The macrophage C-type lectin receptor CLEC5A (MDL-1) expression is associated with early plaque progression and promotes macrophage survival

Abstract

Background: Biomarkers of early plaque progression are still elusive. Myeloid DAP12-associating lectin-1 (MDL-1), also called CLEC5A, is a C-type lectin receptor implicated in the progression of multiple acute and chronic inflammatory diseases. However, the relationship between its level and atherosclerosis is unknown. In this study, we aimed to investigate the correlation between macrophage MDL-1 expression and early atherosclerosis progression.

Methods: Immunofluorescence staining, real-time PCR and western blot were performed to analyze MDL-1 expression in aorta or mice macrophages. The role of MDL-1 in macrophage survival was further investigated by adenovirus infection and TUNEL assay.

Results: Significant MDL-1 expression was found in advanced human and apoE-/- mice atherosclerotic plaques, especially in lesional macrophages. In the model of atherosclerosis regression, we found MDL-1 expression was highly downregulated in lesional macrophages from ldlr-/- mouse regressive plaques, coincident with a reduction in lesional macrophage content and marker of M1 proinflammatory macrophages. Furthermore, we found MDL-1 was significantly expressed in inflammatory M1 subtype polarized bone marrow-derived macrophages. In vitro experiments, the level of MDL-1 was remarkably elevated in macrophages treated with pathophysiological drivers of plaque progression, such as oxidized low-density lipoprotein (ox-LDL) and hypoxia. Mechanistically, we demonstrated that MDL-1 overexpression notably promoted macrophage survival and decreased cleaved caspase-3 expression under ox-LDL stimulation, which suggested that it could maintain lesional macrophage survival and cause its accumulation.

Conclusions: This study firstly demonstrated that MDL-1 is mainly expressed in atherosclerotic lesional macrophages and increased macrophage MDL-1 expression is associated with early plaque progression and promotes macrophage survival.

Keywords: Apoptosis; Atherosclerosis; Macrophage; Myeloid DAP12-associating lectin-1; Plaque progression.

Fig. 1

MDL-1 is mainly expressed by lesional macrophages in human and apoE−/− mice atherosclerotic plaques. a MDL-1 (green) or CD68 (red) were stained in human femoral arterial plaques (top panel) and normal internal thoracic arteries (bottom panel). Areas of colocalization are shown in yellow in the merged image. Images are representative of 3 subjects. b MDL-1 (green) or MOMA-2 (red) were stained in apoE−/− mice aortic root sections from western diet group (top panel) and chow diet group (bottom panel). Areas of colocalization are exhibited in yellow in the merged image. Images are representative of 3 mice

Fig. 2

The serum total and LDL cholesterol levels were significantly lowered in LDLr−/− mice when switching to a chow diet. LDLr−/− mice fed a 8-week HFD were either sacrificed for baseline or switched to a chow diet for another 4 weeks. Total cholesterol and LDL/VLDL cholesterol concentration were decreased and HDL cholesterol was elevated in switching group compared with baseline group mice (n = 4–5 mice/group). *p < 0.05 compared with baseline group mice. LDLr low-density lipoprotein receptor, HFD high-fat diet, LDL/VLDL low-density lipoprotein/very low-density lipoprotein

Fig. 3

MDL-1 expression is downregulated in regressive plaques and has a proinflammatory phenotype in the lesion. a Immunofluorescent staining of MDL-1 (green) and MOMA-2 (red) in aortic sinus plaques in a progressive (ldlr−/− mice fed a 8-week HFD) or regressive (ldlr−/− mice fed a 8-week HFD and then switched to 4-week chow diet) environment. b Immunofluorescent staining of iNOS (M1 subtype macrophage marker, red) or Arg-1 (M2 subtype macrophage marker, red) in aortic sinus plaques both in progressive and regressive context. c Quantification of MDL-1 positive macrophages in a. d Real-time PCR analysis of MDL-1 mRNA in bone marrow-derived macrophages (BMDMs) polarized toward M1, M2, or unpolarized (M0). e Western-blot analysis of aortic lysates from apoE−/− mice fed a 8-week HFD or chow diet. All images are representative of at least three mice per group. Values are expressed as mean ± SEM. *p < 0.05 compared with baseline group mice or unpolarized BMDMs

Fig. 4

MDL-1 expression is regulated by pathophysiological agents implicated in atherosclerotic progression and regression in vitro. Western blot analyses of MDL-1 protein level in C57BL/6 mice peritoneal macrophages treated with a 50 μg/mL ox-LDL for the indicated times or b CoCl₂ at the concentrations indicated for 24 h. Band densitometry are quantified. c Macrophages were stimulated with 50 μg/mL ox-LDL with or without pretreatment with 100 μmol/L HIF-1α inhibitor. d Macrophages were stimulated with 200 μmol/L CoCl₂ with or without pretreatment with 100 μmol/L HIF-1α inhibitor. Band densitometry quantification are shown. e Macrophages were treated with 50 μg/mL ox-LDL with or without pretreatment with 10 μg/mL HDL. Images are representative of three independent experiments. Values are expressed as mean ± SEM. *p < 0.05; ^# p < 0.01

Fig. 5

MDL-1 overexpression promotes macrophage survival under ox-LDL stimulation. a Western-blot analysis of MDL-1 expression in peritoneal macrophages after infected with adenovirus overexpressing MDL-1 (Ad-MDL-1) or control virus (Ad-null). b Macrophage apoptosis (green) measured by TUNEL assay of peritoneal macrophages stimulated with 50 μg/mL ox-LDL with or without pretreatment with Ad-MDL-1 or Ad-null in RPMI 1640 media. Nuclei were costained with DAPI (blue). Quantification of tunel assay from each group (n ≥ 3/group) was performed in (c). Values are expressed as mean ± SEM. *p < 0.05 compared with ox-LDL with Ad-null treatment group; ^# p < 0.01 compared with PBS or Ad-null group

Fig. 6

MDL-1 overexpression reduces apoptosis-associated molecule expression in macrophages under ox-LDL stimulation. a Representative Western blot analyses of cleaved caspase-3 and caspase-3 expression in cell lysates from peritoneal macrophages treated with 50 μg/mL ox-LDL with or without pretreatment of adenovirus overexpressing MDL-1 (Ad-MDL-1) or control virus (Ad-null) in RPMI 1640 media. Images are representative of three independent experiments. Quantification of band densitometry is shown in b. Values are expressed as mean ± SEM. *p < 0.05 compared with Ad-null or ox-LDL with Ad-null treatment group