Autophagy in Advanced Atherosclerosis

描述（由申请人提供）：动脉粥样硬化临床进展中的一个关键致病事件是斑块坏死，它会引发斑块破裂和急性血栓形成。坏死斑块形成的一个主要过程是内质网（ER）应激诱导的巨噬细胞（Mf）凋亡，以及这些凋亡的巨噬细胞（efferocytosis）的吞噬清除缺陷。病变Mfs中发生的另一个基本细胞过程是自噬，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期间被激活，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在晚期动脉粥样硬化中出错的代偿细胞生存途径。有趣的是，自噬可能影响内质网应激诱导的细胞凋亡和有缺陷的efferocytosis。因此，我们建议在体内测试与这些想法相关的分子细胞机制以及与晚期动脉粥样硬化的相关性。在Aim I中，我们将探讨内质网应激诱导的自噬最初通过调节NADPH氧化酶诱导的活性氧（ROS）的机制具有保护作用的假设。我们将使用多种工具，包括来自缺乏关键自噬介质ATG5的条条性基因靶向小鼠的Mfs，来验证这一假设和相关假设，并研究其机制。我们还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Aim II中，我们将验证凋亡的Mfs中自噬的抑制会抑制其红细胞清除的假设。我们将利用各种自噬抑制的凋亡细胞模型，监测其被Mf细胞识别和吞噬的能力，并研究其机制。在Aim III中，我们将使用Atg5flox/flox小鼠与LysMCre和Ldlr-/-小鼠杂交，在体内测试这些想法。在其他模型中，LysMCre导致病变Mfs中floxed基因的非常有效的缺失，在初步研究中，我们已经表明来自Atg5flox/flox的Mfs；Lysmcre小鼠抑制自噬，增加ROS，加速细胞凋亡。我们将研究在Ldlr /-背景下，对照组与Mf-ATGF5-缺陷小鼠的斑块参数和与晚期动脉粥样硬化进展和自噬相关的分子。我们假设Mf- atg5缺乏会导致Mf自噬被抑制，ROS和细胞凋亡增强，可能导致efferocysis缺陷和炎症增加，以及斑块坏死加速。相反，在Mfs通过基因过表达Bcn1 （Beclin-1）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，我们希望有机制和体内因果数据支持自噬在动脉粥样硬化中的保护作用，进而可能提出新的治疗策略，以防止动脉粥样硬化的临床进展。

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.