Autophagy in Advanced Atherosclerosis

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，它会触发斑块。 破裂和急性血栓形成。内质网(ER)是坏死斑形成的主要过程。 应激诱导的巨噬细胞(MF)凋亡与吞噬功能缺陷对这些凋亡的清除 MFS(“泡沫症”)。在皮损性MFS中发生的另一个基本细胞过程是自噬，由此 细胞为了保护目的而降解蛋白质或细胞器。尽管已知自噬被激活 在动脉粥样硬化期间，这一领域的研究大多是描述性的，缺乏明确的认识。 假说、机械洞察和分子遗传学在活体内的因果证明。基于已发表的关于 其他细胞类型和我们自己的初步数据，我们假设自噬是一种补偿性的细胞生存 在晚期动脉粥样硬化中出现问题的途径。有趣的是，自噬可能会影响内质网应激- 诱导细胞凋亡和有缺陷的泡腾。因此，我们建议测试分子-细胞 与这些想法相关的机制以及与体内晚期动脉粥样硬化的相关性。在Aim I中，我们将 探索内质网应激诱导的自噬最初是通过一种机制起保护作用的假设 调节NADPH氧化酶诱导的活性氧物种(ROS)。我们将检验这一假设和相关的 一个，并研究机制，使用各种工具，包括条件基因靶向小鼠的MFS 缺乏关键的自噬调节因子ATG5。我们还将探讨内质网应激诱导的机制 自噬以及自噬失败是否先于最终的细胞凋亡。在AIM II中，我们将测试 假设抑制自噬在凋亡的MFS中会抑制其泡出细胞的清除。我们将使用各种 自噬抑制的凋亡细胞模型用于监测其被MF识别和吞噬的能力 并对其作用机制进行了研究。在Aim III中，我们将使用Atg5Flox/FLOX小鼠在体内测试这些想法 与LysMCre和Ldlr-/-小鼠杂交。在其他模型中，LysMCre导致非常有效的FLOXED删除 基因，在初步研究中，我们已经表明来自Atg5flx/flx；Lysmcre/-小鼠的MFS 抑制自噬，增加ROS，加速细胞凋亡。我们将调查斑块参数 和与晚期动脉粥样硬化进展和自噬相关的分子在对照组与MF-ATGF5- Ldlr-/-背景上的缺陷小鼠。我们假设MF-ATG5缺乏会导致皮损 抑制MF自噬，增强ROS和细胞凋亡，可能有缺陷的泡腾作用，并增加 炎症，加速斑块坏死。相反，在MFS增强自噬的小鼠中 通过Bcn1(Beclin-1)的遗传过表达，我们预测这些参数和 斑块坏死减少。在完成这些研究后，我们希望有机械性的和活体的 因果数据支持自噬在动脉粥样硬化中的保护作用，这反过来可能提出新的 防止动脉粥样硬化症临床进展的治疗策略。

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.