Introduction: 

CLEC4E is a C-type lectin pattern recognition receptor on phagocytotic cells such as monocytes/macrophages. It binds to cellular debris or even cholesterol crystals, and facilitates their incorporation and further intracellular degradation. A recent study has demonstrated the essential protective role of CLEC4E in murine atherosclerosis. We investigated the expression profile of CLEC4E in human patients with aortic valve stenosis (AVS), and its role in phagocytosis of cholesterol crystals. 

Methods and results: 

To find out whether CLEC4E is expressed in human aortic valve stenosis, we first performed immunohistochemical staining with a monoclonal antibody against CLEC4E on stenotic human aortic valves. Aortic valves of patients with aortic valve insufficiency were used as controls. In these experiments human stenotic aortic valves showed increased CLEC4E expression.  To confirm increased CLEC4E expression in human aortic valve stenosis by a second method we additionally performed RT-PCR experiments also illustrating increased CLEC4E expression in stenotic aortic valves. Confocal microscopy of co-stained tissue of human aortic valve stenosis with CLEC4E and markers for smooth muscle cells (ASMAC), endothelial cells (vWF), myocytes (desmin), mesenchymal cells (vimentin) and macrophages (CD68) revealed co-expression of CLEC4E with CD68 verifying CLEC4E to be expressed by macrophages in aortic valve tissue. Next, we investigated CLEC4E expression in circulating monocytes of patients with aortic valve stenosis showing equal expression levels for CLEC4E compared to healthy controls. Flow cytometric measurement of cholesterol crystal phagocytosis detected by switch in sideward scatter revealed significantly increased phagocytosis of cholesterol crystals by CLEC4E positive circulating monocytes compared to CLEC4E negative monocytes. RT-PCR experiments showed lower expression levels of IL-6 an IL-1β in peripheral blood mononuclear cells (PBMC) of patients with aortic valve stenosis compared to healthy controls. 

Conclusions: 

In this study we confirm the presence of CLEC4E in human aortic valve stenosis expressed by macrophages. In contrast CLEC4E expression in blood of patients with aortic valve stenosis showed equal levels compared to controls indicating a specific migration of CLEC4E positive macrophages in stenotic aortic valves or differentiation of macrophages in stenotic aortic valves. Flow cytometric experiments reveled increased phagocytosis of cholesterol crystals by CLEC4E positive monocytes suggesting an important role of CLEC4E in phagocytosis of cholesterol crystals and thereby in development of human aortic valve stenosis. Finally, we observed reduced expression levels of IL-6 and IL-1β in blood of patients with aortic valve stenosis compared to healthy controls most likely by anti-inflammatory processes involved in development of aortic valve stenosis.