TLR4-mediated macropinocytosis promotes smooth muscle cell- derived foam cell formation

Abstract Objective: To investigate whether vascular smooth muscle cells (VSMCs) take up lipids via the Toll-like receptor 4/spleen tyrosine kinase (TLR4/Syk) mediated macropinocytosis pathway and promote the formation of lipid droplets in cells. Methods: Primary wild-type (WT) and TLR4 gene-knockout (TLR4 −/− ) VSMCs were isolated by an adherent tissue culture method. Changes in VSMCs uptake of DiI-labelled native low-density lipoprotein (DiI-nLDL) and Lucifer yellow (LY) were detected by flow cytometry. The colocalization of boron-dipyrromethene (BODIPY)-labelled Neutral lipid and LY-labelled macropinosomes in VSMCs was observed by confocal microscopy. Results: Lipopolysaccharide (LPS)induced nLDL uptake by WT VSMCs, resulting in the accumulation of lipid droplets and the formation of WT VSMC-derived foam cells. Stimulation of WT VSMCs with 200 ng/ml LPS resulted in increase in LY and DiI-nLDL uptake, lipid droplets and macropinocytosis were colocalized in WT VSMCs, but this above effect was significantly reduced in WT VSMCs after treatment with macropinocytosis inhibitors, and was not observed in TLR4 −/− VSMCs. WT VSMCs showed a decrease in LY and DiI-nLDL uptake after treatment with 2 µmol/L R788. Conclusion: LPS induces nLDL uptake by VSMCs via the macropinocytosis pathway to promote lipid droplet aggregation and the formation of VSMC-derived foam cells. TLR4/Syk is an important molecule associated with VSMC uptake of nLDL through the macropinocytosis pathway.