AJP - GI Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 288: G729-G735, 2005. First published November 18, 2004; doi:10.1152/ajpgi.00438.2004
0193-1857/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/4/G729    most recent
00438.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oh, K.
Right arrow Articles by Fujimoto, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oh, K.
Right arrow Articles by Fujimoto, J.

INFLAMMATION/IMMUNITY/MEDIATORS

Ameliorating effect of hepatocyte growth factor on inflammatory bowel disease in a murine model

Koushi Oh,1 Yuji Iimuro,1 Masaharu Takeuchi,1 Yasufumi Kaneda,2 Tsuyoshi Iwasaki,3 Nobuyuki Terada,4 Takayuki Matsumoto,3 Kenji Nakanishi,5 and Jiro Fujimoto1

1First Department of Surgery and Departments of 3Internal Medicine, 4Pathology, and 5Immunology and Medical Zoology, Hyogo College of Medicine, Nishinomiya, Japan; and 2Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka

Submitted 27 September 2004 ; accepted in final form 11 November 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocyte growth factor (HGF), a multifunctional cytokine, accelerates intestinal epithelial proliferation. We studied the effects of HGF in mice with trinitrobenzene sulfonic acid-induced colitis, which shows clinical and molecular resemblance to Crohn's disease. Mice with colitis repeatedly were transfected intramuscularly with human HGF cDNA. Weight, survival, histopathology, proinflammatory cytokine mRNAs, and leukocyte infiltration were assessed. Treatment with HGF cDNA induced tyrosine phosphorylation of intestinal c-Met/HGF receptors, inhibited apoptosis, and promoted mitosis in intestinal epithelial cells, accelerating intestinal epithelial restoration and suppressing inflammation. Transfection with HGF cDNA markedly suppressed intestinal mRNA expression of T-helper 1 cytokines such as interleukin-12 and -1{beta}, interferon-{gamma}, and tumor necrosis factor-{alpha}. Numbers of total and CD4-positive T cells, neutrophils, and myloperoxidase activity in intestinal epithelium were diminished by HGF gene transfer, which also prevented weight loss, and improved survival. HGF might prove useful for controlling inflammatory bowel disease.

2,4,6-trinitrobenzene sulfonic acid; gene therapy

ETIOLOGIES OF BOTH Crohn's disease and ulcerative colitis (UC), idiopathic chronic inflammatory disorders collectively termed inflammatory bowel diseases (IBD), remain obscure. Various experimental animal models have been devised during the last decade to clarify mechanisms underlying IBDs (5, 8, 38). Among these, rectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to immunocompetent mice results in transmural intestinal leukocyte infiltration accompanied by severe diarrhea and weight loss, resembling some characteristics of human Crohn's disease (30). Inflammation in the model is characterized by dense, transmural infiltration by T cells, mainly of the CD4+ phenotype, with excessive production of T-helper cell (Th) 1-derived cytokines (31). Induction of colitis in the mice is begun by local administration of ethanol, which injures the epithelial lining of the intestine, the initial barrier against a variety of toxic agents. An imbalanced mucosal immune response then can be induced by subsequent administration of a hapten reagent, TNBS. Clinical data from patients with IBD similarly suggest the key importance of an impaired epithelial barrier and exposure of intestinal tissues to microbial toxins in initiation and progression of IBD (10). Considering this sequence, accelerating the restoration of the epithelial lining could impede expansion of intestinal inflammation caused by TNBS and also might improve the clinical status of patients with IBD.

Previous studies demonstrated participation of several growth factors and cytokines in the regulation of the intestinal epithelial restoration (6, 20, 36). Hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocytes, has proven to be multifunctional, showing mitogenic, motogenic, morphogenic, angiogenic, and anti-apoptotic activities in a wide variety of cells and organs (3, 28), including those of the gastrointestinal tract (33). HGF has been found to act on intestinal cell populations as a positive growth regulator and to accelerate remodeling after epithelial injury (14, 32, 33). The HGF receptor, c-Met, also has been reported to appear in intestinal mucosal epithelial cells during restoration of continuity (20, 43). Thus intestinal mucosal healing appears to be stimulated by the HGF-c-Met signaling system. Indeed, expression of HGF and its receptor, c-Met, has been reported to increase in a rat model of UC and patients with UC (24, 34). Moreover, the beneficial effect of HGF administration in a rat UC model has also been reported (41). However, the effect of HGF on Crohn's disease remains elusive.

Recent advances in molecular techniques have provided several gene-delivery strategies in vivo that show promise as novel therapeutic approaches using cDNAs encoding beneficial molecules. These techniques variously employ naked plasmid DNA (39), liposomes encapsulating DNA (18), and viral vectors such as adenovirus, retrovirus, and adenoassociated virus (9, 11, 27). Each type of delivery system has individual advantages and limitations (23). A fusigenic liposome with the hemagglutinating virus of Japan (HVJ-liposome) represents an attractive in vivo gene-delivery system (22) characterized by persistent gene expression and low cytotoxicity (17). We used this system in the present study to transduce HGF in mice developing IBD, previously having achieved stable HGF expression in a rat liver cirrhosis model, a rat obstructive nephropathy model, and a mouse graft vs. host disease model (13, 26, 42). We designed the present study to assess the possible therapeutic role of HGF in TNBS-induced colitis, which resembles Crohn's disease in humans. Repeated transfection of the human HGF gene encapsulated in HVJ-liposomes induced phosphorylation of intestinal c-Met/HGF receptors in the mice. We found that HGF transfection inhibited apoptosis in intestinal epithelial cells and promoted constitution of the epithelium, suppressing inflammatory responses. This approach using a gene encoding a multifunctional growth factor may prove useful for the control of IBD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male 12–to 14-wk-old BALB/c mice (SLC, Japan), weighing 24–28 g, were used in this study. They were maintained under specific pathogen-free conditions and fed free autoclaved chow and water ad libitum. Animal care was in accordance with the guidelines of the Hyogo College of Medicine.

Murine colitis was induced by intracolonic administration of a hapten reagent, TNBS (Sigma Chemical, St. Louis, MO), dissolved in ethanol, as described previously (30, 31). Under ether anesthesia, mice received 1.0 mg TNBS dissolved in 100 µl of 50% ethanol via anus using an 18-gauge soft silicon catheter inserted in the proximal colon for 5 cm. They were kept in a vertical position for 1 min. Mice were checked for their body weight and general condition daily.

Preparation of HVJ-liposome. HVJ-liposome was prepared as described previously (22). Briefly, phosphatidylserine, phosphatidylcholine, and cholesterol were mixed in a weight ratio of 1:4.8:2. Dried lipid was hydrated in 200 µl balanced salt solution (BSS: 137 mM NaCl, 5.4 mM KCl, and 10 mM Tris·HCl, pH 7.6). Plasmid DNA (20 µg) was previously incubated with nonhistone chromosomal protein (high-mobility group-1) purified from calf thymus. Liposome was prepared by shake and sonication. Purified HVJ was inactivated by ultraviolet irradiation for 3 min just before use. The liposome suspension was mixed with HVJ (10,000 hemagglutinating units) in a total volume of 1 ml BSS. The mixture was incubated at 4°C for 10 min and then for 30 min with gentle shaking at 37°C. Free HVJ was removed from the HVJ-liposomes by sucrose density gradient centrifugation. In this study, ~20 µg pUC-SR{alpha}/HGF expression plasmid entrapped in HVJ-liposome complex was injected in gluteal muscles for every injection.

Gene transfer and experimental design. Human HGF cDNA (2.2 kb) was inserted between the Not I sites in the pUC-SR{alpha} expression plasmid vector and encapsulated in HVJ-liposome as described. Purified HGF/HVJ-liposome complexes were directly injected in gluteal muscles 3 days before and 4 days after the induction of colitis. According to our previous study, exogenous human HGF expression persisted at least for 7 days after the gene transfer using the HVJ-liposome method with a peak expression at day 4 (42). To maintain a significant level of HGF protein in plasma, we delivered the human HGF gene two times in this experiment. In the control group, they received PBS injection in the same manner. Some mice were killed 3 days after the TNBS treatment, and surviving mice were killed at day 10. Total colonic tissues were collected and used for histopathological analysis, flow cytometric analysis, enzymatic analysis of myeloperoxidase (MPO) activities, analysis of mRNA expression of inflammatory cytokines, and tyrosine phosphorylation of c-Met. In some mice, 100 mg/kg bromodeoxyuridine (BrdU) was injected intraperitoneally 1 h before death for analysis of DNA synthesis in the intestinal epithelium.

Measurement of plasma HGF level. Samples were collected 0, 4, and 10 days after the first injection of HGF cDNA. Exogenous human and endogenous mouse HGF levels were measured in plasma by ELISA kits (Institute of Immunology, Tokyo, Japan).

Western blot and immunoprecipitation analysis. Total colon tissues were removed at day 10, and total protein was extracted from homogenized tissues. The protein samples were adjusted to the same concentration for Western blotting. For immunoprecipitation of c-Met-protein, lysates were incubated with the protein A-agarose beads (Pierce, Rockford, IL) pretreated with an anti-mouse c-met antibody. Each sample containing 20 µg total protein was separated by SDS-PAGE on 7% acrylamide slab gels. After electrophoresis, the gel was transferred to a polyvinylidine difluoride sheet and then probed with the anti-mouse c-met antibody (sc-8057; Santa Cruz Biotechnology, Santa Cruz, CA) or an anti-phosphotyrosine antibody (PY-20; ICN, Transduction Laboratories, Lexington, KY).

Histopathological analysis. At death, the total colon was removed and carefully opened longitudinally on the opposite side to the mesentery. The colonic tissues were washed with PBS and fixed in 10% formalin in PBS overnight at room temperature. After being embedded in paraffin, 4-µm sections were prepared and stained with hematoxylin and eosin (H&E). Histological changes of the colon were evaluated in a blind fashion according to a semiquantitative grading method previously reported elsewhere (30). Moreover, existence of epithelial restoration, namely appearance of growing epithelial cells over the disrupted mucosal layer, was evaluated in the H&E staining in each experimental group.

To access DNA synthesis in the intestinal epithelium, BrdU incorporation in the epithelial cells was determined in mice treated with BrdU 1 h before death. The deparaffinized sections were incubated in 4 N HCl for 20 min at 37°C and washed several times with dH2O and 1x PBS. After blocking of endogenous peroxidase with peroxidase blocking agent, sections were incubated with mouse monoclonal anti-BrDU antibody (1:200, clone Bu20a 1:30; DACO, Kyoto, Japan) for 10 min at 25°C. After being washed, the sections were incubated for 60 min with biotin-conjugated anti-mouse antibody, labeled with peroxidase streptavidin, and examined after being incubated with diaminobenzidine-H2O2 substrate.

Detection of apoptotic cells in large intestine. To detect apoptotic cells in large intestine, the terminal deoxinucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) method, which detects in situ DNA fragmentation, was applied using the Apo Taq in situ Apoptosis Detection System according to the standard protocol (Oncor, Gaithersburg, MD).

Measurement of MPO activities. MPO activity is an index of neutrophils, monocytes, and macrophages infiltrating into the inflamed colonic tissue. A modified method of MPO activity measurement, which was previously described, was used (25).

Analysis of cytokine mRNA expression by RNase protection assay. Purified total RNA was extracted from total colonic tissue at day 10 using a standard acid-guanidium-phenol-chloroform method (1). Cytokine mRNAs were detected using a RiboQuant MultiProbe RNase protection assay system (PharMingen, San Diego, CA) by standard protocols (37). Briefly, multiprobes were 32P labeled and synthesized by T7 RNA polymerase. Purified riboprobes were then hybridized overnight with 10 µg of the RNA samples. The hybridized RNA was treated with RNase, and single-strand RNA was digested. Remained darn samples were then electrophoresed in 6% polyacrylamide-Tris-borate-EDTA-urea minigels (Novex, San Diego, CA). The gels were dried, exposed, and quantitated in a PhosphorImager (BAS 2000; Fuji, Tokyo, Japan) using ImageQuant software (Fuji).

Flow cytometric analysis. Mouse colonic mucosal mononuclear cells were prepared as described previously (15), with minor modification. Briefly, total colonic tissues were removed, and the content was washed with PBS at day 10. The mucosa was dissected from the underlying muscularis using iris scissors, and cells were passed through the mesh and suspended in PBS. Cell suspensions were prepared in PBS containing 1% FCS and 0.1% sodium azide. The cells were incubated with anti-Fc receptor mAb (2.4G2) for 10 min at 4°C and then incubated with FITC-conjugated mAb and PE-conjugated mAb for 30 min. The stained cells were washed two times, resuspended, and analyzed using FACScan (Becton-Dickinson, Mountain View, CA).

Statistical analysis. Results for the group were compared by Mann-Whitney's U-test and X-test. Survival data were plotted by the Kaplan-Meier method and analyzed by Long-Rank test. A P value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of HGF and its biological activity in colonic epithelium. We previously reported that repeated transfection of human HGF gene in skeletal muscle resulted in a sustained high serum concentration of human and endogenous rat or mouse HGF (13, 26, 42). HGF expression initially was transient, but repeated HGF gene injections showed a booster effect, and stable HGF production was obtained. Human and mouse HGF both were detected by ELISA in sera from mice transfected with the human HGF gene in this TNBS-induced colitis model. The total concentration of HGF (human and mouse) in serum was 0.20 ng/ml before injection, 1.70 ng/ml at 4 days after the first injection of HGF cDNA, and 1.63 ng/ml at 10 days. Amounts of c-Met/HGF receptor protein in the intestine increased in TNBS-treated control mice compared with untreated normal mice, whereas expression was relatively low in HGF-treated mice exposed to TNBS (Fig. 1A). The receptor contains an intracellular tyrosine kinase domain, and biological activities of HGF are produced by tyrosine residue phosphorylation in c-Met. Tyrosine phosphorylation of c-Met was barely detectable in intestine from PBS-treated control mice given TNBS, although strong tyrosine phosphorylation of c-Met was observed in intestine from mice given the HGF gene and TNBS (Fig. 1A). These results indicate that introduction of the HGF gene produced biologically effective amounts of HGF protein and activated the c-Met receptor in the intestine. Thus HGF could exert its biological effect in the intestines of mice with TNBS-induced colitis.



View larger version (66K):
[in this window]
[in a new window]
  		Fig. 1. A: expression of c-Met, the hepatocyte growth factor (HGF) receptor, and its phosphorylation (Western blot) are shown. The c-Met protein was overexpressed in the colonic tissues in the PBS-treated group, whereas the expression was relatively low in HGF gene-treated animals. However, strong tyrosine phosphorylation of c-Met was observed only in the HGF gene-treated group, suggesting significant biological activity of HGF in this group. B-G: histological analysis of the large intestine after the induction of colitis was performed at day 10 [hematoxylin and eosin (H&E), original magnification x100 (B, D, and F) and x400 (C, E, and G)]. The large intestines from untreated normal mice (B and C), mice treated with PBS/2,4,6-trinitrobenzene sulfonic acid (TNBS) (D and E), and mice treated with HGF/TNBS (F and G) are shown. Severe transmural infiltration by inflammatory cells and thick intestinal walls accompanied by disruption of the mucosal layer were observed in the PBS/TNBS group (D and E). B–G are representative of 35–37 individual animals among the TNBS-treated mice. H: histological grading scores, which are semiquantitatively calculated at day 10, are presented. PY, phosphotyrosine.

 
HGF gene-mediated amelioration of colitis and weight loss. Consistent with previous reports, TNBS-induced colitis in our mice was evident as severe inflammation by 2–3 days after TNBS administration and persisted for 3 wk (8). In control mice given PBS rather than the transgene, massive infiltration of lymphocytes in the submucosal layer accompanied by disruption of the mucosal layer was observed in the intestine at day 10 (Fig. 1, D and E). In contrast, HGF gene induction resulted in a significant decrease in inflammation and also attenuated the disruption of the mucosal layer (Fig. 1, F and G). The effect of HGF on epithelial injury was accessed using a grading system described in MATERIALS AND METHODS (30). As shown in Fig. 1H, transfection with the HGF gene significantly improved scores at 10 days after TNBS administration. Some mice were killed 3 days after TNBS administration to examine whether ethanol/TNBS caused the same degree of early intestinal epithelial injury in PBS-treated and HGF-treated mice, which proved to be true. Both groups showed disruption of the epithelial layer and transmural infiltration of inflammatory cells. Mean scores at day 3 were 3.5 ± 0.5 in PBS-treated and 3.6 ± 0.5 in HGF gene-treated mice (no significant difference).

Reconstruction of colonic epithelium often was observed in specimens from HGF gene-treated mice at day 10, with epithelium extending over previously disrupted regions (Fig. 2, A and B). In PBS control mice given TNBS, restoration of epithelium was found in 14 of 25 mice (56%), whereas restoration was evident in 17 of 20 mice treated with HGF (85%, P = 0.036, Fig. 2G). Because HGF is a potent mitogen for epithelial cells, we assessed BrdU incorporation in the colonic epithelium. The number of BrdU-positive cells in the intestine was much greater in mice treated with HGF than in PBS-treated control mice (Fig. 2, C and D). Apoptosis of intestinal epithelium has been reported in the TNBS-induced colitis model (12). When apoptosis was investigated with the TUNEL method for detecting DNA fragmentation in situ, a number of apoptotic cells were observed in the epithelial crypts of PBS-treated control mice (Fig. 2E), whereas HGF gene treatment inhibited this apoptosis (Fig. 2F). Thus HGF gene transfection appeared to improve recovery from intestinal damage through its anti-apoptotic effect and through accelerated restoration of epithelial continuity after injury.



View larger version (73K):
[in this window]
[in a new window]
  		Fig. 2. HGF gene transfer stimulated intestinal epithelial restoration and prevented apoptosis after the TNBS treatment. A and B: large intestine from mice treated with HGF/TNBS [H&E, original magnification x100 (A) and x400 (B)]. In HGF-treated mice, newly proliferating epithelial cells, covering the disrupted mucosal layer, are often observed at day 10. C and D: bromodeoxyuridine (BrDU) staining of the large intestine from the PBS/TNBS-treated (C) and the HGF/TNBS-treated (D) mice. In the HGF gene-treated mice, a significant number of BrDU-incorporating cells is detected mainly in the basal layer of the epithelium. G: incidence of intestinal epithelial restoration is also calculated. The number of animals accompanied by intestinal epithelial restoration was significantly greater in the HGF-treated mice (P < 0.05). E and F: TUNEL staining of the inflamed epithelium. A number of apoptotic epithelial cells are detected in the epithelial crypts in the PBS/TNBS-treated mice (E), whereas HGF gene transfer almost prevented apoptosis in the epithelium (F). H: survival rates of mice after induction of colitis. More than 50% of mice treated with PBS/TNBS died within 2 wk. In contrast, mice treated with HGF gene transfer exhibited a significantly higher survival rate (*P < 0.05). I: changes in body weight of mice after TNBS treatment. Each point represents pooled average body weight with an error bar (SE; n = 10–32). Mice that received HGF gene transfer began to gain body weight at day 3 and failed to develop wasting.

 
The main outward signs of TNBS-induced colitis in this experiment were severe diarrhea and loss of body weight; some mice died of extensive wasting. Characteristic signs of potentially lethal colitis persisted up to 3 wk. Survival rates with such involvement are shown in Fig. 2H. More than 50% of mice treated with PBS died within 2 wk, whereas mice treated with the HGF gene exhibited a significantly higher survival rate (Fig. 2H; 50 vs. 75%, P < 0.05). PBS-treated control animals gradually lost weight (Fig. 2I), whereas mice receiving the HGF gene began to gain weight at day 3 and failed to develop wasting, appearing healthy from that time (Fig. 2I).

Downregulation of the inflammatory response by HGF. Intrarectal administration of TNBS induces chronic active inflammation, evident as dense infiltration of the colon by inflammatory cells, among which CD4+ T cells are considered to predominate (30). The population of mucosal mononuclear cells isolated from the lamina propria of the large intestine was analyzed using FACS (Fig. 3A). HGF gene treatment significantly decreased the total number of T cells (CD3+) in the lamina propria, from 6.1 x 105 to 2.1 x 105, at 10 days after TNBS administration. HGF gene therapy also significantly decreased the number of CD4+ T cells from 4.7 x 105 to 1.3 x 105. Numbers of CD8+ T cells and neutrophils also were decreased significantly by HGF treatment. As a quantitative assessment of neutrophil infiltration in the colon, we assayed MPO activity in colonic extracts. In TNBS-treated mice, MPO activity was seven times that in control mice without colitis, whereas HGF treatment resulted in a significant reduction in MPO activity (P < 0.05, Fig. 3B).



View larger version (33K):
[in this window]
[in a new window]
  		Fig. 3. HGF gene transfer prevented infiltration of inflammatory cells and production of inflammatory cytokines. A: FACS scan analysis of the population of mucosal mononuclear cells isolated from lamina propria of the large intestine. The number of infiltrating CD4+, CD8+ T cells, and neutrophils is significantly lowered with the HGF gene transfer (*P < 0.05 compared with normal mice; #P < 0.05 compared with TNBS + PBS treated mice). B: myeloperoxidase (MPO) activity of the total colonic tissue. Increased MPO activity in the PBS/TNBS-treated mice was dramatically attenuated with the HGF gene treatment (*P < 0.05). C: expression of inflammatory cytokine mRNAs accessed by RNase protection assay. The mRNA expressions of TNF-{alpha}, IL-12, IFN-{gamma}, and IL-1{beta} are markedly increased with the TNBS treatment, whereas HGF gene transfer obviously reduced the expression.

 
Previous studies of cytokine production in TNBS-induced colitis demonstrated that Th1 cytokines were dominant in this model (7, 12, 30). First, we tried to measure serum concentrations of Th1 cytokines using ELISA kits, but failed to detect the expression accurately at day 10. Next, we analyzed expression of mRNAs encoding cytokines, including TNF-{alpha}, IL-12, IFN-{gamma}, and IL-1{beta}, in the large intestine using RNase protection assays (Fig. 3C). TNBS treatment enhanced expression of TNF-{alpha}, IL-12, IFN-{gamma}, and IL-1{beta} mRNA beyond that in untreated control mice. HGF treatment strongly suppressed colonic mRNA expression for these cytokines after TNBS treatment.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recently, various anti-cytokine strategies against TNBS-induced colitis, a murine model of human IBD, have been reported to reduce colitis (2, 12, 19, 30, 31). HGF, originally identified as a potent mitogen for hepatocytes, has a variety of effects (3, 28, 29), including anti-apoptotic activities in a wide variety of organs, including those of the gastrointestinal tract (26, 33, 40). In the present study, we demonstrated a beneficial effect of HGF gene transfer in mice with TNBS-induced colitis, which resembles human Crohn's disease.

The first goal of this experiment was to obtain a stable, persistent HGF concentration in plasma. HVJ- and liposome-mediated gene delivery has been characterized as providing persistent gene expression with low cytotoxicity (17). Based on our previous study (26, 42), expression of exogenous human HGF in plasma was obtained by injection of the human HGF gene, encapsulated in the HVJ-liposome, in the gluteal muscles of the mice. The receptor for HGF is a tyrosine kinase receptor encoded by c-met, and the membrane-spanning {beta}-chain of the c-Met receptor contains an intracellular tyrosine kinase domain (4). Although c-Met protein was expressed more strongly in inflamed colonic tissue after TNBS treatment (Fig. 1A), the biological activity of HGF, which was demonstrated by tyrosine residue phosphorylation in c-Met, was very low. In contrast, in HGF gene-treated mice, expression of c-Met was relatively low, but the degree of tyrosine phosphorylation of c-Met was very high, indicating functional effectiveness of HGF gene transfer. We had some difficulty in explaining the lower expression of c-Met protein in HGF-treated mice. Considering that control mice without colitis also had relatively low expression of c-Met, lower expression in HGF-treated mice may have indirectly reflected nearly complete epithelial reconstitution at the time point examined. Alternatively, downregulation of c-Met protein expression after stimulation by HGF possibly may be involved (16).

The mechanism of TNBS-induced colitis is believed to involve disruption of the protective epithelial barrier, followed by imbalance of the mucosal immune response to TNBS. No difference in inflammatory scores was seen between the PBS/TNBS-treated and the HGF gene/TNBS-treated mice at day 3, suggesting that cytoprotective and anti-inflammatory effects of HGF against ethanol/TNBS injury may not be the most important factors in this model. Instead, when HGF gene transfer later was seen to significantly attenuate transmural inflammation at day 10, this was accompanied by enhanced repair of the epithelial lining. Transmural infiltration of leukocytes, including the lamina propria, and elevated MPO activity in the inflamed colonic tissue after the TNBS treatment instead were greatly attenuated by HGF gene transfer (Fig. 1, D-H, and Fig. 3B). Flow cytometric analysis in PBS/TNBS mice demonstrated a large number of T cells, especially CD4+ T cells, infiltrating in colonic tissues after TNBS treatment, resembling findings in human Crohn's disease (7, 35). Infiltration of CD4+ and CD8+ T cells, as well as neutrophils, was reduced significantly by HGF gene transfer (Fig. 3A). More detailed histological analysis demonstrated that HGF accelerated proliferation of the intestinal epithelium by day 10 (Fig. 2, A and B), which was confirmed by comparing BrDU incorporation in proliferating epithelial cells (Fig. 2D). Importantly, numbers of apoptotic cells in the intestinal epithelium after TNBS administration clearly were reduced by HGF gene transfer (Fig. 2F). These results suggest that HGF stimulates proliferation and retards apoptosis in the disrupted intestinal epithelial barrier, thus restoring continuity of epithelial cells and in turn lessening subsequent inflammation caused by TNBS and intestinal bacterial toxins.

In most experimental models of chronic intestinal inflammation, including TNBS-induced colitis, various inflammatory cytokines, such as IL-12, INF-{gamma}, and TNF-{alpha}, were shown to be overexpressed in inflamed intestinal tissue. In the present analysis, elevated mRNA expression for Th1 cytokines, such as IL-12 and INF-{gamma}, was detected after TNBS exposure. Furthermore, mRNA expression of proinflammatory cytokines, such as TNF-{alpha} and IL-1{beta}, was upregulated in this experimental colitis, although we failed to detect serum expression of these cytokines by ELISA at day 10. HGF gene transfer markedly suppressed mRNA expression of these cytokines (Fig. 3C). An anti-inflammatory effect of HGF has been reported previously (21). However, a direct suppressive effect of HGF on inflammatory cytokine production is still obscure. We speculate that, instead, the ameliorating effect of HGF observed in this study reflected mitogenic, morphogenic, and anti-apoptotic effects on epithelial cells rather than direct anti-inflammatory activity. Early restoration of the injured mucosal barrier could prevent prolonged exposure of the submucosal layer to bacterial toxins, leading to reduction in infiltration by inflammatory cells and in production of inflammatory cytokines.

In summary, stable, persistent HGF expression in mice was obtained by repeated intramuscular injections of a vector containing the human HGF gene, ameliorating potentially lethal colitis induced by TNBS. Although the mechanisms underlying this beneficial effect of HGF have not yet been firmly established, treatment with HGF may represent a promising new therapeutic approach to human Crohn's disease.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Fujimoto, First Dept. of Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan (E-mail: surg-1{at}hyo-med.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Akin A, Wu CC, and Lin TL. A comparison of two RNA isolation methods for double-stranded RNA of infectious bursal disease virus. J Virol Methods 74: 179–184, 1998.[CrossRef][ISI][Medline]
  2. Atreya R, Mudter J, Finotto S, Mullberg J, Jostock T, Wirtz S, Schutz M, Bartsch B, Holtmann M, Becker C, Strand D, Czaja J, Schlaak JF, Lehr HA, Autschbach F, Schurmann G, Nishimoto N, Yoshizaki K, Ito H, Kishimoto T, Galle PR, Rose-John S, and Neurath MF. Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo. Nat Med 6: 583–588, 2000.[CrossRef][ISI][Medline]
  3. Boros P and Miller CM. Hepatocyte growth factor: a multifunctional cytokine. Lancet 345: 293–295, 1995.[CrossRef][ISI][Medline]
  4. Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Woude GF, and Aaronson SA. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251: 802–804, 1991.[Abstract/Free Full Text]
  5. Cooper HS, Murthy SN, Shah RS, and Sedergran DJ. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest 69: 238–249, 1993.[ISI][Medline]
  6. Dignass AU. Mechanisms and modulation of intestinal epithelial repair. Inflamm Bowel Dis 7: 68–77, 2001.[ISI][Medline]
  7. Dohi T, Fujihashi K, Kiyono H, Elson CO, and McGhee JR. Mice deficient in Th1- and Th2-type cytokines develop distinct forms of hapten-induced colitis. Gastroenterology 119: 724–733, 2000.[CrossRef][ISI][Medline]
  8. Elson CO, Sartor RB, Tennyson GS, and Riddell RH. Experimental models of inflammatory bowel disease. Gastroenterology 109: 1344–1367, 1995.[CrossRef][ISI][Medline]
  9. Ferry N, Duplessis O, Houssin D, Danos O, and Heard JM. Retroviral-mediated gene transfer into hepatocytes in vivo. Proc Natl Acad Sci USA 88: 8377–8381, 1991.[Abstract/Free Full Text]
  10. Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 115: 182–205, 1998.[CrossRef][ISI][Medline]
  11. Flotte TR and Carter BJ. Adeno-associated virus vectors for gene therapy. Gene Ther 2: 357–362, 1995.[ISI][Medline]
  12. Fuss IJ, Marth T, Neurath MF, Pearlstein GR, Jain A, and Strober W. Anti-interleukin 12 treatment regulates apoptosis of Th1 T cells in experimental colitis in mice. Gastroenterology 117: 1078–1088, 1999.[CrossRef][ISI][Medline]
  13. Gao X, Mae H, Ayabe N, Takai T, Oshima K, Hattori M, Ueki T, Fujimoto J, and Tanizawa T. Hepatocyte growth factor gene therapy retards the progression of chronic obstructive nephropathy. Kidney Int 62: 1238–1248, 2002.[CrossRef][ISI][Medline]
  14. Goke M and Podolsky DK. Regulation of the mucosal epithelial barrier. Baillieres Clin Gastroenterol 10: 393–405, 1996.[CrossRef][ISI][Medline]
  15. Hakim FT, Sharrow SO, Payne S, and Shearer GM. Repopulation of host lymphohematopoietic systems by donor cells during graft-versus-host reaction in unirradiated adult F1 mice injected with parental lymphocytes. J Immunol 146: 2108–2115, 1991.[Abstract]
  16. Hammond DE, Urbe S, Vande Woude GF, and Clague MJ. Down-regulation of MET, the receptor for hepatocyte growth factor. Oncogene 20: 2761–2770, 2001.[CrossRef][ISI][Medline]
  17. Hirano T, Fujimoto J, Ueki T, Yamamoto H, Takeuchi M, Okamoto E, Takahashi H, Morisita R, Sawa Y, and Kaneda Y. HVJ-liposome mediated gene transfer into hepatocytes in vivo. J Hepatol 29: 910–914, 1998.[CrossRef][ISI][Medline]
  18. Hofland HE, Shephard L, and Sullivan SM. Formation of stable cationic lipid/DNA complexes for gene transfer. Proc Natl Acad Sci USA 93: 7305–7309, 1996.[Abstract/Free Full Text]
  19. Hogaboam CM, Vallance BA, Kumar A, Addison CL, Graham FL, Gauldie J, and Collins SM. Therapeutic effects of interleukin-4 gene transfer in experimental inflammatory bowel disease. J Clin Invest 100: 2766–2776, 1997.[ISI][Medline]
  20. Howarth GS and Shoubridge CA. Enhancement of intestinal growth and repair by growth factors. Curr Opin Pharmacol 1: 568–574, 2001.[CrossRef][Medline]
  21. Jeschke MG, Wolf SE, DebRoy MA, and Herndon DN. The combination of growth hormone with hepatocyte growth factor alters the acute phase response. Shock 12: 181–187, 1999.[ISI][Medline]
  22. Kaneda Y, Iwai K, and Uchida T. Increased expression of DNA cointroduced with nuclear protein in adult rat liver. Science 243: 375–378, 1989.[Abstract/Free Full Text]
  23. Kim CK, Haider KH, and Lim SJ. Gene medicine : a new field of molecular medicine. Arch Pharmacol Res (Seoul) 24: 1–15, 2001.
  24. Kitamura S, Kondo S, Shinomura Y, Isozaki K, Kanayama S, Higashimoto Y, Minami T, Kiyohara T, Yasunaga Y, Ishikawa H, Ohtani T, Ishiguro S, and Matsuzawa Y. Expression of hepatocyte growth factor and c-met in ulcerative colitis. Inflamm Res 49: 320–324, 2000.[CrossRef][ISI][Medline]
  25. Krawisz JE, Sharon P, and Stenson WF. Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models. Gastroenterology 87: 1344–1350, 1984.[ISI][Medline]
  26. Kuroiwa T, Kakishita E, Hamano T, Kataoka Y, Seto Y, Iwata N, Kaneda Y, Matsumoto K, Nakamura T, Ueki T, Fujimoto J, and Iwasaki T. Hepatocyte growth factor ameliorates acute graft-versus-host disease and promotes hematopoietic function. J Clin Invest 107: 1365–1373, 2001.[ISI][Medline]
  27. Li Q, Kay MA, Finegold M, Stratford-Perricaudet LD, and Woo SL. Assessment of recombinant adenoviral vectors for hepatic gene therapy. Hum Gene Ther 4: 403–409, 1993.[ISI][Medline]
  28. Michalopoulos GK and DeFrances MC. Liver regeneration. Science 276: 60–66, 1997.[Abstract/Free Full Text]
  29. Nakamura T, Tomita Y, Hirai R, Yamaoka K, Kaji K, and Ichihara A. Inhibitory effect of transforming growth factor-beta on DNA synthesis of adult rat hepatocytes in primary culture. Biochem Biophys Res Commun 133: 1042–1050, 1985.[CrossRef][ISI][Medline]
  30. Neurath MF, Fuss I, Kelsall BL, Stuber E, and Strober W. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J Exp Med 182: 1281–1290, 1995.[Abstract/Free Full Text]
  31. Neurath MF, Pettersson S, Meyer zum Buschenfelde KH, and Strober W. Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice. Nat Med 2: 998–1004, 1996.[CrossRef][ISI][Medline]
  32. Nishimura S, Takahashi M, Ota S, Hirano M, and Hiraishi H. Hepatocyte growth factor accelerates restitution of intestinal epithelial cells. J Gastroenterol 33: 172–178, 1998.[CrossRef][ISI][Medline]
  33. Nusrat A, Parkos CA, Bacarra AE, Godowski PJ, Delp-Archer C, Rosen EM, and Madara JL. Hepatocyte growth factor/scatter factor effects on epithelia. Regulation of intercellular junctions in transformed and nontransformed cell lines, basolateral polarization of c-met receptor in transformed and natural intestinal epithelia, and induction of rapid wound repair in a transformed model epithelium. J Clin Invest 93: 2056–2065, 1994.[ISI][Medline]
  34. Ortega-Cava CF, Ishihara S, Kawashima K, Rumi MA, Kazumori H, Adachi K, and Kinoshita Y. Hepatocyte growth factor expression in dextran sodium sulfate-induced colitis in rats. Dig Dis Sci 47: 2275–2285, 2002.[CrossRef][ISI][Medline]
  35. Palmen MJ, Wijburg OL, Kunst IH, Kroes H, and van Rees EP. CD4+ T cells from 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis rodents migrate to the recipient's colon upon transfer; down-regulation by CD8+ T cells. Clin Exp Immunol 112: 216–225, 1998.[CrossRef][ISI][Medline]
  36. Podolsky DK. Mechanisms of regulatory peptide action in the gastrointestinal tract: trefoil peptides. J Gastroenterol 35: 69–74, 2000.[CrossRef][ISI][Medline]
  37. Sandler A, Scott D, Azuhata T, Takamizawa S, and O'Dorisio S. The survivin:Fas ratio is predictive of recurrent disease in neuroblastoma. J Pediatr Surg 37: 507–511, 2002.[CrossRef][ISI][Medline]
  38. Sartor RB. Review article: How relevant to human inflammatory bowel disease are current animal models of intestinal inflammation? Aliment Pharmacol Ther 11, Suppl 3: 89–96, 1997.
  39. Sekiguchi K, Yasuzumi F, and Morishita R. Exogenous expression of hepatocyte growth factor (HGF) in rat striatum by naked plasmid DNA. Neurosci Res 45: 173–180, 2003.[CrossRef][ISI][Medline]
  40. Sturm A, Schulte C, Schatton R, Becker A, Cario E, Goebell H, and Dignass AU. Transforming growth factor-beta and hepatocyte growth factor plasma levels in patients with inflammatory bowel disease. Eur J Gastroenterol Hepatol 12: 445–450, 2000.[ISI][Medline]
  41. Tahara Y, Ido A, Yamamoto S, Miyata Y, Uto H, Hori T, Hayashi K, and Tsubouchi H. Hepatocyte growth factor facilitates colonic mucosal repair in experimental ulcerative colitis in rats. J Pharmacol Exp Ther 307: 146–151, 2003.[Abstract/Free Full Text]
  42. Ueki T, Kaneda Y, Tsutsui H, Nakanishi K, Sawa Y, Morishita R, Matsumoto K, Nakamura T, Takahashi H, Okamoto E, and Fujimoto J. Hepatocyte growth factor gene therapy of liver cirrhosis in rats. Nat Med 5: 226–230, 1999.[CrossRef][ISI][Medline]
  43. Xian CJ, Couper R, Howarth GS, Read LC, and Kallincos NC. Increased expression of HGF and c-met in rat small intestine during recovery from methotrexate-induced mucositis. Br J Cancer 82: 945–952, 2000.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
Y. Yoshida, T. Hirano, G. Son, Y. Iimuro, T. Imado, T. Iwasaki, and J. Fujimoto
Allogeneic bone marrow transplantation for hepatocellular carcinoma: hepatocyte growth factor suppresses graft-vs.-host disease
Am J Physiol Gastrointest Liver Physiol, December 1, 2007; 293(6): G1114 - G1123.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M. Kanayama, T. Takahara, Y. Yata, F. Xue, E. Shinno, K. Nonome, H. Kudo, K. Kawai, T. Kudo, Y. Tabuchi, et al.
Hepatocyte growth factor promotes colonic epithelial regeneration via Akt signaling
Am J Physiol Gastrointest Liver Physiol, July 1, 2007; 293(1): G230 - G239.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
T. Yuki, S. Ishihara, M. A. K. Rumi, C. F. Ortega-Cava, Y. Kadowaki, H. Kazumori, N. Ishimura, Y. Amano, N. Moriyama, and Y. Kinoshita
Increased expression of midkine in the rat colon during healing of experimental colitis.
Am J Physiol Gastrointest Liver Physiol, October 1, 2006; 291(4): G735 - G743.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
R. Gong, A. Rifai, and L. D. Dworkin
Anti-Inflammatory Effect of Hepatocyte Growth Factor in Chronic Kidney Disease: Targeting the Inflamed Vascular Endothelium
J. Am. Soc. Nephrol., September 1, 2006; 17(9): 2464 - 2473.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/4/G729    most recent
00438.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oh, K.
Right arrow Articles by Fujimoto, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oh, K.
Right arrow Articles by Fujimoto, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.