Macrophage sortilin promotes LDL uptake, foam cell formation, and atherosclerosis

Abstract

Rationale: Noncoding gene variants at the SORT1 locus are strongly associated with low-density lipoprotein cholesterol (LDL-C) levels, as well as with coronary artery disease. SORT1 encodes a protein called sortilin, and hepatic sortilin modulates LDL metabolism by targeting apolipoprotein B-containing lipoproteins to the lysosome. Sortilin is also expressed in macrophages, but its role in macrophage uptake of LDL and in atherosclerosis independent of plasma LDL-C levels is unknown.

Objective: To determine the effect of macrophage sortilin expression on LDL uptake, foam cell formation, and atherosclerosis.

Methods and results: We crossed Sort1(-/-) mice onto a humanized Apobec1(-/-); hAPOB transgenic background and determined that Sort1 deficiency on this background had no effect on plasma LDL-C levels but dramatically reduced atherosclerosis in the aorta and aortic root. To test whether this effect was a result of macrophage sortilin deficiency, we transplanted Sort1(-/-);LDLR(-/-) or Sort1(+/+);LDLR(-/-) bone marrow into Ldlr(-/-) mice and observed a similar reduction in atherosclerosis in mice lacking hematopoetic sortilin without an effect on plasma LDL-C levels. In an effort to determine the mechanism by which hematopoetic sortilin deficiency reduced atherosclerosis, we found no effect of sortilin deficiency on macrophage recruitment or lipopolysaccharide-induced cytokine release in vivo. In contrast, sortilin-deficient macrophages had significantly reduced uptake of native LDL ex vivo and reduced foam cell formation in vivo, whereas sortilin overexpression in macrophages resulted in increased LDL uptake and foam cell formation.

Conclusions: Macrophage sortilin deficiency protects against atherosclerosis by reducing macrophage uptake of LDL. Sortilin-mediated uptake of native LDL into macrophages may be an important mechanism of foam cell formation and contributor to atherosclerosis development.

Keywords: atherosclerosis; foam cell; low-density lipoprotein cholesterol; macrophage; receptor-mediated endocytosis.

Figure 1. Whole body Sort1 deficiency reduces atherosclerosis independent of plasma lipid level

A. Eight week old Apobec −/−; hApob Tg and Sort −/−;Apobec −/−; hApobTg mice (n=10 per group) placed on a western diet for 18 weeks and retroorbital bleeds were performed after a 4 hr fast. Plasma was isolated by centrifugation and samples were run individually on a Cobas Mira Autoanalyzer (Roche Diagnostic Systems). B. Samples were pooled and run on FPLC. C. Whole aortas were dissected and tissues harvested. Aortas were stained with Oil Red O. D. Quantification of lesions on aorta E. Aortic roots were sectioned & stained with hematoxylin eosin (H&E) F. Quantification of atherosclerotic lesion area at aortic root P value <0.01

Figure 2. Sort1 hematopoietic deficiency reduces atherosclerosis

A. After 18 weeks on western diet recipient Ldlr−/− mice carrying donor Ldlr−/− or Sort1−/−;Ldlr−/− (n=11 per group) bone marrow transplanted mice aortas were dissected and stained with Oil Red O B. Quantification of lesions on aorta P<0.00001 C. Representative aortic roots stained with H&E D. Quantification of aortic roots P value <0.01

Figure 3. Sort1 deficiency reduces macrophage cellular cholesterol levels and Oil Red O staining in vivo and in vitro

A. Bone marrow-derived macrophages were stained with Oil Red O (n=3) B. Cellular total cholesterol C. Free cholesterol D. Cholesterol ester measured by gas chromatography (GC/MS). E. Peritoneal macrophages from mice fed a western diet for 18 weeks were isolated and stained with Oil Red O F. Cellular total cholesterol of peritoneal macrophages measured by GC/MS. All experiments were replicated.

Figure 4. LDL loading increases sortilin in macrophages, where it mediates uptake of LDL

A. Thio-elicited peritoneal macrophages were incubated in conditions listed above. RNA expression and B. Westerns C. I-125 LDL uptake study from thioglycollate-elicited macrophages from Sort1 +/+ and Sort1 −/− mice (n=6) D. I-125 LDL uptake study from bone marrow derived macrophages from Sort1+/+ and Sort1−/− mice (n=6)”. and F. M-CSF differentiated macrophages from Ldlr −/− and Sort1−/−; Ldlr −/− mice. 250ug/ml LDL was incubated and 4ug/ml cytochalasin D (as indicated) for 5 hours (n=6).