Intestinal fibrosis in Crohn's disease: role of microRNAs as fibrogenic modulators, serum biomarkers, and therapeutic targets.

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Intestinal Fibrosis in Crohn’s Disease: Role of microRNAs as Fibrogenic Modulators, Serum Biomarkers, and Therapeutic Targets Amy Lewis, MRes, PhD,* Anke Nijhuis, MSc,* Shameer Mehta, MBBS, BSc, MRCP,* Tomoko Kumagai, MBBS, MRCP, MSc,* Roger Feakins, MB BCh BAO, MD, FRCPI, FRCPath,† James O. Lindsay, BM BCh, FRCP, PhD,* and Andrew Silver, BSc, PhD*

Abstract: Inflammation often precedes fibrosis and stricture formation in patients with Crohn’s disease. Established medical therapies reduce inflam-

mation, but there are currently no specific therapies to prevent fibrosis or treat established fibrosis. Our understanding of the pathogenic processes underpinning fibrogenesis is limited compared with our knowledge of the events initiating and propagating inflammation. There are several biomarkers for intestinal inflammation, but there are none that reflect the development of fibrosis. MicroRNAs (miRNAs) are regulators of cellular activities including inflammation and fibrosis and may serve as biomarkers of disease processes. Differential serum and mucosal miRNA expression profiles have been identified between patients with inflammatory bowel disease with active and inactive inflammatory disease. In contrast, studies in patients with fibrotic phenotypes are comparatively few, although specific miRNAs have defined roles in the development of fibrosis in other organ systems. Here, we discuss the most recent research on miRNA and fibrogenesis with a particular emphasis on Crohn’s disease. We also anticipate the potential of miRNAs in fulfilling current unmet translational needs in this patient group by focusing on the role of miRNAs as modulators of fibrogenesis and on their potential value as serum biomarkers and therapeutic targets in the management of fibrosis. (Inflamm Bowel Dis 2015;21:1141–1150) Key Words: fibrosis, microRNA, Crohn’s disease, biomarkers, therapy targets

he fibrostenosing phenotype of Crohn’s disease (CD) has significant implications for patient health. For example, the estimated lifetime risk of surgery for patients with CD is as high as 70%,1 and fibrostenosing disease is the most common indication for surgery. The surgical burden of disease has diminished since the introduction of biological therapy, but the costs related to surgery still account for one-fifth of the total direct costs associated with management of CD.2 Furthermore, surgery is associated with a significantly higher risk of unemployment and poorer quality of life scores.3 However, our understanding of the key initiatory and regulatory steps in the development of intestinal fibrosis is very limited, particularly when compared with our knowledge of the processes underpinning inflammatory cascades in CD.

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Received for publication September 30, 2014; Accepted October 26, 2014. From the *Centre for Digestive Diseases and National Centre for Bowel Research and Surgical Innovation, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, United Kingdom; and †Department of Cellular Pathology, The Royal London Hospital, London, United Kingdom. The authors have no conflicts of interest to disclose. A. Lewis, A. Nijhuis, S. Mehta, and T. Kumagai contributed equally. Reprints: Andrew Silver, Centre for Digestive Diseases, Blizard Institute, Barts and The London School of Medicine and Dentistry, 4 Newark Street, Whitechapel, London E1 2AT, United Kingdom (e-mail: [email protected]). Copyright © 2015 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000298 Published online 29 January 2015.

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The principle cell types involved in intestinal fibrogenesis are those of mesenchymal origin: myofibroblasts, fibroblasts, and smooth muscle cells (Table 1). Myofibroblasts actively produce and secrete extracellular matrix (ECM) that results in stricture formation. However, the linear concept of fibrogenesis solely characterized by the activation of mesenchymal cells by various profibrotic mediators to produce ECM seems to be outdated. Rather, there exists a complex interplay between mesenchymal cells, inflammatory cascades, intestinal microbiota, profibrotic mediators, and the ECM itself that suggests fibrogenesis, once initiated, is a self-potentiating process (Fig. 1). There remain significant gaps in our knowledge of the complex interplay involved, including how the cellular and molecular processes responsible for fibrogenesis in the intestine are regulated. This review seeks to highlight a potential role for microRNAs (miRNAs) in the development of intestinal fibrosis in CD through modulation of biologically relevant pathways and to assess their value as serum biomarkers and therapeutic targets.

MICRORNAS A number of reviews dealing with the biology of miRNA are already available,11,12 and so only an outline will only be given here. miRNAs are endogenous small noncoding RNAs consisting of 20 to 23 nucleotides, which are derived from precursor stemloop hairpins. Step-wise processing of these hairpins results in the release of the mature miRNA, which is derived from one arm of the hairpin loop in the cytoplasm. Here, it is incorporated into the www.ibdjournal.org |

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TABLE 1. Types, Characteristics, and Principal Functions of Intestinal Mesenchymal Cells that Contribute to Fibrosis Mesenchymal Cell Type

Characteristics

Principle Function

Fibroblast

Positive for vimentin, FSP-1, and N-cadherin

Myofibroblast

Highly contractile Positive for vimentin and aSMA (6FSP-1)

Smooth muscle cell

Positive for aSMA, desmin and type 1 collagen

Maintenance of structural integrity Regulation of matrix homeostasis Tissue regeneration postinsult Wound contraction Synthesis of high levels of ECM (fibronectin, tenascin, collagen, glycosaminoglycans) Chemokine and cytokine production Collagen production Cytokine production (e.g., IL-6)

Reference 4 5 6 7

8 9 10

Principle function column conveys where significant functional overlap exists between cell types. aSMA, alpha smooth muscle actin; FSP-1, fibroblast-specific protein-1. *CD tissue for first two rows described as chronically active (inflamed) on terminal ileum and sigmoid colon.

miRNA-Induced Silencing Complex (miRISC), which facilitates translational repression of the target mRNA by inhibiting protein translation or less commonly mRNA degradation.13,14 Aberrant expression of miRNAs is associated with multiple disease processes in cancer, immunity, and inflammation.11,15–18

DYSREGULATION OF MIRNAS IS ASSOCIATED WITH CROHN’S DISEASE Several studies have investigated whether there is an association between miRNA expression as measured by quantitative real-time polymerase chain reaction and the different phenotypes of CD. Such studies have most often been done without functional analysis, largely because our understanding of the mRNA targets of specific miRNAs remains incomplete.

miRNA Expression in Health and CD Wu et al19 tested 467 miRNAs in tissue taken from the inflamed site of a small number of patients with chronically active CD (5 patients with colonic and 6 patients with ileal disease) and compared them with samples taken from healthy controls. Differences in miRNA expression were found between biopsy samples from colonic or ileal CD and those from site-matched healthy controls (Table 2). The authors also found differential expression profiles between various segments (terminal ileum to the rectum) of the “healthy” intestine. Although the expression of some miRNAs remained constant throughout, others differed significantly depending on the site from which the biopsy sample was taken. For example, there was significantly increased expression of miR-21, -31, and -215 in the terminal ileum compared with the rest of the sites sampled.19 Fasseu et al20 tested the expression of more than 300 miRNAs in biopsies taken from inflamed and noninflamed sites of 8 patients with CD and compared with biopsies from healthy controls. In this study, the expression of specific miRNAs in both inflamed and

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noninflamed CD tissues differed from that of healthy controls (Table 2). The investigators ensured that the inflamed and noninflamed tissues were taken at least 20 cm apart from each other. However, since the expression of miRNAs is likely to be site specific, these differences between the inflamed and noninflamed tissue could be due to the location of the tissues taken rather than based solely on the activity of the disease.

Potential Functional Relevance of miRNA Dysregulation Several studies have linked specific miRNA with pathologically relevant pathways that have been identified in recent genome-wide associated studies. For example, Brest et al21 have established a connection between variants of the immunity-related GTPase family M (IRGM), and miR-196, which is overexpressed in the intestinal epithelium of patients with active CD; miR-196 downregulates the protective variant of IRGM, but not the riskassociated allele. This miRNA binds to IRGM mRNA transcribed in the presence of the normal IRGM gene variant causing the downregulation of IRGM production and allowing the effective clearing of intracellular bacteria by autophagy. The binding of miR-196 is impaired when IRGM is transcribed in the presence of the disease-associated variant, resulting in maintenance of IRGM levels and subsequent impairment of intracellular bacterial clearing by autophagy.21 In contrast, previous studies showed that IRGM downregulation reduced the efficacy of intracellular bacterial killing of autophagy.22,23 Consequently, Brest et al21 suggested that a fine-tuning of IRGM levels was required for proper autophagy function and that both overexpression and underexpression of IRGM could be potentially harmful. Another study showed that patients with CD with the disease-associated mutation in IL23R (rs10889677 variant) produced significantly more IL23R mRNA and IL23R protein due to a loss of a binding site for miR-7e and miR-7f on the mRNA.24


Intestinal Fibrosis and microRNAs in CD

FIGURE 1. Schematic representation of the principle events responsible for the initiation and potentiation of fibrosis in the intestine. Epithelial injury and increased intestinal permeability result from gut ischemia, oxidative stress, and potentiation of injury by microbes and drugs. This leads to infiltration of immune cells into the intestinal wall, which results in mesenchymal cell activation and proliferation through the secretion of profibrotic cytokines and mediators. This cellular expansion is also under the control of activated TLRs and autocrine activation. Mesenchymal cells produce extracellular matrix, which in turn leads to further mesenchymal activation through matrikine- and integrin-mediated mechano-sensing of ECM stiffness. This mechanism, along with mesenchymal autocrine function, allows the self-potentiation of fibrosis independently of inflammation. Finally, there exists a balance between MMPs and TIMPs to regulate ECM composition. The source of activated mesenchymal cells, the control of the self-potentiation process, and the balance between specific MMPs and TIMPs remain unresolved issues. MMPs, matric metalloproteinases; TIMPs, tissue inhibitors of matrix metalloproteinases; TLRs, toll-like receptors; PAMPs, pathogen-associated molecular patterns.

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TABLE 2. miRNAs in CD Intestinal Tissues Compared with Healthy Controls Increased CD tissue versus healthy tissue* CD tissue versus healthy tissue

Markers of CD sigmoid colon* 23b, 106a, 191 Markers of CD terminal ileal tissue* 16, 21, 223, 594 Markers of CD tissue 21, 22, 26a, 29b, 29c, 30b, 31, 34c-5p, 106a, 126*, 127-3p, 133b, 146a, 146b-5p, 150, 155, 196a, 324-3p Markers of inflamed CD tissue 9, 126, 130a, Markers of noninflamed CD tissue 9*, 30a*, 181c, 375 30c, 223

Decreased Reference 196, 629 — —

19 20

—

CD tissue for first 2 rows described as chronically active [inflamed] from terminal ileum and sigmoid colon.19 For this study healthy controls were matched for site.19

MIRNA

EXPRESSION AND CD INTESTINAL FIBROSIS

The studies noted above confirm that miRNA expression is dysregulated as part of the inflammatory process in the intestinal mucosa of patients with CD. In contrast, our knowledge of the role of miRNAs in fibrostenosing CD is relatively limited. However, research on 2 miRNA families, miR-29 and miR-200, provides early indications that miRNA expression does influence fibrosis in CD (Fig. 2).

miR-29 Family Nijhuis et al25 recently demonstrated a reduction in the expression of the miR-29 family (miR-29a, -29b, and -29c) in the mucosa-overlying strictured gut relative to adjacent nonstrictured areas. The profibrotic signaling molecule transforming growth factor (TGF)-b was found to inhibit miR-29b expression, and suppression of miR-29b was necessary for TGF-b-induced expression of collagen in primary cultures of intestinal fibroblasts isolated from patients with CD (Fig. 2). These data indicate that low miR-29 levels in the mucosa-overlying strictured intestine may have a profibrotic effect in CD (Nijhuis et al25). The data for CD accord with previous studies demonstrating reduced levels of the miR-29 family in association with cardiac, hepatic, and renal fibrosis.26–28 MiR-29 regulates directly the production of levels of ECM molecules by targeting the 30 UTR of various genes including the collagen genes (COL1, COL3, and COL4; miR-29a,26 miR-29b,29 miR-29c).30 Importantly for its role in intestinal fibrosis, miR-29 expression is induced by NOD-2 signaling; NOD-2 is a pattern recognition receptor expressed in the gut that contributes to immunity against bacteria and is implicated in the inflammatory response. Polymorphisms in NOD-2 are associated with increased risk of stricture formation in patients with CD.31 Moreover in dendritic cells, expressing variants of NOD-2 associated with CD miR-29 induction were compromised. Genetic variants in NOD-2 may therefore increase the risk of stricture development by inhibiting miR-29 expression.32 A link between altered miR-29 signaling and inflammation, a key driver of fibrosis in CD, has also been established. For example, miR-29 is inhibited by lipopolysaccharides stimulation

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and tumor necrosis factor-alpha treatment in an NFkb-dependent manner.28 Furthermore, in the dextran sodium sulfate-induced model of experimental colitis targeted deletion of the miR-29a/ b-1, locus in mice was associated with increased IL-23 production and an enhanced intestinal inflammation in the dextran sodium sulfate-induced model of experimental colitis in mice.32 Epigenetic DNA modification may also influence miR-29 expression. One such modification is DNA acetylation, levels of which are modulated by histone deacetylase enzymes (HDACs). HDACs have an established role in the propagation of inflammation in numerous disease contexts and HDAC inhibitors (HDACi) suppress colitis in mice.33,34 The expression of miR-29 is also modulated by histone-deacetylases (HDACs) and in models of Duchenne’s muscular dystrophy where fibrosis is a significant feature of the disease phenotype; low levels of miR-29b are associated with high HDAC-2 expression.35,36 Conversely, miR-29 expression is increased by application of class II HDAC inhibitors (HDACi) in hepatic stellate cell activation.37

miR-200 Family Members of the miR-200 family (miR-141, miR-200a, miR-200b, miR-200c, and miR-429) play a key role in epithelial to mesenchymal transition (EMT),38 a process in which epithelial cells lose polarity and cellular adhesion and gain mesenchymal morphology together with an increased migratory phenotype. EMT is a major contributor to renal,39–41 cardiac,42 pulmonary,43 and hepatic44 fibrosis and has an established role in tumor metastasis.45 Animal studies have supported a potential role for EMT in the development of intestinal fibrosis. For example, Flier et al46 used a TNBS-induced mouse model of colonic fibrosis to make an assessment of EMT in the gut. By generating double transgenic mice in which intestinal epithelial cells were permanently labeled with b-galactosidase, the presence of extraepithelial b-galactosidase-positive cells could be demonstrated using immunohistochemistry. Costaining showed the presence of double-labeled cells positive for fibroblast-specific protein-1 together with E-cadherin and b-galactosidase.46 Subsequent studies have found members of the miR-200 family as potentially protective factors against the development of intestinal



FIGURE 2. The roles of miRNAs in the development of EMT and intestinal fibrosis. Epithelial cells lose epithelial characteristics and gain those of mesenchymal cells as they migrate into the submucosal layers. The majority of studies examining the effects of miRNAs on EMT have been performed in the cancer setting (Table 3). Specific miRNAs inhibit cellular targets such as Par3 and RhoA leading to loss of tight junction and adherens junction proteins. Extracellular signals and environmental triggers promote this process, as do certain pro-EMT transcription factors. This latter mechanism of control is regulated by specific miRNAs, although only the role of the miR-200 family has been defined in the intestine. In relation to intestinal fibrosis, the suppression of miR-29b by various extracellular signals including TGF-b leads to increased deposition of ECM molecules by activated intestinal fibroblasts in an EMT-independent fashion. TJ, tight junction; AJ, adherens junction; *Demonstrated in the intestine.

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TABLE 3. miRNAs and the Regulation of Epithelial to Mesenchymal Transition miRNA

Target

Model

miR-9

E-cadherin

Human mammary epithelial cells

miR-491-5p

Par3

Human and rat renal epithelial cells

miR-155

RhoA

Human mammary epithelial cells

miR-1

SLUG

Human and mouse prostate adenocarcinoma cells

miR-17/92 family

SNAIL, TWIST, ZEB

Canine renal epithelial cells

miR-31

SNAIL, TWIST2, ZEB1


miR-205

SNAIL, TWIST


miR-365

HMGA2

Lung adenocarcinoma cells

Action

Reference

Directly inhibits E-cadherin expression, promoting EMT Reduces Par3 expression leading to epithelial tight junction dissociation Reduces RhoA expression leading to epithelial tight junction dissociation and TGF-b-induced EMT Reduces SLUG expression and suppresses EMT. SLUG causes further miR-1 downregulation resulting in a mutually inhibitory feedback loop Reduces expression of pro-EMT transcription factors, suppressing EMT Reduces expression of pro-EMT transcription factors, suppressing EMT Reduces expression of pro-EMT transcription factors, suppressing EMT Reduces HMGA2 and suppresses EMT through reduction in SNAIL and TWIST activation

50 51 52

53

54 54 54 55

miRNAs have been studied principally in cancer-related EMT models rather than fibrosis-specific settings. The miRNA-200 family is the principal group studied in intestinal fibrosisspecific EMT.

EMT. For instance, TGF-b-induced EMT in the DLD1 colorectal adenocarcinoma cell line was abrogated by the previous transfection of cells with an miR-200b mimic.47 Levels of miR-200b were reduced in the colonic mucosa in areas of active CD compared with areas of inactive CD within an individual patient; this was associated with the loss of E-cadherin in active disease areas. Furthermore, recent array data have highlighted the differential expression of miR-200 family members relevant to EMT in intestinal CD fibrosis.48 Interestingly, the observation that TGF-b-induced EMT was negated by miR-200b transfection was reproduced in noncancerous intestinal epithelial cells.49 The reduction of E-cadherin loss seemed to be effected by repression of ZEB1, which was followed by an increase in expression of the mesenchymal marker vimentin.49 Currently, it is unclear how miRNAs regulate the EMT process, and this requires further investigation. Specific miRNAs have roles in EMT (Fig. 2), although the majority of these studies have been performed in cancer models (Table 3). Of the few miRNAs studied in fibrosis-related EMT, most published data concerns the miR-200 family. Evidence from unilateral ureteral obstruction models of renal fibrosis show that miR-141 and miR200a levels were downregulated in a Smad-3 dependent matter in the early stages of fibrosis development.56 Other evidence suggests that both miR-200a and -141 influence fibrogenesis on multiple levels as they target the 30 UTR or TGF-b directly.57 Specific miRNAs also impact on the control of transcription factors involved in the initiation and propagation of EMT (Fig. 2). For

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example, the miR-200 family directly targets and represses the ZEB transcription factors, which act to suppress E-cadherin; miR200b has also been shown to inhibit Slug in prostate adenocarcinoma cell lines, a transcription factor also implicated in EMT.53 This miRNA facilitated control mechanism seems to be reproducible in human breast cancer cell lines,58 hepatocellular carcinoma models,43 and murine renal fibrosis models.56 In addition, epigenetic modification of miRNAs may be an important factor in their role in the development of EMT. A characteristic miRNA signature associated with the induction of EMT in canine kidney cells lines included the downregulation of the miR-200 family as well as miR-205, miR-31, and miR-17 family. DNA hypermethylation was observed for the 2 miR-200 family loci, as well as for the miR-205 locus, suggesting that there is a functionally relevant epigenetic silencing mechanism at work. This finding was reproduced in DNA methylation studies of the miRNAs in the in vivo model of endometrial carcinosarcoma.59

SEROLOGICAL BIOMARKERS OF INTESTINAL FIBROSIS miRNAs secreted from cells inside exosomes are present in the circulation and resistant to degradation.60 The function of circulating miRNAs is still unclear, although there is growing evidence to suggest a role for these miRNAs in cell–cell signaling.61 Clinically, there is the potential to exploit circulating miRNAs as accurate biomarkers of disease. miRNA-based


biomarkers have several advantages: they are readily detectable, stable in serum (permitting minimal invasive sampling), and the profiling is comparatively cheap, quick, and quantitative.62 To date, studies of circulating miRNAs in CD have demonstrated differential expression between patients with CD, patients with ulcerative colitis, and healthy controls as well as between patients with active disease and those in remission.63–65 Recently, serum miRNA levels were assessed in a cohort of 19 patients with CD during induction therapy with infliximab. Significantly increased levels of 5 miRNAs (let-7d, let-7e, miR-28-5p, miR-221, and miR-224) were observed at week 6 compared with week 0. In addition, levels of let-7d and let-7e were significantly increased in patients who achieved clinical remission by infliximab suggesting these miRNAs might be useful as biomarkers of therapeutic response.66 This study will require validation in a larger independent cohort of patients with CD undergoing treatment with infliximab. Assessment of both serum and biopsy tissue samples in a more extensive trial might be useful. Studies investigating the changes in circulating miRNAs as a result of fibrostenosing CD have been lacking with only 2 small targeted studies performed to date. The first study by Chen et al47 reported an increase in miR-200b in patients with CD with fibrosis relative to those without. The second study demonstrated that levels of miR-29a were reduced in the circulation of patients with CD with stricturing disease relative to patients with an inflammatory phenotype.25 The data for miR-29a in patients with CD with intestinal fibrosis accord well with previous studies demonstrating that low serum levels of miR-29a are associated with advanced liver fibrosis and correlated with end-stage liver disease score.28 Reduced exosomal expression of miR-29a and -29c in urine is also related to renal fibrosis in patients with chronic kidney disease.67 The studies by Chen et al and by Nijhuis et al have both highlighted the potential to exploit serum miRNAs as markers of stricturing disease.25,47 For future studies, it is important to determine the specificity of any putative miRNA biomarker for ongoing fibrogenesis as opposed to an aggressive CD phenotype per se and also to determine changes in expression of candidate biomarker miRNAs over time. A biomarker capable of distinguishing between inflammatory and fibrotic strictures would also represent a significant clinical advancement, as this would help to determine whether medical therapy or surgery is more appropriate. A sensitive biomarker of intestinal fibrosis could also potentially be used to monitor response to treatment and inform future drug trials.68


now warranted to characterize fibrosis in relation to changes in miRNA expression. To aid the identification of relevant miRNA, it is important for groups in the field to standardize the miRNA processing pipeline to enable direct comparisons between studies. To date, a range of RNA isolation methods, miRNA profiling platforms and normalization strategies have been used. In serum, the absence of a widely accepted normalizer for miRNA expression quantification in the circulation severely limits intercomparison of published data.69 Current strategies include normalization to an exogenous nonmammalian “spiked-in” control such as Cel_miR_39 to account for technical variation in sample processing; others have normalized serum miRNA levels to endogenous miRNAs or other small noncoding RNAs.70 The latter method has the advantage of accounting for biological and technical variation. However, the scientific community is yet to reach a consensus on which miRNAs to use as a normalizer: ideally, this would be ubiquitously and stably expressed, readily detectable, and not vary with disease. Further work is required to determine the functional relevance of aberrantly expressed miRNA to intestinal fibrosis. This includes establishing miRNA downstream targets and their response to perturbations in miRNA expression in the gut. It is also essential to determine whether changes in expression are causative or reflect underlying pathologies. Here, conditional mouse models that can induce or downregulate specific miRNAs, or families of miRNAs, would provide much needed experimental opportunities, including assigning causation and testing of novel targeted therapeutic interventions. For future studies, it is important to localize changes in miRNA expression within the complex architecture of the gut, to determine which cell types are likely to be affected and inform target prediction. This is increasingly feasible given the rapid development of miRNA in situ hybridization techniques. Yanget al71 have demonstrated this technique as useful for investigations of ulcerative colitis gut tissue. There also remains an unmet need for characterizing fibrosis at a tissue level with formal histological scoring (no validated scoring system exists for intestinal fibrosis) and assessment of tissue expression of markers of fibrosis, to correlate miRNA profiles and functional studies with different degrees of fibrosis. This is important as intestinal fibrosis that occurs as a continuum effect, rather than in a binary “on/off” fashion. Therefore, correlating miRNA profile differences and functional studies with degrees of fibrosis would be informative.

Therapeutic Implications FUTURE PERSPECTIVES Challenges The targeted studies performed to date, and outlined here, have identified a number of specific miRNAs that are differentially expressed in fibrotic and nonfibrotic tissues in CD, raising the possibility of wider changes in miRNA expression with relevance to intestinal fibrosis. Large microNome-wide studies are

In vivo manipulation of miRNAs is increasingly feasible, making miRNA-based therapeutics an attractive new area for development.72 Effective miRNA knockdown can be achieved through administration of antisense oligo nucleotides also known as antagomirs. Conversely miRNA overexpression can be achieved through delivery of miRNA mimics. miRNA therapeutics are now in preclinical and clinical trials for a range of diseases: Miravisen (a miR-122 inhibitor) in phase-2 trials for the treatment of hepatitis C73 and MRX34 (a miR-34 mimic), in phase-1 trials, as a tumor www.ibdjournal.org |

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suppressor.74 Regarding fibrosis, RG-012, an antagomir that inhibits miR-21, has been identified as a potential antifibrotic candidate.75 Preclinical trials have shown that RG-012 is well tolerated, significantly reduces renal fibrosis, and enhances life span in mice. Phase-1 clinical trials are now expected to commence in 2015 for the treatment of Alport Syndrome, a hereditary nephritis disorder characterized by end-stage kidney disease, using RG-012. Finally, the therapeutic potential of miRNAs for hepatic fibrosis was demonstrated by the successful delivery of artificial miRNAs targeting connective tissue growth factor to rats with hepatic fibrosis using the ultrasound-guided “cationic liposome-bearing microbubble destruction gene delivery system.”76 The artificial miRNAs produced histological improvement of fibrosis and a reduction in the mRNA and protein expression of connective tissue growth factor and TGF-b1. Furthermore, the levels of type I collagen and a-smooth muscle actin were also reduced.76 The results of the first miRNA-based therapeutics trials are awaited. However, a number of concerns about the potential for off-target effects have already been raised, as each miRNA modulates the expression of multiple gene targets.77,78 Ensuring efficient and targeted delivery of miRNAs to the site of disease has also proved problematic, and this is likely to be a particular issue for the gut. For miRNAs to be effective as a therapeutic agent for intestinal fibrosis, they must reach the target cells at the site of intestinal fibrosis. This makes systemic administration of miRNAs challenging unless a delivery system can be devised and optimized to ensure the specific target site is reached at sufficient concentration. This might be achieved by coupling the delivery system with a ligand specific for the target cells.79 Another delivery approach could to use bacterial vectors80 or development of delivery systems such as chitosan modified polynanospheres and thioketal nanoparticles,81 which would potentially allow oral administration of miRNA targeting drug. However, this route would have to take into account the effect of gastric acid and digestive proteases, as well as the motility of the intestine. These factors may vary significantly between individuals making the individual therapeutic dose of miRNA required highly unpredictable. miRNAs would then have to breach the mucus membrane and epithelium to deliver therapeutic benefit. Patients with CD may have anatomical abnormalities such as intraluminal edema, adhesions and fistulae and associated symptoms such as nausea and vomiting which impact on successful delivery of miRNA to the site of fibrosis.81 Finally, miRNA intended to treat fibrosis must not aggravate the luminal inflammation or compromise would healing.

CONCLUSIONS The absence of specific therapies to treat intestinal fibrosis in CD needs to be addressed. Specific miRNAs have defined roles in fibrosis development in organ systems, including the intestine. The problems associated with identifying appropriate targets (the miRNA or family of miRNAs or downstream targets) for any novel therapy and the difficulties in achieving successful delivery are significant. The search for serum

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biomarkers that reflect the development of fibrosis and can be used for clinical management needs to continue. Such studies often present opportunities to identify potential therapy targets and will increase our understanding of the disease mechanism. The latter is of particular importance given our limited understanding of the pathogenic processes underpinning fibrogenesis compared with our knowledge of the events initiating and propagating inflammation in the bowel. Improved understanding of intestinal fibrosis should lead to the design of mouse models significantly more reflective of underlying disease initiation and progression, which could in turn inform more helpfully the evaluation of preclinical testing of novel therapies. The ability to detect miRNA in the circulation and to manipulate miRNA levels in vivo makes these molecules attractive candidates for biomarkers and novel therapies respectively.

SUMMARY OF KEY POINTS • Approximately 50% of patients with CD progress from an inflammatory phenotype at diagnosis to fibrostenosing disease requiring surgery as a result of intestinal stricture formation. This is associated with major morbidity and higher health care costs. • There are currently no noninvasive ways to monitor progression to fibrosis or to treat it once established. • Better understanding of the etiology of fibrosis and identification of the biological molecules involved in its regulation may help to identify novel targets for therapy. • miRNAs are important modulators of cell signaling in health and disease, and aberrant miRNA expression has been associated with the pathogenesis of CD. However, the role of miRNAs in the development of intestinal fibrosis is poorly understood and requires investigation.

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