Flow responsive endothelial Pnpt1: an exoribonuclease that regulates mitochondrial function and vascular disease

流量响应内皮 Pnpt1：一种调节线粒体功能和血管疾病的核糖核酸外切酶

• 批准号: 9750410
• 负责人: Bradford C Berk
• 金额: $ 5.3万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750410
• 关键词: 5' -exoribonuclease; Aneurysm; Apolipoprotein E; Apoptotic; Atherosclerosis; Autophagocytosis; Bioinformatics; Biological Assay; Blood Vessels; CRISPR/Cas technology; Cardiovascular Diseases; Carotid Endarterectomy; Cell Cycle; Cell Line; Cell physiology; Cells; Cellular Metabolic Process; Chromosomes, Human, Pair 11; Congenic Mice; DNA biosynthesis; Data; Data Analyses; Drug Design; Encyclopedias; Endothelial Cells; Enzymes; Event; Exhibits; Exoribonucleases; Fibroblasts; Functional disorder; Gene Expression; Generations; Genes; Genetic Polymorphism; Genetic Transcription; Genome; Goals; Growth; Homeostasis; Human; Hypertension; In Vitro; Inflammation; Inflammatory; Knockout Mice; Lesion; Ligation; Link; Measures; Mediating; Mediator of activation protein; Metabolic; Metabolic Pathway; Metabolism; Mitochondria; Mitochondrial RNA; Modeling; Molecular; Morphology; Mouse Strains; Mus; Oxidative Stress; Pathogenesis; Pathologic Processes; Pathology; Pathway Analysis; Pathway interactions; Pattern; Phosphorylation; Phosphorylation Site; Polyribonucleotide Nucleotidyltransferase; Process; Production; Protein Binding Domain; Proteins; Protocols documentation; Quantitative Trait Loci; RNA Degradation; RNA Processing; Role; Signal Transduction; Site; Smooth Muscle Myocytes; Solid; Specimen; Stress; Time; Transcript; Transcription Factor 3; Transgenic Mice; Tunica Intima; Vascular Diseases; Vascular Smooth Muscle; Vascular remodeling; base; cardiovascular risk factor; cell growth; differential expression; enzyme activity; experimental study; improved; in vivo; intima media; mitochondrial dysfunction; mouse model; mutant; new therapeutic target; novel; programs; therapeutic target; transcription factor; transcriptome sequencing; transcriptomics; vascular inflammation; vascular smooth muscle cell proliferation

This proposal aims to delineate molecular mechanisms that link laminar flow mediated signaling with gene expression, mitochondrial homeostasis and endothelial cell (EC) function. Carotid intima-media thickening (IMT) is caused by intima growth, and is a significant risk factor for cardiovascular diseases (CVD). Intima growth is mediated by EC dysfunction, vascular smooth muscle cell (VSMC) growth as well as inflammatory cell accumulation and activation. These pathological processes are stimulated by a disturbed flow pattern (d- flow), while being minimized by steady s-flow. Using congenic mouse strains, we identified a QTL for intima in C3H/F (no intima) and SJL (high intima) mice on chromosome 11 (Im2) that overlapped with a vascular inflammation QTL. Transcriptomic and bioinformatic analyses revealed significant differences in inflammation, cell cycle and RNA degradation. Using KEGG pathway analysis, a focus on genes in Im2 with polymorphisms that were differentially expressed between C3H/F and SJL identified a single gene: polyribonucleotide nucleotidyltransferase 1 (Pnpt1), a 3'-5' exoribonuclease that is required for import and processing of RNA in mitochondria. High level Pnpt1 expression correlated with decreased intima growth and inflammation in the carotid ligation model suggesting it was protective. The goal of this proposal is to understand how Pnpt1 restricts inflammation and atherosclerosis, focusing on novel transcriptional programs and mechanisms that link d-flow-mediated signaling through mitochondrial homeostasis, mitophagy/autophagy and cellular RNA processing pathways to EC dysfunction and CVD. While intima growth is primarily due to proliferation of VSMC and fibroblast-like cells, we focused on d-flow-mediated effects on EC because we believe these signals are initiating events, and are likely more specific and better therapeutic targets. We hypothesize that Pnpt1 is a mechanoresponsive enzyme that is critical to mitochondrial homeostasis and acts as a negative regulator of vascular inflammation and intima growth, thereby limiting CVD. Exciting preliminary data in support of the hypothesis include 1) inducible EC-specific Cre-loxP Pnpt1 mice that exhibit increased intima formation upon loss of Pnpt1; 2) RNA-Seq analyses of altered Pnpt1 expression under different flow patterns identified a novel and significant role for the TFAP2b/c transcription factor; 3) d-flow inhibited Pnpt1 function in EC; 4) Pnpt1 expression regulated EC inflammatory and apoptotic signaling both in vivo and in vitro and 5) Pnpt1 deficiency exacerbated mitochondrial-stress, as measured by ROS generation and autophagy. Proposed experiments will study changes in vascular remodeling and atherosclerosis in transgenic mouse models; determine the transcriptional program regulated by Pnpt1 focusing on the TFAP2b/c transcription factor; and the mechanisms by which flow regulates Pnpt1 function assayed by expression and enzyme activity. This proposal will characterize for the first time the role of Pnpt1, a major enzyme for mitochondrial RNA import and processing, in mouse models of atherosclerosis and vascular remodeling and in human carotid endarterectomy specimens.

该提案旨在描绘将层流介导的信号传导与基因联系起来的分子机制 表达、线粒体稳态和内皮细胞 (EC) 功能。颈动脉内膜中层增厚 （IMT）是由内膜生长引起的，是心血管疾病（CVD）的重要危险因素。内膜 生长是由 EC 功能障碍、血管平滑肌细胞 (VSMC) 生长以及炎症介导的 细胞积累和激活。这些病理过程是由扰乱的流动模式刺激的（d- 流），同时通过稳定的 s 流最小化。使用同源小鼠品系，我们鉴定了内膜的 QTL 11 号染色体 (Im2) 上与血管重叠的 C3H/F（无内膜）和 SJL（高内膜）小鼠 炎症QTL。转录组学和生物信息学分析揭示了炎症、 细胞周期和 RNA 降解。使用KEGG通路分析，重点关注Im2中具有多态性的基因 C3H/F 和 SJL 之间差异表达的结果鉴定出一个基因：多核糖核苷酸 核苷酸转移酶 1 (Pnpt1)，一种 3'-5' 核糖核酸外切酶，是 RNA 导入和加工所需的 线粒体。高水平 Pnpt1 表达与内膜生长减少和炎症相关 颈动脉结扎模型表明其具有保护作用。该提案的目标是了解 Pnpt1 如何 限制炎症和动脉粥样硬化，重点关注新的转录程序和机制 通过线粒体稳态、线粒体自噬/自噬和细胞 RNA 连接 d-flow 介导的信号传导 EC 功能障碍和 CVD 的加工途径。而内膜生长主要是由于 VSMC 的增殖 和成纤维细胞样细胞，我们关注 d-flow 介导的对 EC 的影响，因为我们相信这些信号是 起始事件，并且可能是更具体和更好的治疗目标。我们假设 Pnpt1 是 机械反应酶，对线粒体稳态至关重要，并作为负调节剂 血管炎症和内膜生长，从而限制CVD。令人兴奋的初步数据支持 假设包括 1) 诱导型 EC 特异性 Cre-loxP Pnpt1 小鼠，其表现出内膜形成增加 Pnpt1 丢失； 2) 对不同流动模式下改变的 Pnpt1 表达进行 RNA-Seq 分析，发现了一种新的 TFAP2b/c 转录因子的重要作用； 3) d-flow抑制EC中Pnpt1功能； 4) 点对点1 表达在体内和体外调节 EC 炎症和细胞凋亡信号传导以及 5) Pnpt1 缺乏 通过ROS生成和自噬测量，线粒体应激加剧。拟议的实验将 研究转基因小鼠模型中血管重塑和动脉粥样硬化的变化；确定 由 Pnpt1 调控的转录程序，重点关注 TFAP2b/c 转录因子；和机制 流通过表达和酶活性调节 Pnpt1 功能。该提案将 首次描述了 Pnpt1 的作用，Pnpt1 是线粒体 RNA 导入和加工的主要酶， 在动脉粥样硬化和血管重塑的小鼠模型以及人颈动脉内膜切除术标本中。