Macrophage death and lipid metabolism in atherosclerosis

巨噬细胞(Mphi)死亡是动脉粥样硬化病变的一个重要特征，但这一事件的生理后果仍存在不确定性。我们的实验室已经证明，培养细胞中的游离胆固醇(FC)负荷可以通过FC诱导的磷脂酰胆碱(PC)生物合成的上调以及通过阻断FC向周围细胞部位(如质膜和线粒体)的运输来防止。在这种背景下，这项建议的总体目标是使用脂类代谢和死亡信号通路已被基因操纵的小鼠来测试关于动脉粥样硬化中MPHI死亡的特定假说。假设Mphi可能(A)通过在病变发展过程中限制Mphi的数量以及可能通过在晚期病变中“安全”地处置濒临死亡的Mphi来起到保护作用；和/或(B)通过促进垂死的Mphi释放有害分子来促进脂质核心的发育。当Mphi死于“凋亡”时，可能会产生保护作用，而当Mphi通过更多的“坏死性”过程死亡时，可能会产生有害的影响。在目标I中，我们将使用载脂蛋白E基因敲除(动脉粥样硬化)的小鼠，这些小鼠的PC生物合成具有Mphi特异性改变(VIA；CTP的遗传操作：磷酸胆碱胞苷转移酶)或Fc转运到外周细胞位置有缺陷(Niemann-Pick C小鼠)。我们的目标将是测试PC和FC代谢在体内Mphi死亡中的作用，并在我们的假设背景下评估Mphi死亡改变对动脉粥样硬化和脂核发育的影响。在AIM II中，我们将专门研究Mphi凋亡应该被阻断的小鼠模型。鉴于初步数据显示Fc介导的细胞凋亡在缺乏Fas死亡受体的Mphi中被阻止，主要的焦点将放在Mphi中Fas缺失的小鼠身上。此外，还将探讨Fas通路在氧化脂蛋白和生长因子停用等较老诱导剂导致Mphi死亡中的作用。此外，鉴于bcl2家族蛋白在某些类型的Fas介导的细胞凋亡以及其他原因导致的死亡中的关键作用，Mphi过表达抗凋亡蛋白Bcl2和Bclxl的小鼠将接受动脉粥样硬化和脂核发育的检查。根据上述假说，阻断皮损Mphi的凋亡可能会对动脉粥样硬化的形成产生不利影响，在坏死性改变中保持不受抑制。综上所述，该项目应有助于阐明Mphi死亡在动脉粥样硬化形成和脂核发育中的基于脂类的机制和生理学后果。

Macrophage (Mphi) death is an important feature of atherosclerotic lesions, yet there are still uncertainties about the physiological consequences of this event. Our laboratory has shown that free cholesterol (FC) loading of cultured can be prevented by FC-induced up-regulation of phosphatidylcholine (PC) biosynthesis and by blockage of FC transport to peripheral cellular sites (e.g., plasma membrane & mitochondria). In this context, the overall objective of this proposal is to test specific hypotheses regarding Mphi death in atherosclerosis using mice in which lipid metabolic and death-signaling pathways have been genetically manipulated. The hypotheses are that Mphi may (a) be protective by limiting the number of Mphi's during lesion development and perhaps by "safely" disposing of dying Mphi's in advanced lesions; and/or (b) contribute to lipid core development by promoting the release of harmful molecules from dying Mphi's. Protective effects may occur when Mphi's die by "apoptosis", whereas harmful effects may occur when Mphi's die by more "necrotic"-like processes. In Aim I, we will use apolipoprotein E knockout (atherosclerotic) mice with Mphi-specific alterations in PC biosynthesis (via; genetic manipulation of CTP: phosphocholine cytidylylyltransferase) or with defective FC transport to peripheral cellular sites (Niemann-Pick C mice). The goal will be to test the role of PC and FC metabolism in Mphi death in vivo and to evaluate, in the context of our hypotheses, the consequences of altered Mphi death on atherogenesis and lipid core development. In Aim II, we will specifically examine mouse models in which Mphi apoptosis should be blocked. In view of preliminary data showing the FC-mediated apoptosis is prevented in Mphi's lacking the Fas death receptor, a major focus will be on mice in which Fas is absent in Mphi's. The role of the Fas pathway in Mphi death caused by older inducers, such as oxidized lipoproteins and growth factor withdrawal, will also be explored. Furthermore, given the key role of bcl- 2 family proteins in certain types of Fas-mediated apoptosis as well as in death due to other causes, mice whose Mphi's over-express the anti- apoptotic proteins Bcl-2 and Bcl-xL will be examined for atherogenesis and lipid core development. Blockage of lesional Mphi apoptosis may, according the above-state hypothesis, adversely affect atherogenesis in necrotic-like changes are left uninhibited. In summary, this project should help elucidate lipid-based mechanisms and physiologic consequences of Mphi death in atherogenesis and lipid core development.