Mechanisms by which Small Nucleolar RNAs Exacerbate Atherosclerosis

小核仁 RNA 加剧动脉粥样硬化的机制

基本信息

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
项目类别：
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

项目摘要

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活性氧（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传导。抗动脉生就性策略但是，靶向O2-生产NADPH氧化物，增加了感染的敏感性。这个项目的目标是辨别限制ROS促进动脉粥样形成的新型机制，同时最大程度地减少对对的不良影响免疫。一种这样的机制可能涉及普遍表达的非编码小核酸（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在体外和体内增加了ROS水平和氧化应激。我们的初步对RPL13A-SNORNA的研究 - / - （Snoko）小鼠和SMC从中得出：（1）Snoko SMC具有与先天性WT SMC相比，ROS，细胞增殖和迁移的水平较低。（2）与WT SMC相比 Snoko SMCS Express Express 5.7倍多的细胞色素C氧化酶亚基4同工型2（Cox4i2），它还原线粒体O2生产。（3）Snoko颈动脉动脉的发展量不如wt颈原位移植到apoe - / - 小鼠中。（4）与apoe - / - 小鼠相比，Snoko/apoe - / - 小鼠开发了减少40％的脑头动脉粥样硬化。（5）与SNORNA+/+脑头动脉或颈动脉移植相比 Snoko动脉表现出较少的SMC到泡沫 - 细胞转分化，这是由ROS潜在的过程。 SNORNA通过反义结构域结合其靶RNA，然后募集酶纤维蛋白，从而影响酶 RNA 2'-O-甲基化。 snornas典型地修饰核糖体RNA；但是，我们发现至少一个 Rpl13a snornas可以瞄准mRNA以2'-O-甲基化 - 一种改变mRNA丰度和的过程翻译。但是，构成RPL13A-SNORNAS促氧化作用的靶标的特定mRNA 保持晦涩。因此特别是通过增强SMC到泡沫细胞的转变，并且该RPL13A-SNORNA引导的mRNA 2'-O-甲基化影响SMC和MφS中关键ROS调节酶的蛋白质表达，包括 Cox4i2。为此，此项目将比较rpl13a-snorna - / - /apoe-/ - vont apoe-/ - 鼠标中的Athero，并使用骨髓移植以辨别Rpl13a-snornas在骨髓衍生细胞中的作用动脉壁源细胞。我们将研究RPL13A-SNORNA如何影响巨噬细胞中的泡沫细胞形成和SMC，并确定COX4I2是否会导致SNOKO SMC中的ROS水平降低和炎症。最后，我们将通过执行SMC和巨噬细胞中RPL13A-SNORNA的mRNA靶标的来自WT和RPL13A-SNORNA的mRNA上2'-O-甲基化位点的全转录组映射 - / - SMC和巨噬细胞，使用Rocoxi-Seq和交联，结扎和测序杂种（碰撞）方法。通过阐明SNORNA在SMC和巨噬细胞中调节ROS的机制，这项目应确定Athero的新治疗靶标。