Autophagy in Advanced Atherosclerosis

描述（由申请人提供）：动脉粥样硬化临床进展中的关键致病事件是斑块坏死，其触发斑块破裂和急性血栓形成。坏死斑形成中的主要过程是内质网（ER）应激诱导的巨噬细胞（Mf）凋亡，其与这些凋亡Mf的吞噬清除缺陷（“吞噬细胞作用”）相结合。另一个发生在损伤性Mfs中的基本细胞过程是自噬，由此细胞降解蛋白质或细胞器以达到保护目的。虽然已知自噬在动脉粥样硬化过程中被激活，但该领域的研究主要是描述性的，缺乏明确的假设，机制见解和体内分子遗传因果证据。基于已发表的其他细胞类型的研究和我们自己的初步数据，我们假设自噬是一种代偿性细胞存活途径，在晚期动脉粥样硬化中出错。有趣的是，自噬可能影响内质网应激诱导的细胞凋亡和缺陷性细胞凋亡。因此，我们建议测试的分子-细胞机制，这些想法，以及相关的先进的动脉粥样硬化在体内。在目的一，我们将探讨这一假设，ER应激诱导的自噬最初是通过调节NADPH氧化酶诱导的活性氧（ROS）的机制，保护。我们将测试这一假设和相关假设，并研究机制，使用各种工具，包括缺乏关键自噬介质ATG 5的条件基因靶向小鼠的Mfs。我们也将研究内质网应激诱导的自噬机制，以及自噬失败是否先于最终的细胞凋亡。在目的II中，我们将测试抑制凋亡Mfs中的自噬抑制其巨噬细胞清除的假设。我们将使用各种自噬抑制凋亡细胞的模型来监测它们被Mf巨噬细胞识别和吞噬的能力，然后研究其机制。在目标III中，我们将通过使用与LysMCre和Ldlr-/-小鼠杂交的Atg 5 flox/flox小鼠在体内测试这些想法。在其他模型中，LysMCre导致损伤Mfs中floxed基因的非常有效的缺失，并且在初步研究中，我们已经表明来自Atg 5 flox/flox;Lysmcre小鼠的Mfs抑制了自噬，增加了ROS，并加速了凋亡。我们将研究斑块参数和分子相关的先进的动脉粥样硬化进展和自噬对照与Mf-ATGF 5缺陷小鼠的Ldlr-/-背景。我们假设Mf-ATG 5缺陷将导致病变，Mf自噬抑制，ROS和凋亡增强，可能缺陷的红细胞增多和炎症增加，以及加速斑块坏死。相反，在Mfs通过Bcn 1（Beclin-1）的遗传过表达增强自噬的小鼠中，我们预测这些参数的改善和斑块坏死的减少。在完成这些研究后，我们希望获得支持自噬在动脉粥样硬化中的保护作用的机制和体内因果关系数据，这反过来可能会提出预防动脉粥样硬化临床进展的新治疗策略。 公共卫生相关性：心脏病发作、中风和心脏病引起的猝死是我们社会中死亡的主要原因是由于血小板栓塞突然切断了这些器官的血液供应而引发的，血小板栓塞的形成是因为该区域的血管发生了称为“动脉粥样硬化”（“动脉硬化”）的疾病过程。鉴于只有某些类型的动脉粥样硬化病变触发血小板栓，本提案的总体目标是增加我们对影响这些危险的动脉粥样硬化病变形成的过程的了解。在这种情况下，我们将研究动脉粥样硬化中巨噬细胞中发生的一个基本过程，称为“自噬”，它可以帮助保护血管免受这些危险病变的发展，从而可能提出新的方法来防止危险的动脉粥样硬化病变的形成。

DESCRIPTION (provided by applicant): A key pathogenic event in the clinical progression of atherosclerosis is plaque necrosis, which triggers plaque disruption and acute thrombosis. A major process in necrotic plaque formation is endoplasmic reticulum (ER) stress-induced macrophage (Mf) apoptosis coupled with defective phagocytic clearance of these apoptotic Mfs ("efferocytosis"). Another fundamental cell process that occurs in lesional Mfs is autophagy, whereby cells degrade proteins or organelles for protective purposes. Although autophagy is known to be activated during atherosclerosis, studies in this area have been largely descriptive and have lacked clear-cut hypotheses, mechanistic insight, and molecular-genetic causal proof in vivo. Based on published studies with other cell types and our own preliminary data, we hypothesize that autophagy is a compensatory cell-survival pathway that goes awry in advanced atherosclerosis. Intriguingly, autophagy may affect both ER stress- induced apoptosis and defective efferocytosis. We therefore propose to test the molecular-cellular mechanisms related to these ideas as well as relevance to advanced atherosclerosis in vivo. In Aim I, we will explore the hypothesis that ER stress-induced autophagy is initially protective through a mechanism that modulates NADPH oxidase-induced reactive oxygen species (ROS). We will test this hypothesis and related ones, and study mechanism, using a variety of tools, including Mfs from conditionally gene-targeted mice lacking the key autophagy mediator ATG5. We will also investigate the mechanisms of ER stress-induced autophagy and whether failure of autophagy precedes eventual apoptosis. In Aim II, we will test the hypothesis that inhibition of autophagy in apoptotic Mfs inhibits their efferocytic clearance. We will use various models of autophagy-inhibited apoptotic cells to monitor their ability to be recognized and engulfed by Mf efferocytes and then to study mechanism. In Aim III, we will test these ideas in vivo by using Atg5flox/flox mice crossed with LysMCre and Ldlr-/- mice. In other models, LysMCre leads to very effective deletion of floxed genes in lesional Mfs, and in preliminary studies we have shown that the Mfs from Atg5flox/flox;Lysmcre mice have inhibited autophagy, increased ROS, and accelerated apoptosis. We will investigate plaque parameters and molecules relevant to advanced atherosclerosis progression and autophagy in control vs. Mf-ATGF5- deficient mice on the Ldlr-/- background. We hypothesize that Mf-ATG5 deficiency will lead to lesions with inhibited Mf autophagy, enhanced ROS and apoptosis, possibly defective efferocytosis and increased inflammation, and accelerated plaque necrosis. Conversely, in mice whose Mfs have enhanced autophagy through genetic overexpression of Bcn1 (Beclin-1), we predict improvement in these parameters and decreased plaque necrosis. Upon the completing of these studies, we hope to have mechanistic and in-vivo causation data supporting a protective role of autophagy in atherosclerosis which, in turn, may suggest novel therapeutic strategies to prevent the clinical progression of atheromata. PUBLIC HEALTH RELEVANCE: Heart attacks, strokes, and sudden death due to heart disease-the leading cause of death in our society-are triggered by a sudden cutting off of the blood supply feeding these organs by platelet plugs, which form because the vessel in that area has a disease process called "atherosclerosis" ("hardening of the arteries"). In view of the fact that only certain types of atherosclerotic lesions trigger platelet plugs, the overall objective of this proposal is to add to our knowledge of what processes influence the formation of these dangerous atherosclerotic lesions. In this context, we will study a fundamental process that occurs in macrophages in atherosclerosis, called "autophagy," which can help protect the vessel from developing these dangerous lesions and thus may suggest new ways to prevent dangerous atherosclerotic lesions from forming.