Scandinavian Journal of Gastroenterology. 2015; 50: 53–65

REVIEW

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Mechanisms of initiation and progression of intestinal fibrosis in IBD GIOVANNI LATELLA1, JACOPO DI GREGORIO2, VINCENZO FLATI2, FLORIAN RIEDER3 & IAN C. LAWRANCE4 1

Department of Life, Health and Environmental Sciences, Gastroenterology Unit, University of L’Aquila, L’Aquila, Italy, Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, L’Aquila, Italy, 3Department of Pathobiology, Department of Gastroenterology, Hepatology and Nutrition, Lerner Research Institute, Digestive Disease Institute, Cleveland Clinic Foundation, Cleveland, OH, USA, and 4University Department of Medicine and Pharmacology, Centre for Inflammatory Bowel Diseases, Fremantle Hospital, University of Western Australia, Fremantle, WA, Australia

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Abstract Intestinal fibrosis is a common complication of the inflammatory bowel diseases (IBDs). It becomes clinically apparent in >30% of patients with Crohn’s disease (CD) and in about 5% with ulcerative colitis (UC). Fibrosis is a consequence of local chronic inflammation and is characterized by excessive extracellular matrix (ECM) protein deposition. ECM is produced by activated myofibroblasts, which are modulated by both, profibrotic and antifibrotic factors. Fibrosis depends on the balance between the production and degradation of ECM proteins. This equilibrium can be impacted by a complex and dynamic interaction between profibrotic and antifibrotic mediators. Despite the major therapeutic advances in the treatment of active inflammation in IBD over the past two decades, the incidence of intestinal strictures in CD has not significantly changed as the current anti-inflammatory therapies neither prevent nor reverse the established fibrosis and strictures. This implies that control of intestinal inflammation does not necessarily affect the associated fibrotic process. The conventional view that intestinal fibrosis is an inevitable and irreversible process in patients with IBD is also gradually changing in light of an improved understanding of the cellular and molecular mechanisms that underline the pathogenesis of fibrosis. Comprehension of the mechanisms of intestinal fibrosis is thus vital and may pave the way for the developments of antifibrotic agents and new therapeutic approaches in IBD.

Key Words: Crohn’s disease, extracellular matrix, inflammatory bowel disease, intestinal fibrosis, matrix metalloproteinases, tissue inhibitors of metalloproteinases, ulcerative colitis

Introduction The inflammatory bowel diseases (IBDs) are chronic relapsing diseases, with acute flares followed by partial or complete healing [1,2]. Approximately one-fifth of patients, however, suffer continuous active disease. Intestinal fibrosis is a common complication of IBDs and can occur in >30% of patients with Crohn’s disease (CD) and in about 5% suffering from ulcerative colitis (UC) [3,4]. Fibrosis is a consequence of local chronic inflammation and is characterized by

excessive extracellular matrix (ECM) protein deposition produced by activated myofibroblasts. Progression of intestinal lesions is highly variable and may range from weeks to decades, and deep ulcers, or transmural fissures, are more likely to result in fibrotic strictures [1,2]. It is difficult, however, to predict which patients will develop a fibrostenosing phenotype and how rapidly it will develop. It is also still unclear which factors trigger disease chronicity, and it is not yet known what factors promote the development of intestinal fibrosis [3,4].

Correspondence: Giovanni Latella, MD, Department of Life, Health and Environmental Sciences, Gastroenterology Unit, University of L’Aquila, Piazza S. Tommasi, 1- Coppito, 67100 L’Aquila, Italy. Tel: +39 0862 434735. Fax: +39 0862 433425. E-mail: [email protected]

(Received 3 September 2014; revised 14 September 2014; accepted 19 September 2014) ISSN 0036-5521 print/ISSN 1502-7708 online  2015 Informa Healthcare DOI: 10.3109/00365521.2014.968863

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Intestinal fibrosis follows the distribution and location of inflammation [5–8]. In UC, ECM deposition is restricted to the mucosal and submucosal layers of the large bowel and can induce structural changes (haustral loss and colonic shortening) and motility disorders of the colon [9]. In CD, fibrosis can involve the full thickness of the bowel wall that includes the mucosa, submucosa, muscularis, muscularis propria and serosa layers. In addition, it can affect any part of the gastrointestinal tract and the fibrosis can result in critical narrowing of the lumen resulting in strictures, or stenosis, that commonly lead to intestinal obstruction that requires surgery. The higher prevalence of clinically apparent fibrosis in CD is probably a consequence of the transmural bowel inflammation, which exposes all the ECM-producing cells to the profibrogenic mediators released by activated immune and nonimmune cells [5–8]. In addition, the high prevalence of CD in the terminal ileum, where the ileocecal valve is a natural narrowing of the intestine, could contribute to a higher occurrence of intestinal obstruction in CD. Of note is that the course and extent of intestinal fibrosis in IBD display significant variability among individual patients, suggesting that susceptibility to intestinal fibrosis may have a genetic component [10]. Wound healing is a “physiological process” triggered by inflammation that may lead to tissue repair with reconstitution of the normal intestinal morphology and function or fibrosis depending on the balance between production and degradation of ECM proteins [11,12]. Fibrosis occurs when regeneration and repair fail to restore normal tissue architecture and leads to permanent scarring, organ malfunction, and, potentially, death [13]. Although fibrosis is increasingly recognized as a major cause of morbidity and mortality, few, if any, treatment strategies are currently available. Evidence suggests that inflammation is necessary for establishing fibrosis but subsequently plays a minor role in its progression. Mechanisms that regulate fibrosis, therefore, appear to be distinct from those regulating inflammation [11,12]. Current antiinflammatory therapies used in IBD control disease activity, but unfortunately do not prevent nor reverse established fibrosis or strictures, which may present years after remission of active inflammation [2]. Despite the therapeutic advances in the treatment of IBD in the past two decades, the incidence of intestinal strictures in CD has not significantly changed [14,15]. This implies that control of intestinal inflammation may only be part of the fibrogenic process. In contrast to the intensive investigation of the immunological mechanisms of intestinal inflammation in IBD, the pathophysiology of fibrosis has

remained largely unexplored. The lack of efficient and well-tolerated antifibrotic drugs is partly due to the fact that the main and specific cellular and molecular pathways leading to fibrosis remain to be identified [16]. Another major obstacle in the development of antifibrotic drugs is the slow evolution of intestinal fibrosis in IBD. A clinical benefit may only be observed after a prolonged period of treatment and thus clinical trials could be long and expensive. There is thus an urgent need for noninvasive methods of measuring the presence of intestinal fibrosis, such as serum markers (e.g. growth factors, ECM turnover products) or imaging techniques (magnetization transfer magnetic resonance imaging [MRI], magnetic resonance elastography, ultrasound elastography, positron emission tomography [PET]-MRI, PET-computed tomography). These could also be used to detect early stages of fibrosis, to quickly quantify changes in fibrosis progression, and, in particular, to assess the response to specific medical treatments [17]. An additional difficulty is the lack of commonly accepted histopathological or clinical definitions of intestinal fibrosis. In this review, we describe the relationship between chronic intestinal inflammation and related fibrosis, the most important mechanisms that contribute to the initiation and progression of intestinal fibrosis in IBD and their implications for the development of new therapeutic approaches. Intestinal inflammation and fibrosis Intestinal injury is almost invariably followed by an acute inflammatory response. In most instances this is followed by physiological healing of the damaged tissue and restoration of normal intestinal structure and function [13]. If this does not occur, chronic inflammation, characterized by continuous events of injury and repair, may lead to the development of fibrosis (Figure 1). Injury to the intestine is not an uncommon phenomenon, even in otherwise healthy individuals. In most instances, wound healing fully resolves with normal restitution and resolution of the tissue damage. In IBD, this does not occur but it is still unclear which factors trigger the road to chronicity [13]. In addition, once intestinal inflammation is chronic, it is not yet understood what sets the stage for the later development of intestinal strictures. It appears to be generally accepted that chronic intestinal inflammation will invariably lead to clinically apparent fibrosis. This process, however, does not occur in all patients and thus may not be inevitable. Chronic intestinal inflammatory diseases exist, such as celiac disease or lymphocytic colitis, that are not complicated by fibrosis and stricture formation.

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Intestinal fibrosis in IBD These findings indicate that distinct mechanisms of inflammation and restitution/fibrosis must exist. It is, thus, crucial to explore this area since various pathways could be targeted separately, which would allow for tailored targeted treatment of wound healing abnormalities, especially in IBD. It is now clear that innate and adaptive immune mechanisms acting in chronic inflammation may divert the healing process toward fibrogenesis. Many elements of the innate and adaptive immune response participate in the differentiation, recruitment, proliferation, and activation of several progenitors of ECM-producing myofibroblasts and then contribute to the onset and progression of fibrosis [11] (Figure 1). Because ECM-producing myofibroblasts are central in the pathogenesis of fibrosis, recent research studies have focused on elucidating the immunological and molecular mechanisms that initiate and maintain these cells in an activated state. Epithelial and endothelial damage leads to the coagulation response, which is the first woundhealing mechanism activated after tissue injury, but at the same time also promotes release of chemotactic factors that recruit innate and adaptive inflammatory cells [11,12]. The former includes monocytes, neutrophils, macrophages, eosinophils, basophils, and mast cells that are important sources of proinflammatory and profibrotic molecules, like interleukin-1b (IL-1b), IL-4, IL-6, IL-13, tumor necrosis factor-a (TNF-a), transforming growth factor-b1 (TGFb1), and platelet-derived growth factor (PDGF) (primarily released by activated platelets during the coagulation response). The latter includes the T-helper cell subsets (Th1, Th2, Th17), regulatory T cells (Tregs), and B cells. The Th17-type immune response is proinflammatory and profibrotic.

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Th2-type immunity, defined by the production of IL-4, -5, and -13, is noted as a potent driver of fibrosis with IL-13 considered the dominant profibrogenic mediator. In contrast, Th1-type immunity expressing interferon-g (INF-g) may have antifibrotic activity. The role of Tregs in fibrogenesis is less clear, although it is plausible that they could suppress Th17- and Th2-driven fibrosis [18]. Further role of the innate and adaptive responses in fibrosis has been extensively discussed in a recent review [11]. The persistence of tissue injury and inflammation leads to persistent myofibroblast activation that then contributes to the progression of fibrosis. Consequently, removal of the inflammatory trigger is the most straightforward way to halt the initiation and progression of tissue remodeling and to allow restoration of the tissue architecture and functions after injury [11]. This can be easy to accomplish when the causes of the tissue damage are known, but in many fibrotic diseases, as in IBD, the damaging causes are either unknown or cannot be easily eliminated. In these cases, the main goal is to identify the key mediators of fibrogenesis that could then be specifically targeted to prevent and/or attenuate the fibrosis. Mechanisms of intestinal fibrogenesis Intestinal fibrosis is characterized by abnormal production and deposition of ECM proteins by activated myofibroblasts, which are modulated by both profibrotic and antifibrotic factors [19]. ECM-producing cells Whereas in other organs the source of ECMproducing myofibroblasts is restricted to a few cell

Epithelial/endothelial injury Adaptive immune activation

Innate immune activation

Th1 Th2 Th17

PMN Macr Eos

Treg

Myofibroblast progenitors

Bas Mast

B Inflammation & cell recruitment

Myofibroblast activation

Intestinal healing

Persistent injury & Inflammation

Persistent myofibroblast activation

Intestinal fibrosis

Figure 1. Schematic representation of the relationship between intestinal injury, inflammation, and fibrogenesis. Abbreviations: Th = T-helper cells; Treg = Regulatory T cells; PMN = Polymorphonuclear leukocytes; Macr = Macrophage; Eos = Eosinophil; Bas = Basophil; Mast = Mast cell. Adapted from Ref. [11].

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types, in the intestine the situation is more complicated as multiple cell types may become activated ECM-producing myofibroblasts [3,4,12]. These cells derive not only from resident mesenchymal cells (fibroblasts, subepithelial myofibroblasts, and smooth muscle cells) but also from epithelial and endothelial cells (via epithelial-mesenchymal transition [EMT]/ endothelial-mesenchymal transition [EndoMT]), stellate cells, pericytes, and bone marrow stem cells [3,4,12] (Figure 2). Myofibroblasts are activated by a variety of mechanisms including paracrine signals derived from immune and nonimmune cells, autocrine factors secreted by myofibroblasts, pathogen-associated molecular patterns derived from microorganisms that interact with pattern recognition receptors such as Toll-like receptors, and the socalled damage-associated molecular patterns derived from injured cells, including DNA, RNA, ATP, highmobility group box proteins, microvesicles, and fragments of ECM molecules [3,4,12,19]. Fibrosis also depends on the balance between the production and degradation of ECM proteins. ECM degradation is mediated by matrix metalloproteinases (MMPs), and the fine balance between MMPs and tissue inhibitor of metalloproteinases (TIMPs) appears to be altered in IBD and related intestinal fibrosis [20]. Apoptosis An exquisite equilibrium between cell proliferation and programmed cell death (apoptosis) in the intestinal wall is required to maintain physiological homeostasis. During the development of fibrosis, there are greater numbers of ECM-producing cells, secondary to increased proliferation and decreased apoptosis [3,4,12]. As apoptosis reduces

myofibroblast numbers during fibrosis resolution, the induction of myofibroblast apoptosis has an antifibrotic effect [21]. The main regulators of apoptosis include the caspases, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), p53 and focal adhesion kinase (FAK). Caspases, a family of cysteine-dependent aspartatedirected proteases, are integral to apoptosis. Caspases are grouped as either initiators or effectors of apoptosis, depending on where they enter the cell death process. Bcl-2 is the prototype anti-apoptotic protein and blocks the recruitment and activation of proapoptotic proteins, such as Bax, to the mitochondria. FAK inhibits the activity of p53 with the transcriptional targets p21, Bax and mouse double-minute 2 homolog through protein–protein interactions. NOD2 and ATG16L1 are also expressed by myofibroblasts and enhance apoptosis through the induction of caspase activation [21]. In CD, variants of these genes are associated with an increased risk of small bowel fibrostenosis [22]. Several studies have emphasized the importance of the TIMPs in fibrosis through their inhibition of matrix degradation. Individual TIMPs, however, may regulate cell division and apoptosis independent of this activity [21]. Of note is that TIMP-1 is overexpressed in CD fibrostenosis and suppresses myofibroblast apoptosis, again highlighting a role of myofibroblast survival in fibrogenesis. Hepatocyte growth factor (HGF), by contrast, reduces fibrosis by increasing apoptosis. HGF is a potent inducer of ECM-degrading enzymes, which are overexpressed during myofibroblast apoptosis [21]. MMPs induce apoptosis in myofibroblasts through the degradation of fibronectin. The antifibrotic effect of HGF is due to upregulation of MMPs

Gut microbiome

Exposome

Epithelial cells

EMT

Inflammatory mediators

Pericytes Bone marrow stem cells

Fibrocytes

Fibroblats Myofibroblasts

Activated myofibroblasts Smooth muscle cells

Endothelial cells

EndoMT

Stellate cells ECM

Figure 2. Schematic representation of the cellular origins of ECM-producing activated myofibroblasts. Abbreviations: EMT = Epithelial–mesenchymal transition; EndoMT = Endothelial–mesenchymal transition; ECM = Extracellular matrix.

Intestinal fibrosis in IBD and MMP-dependent myofibroblast apoptosis. Proliferation and apoptosis of ECM-producing cells could represent important steps in intestinal fibrogenesis and possible new targets for therapeutic intervention [21]. Several compounds have shown potential antifibrogenic efficacy through the regulation of mesenchymal cell proliferation and apoptosis.

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Biological mediators of fibrosis All intestinal cell types that produce ECM proteins act in concert and are under the control of various biological mediators, such as growth factors, cytokines, chemokines, proteolytic enzymes, complement components, vasoactive amines and peptides [11,12]. The most important profibrotic mediators include TGF-b, activins, integrins, connective tissue growth factor (CTGF), PDGF, insulin-like growth factor (IGF)1 and -2, epidermal growth factor, fibroblast growth factors (FGFs), endothelin-1, -2, -3, various cytokines (IL-1, -4, -6, -13, -17, -21, -22, -23, -33) and chemokines CCL2 (monocyte chemoattractant protein-1), CCL3 (macrophage inflammatory protein-1 [MIP1]), CCL4 (MIP-1b), and CCL20 (MIP-3a), products of oxidative stress, components of the renin-angiotensin system like angiotensinconverting enzyme and angiotensin II and angiogenic factors (e.g. vascular endothelial growth factor) [9,10]. Soluble factors with antifibrotic properties have also been identified, including IFN-a, IFN-g, IL-7, IL-10, IL-12, Smad7, and nitric oxide [11,12]. Intestinal healing depends on the balance between the release and actions of profibrotic and antifibrotic factors, whereas the imbalance in favor of profibrotic factors leads to fibrosis (Figure 3). In fibrotic diseases, Antifibrotic factors

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the majority of studies have investigated the molecular mechanisms of profibrotic factors, whereas a very little attention has been directed toward the role of antifibrotic factors. TGF-b Among the profibrotic molecules, TGF-b has been the most widely studied and was noted to be crucial in intestinal fibrosis. Its relevance to fibrogenesis is shared with other organs, such as liver, lung, and kidney [11,12]. Both TGF-b and its receptors are overexpressed, particularly in fibrostenotic CD and in animal models of intestinal fibrosis [23–25]. Adenovirus-mediated overexpression of TGF-b in the murine colon leads to colonic fibrosis [26]; conversely, the loss of Smad3 confers resistance to trinitrobenzene sulfonic acid-induced colorectal fibrosis [27]. Disruption of the TGF-b/Smad signaling pathway, either by the loss of Smad3, or increase of Smad7 expression, can reduce fibrosis in several organs including the intestine [12]. Decreased Smad7, and increased pSmad2/3, expression in intestinal CD strictures additionally supports the profibrogenic role of the TGF-b/Smad pathway [23]. Blockade of TGF-b signaling, either at the extracellular or intracellular level offers a strategy to prevent and/or treat fibrosis [11,12,16]. Since TGF-b, however, is also involved in cellular differentiation, proliferation, transformation, and immunoregulation, its blockade is problematic as TGF-b, Smad2 and Smad4 disruptions are lethal [28–30] due to systemic autoimmunity. Targeting of individual intracellular mediators, however, could lead to the selective blockade of TGF-b fibrotic responses without involving Profibrotic factors

Balance

Healing

Imbalance

Fibrosis

Figure 3. Schematic representation of the intestinal fibrosis as a complex and dysfunctional balancing act between profibrotic and antifibrotic factors.

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physiologically vital TGF-b responses. Disrupting Smad3 results in mice that survive to adulthood and also confers resistance to tissue fibrosis [27]. HGF, bone morphogenetic protein (BMP-7) and decorin are three natural inhibitors of the TGF-b/ Smad pathway and demonstrated antifibrotic effects [31–33]. Although the TGF-b/Smad pathway represents the major driving force of fibrosis, several profibrogenic and antifibrogenic molecules seem to interact directly with the TGF-b/Smad pathway (Figure 4). The effects of these mediators on the TGF-b/Smad “core pathway” have been extensively discussed in a recent review [16].

Emerging modulators of fibrosis In addition to the above most common and wellknown profibrotic mediators including several growth factors, cytokines, chemokines and reactive oxygen species, several other critical factors of induction or progression of fibrosis are emerging. These include both profibrotic molecules (integrins, mammalian target of rapamycin [mTOR], Wnt/b-catenin pathway, Hedgehog (Hh) and Notch signaling, homeodomain-interacting protein kinase 2 [HIPK2] and serotonin) and antifibrotic molecular mechanisms (peroxisome proliferator-activated receptors (PPARs), adiponectin, HGF, BMP-7, Hippo, Klotho, SIRT1, and miRNAs) which all interact with TGFb/Smad pathway or directly activate myofibroblasts (Figure 5). The avb6 integrin activates latent TGF-b, while various genetic and pharmacologic interventions IL-13

+

SMAD3

Antifibrotic molecules

TGF-β Profibrotic molecules

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–

CTGF ECM

Intestinal fibrosis Figure 4. IL 13–TGF-b/Smad3–CTGF forms an integrated “profibrotic pathway” acting as a major driving force of intestinal fibrosis reinforced by profibrotic factors and opposed by antifibrotic factors. Abbreviations: IL-13 = Interleukin-13; TGF-b = Transforming growth factor-b; CTGF = Connective tissue growth factor; ECM = Extracellular matrix. Adapted from Ref. [19].

HIPK2

Hippo & klotho αvβ6 integrin

Wnt HGF & BMP-7

Adiponectin TGFβ/Smad3

miRNAs

Serotonin mTOR

Hedgehog PPARγ

HIPK2

Myofibroblasts

Notch SIRT1

Intestinal fibrosis Figure 5. Schematic representation of the interaction between profibrotic and antifibrotic pathways. Abbreviations: TGF-b = Transforming growth factor-b; mTOR = Mammalian target of rapamycin; Wnt = Wnt/b-catenin pathway; HIPK2 = Homeodomain-interacting protein kinase 2; HGF = Hepatic growth factor; BMP-7 = Bone morphogenetic protein-7; PPAR-g = Peroxisome proliferator activator receptorg; miRNAs = MicroRNAs; SIRT1 = Sirtuin 1.

targeting it reduce TGF-b1 activation and fibrosis [34]. Inhibitors of avb6 significantly reduce tissue levels of profibrogenic transcripts, including a-smooth muscle actin (SMA), procollagen a1, TGF-b1, CTGF, and TIMP-1 [35]. Inhibition of the avb6 integrin can inhibit TGF-b at sites of the injured organ, where avb6 integrin is upregulated without affecting other vital homeostatic TGF-b roles in inflammation and immunity. The mTOR forms at least two distinct complexes. The mTOR complex 1 that controls protein synthesis, cell growth, and proliferation, as well as autophagy, angiogenesis, and fibrosis, and the mTOR complex 2 that involves in cell proliferation and survival, metabolic regulation, and actin cytoskeleton organization [36]. The mTOR inhibitors (mTORis) exert direct antifibrotic activities by reducing fibroblast and myofibroblast numbers and also by downregulating the profibrogenic cytokine production of IL-4, -6, -13, -17, and TGF -b1, and collagen synthesis [36,37]. The antifibrotic effects of mTORis have been reported in several fibrotic diseases. The combined immunosuppressive and antifibrotic action of the mTORi medication rapamycin, and its analogs, sirolimus and everolimus, may thus be promising treatments for intestinal fibrosis in CD [38–40]. Activation of Wnt–b-catenin signaling promotes EMT and is required for TGF-b-mediated fibrosis [41]. Wnt signaling increases ECM synthesis and regulates profibrotic MMP-2 and -9 [42]. Inhibition of Wnt/b-catenin signaling reverses fibrosis and thus Wnt signaling may be a therapeutic target for fibrogenesis modification [43].

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Intestinal fibrosis in IBD The Hh signaling regulates fibrosis and progenitor cell proliferation and differentiation. Hh signaling is profibrotic as it promotes the myofibroblast activation, EMT, release of MMPs and TGF-b1, and the ECM production [44,45]. Conversely, inhibition of Hh signaling is potently antifibrotic in preclinical models of fibrosis [46]. The Notch signaling pathway is essential to normal embryonic development, cellular proliferation, specification, and differentiation. Notch signaling induces myofibroblast activation and fibrosis [47,48]. Blocking Notch signaling by the use of a c-secretase inhibitor significantly attenuates fibrosis and decreases TGF-b1 and EMT [49]. HIPK2 is associated with fibrosis and regulates EMT through various signaling pathways including TGF-b, Wnt/b-catenin, and Notch [50]. HIPK2 also regulates two other key fibrosis-associated process, apoptosis and inflammation, through the p53 and NF-kB pathways. HIPK2 is normally repressed by the ubiquitin SIAH1, but oxidative stress and DNA damage inhibit the expression of SIAH1 and release the suppression of HIPK2. Therefore, treatments targeting HIPK2 could prevent or even ameliorate fibrosis. Serotonin, mainly known as a neurotransmitter in the central nervous system, is involved in the pathogenesis of colitis and the development of fibrosis by inducing myofibroblast activation and TNF-a and TGF-b1 production [51,52]. Serotonin receptor inhibitors are noted to lower the fibrotic response [53,54]. PPAR-g activation strongly correlates with the TGF-b/Smad pathway as it directly antagonizes Smad3 or downregulates CTGF expression [55,56]. Overexpression of PPAR-g prevents tissue fibrosis, whereas its loss increases fibrosis [34,57]. PPAR-g agonists attenuate fibrosis in several organs including the intestine and these antifibrotic effects are abolished by PPAR-g selective antagonists [58,59]. PPAR-g is an innate protector against excessive fibrogenesis. Adiponectin is a member of the adipokines that includes leptin, resistin, and ghrelin and is secreted by adipocytes [60]. Increased expression of these adipokines occurs in IBD mesenteric adipose tissue and serum [61]. The central modulator of adiponectin signaling is AMP kinase which when activated inhibits the canonical TGF-b/Smad pathway and the collagen production, as well as mTOR signaling [62]. Adiponectin appears to be antifibrogenic as extensive fibrosis may develop in adiponectin-knockout mice and this is alleviated by the administration of recombinant adiponectin. By contrast, leptin is profibrogenic, as fibrosis is decreased in leptin- or leptin-receptor-deficient mice [63].

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HGF and BMP-7 are natural inhibitors of the TGF-b/Smad pathway and demonstrate antifibrotic effects [31,32]. HGF exerts several biological activities on myofibroblasts, including the inhibition of growth, suppression of fibrogenic cytokine expression, and enhancement of MMP levels [64]. Administration of recombinant HGF protein, or ectopic HGF expression, ameliorates fibrosis [65]. BMP-7 is an antagonist of TGF-bsignaling that has been shown to reverse EMT [66,67]. BMP-7 downregulates of SMA and phosphorylated Smad2/3. Deletion of BMP-7 receptor, activinlike kinase 3, enhances EMT and macrophage influx and worsens fibrosis [68,69]. The Hippo pathway plays a crucial role in proliferation, apoptosis, and organ size control [70]. The Hippo pathway is also involved in intestinal fibrosis, having a direct link with TGF-b signaling [71]. This link seems to be directly dependent on the cellular density. If it is low, TAZ and YAP (two of the downstream targets of the Hippo signaling) translocate into the nucleus and promote SMAD signaling via direct binding of the complex. If the cellular density is high, the two factors are localized in the cytoplasm and block the SMAD signaling and binding of the SMADs and thus inhibit their translocation into the nucleus. In addition, the Hippo pathway shows a crosstalk with Wnt signaling being the downstream effectors of Hippo, TAZ, and YAP, and is able to inhibit the Wnt/b-catenin pathway by interfering with the b-catenin stabilization and activation [72]. The Hippo pathway potentially could also interact with the Hh pathway, but the mechanisms by which this could occur is unclear [73]. Klotho, a single-pass transmembrane protein, has been recognized as an anti-aging gene but it is also involved in fibrotic diseases as well. Klotho family of membrane proteins function as obligate coreceptors for endocrine FGFs [74]. Secreted Klotho protein directly binds to the type-II TGF-b receptor inhibiting TGF-b1 signaling and TGF-b1-induced EMT [75]. Overexpression of Klotho abolishes the fibrogenic effects of TGF-b1, suppresses myofibroblast activation, reduces ECM deposition, and ameliorates fibrosis [76]. In addition, secreted Klotho inhibits the Wnt and IGF-1 signaling that promotes EMT. Sirt1 is an enzyme that deacetylates several transcription factors and regulates pathways mainly involved in aging and chronic diseases. In particular, an in vivo study highlighted a role of Sirt1 in fibrosisassociated EMT [77]. Sirt1 deacetylates SMAD4 and then blocks TGF-b signaling thereby inhibiting EMT. There are no direct data on the involvement of Sirt1 in intestinal fibrosis, but this enzyme is known for having a role in intestinal homeostasis. In fact, Sirt1-null mice have a disrupted intestinal

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homeostasis, resulting in increased cells proliferation and decreased apoptosis [78]. The miRNAs are small, noncoding RNAs of 18–25 nucleotides that regulate gene and protein expression. Over 100 miRNAs are implicated in fibrosis and differentially expressed during fibrosis and modulate antifibrotic and profibrotic gene expression [79,80]. Some miRNAs like miRNA let-7d, miRNA-133, miRNA-30, miRNA-150, miRNA-194, and miRNA-200a are constitutively expressed in healthy tissues but is downregulated in fibrosis, suggesting an antifibrotic role. Specific miRNAs downregulate Smad-3 activity and ECM expression and prevent TGF-b-dependent EMT. The miRNA-21, miRNA-29, miRNA-192, miRNA-216a, and miRNA-217, as miRNA-21, are key triggers of TGF-b- and Smad3-driven fibrosis and miRNA-200a and miRNA-200b are involved in CD fibrosis [81].

MMPs The excessive ECM accumulation characterizing intestinal fibrosis hinges on the balance between ECM deposition, controlled by the abovementioned profibrotic and antifibrotic factors, and degradation mediated by MMPs. The fine balance between MMPs and TIMPs appears to be disturbed in IBD [20,82,83]. It is unclear, however, which specific MMPs and TIMPs are involved in fibrosis and how they are regulated. In addition to playing a central role in ECM turnover, MMPs proteolytically activate or degrade a variety of non-matrix substrates including chemokines, cytokines, growth factors, and junctional proteins. Thus, they are increasingly recognized as critical players in inflammatory response and fibrogenesis [82,83]. MMPs are a group of calcium-activated and zincdependent endopeptidases that are secreted as preforms (inactive zymogens). They are produced in response to inflammatory stimuli by various cell types, including mesenchymal cells, T-cells, monocytes, macrophages, and neutrophils [82]. Depending on substrate specificity, amino acid similarity, and identifiable sequence modules, the MMPs can be classified into six major subgroups: collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -10, -11), matrilysin (MMP-7), membrane types (MMP-14, -15, -16, -17, -24, -25), and others (MMP-19, -20, -21, -22, -23, 26, -27, -28) [84]. All MMPs become activated via proteolytic cleavage and are regarded as the major enzyme group capable of degrading ECM components such as collagens, laminins and fibronectins, including basement membranes.

MMP activity is controlled by specific and nonspecific inhibitors such as TIMPs and a2-macroglobulin, respectively [82,83]. TIMPs are produced by the same cell types as MMPs and form a 1:1 complex with activated MMPs. The fine balance between MMPs and TIMPs regulates the turnover of ECM both under physiological conditions and in tissue remodeling during inflammation and wound healing. Thereby, the imbalance, due to reduced MMP activity and/or increased expression of TIMPs, may lead to excessive deposition of ECM proteins driving fibrogenesis. Downregulation of some MMPs and overexpression of TIMPs have been reported in experimental intestinal fibrosis and in stricturing CD [23,85–87]. Taken together, all these data strongly support the hypothesis that an imbalance of tissue-degrading enzymes and their inhibitors may cause intestinal fibrosis [88]. MMPs and TIMPs may, therefore, be considered as targets for new antifibrotic treatment approaches. Additional functions of both molecules in regulation of inflammation and related fibrosis, however, need to be considered. New therapeutical approach of intestinal fibrosis The advancements in knowledge of IBD over the past two decades have modified the treatment goals. Although, in the past, the aim of medical treatment was an improvement in IBD symptoms, the current objective is not only to achieve clinical remission but also to achieve the healing of all intestinal lesions. Mucosal healing can be considered appropriate for UC, which is a disease of the mucosa, whereas the term “intestinal healing” would be more correct for CD, which is a transmural disease. Reduction or reversal of intestinal fibrosis is also an important goal to achieve, but this represents an immense challenge. At present, there are no approved, or effective, medical therapies targeting intestinal fibrosis [16]. Therefore, intestinal fibrosis and the associated complications, still remain the major cause of surgical intervention [89,90]. Surgical correction, by means of intestinal resection or stricturoplasty, is necessary in up to 75% of CD patients during the course of their disease [3,4]. Surgical resection, however, does not cure CD and is associated with a high rate of recurrent stricturing disease. The need for repeat surgery is high; therefore, exploration of new therapeutic approaches has now become a priority. The agents currently used for the treatment of IBD (salicylates, antibiotics, steroids, immunosuppressive drugs, biological therapies) may relieve the inflammatory symptoms but do not significantly improve

Intestinal fibrosis in IBD

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Progenitors Profibrotic molecules

Antifibrotic molecules

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Myofibroblasts

Cell number

Proliferation

Death

Cell activation

Suface receptors

Cell secretion

Signaling molecules

mRNA Proteins (ECM, MMPs, TIMPs, etc.)

Figure 6. Schematic representation of the therapeutic targets in intestinal fibrosis. Abbreviations: ECM = Extracellular matrix; MMPs = Matrix metalloproteinases; TIMPs = Tissue inhibitors of metalloproteinases.

fibrosis-related strictures and obstruction [90–92]. There is little doubt that these agents work best when introduced early in the course of the disease, when inflammation predominates and fibrosis may still be at a reversible phase. Nevertheless, the results of medical treatment aimed at stricturing or penetrating CD are still poor, since 64% of these patients ultimately require surgery within 1 year [93]. Although several clinical trials suggest that biological therapies may reduce the need for surgery in the short term, the real impact of biological treatment on the lifetime risk of surgery remains to be established [94–96]. Data from population-based cohorts have shown that in the pre-biological era, the rate of surgery ranged between 27% and 61% within 5 years after diagnosis, and, in the era of anti-TNF-a medications it ranged between 25% and 33% suggesting that the need for surgery still remains high [97]. Controlling intestinal inflammation alone would thus appear not to be sufficient to prevent or eliminate the associated fibrotic response [12,16]. Since there is a strong relationship between intestinal inflammation and fibrosis, in IBD, a new therapeutic approach could be to combine the use of drugs with anti-inflammatory actions and drugs with antifibrotic action. Several antifibrotic drugs (chemical and biological) have been tested in experimental models of tissue fibrosis and are able to inhibit, mitigate, or even reverse the fibrotic process [16]. Their antifibrotic activity seems to be related to different mechanisms of action such as reducing numbers of activated ECM-producing cells and their profibrogenic effects (proliferation, motility, contraction, ECM deposition), promoting ECM-producing cells apoptosis or promoting ECM degradation

(Figure 6). Besides efficacy, the safety of any antifibrotic drug is important. They potentially need to be administered over a long period of time and inhibiting fibrosis could also inhibit wound healing and thus lead to perforating or fistulizing complications. There is a growing list of novel mediators and pathways that could be developed as antifibrotic treatments [11,12,16,98]. These include antagonists and inhibitors of profibrotic molecules, antifibrotic molecules, pro-apoptotic drugs that target myofibroblasts,

Table I. Classes of antifibrogenic agents. TGFb/Smad signaling modifiers Growth factors antagonists Profibrotic cytokine and cytokine receptor antagonists Profibrotic chemokine and chemokine receptor antagonists Integrin/adhesion molecule antagonists Toll-like receptor antagonists Angiogenesis antagonists Vasoactive substance antagonists Angiotensin-converting enzyme and angiotensin II receptor inhibitors Mammalian target of rapamycin inhibitors Matrix metalloproteinase inhibitors Tissue inhibitor of metalloproteinase antagonists Mitogen-activated protein kinase and tyrosine kinases inhibitors 3-hydroxy-3-methyl-glutaryl-coenzyme A inhibitors Reactive oxygen species inhibitors Nitric oxide donors Prostaglandins (PGE2, 15-D-PGJ2), COX2 inhibitors Antifibrotic molecules (cytokines, chemokines, HGF, BMP-7, PPARs modulators) Pro-apoptotic drugs that target myofibroblasts Gene silencing strategies and gene therapy Stem cell transplantation technologies Abbreviations: BMP = Bone morphogenetic protein; COX2: Cyclo-oxygensae-2 enzyme; HGF = Hepatocyte growth factor; PPAR = Peroxisome proliferator-activated receptor.

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gene silencing strategies, and stem cell transplantation technologies (Table I). It should be stressed, however, that most of the evidence indicating a beneficial effect of these drugs have been derived from studies performed in vitro or in animal models of fibrogenesis [3,25,99]. Therefore, the real effectiveness of these agents in humans remains to be defined.

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Conclusion The concept of intestinal fibrosis has changed from being a static and irreversible entity to a dynamic and reversible disease, as seen in other organs. Intestinal fibrosis is a highly complex process involving the dynamic actions of numerous molecules, which regulate the activation of ECM-producing myofibroblasts during intestinal damage and repair. The specific molecules determining the balance between physiological repair and excessive ECM accumulation that characterizes fibrosis, however, remain unknown. Strong evidence indicates that inflammation triggers fibrosis, which, once established, may progress independently and the available anti-inflammatory drugs are ineffective against fibrosis. Defining the cellular and molecular mechanisms involved in intestinal fibrosis is the key to the development of new therapeutic approaches in the IBD treatment. Novel therapeutic strategies are under investigation to target specific steps in the process of fibrogenesis with the aim of reducing or reversing advanced intestinal fibrosis in IBD.

Acknowledgments All authors wrote specific parts of the manuscript, critically reviewed the completed manuscript, and approved the final version. No study sponsors had any involvement in study design, data collection, interpretation, or writing of the manuscript. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References [1] Cosnes J, Gower-Rousseau C, Seksik P, Cortot A. Epidemiology and natural history of inflammatory bowel diseases. Gastroenterology 2011;140:1785–94. [2] Latella G, Papi C. Crucial steps in the natural history of inflammatory bowel disease. World J Gastroenterol 2012;18: 3790–9.

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