Macrophage-specific function of GWAS CAD-associated LIPA alleles in atherosclerosis

GWAS CAD 相关 LIPA 等位基因在动脉粥样硬化中的巨噬细胞特异性功能

基本信息

批准号：
9765372
负责人：
Hanrui Zhang
金额：
$ 24.44万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9765372
关键词：
Acceleration Acid Lipase Address Alleles Apolipoprotein E Arterial Fatty Streak Atherosclerosis Automobile Driving Bone Marrow Transplantation Cell Communication Cholesterol Cholesterol Ester Storage Disease Cholesterol Esters Cleaved cell Clustered Regularly Interspaced Short Palindromic Repeats Competence Coronary Arteriosclerosis Crystal Formation Crystallization Data Endothelial Cells Enzymes Foam Cells Functional disorder Genes Genotype Hepatic Human Hydrolase Hydrolysis Hyperlipidemia Inflammasome Inflammation Knock-in Lesion Lipase Lipids Low-Density Lipoproteins Mediating Messenger RNA Modification Mus Oxidative Stress Phase Phenotype Plasma Proton Pump Research Role Smooth Muscle Myocytes Subfamily lentivirinae Susceptibility Gene System Therapeutic Toxic effect Translational Research Triglycerides Vascular Smooth Muscle atherogenesis causal model extracellular gain of function genome wide association study hepatic lipase in vivo induced pluripotent stem cell loss of function mutation mRNA Expression macrophage migration monocyte mouse model novel overexpression prevent promoter risk variant uptake vacuolar H+-ATPase vascular smooth muscle cell proliferation

项目摘要

DESCRIPTION (provided by applicant): Lysosomal acid lipase (LAL), encoded by the LIPA gene, is the key lysosomal hydrolase that cleaves cholesteryl esters (CE) and triglycerides (TG). Loss-of-function (LOF) mutations in LIPA result in cholesteryl ester storage disease (CESD), which manifests with hyperlipidemia, hepatic and macrophage CE accumulation, and atherosclerosis. Recently, genome-wide association studies (GWASs) identified LIPA as a novel locus for coronary artery disease (CAD). Surprisingly, LIPA CAD-GWAS risk alleles do not associate with altered plasma lipids or hepatic LIPA mRNA levels but actually relate to higher monocyte LIPA mRNA expression. Our preliminary data also reveal a coincident increase in both LIPA mRNA and LAL enzymatic activity in monocyte-derived macrophages (HMDM) of CAD risk allele carriers, suggesting that monocyte/macrophage-specific gain-of-function (GOF) of LIPA may explain the GWAS CAD risk alleles. LIPA mRNA and LAL activity were markedly induced upon HMDM differentiation and mature HMDM secrets LAL. Extracellular LAL is abundant in the neointima of advanced human atherosclerotic lesions where LAL can remain enzymatically active in the lesion acidic microenvironment. Thus, macrophage LIPA GOF may aggravate atherosclerosis through accelerating intracellular LAL-induced lysosomal free cholesterol (FC) toxicity and extracellular LAL actions on low-density lipoprotein (LDL). My working hypotheses are that LIPA CAD risk alleles encode for macrophage-specific LIPA GOF, and therefore CAD risk alleles or macrophage LIPA overexpression will (i) increase intracellular LAL activity resulting in a shift toward greater lysosomal CE hydrolysis and FC accumulation and thus accelerate macrophage lysosomal dysfunction during modified-LDL loading; (ii) increase macrophage LAL secretion and extracellular LAL-mediated LDL modification driving atherogenic phenotypes in vascular smooth muscle cells (VSMC) and endothelial cells (EC); and (iii) macrophage LIPA overexpression will accelerate atherogenesis in ApoE-/- and Ldlr-/- mice. These hypotheses will be addressed in primary HMDM of specific LIPA genotype, through causal modeling in a novel human iPSC- differentiated macrophage (IPSDM) system utilizing CRISPR/Cas knock-in of risk alleles, in HMDM with lentivirus-mediated LIPA overexpression (Lenti-LIPA), and in murine models with macrophage-specific Lipa overexpression by lentivirus-infected bone marrow (BM) transplantation driven by macrophage-specific promoter. These will be accomplished by pursuing the following three aims: Aim 1. K99 Phase: Determine the effects of LIPA CAD risk alleles and LIPA overexpression on intracellular macrophage LIPA expression, LAL activity, and phenotype of human macrophages. Aim 2. R00 Phase: Determine the effects of LIPA CAD risk alleles and LIPA overexpression on macrophage LAL secretion, extracellular LAL-modification of LDL and on atherogenic phenotypes in EC and VSMC. Aim 3. R00 Phase: Determine if macrophage Lipa overexpression accelerates atherosclerosis in ApoE-/- and Ldlr-/- mice.

描述（由申请人提供）：由LIPA基因编码的溶酶体酸性脂肪酶（LAL）是裂解胆固醇酯（CE）和甘油三酯（TG）的关键溶酶体水解酶，导致LIPA结果中的功能丧失（LOF）突变。胆固醇酯贮积病 (CESD)，表现为高脂血症、肝脏和巨噬细胞 CE 积聚，以及最近，全基因组关联研究 (GWAS) 确定 LIPA 是冠状动脉疾病 (CAD) 的一个新基因位点，令人惊讶的是，LIPA CAD-GWAS 风险等位基因与血浆脂质或肝脏 LIPA mRNA 水平无关，但实际上与较高水平相关。我们的初步数据还揭示了 CAD 风险的单核细胞源性巨噬细胞 (HMDM) 中 LIPA mRNA 和 LAL 酶活性的同时增加。等位基因携带者，表明 LIPA 的单核细胞/巨噬细胞特异性功能获得 (GOF) 可以解释 LIPA mRNA 和 LAL 活性在 HMDM 分化时显着诱导，并且成熟的 HMDM 分泌细胞外 LAL。晚期人类动脉粥样硬化病变的新内膜，其中 LAL 可以在病变酸性微环境中保持酶活性，因此，巨噬细胞 LIPA GOF 可能。通过加速细胞内 LAL 诱导的溶酶体游离胆固醇 (FC) 毒性和细胞外 LAL 对低密度脂蛋白 (LDL) 的作用，加剧动脉粥样硬化。我的工作假设是 LIPA CAD 风险等位基因编码巨噬细胞特异性 LIPA GOF，因此也是 CAD 风险等位基因。或巨噬细胞 LIPA 过度表达将 (i) 增加细胞内 LAL 活性，导致更大的溶酶体 CE 水解和 FC 转变累积，从而加速巨噬细胞溶酶体功能障碍；(ii) 增加巨噬细胞 LAL 分泌和细胞外 LAL 介导的 LDL 修饰，驱动血管平滑肌细胞 (VSMC) 和内皮细胞 (EC) 中的动脉粥样硬化表型；以及 (iii) 巨噬细胞LIPA 过度表达将加速 ApoE-/- 和 Ldlr-/- 小鼠的动脉粥样硬化形成，这些假设将通过因果模型在特定 LIPA 基因型的原发性 HMDM 中得到解决。在利用风险等位基因的 CRISPR/Cas 敲入的新型人类 iPSC 分化巨噬细胞 (IPSDM) 系统中，在慢病毒介导的 LIPA 过表达 (Lenti-LIPA) 的 HMDM 中，以及在通过慢病毒介导的巨噬细胞特异性 Lipa 过表达的小鼠模型中由巨噬细胞特异性启动子驱动的感染骨髓（BM）移植将通过追求以下三个目标来实现：目标 1. K99 阶段：确定。 LIPA CAD 风险等位基因和 LIPA 过度表达对细胞内巨噬细胞 LIPA 表达、LAL 活性和人类巨噬细胞表型的影响目标 2. R00 阶段：确定 LIPA CAD 风险等位基因和 LIPA 过度表达对巨噬细胞 LAL 分泌、细胞外 LAL- 的影响。 LDL 的修饰以及 EC 和 VSMC 中致动脉粥样硬化表型的影响目标 3. R00 阶段：确定巨噬细胞是否存在。 Lipa 过度表达会加速 ApoE-/- 和 Ldlr-/- 小鼠的动脉粥样硬化。