Mitochondrial Dysfunction in the Endothelium as a Mediator of Inflammatory Injury

摘要 研究表明，内皮细胞通过调节内流和表型在宿主防御中发挥关键作用。 免疫细胞的数量。项目3的重点是描绘线粒体功能障碍和内皮细胞有丝分裂的作用。 细胞在肺损伤过程中调节肺血管稳态和宿主防御的作用。拟议的研究将确定 内皮细胞线粒体损伤的机制以及适应性有丝分裂如何激活代偿线粒体 生物发生学。项目3还将研究吞噬有丝分裂和线粒体肽的释放如何影响中性粒细胞。 在炎症性肺损伤期间。这项提案将解决的基本问题包括： 肿瘤坏死因子α诱导线粒体损伤和有丝分裂的机制是什么？我们可以使用生物传感器来检测线粒体吗？ 在体外和体内确定线粒体有丝分裂和代偿性生物发生的关键阶段？何以 内皮细胞有丝分裂影响内皮细胞代谢恢复和内皮再生？何以 内皮细胞有丝分裂影响中性粒细胞信号转导、炎性损伤和宿主防御。项目3将测试 总体假设肺内皮细胞有丝分裂是内皮细胞动态平衡的中枢调节因子 免疫反应。这将在两个目标中得到解决。目的1：确定内皮细胞吞噬有丝分裂的作用 代偿性线粒体生物发生恢复内皮细胞再生和动态平衡 炎症性肺损伤。在这里，我们将检验炎症导致线粒体损伤和 肺内皮细胞的适应性有丝分裂，以协调内皮细胞再生和动态平衡。此外， 我们推测，恢复动态平衡的中心机制是通过线粒体的代偿性生物发生。 我们使用活体内皮细胞有丝分裂的双光子成像(核心D)，并使用EC- 线粒体生物发生转录因子pGc1α的特异性基因缺失 和TFAM，以及线粒体磷酸酶PGAM5(将吞噬后信号传递到细胞核) 在人肺内皮细胞中进行机械驱动的体外研究。这些研究将与EC的分析相结合 代谢、线粒体生物发生和EC再生。目的2：确定内皮细胞有丝分裂的作用 在炎症损伤过程中激活肺的主机防御功能。在这里，我们将检验假设 EC-有丝分裂增加了经内皮中性粒细胞的流入，从而通过分泌 甲酰化肽和甲酰肽受体的激活。我们将使用EC特有的PINK1的遗传模型 不同炎症性肺损伤模型(内毒素和肺炎假单胞菌)的缺失及EC分析 由有丝分裂引起的表观遗传和转录调控(以核心B为核心)，并说明这些调控如何有助于 EC和中性粒细胞在EC PINK1下游的宿主防御功能。我们还将评估EC-PINK1如何监管 中性粒细胞肺损伤与N-甲酰化线粒体肽的产生。我们还将确定 肺内皮细胞有丝分裂吞噬对中性粒细胞迁移和细菌吞噬功能的影响 中性粒细胞线粒体甲酰肽下游的信号通路(带核心C)。

ABSTRACT Studies have shown that the endothelium plays a critical role in host defense by regulating the influx and phenotype of immune cells. Project 3 focuses on delineating the role of mitochondrial dysfunction and mitophagy in endothelial cells in regulating lung vascular homeostasis and host-defense during injury. The proposed studies will identify mechanisms of mitochondrial injury in ECs as well as how adaptive mitophagy activates compensatory mitochondrial biogenesis. Project 3 will also examine how mitophagy and the release of mitochondrial peptides impact neutrophils during inflammatory lung injury. The fundamental questions that will be addressed by this proposal include: What are the mechanisms of TNFα induced mitochondrial injury and mitophagy? Can we use biosensors for mitochondria in vitro and in vivo to define key phases of mitophagy and compensatory mitochondrial biogenesis? How does endothelial mitophagy affect restoration of endothelial metabolism and endothelial regeneration? How does endothelial mitophagy impact neutrophil signaling, inflammatory injury and host defense. Project 3 will test the overall hypothesis that lung endothelial mitophagy is a central regulator of endothelial homeostasis and the innate immune response. This will be addressed in two Aims. Aim 1: Determine the role of endothelial mitophagy and compensatory mitochondrial biogenesis to restore endothelial regeneration and homeostasis during inflammatory lung injury. Here we will test the hypothesis that inflammation induces mitochondrial injury and adaptive mitophagy in the lung endothelium to orchestrate endothelial regeneration and homeostasis. Furthermore, we posit that the central mechanism of restoring homeostasis is through the compensatory mitochondrial biogenesis. We use intravital two photon imaging of endothelial mitophagy (with Core D) and perform in vivo studies using EC- specific genetic deletion of the mitophagy mediator PINK1, the mitochondrial biogenesis transcription factors PGC1α and TFAM, and the mitochondrial phosphatase PGAM5 (which transfers post-mitophagy signaling to the nucleus) with mechanistically driven in vitro studies in human lung ECs. These studies will be coupled to analysis of EC metabolism, mitochondrial biogenesis, and EC regeneration. Aim 2: Determine the role of endothelial mitophagy in activating the lung host-defense function during inflammatory injury. Here we will test the hypothesis that EC-mitophagy increases transendothelial neutrophil influx and thereby bacterial killing through secretion of formylated peptides and activation of formyl peptide receptors. We will use genetic models of EC-specific PINK1 deletion in distinct models of inflammatory lung injury (LPS and Pseudomonas pneumonia) and analyze EC epigenetic and transcriptomic regulation (with Core B) wrought by mitophagy and address how these contribute to host-defense function of EC and neutrophils downstream of EC PINK1. We will also assess how EC-PINK1 regulates neutrophil-induced lung injury and generation of N-formylated mitochondrial peptides. We will also determine the effects of lung EC mitophagy on neutrophil transmigration and bacterial phagocytosis (with Core D) and study the signaling pathways (with Core C) downstream of mitochondrial formyl peptides in neutrophils.