Review Article
Biomarkers of intestinal fibrosis – one step towards clinical trials for stricturing inflammatory bowel disease
United European Gastroenterology Journal 2016, Vol. 4(4) 523–530 ! Author(s) 2016 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/2050640616640160 ueg.sagepub.com
Paolo Giuffrida1, Massimo Pinzani2, Gino R Corazza1 and Antonio Di Sabatino1,2
Abstract Intestinal fibrosis, caused by an excessive deposition of extracellular matrix components, and subsequent stricture development are a common complication of inflammatory bowel disease. However, currently there are no biomarkers which reliably predict the risk of developing intestinal strictures or identify early stages of fibrosis prior to clinical symptoms. Candidate biomarkers of intestinal fibrosis, including gene variants (i.e. nucleotide-binding oligomerization domain-2 gene), serum microRNAs (miR-19, miR-29), serum extracellular matrix proteins (i.e. collagen, fibronectin) or enzymes (i.e. tissue inhibitor of matrix metalloproteinase-1), serum growth factors (i.e. basic fibroblast growth factor, YKL-40), serum anti-microbial antibodies (i.e. anti-Saccharomyces cerevisiae) and circulating cells (i.e. fibrocytes) have shown conflicting results on relatively heterogeneous patients’ cohorts, and none of them was proven to be strictly specific for fibrostenosis, but rather predictive of a disease disabling course. In this review we critically reassess the diagnostic and prognostic value of serum biomarkers of intestinal fibrosis in inflammatory bowel disease.
Keywords Anti-microbial antibody, Crohn’s disease, extracellular matrix, growth factor, microRNA Received: 10 January 2016; accepted: 20 February 2016
Introduction Intestinal fibrosis is a frequent complication in the natural history of inflammatory bowel disease (IBD), as up to one-third of Crohn’s disease patients and about 5% of ulcerative colitis patients develop strictures in the clinical course of the disease.1 In particular, transmural damage induced by chronic inflammation in Crohn’s disease causes tissue remodelling of the entire intestinal wall, which is characterized by an excessive deposition of extracellular matrix (ECM) components due to an imbalance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the context of a massive activation of ECM-producing cells responsible for the excessive deposition of fibrillar collagens, thus leading to tissue fibrosis as end-stage complication of Crohn’s disease.2,3 Increased amount of collagen and thickening of the muscularis mucosae have been also identified in the colon of ulcerative colitis patients both with short- and long-standing disease, with consequent shortening and increased rigidity of the colon.4
A number of cytokines have been shown to exert a pro-fibrogenic action in Crohn’s disease. Transcript and protein levels of transforming growth factor (TGF)-b1, which enhances TIMP-1 and reduces MMP-12 (also known as macrophage metalloelastase), are highly produced by Crohn’s disease strictured myofibroblasts, whereas an anti-TGF-b neutralizing antibody enhances MMP-12 production and myofibroblast migration.5 Interleukin (IL)-17A is up-regulated in stricturing Crohn’s disease tissue where it increases collagen production and TIMP-1, MMP-3 and
1
First Department of Internal Medicine, San Matteo Hospital Foundation, University of Pavia, Pavia, Italy 2 Institute for Liver and Digestive Health, University College London, London, UK Corresponding author: Antonio Di Sabatino, Clinica Medica I, Fondazione IRCCS Policlinico San Matteo, Universita` di Pavia, Piazzale Golgi 19, 27100 Pavia, Italy. Email: [email protected]
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MMP-12 secretion by myofibroblasts isolated from Crohn’s disease intestinal strictures.6 Conflicting results have been reported regarding the pro-fibrogenic action of some cytokines such as IL-13,7,8 whereas the profibrogenic role of IL-33 has been demonstrated in murine models but not yet in humans.9
Currently, there are no predictors able to estimate the risk of developing intestinal fibrosis in IBD patients.1 All the proposed noninvasive biomarkers of intestinal fibrosis, including gene polymorphisms or variants, microRNAs (miRs), ECM components, growth factors and anti-microbial antibodies (Figure 1) have limited
Antibodies ASCA Anti-CBir1
Y
Genes
PC
Anti-L
Y
Anti-OmpC Anti-I2
IL23R IL L23R 3R
Anti-C ACCA
B cell
AMCA ALCA
M acrophage Macrophage
Epithelial cells
MMP-3
Stricture
Paneth P Pa neth h cell cells lls NOD2 NOD OD2 OD 2
MDP
Bacteria
ATG16L1 16L1 6L1 TLR4
Lumen
CX3CR1 CX X3CR X 3CR R1
Pre-stenotic dilatation
Growth factors
ECM components components
PDGF PIIINP
Fibronectin
bFGF Platelet
Capillary Macrophage age
FAP
YKL-40
Fibrocyte
Myofibroblast Endothelial cells
ITCP
phil Neutrophil TGF-β
PICP
Fibrotic tissue
Laminin
TIMP-1
microRNAs miR-29b
miR-200b
miR-19b-3p miR-29a
miR-19a-3p miR-29c
Figure 1. Pathogenic mechanisms and candidate molecular biomarkers for intestinal fibrosis. Picture shows pre-stenotic dilatation, stricture with fibrotic tissue, lumen, capillary and candidate biomarkers for intestinal fibrosis: genes (red panel), growth factors (purple panel), extracellular matrix (ECM) components (light blue panel), microRNAs (miRs) (yellow panel) and antibodies (green panel). Nucleotide-binding oligomerization domain (NOD)2 variants, which imply loss of binding between NOD2 and the bacterial cell wall component muramyl dipeptide (MDP), polymorphisms of the chemokine fractalkine receptor CX3CR1, variants in the toll like receptor (TLR)4, in the autophagy-related-16L1 (ATG16L1) and in the interleukin 23 receptor (IL23R) induce chronic inflammation leading to stricture development. Excessive deposition of ECM components, which consist of fibronectin, laminin, collagen and its propeptide or telopeptide – such as N-terminal propeptide of type III collagen (PIIINP), C-terminal propeptide of type I collagen (PICP) and C-terminal telopeptide of type I collagen (ITCP) – and alterated tissue remodelling are also due to a specific single nucleotide polymorphism in matrix metalloproteinase (MMP)-3 gene, which loses its proteolytic activity on ECM, and to abnormal expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1. Myofibroblasts, whose proliferation is sustained by endothelial cell-released basic fibroblast growth factor (bFGF), produce increased amounts of collagen in a YKL-40-dependent fashion. YKL-40 is a growth factor secreted by activated macrophages and neutrophils. The production of collagen is also favoured by platelet-derived growth factor (PDGF), secreted by circulating platelets. Moreover, miR-29b, which is suppressed by myofibroblast-produced transforming growth factor (TGF)-b, inhibits the profibrogenic effects of myofibroblasts themselves. These cells express on their surface fibroblast activation protein (FAP) and may be derived from circulating fibrocytes. The abnormal immune response underlying intestinal fibrosis is characterized by the plasma cell (PC)mediated production of antibodies directed against bacterial components: anti-Escherichia coli outer membrane protein C antibodies (Anti-OmpC), anti-Pseudomonas-associated sequence I2 antibodies (Anti-I2), anti-bacterial flagellin CBir1 antibodies (Anti-CBir1) and anti-glycan antibodies, such as anti-Saccharomyces cerevisiae antibodies (ASCA), anti-chitobioside carbohydrate IgA antibodies (ACCA), anti-mannobioside carbohydrate IgG antibodies (AMCA), anti-laminaribioside IgG antibodies (ALCA), anti-laminarin carbohydrate antibodies (anti-L) and/or anti-chitin carbohydrate antibodies (Anti-C).
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Table 1. Serum biomarkers proposed for intestinal fibrosis. Candidate biomarker
Diagnostic value
miRs miR-200a miR-200b þ miR-29a þ miR-29b miR-29c miR-19a-3p þ miR-19b-3p þ ECM components TIMP-1 PIIINP PICP ITCP Laminin Fibronectin Growth factors bFGF YKL-40 PDGF Antibodies ASCA ACCA AMCA NA Anti-L NA Anti-CBir1 NA Anti-OmpC NA Anti-I2 NA
Prognostic value
Used in clinical trials
References
NA NA NA NA NA NA NA
No No No No No No No
18 18 19 19 19 20 20
NA NA NA NA
Yes No No No No No
21,55 22,23 23 23 26 27,28
NA NA NA
Yes Yes No
29–31,55 32,33,55 34
No No No No No No No
37,41–45,47,48 37,41 37,43 37,43 44–46 44,45,48 44
ACCA: anti-chitobioside carbohydrate antibody; AMCA: anti-mannobioside carbohydrate antibody; anti-CBir1: anti-bacterial flagellin CBir1 antibody; anti-I2: anti-Pseudomonas-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; ECM: extracellular matrix; ITCP: C-terminal telopeptide of type I collagen; miR: microRNA; NA: not assessed; PDGF: platelet-derived growth factor; PICP: C-terminal propeptide of type I collagen; PIIINP: N-terminal propeptide of type III collagen; TIMP: tissue inhibitor of matrix metalloproteinases; þ: consistent evidence; : conflicting evidence; –: no evidence.
diagnostic and prognostic value, and most of the studies so far performed have provided conflicting results (Table 1). Biomarkers of intestinal fibrosis would be useful in order to stratify patients according to their risk of stricture development and to identify early stages of fibrosis with the aim of optimizing the therapeutic management.1 Patients with known risk factors for severe disease course, that is, age below 40 years at diagnosis, early requirement of steroids and perianal disease,10 have an increased rate of fibrostenotic complications, thus they should be more strictly followed up.
Here we review the latest findings on candidate biomarkers of intestinal fibrosis in IBD.
Genes A specific genetic background has been supposed to predispose to fibrostenosing phenotype in Crohn’s disease.1 The first gene identified as predisposing to stricturing Crohn’s disease has been nucleotide-binding oligomerization domain (NOD)2 gene, also known as the caspase recruitment domain 15, involved in a-defensin production and intracellular clearance of bacteria. The presence of various predetermined combinations of NOD2 mutations, which imply loss of binding between NOD2 and the bacterial component muramyl dipeptide, has been shown to predispose to stricturing and/ or penetrating Crohn’s disease, whereas the predictive value of any single NOD2 mutation is low.11 NOD2 gene variants are associated with early ileal strictures and with postoperative recurrence in Crohn’s disease patients.12 Presumably, the high need for surgery in Crohn’s disease patients with NOD2 mutations is due to the ileal location and stricturing phenotype. Patients carrying toll like receptor (TLR) variants, especially TLR4, frequently have small bowel stricturing disease.13 T280M and V249I polymorphisms of the chemokine fractalkine receptor CX3CR1 influence the stricturing phenotype, regardless of NOD2 status.14,15 The 5T5T genotype at the MMP-3 SNP-1613 5 T/6 T increases the percentage of stenotic complications through an unbalanced tissue remodelling, but prevents colonic involvement in Crohn’s disease.16 Crohn’s disease fibrosis is also associated with variants in the autophagy-related-16L1 gene (rs2241879 and rs2241880), implicated in autophagy and bacterial phagocytosis, and in the IL23 receptor gene (TT genotype of rs1004819).17,18 CX3CR1 polymorphisms, TLR4, autophagy-related-16L1 and IL23 receptor variants induce chronic inflammation leading to stricture development. In summary, although gene mutations or polymorphisms are promising biomarkers, they are not used in clinical practice due to their incomplete penetrance and low frequency.
miRs Amongst epigenetic pathways, miRs, which are short noncoding RNA negatively regulating target gene expression at post-transcriptional level, are the most extensively studied in the pathogenesis of intestinal fibrosis. Serum levels of miR-200b, but not those of miR-200a, are increased in stricturing Crohn’s disease compared with the nonstricturing phenotype.19 MiR29a is reduced in the serum of fibrostenosing Crohn’s disease patients in comparison with inflammatory
526 Crohn’s disease.20 Similarly, serum miR-29b and miR29c are lower, although not significantly, in stricturing Crohn’s disease than in inflammatory phenotype.20 MiR-29 family is consistently down-regulated in the intestinal mucosa overlying strictures in Crohn’s disease patients, and miR-29b transfection inhibits TGF-b-induced increase in collagen in Crohn’s disease myofibroblasts.20 MiR-19a-3p and miR-19b-3p are reduced in the serum of stricturing Crohn’s disease patients compared with that of nonstricturing Crohn’s disease.21 Moreover, according to a multivariate analysis, the association between miR-19-3p and stricturing phenotype is independent of confounding clinical variables including ileal location and disease duration.21
ECM components Although TIMP-1 is increased in Crohn’s disease mucosa overlying strictures,5 no association has been observed between serum TIMP-1 and the fibrostenotic phenotype.22 As collagens I and III are considered the two major ECM proteins implicated in intestinal fibrosis,12 their propeptides have been detected in the serum of Crohn’s disease patients. N-terminal propeptide of type III collagen (PIIINP) is higher in stricturing Crohn’s disease before surgery than in controls with a significant reduction of its serum levels six months after successful surgical intervention.23 However, an old study, which did not specify the Crohn’s disease phenotype of the patients recruited, showed no significant difference of PIIINP between Crohn’s disease and controls, whereas serum levels of C-terminal propeptide of type I collagen and C-terminal telopeptide of type I collagen were reported to be significantly reduced and increased, respectively, in Crohn’s disease compared with controls.24 The enhanced liver fibrosis (ELF) test, which is a direct marker of hepatic fibrosis based on the three circulating markers of liver ECM metabolism hyaluronic acid, TIMP-1 and PIIINP,25 is able to discriminate stricturing Crohn’s disease from nonstricturing phenotypes (unpublished data). Additionally, ELF test was identified as a prognostic marker in a cohort of patients with primary sclerosing cholangitis, most of whom were affected by IBD.26 Laminin, which is a component of basement membrane, is higher in the serum of Crohn’s disease patients than in controls and it is associated with disease activity but not with the stricturing behaviour.27 Fibronectin, a high-molecular weight ECM glycoprotein, is increased in the plasma of patients with stricturing Crohn’s disease.28 In addition, its plasma levels significantly decrease in the immediate post-surgery period.29 However, the predictive value of plasma fibronectin for subsequent post-surgery stricture development was not significant in one-year follow-up.29
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Growth factors Serum levels of several growth factors, including basic fibroblast growth factor (bFGF), human chitinase 3-like 1 (also known as YKL-40), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) have been investigated in Crohn’s disease patients as possible biomarkers of intestinal fibrosis. Serum bFGF, which induces wound healing by modulating myofibroblast proliferation, is increased in patients with Crohn’s disease,30-32 and its levels correlate with the bowel wall thickness and the fibrostenotic phenotype.31 Serum levels of YKL-40, which is released by activated macrophages and neutrophils and promotes myofibroblast-induced collagen secretion, are higher in the serum of Crohn’s disease patients than in control subjects.33,34 However, the comparison of serum YKL-40 between stricturing and unstricturing phenotypes produced contradictory results in two studies.33,34 In keeping with the pro-angiogenic and pro-fibrogenic role played in liver fibrosis,2 VEGF is increased in the serum of Crohn’s disease patients31,32 and also correlates with colour Doppler signal intensity, leading to the consideration of VEGF as biomarker of angiogenesis in Crohn’s disease.32 Similarly, the pro-fibrogenic/proangiogenic PDGF, which is able to increase the production of collagen but not that of TIMP-1, is high in the serum of IBD patients.35
Antimicrobial antibodies Several anti-microbial antibodies, which are believed to derive from an abnormal immune response against the luminal microbiota,36 have been measured in the serum of Crohn’s disease patients, including anti-Escherichia coli outer membrane protein C antibodies (anti-OmpC), anti-Pseudomonas-associated sequence I2 antibodies (anti-I2), anti-bacterial flagellin CBir1 antibodies (anti-CBir1) and anti-glycan antibodies, such as anti-Saccharomyces cerevisiae antibodies (ASCAs), anti-chitobioside carbohydrate IgA antibodies (ACCAs), anti-mannobioside carbohydrate IgG antibodies (AMCAs), anti-laminaribioside IgG antibodies (ALCAs), anti-laminarin carbohydrate antibodies (anti-L) and anti-chitin carbohydrate antibodies (antiC).37-40 These biomarkers predict and are qualitatively and quantitatively associated with an aggressive and complicated course of Crohn’s disease, including stricturing and/or penetrating phenotype, both in adult and paediatric patients.13 However, a recent study showed that none of the anti-glycan antibodies ASCA, AMCA, ACCA, ALCA, anti-L and anti-C correlates with disease phenotype.41 Circulating ASCAs, which are directed against a cell wall component of the common baker’s yeast Saccharomyces cerevisiae, are those more widely
Giuffrida et al. investigated in IBD, as they are able to discriminate between Crohn’s disease and ulcerative colitis with a sensitivity of 56% and a specificity of 88%.42 Moreover, Crohn’s disease patients with serological positivity for ASCA develop a fibrostenosing or penetrating phenotype with a sensitivity of 70% and a specificity of 48%, and more frequently present an ileal or ileo-colonic location, thus leading to increased need for surgery.42,43 Circulating ASCAs in Crohn’s disease have been also associated with early disease onset and perianal disease.43 Among the other anti-glycan antibodies, ACCAs are those more frequently associated with a fibrostenosing or fistulizing phenotype with lower sensitivity (43%) but higher specificity (75%) than ASCA.42 Moreover, positivity for ASCA, AMCA and anti-L is associated with a higher risk for an earlier occurrence of complications and surgery,44 whereas serum positivity for ASCA, AMCA, ACCA and anti-L predict a more rapid progression toward complications or surgery.38 The risk of developing a stricturing and/or penetrating phenotype is 11-fold higher in Crohn’s disease children with serological positivity for anti-CBir1, anti-OmpC, anti-I2 and ASCA than in seronegative children.45 These results have been confirmed in a larger prospective study conducted in Crohn’s disease children seropositive for antiCBir1, anti-OmpC and ASCA.46 An Irish study shows that serum anti-CBir1 positivity is significantly associated with a complicated disease behaviour and ileal involvement, but not with increased need for surgery.47 In a population-based cohort of Crohn’s disease patients, among a panel of antibodies directed against antimicrobial peptides, including ASCA IgA and IgG, anti-I2, anti-OmpC, anti-CBir1 and pANCA, only ASCA IgG were significantly associated in a multivariate analysis to a stricturing/penetrating behaviour.48 A recent meta-analysis on 11 studies and based on four antibodies – ASCA, anti-OmpC, anti-I2 and antiCBir1 – identified ASCA as the antibody with the highest sensitivity and anti-OmpC as that with the highest specificity for complications and surgery.49 Moreover, disease progression towards complications was predicted more effectively using at least two antimicrobial antibodies rather than any single one.49 All the antimicrobial antibodies are useful for predicting a disabling course of Crohn’s disease (Table 1), but not for discriminating between stricturing and nonstricturing phenotypes. Further prospective studies are needed to ascertain whether circulating antibodies, alone or in conjunction with other biomarkers, can predict the disease course and the development of fibrosis in IBD.
Other factors Fibroblast activation protein (FAP) is a glycoprotein expressed on activated fibroblasts during wound
527 healing and its up-regulation has been reported in idiopathic pulmonary fibrosis and liver cirrhosis.50,51 In the submucosa and in the muscle layer of Crohn’s disease strictures FAP is up-regulated and its expression on intestinal myofibroblasts from stricturing Crohn’s disease is increased by profibrotic cytokines, such as tumour necrosis factor (TNF)-a and TGF-b1.52 Although serum FAP has been proposed as an index of liver fibrosis,53 there are no data on its utility as a biomarker of intestinal fibrosis in IBD patients. There is evidence that circulating fibrocytes, which are bone marrow-derived mesenchymal progenitor cells, may be considered as a marker of fibrosis in idiopathic pulmonary fibrosis.54 As far as intestinal fibrosis is concerned, there are no studies determining the circulating levels of fibrocytes in patients with fibrostenosing Crohn’s disease. The only data available showed an increased percentage of fibrocytes both in peripheral blood and inflamed gut.55 The demonstration that fibrocytes isolated from the peripheral blood express higher amounts of TNF-a and collagen type I after in vitro stimulation with lipopolysaccharide suggests that their interaction with microbial components may contribute to the fibrogenic process.55
Clinical trials for stricturing IBD Clinical trial design to identify surrogate biomarkers of fibrosis, which are crucial for enabling both a patienttailored anti-fibrotic treatment and an individual risk stratification for fibrostenosing disease, are hampered by a number of limitations, such as (i) the long-lasting evolution of intestinal strictures, which requires clinical trials of long duration and involving large patients’ cohorts, (ii) the common overlap along the disease course of other clinical behaviors (i.e. penetrating),56 (iii) the influence of concomitant immunomodulatory therapies which may mask blood candidate molecule changes, and (iv) the fact that disease duration does not necessarily correlate with the degree of intestinal damage as an early disease can already be a late (stricturing) disease. Moreover, on the basis of the high complexity of the molecular mechanisms underlying the fibrogenic process, it is conceivable that a panel of candidate molecules would be more appropriate than a single biomarker, as has been already shown in other organs (ELF panel).25 A possible study design to validate serum fibrosis biomarkers in a relatively short time period could be to strictly follow-up Crohn’s disease fibrostenosing patients after surgical resection, although the lack of a clear definition of fibrosis and consequently of a gold standard technique for fibrosis represents a limit in the progress in this field. The same limitations can be applied to clinical trials for identifying anti-fibrotic drugs in stricturing IBD. The ideal
528 pilot trial of an anti-fibrotic drug should include restricted populations in whom fibrosis is very common; however, we have no predictor of fibrostenosing complication. Perhaps data from experimental fibrosis could be helpful in stratifying patients according to the risk of fibrosis, in defining the optimal timing to start anti-fibrotic treatment and in understanding how to manage drugs modulating tissue remodelling without inhibiting the desired healing process. Up to now, serum biomarkers of intestinal fibrosis have been measured only in a phase 1 open label trial on Crohn’s disease patients with inflammatory phenotype receiving a seven-day-long treatment with Smad7 antisense oligonucleotide aimed at restoring TGF-b signaling.57 In order to rule out a putative pro-fibrogenic effect exerted by this drug, serum bFGF, YKL-40, TIMP-1, MMP-1, MMP-3 and MMP-9 were detected at baseline and after six months and, in parallel, patients underwent small intestine contrast ultrasonography.57 In the trial no significant change was observed for all these biomarkers in agreement with the small intestine contrast ultrasonography results.57
Conclusions Intestinal fibrosis is a biologically heterogeneous process, involving multiple complex and interactive mechanisms. Major advances in understanding the pathophysiology of intestinal fibrosis have occurred over the past years. Nevertheless, there is insufficient evidence supporting the diagnostic and prognostic value of any biomarker in stricturing IBD. There is an urgent need for effective noninvasive methodologies employing blood samples. The identification of biomarkers will not only impact on the overall management of IBD patients but will also contribute to answering some of the most basic questions of pathogenesis underlying intestinal fibrosis. Candidate biomarkers of intestinal fibrosis should be validated in order to be used in clinical trials testing anti-fibrotic drugs in stricturing IBD patients. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Conflict of interest The authors declare that there is no conflict of interest.
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529 34. Erzin Y, Uzun H, Karatas A, et al. Serum YKL-40 as a marker of disease activity and stricture formation in patients with Crohn’s disease. J Gastroenterol Hepatol 2008; 23: e357–362. 35. Matusiewicz M, Neubauer K, Mierzchala-Pasierb M, et al. Matrix metalloproteinase-9: Its interplay with angiogenic factors in inflammatory bowel diseases. Dis Markers 2014; 2014: 643645. 36. Dotan I, Fishman S, Dgani Y, et al. Antibodies against laminaribioside and chitobioside are novel serologic markers in Crohn’s disease. Gastroenterology 2006; 131: 366–378. 37. Arnott ID, Landers CJ, Nimmo EJ, et al. Sero-reactivity to microbial components in Crohn’s disease is associated with disease severity and progression, but not NOD2/ CARD15 genotype. Am J Gastroenterol 2004; 99: 2376–2384. 38. Reumaux D, Sendid B, Poulain D, et al. Serological markers in inflammatory bowel diseases. Best Pract Res Clin Gastroenterol 2003; 17: 19–35. 39. Rieder F, Schleder S, Wolf A, et al. Serum anti-glycan antibodies predict complicated Crohn’s disease behavior: A cohort study. Inflamm Bowel Dis 2010; 16: 1367–1375. 40. van Schaik FD, Oldenburg B, Hart AR, et al. Serological markers predict inflammatory bowel disease years before the diagnosis. Gut 2013; 62: 683–688. 41. Paul S, Boschetti G, Rinaudo-Gaujous M, et al. Association of anti-glycan antibodies and inflammatory bowel disease course. J Crohns Colitis 2015; 9: 445–451. 42. Kaul A, Hutfless S, Liu L, et al. Serum anti-glycan antibody biomarkers for inflammatory bowel disease diagnosis and progression: A systematic review and meta-analysis. Inflamm Bowel Dis 2012; 18: 1872–1884. 43. Zhang Z, Li C, Zhao X, et al. Anti-Saccharomyces cerevisiae antibodies associate with phenotypes and higher risk for surgery in Crohn’s disease: A meta-analysis. Dig Dis Sci 2012; 57: 2944–2954. 44. Rieder F, Dirmeier A, Strauch U, et al. Association of the novel serologic anti-glycan antibodies anti-laminarin and anti-chitin with complicated Crohn’s disease behavior. Inflamm Bowel Dis 2010; 16: 263–274. 45. Dubinsky MC, Lin YC, Dutridge D, et al. Serum immune responses predict rapid disease progression among children with Crohn’s disease: Immune responses predict disease progression. Am J Gastroenterol 2006; 101: 360–367. 46. Dubinsky MC, Kugathasan S, Mei L, et al. Increased immune reactivity predicts aggressive complicating Crohn’s disease in children. Clin Gastroenterol Hepatol 2008; 6: 1105–1111. 47. O’Donnell S, O’Sullivan M, O’Morain CA, et al. The clinical significance of antimicrobial serologic responses within an Irish Crohn’s disease population. Eur J Gastroenterol Hepatol 2013; 25: 1464–1469. 48. Ryan JD, Silverberg MS, Xu W, et al. Predicting complicated Crohn’s disease and surgery: Phenotypes, genetics, serology and psychological characteristics of a population-based cohort. Aliment Pharmacol Ther 2013; 38: 274–283.
530 49. Xiong Y, Wang GZ, Zhou JQ, et al. Serum antibodies to microbial antigens for Crohn’s disease progression: A meta-analysis. Eur J Gastroenterol Hepatol 2014; 26: 733–742. 50. Acharya PS, Zukas A, Chandan V, et al. Fibroblast activation protein: A serine protease expressed at the remodeling interface in idiopathic pulmonary fibrosis. Hum Pathol 2006; 37: 352–360. 51. Levy MT, McCaughan GW, Abbott CA, et al. Fibroblast activation protein: A cell surface dipeptidyl peptidase and gelatinase expressed by stellate cells at the tissue remodelling interface in human cirrhosis. Hepatology 1999; 29: 1768–1778. 52. Rovedatti L, Di Sabatino A, Knowles CH, et al. Fibroblast activation protein expression in Crohn’s disease strictures. Inflamm Bowel Dis 2011; 17: 1251–1253. 53. Williams KH, Viera de Ribeiro AJ, Prakoso E, et al. Lower serum fibroblast activation protein shows promise
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in the exclusion of clinically significant liver fibrosis due to non-alcoholic fatty liver disease in diabetes and obesity. Diabetes Res Clin Pract 2015; 108: 466–472. Moeller A, Gilpin SE, Ask K, et al. Circulating fibrocytes are an indicator of poor prognosis in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2009; 179: 588–594. Sazuka S, Katsuno T, Nakagawa T, et al. Fibrocytes are involved in inflammation as well as fibrosis in the pathogenesis of Crohn’s disease. Dig Dis Sci 2014; 59: 760–768. Cosnes J, Cattan S, Blain A, et al. Long-term evolution of disease behavior of Crohn’s disease. Inflamm Bowel Dis 2002; 8: 244–250. Zorzi F, Calabrese E, Monteleone I, et al. A phase 1 open-label trial shows that smad7 antisense oligonucleotide (GED0301) does not increase the risk of small bowel strictures in Crohn’s disease. Aliment Pharmacol Ther 2012; 36: 850–857.
Biomarkers of intestinal fibrosis – one step towards clinical trials for stricturing inflammatory bowel disease
United European Gastroenterology Journal 2016, Vol. 4(4) 523–530 ! Author(s) 2016 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/2050640616640160 ueg.sagepub.com
Paolo Giuffrida1, Massimo Pinzani2, Gino R Corazza1 and Antonio Di Sabatino1,2
Abstract Intestinal fibrosis, caused by an excessive deposition of extracellular matrix components, and subsequent stricture development are a common complication of inflammatory bowel disease. However, currently there are no biomarkers which reliably predict the risk of developing intestinal strictures or identify early stages of fibrosis prior to clinical symptoms. Candidate biomarkers of intestinal fibrosis, including gene variants (i.e. nucleotide-binding oligomerization domain-2 gene), serum microRNAs (miR-19, miR-29), serum extracellular matrix proteins (i.e. collagen, fibronectin) or enzymes (i.e. tissue inhibitor of matrix metalloproteinase-1), serum growth factors (i.e. basic fibroblast growth factor, YKL-40), serum anti-microbial antibodies (i.e. anti-Saccharomyces cerevisiae) and circulating cells (i.e. fibrocytes) have shown conflicting results on relatively heterogeneous patients’ cohorts, and none of them was proven to be strictly specific for fibrostenosis, but rather predictive of a disease disabling course. In this review we critically reassess the diagnostic and prognostic value of serum biomarkers of intestinal fibrosis in inflammatory bowel disease.
Keywords Anti-microbial antibody, Crohn’s disease, extracellular matrix, growth factor, microRNA Received: 10 January 2016; accepted: 20 February 2016
Introduction Intestinal fibrosis is a frequent complication in the natural history of inflammatory bowel disease (IBD), as up to one-third of Crohn’s disease patients and about 5% of ulcerative colitis patients develop strictures in the clinical course of the disease.1 In particular, transmural damage induced by chronic inflammation in Crohn’s disease causes tissue remodelling of the entire intestinal wall, which is characterized by an excessive deposition of extracellular matrix (ECM) components due to an imbalance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the context of a massive activation of ECM-producing cells responsible for the excessive deposition of fibrillar collagens, thus leading to tissue fibrosis as end-stage complication of Crohn’s disease.2,3 Increased amount of collagen and thickening of the muscularis mucosae have been also identified in the colon of ulcerative colitis patients both with short- and long-standing disease, with consequent shortening and increased rigidity of the colon.4
A number of cytokines have been shown to exert a pro-fibrogenic action in Crohn’s disease. Transcript and protein levels of transforming growth factor (TGF)-b1, which enhances TIMP-1 and reduces MMP-12 (also known as macrophage metalloelastase), are highly produced by Crohn’s disease strictured myofibroblasts, whereas an anti-TGF-b neutralizing antibody enhances MMP-12 production and myofibroblast migration.5 Interleukin (IL)-17A is up-regulated in stricturing Crohn’s disease tissue where it increases collagen production and TIMP-1, MMP-3 and
1
First Department of Internal Medicine, San Matteo Hospital Foundation, University of Pavia, Pavia, Italy 2 Institute for Liver and Digestive Health, University College London, London, UK Corresponding author: Antonio Di Sabatino, Clinica Medica I, Fondazione IRCCS Policlinico San Matteo, Universita` di Pavia, Piazzale Golgi 19, 27100 Pavia, Italy. Email: [email protected]
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MMP-12 secretion by myofibroblasts isolated from Crohn’s disease intestinal strictures.6 Conflicting results have been reported regarding the pro-fibrogenic action of some cytokines such as IL-13,7,8 whereas the profibrogenic role of IL-33 has been demonstrated in murine models but not yet in humans.9
Currently, there are no predictors able to estimate the risk of developing intestinal fibrosis in IBD patients.1 All the proposed noninvasive biomarkers of intestinal fibrosis, including gene polymorphisms or variants, microRNAs (miRs), ECM components, growth factors and anti-microbial antibodies (Figure 1) have limited
Antibodies ASCA Anti-CBir1
Y
Genes
PC
Anti-L
Y
Anti-OmpC Anti-I2
IL23R IL L23R 3R
Anti-C ACCA
B cell
AMCA ALCA
M acrophage Macrophage
Epithelial cells
MMP-3
Stricture
Paneth P Pa neth h cell cells lls NOD2 NOD OD2 OD 2
MDP
Bacteria
ATG16L1 16L1 6L1 TLR4
Lumen
CX3CR1 CX X3CR X 3CR R1
Pre-stenotic dilatation
Growth factors
ECM components components
PDGF PIIINP
Fibronectin
bFGF Platelet
Capillary Macrophage age
FAP
YKL-40
Fibrocyte
Myofibroblast Endothelial cells
ITCP
phil Neutrophil TGF-β
PICP
Fibrotic tissue
Laminin
TIMP-1
microRNAs miR-29b
miR-200b
miR-19b-3p miR-29a
miR-19a-3p miR-29c
Figure 1. Pathogenic mechanisms and candidate molecular biomarkers for intestinal fibrosis. Picture shows pre-stenotic dilatation, stricture with fibrotic tissue, lumen, capillary and candidate biomarkers for intestinal fibrosis: genes (red panel), growth factors (purple panel), extracellular matrix (ECM) components (light blue panel), microRNAs (miRs) (yellow panel) and antibodies (green panel). Nucleotide-binding oligomerization domain (NOD)2 variants, which imply loss of binding between NOD2 and the bacterial cell wall component muramyl dipeptide (MDP), polymorphisms of the chemokine fractalkine receptor CX3CR1, variants in the toll like receptor (TLR)4, in the autophagy-related-16L1 (ATG16L1) and in the interleukin 23 receptor (IL23R) induce chronic inflammation leading to stricture development. Excessive deposition of ECM components, which consist of fibronectin, laminin, collagen and its propeptide or telopeptide – such as N-terminal propeptide of type III collagen (PIIINP), C-terminal propeptide of type I collagen (PICP) and C-terminal telopeptide of type I collagen (ITCP) – and alterated tissue remodelling are also due to a specific single nucleotide polymorphism in matrix metalloproteinase (MMP)-3 gene, which loses its proteolytic activity on ECM, and to abnormal expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1. Myofibroblasts, whose proliferation is sustained by endothelial cell-released basic fibroblast growth factor (bFGF), produce increased amounts of collagen in a YKL-40-dependent fashion. YKL-40 is a growth factor secreted by activated macrophages and neutrophils. The production of collagen is also favoured by platelet-derived growth factor (PDGF), secreted by circulating platelets. Moreover, miR-29b, which is suppressed by myofibroblast-produced transforming growth factor (TGF)-b, inhibits the profibrogenic effects of myofibroblasts themselves. These cells express on their surface fibroblast activation protein (FAP) and may be derived from circulating fibrocytes. The abnormal immune response underlying intestinal fibrosis is characterized by the plasma cell (PC)mediated production of antibodies directed against bacterial components: anti-Escherichia coli outer membrane protein C antibodies (Anti-OmpC), anti-Pseudomonas-associated sequence I2 antibodies (Anti-I2), anti-bacterial flagellin CBir1 antibodies (Anti-CBir1) and anti-glycan antibodies, such as anti-Saccharomyces cerevisiae antibodies (ASCA), anti-chitobioside carbohydrate IgA antibodies (ACCA), anti-mannobioside carbohydrate IgG antibodies (AMCA), anti-laminaribioside IgG antibodies (ALCA), anti-laminarin carbohydrate antibodies (anti-L) and/or anti-chitin carbohydrate antibodies (Anti-C).
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Table 1. Serum biomarkers proposed for intestinal fibrosis. Candidate biomarker
Diagnostic value
miRs miR-200a miR-200b þ miR-29a þ miR-29b miR-29c miR-19a-3p þ miR-19b-3p þ ECM components TIMP-1 PIIINP PICP ITCP Laminin Fibronectin Growth factors bFGF YKL-40 PDGF Antibodies ASCA ACCA AMCA NA Anti-L NA Anti-CBir1 NA Anti-OmpC NA Anti-I2 NA
Prognostic value
Used in clinical trials
References
NA NA NA NA NA NA NA
No No No No No No No
18 18 19 19 19 20 20
NA NA NA NA
Yes No No No No No
21,55 22,23 23 23 26 27,28
NA NA NA
Yes Yes No
29–31,55 32,33,55 34
No No No No No No No
37,41–45,47,48 37,41 37,43 37,43 44–46 44,45,48 44
ACCA: anti-chitobioside carbohydrate antibody; AMCA: anti-mannobioside carbohydrate antibody; anti-CBir1: anti-bacterial flagellin CBir1 antibody; anti-I2: anti-Pseudomonas-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; ECM: extracellular matrix; ITCP: C-terminal telopeptide of type I collagen; miR: microRNA; NA: not assessed; PDGF: platelet-derived growth factor; PICP: C-terminal propeptide of type I collagen; PIIINP: N-terminal propeptide of type III collagen; TIMP: tissue inhibitor of matrix metalloproteinases; þ: consistent evidence; : conflicting evidence; –: no evidence.
diagnostic and prognostic value, and most of the studies so far performed have provided conflicting results (Table 1). Biomarkers of intestinal fibrosis would be useful in order to stratify patients according to their risk of stricture development and to identify early stages of fibrosis with the aim of optimizing the therapeutic management.1 Patients with known risk factors for severe disease course, that is, age below 40 years at diagnosis, early requirement of steroids and perianal disease,10 have an increased rate of fibrostenotic complications, thus they should be more strictly followed up.
Here we review the latest findings on candidate biomarkers of intestinal fibrosis in IBD.
Genes A specific genetic background has been supposed to predispose to fibrostenosing phenotype in Crohn’s disease.1 The first gene identified as predisposing to stricturing Crohn’s disease has been nucleotide-binding oligomerization domain (NOD)2 gene, also known as the caspase recruitment domain 15, involved in a-defensin production and intracellular clearance of bacteria. The presence of various predetermined combinations of NOD2 mutations, which imply loss of binding between NOD2 and the bacterial component muramyl dipeptide, has been shown to predispose to stricturing and/ or penetrating Crohn’s disease, whereas the predictive value of any single NOD2 mutation is low.11 NOD2 gene variants are associated with early ileal strictures and with postoperative recurrence in Crohn’s disease patients.12 Presumably, the high need for surgery in Crohn’s disease patients with NOD2 mutations is due to the ileal location and stricturing phenotype. Patients carrying toll like receptor (TLR) variants, especially TLR4, frequently have small bowel stricturing disease.13 T280M and V249I polymorphisms of the chemokine fractalkine receptor CX3CR1 influence the stricturing phenotype, regardless of NOD2 status.14,15 The 5T5T genotype at the MMP-3 SNP-1613 5 T/6 T increases the percentage of stenotic complications through an unbalanced tissue remodelling, but prevents colonic involvement in Crohn’s disease.16 Crohn’s disease fibrosis is also associated with variants in the autophagy-related-16L1 gene (rs2241879 and rs2241880), implicated in autophagy and bacterial phagocytosis, and in the IL23 receptor gene (TT genotype of rs1004819).17,18 CX3CR1 polymorphisms, TLR4, autophagy-related-16L1 and IL23 receptor variants induce chronic inflammation leading to stricture development. In summary, although gene mutations or polymorphisms are promising biomarkers, they are not used in clinical practice due to their incomplete penetrance and low frequency.
miRs Amongst epigenetic pathways, miRs, which are short noncoding RNA negatively regulating target gene expression at post-transcriptional level, are the most extensively studied in the pathogenesis of intestinal fibrosis. Serum levels of miR-200b, but not those of miR-200a, are increased in stricturing Crohn’s disease compared with the nonstricturing phenotype.19 MiR29a is reduced in the serum of fibrostenosing Crohn’s disease patients in comparison with inflammatory
526 Crohn’s disease.20 Similarly, serum miR-29b and miR29c are lower, although not significantly, in stricturing Crohn’s disease than in inflammatory phenotype.20 MiR-29 family is consistently down-regulated in the intestinal mucosa overlying strictures in Crohn’s disease patients, and miR-29b transfection inhibits TGF-b-induced increase in collagen in Crohn’s disease myofibroblasts.20 MiR-19a-3p and miR-19b-3p are reduced in the serum of stricturing Crohn’s disease patients compared with that of nonstricturing Crohn’s disease.21 Moreover, according to a multivariate analysis, the association between miR-19-3p and stricturing phenotype is independent of confounding clinical variables including ileal location and disease duration.21
ECM components Although TIMP-1 is increased in Crohn’s disease mucosa overlying strictures,5 no association has been observed between serum TIMP-1 and the fibrostenotic phenotype.22 As collagens I and III are considered the two major ECM proteins implicated in intestinal fibrosis,12 their propeptides have been detected in the serum of Crohn’s disease patients. N-terminal propeptide of type III collagen (PIIINP) is higher in stricturing Crohn’s disease before surgery than in controls with a significant reduction of its serum levels six months after successful surgical intervention.23 However, an old study, which did not specify the Crohn’s disease phenotype of the patients recruited, showed no significant difference of PIIINP between Crohn’s disease and controls, whereas serum levels of C-terminal propeptide of type I collagen and C-terminal telopeptide of type I collagen were reported to be significantly reduced and increased, respectively, in Crohn’s disease compared with controls.24 The enhanced liver fibrosis (ELF) test, which is a direct marker of hepatic fibrosis based on the three circulating markers of liver ECM metabolism hyaluronic acid, TIMP-1 and PIIINP,25 is able to discriminate stricturing Crohn’s disease from nonstricturing phenotypes (unpublished data). Additionally, ELF test was identified as a prognostic marker in a cohort of patients with primary sclerosing cholangitis, most of whom were affected by IBD.26 Laminin, which is a component of basement membrane, is higher in the serum of Crohn’s disease patients than in controls and it is associated with disease activity but not with the stricturing behaviour.27 Fibronectin, a high-molecular weight ECM glycoprotein, is increased in the plasma of patients with stricturing Crohn’s disease.28 In addition, its plasma levels significantly decrease in the immediate post-surgery period.29 However, the predictive value of plasma fibronectin for subsequent post-surgery stricture development was not significant in one-year follow-up.29
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Growth factors Serum levels of several growth factors, including basic fibroblast growth factor (bFGF), human chitinase 3-like 1 (also known as YKL-40), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) have been investigated in Crohn’s disease patients as possible biomarkers of intestinal fibrosis. Serum bFGF, which induces wound healing by modulating myofibroblast proliferation, is increased in patients with Crohn’s disease,30-32 and its levels correlate with the bowel wall thickness and the fibrostenotic phenotype.31 Serum levels of YKL-40, which is released by activated macrophages and neutrophils and promotes myofibroblast-induced collagen secretion, are higher in the serum of Crohn’s disease patients than in control subjects.33,34 However, the comparison of serum YKL-40 between stricturing and unstricturing phenotypes produced contradictory results in two studies.33,34 In keeping with the pro-angiogenic and pro-fibrogenic role played in liver fibrosis,2 VEGF is increased in the serum of Crohn’s disease patients31,32 and also correlates with colour Doppler signal intensity, leading to the consideration of VEGF as biomarker of angiogenesis in Crohn’s disease.32 Similarly, the pro-fibrogenic/proangiogenic PDGF, which is able to increase the production of collagen but not that of TIMP-1, is high in the serum of IBD patients.35
Antimicrobial antibodies Several anti-microbial antibodies, which are believed to derive from an abnormal immune response against the luminal microbiota,36 have been measured in the serum of Crohn’s disease patients, including anti-Escherichia coli outer membrane protein C antibodies (anti-OmpC), anti-Pseudomonas-associated sequence I2 antibodies (anti-I2), anti-bacterial flagellin CBir1 antibodies (anti-CBir1) and anti-glycan antibodies, such as anti-Saccharomyces cerevisiae antibodies (ASCAs), anti-chitobioside carbohydrate IgA antibodies (ACCAs), anti-mannobioside carbohydrate IgG antibodies (AMCAs), anti-laminaribioside IgG antibodies (ALCAs), anti-laminarin carbohydrate antibodies (anti-L) and anti-chitin carbohydrate antibodies (antiC).37-40 These biomarkers predict and are qualitatively and quantitatively associated with an aggressive and complicated course of Crohn’s disease, including stricturing and/or penetrating phenotype, both in adult and paediatric patients.13 However, a recent study showed that none of the anti-glycan antibodies ASCA, AMCA, ACCA, ALCA, anti-L and anti-C correlates with disease phenotype.41 Circulating ASCAs, which are directed against a cell wall component of the common baker’s yeast Saccharomyces cerevisiae, are those more widely
Giuffrida et al. investigated in IBD, as they are able to discriminate between Crohn’s disease and ulcerative colitis with a sensitivity of 56% and a specificity of 88%.42 Moreover, Crohn’s disease patients with serological positivity for ASCA develop a fibrostenosing or penetrating phenotype with a sensitivity of 70% and a specificity of 48%, and more frequently present an ileal or ileo-colonic location, thus leading to increased need for surgery.42,43 Circulating ASCAs in Crohn’s disease have been also associated with early disease onset and perianal disease.43 Among the other anti-glycan antibodies, ACCAs are those more frequently associated with a fibrostenosing or fistulizing phenotype with lower sensitivity (43%) but higher specificity (75%) than ASCA.42 Moreover, positivity for ASCA, AMCA and anti-L is associated with a higher risk for an earlier occurrence of complications and surgery,44 whereas serum positivity for ASCA, AMCA, ACCA and anti-L predict a more rapid progression toward complications or surgery.38 The risk of developing a stricturing and/or penetrating phenotype is 11-fold higher in Crohn’s disease children with serological positivity for anti-CBir1, anti-OmpC, anti-I2 and ASCA than in seronegative children.45 These results have been confirmed in a larger prospective study conducted in Crohn’s disease children seropositive for antiCBir1, anti-OmpC and ASCA.46 An Irish study shows that serum anti-CBir1 positivity is significantly associated with a complicated disease behaviour and ileal involvement, but not with increased need for surgery.47 In a population-based cohort of Crohn’s disease patients, among a panel of antibodies directed against antimicrobial peptides, including ASCA IgA and IgG, anti-I2, anti-OmpC, anti-CBir1 and pANCA, only ASCA IgG were significantly associated in a multivariate analysis to a stricturing/penetrating behaviour.48 A recent meta-analysis on 11 studies and based on four antibodies – ASCA, anti-OmpC, anti-I2 and antiCBir1 – identified ASCA as the antibody with the highest sensitivity and anti-OmpC as that with the highest specificity for complications and surgery.49 Moreover, disease progression towards complications was predicted more effectively using at least two antimicrobial antibodies rather than any single one.49 All the antimicrobial antibodies are useful for predicting a disabling course of Crohn’s disease (Table 1), but not for discriminating between stricturing and nonstricturing phenotypes. Further prospective studies are needed to ascertain whether circulating antibodies, alone or in conjunction with other biomarkers, can predict the disease course and the development of fibrosis in IBD.
Other factors Fibroblast activation protein (FAP) is a glycoprotein expressed on activated fibroblasts during wound
527 healing and its up-regulation has been reported in idiopathic pulmonary fibrosis and liver cirrhosis.50,51 In the submucosa and in the muscle layer of Crohn’s disease strictures FAP is up-regulated and its expression on intestinal myofibroblasts from stricturing Crohn’s disease is increased by profibrotic cytokines, such as tumour necrosis factor (TNF)-a and TGF-b1.52 Although serum FAP has been proposed as an index of liver fibrosis,53 there are no data on its utility as a biomarker of intestinal fibrosis in IBD patients. There is evidence that circulating fibrocytes, which are bone marrow-derived mesenchymal progenitor cells, may be considered as a marker of fibrosis in idiopathic pulmonary fibrosis.54 As far as intestinal fibrosis is concerned, there are no studies determining the circulating levels of fibrocytes in patients with fibrostenosing Crohn’s disease. The only data available showed an increased percentage of fibrocytes both in peripheral blood and inflamed gut.55 The demonstration that fibrocytes isolated from the peripheral blood express higher amounts of TNF-a and collagen type I after in vitro stimulation with lipopolysaccharide suggests that their interaction with microbial components may contribute to the fibrogenic process.55
Clinical trials for stricturing IBD Clinical trial design to identify surrogate biomarkers of fibrosis, which are crucial for enabling both a patienttailored anti-fibrotic treatment and an individual risk stratification for fibrostenosing disease, are hampered by a number of limitations, such as (i) the long-lasting evolution of intestinal strictures, which requires clinical trials of long duration and involving large patients’ cohorts, (ii) the common overlap along the disease course of other clinical behaviors (i.e. penetrating),56 (iii) the influence of concomitant immunomodulatory therapies which may mask blood candidate molecule changes, and (iv) the fact that disease duration does not necessarily correlate with the degree of intestinal damage as an early disease can already be a late (stricturing) disease. Moreover, on the basis of the high complexity of the molecular mechanisms underlying the fibrogenic process, it is conceivable that a panel of candidate molecules would be more appropriate than a single biomarker, as has been already shown in other organs (ELF panel).25 A possible study design to validate serum fibrosis biomarkers in a relatively short time period could be to strictly follow-up Crohn’s disease fibrostenosing patients after surgical resection, although the lack of a clear definition of fibrosis and consequently of a gold standard technique for fibrosis represents a limit in the progress in this field. The same limitations can be applied to clinical trials for identifying anti-fibrotic drugs in stricturing IBD. The ideal
528 pilot trial of an anti-fibrotic drug should include restricted populations in whom fibrosis is very common; however, we have no predictor of fibrostenosing complication. Perhaps data from experimental fibrosis could be helpful in stratifying patients according to the risk of fibrosis, in defining the optimal timing to start anti-fibrotic treatment and in understanding how to manage drugs modulating tissue remodelling without inhibiting the desired healing process. Up to now, serum biomarkers of intestinal fibrosis have been measured only in a phase 1 open label trial on Crohn’s disease patients with inflammatory phenotype receiving a seven-day-long treatment with Smad7 antisense oligonucleotide aimed at restoring TGF-b signaling.57 In order to rule out a putative pro-fibrogenic effect exerted by this drug, serum bFGF, YKL-40, TIMP-1, MMP-1, MMP-3 and MMP-9 were detected at baseline and after six months and, in parallel, patients underwent small intestine contrast ultrasonography.57 In the trial no significant change was observed for all these biomarkers in agreement with the small intestine contrast ultrasonography results.57
Conclusions Intestinal fibrosis is a biologically heterogeneous process, involving multiple complex and interactive mechanisms. Major advances in understanding the pathophysiology of intestinal fibrosis have occurred over the past years. Nevertheless, there is insufficient evidence supporting the diagnostic and prognostic value of any biomarker in stricturing IBD. There is an urgent need for effective noninvasive methodologies employing blood samples. The identification of biomarkers will not only impact on the overall management of IBD patients but will also contribute to answering some of the most basic questions of pathogenesis underlying intestinal fibrosis. Candidate biomarkers of intestinal fibrosis should be validated in order to be used in clinical trials testing anti-fibrotic drugs in stricturing IBD patients. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Conflict of interest The authors declare that there is no conflict of interest.
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