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的活性氧物质（ROS）促进动脉粥样硬化（athero）， 血管平滑肌细胞（SMC）和巨噬细胞炎症信号。抗动脉粥样硬化策略 然而，靶向产生O2的NADPH氧化酶增加了对感染的易感性。该项目的目标是 识别抑制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 免疫力其中一个机制可能涉及广泛表达的非编码小核仁（sno） 来自核糖体蛋白L13 a（Rpl 13 a）基因座的RNA：SNORD 32A、SNORD 33、SNORD 34和SNORD 35 A。 我们发现这些snoRNA在体外和体内增加ROS水平和氧化应激。我们的初步 用Rpl 13 a-snoRNA-/-（snoKO）小鼠和源自它们的SMC的研究显示：（1）snoKO SMC具有以下特性： 较低水平的ROS、细胞增殖和迁移。(2)与WT SMC相比， snoKO SMC表达的细胞色素C氧化酶亚基4同种型2（COX 4 I2）是对照组的5.7倍， 线粒体O2生成。(3)SnoKO颈动脉比WT颈动脉发生更少的动脉粥样硬化， 原位移植到Apoe-/-小鼠中。(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，这是一个由ROS增强的过程。 SnoRNA通过反义结构域与它们的靶RNA结合，然后募集纤维素酶， RNA 2 '-O-甲基化。SnoRNA通常修饰核糖体RNA;然而，我们发现，至少有一个 的Rpl 13 a snoRNA可以靶向mRNA进行2 '-O-甲基化-这一过程改变mRNA丰度， 翻译.尽管如此，构成Rpl 13 a-snoRNA的促氧化作用的靶标的特异性mRNA 保持模糊。因此，该项目将测试Rpl 13 a snoRNA促进动脉粥样硬化的假设， 特别是通过增强SMC向泡沫细胞的转分化，并且Rpl 13 a-snoRNA引导的mRNA 2 '-O-甲基化影响SMC和Mφ中关键ROS调节酶的蛋白表达，包括 COX4I2。为此，该项目将比较Rpl 13 a-snoRNA-/-/Apoe-/-与Apoe-/-小鼠中的动脉粥样硬化，并使用 骨髓移植以辨别Rpl 13 a-snoRNA在骨髓来源的细胞中的作用， 动脉壁来源的细胞。我们将研究Rpl 13 a-snoRNA如何影响巨噬细胞中泡沫细胞的形成 和SMC，并确定COX 4 I2是否在snoKO SMC中产生较低的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Rpl 13 a-snoRNA的mRNA靶点： 来自WT和Rpl 13 a-snoRNA-/-SMC的mRNA上的2 '-O-甲基化位点的全转录组作图，以及 巨噬细胞，通过使用RibOxi-seq和杂交体的交联、连接和测序（CLASH） approach.通过阐明snoRNA调节SMC和巨噬细胞中ROS的机制， 该项目应确定动脉粥样硬化的新治疗靶点。