Macrophage Sortilin Promotes LDL Uptake, Foam Cell Formation, and AtherosclerosisNovelty and Significance
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.
Atherosclerotic cardiovascular disease is the leading cause of morbidity and mortality in the world. A central hallmark of atherosclerosis is the cholesterol-loaded macrophage or foam cell. Despite decades of research, the molecular mechanisms by which arterial macrophages take up cholesterol-rich lipoproteins, such as low-density lipoprotein (LDL), leading to the development of foam cells and atherosclerotic lesions, remain to be fully elucidated. Kruth and colleagues have shown that macrophages internalize native LDL through a process of macropinocytosis, although LDL uptake cannot be fully accounted for by this process.1 Gene deletion of known receptors of modified LDL, such as scavenger receptor A and CD36, do not reduce foam cell formation or the development of atherosclerosis in mice.2 Thus, pathways that mediate macrophage uptake of LDL, leading to foam cell formation and atherosclerosis, remain of substantial interest.
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Unbiased genome-wide association studies of coronary artery disease have the potential to identify new pathways involved in atherosclerosis. In one of the first genome-wide association studies for coronary artery disease, noncoding genetic variants at chromosome 1p13 were reported to be significantly associated with myocardial infarction and coronary artery disease,3 a finding that has been widely replicated.4,5 The same variants have also shown to be significantly associated with plasma levels of LDL cholesterol.6–8 The SORT1 gene, encoding the protein sortilin, seems to be the causal gene at the locus regulating LDL cholesterol levels.9–11 Sortilin is a type I transmembrane trafficking receptor initially characterized by its ability to serve as a receptor for proneurotrophins9–11 and for its role as a sorting receptor for lysosomal hydrolases.12,13 Hepatic sortilin expression modulates VLDL (very low-density lipoprotein) production rates7,9,10; in addition, hepatic sortilin binds LDL and promotes its cellular uptake and lysosomal degradation.10 Sortilin is also expressed in macrophages, but little is known about its function in this cell type or its relationship to atherosclerosis.14,15 We hypothesized that macrophage sortilin mediates macrophage LDL uptake. Through a combination of in vivo mouse studies and ex vivo macrophage studies using Sort1−/− macrophages, we show here that macrophage sortilin promotes macrophage LDL uptake, foam cell formation, and atherosclerosis independent of plasma LDL-C levels.
Detailed descriptions of all Methods can be found in the Online Data Supplement. Following is a summary of the key experimental approaches.
For studies of the effect of total body sortilin deficiency on atherosclerosis, we used the Apobec1−/−; hAPOB transgenic mouse, in which the human apolipoprotein B (apoB) transgene is overexpressed in the liver and, in contrast to the wild-type mouse, is not edited, thus producing only the apoB-100 protein.16–18 These mice were crossed with total body Sort1−/− mice and experiments compared Sort1−/−;Apobec1−/−; hAPOB transgenic mice to Sort1+/+;Apobec1−/−; hAPOB transgenic littermates. Mice were fed a western-type diet for 18 weeks and assessed for atherosclerosis in the aortic roots and the entire aorta by en face quantification. A detailed description of the atherosclerosis methods can be found in the Online Data Supplement.
For studies of hematopoietic sortilin deficiency on atherosclerosis, we transplanted donor Sort1−/−;Ldlr−/− and Sort1+/+;Ldlr−/− bone marrow into irradiated recipient Ldlr−/− mice. Six weeks post bone marrow transplantation, the mice were placed on a western diet for 18 weeks and then assessed for atherosclerosis.
For studies of macrophage LDL uptake, both thioglycollate-elicited peritoneal macrophages and bone marrow–derived macrophages were used. The macrophages were incubated with 125I-LDL for 5 hours and uptake and degradation were assessed.
Statistical analyses were done using 2-tailed paired Student t test and 1-way ANOVA with a Bonferroni correction (for lipopolysaccharide experiment).
Sortilin Deficiency in Hematopoetic Cells Protects Against Atherosclerosis
Total body Sort1 deficiency on an Ldlr−/− background is associated with reduced plasma cholesterol levels, confounding attempts to address its role in atherosclerosis independent of LDL-C levels. We crossed Sort1−/− mice onto the background of an atherosclerosis-prone Apobec1−/−; hAPOB transgenic mouse model, which has a human-like lipoprotein profile, and fed the mice a western-type diet for 18 weeks. On this genetic background, total and LDL cholesterol levels were not different in Sort1−/− mice compared with Sort1+/+ mice (Figure 1A and 1B). After 18 weeks on diet, Sort1−/− mice had a 68% reduction in en face aorta lesion area (P<0.0001; Figure 1C and 1D) and an 87% reduction in aortic root lesion area (P<0.0001; Figure 1E and 1F) compared with Sort1+/+ mice, demonstrating a major effect of sortilin deficiency in reducing atherosclerosis, despite no effect on plasma cholesterol in this model.
Macrophages express sortilin, and we hypothesized that macrophage sortilin deficiency might account specifically for the reduced atherosclerosis. To test this hypothesis, irradiated Ldlr−/− mice were transplanted with bone marrow from Sort1−/−;Ldlr−/− mice or Sort1+/+;Ldlr−/− mice and 6 weeks after transplantation were started on a western-type diet and fed for 18 weeks. Bone marrow engraftment was 74% (Online Figure IA). Body, liver, and spleen weights, plasma cholesterol, peripheral blood counts, and hepatic Sort1 expression were similar between groups (Online Figure I). Mice transplanted with Sort1−/−;Ldlr−/− bone marrow had a 69% reduction in en face aortic lesion area (P<0.00001) and a 34% reduction in aortic root lesion area (P<0.01) compared with mice transplanted with Sort1+/+;Ldlr−/− bone marrow (Figure 2A–2D), suggesting that hematopoietic and potentially macrophage sortilin influences the development of atherosclerotic disease.
Sortilin Deficiency Has no Effect on Thioglycollate-Elicited Monocyte Recruitment or Lipopolysaccharide-Induced Inflammatory Response In Vivo
Monocyte recruitment is a key determinant of the macrophage content of atherosclerotic lesions. To determine whether Sort1 deficiency affects macrophage recruitment, Sort1−/− and Sort1+/+ mice were injected IP with thioglycollate to elicit an inflammatory response. Three days after injection, peritoneal macrophages were harvested and counted. There was no difference in macrophage counts between Sort1+/+ and Sort−/− mice (Online Figure IIA). Monocyte recruitment and atherosclerosis development is strongly influenced by inflammation and cytokine production. To determine whether Sort1−/− mice have reduced cytokine levels, cytokine multiplexing assays were performed on Sort1−/− and Sort1+/+ mice injected with lipopolysaccharide. Cytokine levels post lipopolysaccharide injection were found to be similar between Sort1−/− and Sort1+/+ (Online Figure IIB and IIC).
Macrophage Sortilin Deficiency Reduces LDL Uptake and Foam Cell Formation
To determine whether macrophage sortilin deficiency reduces foam cell formation, primary bone marrow macrophages were isolated from Sort1−/−;Ldlr−/− and control Sort1+/+;Ldlr−/− mice, cells were differentiated with M-CSF (macrophage colony stimulating factor) for 7 days, incubated with 1 mg/mL LDL for 5 hours, and Oil Red O staining was performed. Sort1−/−;Ldlr−/− macrophages had a clear and consistent reduction in Oil Red O staining (Figure 3A). Sort1-deficient macrophages had a significant 28% reduction in total cellular cholesterol, a 25% reduction in free cholesterol, and a 32% reduction in cholesteryl ester (P<0.05; Figure 3B–3D). In vivo foam cell formation assays were performed by isolating thioglycollate-elicited peritoneal macrophages from Sort1+/+;Apobec−/−;hApob transgenic and Sort1−/−;Apobec−/−;hApob transgenic mice fed a western-type diet for 18 weeks. Consistent with the in vitro loading experiments, macrophages isolated from Sort1−/− mice had reduced Oil Red O staining and a significant 33% reduction in cellular cholesterol content compared with macrophages isolated from Sort1+/+ mice (P<0.05; Figure 3E and 3F). These studies indicated that sortilin-deficient macrophages have reduced capacity to form foam cells when exposed to high levels of LDL.
As sortilin can act as a receptor for LDL in hepatocytes, we hypothesized that sortilin promotes the internalization of LDL by macrophages. To test the response of sortilin expression to increasing cholesterol concentration in macrophages, thioglycollate-elicited peritoneal macrophages were isolated from wild-type mice and incubated for 24 hours in lipoprotein-deficient serum, lipoprotein-deficient serum supplemented with 25-hydroxycholesterol to reduce intracellular cholesterol content, or with lipoprotein deficient serum supplemented with high concentrations of LDL. In contrast to the LDL receptor, whose expression was reduced by coincubation with LDL, Sort1 mRNA abundance increased over 400-fold with LDL incubation (P<0.05; Figure 4A) and sortilin protein also increased significantly with LDL incubation (Figure 4B).
To test the hypothesis that sortilin is able to promote the uptake of LDL into macrophages, 125I-LDL uptake studies were performed in thioglycollate-elicited and bone marrow–derived macrophages from Sort1+/+ and Sort1−/− mice. Sort1 deficiency was associated with a 48% and 33% reduction in LDL uptake, respectively (P<0.05 for both; Figure 4C and 4D). We next tested whether this effect on LDL uptake was independent of the LDL receptor. Bone marrow–derived macrophages were isolated from Sort1+/+;Ldlr−/− and Sort1−/−;Ldlr−/− mice, and 125I-LDL uptake studies were performed. Sort1 deficiency was associated with a 39% reduction in LDL uptake in the absence of the LDLR (P<0.05; Figure 4F).
To further confirm that sortilin deficiency confers atheroprotection by eliminating a receptor-dependent pathway for LDL uptake and not by modulating macropinocytosis, LDL uptake studies were performed in bone marrow–derived macrophages in the presence of cytochalasin D, a potent inhibitor of actin polymerization and macropinocytosis. Under these conditions, although LDL uptake is reduced, substantial residual LDL uptake still takes place.1 Sort1 deficiency was associated with a 38% reduction in LDL uptake in the presence of cytochalasin D (P<0.05; Figure 4F). These studies indicate that macrophage sortilin deficiency reduces macrophage uptake of LDL and formation of foam cells, and this effect is independent of the LDL receptor and of macropinocytosis.
Finally, to determine whether increased macrophage sortilin results in increased LDL uptake, J774 cells were transduced with lentivirus encoding green fluorescent protein or Sort1, and LDL uptake studies were performed. Sort1 overexpression in macrophages led to a 29% increase in LDL uptake (P<0.05; Figure 4E).
Genetic variation at the 1p13 SORT1 locus is strongly associated both with coronary artery disease, as well as with plasma LDL-C levels. We have previously shown that sortilin is a cell surface receptor for LDL on hepatocytes, and its elevated expression in liver reduces LDL-C at least in part by facilitating LDL clearance from blood. Sortilin is expressed in macrophages, which actively take up LDL, leading us to investigate the role of macrophage sortilin in LDL uptake, foam cell formation, and atherosclerosis. After a series of studies of atherosclerosis in mice and LDL uptake in macrophages, we conclude that macrophage sortilin promotes LDL uptake, foam cell formation, and atherosclerosis and that deficiency is protective against atherosclerosis at least in part by reducing LDL uptake.
Macrophage uptake of modified LDL can be mediated by scavenger receptors, such as scavenger receptor A and CD36. However, deletion of scavenger receptor A or CD36 does not reduce macrophage uptake of native LDL,1 nor does it ameliorate atherosclerosis in hypercholesterolemic mice.19 Even CD36−/−; SRA−/− mice still contain abundant lipid laden macrophages in vessel wall and develop atherosclerosis.20 Kruth has shown that macrophages can take up native LDL through fluid-phase macropinocytosis, but there remains substantial LDL uptake even when this pathway is inhibited.1 Our data establish macrophage sortilin as the first receptor-mediated pathway of uptake of native LDL, leading to foam cell formation and promoting atherosclerosis development. We also found that increasing concentrations of extracellular LDL causes an upregulation of macrophage Sort1 mRNA and protein. Because a function of macrophages is to phagocytose LDL, it is possible that increased exposure of macrophages to LDL triggers the transcriptional upregulation of sortilin, which then mediates increased LDL uptake. The mechanisms of this upregulation of macrophage Sort1 by LDL require further exploration.
Although this article was under preparation, Mortensen et al reported that sortilin deficiency reduced atherosclerosis in the ApoE−/− mouse model.21 Although the fundamental observation is consistent with our data, these authors suggested a different mechanism, namely that decreased proinflammatory cytokines may have been responsible for the reduced atherosclerosis. We performed our cytokine assays before initiation of atherosclerotic disease, whereas Mortensen et al measured the cytokine profile after disease was present. In addition, these authors did not see a reduction in LDL uptake by sortilin-deficient macrophages, although they used an ATTO dye conjugated to the LDL that may have influenced the interaction with sortilin. We also used a different mouse model, the Apobec1−/−; hAPOB transgenic mouse, in which human apoB-100 containing LDL is the dominant lipoprotein, in a human-like lipoprotein profile, whereas Mortensen et al used the Apoe−/− mouse, which is characterized primarily by mouse apoB-48 containing remnant lipoproteins. Overall, the top-line results of the 2 studies, which used different mouse atherosclerosis models, are highly comparable, whereas the mechanisms responsible for the reduced atherosclerosis may be complex and multifactorial.
In summary, our findings indicate that SORT1 deficiency in macrophages reduces LDL uptake and macrophage cholesterol loading, independent of the LDL receptor or macropinocytosis, and protects against the development of atherosclerosis. The macrophage sortilin pathway is a novel pathway of macrophage cholesterol loading that quantitatively contributes to atherosclerosis.
We thank Aisha Wilson, Edwige Edoard, Mao-Sen Sun, Amrith Rodrigues, and Antonino Picataggi for animal/cell help; Kiran Musunuru and Qiurong Ding for human SORT1 and green fluorescent protein (GFP) plasmids. Deborah Cromley for help with lipid measurements; Margaret Nickel and Kevin Trindade for LDL isolation; Human Immunology Core for running Bioplex Pro Assay; Karen Vo for help with Hemavet; Edward Fisher, Peter Davies, Ellen Pure, Richard Assoian, Sissel Lund-Katz, Michael Phillips, Yanqing Gong, Robert Bauer, Sumeet Khetarpal, and Sony Tuteja-Stevens for scientific discussions.
Sources of Funding
This work was supported by R01HL109489 from the National Heart, Lung, and Blood Institute (D.J. Rader) and T32GM008076-30 from the National Institute of General Medical Sciences (K.M. Patel).
In December 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.47 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.116.305811/-/DC1
- Nonstandard Abbreviations and Acronyms
- apolipoprotein B
- low-density lipoprotein cholesterol
- low-density lipoprotein receptor
- Received December 8, 2014.
- Revision received January 9, 2015.
- Accepted January 15, 2015.
- © 2015 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Genetic variants at the SORT1 locus are associated with plasma low-density lipoprotein cholesterol levels, as well as myocardial infarction and coronary artery disease.
The gene SORT1 encodes a protein called sortilin, which in hepatocytes can mediate the uptake and degradation of low-density lipoprotein.
What New Information Does This Article Contribute?
Deletion of sortilin in macrophages reduces foam cell formation and atherosclerosis without influencing plasma low-density lipoprotein cholesterol levels.
Macrophage sortilin is a new receptor-mediated pathway promoting uptake of native low-density lipoprotein by macrophages promoting foam cell formation and atherosclerosis.
The mechanisms linking genetic variation at the SORT1 locus with coronary artery disease have not been fully elucidated. Using atherosclerosis-prone mice with a human-like lipoprotein profile, we found that deletion of sortilin in the whole body or specifically in macrophages substantially reduced atherosclerosis without affecting plasma cholesterol. We found that macrophage sortilin mediates the uptake of native (unmodified) low-density lipoprotein, leading to foam cell formation, a novel receptor-mediated process for macrophage uptake of low-density lipoprotein. This pathway could potentially be targeted as an approach to reducing risk of atherosclerosis.