Effect Of Deletion Of CUL4B In Myeloid Cells On Atherosclerotic Progression

Backgroud and Objective:Atherosclerosis(AS),as a lipid-driven intimal inflammatory disease,is the main pathological basis of cardiovascular disease.Macrophages are the most abundant subset of white blood cells in atherosclerotic lesions,and their activation determines the inflammatory environment in plaques and is influenced by lipids and their derivatives in the environment.Cullin 4B(CUL4B)protein,a scaffold protein in the Cullin-4B RING E3 ligase complex(CRL4B),is involved in multiple biological processes.Previous studies suggest that CUL4B regulates the differentiation and function of myeloid cells.However,whether CUL4B affects the progression of atherosclerosis via regulating macrophage functions is unclear.In this study,myeloid cell-specific Cul4b gene knockout mice were used to study the role and mechanism of CUL4B in atherosclerosis of ApoE-deficient mice.Methods and Results:1.Western Blotting was used to detect the expression of CUL4B protein in the mouse peritoneal macrophage(PM)and THP-1 cells stimulated by oxidized low density lipoprotein(oxLDL).Western Blotting showed that the expression of CUL4B was upregulated by oxLDL in primary macrophages and THP-1 cells in a time and concentration-dependent manner.2.Myeloid cell-specific Cul4b knockout ApoE-deficient mice(Cul4bflox/Y;LysMCre+/-;ApoE-/-)and control mice(Cul4bflox/Y;LysM-Cre-/-;ApoE-/-)were generated and given Western diet for 16 weeks,and their growth status and biochemical indicators such as blood lipids were evaluated.The aortic arch from knoctkout mice and control mice were analyzed using H&E,oil red O,and immunofluorescence staining.The results indicated that depletion of Cul4b in myeloid cells alleviated the pathogenic progression of ApoE-deficient mice,as demonstrated by reduced blood lipid level,plaque area,lipid accumulation as well as infiltration of macrophages.3.Transwell experiment was used to examine the migration ability of macrophages.qRT-PCR and ELISA were used to examine the level of inflammation.These assays showed that depletion of Cul4b inhibited the migration ability and inflammatory response of macrophages.4.Lipidomics was performed on oxLDL-stimulated peritoneal macrophages from knockout and control mice,and showed that depletion of CUL4B in myeloids downregulated hemolytic phosphatidylcholine(LPC).Western Blotting showed that the expression of ER stress-related proteins was downregulated.Conclusions:Depletion of Cul4b in myeloid cells alleviates pathogenic progression of ApoEdeficient mice by inhibiting macrophage migration and alleviating macrophage ER stress and inflammation.