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

 描述（由申请方提供）：由利帕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。利帕中的功能丧失（LOF）突变导致胆固醇酯累积病（CESD），其表现为高脂血症、肝和巨噬细胞CE积聚和动脉粥样硬化。最近，全基因组关联研究（GWASs）将利帕确定为冠状动脉疾病（CAD）的新位点。令人惊讶的是，利帕CAD-GWAS风险等位基因与血脂或肝脏利帕mRNA水平的改变无关，但实际上与较高的单核细胞利帕mRNA表达有关。我们的初步数据还揭示了冠心病风险等位基因携带者单核细胞衍生的巨噬细胞（HMDM）中利帕mRNA和LAL酶活性的同时增加，表明利帕的单核细胞/巨噬细胞特异性功能获得（GOF）可能解释GWAS CAD风险等位基因。利帕mRNA和LAL活性在HMDM分化和成熟HMDM分泌LAL时被显著诱导。细胞外LAL在晚期人类动脉粥样硬化病变的新生内膜中丰富，其中LAL可以在病变酸性微环境中保持酶活性。因此，巨噬细胞利帕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）的作用而加重动脉粥样硬化。我的工作假设是利帕CAD风险等位基因编码巨噬细胞特异性利帕GOF，因此CAD风险等位基因或巨噬细胞利帕过表达将（i）增加细胞内LAL活性，导致向更大的溶酶体CE水解和FC积累转变，从而在修饰的LDL负荷期间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，从而驱动血管平滑肌细胞（VSMC）和内皮细胞（EC）中的致动脉粥样硬化表型;和（iii）巨噬细胞利帕过表达将加速ApoE-/-和Ldlr-/-小鼠中的动脉粥样硬化形成。这些假设将在特定LIPA基因型的原发性HMDM中，通过在利用CRISPR/Cas敲入风险等位基因的新型人iPSC分化的巨噬细胞（IPSDM）系统中的因果建模，在具有慢病毒介导的LIPA过表达（Lenti-LIPA）的HMDM中，以及在具有巨噬细胞特异性利帕过表达的鼠模型中，通过由巨噬细胞特异性启动子驱动的慢病毒感染的骨髓（BM）移植来解决。这些目标将通过实现以下三个目标来实现：K99阶段：确定利帕CAD风险等位基因和利帕过表达对细胞内巨噬细胞利帕表达、LAL活性和人巨噬细胞表型的影响。目标二。R 00阶段：确定利帕CAD风险等位基因和利帕过表达对巨噬细胞LAL分泌、LDL的细胞外LAL修饰以及EC和VSMC中致动脉粥样硬化表型的影响。目标3：R 00阶段：确定巨噬细胞利帕过表达是否加速ApoE-/-和Ldlr-/-小鼠中的动脉粥样硬化。

 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.