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种(ROS)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。抗动脉粥样硬化策略 然而，以产生O2-的NADPH氧化酶为靶标会增加感染的敏感性。这个项目的目标是 识别抑制ROS促进的动脉粥样硬化的新机制，同时最大限度地减少对 豁免权。一种这样的机制可能涉及普遍表达的非编码小核仁(Sno)。 核糖体蛋白L13a(Rpl13a)基因座的RNA：SNORD32A、SNORD33、SNORD34和SNORD35A。 我们发现，这些snoRNAs在体外和体内都能增强ROS水平和氧化应激。我们的预赛 对Rpl13a-snoRNA-/-(SnoKO)小鼠及其来源的SMC的研究表明：(1)SnoKO SMC具有 ROS水平、细胞增殖和迁移能力低于同种WT SMC。(2)与WT SMCS相比， SnoKO SMC表达的细胞色素C氧化酶亚基4异构体2(COX4I2)增加5.7倍，从而减少 线粒体产生O2-。(3)与WT颈动脉相比，SnoKO颈动脉的动脉粥样硬化程度较低 原位移植到APOE-/-小鼠体内。(4)与APOE-/-小鼠相比，snoKO/APOE-/-小鼠 头臂动脉粥样硬化减少40%。(5)与snoRNA+/+头臂动脉或颈动脉相比， SnoKO动脉表现出较少的SMC到泡沫细胞的转分化，这一过程被ROS加强。 SnoRNAs通过反义结构域与其目标RNA结合，然后招募纤维素酶，从而影响 RNA2‘-O-甲基化。SnoRNAs典型地修饰核糖体RNA；然而，我们发现至少有一个 在Rpl13a中，snoRNAs可以靶向mRNA进行2‘-O-甲基化-这一过程改变了mRNA的丰度和 翻译。然而，构成Rpl13a-snoRNAs促氧化作用靶点的特定mRNAs 仍然默默无闻。因此，该项目将测试Rpl13a snoRNAs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MφS中关键ROS调节酶(S)的蛋白表达，包括 COX4I2。为此，该项目将比较Rpl13a-snoRNA-/-/APOE-/-和APOE-/-小鼠的动脉粥样硬化，并使用 骨髓移植对比研究Rpl13a-snoRNAs在骨髓源性细胞中的作用 动脉壁来源的细胞。我们将研究Rpl13a-snoRNAs如何影响巨噬细胞中泡沫细胞的形成 并确定COX4I2是否会降低snoKO SMC中的ROS水平和炎症反应。 最后，我们将确定rpl13a-snoRNAs在SMC和巨噬细胞中的mrna靶点，通过执行 WT和Rpl13a-snoRNA-/-SMC和RPL13a-snoRNA-/-SMC的2‘-O-甲基化位点的转录组定位 巨噬细胞，通过使用RibOxi-seq和杂交的交联物、连接和测序(CLASH) 接近。通过阐明snoRNAs调节SMC和巨噬细胞中ROS的机制，这 该项目应确定动脉粥样硬化的新治疗靶点。