Dataset Information

Quantitative Trait Locus Mapping of Macrophage Cholesterol Metabolism and CRISPR/Cas9 Editing Implicate an ACAT1 Truncation as a Causal Modifier Variant.

ABSTRACT: OBJECTIVE:Cholesterol metabolism is a dynamic process involving intracellular trafficking, cholesterol esterification, and cholesterol ester hydrolysis. Our objective was to identify genes that regulate macrophage cholesterol metabolism. APPROACHES AND RESULTS:We performed quantitative trait loci mapping of free and esterified cholesterol levels and the ratio of esterified to free cholesterol in acetylated low-density lipoprotein-loaded bone marrow-derived macrophages from an AKR×DBA/2 strain intercross. Ten distinct cholesterol modifier loci were identified, and bioinformatics was used to prioritize candidate genes. The strongest locus was located on distal chromosome 1, which we named Mcmm1 (macrophage cholesterol metabolism modifier 1). This locus harbors the Soat1 (sterol O-acyltransferase 1) gene, encoding Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), which esterifies free cholesterol. The parental AKR strain has an exon 2 deletion in Soat1, which leads to a 33 amino acid N-terminal truncation in ACAT1. CRISPR/Cas9 editing of DBA/2 embryonic stem cells was performed to replicate the AKR strain Soat1 exon 2 deletion, while leaving the remainder of the genome unaltered. DBA/2 stem cells and stem cells heterozygous and homozygous for the Soat1 exon 2 deletion were differentiated into macrophages and loaded with acetylated low-density lipoprotein. DBA/2 stem cell-derived macrophages accumulated less free cholesterol and more esterified cholesterol relative to cells heterozygous and homozygous for the Soat1 exon 2 deletion. CONCLUSIONS:A Soat1 deletion present in AKR mice, and resultant N-terminal ACAT1 truncation, was confirmed to be a significant modifier of macrophage cholesterol metabolism. Other Mcmm loci candidate genes were prioritized via bioinformatics.

SUBMITTER: Hai Q

PROVIDER: S-EPMC5746475 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Quantitative Trait Locus Mapping of Macrophage Cholesterol Metabolism and CRISPR/Cas9 Editing Implicate an ACAT1 Truncation as a Causal Modifier Variant.

Hai Qimin Q Ritchey Brian B Robinet Peggy P Alzayed Alexander M AM Brubaker Greg G Zhang Jinying J Smith Jonathan D JD

Arteriosclerosis, thrombosis, and vascular biology 20171102 1

<h4>Objective</h4>Cholesterol metabolism is a dynamic process involving intracellular trafficking, cholesterol esterification, and cholesterol ester hydrolysis. Our objective was to identify genes that regulate macrophage cholesterol metabolism.<h4>Approaches and results</h4>We performed quantitative trait loci mapping of free and esterified cholesterol levels and the ratio of esterified to free cholesterol in acetylated low-density lipoprotein-loaded bone marrow-derived macrophages from an AKR× ...[more]

PMID: 29097366

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Project description:BackgroundWe have investigated the efficacy of a new strategy to limit pathological retinal neovascularization (RNV) during ischemic retinopathy by targeting the cholesterol metabolizing enzyme acyl-coenzyme A: cholesterol transferase 1 (ACAT1). Dyslipidemia and cholesterol accumulation have been strongly implicated in promoting subretinal NV. However, little is known about the role of cholesterol metabolism in RNV. Here, we tested the effects of inhibiting ACAT1 on pathological RNV in the mouse model of oxygen-induced retinopathy (OIR).MethodsIn vivo studies used knockout mice that lack the receptor for LDL cholesterol (LDLR-/-) and wild-type mice. The wild-type mice were treated with a specific inhibitor of ACAT1, K604 (10 mg/kg, i.p) or vehicle (PBS) during OIR. In vitro studies used human microglia exposed to oxygen-glucose deprivation (OGD) and treated with the ACAT1 inhibitor (1 μM) or PBS.ResultsAnalysis of OIR retinas showed that increased expression of inflammatory mediators and pathological RNV were associated with significant increases in expression of the LDLR, increased accumulation of neutral lipids, and formation of toxic levels of cholesterol ester (CE). Deletion of the LDLR completely blocked OIR-induced RNV and significantly reduced the AVA. The OIR-induced increase in CE formation was accompanied by significant increases in expression of ACAT1, VEGF and inflammatory factors (TREM1 and MCSF) (p < 0.05). ACAT1 was co-localized with TREM1, MCSF, and macrophage/microglia makers (F4/80 and Iba1) in areas of RNV. Treatment with K604 prevented retinal accumulation of neutral lipids and CE formation, inhibited RNV, and decreased the AVA as compared to controls (p < 0.05). The treatment also blocked upregulation of LDLR, ACAT1, TREM1, MCSF, and inflammatory cytokines but did not alter VEGF expression. K604 treatment of microglia cells also blocked the effects of OGD in increasing expression of ACAT1, TREM1, and MCSF without altering VEGF expression.ConclusionsOIR-induced RNV is closely associated with increases in lipid accumulation and CE formation along with increased expression of LDLR, ACAT1, TREM1, and MCSF. Inhibiting ACAT1 blocked these effects and limited RNV independently of alterations in VEGF expression. This pathway offers a novel strategy to limit vascular injury during ischemic retinopathy.

Dataset Information

Quantitative Trait Locus Mapping of Macrophage Cholesterol Metabolism and CRISPR/Cas9 Editing Implicate an ACAT1 Truncation as a Causal Modifier Variant.

Publications

Quantitative Trait Locus Mapping of Macrophage Cholesterol Metabolism and CRISPR/Cas9 Editing Implicate an ACAT1 Truncation as a Causal Modifier Variant.

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