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

这一建议旨在描述层流介导的信号与基因之间的分子机制。 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)是由血管内膜生长引起的，是心血管疾病(CVD)的重要危险因素。内膜 血管内皮细胞功能障碍、血管平滑肌细胞(VSMC)的生长以及炎症都参与了生长 细胞的积累和激活。这些病理过程是由扰动的流型(d- 流)，同时被稳定的S流最小化。利用同源小鼠品系，我们确定了一个与血管内膜有关的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。转录和生物信息学分析显示，在炎症方面存在显著差异， 细胞周期和RNA降解。利用KEGG途径分析IM2基因的多态性 在C3H/F和SJL之间差异表达的基因鉴定出一个单基因：多核苷酸 核苷酸转移酶1(Pnpt1)是一种3‘-5’外切核糖核酸酶，在核糖核酸的导入和加工中是必需的。 线粒体。Pnpt1的高表达与血管内膜生长和炎症的减少有关 颈动脉结扎模型表明它是保护性的。该提案的目标是了解Pnpt1如何 抑制炎症和动脉粥样硬化，专注于新的转录程序和机制， D-Flow通过线粒体稳态、有丝分裂/自噬和细胞RNA介导的信号转导 EC功能障碍和心血管疾病的处理途径。而血管内膜的生长主要是由于VSMC的增殖 和成纤维细胞样细胞，我们专注于d-flow对EC的影响，因为我们相信这些信号是 启动事件，并可能是更具体和更好的治疗目标。我们假设Pnpt1是一个 一种机械反应酶，对线粒体的动态平衡至关重要，是一种负性调节因子。 血管炎症和血管内膜生长，从而限制心血管疾病。令人振奋的初步数据支持 假说包括1)可诱导的EC特异性Cre-loxP Pnpt1小鼠，在 Pnpt1的丢失；2)对不同流动模式下改变的Pnpt1表达的RNA-Seq分析确定了一个新的 和TFAP2b/c转录因子的显著作用；3)d-flow抑制Pnpt1在EC中的功能；4)Pnpt1 体内和体外表达调控EC炎症和凋亡信号及Pnpt1缺陷 通过ROS的产生和自噬来衡量，加剧了线粒体的应激。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 Pnpt1调控TFAP2b/c转录因子的转录程序及其机制 Flow通过Pnpt1的表达和酶活性来调节Pnpt1的功能。这项提议将 首次表征Pnpt1的作用，Pnpt1是线粒体RNA输入和加工的主要酶， 在小鼠动脉粥样硬化和血管重塑模型中以及在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.