Endothelial lipase: a modulator of HDL metabolism and atherosclerosis in humans

内皮脂肪酶：人类 HDL 代谢和动脉粥样硬化的调节剂

• 批准号: 7545618
• 负责人: Andrew Charles Edmondson
• 金额: $ 2.84万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7545618
• 关键词: Affect; Alleles; Animal Model; Atherosclerosis; Candidate Disease Gene; Cells; Code; Coronary heart disease; DNA Resequencing; Depth; Endothelial Cells; Extracellular Protein; Family; Firefly Luciferases; Frequencies; Genes; Genetic Transcription; High Density Lipoprotein Cholesterol; High Density Lipoproteins; Human; Hydrolysis; LDL Cholesterol Lipoproteins; Lipase; Lipoproteins; Low-Density Lipoproteins; Metabolism; Mus; Phospholipids; Plasmids; Proteins; Rate; Site-Directed Mutagenesis; Testing; Variant; Week; Woman; base; cardiovascular risk factor; hepatic lipase; human LIPG protein; in vitro Assay; in vivo; lipoprotein lipase; member; men; mortality; promoter; vector

DESCRIPTION (provided by applicant): Endothelial lipase (EL) is an extracellular protein with significant hydrolysis activity against HDL. Studies in model organisms suggest that EL is important in HDL metabolism; however, the significance of EL in human HDL metabolism is unknown. The objective of this proposal is to investigate variation in human EL and the effects of this variation on HDL levels. Deep resequencing of EL in subjects with high HDL has identified potentially functional nonsynonymous and promoter variants. We hypothesize that rare EL coding variants increase HDL by decreasing EL activity. We also hypothesize that EL promoter variants modulate HDL levels by affecting levels of EL protein via alterations in transcriptional efficiency. Specific Aim 1: To determine if coding variants of EL increase HDL by decreasing EL activity. To test for decreased activity, coding variants will be recreated in an EL expression plasmid via site-directed mutagenesis, expressed in 293 cells, and their specific activities assayed in vitro against synthetic phospholipids substrates and native HDL. To test for an influence on HDL levels in vivo, the coding variants will be cloned into AAV-based vectors, injected into EL-KO mice, and HDL levels assessed over 6 weeks. Association of the variants with human HDL levels will be tested statistically by comparing frequencies among subjects from HDL extremes and by analyzing cosegregation with HDL levels within families. Specific Aim 2: To determine if promoter variants of EL modulate HDL levels through their influence on the rate of transcription of the EL gene. To test for altered rates of transcription, promoter variants will be recreated via site-directed mutagenesis in a firefly luciferase expression plasmid driven by the EL promoter, and transfected into HUVEC cells. Expression results will be validated by allele-specific HaploChIP analysis on endothelial cells isolated from heterozygous subjects. Association of the variants with human HDL levels will be tested statistically by comparing frequencies among subjects from HDL extremes and by analyzing cosegregation with HDL levels within families. HDL levels correlate with protection from coronary heart disease, the leading cause of mortality among men and women. This proposal will attempt to determine the significance of endothelial lipase in human HDL metabolism, and validate it as a target for pharmacologic inhibition to raise HDL levels.

描述（申请人提供）：内皮脂肪酶（EL）是一种细胞外蛋白，对 HDL 具有显着的水解活性。对模式生物的研究表明，EL 在 HDL 代谢中很重要；然而，EL 在人类 HDL 代谢中的重要性尚不清楚。该提案的目的是研究人类 EL 的变化以及这种变化对 HDL 水平的影响。对高 HDL 受试者的 EL 进行深度重测序已鉴定出潜在的功能性非同义变异和启动子变异。我们假设罕见的 EL 编码变体通过降低 EL 活性来增加 HDL。我们还假设 EL 启动子变体通过改变转录效率影响 EL 蛋白的水平来调节 HDL 水平。具体目标 1：确定 EL 的编码变体是否通过降低 EL 活性来增加 HDL。为了测试活性降低，将通过定点诱变在 EL 表达质粒中重新创建编码变体，在 293 细胞中表达，并在体外针对合成磷脂底物和天然 HDL 测定其特定活性。为了测试对体内 HDL 水平的影响，编码变体将被克隆到基于 AAV 的载体中，注射到 EL-KO 小鼠中，并在 6 周内评估 HDL 水平。通过比较受试者中 HDL 极端值的频率并分析家庭内 HDL 水平的共分离，可以对变异与人类 HDL 水平的关联进行统计测试。具体目标 2：确定 EL 启动子变体是否通过影响 EL 基因的转录速率来调节 HDL 水平。为了测试转录速率的改变，将通过在 EL 启动子驱动的萤火虫荧光素酶表达质粒中进行定点诱变来重新创建启动子变体，并转染到 HUVEC 细胞中。表达结果将通过对从杂合受试者中分离的内皮细胞进行等位基因特异性 HaploChIP 分析来验证。通过比较受试者中 HDL 极端值的频率并分析家庭内 HDL 水平的共分离，可以对变异与人类 HDL 水平的关联进行统计测试。高密度脂蛋白水平与预防冠心病相关，冠心病是男性和女性死亡的主要原因。该提案将尝试确定内皮脂肪酶在人类 HDL 代谢中的重要性，并验证其作为药物抑制以提高 HDL 水平的靶标。