Masahiro Ikesue

Interleukin-17A Deficiency Accelerates Unstable Atherosclerotic Plaque Formation in Apolipoprotein E-Deficient Mice

Objective—Interleukin(IL)-17A, an inflammatory cytokine, has been implicated in atherosclerosis, in which inflammatory cells within atherosclerotic plaques express IL-17A. However, its role in the development of atheroscelrosis remains to be controversial. Methods and Results—To directly examine the role of IL-17A in atherosclerosis, we generated apolipoprotein E (ApoE)/IL-17A double-deficient (ApoE−/−IL-17A−/−) mice. Mice were fed with high-fat diet (HFD) for either 8 or 16 weeks, both starting at ages of 6 to 8 weeks. We found that splenic CD4+ T-cells produced high amounts of IL-17A in ApoE−/− mice after HFD feeding for 8 weeks. Atherosclerosis was significantly accelerated in HFD-fed ApoE−/−IL-17A−/− mice compared with ApoE−/− mice. Splenic CD4+ T-cells of ApoE−/−IL-17A−/− mice after HFD feeding for 8 weeks, but not for 16 weeks, exhibited increased interferon gamma and decreased IL-5 production. Importantly, formation of vulnerable plaque as evidenced by reduced numbers of vascular smooth muscle cells and reduced type I collagen deposition in the plaque was detected in ApoE−/−IL-17A−/− mice after HFD feeding for 8 weeks. Conclusion—These results suggest that IL-17A regulates the early phase of atherosclerosis development after HFD feeding and plaque stability, at least partly if not all by modulating interferon gamma and IL-5 production from CD4+ T-cells.

Syndecan-4 Prevents Cardiac Rupture and Dysfunction After Myocardial Infarction

Rationale: Syndecan-4 (Syn4), a cell-surface heparan sulfate proteoglycan, has been detected in the infarct region after myocardial infarction (MI), but its functional significance has not been elucidated. Objective: We examined whether and how Syn4 regulates the cardiac healing process after MI. Methods and Results: Although the heart in Syn4-deficient (Syn4−/−) mice was morphologically and functionally normal, Syn4−/− mice exhibited impaired heart function and increased mortality rate as a result of cardiac ruptures after MI. Cardiac ruptures in Syn4−/− mice were associated with reduced inflammatory reaction and impaired granulation tissue formation during the early phase of MI, as evidenced by reduced numbers of leukocytes, fibroblasts, myofibroblasts, macrophages, and capillary vessels, along with reduced extracellular matrix protein deposition in the infarct region after MI. Transforming growth factor-&bgr;1–dependent cell signaling was preserved, whereas cell migration, fibronectin-induced cell signaling, and differentiation into myofibroblasts were defective in Syn4−/− cardiac fibroblasts. We also found that Syn4 was involved in basic fibroblast growth factor–dependent endothelial cell signaling, cell proliferation, and tube formation. Finally, overexpression of the shed form of Syn4 before MI creation led to an increase in mortality due to cardiac rupture via its action as a dominant-negative inhibitor of endogenous Syn4 signaling, which suggested a protective role of Syn4 signaling in MI. Conclusions: These results suggest that Syn4 plays an important role in the inflammatory response and granulation tissue formation, thereby preventing cardiac rupture and dysfunction after MI.

Syndecan-4 protects against osteopontin-mediated acute hepatic injury by masking functional domains of osteopontin

Osteopontin (OPN) is a T helper type 1 immunoregulatory cytokine that plays a critical role in various inflammatory disorders. OPN exerts proinflammatory reactions through interaction with integrin receptors. OPN function can be modulated by protease digestion. However, the molecular mechanisms that regulate OPN function in vivo have not been elucidated. There are two putative heparin-binding domains (HBDs) within the OPN molecule, which may bind both heparin and heparin-like glycosaminoglycans such as syndecan. We show that expression of OPN and syndecan-4 is significantly up-regulated after concanavalin-A (ConA) injection. Syndecan-4 binds to one of the HBDs of OPN, which overlaps with the thrombin cleavage site of OPN. When OPN is associated with syndecan-4, syndecan-4 masks both the thrombin cleavage and the integrin binding sites within OPN. Importantly, syndecan-4–deficient (Syn4KO) mice are more susceptible to hepatic injury, and the thrombin-cleaved form of OPN is significantly elevated in Syn4KO mice as compared with wild-type mice after ConA injection. Finally, we demonstrate that administration of purified syndecan-4 protects mice from ConA-induced hepatic injury. Thus, syndecan-4 is a critical intrinsic regulator of inflammatory reactions via its effects on OPN function and is a potential novel therapeutic tool for treating inflammatory diseases.

α9β1 Integrin-Mediated Signaling Serves as an Intrinsic Regulator of Pathogenic Th17 Cell Generation

The interaction between matricellular proteins such as tenascin-C (TN-C) and osteopontin (OPN) and integrins has been implicated in the pathology of rheumatoid arthritis in which Th17 cells are recognized as primary pathogenic cells. The differentiation of Th17 cells is tightly regulated by cytokines derived from APCs, receiving various signals including TLR stimuli. In this study, we used a collagen-induced arthritis model and found that increased numbers of α9 integrin-positive conventional dendritic cells and macrophage were detectable in the draining lymph node (dLN) shortly following first immunization, and these cells produced both TN-C and OPN, ligands for α9 integrin. α9 integrin-mediated signaling, induced by TN-C and OPN, promoted the production of Th17-related cytokines by conventional dendritic cells and macrophages in synergy with TLR2 and 4 signaling. This led to the Th17 cell differentiation and arthritis development. Moreover, Th17 cells generated under blocking of α9 integrin-mediated signaling showed low level of CCR6 expression and impaired migration ability toward CCL20. Thus, we have identified α9 integrin-mediated signaling by TN-C and OPN as a novel intrinsic regulator of pathogenic Th17 cell generation that contributes to the development of rheumatoid arthritis.

Syndecan-4 Deficiency Limits Neointimal Formation After Vascular Injury by Regulating Vascular Smooth Muscle Cell Proliferation and Vascular Progenitor Cell Mobilization

Objective—Syndecan-4 (Syn4) is a heparan sulfate proteoglycan and works as a coreceptor for various growth factors. We examined whether Syn4 could be involved in the development of neointimal formation in vivo. Methods and Results—Wild-type (WT) and Syn4-deficient (Syn4−/−) mice were subjected to wire-induced femoral artery injury. Syn4 mRNA was upregulated after vascular injury in WT mice. Neointimal formation was attenuated in Syn4−/− mice, concomitantly with the reduction of Ki67-positive vascular smooth muscle cells (VSMCs). Basic-fibroblast growth factor– or platelet-derived growth factor-BB–induced proliferation, extracellular signal-regulated kinase activation, and expression of cyclin D1 and Bcl-2 were impaired in VSMCs from Syn4−/− mice. To examine the role of Syn4 in bone marrow (BM)–derived vascular progenitor cells (VPCs) and vascular walls, we generated chimeric mice by replacing the BM cells of WT and Syn4−/− mice with those of WT or Syn4−/− mice. Syn4 expressed by both vascular walls and VPCs contributed to the neointimal formation after vascular injury. Although the numbers of VPCs were compatible between WT and Syn4−/− mice, mobilization of VPCs from BM after vascular injury was defective in Syn4−/− mice. Conclusion—Syn4 deficiency limits neointimal formation after vascular injury by regulating VSMC proliferation and VPC mobilization. Therefore, Syn4 may be a novel therapeutic target for preventing arterial restenosis after angioplasty.

Establishment of the enzyme‐linked immunosorbent assay system to detect the amino terminal secretory form of rat Erc/Mesothelin

By representational difference analysis, we previously identified the rat Erc (Expressed in renal carcinoma) gene that was more abundantly expressed in the renal carcinoma tissues of Eker rats than in the rat normal kidney. In this study, we raised antibodies against the amino‐terminal portion of the rat Erc, and demonstrated the existence of a ∼30‐kDa secretory form in the supernatant of cultured cells derived from rat renal carcinoma. The enzyme‐linked immunosorbent assay (ELISA) system using these antibodies detected high concentrations of this form in the sera of Eker rats bearing renal carcinomas, and in the sera of rats transplanted with mesothelioma cells. Mesothelin, a human homolog of the rat Erc, was recently reported to be a serum marker of malignant mesothelioma. The prognosis of mesothelioma is poor and there is no effective treatment at present. There are several rat model systems of mesothelioma that may be promising tools in the development of an antimesothelioma treatment. We hope our ELISA to detect the soluble form of rat Erc/Mesothelin is useful in the rat model system to exploit the antimesothelioma therapy to be used in human cases. (Cancer Sci 2007; 98: 659–664)

Death receptor 6 contributes to autoimmunity in lupus-prone mice

Expansion of autoreactive follicular helper T (Tfh) cells is tightly restricted to prevent induction of autoantibody-dependent immunological diseases, such as systemic lupus erythematosus (SLE). Here we show expression of an orphan immune regulator, death receptor 6 (DR6/TNFRSF21), on a population of Tfh cells that are highly expanded in lupus-like disease progression in mice. Genome-wide screening reveals an interaction between syndecan-1 and DR6 resulting in immunosuppressive functions. Importantly, syndecan-1 is expressed specifically on autoreactive germinal centre (GC) B cells that are critical for maintenance of Tfh cells. Syndecan-1 expression level on GC B cells is associated with Tfh cell expansion and disease progression in lupus-prone mouse strains. In addition, Tfh cell suppression by DR6-specific monoclonal antibody delays disease progression in lupus-prone mice. These findings suggest that the DR6/syndecan-1 axis regulates aberrant GC reactions and could be a therapeutic target for autoimmune diseases such as SLE.

Oral administration of the Aureobasidium pullulans -derived β-glucan effectively prevents the development of high fat diet-induced fatty liver in mice

Aureobasidium pullulans-derived β-glucan (AP-PG) consisting of a β-(1,3)-linked glucose main chain and β-(1,6)-linked glucose branches is taken as a supplement to improve health. This study demonstrates that oral administration of AP-PG is effective to prevent the development of high-fat diet (HFD)-induced fatty liver in mice. Here, C57BL/6N mice were fed with a normal diet or HFD, and AP-PG diluted in drinking water was administered orally. After 16 weeks, the serological analysis showed that HFD-induced high blood cholesterol and triglyceride levels were reduced by the oral administration of AP-PG. Further, HFD induced-fatty liver was significantly reduced by the oral administration of AP-PG. The triglyceride accumulation in the liver was also significantly reduced in mice administered AP-PG. Liver injury as indicated by an increase in serum alanine aminotransferase (ALT) in the HFD-fed mice was significantly reduced in the mice administered AP-PG orally, and the gene expression of cholesterol 7 alpha-hydroxylase (CYP7A1) which is known to be involved in cholesterol degradation in the liver was significantly increased in the AP-PG administered mice. These results suggest the possibility that the oral administration of AP-PG is effective to prevent the development of non-alcoholic fatty liver disease (NAFLD).

Syndecan 4 Regulation of the Development of Autoimmune Arthritis in Mice by Modulating B Cell Migration and Germinal Center Formation

Syndecan 4 has been implicated as a critical mediator of inflammatory responses because of its functions as a coreceptor and reservoir for growth factors and chemokines. Although syndecan 4 is known to be expressed on B cells, its role in immune responses remains unclear. The purpose of this study was to investigate the contribution of syndecan 4 to the development of immune arthritis in murine models.