VEGF-C/VEGFR3 AND LYMPHATIC TRANSPORT OF CHOLESTEROL FROM ATHEROSCLEROTIC PLAQUE

描述（由申请人提供）：我们的长期目标是了解如何从斑块中去除巨噬细胞和胆固醇，以促进疾病消退。我们最近发现，淋巴管是胆固醇从动脉壁和其他组织中排出的管道。胆固醇逆向转运（RCT）是胆固醇从体内动员并通过粪便排泄的过程。关于动脉粥样硬化，胆固醇从巨噬细胞中的动员用于去除和排泄是最相关的。多年来，出现了许多关于胆固醇如何从巨噬细胞动员到HDL（HDL-C）上的细节。然而，人们对HDL-胆固醇随后如何从组织（包括动脉壁的动脉粥样硬化斑块等部位）中排出，在进入排泄途径之前返回血浆知之甚少。在20世纪80年代早期，一些临床或实验观察导致一些医生提出淋巴转运受损和动脉粥样硬化之间的联系。然而，除了一项令人信服的研究估计HDL-C通过人体淋巴的净流量很大外，尚未检查RCT中淋巴管的定量重要性。最近，我们利用实验小鼠模型，其中淋巴流的通畅性可以通过手术或遗传方法来调节。在皮肤中，我们能够完全消除淋巴流动，这使我们能够证明，在巨噬细胞RCT后，代谢物是胆固醇动员到血浆中的主要途径。在动脉粥样硬化影响的主动脉壁中，我们还使用了手术方法以脉冲追踪方式追踪斑块中的氘标记胆固醇（[2 H] D 6-胆固醇; D 6-胆固醇）。将主动脉手术移植到接受者中，用抗VEGFR 3 mAb阻断或不阻断淋巴管系统的再吻合。这种阻断显著地保留了动脉粥样硬化主动脉中的D 6-胆固醇，表明其在动脉粥样硬化中的关键作用。 对于淋巴管，如在皮肤中观察到的，胆固醇从主动脉动员。在目标1中，我们将采取关键的下一步改进方法，使我们能够更好地量化从主动脉清除胆固醇的淋巴管的roe，并评估对血管的阻断是否真正在主动脉壁局部起作用。在初步数据中，我们表明，治疗apoE-/-小鼠与VEGF-C，VEGFR 3的配体，恢复受损的淋巴转运发生后高胆固醇血症，使我们能够测试的假设，VEGF-C作用于淋巴管治疗维持一个关键的途径胆固醇运输出斑块后，巨噬细胞胆固醇流出刺激。这一假说提出了一个尚未在该领域得到解决的基本问题：有效的斑块消退是否真正取决于从斑块中去除胆固醇，仅取决于从巨噬细胞中去除胆固醇，或者两者都不依赖？我们的研究设计是解决这一基本问题的理想选择，同时深入挖掘支持淋巴管功能可能有助于解决动脉粥样硬化斑块炎症的新概念。

DESCRIPTION (provided by applicant): Our long-term goal is to understand how to remove macrophages and cholesterol from plaques to promote disease regression. We recently discovered that lymphatic vessels serve as the conduits by which cholesterol is removed from the artery wall and other tissues. Reverse cholesterol transport (RCT) is the process by which cholesterol is mobilized from the body for excretion through the feces. With respect to atherosclerosis, the mobilization of cholesterol from macrophages for removal and excretion is most relevant. Over the years, many details emerged regarding how cholesterol is mobilized from macrophages to be loaded onto HDL (HDL-C). However, little was known about how HDL-cholesterol subsequently makes its way out of tissues, including sites like atherosclerotic plaques of the artery wall, to return to plasma before entering pathways for excretion. A handful of clinical or experimental observations led a few physicians in the early 1980's to propose a connection between impaired lymphatic transport and atherosclerosis. However, the quantitative importance of lymphatic vessels in RCT had not been examined, aside from a compelling study that estimated that the net flux of HDL-C through human lymph is substantial. Recently, we utilized experimental mouse models where the patency of lymphatic flow could be modulated by surgical or genetic methods. In skin, we were able to fully abrogate lymphatic flow allowing us to demonstrate that lymphatics are quantitatively the major route for cholesterol mobilization to plasma following macrophage RCT. In atherosclerosis-affected aortic walls, we also used a surgical approach to track deuterium-labeled cholesterol ([2H] D6-cholesterol; D6-cholesterol) from plaques in a pulse-chase manner. Aortas were surgically transplanted into recipients with re-anastomosis of the lymphatic vasculature blocked or not with anti-VEGFR3 mAb. This blockade significantly retained D6- cholesterol in the atherosclerotic aorta, suggesting a key role for lymphatic vessels in cholesterol mobilization from the aorta as observed in skin. In aim 1, we will take a critical next step with refined approaches that will allow us to better quantify the roe of lymphatic vessels in cholesterol removal from the aorta and to assess whether the blockade on lymphatics is truly acting locally at the aortic wall. In preliminary data, we show that treatin apoE-/- mice with VEGF-C, the ligand for VEGFR3, restores impaired lymphatic transport that occurs following hypercholesterolemia, allowing us to test the hypothesis that VEGF-C acts on lymphatic vessels to therapeutically sustain a critical route for cholesterol transport out of plaques after macrophage cholesterol efflux is stimulated. This hypothesis raises a fundamental question not yet addressed in the field: does effective plaque regression truly depend upon cholesterol removal from plaques, only upon cholesterol removal from macrophages, or perhaps neither? Our study design is ideal to address this fundamental issue while simultaneously digging deeply into a new concept that supporting lymphatic vessel function may help resolve inflammation in the atherosclerotic plaque.