Transcription Factor-EB and Postischemic Angiogenesis

摘要 外周动脉疾病（PAD）是一种高度流行的疾病，越来越多地被认为是导致动脉粥样硬化的主要原因。 心血管疾病（CVD）和公共卫生负担。严重肢体缺血（CLI）是最常见的 先进的PAD到目前为止，除了血管内或外科治疗外，很少有治疗方法可以恢复血管内的血管。 缺血组织中的血流是可用的。因此，对小说的鉴别具有重要意义 治疗体内缺血性血管损伤的策略。转录因子-EB（TFEB）是一种重要的调节因子， 溶酶体生物发生和自噬。 然而，TFEB在血管疾病中的作用仍有待进一步研究。 探讨了 一氧化氮依赖性脂肪酸硝化产物的临床检测与定量 （硝基烯烃），引发了对新型抗炎脂质的兴趣。游离和酯化硝基脂肪酸 已经在人和动物血浆中检测到纳摩尔范围的衍生物， 在预防心血管疾病的不同病理生理学方面具有深远的意义。重大进展 代谢组学和脂质组学策略确定共轭亚油酸（CLA）为优先底物 脂肪酸硝化作用的关键。在这里，我们表明，硝基共轭亚油酸（硝基CLA）的形成， 很容易转化为脉管系统中的保护机制。我们的初步数据表明硝基共轭亚油酸 通过TFEB增强内皮细胞（EC）中的自噬。内皮TFEB显著增加 体内缺血后血管生成。最后，硝基-CLA调节TFEB中的自噬和管形成。 依赖的方式。根据这些证据，该项目将检验中心假设，即增强 内源性硝基共轭亚油酸的产生通过促进TFEB-3的表达来保护缺血性血管损伤。 介导的自噬导致增强的缺血后血管生成。提出了两个具体目标。 目的1：确定内皮TFEB对于硝基-CLA依赖性保护性自噬是必需的， 体外促血管生成表型。这将通过培养的细胞获得和丧失功能的方法来解决。 在硝基-CLA处理的存在下的原代EC。目的2：确定内源性硝基-CLA保护 通过内皮TFEB的体内缺血性血管损伤。口服治疗策略，以促进内源性 将建立硝基-CLA形成。将使用独特的EC选择性TFEB转基因和基因敲除小鼠 以确定TFEB功能是硝基-CLA促进体内血流恢复所必需的。预计 有了这个提议，我们将更好地确定TFEB在硝基-CLA调节的EC促血管生成中的重要作用， 表型和缺血后血管生成，并建立口服生物利用度的硝基共轭亚油酸作为一种新的 缺血性血管损伤的治疗策略。这一机理研究将为 硝基-CLA的临床应用，并导致治疗或/和预防缺血性疾病的重大突破 通过有效增加缺血后血管生成来治疗血管疾病。

ABSTRACT Peripheral arterial disease (PAD) is highly prevalent and is increasingly recognized as a major contributor to the cardiovascular disease (CVD) and public health burden. Critical limb ischemia (CLI) is one of the most advanced PAD. To date, beside endovascular or surgical treatment, few therapeutic alternatives to restore the blood flow in ischemic tissues are available. Therefore, it would be of high significance to identify novel strategies to treat ischemic vascular injury in vivo. Transcription factor-EB (TFEB) is a crucial regulator of lysosomal biogenesis and autophagy. However, the functions of TFEB in vascular disease remain to be explored. The clinical detection and quantitation of nitric oxide (NO)-dependent fatty acid nitration products (nitroalkenes), has sparked the interest on novel anti-inflammatory lipids. Free and esterified nitro-fatty acid derivatives have been detected in human and animal plasma in the nanomolar range and shown to have profound implications in the prevention of diverse pathophysiological aspects of CVDs. Significant advances in metabolomics and lipidomics strategies identified conjugated linoleic acid (CLA) as the preferential substrate for fatty acid nitration in humans. Herein, we show that nitro-conjugated linoleic acid (nitro-CLA) formation readily translates into protective mechanisms in the vasculature. Our preliminary data indicate that nitro-CLA enhances autophagy through TFEB in endothelial cells (ECs). Endothelial TFEB significantly increases postischemic angiogenesis in vivo. Finally, nitro-CLA regulates autophagy and tube formation in a TFEB- dependent manner. Based on this evidence, the project will test the central hypothesis that enhancing the endogenous production of nitro-CLA protects against ischemic vascular injury by promoting TFEB- mediated autophagy leading to enhanced postischemic angiogenesis. Two Specific Aims are proposed. Aim 1: Establish that endothelial TFEB is essential for nitro-CLA-dependent protective autophagy and proangiogenic phenotype in vitro. This will be addressed by gain- and loss-of-function approaches in cultured primary ECs in the presence of nitro-CLA treatment. Aim 2: Establish that endogenous nitro-CLA protects from ischemic vascular injury in vivo through endothelial TFEB. An oral therapeutic strategy to promote endogenous nitro-CLA formation will be established. Unique EC-selective TFEB transgenic and knockout mice will be used to establish that TFEB function is required for nitro-CLA-promoting blood flow recovery in vivo. It is expected that with this proposal we will better define the essential role of TFEB in nitro-CLA-regulated EC proangiogenic phenotype and postischemic angiogenesis, and establish oral bioavailability of nitro-CLA as a novel therapeutic strategy against ischemic vascular injury. This mechanistic research will set a solid foundation for clinical utilization of nitro-CLA and lead to a major breakthrough for treating or/and preventing ischemic vascular disease by efficiently increasing postischemic angiogenesis.