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，它会引发斑块 破坏和急性血栓形成。坏死斑形成的主要过程是内质网（ER） 应激诱导的巨噬细胞 (Mf) 凋亡以及这些凋亡细胞的吞噬清除缺陷 Mfs（“胞吞作用”）。病变 Mfs 中发生的另一个基本细胞过程是自噬，其中 细胞出于保护目的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，该领域的研究大多是描述性的，缺乏明确的 假设、机制洞察和体内分子遗传学因果证明。根据已发表的研究 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细胞生存 晚期动脉粥样硬化中出现问题的通路。有趣的是，自噬可能影响 ER 应激 诱导细胞凋亡和有缺陷的胞吞作用。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。在目标一中，我们将 探索内质网应激诱导的自噬最初通过以下机制发挥保护作用的假设： 调节 NADPH 氧化酶诱导的活性氧 (ROS)。我们将检验这个假设和相关的 以及研究机制，使用各种工具，包括来自条件基因靶向小鼠的 Mfs 缺乏关键的自噬介质 ATG5。我们还将研究 ER 应激诱导的机制 自噬以及自噬失败是否先于最终凋亡。在目标 II 中，我们将测试 假设抑制凋亡 Mfs 中的自噬会抑制其细胞清除。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 然后研究传热细胞的机制。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 可以非常有效地删除 floxed 病变 Mfs 中的基因，并且在初步研究中我们已经表明来自 Atg5flox/flox;Lysmcre /- 小鼠的 Mfs 抑制自噬、增加ROS并加速细胞凋亡。我们将研究斑块参数 以及与对照中的晚期动脉粥样硬化进展和自噬相关的分子与 Mf-ATGF5- 的比较 Ldlr-/- 背景上有缺陷的小鼠。我们假设 Mf-ATG5 缺乏会导致以下病变： 抑制 Mf 自噬，增强 ROS 和细胞凋亡，可能有缺陷的胞吞作用并增加 炎症，并加速斑块坏死。相反，在 Mfs 增强自噬的小鼠中 通过 Bcn1 (Beclin-1) 的遗传过度表达，我们预测这些参数将得到改善，并且 斑块坏死减少。完成这些研究后，我们希望能够获得机制和体内 因果数据支持自噬在动脉粥样硬化中的保护作用，这反过来可能表明新的 预防动脉粥样硬化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.