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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Summary, General Discussion,
Conclusions, and Perspectives
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SUMMARY AND GENERAL DISCUSSION
Intestinal fibrosis is a chronic disease caused by persistent injury and inflammation of the intestine, which will ultimately result in loss of organ function.1 Understanding the development of intestinal fibrosis is important to be able to detect the disease at an early stage and to prevent surgical intervention. Furthermore, knowledge about the pathophysiology of intestinal fibrosis will also benefit the development of antifibrotic drugs.
This thesis delineates the use of precision-cut intestinal slice (PCIS) as a tool to study the pathophysiology of intestinal fibrosis, as well as a drug screening platform. The thesis consists of two parts; the first part focuses on the exploration of several drug candidates for the treatment of fibrosis by using the precision-cut tissue slices (PCTS). The second part is devoted to the use of PCIS to study the regional differences in the early onset of intestinal fibrosis and intestinal drug metabolism.
IBD, Crohn’s disease, and intestinal fibrosis
Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the intestinal tract. IBD has been associated with an imbalance of the intestinal microbiota, genetics as well as external and environmental factors (including smoking, diet, and lifestyle).2–4 IBD is traditionally considered as a disease of Westernized nations (North America, Europe, and Oceania). However, the epidemiology of IBD changed throughout the world at the turn of the 21st century. There is an increase in the incidence of IBD in South America, Eastern Europe, Asia, and Africa.5Over 1 million residents in the USA and 2.5 million in Europe are estimated to have IBD.6 Crohn's disease (CD) and ulcerative colitis (UC) are the most widely known types of IBD.2In Indonesia, the IBD incidence rate has been reported to be 1.7 per 100,000 persons and 0.33 per 100,000 persons are categorized as CD patients.7 Over their lifetime, more than 50% of CD patients have clinically apparent intestinal obstruction due to fibrostenosis.8Although the pathophysiological mechanism of intestinal fibrosis is not fully understood, the main effector cells for fibrogenesis are mesenchymal cells, which holds true for all types of organ fibrosis. The mesenchymal cells consist of three distinct but interrelated forms: the fibroblast, the myofibroblast and the smooth muscle cell.9 Regional differences in the cellular composition of the intestinal tract might influence
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the development of intestinal fibrosis in different parts of the intestine. The main pathway driving fibrogenesis in CD is related to transforming growth factor-β1 (TGF-β1).10–12When TGF-β1 is released, mesenchymal cells in the intestine are activated and start to produce extracellular matrix (ECM).13Besides TGF-β1, various cytokines are elevated in inflamed regions, including platelet-derived growth factor (PDGF),14–16 which will ultimately lead to the development of intestinal fibrosis.
Pro-inflammatory markers in fibrosis
In IBD, the initiation and continuance of intestinal inflammation and fibrogenesis are depended on the persistent and/or recurrent epithelial injury.1 Epithelial and endothelial damage results in the release of chemotactic factors that promote the recruitment and activation of innate and adaptive immune cells.17 Activated innate immune cells, including monocytes, neutrophils, and mast cells, produce pro-inflammatory and pro-fibrotic molecules like interleukins (ILs), tumor necrosis factor-α (TNFα), TGF-β1 and PDGF.1 Moreover, innate immune signaling pathways are important drivers of myofibroblast transdifferentiation.18
IL-1 is a pro-fibrotic cytokine that promotes myofibroblast activation, stimulates the production and secretion of chemokines and matrix metalloproteinases (MMP) by myeloid cells.19,20IL-6 is a potent pro-inflammatory cytokine, which plays an important role in the pathogenesis of IBD.21,22It stimulates fibrogenetic mesenchymal cells, increases the expression of TGF-β and TGF-β receptor 2 in the epidermal, and promotes fibroblast proliferation.23 IL-8 is a chemoattractant for immune cells, promotes cell proliferation, motility, invasion, and epithelial-mesenchymal transition, and has pro-angiogenesis functions.24,25In Chapter 3, some of these pro-inflammatory markers were investigated namely IL-1, IL-6, and IL-8 (CXCl1/KC). We demonstrated that Il-6 was up-regulated in murine precision-cut liver slices (PCLS). Moreover, all of the tested pro-inflammatory markers were up-regulated in human PCIS, as well as Il-6 and Cxcl/kc or Il-8 in murine PCIS and rat PCIS. Our findings support the notion that IL-6 plays an important role in the pathogenesis of fibrosis in the intestine,26,27liver,28,29 kidney,30,31and lung.32,33Therefore, IL-6 might possibly be used as fibrosis biomarker. However, more studies are needed to unveil sensitivity and specificity.
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Markers for intestinal fibrosis
To elucidate the pathophysiology of intestinal fibrosis, it is important to develop a model that recapitulates intestinal fibrosis in vivo. To this end, PCIS hold great potential. In this ex vivo model, all the different cells that constitute an organ are maintained in their original tissue environment allowing for cell-cell and cell-matrix interactions.34 Moreover, in PCIS the organization of the intestinal villi and microvilli
is well conserved.35 Also, the method is highly efficient, since a large number of slices
can be prepared from a small tissue sample, and the slices are relatively easy to process.36 However, we are still in need of good markers that allow us to monitor
fibrogenesis in PCIS. In Chapter 2, 3, and 4 we used markers that were previously described by Pham and colleagues, the majority of which are directly regulated by TGF-β1.
TGF-β1 is produced by macrophages and fibroblasts. Activation of TGF-β1 signaling will increase the production of ECM proteins (e.g. collagen, fibronectin, tenascin, laminin, and entactin).37 Moreover, TGF-β1 is the most potent inductor of
αSMA in the intestine.1 αSMA-positive myofibroblasts is the main cell type that
produces ECM during intestinal fibrosis. Interestingly, in Chapter 4, αSMA gene expression was decreased in both murine and human jejunum PCIS. This might indicate that there is a loss of fibroblasts. The reduction in αSMA gene expression is also observed in slices prepared from other tissues.38 However, in these slices, the
protein level of αSMA remained constant over time.39–41 Further studies are needed to
elucidate if this is also the case in PCIS. In Chapter 2, we showed upregulation of αSma during culture with TGF-β1 in murine PCIS (mPCIS) of the jejunum. In contrast, the expression of αSma remained unaffected by TGF-β1 in ileum and colon mPCIS (Chapter 4). This might indicate that there are regional differences in the sensitivity to TGF-β. However additional studies are needed to elucidate the impact of TGF-β1 on αSma protein expression in mPCIS.
TGF-β and its receptors are over-expressed particularly in fibrostenotic CD and in animal models of intestinal fibrosis42–44, the expression of these receptors still has
to be clarified in murine and human PCIS. Activation of the TGF-β signaling pathway will induce the expression of plasminogen activator inhibitor 1 (PAI-1) and connective tissue growth factor (CTGF).1,45 In Chapter 2, we clearly demonstrated that Pai-1 and
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Ctgf expression was induced in jejunum mPCIS following treatment with TGF-β1. Our
results were in line with the previous study by Pham et al., they discovered that CTGF and PAI-1 expression was up-regulated in the jejunum of human, rat, and mouse PCIS.35 Furthermore, activation of the TGF-β signaling pathway increases the expression of pro-collagen 1α1 (Col1α1) and heat shock protein 47 (Hsp47), a collagen-specific molecular chaperone that plays a role in the maturation, biosynthesis, and secretion of various types of collagens.46 In Chapter 2, we showed that the gene expression of all several fibrosis markers (Col1α1, Hsp47, and Fn2) was induced during culture with TGF-β in jejunum mPCIS. Moreover, as described in Chapter 4, exposure of colon mPCIS to TGF-β1 increased the expression of all the tested fibrosis markers, and in ileum mPCIS, we observed a significant increase of Col1α1. In contrast, exposure of human PCIS to TGF-β1 did not alter the gene expression of any of the tested genes. Taken together, these results indicate that TGF-β1 is a major profibrotic stimulus in mPCIS, but not in hPCIS. This might be due to species differences in the sensitivity to TGF-β1.
Exploration of antifibrotic drugs
In Chapter 2 and 3, we studied the antifibrotic efficacy of several compounds. Of the various compounds we tested, only sunitinib showed potential antifibrotic effects (Chapter 2). Sunitinib reduced the gene expression of Pai-1 and Ctgf suggesting that it has an inhibitory effect upstream of the molecular signaling pathways, most likely by blocking the PDGF-α and PDGF-β receptors.47These findings also indicate that sunitinib might target the TGF-β pathway. This potential dual effect on both the TGF-β and PDGF signaling pathway makes sunitinib an interesting drug candidate for the treatment of intestinal fibrosis. Still, more studies are needed to fully unveil the therapeutic effectiveness of sunitinib.
Many of the compounds tested in Chapter 2 were demonstrated to have an antifibrotic effect in precision-cut liver slices; however, only sunitinib was effective in PCIS. A similar observation was made with rosmarinic acid. As detailed in Chapter 3 rosmarinic acid only showed antifibrotic properties in rodent and human liver slices,48 but not in PCIS. Thus, the antifibrotic effects of putative drugs are clearly organ- and species-specific.
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Regional, sex, species, and organ differences in the ex vivo model for fibrosis
It is important to study intestinal fibrosis in different parts of the intestine, because regional differences in the cellular composition of the gut may influence the local fibrotic process. In Chapter 4 we treated murine and human PCIS with 5 ng/ml TGF-β1. This concentration did not elicit toxicity in colon hPCIS, while it was toxic in colon mPCIS (Chapter 4). In addition, Pham et al. showed that the same concentration of TGF-β1 was not toxic for jejunum mPCIS and hPCIS,35 and others have
demonstrated that it was not toxic for liver,49 kidney,50 and lung slices (Putri et al.,
manuscript in preparation). Why colon mPCIS are the only slices prone to TGF-β1-induced toxicity requires further study.
Our results further demonstrated that Hsp47 and Fn2 expression levels were only increased during incubation in colon and jejunum mPCIS, 35 but not in ileum
mPCIS nor in ileum and colon hPCIS. Moreover, we showed that TGF-β1 only stimulated fibrosis in jejunum, ileum and colon mPCIS, and not in hPCIS (Chapter 2
and 4). These findings illustrate that the fibrotic process differs per intestinal region
and species. Therefore, it is of the utmost importance to take regional differences into consideration during drug development.
It is well known from rodent studies that there are regional differences in intestinal phase I and phase II drug metabolism.36 Chapter 5 describes the first-ever
study on intestinal drug metabolism in matched human ileum and proximal colon PCIS. Our results demonstrated that phase I metabolism of testosterone predominantly occurred in ileum PCIS, while phase II metabolism of 7-hydroxycoumarin glucuronide, mostly took place in proximal colon PCIS. These results are in line with a previous study showing that protein levels of cytochrome P450 (CYP)3A, which exhibits testosterone 6β-hydroxylase activity,51 is highest in the proximal region of the intestine and declines
in the distal part.51–54 In addition, Van de Kerkhof et al. showed that phase II
(7-hydroxycoumarin glucuronidation) metabolism predominantly occurred in the proximal jejunum and colon.55 In addition to regional differences in intestinal
metabolism, we also found sex differences (Chapter 5). In both regions, we observed that the rate of phase II metabolism was higher in PCIS prepared from male patients as compared to PCIS prepared from females. To the best of our knowledge, this has not
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been reported before. However, our study only included a small number of patients, so more research is needed to completely characterize sex differences in intestinal drug metabolism.
Taken together, this research has made it very clear that organ, sex, and species differences should be taken into account during the search for novel therapeutic agents.
CONCLUSIONS AND PERSPECTIVES
Previously, our group successfully established PCIS as an ex vivo model for the early onset of fibrosis.56In this thesis, we successfully used this model for testing the antifibrotic potential of putative drugs. Furthermore, we also studied regional differences in fibrogenesis and drug metabolism.
Since CD is heterogeneous in its pathology, and mostly present in the ileum and colon, it is essential to study the early onset of fibrosis and the effect of antifibrotic compounds in different intestinal regions. Due to intra-individual differences in drug responses, one would, ideally, use different gut regions from the same patient for drug testing, which is arduous. Therefore, these studies are lacking. However, we were able to use matched ileum and colon PCIS, which were prepared from the same patient. Thus, this approach is feasible.
Moreover, it is essential to study the efficacy of antifibrotic drugs in established fibrosis. Therefore, future studies should include hPCIS prepared from fibrotic tissue. These studies are ongoing but are hampered by the fact that human intestinal stricture samples are still relatively scarce. It is possible to isolate fibroblasts from strictures of CD patients.57–59However, these cells will not fully recapitulate in vivo pathogenesis and drug responses.
In our study with human PCIS, TGF-β1 did not elicit a fibrotic response in the ileum and colon. This could be due to the fact that an additional trigger (like PDGF) is necessary to induce fibrosis in human PCIS. Previous work from our group showed that both TGF-β1 and PDGF were necessary to promote fibrogenesis in human liver slices.49 To investigate whether the same holds true for hPCIS studies are currently being performed in which human PCIS are treated with the combination of TGF-β1 and PDGF.
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The studies described in this thesis mainly investigated the gene expression of fibrosis markers. However, it is known that mRNA levels not always directly reflect protein expression due to the time needed for transcription and translation.39–41One disadvantage of the PCTS model is that the slices remain viable and functional for a limited period of time.56,60–62If the culture period could be extended, this would allow for more detailed studies on (ECM) protein expression. Therefore, new culture strategies, such as already implemented for PCLS,61 or by using organoid medium63 (Bigaeva et al., manuscript in preparation), could be utilized to prolong PCIS culture.
Furthermore, this human model, which is highly efficient and relatively easy to use, may become an effective tool in exploring new antifibrotic drug candidates. In addition, PCIS can also be used to evaluate the safety of drug candidates and can be utilized to assess if the candidate drug is delivered to the site action. Finally, PCIS are in good compliance with the 3Rs (replacement, reduction, and refinement), most notably with reduction, because the number of animals needed can be considerably reduced when using PCIS. Hopefully, human PCIS will ultimately eliminate the need for animal studies in the field of intestinal fibrosis.
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