Exploring the Regional Characteristics of Intestinal Drug Metabolism and Fibrogenesis Iswandana, Raditya
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Iswandana, R. (2019). Exploring the Regional Characteristics of Intestinal Drug Metabolism and Fibrogenesis. University of Groningen.
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1
General Introduction,
Scope, and Aim of the Thesis
Chapter 1
GENERAL INTRODUCTION
Chronic tissue inflammation or injury leads to fibrosis, a pathological process that is characterized by the excessive accumulation of extracellular matrix (ECM) in an organ, which will ultimately lead to loss of organ function. Fibrosis can affect almost all tissues and organs.1 In fact, 45% of deaths worldwide can be attributed to fibrotic disorders.1,2
In this thesis, the focus is mainly on intestinal fibrosis. The intestines play an important role in the absorption of, among others, nutrients and water and intestinal malfunctioning will lead to a systemic imbalance of nutrients and water. Inflammatory bowel diseases (IBD) are related to an immunological disruption of the intestinal mucosa, mainly associated with cells of the adaptive immune system, which respond to the self-antigen producing chronic inflammatory conditions in these patients.3 Crohn's disease (CD) and ulcerative colitis (UC) are the most widely known types of IBD and have been the focus of attention due to their increasing incidence.3 CD generally involves the ileum and colon, but it can affect any region of the gastrointestinal tract, often discontinuously. UC involves the rectum and may affect a part of or the entire colon (pancolitis) in an uninterrupted pattern.4
Intestinal fibrosis is predominantly found in patients with CD.5,6More than 50% of CD patients have over their lifetime clinically apparent intestinal obstruction due to fibrostenosis, while for UC patients, reports of stenosis in the colon vary between approximately 1-11%.6,7CD is a life-long, incurable, disabling inflammatory disorder frequently diagnosed between the age of 15 and 35 years, and the incidence continues to increase worldwide.8 In CD, fibrosis is the end product of chronic transmural inflammation and dysregulated healing mechanisms, resulting in excessive and abnormal deposition of ECM.9The main purpose of CD therapy is to avoid endoscopic or surgical intervention; however, most patients will end up having at least one surgery during their life time because of stricture formation.6 After several years, these CD patients usually have recurrence of the stricturing disease and will need to undergo surgery again.6 Since CD is known as a chronic inflammatory disease, improved therapies to control inflammation have been developed.6,10 However, this did not change the incidence of strictures (i.e. intestinal fibrosis) in CD patients. Thus, there is
1
an urgent need to find an effective therapy for intestinal fibrosis, targeting molecular mechanisms other than inflammation.
Intestinal fibrosis is characterized by abnormal ECM deposition. The ECM is mainly produced by activated myofibroblasts1. The main effector cells in intestinal fibrosis, are mesenchymal cells, which exists in three distinct but interrelated forms: the fibroblast, the myofibroblast, and the smooth muscle cell.11Regional differences in the cellular composition of the intestinal tract might influence the development of intestinal fibrosis in different parts of the intestine and may partly explain why fibrosis is predominantly found in the ileum and ileocolonic region, as compared to other parts of the intestine.11,12 Activation of collagen-secreting myofibroblasts is ultimately responsible for increased tissue stiffness and progressive organ dysfunction.13 This process is further enhanced by both the innate and adaptive immune system, and includes pro-inflammatory and profibrotic molecules such as interleukins (ILs), tumor necrosis factor-α (TNFα), transforming growth factor (TGF)-β1 and platelet-derived growth factors (PDGF).13 Fibrogenesis is further boosted through the differentiation, recruitment, proliferation, and activation of ECM-producing myofibroblast progenitors as well as dedifferentiation of epithelial cells by epithelial-mesenchymal transition (EMT)14 and endothelial cells via epithelial/endothelial–mesenchymal transition. Other players include stellate cells, pericytes, and bone marrow stem cells1,13,15(Figure
1).
The etiology of intestinal fibrosis is diverse and disease development can occur in patients after radiation therapy,16 due to internal factors like gut microbiota or fibrogenesis can be triggered by external and environmental factors.17
Irrespective of the initial trigger, numerous molecules are associated with the development of fibrosis. To date, the most important molecular mediators for intestinal fibrosis are TGF-β, PDGF, activins, connective tissue growth factor (CTGF), insulin-like growth factor (IGF)I, epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), various cytokines (e.g. interleukin [IL]-6, TNF-α and interferon [IFN]-γ), various chemokines (e.g. CCL2 [monocyte chemoattractant protein-1; MCP1], CCL3 [macrophage inflammatory protein-1; MIP1], CCL4 [MIP-1β] and CCL20 [MIP-3α]) as well as reactive oxygen species (ROS) and associated products (Figure 2).13
Figure 2. Major molecular mediators in the intestinal fibrosis through activated myofibroblasts. Pro- or
antifibrogenic effect (adapted from Lawrance et al., 2017).13
Some of these molecules are being investigated as potential targets of antifibrotic drugs. For instance, the growth factors TGF-β, PDGF, and CTGF, mediators that activate myofibroblasts, play an important role in wound healing, fibrosis, cell proliferation, and ECM production.13,18 Also, cytokines like IL-1 contribute to fibrosis during chronic intestinal inflammation. IL-1, in combination with TNF and IFN-γ, also
1
increases TGF-β-induced EMT, which promotes fibrogenesis through myofibroblast activation, and chemokine and matrix metalloprotease (MMP) secretion.19
TGF-β is primarily produced by several tissue-resident and blood-derived cells.20 TGF-β has a critical role in intestinal mesenchymal cell activation and ECM production.21In general, TGF-β increases collagen, fibronectin, tenascin, laminin, and entactin production.13 It also upregulates the tissue inhibitor of metalloproteinases (TIMP) expression and is a potent inductor of α-smooth muscle actin (α-SMA), a marker of myofibroblast.13 The intracellular TGF-β signal transduction pathway is mediated via Smad proteins. This TGF-β/Smad pathway is important in intestinal fibrosis since both TGF-β, and its receptors are overexpressed in fibrostenotic CD and animal models of intestinal fibrosis.1,22 Therefore, inhibiting TGF-β signaling seems a promising strategy for fibrosis prevention.23However, since TGF-β is also involved in several vital cellular functions, e.g. differentiation, chemotaxis, proliferation, and activation of many immune cells,24 fully blocking this pathway is associated with severe side effects. Still, selective inhibition of mediators in the TGF-β/Smad pathway can be useful to prevent the fibrotic process in the intestine (Chapter 2 and Chapter
4).
PDGF is produced by, among others, macrophages, endothelial cells, fibroblasts, glial cells, astrocytes, myoblasts, and smooth muscle cells.25 PDGF has a crucial role during the development of fibrosis in several organs, including the intestine. The levels of PDGF are increased in the inflamed mucosa of IBD patients, especially in the intestinal mucosa of CD patients. PDGF increases the migratory capacity of fibroblast and myofibroblast and promotes ECM deposition.13 In addition, proteins from the PDGF family, mainly PDGF-BB, promote the proliferation of intestinal fibroblasts and subepithelial myofibroblasts.1 Several groups have shown that inhibiting the PDGF pathway in the liver, lung, and kidney successfully decreased fibrogenesis.26–28 Moreover, we were the first to show that inhibitors of the PDGF pathway could have antifibrotic effects in an ex vivo model of intestinal fibrosis (Chapter 2).
CTGF is a downstream mediator of TGF-β, CTGF stimulates cell proliferation and ECM synthesis.13Its expression is controlled by TGF-β in a Smad-dependent manner.29 Besides TGF-β, there are also other regulators of CTGF expression, such as VEGF, TNF-α, and ROS.30CTGF could be a useful target for antifibrotic therapies since inhibitors of
CTGF will block the profibrotic effects of TGF-β without affecting the immunosuppressive and anti-inflammatory functions of TGF-β.31 In Chapter 2, the effect of several antifibrotics was investigated on the CTGF gene expression in intestinal slices.
Numerous in vitro and animal models are available to study intestinal fibrosis.32 To date, in vitro models include fibroblast cultures,33co-cultures,34three-dimensional culture models (between enterocytes, monocytes and dendritic cells),35and intestinal organoids36. In addition, there are different animal models of intestinal fibrosis, i.e. spontaneous, gene-targeted, chemical-, immune-, bacteria-, radiation-induced, and postoperative fibrosis.32
In the animal models that spontaneously develop intestinal fibrosis, the inflammatory and fibrotic responses occur without any exogenous manipulation. The senescence-accelerated mouse (SAM)P1/Yit and SAMP1/YitFc mice were generated by the mating of a senescence-accelerated mouse line.37,38 These mice show impaired immune functions with age, resulting in enteritis manifesting as a discontinuous lesion in the terminal ileum and caecum.37Since the terminal ileum is the primary location of strictures in CD patients, this murine model can be used as a model to study the pathogenesis of intestinal stricture formation. However, these mice have a low breeding rate and are not commercially available, therefore these mice cannot be used on a large scale.
Manipulation of selected inflammation-associated genes that may lead to fibrosis is also used to create experimental models, i.e. IL-10 deficiency,39 TGF-β overexpression,40 and monocyte chemoattractant protein (MCP)-1 overexpression41. These murine models are only useful to study the effect of antifibrotic compounds that target the specific pathways that have been manipulated. Because of this limitation, these models have not been used extensively to study intestinal fibrosis.
Healthy wild-type animals can develop intestinal fibrosis by exposing them to exogenous agents that stimulate a local inflammatory response. Compounds that can be used to induce intestinal fibrosis include dextran sodium sulfate (DSS),42 trinitrobenzene sulfonic acid (TNBS)43 and peroxynitrite.44 These agents are widely used since they cause epithelial cell injury followed by acute and chronic inflammatory
1
responses. However, the clinical relevance of these models regarding CD-associated intestinal inflammation and fibrosis is questionable.
Immune-mediated models are developed to investigate the role of the intestinal mucosa during gut inflammation. One example is the T-cell naive CD45RBhigh CD4+ transfer model.45This model is associated with an increase in fibroblast and narrowing of the intestinal lumen. Since T-cells are also involved in chronic intestinal inflammation and fibrosis seen in human IBD, these models seem promising.
In other models, microbial components are used to induce gut inflammation.46 Intestinal inflammation in these animals can be induced by injecting bacterial cell wall polymer peptidoglycan-polysaccharide directly into the bowel wall.47 Another approach is injecting the colonic wall with a fecal suspension of luminal contents or selected aerobic and anaerobic strains isolated from the same animals,32or by infecting the animal with live bacteria (Salmonella enterica) via oral delivery.48 All of these protocols lead to an increased expression of TGF-β1, IGF-I, CTGF, and collagen. It is a widely held belief that CD is closely linked with immune responses against the gut microbiota, therefore these models seem to be useful to better understand one of the key steps in the development of intestinal fibrosis.
Exposure of the intestines to therapeutic doses of radiation will cause the development of intestinal inflammation and subsequent intestinal fibrosis.16Based on this observation, experimental models of radiation-induced intestinal fibrosis in rats and mice were developed. In these models, upregulation of TGF-β1, CTGF, ECM proteins were observed as well as thickening of a submucosal and serosal layer of the intestine, thus mimicking radiation-induced human intestinal fibrosis.16,49
As mentioned before, 50% of CD patients need surgery because of stricture formation, and in up to 40% of patients, there will be a symptomatic recurrence after 4 years.7,50 Currently, it cannot be predicted which patients are at risk for the recurrence of strictures. Therefore, experimental models are needed to study clinically relevant postoperative recurrence. An attempt was made with ileocecal resection in IL-10-/-mice, this mouse model develops inflammation and intestinal fibrosis.51
The available in vitro and animal models of intestinal fibrosis help to unravel the pathophysiological mechanisms underlying disease development and progression, and are used in preclinical therapeutic studies of fibrosis. However, none of these
models is regarded as the golden standard, because they all fail to recapitulate the numerous pathophysiological and clinical features of human intestinal fibrosis. Furthermore, in most animal models, intestinal fibrosis spontaneously reverses as soon as the inducing stimulus is removed, unlike the actual human situation.32 To tackle these issues, a new ex vivo model was developed -precision-cut tissue slices (PCTS)- as an ideal model to study the multicellular process of fibrosis. This model is a useful tool to study organ fibrosis in both human and animal tissue.52–54 Human precision-cut intestinal slices (PCIS) may better reflect the clinical condition because the slices contain all the different cells in their original organ environment allowing for cell-cell and cell-matrix interactions, also the organization of the intestinal villi and microvilli is well conserved.55Furthermore, this technique is highly efficient, since a large number of intestinal slices can be prepared from a small tissue sample, and the slices are relatively easy to process and culture.56Thus, by using PCIS, we can test novel antifibrotic drugs (Chapter 2 and Chapter 3), study the factors that play a role in fibrogenesis (Chapter 4) and intestinal drug metabolism (Chapter 5).
SCOPE AND AIM OF THE THESIS
Our group is working on the forefront of translational disease models and has been greatly involved in the development of PCIS as intestinal fibrosis model. Previously, Pham et al. studied the early onset of intestinal fibrosis in PCIS prepared from the jejunum of mouse, rat, and human.53 In this thesis, this body of work was expanded by further characterizing the use of jejunum PCIS for the study of fibrosis and the testing of antifibrotic compounds. In Chapter 2 mouse jejunum slices were used to study the antifibrotic efficacy of various TGF-β pathway inhibitors including valproic acid, tetrandrine, pirfenidone, SB203580, and LY2109761 as well as PDGF pathway inhibitors (e.g. imatinib, sorafenib, and sunitinib). This chapter demonstrated the usefulness of PCIS as drug testing platform.
Previously, Westra et al. studied the antifibrotic efficacy of rosmarinic acid in human and rat liver slices.52In Chapter 3, this study was complimented by evaluating the antifibrotic efficacy of rosmarinic acid in mouse liver slices, as well as in mouse, rat, and human jejunum PCIS. Moreover, the anti-inflammatory effects of rosmarinic acid in tissue slices were also evaluated.
1
However, in CD, the ileum and colon are mainly affected. Therefore, in Chapter
4, it was investigated whether human and mouse PCIS could be used to study the early
onset of intestinal fibrosis in ileum and colon. In Chapter 5 the regional differences were studied in intestinal metabolic activity by using human ileum and colon PCIS.
The aim of this thesis was to identify potential antifibrotic compounds for the treatment of fibrosis, including intestinal fibrosis, by using PCTS. Moreover, we aimed to elucidate regional differences in the early onset of intestinal fibrosis as well as intestinal drug metabolism by using PCIS.
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