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.

该建议旨在描述将层流介导的信号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。颈动脉内膜媒体增厚 （IMT）是由内膜增长引起的，是心血管疾病（CVD）的重要危险因素。 intima EC功能障碍，血管平滑肌细胞（VSMC）生长以及炎症介导 细胞积累和激活。这些病理过程受到干扰流动模式的刺激（d- 流动），同时被稳定的S-Flow最小化。使用先天小鼠菌株，我们确定了Intima的QTL C3H/F（无内膜）和SJL（高内膜）小鼠在11（IM2）上与血管重叠 炎症QTL。转录组和生物信息学分析显示，炎症有显着差异， 细胞周期和RNA降解。使用KEGG途径分析，重点是具有多态性的IM2基因 在C3H/F和SJL之间差异表达的单个基因：polyribibonucleotide 核苷酸转移酶1（PNPT1），一种3'-5'驱核核酸酶，在进口和加工RNA中需要 线粒体。高水平的PNPT1表达与内膜生长的降低和炎症相关 颈动脉连接模型表明它具有保护性。该提议的目的是了解PNPT1如何 限制炎症和动脉粥样硬化，重点是新的转录程序和机制 通过线粒体稳态，线粒体/自噬和细胞RNA链接D-Flow介导的信号传导 处理EC功能障碍和CVD的处理途径。虽然Intima的生长主要是由于VSMC的增殖 和成纤维细胞样细胞，我们专注于D-Flow介导的对EC的影响，因为我们认为这些信号是 发起事件，可能是更具体，更好的治疗靶标。我们假设PNPT1是 机械响应酶对线粒体稳态至关重要，并且是负调节剂 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）可诱导的EC特异性CRE-loxp PNPT1小鼠，其内膜形成增加 损失PNPT1； 2）在不同流动模式下改变PNPT1表达的RNA-seq分析确定了一种新型 TFAP2B/C转录因子的重要作用； 3）D-Flow抑制EC中的PNPT1功能； 4）PNPT1 表达调节EC的EC炎症和凋亡信号在体内和体外均和5）PNPT1缺乏症 通过ROS的产生和自噬来测量的线粒体压力加剧。建议的实验将 研究转基因小鼠模型中血管重塑和动脉粥样硬化的研究变化；确定 由PNPT1调节的转录程序重点是TFAP2B/C转录因子；和机制 通过表达和酶活性测定的流动调节PNPT1功能。该提议将 首次表征PNPT1的作用，PNPT1是线粒体RNA进口和加工的主要酶， 在动脉粥样硬化和血管重塑的小鼠模型中以及人颈动脉内膜切除术标本中。