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)

Indiana University-Purdue University Indianapolis (IUPUI) / Pneumocystis pneumonia (PCP) is the leading opportunistic disease in immunocompromised individuals, particularly in AIDS patients. The alveolar macrophage (AM) is the major type of cell responsible for the clearance of Pneumocystis organisms; however, they undergo a high rate of apoptosis during PCP due to increased intracellular polyamine levels. This study examined the mechanism of this polyamine mediated apoptosis and investigated an alternative therapy for PCP by targeting this mechanism. The elevated polyamine levels were determined to be caused by increased polyamine synthesis and uptake. Increased polyamine uptake was found to be AM-specific, and recruited inflammatory cells including monocytes, B cells, and CD8+ T cells were found to be a potential source of polyamines. The expression of the antizyme inhibitor (AZI), which regulates both polyamine synthesis and uptake, was found to be greatly up-regulated in AMs during PCP. AZI overexpression was confirmed to be the cause of increased polyamine synthesis and uptake and apoptosis of AMs during PCP by gene knockdown assays. Pneumocystis organisms and zymosan were found to induce AZI overexpression in AMs, suggesting that the β-glucan of the Pneumocystis cell wall is responsible for this AZI up-regulation. In addition, levels of mRNA, protein, and activity of polyamine oxidase (PAO) were also found to be increased in AMs during PCP, and its substrates N1-acetylspermidine and N1-acetylspermine were found to induce its up-regulation. These results indicate that the H2O2 generated during PAO-mediated polyamine catabolism caused AMs to undergo apoptosis. Since increased polyamine uptake was demonstrated to be a pathogenic mechanism of PCP in this study, the potential therapeutic activity of five putative polyamine transport inhibitors against PCP was tested. Results showed that compound 44-Ant-44 significantly decreased pulmonary inflammation, organism burden, and macrophage apoptosis, and prolonged the survival of rats with PCP. In summary, this study demonstrated that Pneumocystis organisms induce AZI overexpression, leading to increased polyamine synthesis, uptake, and apoptosis rate in AMs and that targeting polyamine transport is a viable therapeutic approach against PCP.

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