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

描述(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，淋巴管是从动脉壁和其他组织中清除胆固醇的管道。胆固醇反向转运(RCT)是胆固醇从体内动员并通过粪便排泄的过程。对于动脉粥样硬化，从巨噬细胞中动员胆固醇以清除和排泄是最相关的。多年来，关于胆固醇是如何从巨噬细胞中动员到高密度脂蛋白(HDL-C)上的，出现了许多细节。然而，关于高密度脂蛋白-胆固醇随后如何从组织中出来，包括动脉壁的动脉粥样硬化斑块，在进入排泄途径之前回到血浆中，人们知之甚少。20世纪80年代初，一些临床或实验观察使一些内科医生提出淋巴运输受损与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通过人类淋巴的高密度脂蛋白-C的净流量是可观的之外，还没有研究淋巴管在随机对照试验中的定量重要性。最近，我们使用了实验小鼠模型，其中淋巴流动的通畅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管流动，使我们能够证明淋巴管是巨噬细胞RCT后胆固醇动员到血浆的主要途径。在动脉粥样硬化影响的主动脉壁中，我们还使用了一种外科方法，以脉搏追逐的方式追踪斑块中的氚标记胆固醇([2H]D6-胆固醇；D6-胆固醇)。用抗血管内皮生长因子受体3单抗阻断或不阻断淋巴管重新吻合的情况下，将主动脉移植到受体。这种阻断显著地保留了动脉粥样硬化动脉中的D6-胆固醇，这表明D6-胆固醇起着关键作用 对于淋巴管中的胆固醇从主动脉动员，如在皮肤中观察到的。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淋巴管在从主动脉中清除胆固醇方面的ROE，并评估淋巴管的阻断是否真的在主动脉壁起作用。在初步数据中，我们显示，VEGFR3的配基--血管内皮生长因子-C治疗载脂蛋白E-/-小鼠，可以恢复高胆固醇血症后受损的淋巴运输，使我们能够检验这样的假设：在巨噬细胞胆固醇外流被刺激后，血管内皮生长因子-C作用于淋巴管，在治疗上维持胆固醇从斑块中运输出去的关键途径。这一假说提出了一个尚未在该领域解决的根本问题：有效的斑块消退真的依赖于斑块中胆固醇的去除，仅仅依赖于巨噬细胞中胆固醇的去除，还是两者都不依赖？我们的研究设计理想地解决了这一根本问题，同时深入挖掘了一个新的概念，即支持淋巴管功能可能有助于解决动脉粥样硬化斑块中的炎症。

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.