Shigeaki Nagao

Free cholesterol accumulation in hepatic stellate cells: Mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice

Although nonalcoholic steatohepatitis (NASH) is associated with hypercholesterolemia, the underlying mechanisms of this association have not been clarified. We aimed to elucidate the precise role of cholesterol in the pathophysiology of NASH. C57BL/6 mice were fed a control, high‐cholesterol (HC), methionine‐choline‐deficient (MCD), or MCD+HC diet for 12 weeks or a control, HC, high‐fat (HF), or HF+HC diet for 24 weeks. Increased cholesterol intake accelerated liver fibrosis in both the mouse models without affecting the degree of hepatocellular injury or Kupffer cell activation. The major causes of the accelerated liver fibrosis involved free cholesterol (FC) accumulation in hepatic stellate cells (HSCs), which increased Toll‐like receptor 4 protein (TLR4) levels through suppression of the endosomal‐lysosomal degradation pathway of TLR4, and thereby sensitized the cells to transforming growth factor (TGF)β‐induced activation by down‐regulating the expression of bone morphogenetic protein and activin membrane‐bound inhibitor. Mammalian‐cell cholesterol levels are regulated by way of a feedback mechanism mediated by sterol regulatory element‐binding protein 2 (SREBP2), maintaining cellular cholesterol homeostasis. Nevertheless, HSCs were sensitive to FC accumulation because the high intracellular expression ratio of SREBP cleavage‐activating protein (Scap) to insulin‐induced gene (Insig) disrupted the SREBP2‐mediated feedback regulation of cholesterol homeostasis in these cells. HSC activation subsequently enhanced the disruption of the feedback system by Insig‐1 down‐regulation. In addition, the suppression of peroxisome proliferator‐activated receptor γ signaling accompanying HSC activation enhanced both SREBP2 and microRNA‐33a signaling. Consequently, FC accumulation in HSCs increased and further sensitized these cells to TGFβ‐induced activation in a vicious cycle, leading to exaggerated liver fibrosis in NASH. Conclusion: These characteristic mechanisms of FC accumulation in HSCs are potential targets to treat liver fibrosis in liver diseases including NASH. (Hepatology 2014;58:154–169)

Reduced sensitivity of inducible nitric oxide synthase-deficient mice to chronic colitis.

Abstract Background: Overproduction of nitric oxide by the inducible form of nitric oxide synthase (iNOS) has been implicated in colitis. Different authors have postulated both toxic and protective effects of nitric oxide (NO) in the pathophysiology of active inflammation. The objective of this study was to examine the role of iNOS in experimental chronic colitis using iNOS-deficient mice. Methods: For induction of colitis, mice received three cycles of 2% of dextran sodium sulfate (DSS) (M.W. 40,000) treatment in drinking water. The degree of colonic inflammation, leukocyte infiltration, and the expression of cell adhesion molecules were determined. INOS expression and nitrotyrosine were also determined by immunohistochemistry. Results: After DSS treatment, a moderate colitis with marked cell infiltration was observed. Intense expression of iNOS was observed on infiltrating cells as well as on the colonic mucosal epithelium in these animals. In the iNOS-deficient mice, tissue damage was significantly diminished. No iNOS or nitrotyrosine staining was found in iNOS-deficient mice. The number of infiltrating cells and the expression of mucosal adressin cell adhesion molecule-1 were significantly attenuated in the DSS-treated colon of iNOS-deficient mice. Conclusion: Induction of iNOS seems to act as a critical toxic effector molecule in the pathogenesis of chronic colonic inflammation.

Propionibacterium freudenreichii component 1.4‐dihydroxy‐2‐naphthoic acid (DHNA) attenuates dextran sodium sulphate induced colitis by modulation of bacterial flora and lymphocyte homing

Background and aim: 1.4-Dihydroxy-2-naphthoic acid (DHNA), a bifidogenic growth stimulator from Propionibacterium freudenreichii, is thought to have a beneficial effect as a prebiotic; however, its in vivo effect on intestinal inflammation remains unknown. The aim of this study was to determine whether oral administration of DHNA can ameliorate dextran sodium sulphate (DSS) induced colitis and to determine the possible underlying mechanisms. Method: Colitis was induced in mice by treatment with 2.0% DSS for seven days. DHNA (0.6 or 2.0 mg/kg) was given in drinking water prior to (preventive study) or after (therapeutic study) DSS administration. Colonic damage was histologically scored, and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expression and β7 positive cell infiltration were determined by immunohistochemistry. mRNA levels of proinflammatory cytokines (interleukin (IL)-1β, IL-6 and tumour necrosis factor α (TNF-α)) were determined by quantitative real time polymerase chain reaction. In addition, bacterial flora in the caecum, concentrations of short chain acids, and luminal pH were examined. Results: DHNA improved survival rate and histological damage score in mice administered DSS in both the preventive and therapeutic studies. DHNA significantly attenuated the enhanced expression of MAdCAM-1, the increased β7 positive cell number, and the increased mRNA levels of IL-1β, IL-6, and TNF-α in DSS treated colon. In addition, the decreased number of Lactobacillus and Enterobacteriaceae induced by DSS was recovered by DHNA. Preventive effects on decrease in butyrate concentration and decrease in pH level in mice administered DSS were also observed in the DHNA preventive study. Conclusion: DHNA, a novel type of prebiotic, attenuates colonic inflammation not only by balancing intestinal bacterial flora but also by suppressing lymphocyte infiltration through reduction of MAdCAM-1.

Blockade of PSGL-1 attenuates CD14+ monocytic cell recruitment in intestinal mucosa and ameliorates ileitis in SAMP1/Yit mice.

The pathogenesis of Crohn’s disease (CD) is not known. However, monocytes and macrophages are thought to play important roles in the development of mucosal inflammation. Therefore, in this study, we examined the role of monocyte‐endothelial cell interactions in senescence‐accelerated mouse P1 (SAMP1)/Yit mice, a murine model of spontaneous ileitis. Fluorescence‐labeled CD14+ monocytic cells isolated from the spleen and mesenteric lymph nodes of AKR/J (control) mice were injected into the tail veins of recipient (AKR/J and SAMP1/Yit) mice, and migration in the postcapillary venules (PCV) of Peyer’s patches, submucosal venules, and villus microvessels of the terminal ileum was monitored by using an intravital microscope. Rolling and adhesion of CD14+ monocytic cells in the PCV of Peyer’s patches and microvessels of the terminal ileum were increased in SAMP1/Yit mice. An imunohistochemical study showed increased expression of P‐selectin glycoprotein‐1 (PSGL‐1), P‐selectin, and vascular cell adhesion molecule‐1 in the terminal ileum of SAMP1/Yit mice. Antibodies against these three adhesion molecules significantly inhibited adhesion of CD14+ monocytic cells to the PCV of Peyer’s patches and microvessels of the terminal ileum, treatment with an anti‐PSGL‐1 monoclonal antibody (mAb) showing the strongest suppressive effect. Anti‐PSGL‐1 mAb also attenuated T cell adhesion in microvessels of intestinal mucosa. In addition, periodical administration of an anti‐PSGL‐1 mAb for 7 weeks significantly ameliorated ileitis of SAMP1/Yit mice. The results suggest that PSGL‐1‐P‐selectin interaction plays an important role in monocyte‐endothelial cell interactions and the development of ileitis in a murine model of CD and that the blockade of this adhesion molecule may be a novel strategy for treating CD.

Omega‐3 fatty acids exacerbate DSS‐induced colitis through decreased adiponectin in colonic subepithelial myofibroblasts

Background: Although the immunoregulatory effects of &ohgr;‐3 fatty acid and adiponectin have been postulated, their role in intestinal inflammation is controversial. The aim of this study was to determine whether dietary fat intake influences activity of colonic inflammation through modulating this system. Methods: C57BL/6 mice received dextran sulfate sodium for induction of colitis. Mice were fed a control diet, &ohgr;‐3 fat‐rich diet, &ohgr;‐6 fat‐rich diet, or saturated fat‐rich diet. Some mice were administered a peroxisome proliferator activated receptor‐gamma; agonist, pioglitazone. Messenger RNA expression of adiponectin and its receptors were analyzed. Adiponectin expression in colonic mucosa of ulcerative colitis patients was also analyzed. Results: The receptors for adiponectin were found to be ubiquitously expressed in epithelial cells, intraepithelial lymphocytes, lamina proprial mononuclear cells, and subepithelial myofibroblasts from colonic tissue, but adiponectin was only expressed in myofibroblasts. Induction of colitis significantly decreased the expression of adiponectin in colonic mucosa. The &ohgr;‐3 fat diet group, but not the other fat diet groups, showed exacerbated colitis with a further decrease of adiponectin expression. Pioglitazone treatment ameliorated the level of decrease in adiponectin expression and improved colonic inflammation induced by the &ohgr;‐3 fat‐rich diet. In patients with ulcerative colitis, the expression level of adiponectin in colonic mucosa was also decreased compared with that in control mucosa. Conclusions: Adiponectin was found to be expressed in myofibroblasts. Adiponectin expression was significantly suppressed by induction of colitis, and aggravation of colitis after exposure to &ohgr;‐3 fat may be due to a further decrease in the expression level of adiponectin.

In vivo demonstration of T lymphocyte migration and amelioration of ileitis in intestinal mucosa of SAMP1/Yit mice by the inhibition of MAdCAM-1

The aetiology of Crohns disease (CD) remains unknown. Since SAMP1/Yit mice have been reported to develop CD‐like spontaneous enteric inflammation, such mice have been studied as an animal model of CD. In this study, using this model we examined T lymphocyte migration in microvessels of intestinal mucosa in vivo and the expression of adhesion molecules by immunohistochemistry. Fluorescence‐labelled T lymphocytes isolated from AKR/J (control) mice were injected into the tail veins of recipient mice, and T lymphocyte migration in the postcapillary venules of Peyers patches, submucosal microvessels, and villus capillaries of the terminal ileum was monitored using an intravital microscope. Adhesion of T lymphocytes was significantly increased in 35 week old SAMP1/Yit mice compared with that in AKR/J or 15 week old SAMP1/Yit mice. Immunohistochemical study showed increased infiltration of CD4, CD8 and β7‐integrin‐positive cells and increased expression of MAdCAM‐1 and VCAM‐1 in the terminal ileum of SAMP1/Yit mice. Antibodies against MAdCAM‐1 and VCAM‐1 significantly inhibited adhesion of T lymphocytes to microvessels of the terminal ileum, and anti‐MAdCAM‐1 antibody showed stronger suppressive effect than the anti‐VCAM‐1 antibody. Periodical administration of anti‐MAdCAM‐1 antibody twice a week for 7 weeks significantly ameliorated ileitis of SAMP1/Yit mice, but submucosal hypertrophy was not significantly suppressed. Anti‐VCAM‐1 antibody treatment failed to show significant resolution of ileitis. In addition, anti‐MAdCAM‐1 antibody treatment also attenuated established ileitis. The results demonstrate that, although MAdCAM‐1 and VCAM‐1 play an important role in T lymphocyte–endothelial cell interactions in SAMP1/Yit mice, MAdCAM‐1 may be a more appropriate target for therapeutic modulation of chronic ileitis.

Expression of bone morphogenetic proteins in colon carcinoma with heterotopic ossification.

Here we report the case of a 50‐year‐old woman with adenocarcinoma of the colon, showing heterotopic ossification. The patient was referred to our hospital for investigation of anemia secondary to occult gastrointestinal blood loss. By colonoscopy, an irregular polypoid mass was found in the ascending colon. A biopsy of the lesion revealed moderately to poorly differentiated adenocarcinoma with heterotopic ossification. A right hemicolectomy was done and revealed areas of heterotopic bone within the tumor, but no ossification was evident in the metastatic lesions within the mesenteric lymph nodes. The formation of heterotopic bone in gastrointestinal tumors is rare and its exact mechanism is unknown. Immunohistochemical localization of bone morphogenetic proteins (BMP), known to be primary inducers of new bone formation, was determined. BMP‐5 and ‐6 were prominent in the cytoplasm of tumor cells, and they stained weakly in osteoblast‐like cells adjacent to newly formed bone. Cytoplasmic staining for BMP‐2 and ‐4 was weak in tumor cells, osteoblast‐like cells, and stromal fibroblast cells. BMP may play an important role in heterotopic ossification in colon adenocarcinoma.

Expression of PD-1, PD-L1, and PD-L2 in the liver in autoimmune liver diseases

OBJECTIVES:PD-L1 (also B7-H1) and PD-L2 (also B7-DC) are ligands for programmed death-1 (PD-1), which is a member of the CD28/B7 superfamily of costimulatory molecules and plays an inhibitory role on the periphery. Impaired regulation of this system may cause disruption to self-tolerance leading to autoimmunity; however, the role of these molecules in the liver is unknown. Therefore, we examined the expression of PD-1, PD-L1, and PD-L2 in the liver in autoimmune liver diseases.METHODS:We examined the liver expression of these molecules in autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) with no previous medical treatment using immunohistochemical staining and real-time PCR, and compared with chronic hepatitis type C (CHC) as a control.RESULTS:Although PD-1, PD-L1, and PD-L2 were expressed in the liver in AIH, PBC, as well as CHC, the expressions were relatively lower in PBC. In AIH, despite more severe inflammation than in CHC, the expression of these molecules was not greater than in CHC, and when compared with the relative expression of PD-L1, PD-L2 was lower in AIH. PD-L1 and PD-L2 expressions were well correlated with the level of IFN-γ; however, relatively decreased induction for PD-L1 and PD-L2 by IFN-γ was observed in AIH or PBC than in CHC.CONCLUSION:Modulation of PD-1/PD-L1 and PD-L2 systems may play a role in the development of autoimmune liver diseases.

Involvement of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the pathogenesis of granulomatous colitis in rats

Although increased expression of mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) has been demonstrated in inflammatory sites of various diseases, its role in colitis remains unknown. In this study, we examined whether MAdCAM‐1 is involved in the pathogenesis of granulomatous colitis induced by peptidoglycan‐polysaccharide (PG‐PS). Experimental colitis was induced by intramural injection of PG‐PS to rat colon. After 3 weeks the colon was removed and the mucosal inflammation was assessed. The area of MAdCAM‐1‐positive venules and the subsets of infiltrating cells were determined in colonic mucosa by immunohistochemistry. In another experiment, monoclonal antibody against MAdCAM‐1 was administered intraperitoneally to examine its attenuating effect on colitis. The intramural injection of PG‐PS induced significant colonic inflammation with granuloma formation. The submucosa was drastically thickened with the infiltration of CD4 positive lymphocytes and ED‐1 positive macrophages. Intense MAdCAM‐1 expression was observed on endothelium of the submucosal venules in inflamed mucosa. Administration of anti‐MAdCAM‐1 antibody significantly attenuated the PG‐PS‐induced colonic damage and cell infiltration. Enhanced expression of MAdCAM‐1 was demonstrated in venular endothelium of the inflamed colon in PG‐PS‐induced colitis. The attenuating effect of anti‐MAdCAM‐1 suggests the importance of the MAdCAM‐1‐dependent process in the formation of chronic granulomatous colitis.

Changes in regulatory molecules for lymphangiogenesis in intestinal lymphangiectasia with enteric protein loss

Background and Aim:  Vascular endothelial growth factor receptor 3 (VEGFR3) and LYVE‐1 are specifically expressed in the endothelium of the lymphatic systems. VEGF‐C, D, FOXC2, Prox 1, and SOX18 are known to play central roles in lymphatic development. We investigated the expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa of idiopathic intestinal lymphangiectasia.