Abstract
BackgroundThe accumulation of lipid-laden macrophages, foam cells, within sub-endothelial intima is a key feature of early atherosclerosis. Siglec-E, a mouse orthologue of human Siglec-9, is a sialic acid binding lectin predominantly expressed on the surface of myeloid cells to transduce inhibitory signal via recruitment of SH2-domain containing protein tyrosine phosphatase SHP-1/2 upon binding to its sialoglycan ligands. Whether Siglec-E expression on macrophages impacts foam cell formation and atherosclerosis remains to be established.MethodsApoE-deficient (apoE−/−) and apoE/Siglec-E-double deficient (apoE−/−/Siglec-E−/−) mice were placed on high fat diet for 3 months and their lipid profiles and severities of atherosclerosis were assessed. Modified low-density lipoprotein (LDL) uptake and foam cell formation in wild type (WT) and Siglec-E−/−- peritoneal macrophages were examined in vitro. Potential Siglec-E-interacting proteins were identified by proximity labeling in conjunction with proteomic analysis and confirmed by coimmunoprecipitation experiment. Impacts of Siglec-E expression and cell surface sialic acid status on oxidized LDL uptake and signaling involved were examined by biochemical assays.ResultsHere we show that genetic deletion of Siglec-E accelerated atherosclerosis without affecting lipid profile in apoE−/− mice. Siglec-E deficiency promotes foam cell formation by enhancing acetylated and oxidized LDL uptake without affecting cholesterol efflux in macrophages in vitro. By performing proximity labeling and proteomic analysis, we identified scavenger receptor CD36 as a cell surface protein interacting with Siglec-E. Further experiments performed in HEK293T cells transiently overexpressing Siglec-E and CD36 and peritoneal macrophages demonstrated that depletion of cell surface sialic acids by treatment with sialyltransferase inhibitor or sialidase did not affect interaction between Siglec-E and CD36 but retarded Siglec-E-mediated inhibition on oxidized LDL uptake. Subsequent experiments revealed that oxidized LDL induced transient Siglec-E tyrosine phosphorylation and recruitment of SHP-1 phosphatase in macrophages. VAV, a downstream effector implicated in CD36-mediated oxidized LDL uptake, was shown to interact with SHP-1 following oxidized LDL treatment. Moreover, oxidized LDL-induced VAV phosphorylation was substantially lower in WT macrophages comparing to Siglec-E−/− counterparts.ConclusionsThese data support the protective role of Siglec-E in atherosclerosis. Mechanistically, Siglec-E interacts with CD36 to suppress downstream VAV signaling involved in modified LDL uptake.
Highlights
The accumulation of lipid-laden macrophages, foam cells, within sub-endothelial intima is a key feature of early atherosclerosis
To confirm the increased lipid accumulation in aortic lesions in apoE−/−/SE−/−mice is resulted from enhanced foam cell formation, the peritoneal macrophages were isolated from high-fat diet (HFD)-fed apoE−/− and apoE−/−/SE−/− mice and stained with oil red dye (ORD)
When the immunoprecipitated proteins were subjected to Western blot analysis, the results showed that both wild type (WT) and mutant HA-SE were coeluted with Flag-CD36, suggesting that sialic acid is not involved in the interaction between CD36 and Siglec-E (Fig. 4a)
Summary
The accumulation of lipid-laden macrophages, foam cells, within sub-endothelial intima is a key feature of early atherosclerosis. The monocytes differentiate into macrophages and uptake modified LDLs to become foam cells with a feature of intracellular cholesterol accumulation as lipid droplets, which is the hallmark of early lesion [2,3,4] These foam cells in turn produce more inflammatory cytokines and growth factors to promote the migration of the quiescent vascular smooth muscle cells (VSMCs) in the medial layer to intima and activate VSMC proliferation and increased synthesis of extracellular matrix proteins, resulting in the thickening of intima and occlusion of the blood vessel [2,3,4]. At the advanced stage foam cells are unable to fully process the internalized cholesterol and eventually die, causing the deposition of cholesterol and the formation of lipid necrotic core in the arterial wall This triggers further immune reaction to exacerbate the disease progression and provoke coronary rupture underlying heart attack [2,3,4]. It is apparent that macrophage-derived foam cells play a key role in the initiation and progression of atherosclerosis
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