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

描述（由申请人提供）：内皮脂肪酶（EL）是一种细胞外蛋白，对HDL具有显著的水解活性。模式生物的研究表明，EL在HDL代谢中很重要；然而，EL在人体高密度脂蛋白代谢中的意义尚不清楚。本提案的目的是研究人类EL的变化及其对HDL水平的影响。高HDL受试者的EL深度重测序已经确定了潜在的功能非同义和启动子变体。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启动子变异通过改变转录效率影响EL蛋白水平来调节HDL水平。具体目的1：确定EL编码变体是否通过降低EL活性来增加HDL。为了检测活性降低，编码变异体将通过位点定向诱变在EL表达质粒中重建，在293细胞中表达，并在体外测定其对合成磷脂底物和天然HDL的特异性活性。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。这些变异与人类高密度脂蛋白水平的关系将通过比较高密度脂蛋白极端的受试者之间的频率和分析家族内高密度脂蛋白水平的共分离来进行统计检验。具体目的2：确定EL启动子变异是否通过影响EL基因的转录速率来调节HDL水平。为了测试转录率的改变，启动子变体将通过在EL启动子驱动的萤火虫荧光素酶表达质粒中进行定点突变来重建，并转染到HUVEC细胞中。表达结果将通过对从杂合受试者分离的内皮细胞进行等位基因特异性HaploChIP分析来验证。这些变异与人类高密度脂蛋白水平的关系将通过比较高密度脂蛋白极端的受试者之间的频率和分析家族内高密度脂蛋白水平的共分离来进行统计检验。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。本提案将试图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提高HDL水平的靶标。

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.