Investigating Mechanisms for Lipid Transport in Health and Disease

摘要 我们的实验室专注于脂蛋白脂酶（LPL）介导的富含磷脂酰肌醇脂蛋白（TRL）的加工 在毛细血管中。这个过程，血管内脂解，对于将脂质营养素递送到重要组织（例如， 心脏）并且与血浆脂质水平和冠状动脉疾病风险高度相关。我们发现了一 血管内脂解所需的仅在毛细血管内皮细胞中表达的蛋白质，GPIHBP 1。 GPIHBP 1在间质空间内结合LPL，并将其穿梭于内皮细胞至其作用位点， 毛细血管腔。GPIHBP 1也是毛细血管中TRL的边缘化和保存血管内皮细胞所必需的。 LPL的催化活性。这些发现已经改变了教科书对脂解作用的描述， 许多挑战依然存在。一个是定义脂肪酸（FA） TRL加工的产物穿过内皮细胞并进入实质细胞。没有人理解 这一过程，部分原因是没有可视化FA进入和穿过毛细血管的方法 内皮细胞提出关于FA在组织内运动机制的假设，并检验 在这个过程中特定基因和代谢物的作用，我们现在用NanoSIMS成像组织。 NanoSIMS使用Cs+束轰击组织切片，释放出可收集的二次离子， 定量，并用于创建仅基于其同位素含量的细胞和组织的高分辨率图像。 我们常规地制备富含13 C或2 H标记的甘油三酯的新鲜TRL，将其静脉注射到 小鼠，然后使用NanoSIMS创建13 C-和2 H-FA的高分辨率图像，因为它们移动到 穿过毛细血管内皮细胞。我们获得背散射电子（BSE）的图像上相同的部分。我们 在脂质代谢和血管生物学领域，相关成像方法是独一无二的， 将NanoSIMS的化学信息与BSE提供的超微结构形态相匹配 图像.我们现在定位于确定脂质运动的细胞和分子机制， 重要组织第二个挑战是鉴定毛细血管内皮细胞中的其他蛋白质， 相关的脂质代谢;一个相关的问题是确定是否积极TRL加工改变基因表达， 毛细血管内皮细胞幸运的是，我们的GPIHBP 1特异性单克隆抗体使其成为可能， 从复杂的细胞混合物中纯化毛细血管内皮细胞，便于分析细胞中的基因表达。 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挑战--是 探讨GPIHBP 1和毛细血管内皮细胞在人高脂血症中的作用。我们 在多名高脂血症患者的血浆中发现了抗GPIHBP 1的自身抗体;这些 自身抗体通过阻断LPL与GPIHBP 1的结合而引起疾病。GPIHBP 1自身抗体 需要表征，这种新的自身免疫性/代谢性疾病综合征的频率需要 定义了凭借我们的试剂和检测试剂，我们在解决这些问题方面具有独特的优势。

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.