© Birkhäuser Verlag, Basel, 2008 Inflamm. res. 57 (2008) 84–91 1023-3830/08/020084-8 DOI 10.1007/s00011-007-7098-y
Inflammation Research
Dextran sulfate sodium-induced acute colonic inflammation in angiotensin II type 1a receptor deficient mice K. Katada1,5, N. Yoshida2, T. Suzuki1, T. Okuda1, K. Mizushima1, T. Takagi3, H. Ichikawa1, Y. Naito4, G. Cepinskas5 and T. Yoshikawa1,3,4 1
Inflammation and Immunology Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602-8566 Japan, Fax: ++81 75 251 0710, e-mail:
[email protected] 3 Biomedical Safety Science 4 Medical Proteomics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan 5 Centre for Critical Illness Research, Lawson Health Research Institute, London, Ontario, Canada 2
Received 11 May 2007; returned for revision 12 July 2007; accepted by M. Katori 19 September 2007
Abstract. Objective: Angiotensin II (Ang II) receptor blockers have been reported to contribute to cytoprotective effects in various organs. However, the role of renin-angiotensin system (RAS) in modulation of the inflammatory bowel disease (IBD) remains unclear. In this study we assessed the role of angiotensin II type 1a (AT1a) receptor on the outcome of dextran sulfate sodium (DSS)-induced acute colitis by employing AT1a receptor deficient mice. Materials and methods: The acute colitis was induced in wild type (WT) and AT1a receptor deficient mice by giving orally 3 % DSS in drinking water for 7 days. Results: Induction of DSS colitis resulted in up-regulation of Ang II and AT1a receptor in the colonic mucosa of WT mice. In parallel, loss of body weight, an increase in disease activity index (DAI), and the shortening of colon were found in DSS-challenged WT mice. In addition, an increase in thiobarbituric acid (TBA)-reactive substances and myeloperoxidase (MPO) activity, along with the up-regulation of tumor necrosis factor (TNF)-a were detected in the colonic mucosa of DSS-challenged WT mice. The endpoints mentioned above were significantly ameliorated in DSS-challenged AT1a receptor deficient mice. Conclusions: RAS is involved in the pathophysiology of DSS-induced colitis and AT1a receptor may be a novel therapeutic target for the treatment of IBD. Key words: Experimental colitis – AT1a receptor – Angiotensin II – Inflammation – TNF-a.
Correspondence to: N. Yoshida.
Introduction Renin-angiotensin system (RAS) plays a major role in regulating the blood pressure and body fluid homeostasis [1]. Angiotensin II (Ang II), the main peptide of RAS, is considered as a proinflammatory mediator that participates in inflammatory responses such as apoptosis, angiogenesis, vascular remodeling, and inflammation [2–5]. In regard to the latter it has been shown that Ang II modulates the expression of proinflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-a, and is capable of inducing production of reactive oxygen species (ROS) [6–8]. Ang II is converted from angiotensin I by angiotensinconverting enzyme (ACE) and binds to its specific membrane receptors, angiotensin II type 1 (AT1) or type 2 (AT2) receptors. In mice, the AT1 receptor is further subdivided into AT1a and AT1b receptors. The studies addressing the role of RAS indicate that interfering with RAS function either by using ACE inhibitors or AT1 specific Ang II receptor blockers (ARB) attenuates inflammatory response with respect to pro-inflammatory cytokine production [9–11]. Similar observations were also made employing AT1a receptor deficient mice [2, 12–18]. With respect to the latter it has been shown that the absence of AT1a receptor has relevance to hypotension, hyperreninemia [12] and renal growth and development [13]. In addition, recent studies indicate that AT1a receptor plays a significant role during inflammatory conditions such as anti-glomerular basement membrane nephritis [14], renal interstitial fibrosis [15], atherosclerosis [16], hepatic inflammation [2], cardiac remodeling [17] and tumor angiogenesis [18]. Unfortunately, there is little known in regard to the role of RAS in the development of inflammation in the gut. To assess the potential role of RAS in gut inflammation we employed a model of dextran sulfate sodium (DSS)-induced
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colitis in mice. DSS-induced colitis model used in this study has been proven to be similar to ulcerative colitis (UC) in human [19]. Although the pathogenesis of DSS-induced colitis is not entirely clear, its induction may result from the toxic effects of DSS on colonic epithelial cells [19]. In addition, the cytokine expression in DSS-induced colitis has been shown to be similar to those observed in human inflammatory bowel disease (IBD) [20, 21]. Our previous studies also indicate that DSS-induced colitis is accompanied by the increase in mucosal proinflammatory cytokine, such as IL-6 and TNF-a, production and lipid peroxidation [22–24]. Therefore, the aim of this study was to assess the role of RAS (AT1a receptor) in the development of colitis by employing a model of DSS-induced acute colitis in WT and AT1a receptor deficient mice. Materials and methods Chemicals All chemicals were prepared immediately before use. Thiobarbituric acid (TBA) and 3,3’,5,5’-tetramethylbenzidine were obtained from Wako (Osaka, Japan). 1,1,3,3-tetramethoxypropane was obtained from Tokyo Kasei (Tokyo, Japan). An enzyme-linked immunosorbent assay kits for mouse Ang II and TNF-a were obtained from SPI Bio (Montigny, France) and BioSource International (Camarillo, CA). All other chemicals used were of reagent grade.
85 The colon was placed on a nonabsorbent surface and measured with a ruler. The entire colon was fixed in 10 % neutral buffered formalin. After fixation, the samples were embedded in paraffin, cut into 7-µm sections, and stained with hematoxylin and eosin.
Measurements of TBA-reactive substances and MPO activity The concentrations of TBA-reactive substances were measured in the colon mucosa using the method of Ohkawa et al. [26] as an index of lipid peroxidation. The animals were sacrificed by exsanguinations from the abdominal aorta after the experiments, the colons were removed, and the colon mucosa was scraped off using two glass slides, then homogenized with 1.5 mL of 10 mM potassium phosphate buffer (pH 7.8) containing 30 mM KCl in a Teflon Potter-Elvehjem homogenizer. The levels of TBA-reactive substances in the mucosal homogenates were expressed as nanomoles of malondialdehyde per milligram of protein using 1,1,3,3tetramethoxypropane as the standard. Total protein in the tissue homogenates was measured by the method of Lowry [27]. Tissue-associated MPO activity was determined by a modification of the method of Grisham et al. [28] as an index of neutrophil accumulation. Two milliliters of mucosa homogenates was centrifuged at 20,000 × g for 15 min at 4 ºC to pellet the insoluble cellular debris. The pellet was then re-homogenized in an equivalent volume of 0.05 M potassium phosphate buffer (pH 5.4), containing 0.5 % hexadecyltrimethylammonium bromide. The samples were centrifuged at 20,000 × g for 15 min at 4 ºC, and the supernatants were saved. MPO activity was assessed by measuring the H2O2-dependent oxidation of 3,3’,5,5’-tetramethylbenzidine. One unit of enzyme activity was defined as the amount of MPO present causing a change in absorbance of 1.0/min at 655 nm and 25 ºC.
Experimental design Determination of Colonic mucosal Ang II and TNF-a Eight-week-old male AT1a receptor deficient mice were obtained from Discovery Research Laboratory (Tanabe Seiyaku Co., Ltd., Osaka, Japan). Appropriate background strain male WT (C57BL/6) mice were purchased from Shimizu Experimental Animal (Osaka, Japan). The mice were housed individually in cages in a room kept at 18–24 °C and 40 % to 70 % relative humidity, with a 12-h light/dark cycle. They were allowed free access to their food and drinking water. During their acclimatization period, they were fed laboratory chow (Nihon Clea, Tokyo, Japan) for 7 days. Acute colitis was induced by oral administration of 3.0 % (w/vol) DSS (mol. wt 5,000, Wako Pure Chemical Industries, Ltd., Osaka, Japan) in drinking water for 7 days. Control mice received drinking water ab libitum. For the kinetics study, the animals were sacrificed on day 0, 3, 5, and 7.
Evaluation of Colitis Severity The parameters recorded in the experiments were the body weight change, the disease activity index (DAI), colon length, and histology. For determining DAI scores, body weight, occult and rectal bleeding, and stool consistency were monitored daily. Occult bleeding was tested using a commercial kit based on the detection of the peroxidase activity of heme in stool (occult Blood Slide 5 Shionogi, Shionogi&Co., Osaka, Japan). DAI was determined by scoring changes in weight, occult blood positivity, and gross stool consistency, as described previously [25]. This method has been shown to correlate well with histological measures of inflammatory and crypt damage. We used five grade score for weight loss evaluation (0 – no loss; 1 – 1 % to 5 %; 2 – 5 % to 10 %; 3 – 10 % to 20 %; 4, loss of more than 20 %), three grade score for stool consistency (0, normal; 2, loose; and 4, diarrhea), and three grade score for occult blood (0, normal; 2, occult blood-positive; 4, gross bleeding) evaluation. The combined scores in each group were divided by 3 to obtain final DAI score. After determining DAI, mice were sacrificed by cervical dislocation, and the colon was resected between the ileo-cecal junction and the proximal rectum, close to its passage under the pelvisternum.
The concentrations of Ang II and TNF-a in the supernatants of mucosal homogenate were determined by ELISA specific for mouse Ang II and TNF-a. The assay was performed according to the manufacturer’s instructions. After color development, optimal densities were measured at 405 nm for Ang II and 450 nm for TNF-a with a microplate reader (MPR A4i, Tosoh, Tokyo, Japan). The concentrations of Ang II and TNF-a were expressed as pg per mg protein.
Real-time PCR analysis for AT1a receptor and TNF-a mRNA expression The colonic mucosa was scraped off using two glass slides and then total RNA was isolated with the guanidinium phenol chloroform method using an ISOGEN kit (Nippon Gene, Tokyo, Japan) according to the manufacturer’s suggested protocol. RNA concentrations were determined by their absorbance at 260 nm in relation to the absorbance at 280 nm. RNA was stored at –70 ºC until real-time PCR was performed. An aliquot (1 µg) of extract RNA was reverse-transcribed into first-strand complementary DNA (cDNA) at 42 °C for 40 min, using 100 U/ml reverse-transcriptase (Takara Biochemicals, Shiga, Japan) and 0.1 µM of oligo (dT)-adapter primer (Takara Biochemicals, Shiga, Japan) in a 50 µl reaction mixture. Real-time polymerase chain reaction (PCR) was carried outwith a 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA) using the DNA-binding dye SYBER Green I for the detection of PCR products. The reaction mixture (RT-PCR kit, Code RRO43A, Takara Biochemicals, Shiga, Japan) contained 12.5 µl Premix Ex Taq, 2.5 µl SYBER Green I, custom-synthesized primers, ROX reference dye, and cDNA (equivalent to 20 ng total RNA) to give a final reaction volume of 25 µl. The primers had the following sequences: for AT1a receptor, sense 5’-TCGCTACCTGGCCATTGTC-3’ and antisense 5’-TGACTTTGGCCACCAGCAT-3’, for TNF-a, sense 5’-ATCCGCGACGTGGAACTG-3’ and antisense 5’ACCGCCTGGAGTTCTGGAA-3’ and for b-actin, sense 5’-TGTCCACCTTCCAGCAGATGT-3’ and antisense 5’-AGCTCAGTAACAGTC-
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Fig. 1. Expression of Ang II protein and AT1a receptor mRNA in colonic mucosa of WT mice following administration of DSS. (A) Ang II protein in the supernatants of mucosal homogenate was determined by ELISA. Each value indicates the mean/SEM. n = 6. #p < 0.05 compared to control mice. (B) AT1a receptor expression was assessed by RT-PCR and is presented as relative change in comparison to day 0. n = 6. †p < 0.05 compared to day 0.
CGCCTAGA-3’. The PCR settings were as follows: initial denaturation of 15 s at 95 ºC was followed by 40 cycles of amplification for 3 s at 95 ºC and 31 s at 60 ºC, with subsequent melting curve analysis increasing the temperature from 60 to 95 ºC. Relative quantification of gene expression with real-time PCR data was calculated relative to b-actin.
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The results presented are the mean/SEM. For the statistical comparison the data were analyzed using two-way analysis of variance (ANOVA) with the exception being Figure 1A, where the data were analyzed using one-way ANOVA. The differences were considered significant if the p value was less than 0.05 based on Scheff’s multiple comparison test. Samples for non-parametric analysis (mRNA expression and DAI score) were analyzed using Mann-Whitney U-test and expressed in a form of the box graphs. The differences between the groups were considered significant if the p-values were less than 0.05. All analyses were performed using the Stat View 5.0 program (Abacus Concepts Inc. Berkeley, CA).
Ethical considerations Maintenance of animals and experimental procedures were carried out in accordance with the NIH guidelines for the use of experimental animals. All protocols of the experiments were approved by the Kyoto Prefectural University of Medicine, Animal Care Committee (Kyoto, Japan).
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Fig. 2. Changes in body weight in DSS-challenged WT and AT1a (–/–) mice. The body weight change was assessed on days 0, 3, 5 and 7 following DSS administration. Each value indicates the mean/SEM. n = 10. #p < 0.05 compared to the same genetic background control mice. *p < 0.05 compared to DSS-treated WT mice on the corresponding day.
Results Up-regulation of Ang II and AT1a Receptor in colonic mucosa by DSS Administration The concentrations of Ang II in the supernatant of mucosal homogenate were determined by ELISA. The mucosal levels of Ang II protein were significantly up-regulated in DSS-
induced WT mice (Fig. 1A). In parallel, DSS administration also resulted in up-regulation of AT1a receptor mRNA expression in WT mice in a time-dependent manner (Fig. 1B). The significant increase in AT1a receptor expression was found on days 5 and 7 following DSS administration. On the contrary, there was no expression of AT1a receptor in AT1a receptor deficient mice (data not shown).
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Fig. 3. Disease activity index (DAI) in WT and AT1a (–/–) mice after DSS administration. DAI score was assessed on days 0, 3, 5 and 7 following DSS administration. n = 10. †p < 0.05 compared to the same genetic background mice on day 0. *p < 0.05 compared to DSS-treated WT mice on the corresponding day.
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Fig. 4. Colon length in DSS-challenged WT and AT1a (–/–) mice. The colon length was measured on day 7 after administration of DSS. Results are mean/SEM. n = 10. #p < 0.05 compared to control mice. *p < 0.05 compared to DSS-treated WT counterpart mice.
Functional and morphological characteristics of DSSinduced Colitis in WT and AT1a Receptor Deficient Mice The WT mice challenged with 3 % DSS developed symptoms of acute colitis, with diarrhea being observed first, followed by rectal bleeding and severe weight loss. The changes mentioned above were significantly ameliorated in AT1a receptor deficient mice (significant from days 5–7; Fig. 2). Important to note that there was no difference in the consumption of
Fig. 5. Histological appearance of the colon in DSS-treated mice. (A, C) Appearance of the colon in control WT and AT1a (–/–) mice, respectively. No signs of inflammation or changes in cytoarchitectural arrangement of the colon mucosa were noted. (B, D) Administration of 3% DSS for 7 days to both, WT (B) and AT1a receptor deficient mice (D) resulted in a loss of epithelial cells from villus tips (arrows), formation of edema and a prominent infiltration of inflammatory cells in to mucosa (arrow heads). Note that the above changes were less prominent in the colon of DSS-challenged AT1a receptor deficient mice in comparison to the WT mice. Shown are representative images from six mice. Hematoxylin and eosin staining. Magnification, ×40.
drinking water between WT and AT1a receptor deficient mice challenged with DSS (data not shown). DAI scores, determined by weight loss, stool consistency, and blood in stool were also significantly lower in DSS-treated AT1a receptor deficient mice in comparison to their WT counterparts (significant from days 3–7; Fig. 3). Control WT and control AT1a receptor deficient mice did not develop symptoms associated to DSS-induced colitis through the whole period of the experiment (days 0–7; data not shown). In parallel, challenging mice with 3 % DSS for 7 days resulted in a significant decrease in colon length in both, WT and AT1a receptor deficient mice (Fig. 4). However, the latter change was significantly lesser in AT1a receptor deficient mice in comparison to the WT counterparts (Fig. 4.) Histological examination of the colon samples obtained from the control WT and AT1a receptor deficient mice showed no signs of inflammation or changes in cytoarchitectural arrangement of the colon mucosa (Figs. 5A and 5C). Administration of 3 % DSS for 7 days in both, WT and AT1a receptor deficient mice resulted in an induction of alteration of the mucosal epithelial cell integrity (loss of epithelial cells from villus tips; Figs. 5B and 5D, arrows) and formation of edema with a prominent infiltration of inflammatory cells throughout the mucosa (arrow heads). However, the morphological changes mentioned above (i. e. edema formation, loss of epithelial cells and infiltration of inflammatory cells) were less prominent in the colon of DSS-challenged AT1a receptor deficient mice in comparison to the WT mice (compare Figs. 5B and 5D).
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Fig. 6. Markers of inflammation in colon mucosa of WT and AT1a (–/–) mice challenged with DSS. (A) Accumulation of neutrophilic leukocytes (MPO assay) and (B) lipid peroxidation (production of TBA-reactive substances) in colonic mucosa were assessed on day 7 after administration of DSS. Each value is mean/SEM. n = 6. #p < 0.05 compared to control mice. *p < 0.05 compared to DSS-treated WT counterpart mice.
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Fig. 7. Expression of TNF-a mRNA and protein in colon mucosa of DSS-challenged WT and AT1a (–/–) mice. (A) Mice were challenged with DSS for 0, 3, 5 and 7 days. TNF-a gene expression was assessed by RT-PCR. Results presented are relative change in comparison to day 0. n = 6. †p < 0.05 compared to the same genetic background mice on day 0. *p < 0.05 compared to DSS-treated WT mice on the corresponding day. (B) Mice were challenged with DSS for 7 days and TNF-a protein in the supernatants obtained from the mucosal homogenates was assessed by ELISA. Each value indicates the mean/SEM. n = 6. #p < 0.05 compared to control mice. *p < 0.05 compared to DSS-treated WT counterpart mice.
MPO Activity and TBA-reactive substances in DSS-induced WT and AT1a Receptor Deficient Mice Neutrophil accumulation was evaluated by measuring the MPO activity in colonic mucosal homogenates. Tissue-associated MPO activity in the colonic mucosa was significantly increased on day 7 after DSS administration (Fig. 6A). The increase in MPO activity in the colonic mucosa seen in WT
mice after DSS administration was significantly lower in AT1a receptor deficient mice, confirming the results of the histological analysis (Fig. 5). In parallel, production of TBA-reactive substances in the colonic mucosa of WT mice was also significantly increased 7 days after DSS administration (Fig. 6B). The latter change was significantly reduced in AT1a receptor deficient mice in comparison to the WT counterparts.
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The levels of TNF-a mRNA and Protein in colonic mucosa of DSS-induced WT and AT1a Receptor Deficient Mice We analyzed intestinal expression of TNF-a mRNA in the colon of WT and AT1a receptor deficient mice on days 0, 3, 5, and 7. As shown in Figure 7A, the expression of TNF-a mRNA in the colon of DSS-treated WT mice was significantly up-regulated in a time-dependent manner (days 3–7) with the most profound increase seen on day 7 after DSS administration. The levels of TNF-a mRNA in the colon of AT1a receptor deficient mice assessed on the same day of the treatment were significantly lower in comparison to their WT counterparts. In parallel, the levels of TNF-a protein in the colon of DSS-challenged WT mice were also significantly up-regulated on day 7 (Fig. 7B). Similarly to the changes with respect to TNF-a mRNA levels, the levels of TNF-a protein were also significantly reduced in DSS-treated AT1a receptor deficient mice. Discussion The present study demonstrates that AT1a receptor plays a prominent role in the development of DSS-induced colitis. DSS administration in WT mice results in up-regulation of Ang II and AT1a receptor expression and induction of colonic inflammation as evidenced by the morphological changes in the colon. Moreover, DSS administration in WT mice caused severe body weight loss, severe shortening of colon length and an increase in DAI score. These changes are accompanied by an increase in mucosal pro-inflammatory cytokine (TNF-a) expression, MPO activity and lipid peroxidation. Interestingly, the changes mentioned above were significantly ameliorated in AT1a receptor deficient mice. The results of our study are the first to demonstrate the role of RAS in the development of DSS-induced colitis by employing AT1a receptor deficient mice and are complementary to the recently published findings indicating that pharmacologic blockade of Ang II with ACE inhibitor also reduces the severity of DSSinduced colitis in mice [29]. This strongly suggests that individual targeting of either Ang II or AT1a receptor can be used to interfere with the development of DSS–induced colitis. Currently, it has been demonstrated that RAS plays an important role not only in the development of experimental colitis but also in the pathogenesis of IBD, such as UC and Crohn’s disease (CD) in human [21, 30]. It has been shown that mucosal levels of Ang I and II were elevated in CDpatients in comparison to the healthy subjects [21]. In parallel, elevation in Ang II expression in colon mucosa was also demonstrated in an animal model of trinitrobenzene sulphonic acid (TNBS)-induced experimental colitis. These changes were prevented by interfering with angiotensinogen gene expression (angiotensinogen deficient mice) or pharmacological blockade of Ang II or its receptor by ACE inhibitor and ARB, respectively [9–11]. It is important to mention, that most of the studies addressing the molecular mechanisms of colitis were/are performed employing two well accepted, but different models of pharmacologically-induced colon inflammation (e. g. DSS- vs TNBS-induced colitis). TNBS-induced colitis de-
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velops as a delayed-type hypersensitivity reaction to haptenized proteins by intrarectal application of TNBS [31], while the mechanisms associated with the development of DSSinduced colitis are not entirely clear. It has been suggested that DSS-colitis is a result of the change in epithelial barrier function [32], alterations of luminal bacterial flora [19] or increases in oxidative and nitrosative stress [22] following oral administration of DSS. Although the mechanisms associated with the development of experimental colitis are apparently different, Ang II has been shown to be involved in the pathogenesis of both TNBS- and DSS-induced colitis [9–11, 29]. It is worthwhile to note, however, that the role of Ang II in colon inflammation was primarily assessed employing the TNBS-induced model of colitis, leaving the role of Ang II in DSS-induced colitis very poorly investigated as yet. The results of our present study contribute to the latter and indicate that Ang II plays an important role not only in the development of TNBS-induced colitis but also in the pathogenesis of DSS-induced colitis. In general, one of the key roles in the development of colitis have been attributed to the pro-inflammatory cytokines such as TNF-a, IFN-g, IL-6, IL-12, and IL-15 [23, 24, 33]. Our previously published results are in agreement with the above and indicate that TNF-a is up-regulated in DSS-challenged WT mice in a time-dependent manner [23]. Also, some other studies indicate that TNF-a and IL-6 production can be directly induced by Ang II both, in vitro and in vivo models of inflammation [6, 7]. The findings of the current study indicate that TNF-a levels are up-regulated in the colon mucosa of DSS-challenged WT mice, and that the latter change is significantly suppressed in DSS-challenged AT1a receptor deficient mice. Numerous studies have demonstrated that ROS are implicated in tissue injury and contribute to the development of inflammation [34, 35]. In addition, several reports also indicate that neutrophils are the prime source of ROS during colonic inflammation [36, 37]. In this regard, an increase in neutrophil infiltration and lipid peroxidation has been determined in both TNBS- [11, 38] and DSS-induced [22–24] models of colitis. In addition, recent studies have also shown that ACE inhibitors prevented neutrophil infiltration and lipid peroxidation in the gut mucosa during TNBS-induced colitis [11]. The results of our current study are in agreement with the latter and demonstrate that the magnitude of neutrophil infiltration and lipid peroxidation in the colonic tissue is significantly lower in AT1a receptor deficient mice. However, one of the challenges in addressing the role of RAS in different pathologies is to determine the cellular source of AT1a receptors. At the organ level, tissue-specific RAS has been identified in kidney, brain, aorta, adrenal gland, heart, stomach, and colon [39–41]. Furthermore, AT1a receptor has been recently detected in neutrophilic leukocytes and monocytes [42] and in the blood vessel walls, myofibroblasts, macrophages, and surface epithelium of human colon [39], indicating the ability of different cell types to respond to Ang II stimulation. In addition, it has been shown that upregulation of AT1a receptor is associated with cuff-induced vascular injury [43] and cytokine-induced endothelial dysfunction [44]. In the present study, we demonstrate that AT1a receptor expression (at the mRNA level) was up-regulated in the colonic mucosa of DSS-challenged WT mice. However,
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the specific cell-type source of AT1a receptor (e. g. epithelial cell, endothelial cell or leukocyte) in the mucosa of the colon remains to be determined in the future. In addition, the detailed signal transduction in the RAS-mediated colonic inflammation also remains unclear, though there are several possible hypotheses. It has been reported that Ang II-AT1 pathway activates nuclear factor kB (NFkB) [45, 46] and that activation of NFkB in turn is responsible for the increase in cytokine and chemokine synthesis [46]. Furthermore, recent findings indicate that Ang II induces production of ROS through vascular [8, 47, 48] and neutrophil NADPH oxidases [42], and that the increased production of ROS in turn regulates the subsequent amplification of the proinflammatory response in the cell [47, 48]. In addition, it has been demonstrated that interfering with Ang II-AT1 pathway (inhibition of ACE or AT1a receptor) offers an anti-inflammatory effect with respect to the reduced leukocyte-accumulation in the gut [49, 50]. Taken altogether, the results of this study indicate that up-regulation of both Ang II and AT1a receptor in the colonic mucosa are associated with the onset of mucosal inflammation induced by oral DSS administration. It also suggests that AT1a receptor may be a novel therapeutic target for the treatment of IBD. Further studies are warranted to define the cellular sources and detailed signal transduction in RAS-mediated colonic inflammation. Acknowledgements. We are grateful to Discovery Research Laboratory (Tanabe Seiyaku Co., Ltd.) for providing AT1a receptor deficient mice. This work was supported by a Grant-in-Aid for Scientific Research (14570493 YN, 15590671 NY, 15390178 TY) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a grant from the Bio-Oriented Technology Research Advancement Institution, and by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan.
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