Defining mechanisms for lipid transport across capillary endothelial cells

 描述（由申请人提供）： 项目名称：“定义跨毛细血管内皮细胞的脂质转运机制” 我们的目标是了解富含甘油三酯的脂蛋白 (TRL) 中的脂质如何跨毛细血管内皮细胞流向实质细胞。我们对这个主题（血浆甘油三酯代谢中最不为人所知的领域）的兴趣源于我们努力了解 TRL 血管内加工的分子机制。在过去的几年中，我们发现 GPIHBP1（一种毛细血管内皮细胞的 GPI 锚定蛋白）单独负责将脂蛋白脂肪酶 (LPL) 从细胞间隙运送到毛细血管腔中的作用位点。最近，我们发现 LPL-GPIHBP1 复合物对于沿着毛细血管的 TRL 边缘化至关重要（以便 LPL 介导的 TRL 处理能够继续进行）。脂质营养物质穿过毛细血管到实质细胞的运动对于将燃料输送到重要器官以及储存在脂肪组织中的脂质至关重要。不幸的是，人们对这个过程的了解很少。没有人了解：（1）脂肪分解的脂肪酸产物是否简单地扩散到内皮细胞； (2) 脂质是否在穿梭 GPIHBP1 和 LPL 的同一囊泡中穿过内皮细胞； (3)完整的TRL是否穿过内皮细胞到达内皮下空间； (4) 脂质跨毛细血管转运是否需要 CD36 与内皮细胞上的脂肪酸结合。此外，没有人了解脂质如何穿过其他脊椎动物（例如鸟类、鱼类）的毛细血管。在这些生物体中，GPIHBP1 不存在，并且 LPL 似乎大部分（如果不是全部）位于血管外空间（即，它不是 与毛细血管有关）。在其他脊椎动物中，我们怀疑 TRL 可能穿过毛细血管运输到内皮下空间，即 LPL 所在的位置。对其他脊椎动物 TRL 代谢的进一步了解可能会深入了解哺乳动物 TRL 处理的辅助机制。对脂质跨毛细血管运输的了解进展缓慢的主要原因之一是没有办法可视化脂质运输。 “观察”脂质如何穿过毛细血管对于破译分子机制和设计可检验的假设至关重要。幸运的是，我们已经克服了“成像障碍”。在过去的两年中，我们使用 NanoSIMS 和背散射电子 (BSE) 成像来创建 TRL 的高分辨率图像，因为它们沿着毛细血管边缘，并且当 TRL 脂质穿过内皮细胞到达实质细胞时。这些研究表明，一些 TRL 脂质在囊泡中穿过内皮细胞，但需要额外的高分辨率成像研究来确定是否涉及脂肪酸沿质膜的扩散或脂质穿过细胞质的运输。相同的方法可用于定义特定蛋白质（例如 CD36）在内皮细胞脂质转运中的作用。在接下来的五年中，我们将使用 NanoSIMS 和 BSE 成像来定义脂质跨毛细血管运输的细胞和分子机制。我们还将定义 CD36 与 TRL 衍生脂质跨毛细血管内皮细胞转运的体内功能相关性。

 DESCRIPTION (provided by applicant): Title of Project: "Defining mechanisms for lipid transport across capillary endothelial cells" Our objective is to understand how lipids from triglyceride-rich lipoproteins (TRLs) move across capillary endothelial cells towards parenchymal cells. Our interest in this topic-the least understood area within plasma triglyceride metabolism-arose from our efforts to understand molecular mechanisms for the intravascular processing of TRLs. During the past few years, we showed that GPIHBP1, a GPI-anchored protein of capillary endothelial cells, is solely responsible for shuttling lipoprotein lipase (LPL) from the interstitial spaces to its site of action in the capillary lumen. More recenty, we showed that the LPL-GPIHBP1 complex is critical for the margination of TRLs along capillaries (so that the LPL-mediated processing of TRLs can proceed). The movement of lipid nutrients across capillaries to parenchymal cells is crucial for delivering fuel to vital organs an lipids for storage in adipose tissue. Unfortunately, there are few insights into this process. No one understands: (1) whether the fatty acid products of lipolysis simply diffuse across endothelial cells; (2) whether lipids move across endothelial in the very same vesicles that shuttle GPIHBP1 and LPL; (3) whether intact TRLs move across endothelial cells to the subendothelial spaces; and (4) whether binding of fatty acids by CD36 on endothelial cells is required for lipid transport across capillaries. Also, no one understands how lipids move across capillaries in other vertebrates (e.g., birds, fish). In those organisms, GPIHBP1 is absent and LPL appears to be located largely, if not exclusively, in the extravascular spaces (i.e., it is not associated with capillaries). In other vertebrates, we suspect that the TRLs might be transported across capillaries to the subendothelial spaces-to where the LPL is located. An improved understanding of TRL metabolism in other vertebrates will likely yield insights into accessory mechanisms for TRL processing in mammals. One of the main reasons for the slow progress in understanding lipid transport across capillaries is that there has been no way to visualize lipid transport. "Seeing" how lipids move across capillaries is crucial for deciphering molecular mechanisms and designing testable hypotheses. Fortunately, we have overcome the "imaging roadblock." During the past two years, we have used NanoSIMS and backscattered electron (BSE) imaging to create high-resolution images of TRLs as they marginate along capillaries and as the TRL lipids move across endothelial cells to parenchymal cells. These studies have demonstrated that some TRL lipids move across endothelial cells in vesicles, but additional high-resolution imaging studies are required to determine if diffusion of fatty acids along plasma membranes-or transport of lipids across the cytosol-is involved. The same methods can be used to define the role of specific proteins (e.g., CD36) in lipid transport across endothelial cels. For the next five years, we will use NanoSIMS and BSE imaging to define the cellular and molecular mechanisms for lipid transport across capillaries. We will also define the in vivo functional relevance of CD36 for the transport of TRL-derived lipids across capillary endothelial cells.