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Epithelial FZD4 expression and WNT signalling are increased in intestinal tissue from CD patients with a penetrating behaviour. Surgical resections from control, B2-and B3-CD patients were obtained, fixed for immunohistochemical analysis, or frozen for both WB and qRT-PCR analysis. A] Graphs show the mRNA [n = 25] and protein [n = 4] expression of FZD4 in intestinal tissue and the mRNA expression of the same gene in intestinal crypts [n = 5]; a representative WB showing protein levels of FZD4 [n = 4]. B] Representative images showing β-CATENIN immunostaining in intestinal tissue [scale bars 100 µm] [n = 5] and a graph and a representative WB of a total of four showing protein levels of β-CATENIN in intestinal crypts. C] Graphs show the mRNA expression of WNT targets in intestinal tissue [n = 25] and intestinal crypts [n = 5]. In all cases data correspond to relative values vs the expression of the housekeeping gene, and are calculated as fold induction vs non-IBD group or as arbitrary units. Bars represent mean ± SEM of at least four patients per experimental group. Significant differences vs the non-IBD group are shown by *p < 0.05, and vs the B2-CD samples by # p < 0.05. CD, Crohn's disease; qRT-PCR, quantitative real-time polymerase chain reaction; IBD, inflammatory bowel disease; SEM, standard error of the mean; WB, Western blot.

Epithelial FZD4 expression and WNT signalling are increased in intestinal tissue from CD patients with a penetrating behaviour. Surgical resections from control, B2-and B3-CD patients were obtained, fixed for immunohistochemical analysis, or frozen for both WB and qRT-PCR analysis. A] Graphs show the mRNA [n = 25] and protein [n = 4] expression of FZD4 in intestinal tissue and the mRNA expression of the same gene in intestinal crypts [n = 5]; a representative WB showing protein levels of FZD4 [n = 4]. B] Representative images showing β-CATENIN immunostaining in intestinal tissue [scale bars 100 µm] [n = 5] and a graph and a representative WB of a total of four showing protein levels of β-CATENIN in intestinal crypts. C] Graphs show the mRNA expression of WNT targets in intestinal tissue [n = 25] and intestinal crypts [n = 5]. In all cases data correspond to relative values vs the expression of the housekeeping gene, and are calculated as fold induction vs non-IBD group or as arbitrary units. Bars represent mean ± SEM of at least four patients per experimental group. Significant differences vs the non-IBD group are shown by *p < 0.05, and vs the B2-CD samples by # p < 0.05. CD, Crohn's disease; qRT-PCR, quantitative real-time polymerase chain reaction; IBD, inflammatory bowel disease; SEM, standard error of the mean; WB, Western blot.

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Background and Aims Epithelial-mesenchymal transition (EMT) has been related to fibrosis and fistula formation, common complications associated to Crohn´s disease (CD). The WNT signaling pathway mediates EMT and specific WNT/FZD interactions have been related with the activation of this process in several diseases. We aim to analyze the relevance o...

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... analysis of the mRNA expression of FZD receptors in human intestine showed non-significant differences between control and CD patients, and only a significant decrease in FZD5 was detected in tissue from CD patients [Supplementary Table S1A, available as Supplementary data at ECCO-JCC online]. Protein expression of FZD4 was significantly higher in tissue from patients with penetrating behaviour than in that from controls or B2-CD patients [ Figure 2A]. In isolated intestinal crypts, results revealed a significant increase in the mRNA expression of FZD4 in cells obtained from B3-CD patients compared with that observed in samples from B2-CD patients or control subjects [Supplementary Table S1B, Figure 2A]. ...
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... expression of FZD4 was significantly higher in tissue from patients with penetrating behaviour than in that from controls or B2-CD patients [ Figure 2A]. In isolated intestinal crypts, results revealed a significant increase in the mRNA expression of FZD4 in cells obtained from B3-CD patients compared with that observed in samples from B2-CD patients or control subjects [Supplementary Table S1B, Figure 2A]. ...
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... WNT signalling pathway was analysed by the determination of both protein levels of β-CATENIN and the mRNA expression of WNT target genes. Immunohistochemical experiments revealed presence of β-CATENIN mainly in epithelial cells of intestinal tissue [ Figure 2B], and a WB analysis revealed that protein levels of β-CATENIN were higher in intestinal crypts from B3-than in those from B2-CD patients or controls [ Figure 2B]. The mRNA expression Table 3. Primer sequences of specific polymerase chain reaction [PCR] products for each gene analysed. ...
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... WNT signalling pathway was analysed by the determination of both protein levels of β-CATENIN and the mRNA expression of WNT target genes. Immunohistochemical experiments revealed presence of β-CATENIN mainly in epithelial cells of intestinal tissue [ Figure 2B], and a WB analysis revealed that protein levels of β-CATENIN were higher in intestinal crypts from B3-than in those from B2-CD patients or controls [ Figure 2B]. The mRNA expression Table 3. Primer sequences of specific polymerase chain reaction [PCR] products for each gene analysed. ...
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... B2 B3 of the WNT target genes, CMYC, LGR5, DKK1, and CYCLIN D1 was significantly higher in both intestinal tissue and intestinal crypts from B3-CD patients than in those from control or B2-CD patients [ Figure 2C]. ...
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... failed to significantly modify the expression of VIMENTIN, SNAIL1, and SNAIL2 in primary fibroblasts isolated from human intestine [Supplementary Figure S2, available as Supplementary data at ECCO-JCC online]. Of interest, WNT2b failed to modulate in these cells the expression of CMYC, a WNT target gene. ...
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... analysis of the mRNA expression of FZD receptors in human intestine showed non-significant differences between control and CD patients, and only a significant decrease in FZD5 was detected in tissue from CD patients [Supplementary Table S1A, available as Supplementary data at ECCO-JCC online]. Protein expression of FZD4 was significantly higher in tissue from patients with penetrating behaviour than in that from controls or B2-CD patients [ Figure 2A]. In isolated intestinal crypts, results revealed a significant increase in the mRNA expression of FZD4 in cells obtained from B3-CD patients compared with that observed in samples from B2-CD patients or control subjects [Supplementary Table S1B, Figure 2A]. ...
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... expression of FZD4 was significantly higher in tissue from patients with penetrating behaviour than in that from controls or B2-CD patients [ Figure 2A]. In isolated intestinal crypts, results revealed a significant increase in the mRNA expression of FZD4 in cells obtained from B3-CD patients compared with that observed in samples from B2-CD patients or control subjects [Supplementary Table S1B, Figure 2A]. ...
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... WNT signalling pathway was analysed by the determination of both protein levels of β-CATENIN and the mRNA expression of WNT target genes. Immunohistochemical experiments revealed presence of β-CATENIN mainly in epithelial cells of intestinal tissue [ Figure 2B], and a WB analysis revealed that protein levels of β-CATENIN were higher in intestinal crypts from B3-than in those from B2-CD patients or controls [ Figure 2B]. The mRNA expression Table 3. Primer sequences of specific polymerase chain reaction [PCR] products for each gene analysed. ...
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... WNT signalling pathway was analysed by the determination of both protein levels of β-CATENIN and the mRNA expression of WNT target genes. Immunohistochemical experiments revealed presence of β-CATENIN mainly in epithelial cells of intestinal tissue [ Figure 2B], and a WB analysis revealed that protein levels of β-CATENIN were higher in intestinal crypts from B3-than in those from B2-CD patients or controls [ Figure 2B]. The mRNA expression Table 3. Primer sequences of specific polymerase chain reaction [PCR] products for each gene analysed. ...
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... B2 B3 of the WNT target genes, CMYC, LGR5, DKK1, and CYCLIN D1 was significantly higher in both intestinal tissue and intestinal crypts from B3-CD patients than in those from control or B2-CD patients [ Figure 2C]. ...
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... failed to significantly modify the expression of VIMENTIN, SNAIL1, and SNAIL2 in primary fibroblasts isolated from human intestine [Supplementary Figure S2, available as Supplementary data at ECCO-JCC online]. Of interest, WNT2b failed to modulate in these cells the expression of CMYC, a WNT target gene. ...

Citations

... In recent years, the relevance of epithelial-mesenchymal transition (EMT), a cellular trans-differentiation programme by which epithelial cells acquire mesenchymal features, has become apparent in CD complications [5,6]. The WNT signalling pathway plays a fundamental role in EMT, and we have recently reported increased EMT associated with enhanced WNT2b/FZD4 interaction in intestinal tissue from CD patients with penetrating behaviour, thus demonstrating that WNT2b induces EMT through the activation of FZD4 [7]. However, the source of this enhanced WNT2b is yet to be established. ...
... These observations, together with the fact that these cells usually accumulate when there is chronic inflammation, point to the relevance of macrophage-derived WNT ligands in mucosal regeneration and in complications such as fibrosis and cancer. However, we have recently reported that the WNT2b ligand is expressed mainly in epithelial cells and not so much in the lamina propria of the intestine [7]. All these observations have led us to analyse the macrophage phenotype that is mainly expressed in the intestine of CD patients with stenotic or penetrating complications and to determine the role that these cells play in the regulation of the WNT signalling pathway and, in turn, in EMT. ...
... Alternatively, HT29 cells were transfected with the following vector-targeting human FZD4 (miFZD4, targeting sequences: 5 -CGGCATGTGTCTTTCAGTCAA-3 and 5 -GTCACTCTGTGGGAACCAATT-3 (GenBank Accession No. NM_012193), as described previously [7]. Twenty-four hours post-transfection, the cells were co-cultured with IFNγtreated U937 cells for 72 h. ...
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Background: Fibrosis is a common complication of Crohn's disease (CD) in which macrophages play a central role. Epithelial-mesenchymal transition (EMT) and the WNT pathway have been associated with fibrosis. We aim to analyse the relevance of the tissue microenvironment in macrophage phenotype and the EMT process. Methods: Intestinal surgical resections are obtained from control and CD patients with stenotic or penetrating behaviour. Cytokine's expression, macrophage phenotype, EMT markers and WNT signalling pathway are determined by WB, RT-PCR, ELISA or Cytometry. U937 cells are treated with IFNγ, TNFα, IL1β, IL4 or IL10 and co-cultured with HT29 cells and, in some cases, are treated with XAV939 or miFZD4. The expression of macrophage, EMT and WNT pathway markers in U937 or HT29 cells is analysed by WB or RT-PCR. Results: IFNγ, WNT6, CD16 and CD86 are increased in the intestinal tissue of CD patients. IFNγ-treated U937 activated the EMT process and WNT pathway in HT29 cells, and the EMT process is mediated by FZD4. Conclusions: An IFNγ-rich microenvironment polarises macrophages, which induces EMT through the WNT pathway.
... This TGF-β-driven process is called epithelialor endothelial-to-mesenchymal transition (EMT) and represents the loss of typical epithelial/endothelial markers together with the upregulation of fibroblast markers, such as αSMA, vimentin, fibronectin, and collagens, and the acquisition of a fibroblast-like morphology [103,114]. Although the occurrence of EMT in IBD-related intestinal fibrosis was previously reported [115,116], our understanding of its functional role and the exact mechanism is still in its infancy. ...
Article
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The gastrointestinal tract is the largest mucosal surface in our body and accommodates the majority of the total lymphocyte population. Being continuously exposed to both harmless antigens and potentially threatening pathogens, the intestinal mucosa requires the integration of multiple signals for balancing immune responses. This integration is certainly supported by tissue-resident intestinal mesenchymal cells (IMCs), yet the molecular mechanisms whereby IMCs contribute to these events remain largely undefined. Recent studies using single-cell profiling technologies indicated a previously unappreciated heterogeneity of IMCs and provided further knowledge which will help to understand dynamic interactions between IMCs and hematopoietic cells of the intestinal mucosa. In this review, we focus on recent findings on the immunological functions of IMCs: On one hand, we discuss the steady-state interactions of IMCs with epithelial cells and hematopoietic cells. On the other hand, we summarize our current knowledge about the contribution of IMCs to the development of intestinal inflammatory conditions, such as infections, inflammatory bowel disease, and fibrosis. By providing a comprehensive list of cytokines and chemokines produced by IMCs under homeostatic and inflammatory conditions, we highlight the significant immunomodulatory and tissue niche forming capacities of IMCs.
... Recently, Lenoir et al., have demonstrated that F. prausnitzii supernatant containing butyrate could mediate anti-inflammatory responses by promoting the expression of dact3, a gene which negatively regulates the Wnt/JNK pathway [120]. Recently, Wnt signaling pathways has been suggested to be essential in the onset of intestinal fibrosis [121]. Moreover, macrophages (especially those with the M2 phenotype) have been described as a putative source of Wnt ligands and relevant to mucosal integrity [122,123]. ...
Article
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Intestinal fibrosis is a common complication in inflammatory bowel disease (IBD) without specific treatment. As macrophages are the key actors in inflammatory responses and the wound healing process, they have been extensively studied in chronic diseases these past decades. By their exceptional ability to integrate diverse stimuli in their surrounding environment, macrophages display a multitude of phenotypes to underpin a broad spectrum of functions, from the initiation to the resolution of inflammation following injury. The hypothesis that distinct macrophage subtypes could be involved in fibrogenesis and wound healing is emerging and could open up new therapeutic perspectives in the treatment of intestinal fibrosis. Gut microbiota and diet are two key factors capable of modifying intestinal macrophage profiles, shaping their specific function. Defects in macrophage polarisation, inadequate dietary habits, and alteration of microbiota composition may contribute to the development of intestinal fibrosis. In this review, we describe the intriguing triangle between intestinal macrophages, diet, and gut microbiota in homeostasis and how the perturbation of this discreet balance may lead to a pro-fibrotic environment and influence fibrogenesis in the gut.
... Patients with penetrating Crohn's disease also display increased activation of WNT family member 2B (WNT2b). Interaction of WNT2b with activated frizzled-4 (FZD4) induces epithelial-mesenchymal transition in vitro, highlighting another signalling axis that promotes Crohn's disease-associated fibrosis 59 . ...
Article
Intestinal fibrosis, which is usually the consequence of chronic inflammation, is a common complication of inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis. In the past few years, substantial advances have been made in the areas of pathogenesis, diagnosis and management of intestinal fibrosis. Of particular interest have been inflammation-independent mechanisms behind the gut fibrotic process, genetic and environmental risk factors (such as the role of the microbiota), and the generation of new in vitro and in vivo systems to study fibrogenesis in the gut. A huge amount of work has also been done in the area of biomarkers to predict or detect intestinal fibrosis, including novel cross-sectional imaging techniques. In parallel, researchers are embarking on developing and validating clinical trial end points and protocols to test novel antifibrotic agents, although no antifibrotic therapies are currently available. This Review presents the state of the art on the most recently identified pathogenic mechanisms of this serious IBD-related complication, focusing on possible targets of antifibrotic therapies, management strategies, and factors that might predict fibrosis progression or response to treatment.
... TGF-b-induced pro-fibrotic signaling in part by the Frizzled-8 receptor in lung injury [46]. In Penetrating Crohns Disease, Wnt2b induces epithelial-mesenchymal transition (EMT) via Fzd4 receptor [47]. Fzd4 and Fzd2 have been found to be abundant in rat cardiac fibroblasts [48]. ...
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Background Acute myocardial infarction (AMI)-induced excessive myocardial fibrosis exaggerates cardiac dysfunction. However, serum Wnt2 or Wnt4 level in AMI patients, and the roles in cardiac fibrosis are largely unkown. Methods AMI and non-AMI patients were enrolled to examine serum Wnt2 and Wnt4 levels by ELISA analysis. The AMI patients were followed-up for one year. MI mouse model was built by ligation of left anterior descending branch (LAD). Findings Serum Wnt2 or Wnt4 level was increased in patients with AMI, and the elevated Wnt2 and Wnt4 were correlated to adverse outcome of these patients. Knockdown of Wnt2 and Wnt4 significantly attenuated myocardial remodeling and cardiac dysfunction following experimental MI. In vitro, hypoxia enhanced the secretion and expression of Wnt2 and Wnt4 in neonatal rat cardiac myocytes (NRCMs) or fibroblasts (NRCFs). Mechanistically, the elevated Wnt2 or Wnt4 activated β-catenin /NF-κB signaling to promote pro-fibrotic effects in cultured NRCFs. In addition, Wnt2 or Wnt4 upregulated the expression of these Wnt co-receptors, frizzled (Fzd) 2, Fzd4 and (ow-density lipoprotein receptor-related protein 6 (LRP6). Further analysis revealed that Wnt2 or Wnt4 activated β-catenin /NF-κB by the co-operation of Fzd4 or Fzd2 and LRP6 signaling, respectively. Interpretation Elevated Wnt2 and Wnt4 activate β-catenin/NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 in fibroblasts, which contributes to adverse outcome of patients with AMI, suggesting that systemic inhibition of Wnt2 and Wnt4 may improve cardiac dysfunction after MI.
... However, FZD4 also plays a role in tumor progression, migration, and invasion [58]. The role of FZD4 in EMT was first identified in patients suffering from penetrating Crohn's disease, a common risk factor for CRC [59]. Penetrating Crohn's disease patients have elevated levels of WNT2b and FZD4, as well as n-cadherin and vimentin, suggesting FZD4 contributes to EMT in the colon [60]. ...
... FZD4 also contributes to EMT in non-small cell lung cancer (NSCLC) both in vitro and in vivo by activating the TCF/LEF/WNT/β-catenin pathway [58]. Although studies in premalignant lesions are minimal, FZD4 is known to play a role in mesothelial hyperplasia and Crohn's disease, a risk factor for colorectal carcinoma [59,64]. The presence of FZD4 in premalignant tissues along with the ability of FZD4 to induce cancer cell EMT highlights FZD4's potential in future chemopreventive studies. ...
Article
Full-text available
Frizzled (FZD) transmembrane receptors are well known for their role in β-catenin signaling and development and now understanding of their role in the context of cancer is growing. FZDs are often associated with the process of epithelial to mesenchymal transition (EMT) through β-catenin, but some also influence EMT through non-canonical pathways. With ten different FZDs, there is a wide range of activity from oncogenic to tumor suppressive depending on the tissue context. Alterations in FZD signaling can occur during development of premalignant lesions, supporting their potential as targets of chemoprevention agents. Agonizing or antagonizing FZD activity may affect EMT, which is a key process in lesion progression often targeted by chemoprevention agents. Recent studies identified a specific FZD as important for activity of an EMT inhibiting chemopreventive agent and other studies have highlighted the previously unrecognized potential for targeting small molecules to FZD receptors. This work demonstrates the value of investigating FZDs in chemoprevention and here we provide a review of FZDs in cancer EMT and their potential as chemoprevention targets.
... WNT2B induced the mRNA expression of cMYC, VIM, SNAI1, and SNAI2, but decreasing CDH1 one. In addition, WNT2B induced the phosphorylation of STAT3 in cells derived from intestinal crypts [40]. In patients with stricturing CD, the greater Wnt activation through cytoskeleton rearrangement and cell movement [10], might be also involved in the gain of EMT detection. ...
Article
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Crohn’s Disease (CD) is a chronic inflammatory disorder in which up to 50% of patients develop fistula within 20 years after the initial diagnosis, and half of these patients suffer perianal fistulizing disease. The etiopathogenesis of CD-related perianal fistula is still unclear, and its phenotypical and molecular characteristics are even more indefinite. A better understanding would be crucial to develop targeted and more effective therapeutic strategies. At present, the most accredited theory for the formation of CD-related fistula identifies the epithelial-to-mesenchymal transition (EMT) as the driving force. It has been well recognized that CD carries an increased risk of malignancy, particularly mucinous adenocarcinoma is often associated with long-standing fistula in CD patients. Despite the availability of multiple treatment options, perianal fistulizing CD represents a therapeutic challenge and is associated with an important impact on patients’ quality of life. To date, the most effective management is multidisciplinary with the cooperation of gastroenterologists, surgeons, radiologists, and nutritionists and the best recommended treatment is a combination of medical and surgical approaches.
... 50 A recent study demonstrated the occurrence of EMT in fibrotic intestinal CD tissue independent of the effect of inflammation. 51 Another report de- 52 The functional role of EMT in IBD still needs to be determined. ...
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The incidence of inflammatory bowel diseases (IBD) worldwide has resulted in a global public health challenge. Intestinal fibrosis leading to stricture formation and bowel obstruction is a frequent complication in Crohn's disease (CD), and the lack of anti‐fibrotic therapies makes elucidation of fibrosis mechanisms a priority. Progress has shown that mesenchymal cells, cytokines, microbial products, and mesenteric adipocytes are jointly implicated in the pathogenesis of intestinal fibrosis. This recent information puts prevention or reversal of intestinal strictures within reach through innovative therapies validated by reliable clinical trial endpoints. Here, we review the role of immune and non‐immune components of the pathogenesis of intestinal fibrosis, including new cell clusters, cytokine networks, host‐microbiome interactions, creeping fat, and their translation for endpoint development in anti‐fibrotic clinical trials.
... Exploration into the origins of myofibroblasts may provide an opportunity to develop effective treatments in intestinal fibrosis. An increasing number of studies indicate that epithelial to mesenchymal transition (EMT) is involved in intestinal fibrosis [9]. During this process, epithelial cells lose polarity as well as tight junctions, transform into interstitial cells, and produce a large amount of ECM deposited in the intestine, resulting in intestinal lumen narrowing [10]. ...
Article
Full-text available
Background and Aims. Recent evidences reveal that epithelial to mesenchymal transition (EMT) exacerbates the process of intestinal fibrosis. Tumor necrosis factor-like ligand 1A (TL1A) is a member of the tumor necrosis family (TNF), which can take part in the development of colonic inflammation and fibrosis by regulating immune response or inflammatory factors. The purpose of this study was to elucidate the possible contribution of TL1A in onset and progression of intestinal inflammation and fibrosis through EMT. Methods. Colonic specimens were obtained from patients with inflammatory bowel disease (IBD) and control individuals. The expression levels of TL1A and EMT-related markers in intestinal tissues were evaluated. Furthermore, the human colorectal adenocarcinoma cell line, HT-29, was stimulated with TL1A, anti-TL1A antibody, or BMP-7 to assess EMT process. In addition, transgenic mice expressing high levels of TL1A in lymphoid cells were used to further investigate the mechanism of TL1A in intestinal fibrosis. Results. High levels of TL1A expression were detected in the intestinal specimens of patients with ulcerative colitis and Crohn’s disease and were negatively associated with the expression of an epithelial marker (E-cadherin), while it was positively associated with the expression of interstitial markers (FSP1 and α-SMA). Transgenic mice with high expression of TL1A were more sensitive to dextran sodium sulfate and exhibited severe intestinal inflammation and fibrosis. Additionally, the TGF-β1/Smad3 pathway may be involved in TL1A-induced EMT, and the expression of IL-13 and EMT-related transcriptional molecules (e.g., ZEB1 and Snail1) was increased in the intestinal specimens of the transgenic mice. Furthermore, TL1A-induced EMT can be influenced by anti-TL1A antibody or BMP-7 in vitro. Conclusions. TL1A participates in the formation and process of EMT in intestinal fibrosis. This new knowledge enables us to better understand the pathogenesis of intestinal fibrosis and identify new therapeutic targets for its treatment. 1. Introduction In patients with inflammatory bowel disease (IBD), recurrent intestinal inflammation triggers mucosal healing reactions, leading to extracellular matrix (ECM) deposition in the intestine to form intestinal fibrosis [1]. As many as one-third of patients with Crohn’s disease (CD) develop end-stage fibrotic disease characterized by stenosis and organ failure [2, 3], and 80% of cases require surgical resection of the fibrotic intestinal tissue. Unfortunately, the recurrence rate is as high as 70% [4]. Ulcerative colitis (UC) has long been believed to be a nonfibrotic disease; however, recent studies have found a certain degree of submucosal fibrosis in almost all colon resection specimens from patients with UC [5–7]. Identifying effective treatment for intestinal fibrosis-induced by IBD has become the focus of investigations worldwide. Myofibroblasts are important effector cells for the deposition of ECM in intestinal fibrosis, and their sources are varied [8]. Exploration into the origins of myofibroblasts may provide an opportunity to develop effective treatments in intestinal fibrosis. An increasing number of studies indicate that epithelial to mesenchymal transition (EMT) is involved in intestinal fibrosis [9]. During this process, epithelial cells lose polarity as well as tight junctions, transform into interstitial cells, and produce a large amount of ECM deposited in the intestine, resulting in intestinal lumen narrowing [10]. Experiments with a transgenic mouse model with epithelial-driven expression of fluorescent reporters, indicate that about one-third of FSP1⁺ fibroblasts are derived from intestinal epithelial cells [11]. EMT can also be observed in vitro. For example, rat intestinal epithelial cells (IEC-6) displayed an irregular polygonal shape in control medium but obtained a spindle-shaped morphology after exposure to TGF-β1 for seven days [11]. Another study showed that parathyroid hormone-like hormone (PTHLH) was capable of increasing vimentin and FSP1 expression and reducing E-cadherin expression in a concentration- and time-dependent manner. Furthermore, exposure to PTHLH leads to collagen deposition [12]. All of these studies suggest that EMT may be an important mechanism in the development of intestinal fibrosis. The process of EMT is regulated by a complex cell signaling pathway and gene regulation. The TGF-β1/Smad3 pathway is the most classical pathway involving TGF-β1 [13]. However, exploring when and how it occurs may lead to effective treatment strategies for intestinal fibrosis. Genome-wide association studies (GWAS) have emphasized that the gene encoding tumor necrosis factor-like ligand 1A (TL1A) is associated with susceptibility to UC and CD and is upregulated in both UC and CD patients [14–16]. In recent years, studies have shown that TL1A participates in the development of intestinal inflammation and fibrosis [17, 18]. Moreover, mice with high expression of TLIA can develop spontaneous ileitis, proximal colitis, and even fibrosis, which is associated with elevated levels of fibrotic factor interleukin-13 (IL-13). Elevated secretion of IL-13 is tightly associated with the expression of TL1A in the transgenic mouse model described above [19, 20], and the fourth European Crohn’s and Colitis Organisation (ECCO) guidelines state that the TGF-β1/Smad3 pathway activated by IL-13 is a central process in the formation of intestinal fibrosis [21]. Further study has shown that in colitic mice with adoptively transferred T cells, intestinal inflammation and fibrosis could be alleviated with the treatment of anti-TL1A antibodies, which reduce the expression of α-SMA and vimentin and inhibit the TGF-β1/smad3 pathway [22]. Recombinant human bone morphogenetic protein-7 (BMP-7), which belongs to the TGF-β superfamily, reverses TGF-β1-induced EMT in intestinal fibrosis both in vitro and in vivo [11]. Therefore, we hypothesized that TL1A promotes intestinal fibrosis by inducing EMT. In this study, we evaluated the correlation between TL1A expression and changes in EMT-related markers in patients with UC or CD. Furthermore, wild-type (WT) mice and TL1A transgenic (Tg) mice were used to establish a chronic colitis-associated intestinal fibrosis model. Moreover, HT-29 cells were stimulated with TL1A, anti-TL1A antibodies, and BMP-7, and the changes in EMT-related markers were evaluated. The effects and mechanisms of TL1A on EMT are discussed in order to bring forward new theoretical bases for the treatment of intestinal fibrosis. 2. Methods 2.1. Human Tissues Twelve patients with UC (male/female: 6/6) and ten patients with CD (male/female: 4/6) were enrolled in this study from the Second Hospital of Hebei Medical University (Shijiazhuang, China). The diagnosis of IBD was based on standard clinical, endoscopic, radiological, and histological findings. Normal colonic samples were taken from 8 control individuals (male/female: 4/4), who underwent colonoscopy for other reasons and were found to be normal in examination and histology. The clinical characteristics and inflammatory markers of the three groups are shown in Tables 1 and 2. The age, sex, and other relevant markers of the three groups were matched. This study has been approved by the Ethics Committee of Hebei Medical University. Controls () UC () CD () Mean age (year, range) Females 4 (50%) 6 (50%) 6 (60%) Males 4 (50%) 6 (50%) 4 (40%) Mean disease duration (year, range) 0 Disease activity No 8 (100%) 0 (0%) 0 (0%) Low 0 3 (25%) 3 (30%) Moderate 0 5 (41.67%) 5 (50%) Strong 0 4 (33.33%) 2 (20%) UC: ulcerative colitis; CD: Crohn’s disease. Disease activity based on modified Mayo score. Data are expressed as .
... We evaluated the effects of the VDR gene on the human intestinal epithelial tumor cell line HT29 by establishing a well-characterized model of TGF-β1-induced EMT (27,28). In HT29 cells, knockdown of VDR expression decreased epithelial integrity ( Fig. 5D; Fig. S5A). ...
Article
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We previously showed that vitamin D receptor (VDR) plays a crucial role in acute inflammatory bowel disease and that intestinal fibrosis is a common complication of Crohn’s disease (CD). Epithelial-to-mesenchymal transition (EMT) is an important hallmark of fibrogenesis through which epithelial cells lose their epithelial phenotype and transform into mesenchymal cells. It is known that VDR plays an essential role in epithelial integrity and mitochondrial function, but its role in intestinal fibrosis remains unknown. Here, we investigated whether VDR is involved in epithelial mitochondrial dysfunction that results in EMT in intestinal fibrosis. Using human CD samples, intestinal-specific VDR-knockout (VDRIEC-KO) mice, and fibroblast cellular models, we showed that the expression of VDR was significantly lower in intestinal stenotic areas than in nonstenotic areas in patients with chronic Crohn’s disease. Genetic deletion of VDR in the intestinal epithelium exacerbated intestinal fibrosis in mice administered with DSS or TNBS, two experimental colitis inducers. Additionally, we found that vitamin D dietary intervention regulated intestinal fibrosis by modulating the intestinal expression of VDR. Mechanistically, knocking down VDR in both CCD-18Co cells and human primary colonic fibroblasts promoted fibroblast activation, while VDR overexpression or VDR agonist administration inhibited fibroblast activation. Further analysis illustrated that VDR inhibited EMT in the HT29 cell model and that mitochondrial dysfunction mediated epithelial integrity and barrier function in VDR-deficient epithelial cells. Together, our data for the first time demonstrate that VDR activation alleviates intestinal fibrosis by inhibiting fibroblast activation and epithelial mitochondria-mediated EMT.