in intestinal fibrosis should be investigated in situ and ex vivo, thus avoiding artefacts due to prolonged culture on plastic. The use of experimental models of fibrosis should be reconsidered, as the positive effect of anti-fibrotic strategies in animals may not occur in humans. Moreover, it is difficult to translate the few basic notions available on 8

Molecular Aspects of Medicine xxx (xxxx) xxx–xxx

M.V. Lenti, A. Di Sabatino

Fumery, M., et al., 2015. Detection of dysplasia or cancer in 3.5% of patients with inflammatory bowel disease and colonic strictures. Clin. Gastroenterol. Hepatol. 13 (10), 1770–1775. https://doi:10.1016/j.cgh.2015.04.185. Gangwar, R.S., et al., 2018. Noncoding RNAs in cardiovascular disease: pathological relevance and emerging role as biomarkers and therapeutics. Am. J. Hypertens. 31 (2), 150–165. https://doi:10.1093/ajh/hpx197. Giuffrida, P., et al., 2016. Biomarkers of intestinal fibrosis - one step towards clinical trials for stricturing inflammatory bowel disease. United. European. Gastroenterol. J. 4 (4), 523–530. https://doi:10.1177/2050640616640160. Gomollón, F., et al., 2017. EUROPEAN Evidence-based consensus on the diagnosis and management of Crohn's disease 2016: Part 1: diagnosis and medical management. J. Crohns. Colitis 11 (1), 3–25. https://doi:10.1093/ecco-jcc/jjw168. Gordon, I.O., et al., 2018. Fibrosis in ulcerative colitis is directly linked to severity and chronicity of mucosal inflammation. Aliment. Pharmacol. Ther. 47 (7), 922–939. https://doi:10.1111/apt.14526. Griga, T., et al., 1998. Increased serum levels of vascular endothelial growth factor in patients with inflammatory bowel disease. Scand. J. Gastroenterol. 33 (5), 504–508. Griga, T., et al., 1999. Increased production of vascular endothelial growth factor by intestinal mucosa of patients with inflammatory bowel disease. HepatoGastroenterology 46 (26), 920–923. Harbord, M., et al., 2016. The first European evidence-based consensus on extra-intestinal manifestations in inflammatory bowel disease. J. Crohns. Colitis. 10 (3), 239–254. https://doi:10.1093/ecco-jcc/jjv213. Hauer-Jensen, M., Denham, J.W., Andreyev, H.J., 2014. Radiation enteropathy–pathogenesis, treatment and prevention. Nat. Rev. Gastroenterol. Hepatol. 11 (8), 470–479. https://doi:10.1038/nrgastro.2014.46. Holvoet, T., et al., 2017. Treatment of intestinal fibrosis in experimental inflammatory bowel disease by the pleiotropic actions of a local rho kinase inhibitor. Gastroenterology 153 (4), 1054–1067. https://doi:10.1053/j.gastro.2017.06.013. Huang, Y., Xiao, S., Jiang, Q., 2015. Role of Rho kinase signal pathway in inflammatory bowel disease. Int. J. Clin. Exp. Med. 8 (3), 3089–3097. Hwang, J., et al., 2017. Cryptogenic multifocal ulcerous stenosing enteritis: radiologic features and clinical behavior. World J. Gastroenterol. 23 (25), 4615–4623. https:// doi:10.3748/wjg.v23.i25.4615. Jiang, H., Shen, J., Ran, Z., 2018. Epithelial-mesenchymal transition in Crohn's disease. Mucosal Immunol. 11 (2), 294–303. https://doi:10.1038/mi.2017.107. Johnson, L.A., et al., 2012. Intestinal fibrosis is reduced by early elimination of inflammation in a mouse model of IBD: impact of a "Top-Down" approach to intestinal fibrosis in mice. Inflamm. Bowel Dis. 18 (3), 460–471. https://doi:10.1002/ibd. 21812. Juillerat-Jeanneret, L., Gerber-Lemaire, S., 2009. The prolyl-aminodipeptidases and their inhibitors as therapeutic targets for fibrogenic disorders. Mini Rev. Med. Chem. 9 (2), 215–226. Kanazawa, S., et al., 2001. VEGF, basic-FGF, and TGF-beta in Crohn's disease and ulcerative colitis: a novel mechanism of chronic intestinal inflammation. Am. J. Gastroenterol. 96 (3), 822–828. https://doi:10.1111/j.1572-0241.2001.03527.x. Kaplan, G.G., 2015. The global burden of IBD: from 2015 to 2025. Nat. Rev. Gastroenterol. Hepatol. 12 (12), 720–727. https://doi:10.1038/nrgastro.2015.150. Kapsoritakis, A.N., et al., 2008. Imbalance of tissue inhibitors of metalloproteinases (TIMP) - 1 and - 4 serum levels, in patients with inflammatory bowel disease. BMC Gastroenterol. 8, 55. https://doi:10.1186/1471-230X-8-55. Kisseleva, T., Brenner, D.A., 2008. Mechanisms of fibrogenesis. Exp. Biol. Med. 233 (2), 109–122. https://doi:10.3181/0707-MR-190. Koga, H., et al., 2008. Transanal delivery of angiotensin converting enzyme inhibitor prevents colonic fibrosis in a mouse colitis model: development of a unique mode of treatment. Surgery 144 (2), 259–268. https://doi:10.1016/j.surg.2008.03.043. Konkel, J.E., Chen, W., 2011. Balancing acts: the role of TGF-beta in the mucosal immune system. Trends Mol. Med. 17 (11), 668–676. https://doi:10.1016/j.molmed.2011.07. 002. Koo, J.B., et al., 2017. Anti-fibrogenic effect of PPAR-γ agonists in human intestinal myofibroblasts. BMC Gastroenterol. 17 (1), 73. https://doi:10.1186/s12876-0170627-4. Kopylov, U., et al., 2018. Effectiveness and safety of vedolizumab in anti-TNF-naïve patients with inflammatory bowel disease-a multicenter retrospective European study. Inflamm. Bowel Dis. 24 (11), 2442–2451. Kulkarni, A.B., Karlsson, S., 1993. Transforming growth factor-beta 1 knockout mice. A mutation in one cytokine gene causes a dramatic inflammatory disease. Am. J. Pathol. 143 (1), 3–9. Kurahara, L.H., et al., 2015. Intestinal myofibroblast TRPC6 channel may contribute to stenotic fibrosis in Crohn's disease. Inflamm. Bowel Dis. 21 (3), 496–506. https://doi: 10.1097/MIB.0000000000000295. Latella, G., et al., 2014. Results of the 4th scientific workshop of the ECCO (I): pathophysiology of intestinal fibrosis in IBD. J. Crohns. Colitis. 8 (10), 1147–1165. https:// doi:10.1016/j.crohns.2014.03.008. Lawrance, I.C., Maxwell, L., Doe, W., 2001. Altered response of intestinal mucosal fibroblasts to profibrogenic cytokines in inflammatory bowel disease. Inflamm. Bowel Dis. 7 (3), 226–236. Leask, A., Abraham, D.J., 2004. TGF-beta signaling and the fibrotic response. FASEB. J. 18 (7), 816–827. https://doi:10.1096/fj.03-1273rev. Leeb, S.N., et al., 2004. Autocrine fibronectin-induced migration of human colonic fibroblasts. Am. J. Gastroenterol. 99 (2), 335–340. Lenti, M.V., Selinger, C.P., 2017. Medication non-adherence in adult patients affected by inflammatory bowel disease: a critical review and update of the determining factors, consequences and possible interventions. Expet Rev. Gastroenterol. Hepatol. 11 (3), 215–226. https://doi:10.1080/17474124.2017.1284587. Lewis, A., et al., 2015. Low serum levels of MicroRNA-19 are associated with a stricturing

disease. Eur. J. Immunol. 44 (2), 370–385. https://doi:10.1002/eji.201343524. Blomberg, B., Rolny, P., Järnerot, G., 1991. Endoscopic treatment of anastomotic strictures in Crohn's disease. Endoscopy 23 (4), 195–198. https://10.1055/s-20071010654. Booya, F., et al., 2006. Active Crohn disease: CT findings and interobserver agreement for enteric phase CT enterography. Radiology 241 (3), 787–795. https://doi:10.1148/ radiol.2413051444. Bouhnik, Y., et al., 2018. Efficacy of adalimumab in patients with Crohn's disease and symptomatic small bowel stricture: a multicentre, prospective, observational cohort (CREOLE) study. Gut 67 (1), 53–60. https://doi:10.1136/gutjnl-2016-312581. Bousvaros, A., et al., 1997. Serum basic fibroblast growth factor in pediatric Crohn's disease. Implications for wound healing. Dig. Dis. Sci. 42 (2), 378–386. Bousvaros, A., et al., 1999. Elevated serum vascular endothelial growth factor in children and young adults with Crohn's disease. Dig. Dis. Sci. 44 (2), 424–430. Brain, O., et al., 2013. The intracellular sensor NOD2 induces microRNA-29 expression in human dendritic cells to limit IL-23 release. Immunity 39 (3), 521–536. https:// doi:10.1016/j.immuni.2013.08.035. Bruining, D.H., et al., 2018. Consensus recommendations for evaluation, interpretation, and utilization of computed tomography and magnetic resonance enterography in patients with small bowel Crohn's disease. Gastroenterology 154 (4), 1172–1194. https://doi:10.1053/j.gastro.2017.11.274. Burke, J.P., et al., 2011. N-cadherin is overexpressed in Crohn's stricture fibroblasts and promotes intestinal fibroblast migration. Inflamm. Bowel Dis. 17 (8), 1665–1673. https://doi:10.1002/ibd.21543. Cantoro, L., et al., 2017. The time course of diagnostic delay in inflammatory bowel disease over the last sixty years: an Italian multicentre study. J. Crohns. Colitis. 11 (8), 975–980. https://doi:10.1093/ecco-jcc/jjx041. Casanova, M.J., et al., 2017. Evolution after anti-TNF discontinuation in patients with inflammatory bowel disease: a multicenter long-term follow-up study. Am. J. Gastroenterol. 112 (1), 120–131. https://doi:10.1038/ajg.2016.569. Castiglione, F., et al., 2013. Noninvasive diagnosis of small bowel Crohn's disease: direct comparison of bowel sonography and magnetic resonance enterography. Inflamm. Bowel Dis. 19 (5), 991–998. https://doi:10.1097/MIB.0b013e3182802b87. Chen, Y., et al., 2015. Robust bioengineered 3D functional human intestinal epithelium. Sci. Rep. 5, 13708. https://doi:10.1038/srep13708. Chu, S.J., et al., 2017. Effect of bevacizumab on the expression of fibrosis-related inflammatory mediators in ARPE-19 cells. Int. J. Ophthalmol. 10 (3), 366–371. https:// doi:10.18240/ijo.2017.03.07. Costello, C.M., et al., 2014. Synthetic small intestinal scaffolds for improved studies of intestinal differentiation. Biotechnol. Bioeng. 111 (6), 1222–1232. https://doi:10. 1002/bit.25180. Cronin, C.G., et al., 2010. Normal small bowel wall characteristics on MR enterography. Eur. J. Radiol. 75 (2), 207–211. Epub 2009 Jun 4. https://doi:10.1016/j.ejrad.2009. 04.066. Daperno, M., et al., 2004. Development and validation of a new, simplified endoscopic activity score for Crohn's disease: the SES-CD. Gastrointest. Endosc. 60 (4), 505–512. de Bruyn, J.R., et al., 2015. Development of fibrosis in acute and longstanding ulcerative colitis. J. Crohns. Colitis. 9 (11), 966–972. https://doi:10.1093/ecco-jcc/jjv133. de Buck van Overstraeten, A., Wolthuis, A., D'Hoore, A., 2012. Surgery for Crohn's disease in the era of biologicals: a reduced need or delayed verdict? World. J. Gastroenterol. 18 (29), 3828–3832. https://doi:10.3748/wjg.v18.i29.3828. di Mola, F.F., et al., 1999. Transforming growth factor-betas and their signaling receptors are coexpressed in Crohn's disease. Ann. Surg. 229 (1), 67–75. Danese, S., et al., 2018. Identification of endpoints for development of antifibrosis drugs for treatment of Crohn's disease. Gastroenterology 155 (1), 76–87. Di Sabatino, A., et al., 2004. Serum bFGF and VEGF correlate respectively with bowel wall thickness and intramural blood flow in Crohn's disease. Inflamm. Bowel Dis. 10 (5), 573–577. Di Sabatino, A., Corazza, G.R., 2009. Coeliac disease. Lancet 373 (9673), 1480–1493. https://doi:10.1016/S0140-6736(09)60254-3. Di Sabatino, A., et al., 2009. Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures. Gut 58 (6), 777–789. https://doi:10.1136/gut.2008.149096. Di Sabatino, A., et al., 2013. Recent advances in understanding Crohn's disease. Intern. Emerg. Med. 8 (2), 101–113. https://doi:10.1007/s11739-011-0599-2. Di Sabatino, A., et al., 2015. New insights into immune mechanisms underlying autoimmune diseases of the gastrointestinal tract. Autoimmun. Rev. 14 (12), 1161–1169. https://doi:10.1016/j.autrev.2015.08.004. Di Sabatino, A., et al., 2017. Clinical usefulness of serum antibodies as biomarkers of gastrointestinal and liver diseases. Dig. Liver Dis. 49 (9), 947–956. https://doi:10. 1016/j.dld.2017.06.010. Dubuquoy, L., et al., 2006. PPARgamma as a new therapeutic target in inflammatory bowel diseases. Gut 55 (9), 1341–1349. https://doi:10.1136/gut.2006.093484. Engel, T., et al., 2018. Vedolizumab in IBD-Lessons from real-world experience; a systematic review and pooled analysis. J. Crohns. Colitis. 12 (2), 245–257. https:// doi:10.1093/ecco-jcc/jjx143. Fiorino, G., et al., 2017. Prevalence of bowel damage assessed by cross-sectional imaging in early Crohn's disease and its impact on disease outcome. J. Crohns. Colitis. 11 (3), 274–280. https://doi:10.1093/ecco-jcc/jjw185. Fischer, S., Neurath, M.F., 2017. Precision medicine in inflammatory bowel diseases. Clin. Pharmacol. Ther. 102 (4), 623–632. https://doi:10.1002/cpt.793. Freeman, H.J., 2009. Multifocal stenosing ulceration of the small intestine. World J. Gastroenterol. 15 (39), 4883–4885. Fries, W., et al., 2017. Disease patterns in late-onset ulcerative colitis: results from the IGIBD "AGED study. Dig. Liver Dis. 49 (1), 17–23. https://doi:10.1016/j.dld.2016.09. 006.

9

Molecular Aspects of Medicine xxx (xxxx) xxx–xxx

M.V. Lenti, A. Di Sabatino

1016/j.crohns.2008.05.009. Rieder, F., et al., 2011. Inflammation-induced endothelial-to-mesenchymal transition: a novel mechanism of intestinal fibrosis. Am. J. Pathol. 179 (5), 2660–2673. https:// doi:10.1016/j.ajpath.2011.07.042. Rieder, F., Fiocchi, C., Rogler, G., 2017. Mechanisms, management, and treatment of fibrosis in patients with inflammatory bowel diseases. Gastroenterology 152 (2), 340–350. https://doi: 10.1053/j.gastro.2016.09.047. Rieder, F., et al., 2018. An expert consensus to standardise definitions, diagnosis and treatment targets for anti-fibrotic stricture therapies in Crohn's disease. Aliment. Pharmacol. Ther. 48 (3), 347–357. https://doi: 10.1111/apt.14853. Rodansky, E.S., et al., 2015. Intestinal organoids: a model of intestinal fibrosis for evaluating anti-fibrotic drugs. Exp. Mol. Pathol. 98 (3), 346–351. https://doi:10.1016/j. yexmp.2015.03.033. Rovedatti, L., et al., 2011. Fibroblast activation protein expression in Crohn's disease strictures. Inflamm. Bowel Dis. 17 (5), 1251–1253. https://doi:10.1002/ibd.21446. Shimada, H., Staten, N.R., Rajagopalan, L.E., 2011. TGF-β1 mediated activation of Rho kinase induces TGF-β2 and endothelin-1 expression in human hepatic stellate cells. J. Hepatol. 54 (3), 521–528. https://doi:10.1016/j.jhep.2010.07.026. Shull, M.M., et al., 1992. Targeted disruption of the mouse transforming growth factorbeta 1 gene results in multifocal inflammatory disease. Nature 359 (6397), 693–699. Siddiqui, U.D., et al., 2013. Tools for endoscopic stricture dilation. Gastrointest. Endosc. 78 (3), 391–404. https://doi:10.1016/j.gie.2013.04.170. Silverberg, M.S., et al., 2005. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can. J. Gastroenterol. 19, 5A–36A. Silverstein, M.D., et al., 1999. Clinical course and costs of care for Crohn's disease: markov model analysis of a population-based cohort. Gastroenterology 117 (1), 49–57. Singh, A., et al., 2017. Efficacy, safety, and long-term outcome of serial endoscopic balloon dilation for upper gastrointestinal Crohn's disease-associated strictures-a cohort study. J. Crohns. Colitis. 11 (9), 1044–1051. https://doi:10.1093/ecco-jcc/jjx078. Stempien-Otero, A., Kim, D.H., Davis, J., 2016. Molecular networks underlying myofibroblast fate and fibrosis. J. Mol. Cell. Cardiol. 97, 153–161. https://doi:10.1016/j. yjmcc.2016.05.002. Taha, Y., et al., 2004. Vascular endothelial growth factor (VEGF)–a possible mediator of inflammation and mucosal permeability in patients with collagenous colitis. Dig. Dis. Sci. 49 (1), 109–115. Thörn, M., et al., 2000. Intestinal mucosal secretion of basic fibroblast growth factor in patients with ulcerative colitis. Scand. J. Gastroenterol. 35 (4), 408–412. Truffi, M., et al., 2018. Inhibition of fibroblast activation protein restores a balanced extracellular matrix and reduces fibrosis in Crohn's disease strictures ex vivo. Inflamm. Bowel Dis. 24 (2), 332–345. https://doi:10.1093/ibd/izx008. Tse, C.S., et al., 2018. Effects of vedolizumab, adalimumab and infliximab on biliary inflammation in individuals with primary sclerosing cholangitis and inflammatory bowel disease. Aliment. Pharmacol. Ther. 48 (2), 190–195. Vanoli, A., et al., 2017. Small bowel carcinomas in coeliac or Crohn's disease: clinicopathological, molecular, and prognostic features. a study from the small bowel cancer Italian consortium. J. Crohns. Colitis. 11 (8), 942–953. https://doi:10.1093/ecco-jcc/ jjx031. Vavricka, S.R., et al., 2015. Extraintestinal manifestations of inflammatory bowel disease. Inflamm. Bowel Dis. 21 (8), 1982–1992. https://doi:10.1097/MIB. 0000000000000392. Wahid, F., et al., 2010. MicroRNAs: synthesis, mechanism, function, and recent clinical trials. Biochim. Biophys. Acta 1803 (11), 1231–1243. https://doi: 10.1016/j.bbamcr. 2010.06.013. Wei, J., et al., 2010. PPARγ downregulation by TGFß in fibroblast and impaired expression and function in systemic sclerosis: a novel mechanism for progressive fibrogenesis. PloS One 5 (11), e13778. https://doi:10.1371/journal.pone.0013778. Wells, R.G., 2013. Tissue mechanics and fibrosis. Biochim. Biophys. Acta 1832 (7), 884–890. https://doi:10.1016/j.bbadis.2013.02.007. Yamamoto, T., Fazio, V.W., Tekkis, P.P., 2007. Safety and efficacy of strictureplasty for Crohn's disease: a systematic review and meta-analysis. Dis. Colon Rectum 50 (11), 1968–1986. https://doi:10.1007/s10350-007-0279-5. Yu, Y., et al., 2016. Decellularized scaffolds in regenerative medicine. Oncotarget 7 (36), 58671–58683. https://doi:10.18632/oncotarget.10945.

Crohn's disease phenotype. Inflamm. Bowel Dis. 21 (8), 1926–1934. https://doi:10. 1097/MIB.0000000000000443. Li, C., et al., 2013. Increased activation of latent TGF-β1 by αVβ3 in human Crohn's disease and fibrosis in TNBS colitis can be prevented by cilengitide. Inflamm. Bowel Dis. 19 (13), 2829–2839. https://doi:10.1097/MIB.0b013e3182a8452e. Li, X.H., et al., 2018. Characterization of degree of intestinal fibrosis in patients with crohn disease by using magnetization transfer MR imaging. Radiology 287 (2), 494–503. https://doi:10.1148/radiol.2017171221. Ma, C., et al., 2017. Clinical, endoscopic and radiographic outcomes with ustekinumab in medically-refractory Crohn's disease: real world experience from a multicentre cohort. Aliment. Pharmacol. Ther. 45 (9), 1232–1243. https://doi:10.1111/apt.14016. Ma, C., et al., 2018. Corrigendum: surgical rates for Crohn's disease are decreasing: a population-based time trend analysis and validation study. Am. J. Gastroenterol. 113 (2), 310. https://doi:10.1038/ajg.2017.468. Masterson, J.C., et al., 2015. Eosinophils and IL-33 perpetuate chronic inflammation and fibrosis in a pediatric population with stricturing Crohn's ileitis. Inflamm. Bowel Dis. 21 (10), 2429–2440. https://doi:10.1097/MIB.0000000000000512. Matsumoto, T., et al., 2007. Chronic nonspecific multiple ulcers of the small intestine: a proposal of the entity from Japanese gastroenterologists to Western enteroscopists. Gastrointest. Endosc. 66 (3 Suppl. l), S99–S107. https://10.1016/j.gie.2007.01.004. Mazza, G., et al., 2015. Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation. Sci. Rep. 5, 13079. https://doi:10.1038/ srep13079. McKaig, B.C., et al., 2002. Differential expression of TGF-beta isoforms by normal and inflammatory bowel disease intestinal myofibroblasts. Am. J. Physiol. Cell Physiol. 282 (1), C172–C182. https://10.1152/ajpcell.00048.2001. Mehta, S.J., et al., 2018. Epithelial down-regulation of the miR-200 family in fibrostenosing Crohn's disease is associated with features of epithelial to mesenchymal transition. J. Cell Mol. Med. 22 (11), 5617–5628. Monteleone, G., et al., 2001. Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease. J. Clin. Invest. 108 (4), 601–609. Navaneethan, U., et al., 2016. Endoscopic balloon dilation in the management of strictures in Crohn's disease: a systematic review and meta-analysis of non-randomized trials. Surg. Endosc. 30 (12), 5434–5443. https://10.1007/s00464-016-4902-1. Neufeld, G., et al., 1999. Vascular endothelial growth factor (VEGF) and its receptors. FASEB. J. 13 (1), 9–22. Nijhuis, A., et al., 2014. In Crohn's disease fibrosis-reduced expression of the miR-29 family enhances collagen expression in intestinal fibroblasts. Clin. Sci. (Lond.) 127 (5), 341–350. https://doi:10.1042/CS20140048. Orlando, S., et al., 2018. Ultrasound elasticity imaging predicts therapeutic outcomes of patients with Crohn's disease treated with anti-tumour necrosis factor antibodies. J. Crohns. Colitis. 12 (1), 63–70. https://doi:10.1093/ecco-jcc/jjx116. Pakshir, P., Hinz, B., 2018. The big five in fibrosis: macrophages, myofibroblasts, matrix, mechanics, and miscommunication. Matrix Biol. 68-69, 81–93. Pariente, B., et al., 2011. Development of the Crohn's disease digestive damage score, the Lémann score. Inflamm. Bowel Dis. 17 (6), 1415–1422. https://doi:10.1002/ibd. 21506. Park, M.J., Lim, J.S., 2013. Computed tomography enterography for evaluation of inflammatory bowel disease. Clin. Endosc. 46 (4), 327–366. https://doi:10.5946/ce. 2013.46.4.327. Paul, M., Poyan Mehr, A., Kreutz, R., 2006. Physiology of local renin-angiotensin systems. Physiol. Rev. 86 (3), 747–803. https://doi:10.1152/physrev.00036.2005. Paul, S., et al., 2015. Association of anti-glycan antibodies and inflammatory bowel disease course. J. Crohns. Colitis. 9 (6), 445–451. https://doi:10.1093/ecco-jcc/jjv063. Pinzani, M., 2010. Fibrosis in the GI tract: pathophysiology, diagnosis and treatment options. In: Mayerle, J., Tilg, H. (Eds.), Clinical Update on Inflammatory Disorders of the Gastrointestinal Tract. Front. Gastrointest. Res., Basel, Karger, pp. 15–31. Piscaglia, F., et al., 2012. The EFSUMB guidelines and recommendations on the clinical practice of contrast enhanced ultrasound (CEUS): update 2011 on non-hepatic applications. Ultraschall der Med. 33 (1), 33–59. https://doi:10.1055/s-0031-1281676. Regan, M.C., et al., 2000. Stricture formation in Crohn's disease: the role of intestinal fibroblasts. Ann. Surg. 231 (1), 46–50. Rieder, F., Fiocchi, C., 2008. Intestinal fibrosis in inflammatory bowel disease – current knowledge and future perspectives. J. Crohns. Colitis. 2 (4), 279–290. https://doi:10.

10