Investigating Mechanisms for Lipid Transport in Health and Disease

摘要 本实验室致力于脂蛋白脂肪酶(LPL)介导的富含甘油三酯的脂蛋白(TRL)的加工 在毛细血管中。这一过程，血管内脂肪分解，对于将脂肪营养输送到重要组织是必不可少的(例如， 心脏)，并与血脂水平和冠状动脉疾病风险高度相关。我们发现了一个 仅在毛细血管内皮细胞GPIHBP1中表达的蛋白质，是血管内脂肪分解所必需的。 GPIHBP1与间质间隙内的LPL结合，并将其穿梭于内皮细胞之间，到达其作用部位 毛细血管管腔。GPIHBP1也是毛细血管内TRL的边际形成和保存 LPL的催化活性。这些发现已经改变了教科书中对脂肪分解的描述，但 许多挑战依然存在。其一是确定脂肪酸(FA)的细胞和分子机制。 TRL加工产物穿过内皮细胞，进入实质细胞。没有人能理解 这一过程，部分是因为没有方法可视化FA进入和穿过毛细管的运动 内皮细胞。提出关于FA在组织内运动机制的假说，并验证 特定基因和代谢物在这一过程中的作用，我们现在正在用NanoSIMS对组织进行成像。 NanoSIMS使用Cs+束轰击组织切片，释放可以收集的二次离子， 仅根据细胞和组织的同位素含量对其进行量化，并用于创建高分辨率图像。 我们常规制备富含13C或2H标记甘油三酯的新鲜TRL，将它们静脉注射到 然后使用NanoSIMS创建高分辨率的13C和2H-FA图像，当它们进入和 穿过毛细血管内皮细胞。我们在同一截面上获得了背散射电子(BSE)图像。我们的 相关成像方法在脂质代谢和血管生物学领域是独一无二的，它使我们能够 将来自NanoSIMS的化学信息与BSE提供的超微结构形态进行匹配 图像。我们现在可以确定脂质运动的细胞和分子机制。 重要组织。第二个挑战是在毛细血管内皮细胞中识别额外的蛋白质 与脂质代谢有关；一个相关的问题是确定主动的TRL处理是否改变了 毛细血管内皮细胞。幸运的是，我们的GPIHBP1特异性单抗使我们有可能 从复杂的细胞混合物中纯化毛细血管内皮细胞，促进基因表达的分析 毛细血管内皮细胞。第三个挑战--也是与临床医学特别相关的--是 探讨GPIHBP1和毛细血管内皮细胞在人类高甘油三酯血症中的作用。我们 在多名高甘油三酯血症患者的血浆中发现抗GPIHBP1的自身抗体；这些 自身抗体通过阻断LPL与GPIHBP1的结合而致病。GPIHBP1自身抗体的研究进展 需要定性，这种新的自身免疫/代谢疾病综合征的频率需要 已定义。有了我们的试剂和分析，我们在解决这些问题上处于独特的地位。

Abstract Our laboratory focuses on lipoprotein lipase (LPL)–mediated processing of triglyceride-rich lipoproteins (TRLs) in capillaries. This process, intravascular lipolysis, is essential for delivering lipid nutrients to vital tissues (e.g., the heart) and is highly relevant to plasma lipid levels and coronary artery disease risk. We discovered a protein expressed exclusively in capillary endothelial cells, GPIHBP1, that is required for intravascular lipolysis. GPIHBP1 binds LPL within the interstitial spaces and shuttles it across endothelial cells to its site of action in the capillary lumen. GPIHBP1 is also required for the margination of TRLs in capillaries and for preserving the catalytic activity of LPL. These discoveries have already transformed textbook descriptions of lipolysis, but many challenges remain. One is to define the cellular and molecular mechanisms by which the fatty acid (FA) products of TRL processing traverse endothelial cells and move into parenchymal cells. No one understands this process, in part because there were no methods for visualizing FA movement into and across capillary endothelial cells. To formulate hypotheses about the mechanisms for FA movement within tissues and to test the roles of specific genes and metabolites in this process, we are now imaging tissues with NanoSIMS. NanoSIMS uses a Cs+ beam to bombard a tissue section, releasing secondary ions that can be collected, quantified, and used to create high-resolution images of cells and tissues based solely on their isotopic content. We routinely prepare fresh TRLs enriched in 13C- or 2H-labeled triglycerides, inject them intravenously into mice, and then use NanoSIMS to create high-resolution images of 13C- and 2H-FAs as they move into and across capillary endothelial cells. We obtain backscattered electron (BSE) images on the same section. Our correlative imaging approach, which is unique in the fields of lipid metabolism and vascular biology, allows us to match the chemical information from NanoSIMS to the ultrastructural morphology provided by the BSE images. We are now positioned to identify the cellular and molecular mechanisms for the movement of lipids to vital tissues. A second challenge has been to identify additional proteins in capillary endothelial cells that are relevant to lipid metabolism; a related issue is to determine if active TRL processing alters gene expression in capillary endothelial cells. Fortunately, our GPIHBP1-specific monoclonal antibodies have made it possible to purify capillary endothelial cells from complex mixtures of cells, facilitating analyses of gene expression in capillary endothelial cells. A third challenge—and one that is particularly relevant to clinical medicine—is to explore the importance of GPIHBP1 and capillary endothelial cells to human hypertriglyceridemia. We discovered autoantibodies against GPIHBP1 in the plasma of multiple patients with hypertriglyceridemia; these autoantibodies cause disease by blocking the binding of LPL to GPIHBP1. The GPIHBP1 autoantibodies now need characterization, and the frequency of this new autoimmune/metabolic disease syndrome needs to be defined. With our reagents and assays, we are uniquely positioned to address these issues.