ミクログリアのEP4受容体関連蛋白EPRAPは脳内で炎症を促進する

京都大学0048新制・課程博士博士(医学)甲第20237号医博第4196号新制||医||1019(附属図書館)京都大学大学院医学研究科医学専攻(主査)教授 松原 和夫, 教授 渡邉 大, 教授 伊佐 正学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDGA

CPT-11-Induced Delayed Diarrhea Develops via Reduced Aquaporin-3 Expression in the Colon

While irinotecan (CPT-11) has a potent anti-cancer effect, it also causes serious diarrhea as an adverse reaction. In this study, we analyzed the pathogenic mechanism of CPT-11-induced delayed diarrhea by focusing on water channel aquaporin-3 (AQP3) in the colon. When rats received CPT-11, the expression level of AQP3 was reduced during severe diarrhea. It was found that the expression levels of inflammatory cytokines and the loss of crypt cells were increased in the colon when CPT-11 was administered. When celecoxib, an anti-inflammatory drug, was concomitantly administered, both the diarrhea and the reduced expression of AQP3 induced by CPT-11 were suppressed. The inflammation in the rat colon during diarrhea was caused via activated macrophage by CPT-11. These results showed that when CPT-11 is administered, the expression level of AQP3 in the colon is reduced, resulting in delayed diarrhea by preventing water transport from the intestinal tract. It was also suggested that the reduced expression of AQP3 might be due to the inflammation that occurs following the loss of colonic crypt cells and to the damage caused by the direct activation of macrophages by CPT-11. Therefore, it was considered that anti-inflammatory drugs that suppress the reduction of AQP3 expression could prevent CPT-11-induced delayed diarrhea

Expression of Vasohibin-1 in Human Carotid Atherosclerotic Plaque

[Aim]: In patients with carotid plaque, intraplaque hemorrhage arising from ruptured neovascular vessels within the neointima is an important cause of stroke. The expression of Vasohibin-1 (VASH1), a negative feedback regulator of angiogenesis, occurs in the microvessel endothelial cells of various solid tumors and the arterial wall. However, the roles of VASH1 in the pathogenesis of atherosclerotic diseases remain unclear. The present study aimed to clarify the relevance of the VASH1 expression and plaque instability in human carotid plaques. [Methods]: We used quantitative real-time PCR and immunostaining to examine 12 atheromatous plaque specimens obtained via carotid endarterectomy. The distal areas of specimens lacking macroscopic atherosclerotic lesions served as controls. [Results]: Compared with that observed in the controls, the VASH1 gene expression increased significantly in the atheromatous plaque (p=0.018). Moreover, the VASH1 mRNA levels correlated positively with those of VEGFA, CD31 and VCAM1 (r=0.788, p=0.004; r=0.99, p＜0.001; r=0.94, p＜0.001, respectively). Finally, the immunohistochemical analyses revealed the VASH1 expression in the neointimal microvessel endothelial cells of carotid plaque. [Conclusions]: The VASH1 expression levels in atheroma reflect both enhanced neovascularization and the inflammatory burden. Therefore, the VASH1 level may be a novel biomarker for evaluating plaque instability in patients with carotid arteriosclerosis and predicting ischemic stroke

The Prostaglandin E2 Receptor EP4 Regulates Obesity-Related Inflammation and Insulin Sensitivity

<div><p>With increasing body weight, macrophages accumulate in adipose tissue. There, activated macrophages secrete numerous proinflammatory cytokines and chemokines, giving rise to chronic inflammation and insulin resistance. Prostaglandin E₂ suppresses macrophage activation via EP4; however, the role of EP4 signaling in insulin resistance and type 2 diabetes mellitus remains unknown. In this study, we treated <i>db/db</i> mice with an EP4-selective agonist, ONO-AE1-329, for 4 weeks to explore the role of EP4 signaling in obesity-related inflammation <i>in vivo</i>. Administration of the EP4 agonist did not affect body weight gain or food intake; however, in the EP4 agonist–treated group, glucose tolerance and insulin resistance were significantly improved over that of the vehicle–treated group. Additionally, administration of the EP4 agonist inhibited the accumulation of F4/80-positive macrophages and the formation of crown-like structures in white adipose tissue, and the adipocytes were significantly smaller. The treatment of the EP4 agonist increased the number of anti-inflammatory M2 macrophages, and in the stromal vascular fraction of white adipose tissue, which includes macrophages, it markedly decreased the levels of proinflammatory cytokines and chemokines. Further, EP4 activation increased the expression of adiponectin and peroxidase proliferator–activated receptors in white adipose tissue. Next, we examined <i>in vitro</i> M1/M2 polarization assay to investigate the impact of EP4 signaling on determining the functional phenotypes of macrophages. Treatment with EP4 agonist enhanced M2 polarization in wild-type peritoneal macrophages, whereas EP4-deficient macrophages were less susceptible to M2 polarization. Notably, antagonizing peroxidase proliferator–activated receptor δ activity suppressed EP4 signaling-mediated shift toward M2 macrophage polarization. Thus, our results demonstrate that EP4 signaling plays a critical role in obesity-related adipose tissue inflammation and insulin resistance by regulating macrophage recruitment and polarization. The activation of EP4 signaling holds promise for treating obesity and type 2 diabetes mellitus.</p></div

EP4 Receptor–Associated Protein in Macrophages Ameliorates Colitis and Colitis-Associated Tumorigenesis

Prostaglandin E2 plays important roles in the maintenance of colonic homeostasis. The recently identified prostaglandin E receptor (EP) 4–associated protein (EPRAP) is essential for an anti-inflammatory function of EP4 signaling in macrophages in vitro. To investigate the in vivo roles of EPRAP, we examined the effects of EPRAP on colitis and colitis-associated tumorigenesis. In mice, EPRAP deficiency exacerbated colitis induced by dextran sodium sulfate (DSS) treatment. Wild-type (WT) or EPRAP-deficient recipients transplanted with EPRAP-deficient bone marrow developed more severe DSS-induced colitis than WT or EPRAP-deficient recipients of WT bone marrow. In the context of colitis-associated tumorigenesis, both systemic EPRAP null mutation and EPRAP-deficiency in the bone marrow enhanced intestinal polyp formation induced by azoxymethane (AOM)/DSS treatment. Administration of an EP4-selective agonist, ONO-AE1-329, ameliorated DSS-induced colitis in WT, but not in EPRAP-deficient mice. EPRAP deficiency increased the levels of the phosphorylated forms of p105, MEK, and ERK, resulting in activation of stromal macrophages in DSS-induced colitis. Macrophages of DSS-treated EPRAP-deficient mice exhibited a marked increase in the expression of pro-inflammatory genes, relative to WT mice. By contrast, forced expression of EPRAP in macrophages ameliorated DSS-induced colitis and AOM/DSS-induced intestinal polyp formation. These data suggest that EPRAP in macrophages functions crucially in suppressing colonic inflammation. Consistently, EPRAP-positive macrophages were also accumulated in the colonic stroma of ulcerative colitis patients. Thus, EPRAP may be a potential therapeutic target for inflammatory bowel disease and associated intestinal tumorigenesis

Cytokine and chemokine gene expression in SVF.

<p>Relative expression of <i>Tnfα</i>, <i>Il-6</i>, <i>Mcp-1</i>, and <i>Ip-10</i> mRNA in SVF isolated from <i>db/db</i> mice administered EP4 agonist (black bar) or vehicle (white bar). All values are mean ± SEM (n = 3 each). * p<0.05; ♯ p<0.01 vs. vehicle. SVF, stromal vascular fraction; Tnfα, tumor necrosis factor α; Il-6, interleukin-6; Mcp-1, monocyte chemotactic protein-1; Ip-10, interferon gamma–induced protein 10.</p

Administration of EP4 agonist alters adipose tissue macrophage polarization.

<p>(A) Relative expression of marker genes for M1 (<i>Cd11c</i>) and M2 (<i>MR</i>, <i>Cd163</i>) macrophages in epididymal fat tissues from <i>db/db</i> mice administered EP4 agonist (black bar) or vehicle (white bar). All values are mean ± SEM (n = 4–5 each). ♯ p<0.01 vs. vehicle. (B) Epididymal adipose tissues of EP4 agonist–or vehicle–treated <i>db/db</i> mice were double stained with anti-F4/80 (green), and anti-CD11c (red, upper panel) or anti-CD163 (red, lower panel) antibodies. Arrows indicate double-positive cells. Scale bar: 100 μm. MR, Mannose Receptor.</p