Mechanisms by which Small Nucleolar RNAs Exacerbate Atherosclerosis

小核仁 RNA 加剧动脉粥样硬化的机制

• 批准号: 10670399
• 负责人: NEIL J. FREEDMAN
• 金额: $ 58.7万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670399
• 关键词: Adverse effects; Affect; Age; Antiatherogenic; Antioxidants; Antisense Oligonucleotides; Aorta; Apolipoprotein E; Arteries; Atherosclerosis; Binding; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Carotid Arteries; Cell Proliferation; Cells; Clinical Trials; Congenic Mice; Embryo; Enzymes; Foam Cells; Genetic; Genetic Complementation Test; Goals; Guide RNA; Hepatic; Human; Hybrids; Immunity; In Vitro; Infection; Inflammation; Inflammatory; Insulin Resistance; Introns; Knowledge; Lecithin; Ligation; Listeriosis; Macrophage; Maps; Mediating; Messenger RNA; Methods; Methylation; Methyltransferase; Mission; Mitochondria; Molecular Chaperones; Mus; Mutagenesis; Myocardial Infarction; NADPH Oxidase; Oxidases; Oxidative Stress; Predisposition; Process; Production; Protein Isoforms; Proteins; RNA; Reactive Oxygen Species; Research; Ribosomal Proteins; Ribosomal RNA; Role; Signal Transduction; Site; Small Interfering RNA; Small Nucleolar RNA; Smooth Muscle Myocytes; Stroke; Superoxides; TNF gene; Testing; Tissues; Transgenes; Translations; Transplantation; United States National Institutes of Health; Untranslated RNA; Vascular Smooth Muscle; atherogenesis; burden of illness; catalase; cell motility; cohort; congenic; crosslink; cytochrome c oxidase; disability; fibrillarin; in vivo; mRNA Precursor; new therapeutic target; novel; novel therapeutic intervention; oxidized low density lipoprotein; protein expression; recruit; transcriptome; transdifferentiation; western diet

Superoxide and other derivative reactive oxygen species (ROS) promote atherosclerosis (athero) as well as vascular smooth muscle cell (SMC) and macrophage inflammatory signaling. Anti-atherogenic strategies targeting O2--producing NADPH oxidases, however, increase susceptibility to infection. This project’s goal is to discern novel mechanisms for constraining ROS-promoted atherogenesis while minimizing adverse effects on immunity. One such mechanism may involve the ubiquitously expressed noncoding small nucleolar (sno) RNAs from the ribosomal protein L13a (Rpl13a) locus: SNORD32A, SNORD33, SNORD34, and SNORD35A. We found that these snoRNAs augment ROS levels and oxidative stress in vitro and in vivo. Our Preliminary Studies with Rpl13a-snoRNA-/- (snoKO) mice and SMCs derived from them show: (1) snoKO SMCs have lower levels of ROS, cell proliferation and migration than congenic WT SMCs. (2) Compared with WT SMCs, snoKO SMCs express 5.7-fold more cytochrome C oxidase subunit 4 isoform 2 (COX4I2), which reduces mitochondrial O2- production. (3) SnoKO carotid arteries develop less athero than WT carotids when transplanted orthotopically into Apoe-/- mice. (4) Compared with Apoe-/- mice, snoKO/Apoe-/- mice develop 40% less brachiocephalic athero. (5) Compared with snoRNA+/+ brachiocephalic arteries or carotid grafts, snoKO arteries demonstrate less SMC-to-foam-cell transdifferentiation, a process potentiated by ROS. SnoRNAs bind to their target RNAs via an antisense domain, then recruit the enzyme fibrillarin, which effects RNA 2’-O-methylation. SnoRNAs canonically modify ribosomal RNA; however, we discovered that at least one of the Rpl13a snoRNAs can target mRNA for 2’-O-methylation—a process that alters mRNA abundance and translation. Nonetheless, specific mRNAs that constitute targets for pro-oxidant effects of Rpl13a-snoRNAs remain obscure. This project will therefore test the hypotheses that Rpl13a snoRNAs promote athero, particularly by potentiating SMC-to-foam cell transdifferentiation, and that that Rpl13a-snoRNA-guided mRNA 2’-O-methylation affects protein expression of key ROS-regulating enzyme(s) in SMCs and Mφs, including COX4I2. To do so, this project will compare athero in Rpl13a-snoRNA-/-/Apoe-/- versus Apoe-/- mice, and use bone marrow transplantation to discern the roles of Rpl13a-snoRNAs in bone marrow-derived cells versus arterial wall-derived cells. We will investigate how Rpl13a-snoRNAs affect foam cell formation in macrophages and SMCs, and determine whether COX4I2 engenders lower ROS levels and inflammation in snoKO SMCs. Finally, we will identify mRNA targets of Rpl13a-snoRNAs in SMCs and macrophages, by performing transcriptome-wide mapping of 2’-O-methylation sites on mRNA from WT and Rpl13a-snoRNA-/- SMCs and macrophages, by using the RibOxi-seq and crosslinking, ligation, and sequencing of hybrids (CLASH) approach. By elucidating mechanisms by which snoRNAs regulate ROS in SMCs and macrophages, this project should identify new therapeutic targets for athero.

超氧化物和其他衍生活性氧 (ROS) 会促进动脉粥样硬化 (athero) 以及 血管平滑肌细胞（SMC）和巨噬细胞炎症信号传导。抗动脉粥样硬化策略 然而，针对产生 O2 的 NADPH 氧化酶会增加感染的易感性。该项目的目标是 发现抑制 ROS 促进的动脉粥样硬化形成的新机制，同时最大限度地减少对动脉粥样硬化的不利影响 免疫。其中一种机制可能涉及普遍表达的非编码小核仁（sno） 来自核糖体蛋白 L13a (Rpl13a) 位点的 RNA：SNORD32A、SNORD33、SNORD34 和 SNORD35A。 我们发现这些 snoRNA 在体外和体内都会增加 ROS 水平和氧化应激。我们的初步 对 Rpl13a-snoRNA-/- (snoKO) 小鼠及其衍生的 SMC 的研究表明：(1) snoKO SMC 具有 与同源 WT SMC 相比，ROS、细胞增殖和迁移水平较低。 (2) 与WT SMC相比， snoKO SMC 表达的细胞色素 C 氧化酶亚基 4 亚型 2 (COX4I2) 增加了 5.7 倍，从而减少 线粒体 O2- 产生。 (3) 当以下情况时，SnoKO 颈动脉比 WT 颈动脉产生更少的动脉粥样硬化 原位移植到 Apoe-/- 小鼠体内。 (4)与Apoe-/-小鼠相比，snoKO/Apoe-/-小鼠发育 头臂动脉粥样硬化减少 40%。 (5)与snoRNA+/+头臂动脉或颈动脉移植物相比， snoKO 动脉表现出较少的 SMC 到泡沫细胞的转分化，这一过程由 ROS 增强。 SnoRNA 通过反义结构域与其靶 RNA 结合，然后招募原纤维蛋白酶，该酶会影响 RNA 2’-O-甲基化。 SnoRNA 典型地修饰核糖体 RNA；然而，我们发现至少有一个 的 Rpl13a snoRNA 可以靶向 mRNA 进行 2'-O-甲基化，这一过程会改变 mRNA 丰度并 翻译。尽管如此，构成 Rpl13a-snoRNA 促氧化作用靶标的特定 mRNA 保持模糊。因此，该项目将测试 Rpl13a snoRNA 促进动脉粥样硬化的假设， 特别是通过增强 SMC 到泡沫细胞的转分化，以及 Rpl13a-snoRNA 引导的 mRNA 2'-O-甲基化影响 SMC 和 Mφ 中关键 ROS 调节酶的蛋白质表达，包括 COX4I2。为此，该项目将比较 Rpl13a-snoRNA-/-/Apoe-/- 与 Apoe-/- 小鼠中的动脉粥样硬化，并使用 骨髓移植以辨别 Rpl13a-snoRNA 在骨髓来源细胞与 动脉壁来源的细胞。我们将研究 Rpl13a-snoRNA 如何影响巨噬细胞中泡沫细胞的形成 和 SMC，并确定 COX4I2 是否会降低 snoKO SMC 中的 ROS 水平和炎症。 最后，我们将通过执行以下操作来识别 SMC 和巨噬细胞中 Rpl13a-snoRNA 的 mRNA 靶标： WT 和 Rpl13a-snoRNA-/- SMC mRNA 上 2'-O-甲基化位点的全转录组图谱 巨噬细胞，通过使用 RibOxi-seq 和杂交体的交联、连接和测序 (CLASH) 方法。通过阐明 snoRNA 调节 SMC 和巨噬细胞中 ROS 的机制， 项目应确定动脉粥样硬化的新治疗靶点。