Defining mechanisms for lipid transport across capillary endothelial cells

 描述(申请人提供)：项目标题：“定义跨毛细血管内皮细胞的脂质传输机制”我们的目标是了解富含甘油三酯的脂蛋白(TRL)的脂类如何通过毛细血管内皮细胞向实质细胞移动。我们对这个主题的兴趣--血浆甘油三酯代谢中最不被了解的领域--源于我们努力了解TRL血管内处理的分子机制。在过去的几年里，我们发现GPIHBP1是一种GPI锚定的毛细血管内皮细胞蛋白，它只负责将脂蛋白脂酶(LPL)从间质间隙运送到毛细血管管腔中的作用部位。最近，我们证明LPL-GPIHBP1复合体对于TRL沿着毛细血管的边际形成是关键的(以便LPL介导的TRL的处理可以进行)。脂类营养物质通过毛细血管进入实质细胞，对于将燃料输送到重要器官和脂肪组织中储存的脂类至关重要。不幸的是，对这一过程的洞察很少。没有人知道：(1)脂解的脂肪酸产物是否只是在内皮细胞之间扩散；(2)脂类是否在穿梭GPIHBP1和LPL的相同小泡中跨内皮细胞移动；(3)完整的TRL是否可以跨内皮细胞移动到内皮下间隙；以及(4)是否需要CD36与内皮细胞的脂肪酸结合才能跨毛细血管进行脂类运输。此外，没有人了解其他脊椎动物(如鸟类、鱼类)中的脂质是如何通过毛细血管移动的。在这些生物中，GPIHBP1不存在，而LPL似乎主要(如果不是唯一的话)位于血管外间隙(即，它不是 与毛细血管相关)。在其他脊椎动物中，我们怀疑TRL可能通过毛细血管运输到内皮下间隙-LPL所在的位置。对其他脊椎动物TRL代谢的更好理解可能会对哺乳动物TRL处理的辅助机制有更深入的了解。理解脂类通过毛细血管的运输进展缓慢的主要原因之一是一直没有办法可视化脂类的运输。“观察”脂质如何在毛细血管中移动，对于破译分子机制和设计可验证的假说至关重要。幸运的是，我们已经克服了“成像障碍”。在过去的两年中，我们使用了NanoSIMS和背向散射电子(BSE)成像技术来创建TRL沿毛细血管边缘和TRL脂类跨内皮细胞向实质细胞移动时的高分辨率图像。这些研究已经证明，一些TRL脂质以囊泡的形式跨内皮细胞移动，但需要进行额外的高分辨率成像研究，以确定是否涉及脂肪酸在质膜上的扩散-或脂类在胞浆中的运输。同样的方法也可以用来确定特定蛋白质(例如，CD36)在内皮细胞间脂类运输中的作用。在接下来的五年里，我们将使用NanoSIMS和BSE成像来确定脂类通过毛细血管运输的细胞和分子机制。我们还将确定CD36与TRL衍生的脂类通过毛细血管内皮细胞运输的体内功能相关性。

 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.