Deletion of the asialyloglycoprotein receptor-1 (ASGR1) causes atheroprotective effects in vitro and in vivo.

Abstract

Introduction: The asialoglycoprotein receptor 1 (ASGR1) is a hepatic receptor that clears desialylated glycoproteins from the circulation. A loss-of-function mutation in ASGR1 was reported to associate with a 34% reduction in coronary artery disease (CAD) risk, suggesting a role for ASGR1 in CAD. Aim(s): To determine the role of ASGR1 in atherosclerosis and whether deletion of ASGR1 elicits atheroprotective effects. Method(s): Western blotting, mass spectrometry and flow cytometry assessed ASGR1 expression in cultured macrophages or immune cells collected from the blood and aortas of wildtype mice. Bone marrow-derived macrophages (BMDMs) from wildtype and Asgr1-/- mice were subjected to cholesterol efflux and oxidised low-density lipoprotein (oxLDL) uptake in vitro functionalassays. ELISA and qPCR measured changes in lipid regulators in BMDMs. Asgr1-/- mice were crossed with atherosclerosis-prone apolipoprotein (Apo)e-/- mice (Apoe-/-Asgr1-/-). Stable plaque area was assessed histologically in the aortic sinuses of mice fed a high-cholesterol diet for 8 weeks (n=16/group). Using the tandem stenosis surgical model, unstable plaque was assessed in carotid arteries (n=10/group). Human ASGR1 was measured by ELISA in peripheral blood mononuclear cells (PBMCs) isolated from patient samples (n=18). Result(s): Western blotting and mass spectrometry discovered ASGR1 is expressed in macrophages. Immunofluorescence identified the presence of ASGR1 in aortic sinus plaque. Flow cytometry revealed ASGR1 is expressed in both inflammatory and patrolling monocytes, neutrophils, macrophages and dendritic cells of blood and aortas. Asgr1-/- BMDMs elicited greater cholesterol efflux (+39%, P<0.05) and decreased oxLDL uptake (-15%, P<0.05), compared to wildtype BMDMs. Moreover, Asgr1-/- BMDMs had significantly higher LDL receptor protein levels (+2-fold), and Lxra (+2-fold) and Pparg (+1.5-fold) mRNA levels than wildtype BMDMs, P<0.001. Plasma from Asgr1-/- mice had lower total (-25%, P<0.05) and LDL (-36%, P<0.05) cholesterol concentrations than wildtype mice. Apoe-/-Asgr1-/- mice developed less stable plaque in aortic sinuses (-37%, P<0.001) than Apoe-/- controls, as well as smaller unstable plaques in carotid arteries (-49%, P<0.05). Finally, in a human population, ASGR1 protein levels were higher in PBMCs from patients with coronary plaque (+61%, P<0.05), than those without plaque, determined from coronary angiograms. Conclusion(s): ASGR1 is expressed on monocytes, macrophages and in plaques, and human PBMC ASGR1 associates with coronary plaque. Deletion of ASGR1 causes atheroprotective functions in macrophages in vitro and reduced plaque burden in vivo. Our findings demonstrate ASGR1 is important in atherosclerosis with implications for ASGR1 as a therapeutic target.