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

性状（由申请方提供）：内皮脂肪酶（EL）是一种对HDL具有显著水解活性的细胞外蛋白。在模式生物中的研究表明，EL是重要的HDL代谢，然而，EL在人体HDL代谢的意义是未知的。这项建议的目的是调查人类EL的变化和这种变化对HDL水平的影响。高HDL受试者EL的深度重测序已经确定了潜在的功能性非同义和启动子变体。我们推测，罕见的EL编码变异增加HDL降低EL活动。我们还假设EL启动子变体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的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，将通过定点诱变在EL表达质粒中重建编码变体，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的比活性。为了测试对体内HDL水平的影响，将编码变体克隆到基于AAV的载体中，注射到EL-KO小鼠中，并在6周内评估HDL水平。将通过比较受试者中HDL极值的频率和分析家族内HDL水平的共分离，对变异体与人HDL水平的关联进行统计学检验。具体目标2：确定EL的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，将在由EL启动子驱动的萤火虫荧光素酶表达质粒中通过定点诱变重建启动子变体，并转染到HUVEC细胞中。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分析来验证表达结果。将通过比较受试者中HDL极值的频率和分析家族内HDL水平的共分离，对变异体与人HDL水平的关联进行统计学检验。高密度脂蛋白水平与预防冠心病有关，冠心病是男性和女性死亡的主要原因。该建议将试图确定内皮脂肪酶在人类HDL代谢中的重要性，并验证其作为药理学抑制以提高HDL水平的靶点。

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.