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

该建议旨在阐明层流介导的信号转导与基因表达之间的分子机制。 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，并且是心血管疾病（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。 细胞积累和活化。这些病理过程受到扰动流型（d- 流），同时通过稳定的S流使其最小化。使用同源小鼠品系，我们确定了内膜的QTL， C3 H/F（无内膜）和SJL（高内膜）小鼠11号染色体（Im 2）上与血管重叠， 炎症QTL转录组学和生物信息学分析揭示了炎症的显著差异， 细胞周期和RNA降解。利用KEGG通路分析，重点分析Im 2中具有多态性的基因 C3 H/F和SJL之间的差异表达鉴定了一个单一的基因：多核苷酸 核苷酸转移酶1（Pnpt 1），一种3 '-5'核糖核酸外切酶，其是细胞中RNA的输入和加工所需的。 线粒体高水平的Pnpt 1表达与血管内膜生长和炎症的减少相关。 颈动脉结扎模型提示其具有保护作用。本提案的目标是了解Pnpt 1 限制炎症和动脉粥样硬化，专注于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-流介导的信号传导 EC功能障碍和CVD的处理途径。而内膜生长主要是由于VSMC的增殖 和成纤维细胞样细胞，我们专注于d-流介导的EC效应，因为我们相信这些信号是 启动事件，并且可能是更特异和更好的治疗靶点。我们假设Pnpt 1是一个 一种机械反应酶，对线粒体内稳态至关重要，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）可诱导EC特异性Cre-loxPPnpt 1小鼠， Pnpt 1的丢失; 2）在不同流动模式下改变的Pnpt 1表达的RNA-Seq分析鉴定了一种新的 TFAP 2b/c转录因子在EC中起重要作用; 3）d-flow抑制Pnpt 1的功能; 4）Pnpt 1 在体内和体外表达调节EC炎症和凋亡信号传导; 5）Pnpt 1缺陷 通过ROS生成和自噬来测量，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; Pnpt 1对TFAP 2b/c转录因子调控的转录程序及其机制 通过表达和酶活性测定，flow调节Pnpt 1功能。这项建议会 首次描述了Pnpt 1的作用，Pnpt 1是线粒体RNA输入和加工的主要酶， 在动脉粥样硬化和血管重塑的小鼠模型和人颈动脉内膜切除术标本中。

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.