Deficiency of ASGR1 in pigs recapitulates reduced risk factor for cardiovascular disease in humans.

Dengke Pan,Minjie Du,Baocai Xie,Shaoping Deng,Xiaochen Shi,Yan Li,Xiangyang Xing,Jia Zhou,Fernando Alvarez,Gregory S Barsh,Liang Bai,Jiangwei Wu,Enqi Liu,Mingzhou Li,Grant A Mitchell,Bo Xia,Long Jin

doi:10.1371/journal.pgen.1009891

Abstract

Genetic variants in the asialoglycoprotein receptor 1 (ASGR1) are associated with a reduced risk of cardiovascular disease (CVD) in humans. However, the underlying molecular mechanism remains elusive. Given the cardiovascular similarities between pigs and humans, we generated ASGR1-deficient pigs using the CRISPR/Cas9 system. These pigs show age-dependent low levels of non-HDL-C under standard diet. When received an atherogenic diet for 6 months, ASGR1-deficient pigs show lower levels of non-HDL-C and less atherosclerotic lesions than that of controls. Furthermore, by analysis of hepatic transcriptome and in vivo cholesterol metabolism, we show that ASGR1 deficiency reduces hepatic de novo cholesterol synthesis by downregulating 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and increases cholesterol clearance by upregulating the hepatic low-density lipoprotein receptor (LDLR), which together contribute to the low levels of non-HDL-C. Despite the cardioprotective effect, we unexpectedly observed mild to moderate hepatic injury in ASGR1-deficient pigs, which has not been documented in humans with ASGR1 variants. Thus, targeting ASGR1 might be an effective strategy to reduce hypercholesterolemia and atherosclerosis, whereas further clinical evidence is required to assess its hepatic impact.

Highlights

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide with a major risk factor of elevated plasma levels of non-high-density lipoprotein cholesterol [1]
Based on the finding that variant asialoglycoprotein receptor 1 (ASGR1) in humans is associated with reduced levels of nonHDL-C and coronary artery disease (CAD) [15], we generated an ASGR1 knockout pig model using the CRISPR/Cas9 gene-editing approach
Deficiencies of ASGR1 significantly reduce plasma non-high-density lipoprotein cholesterol (HDL-C) and TG levels, and ASGR1+/- pigs were strongly protected against high-fat and high-cholesterol (HFHC) diet-induced atherosclerosis

Summary

Introduction

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide with a major risk factor of elevated plasma levels of non-high-density lipoprotein cholesterol (nonHDL-C) [1]. HMGCR, NPC1L1 and PCSK9 are well-established clinical targets for hypercholesterolemia [8,9,10,11] Mutations in these genes, despite conferring high risk, are rare and explain only a small portion of familial clustering of hypercholesterolemia [12], implying that other genetic factors remain to be identified [13]. Two loss-offunction variants in ASGR1 (12 bp deletion in the fourth intron that causes a frameshift mutation and a premature stop codon [carried by 1 in 120 persons], and a 4 bp insertion that introduces a stop codon at position 158 in the protein [carried by 1 in 1850 persons]) were identified in an Iceland population, and ASGR1 haploinsufficiency was associated with reduced levels of non-HDL-C and a reduced risk of coronary artery disease (CAD) [15].

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Results

Conclusion