Atherosclerosis is a chronic inflammatory disease affecting large- and medium-sized arteries and is considered the major cause behind cardiovascular diseases (CVD). Elevated low-density lipoprotein (LDL)-cholesterol and low high-density lipoprotein (HDL)-cholesterol are considered major risk factors for the CVD. HDL mediates a variety of atheroprotective actions, many of them involving the interaction with the scavenger receptor class B, type 1 (SR-B1). Despite the efforts placed in raising HDL-cholesterol, no improvement has been achieved in reducing CVD risk, suggesting that other components of the HDL particles may be responsible for HDL-mediated atheroprotection. One of these may be sphingosine-1-phospate (S1P). In this thesis, the role of S1P receptors (S1PRs) in atherosclerosis is explored, with emphasis in macrophage apoptosis. In particular, the importance of the macrophage S1PR1 and its role in apoptosis and atherosclerosis was evaluated. We demonstrated that diabetes exacerbates atherosclerosis development and myocardial infarction in SR-B1 KO/apoE-hypomorphic mice and that treatment with FTY720, a S1PR agonist, protects against diabetes pro-atherogenic effects. We also show that S1PR1 agonists protected macrophages against apoptosis through phosphoinositide 3-kinase (PI3K)/AKT, and that HDL failed to protect S1PR1 deficient macrophages against apoptosis. In vivo, macrophage S1PR1 deficiency translated into increased atherosclerosis, necrotic core formation and numbers of apoptotic cells in the atherosclerotic plaque. BIM deficiency in BM cells was protective against atherosclerosis development and HDL treatment reduced BIM protein levels in cells exposed to ER stressors, suggesting that the pro-apoptotic protein may be an important target for HDL in macrophages. We conclude that signaling through the S1PRs, in particular S1PR1 is important in controlling macrophage apoptosis and atherosclerosis development. Our data suggests that S1PR1 signaling axis and the pro-apoptotic protein BIM play an important role in mediating HDL anti-apoptotic signaling, however further studies are required to clarify the interaction between all of these factors. / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24829号 / 医博第4997号 / 新制||医||1067(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 森信, 暁雄, 教授 竹内, 理, 教授 濵﨑, 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24834号 / 医博第5002号 / 新制||医||1067(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 竹内, 理, 教授 上杉, 志成, 教授 金子, 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Spinal cord injury (SCI) often leads to irreversible damage, and permanent paralysis inferior to the injury is common (Leibinger et al., 2013). Injury to the spinal cord occurs in two phases. In the first phase, components of the spinal cord are subject to mechanical trauma causing direct damage. In the second phase, damage spreads from the area of injury through molecular processes. Several studies have linked M1 "pro-inflammatory" macrophages to exacerbation of damage by inducing dieback of dystrophic axons, but not healthy axons, through direct cellular contact. Several studies have identified the presence of macrophage subtypes at specific time. A literature review was conducted in order to summarize these findings (Busch, Horn, Silver, & Silver, 2009; Evans et al., 2014; Horn, Busch, Hawthorne, van Rooijen, & Silver, 2008; Kigerl et al., 2009; Shechter et al., 2013). Although the full mechanism behind the process of M1 macrophage-mediated dieback of dystrophic axons is unclear, matrix metalloproteinase-9 (MMP-9) produced by these macrophages has been shown to play a role. However, the specific interaction between MMP-9 and neurons is under investigation. The research described explores the relationship between MMP-9 and fractalkine (CX3CL1), a surface protein expressed by CNS neurons. SDS-PAGE and western blot were used to determine whether the presence of MMP-9 increases the cleavage of fractalkine at several time intervals. At a concentration of 300ng/ml, MMP-9 was not found to demonstrate cleavage of fractalkine.

Orthopedic metal implant materials may mediate a variety of adverse tissue reactions by releasing ions through corrosion. Adverse tissue reactions include inflammation, fibrosis and hypersensitivity. All of these reactions eventually lead to implant failure. The goal of this study was to provide a better understanding of the cellular-material interaction at the metal surface. The hypotheses were that 1. the attachment of cells and their released reactive inflammatory compounds (e.g. hydrogen peroxide H2O2, superoxide O2. and nitric oxide NO.) on the surfaces alter the alloys? corrosion and surface properties and 2. the changes in corrosion and chemical properties of the surfaces affect cell behavior. To evaluate the hypotheses, a custom-made electrochemical corrosion cell was used to evaluate how cell culture medium, macrophage cells and macrophage cells activated to simulate inflammation affected the corrosion and surface properties of Co-Cr-Mo and Ti-6Al-4V alloys and how released alloy corrosion products affected cell behaviors. The macrophage cell line used was known to produce reactive species H2O2, O2. and NO. when activated by antigen and interferon. The alloy corrosion properties were enhanced by observing the open circuit potential (OCP), charge transfer, metal ion release, and changes in surface oxides. Proliferation, viability and metabolism were used to evaluate effects of corrosion on the cells. The OCP of Co-Cr-Mo remained unchanged whereas that of Ti-6Al-4V increased over three days for all three test conditions. Both alloys cultured with medium had the lowest OCP among all conditions. With activated macrophage cells, both alloys had the lowest total charge transfer and concentrations of metal ion released. This improved corrosion resistance was mostly due to an enhancement of the surface oxide due to the reactive species released from activated cells, as indicated from the surface analyses. Both alloys were found to have increased percentage (in peak intensity) of O and Ti or Cr peaks, which indicated an increase of Ti and Cr oxides on Ti-6Al-4V and Co-Cr-Mo alloys respectively. The improved corrosion properties resulted in less metal ion release than those without enhanced surface oxides, thus alloys did not further activate cell immune responses in the three day period. The non-activated or activated cells with released metal ions did not exhibit any degradation in their viability, intracellular ATP, NO and IL-1b release as compared to controls. This is consistent with the generally accepted good biocompatibility of these alloys.

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