Activation of receptor-independent fluid-phase pinocytosis promotes foamy monocyte formation in atherosclerotic mice

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of death worldwide. Clinical and experimental data demonstrated that circulating monocytes internalize plasma lipoproteins and become lipid-laden foamy cells in hypercholesterolemic subjects. This study was designed to identify the endocytic mechanisms responsible for foamy monocyte formation, perform functional and transcriptomic analysis of foamy and non-foamy monocytes relevant to ASCVD, and characterize specific monocyte subsets isolated from the circulation of normocholesterolemic controls and hypercholesterolemic patients. We hypothesized that activation of fluid-phase macropinocytosis contributes to foamy monocyte formation in vitro and in hypercholesterolemic mice in vivo . High resolution scanning electron microscopy (SEM) and quantification of FITC/TRITC-dextran internalization demonstrated macropinocytosis stimulation in human (THP-1) and wild type murine monocytes. Stimulation of macropinocytosis induced foamy monocyte formation in the presence of unmodified, native LDL (nLDL) and oxidized LDL (ox-LDL) in vitro . Genetic blockade of macropinocytosis ( LysMCre + Nhe1 f/f ) inhibited foamy monocyte formation in hypercholesterolemic mice in vivo and attenuated monocyte adhesion to atherosclerotic aortas ex vivo . Mechanistic studies identified NADPH oxidase 2 (Nox2)-derived superoxide (O 2 ⋅− ) as an important downstream signaling molecule stimulating macropinocytosis in monocytes. qRT-PCR identified CD36 as a major SR that increases in response to lipid loading in monocytes and deletion of CD36 ( Cd36 -/- ) inhibited foamy monocyte formation in hypercholesterolemic mice. Bulk RNA-sequencing characterized transcriptional differences between non-foamy and foamy monocytes versus macrophages. Finally, flow cytometry analysis of CD14 and CD16 expression demonstrated a significant increase in intermediate monocytes in hypercholesterolemic patients compared to normocholesterolemic controls. These results provide novel insights into the mechanisms of foamy monocyte formation and potentially identify new therapeutic targets for the treatment of atherosclerosis. • Stimulation of macropinocytosis in human and murine monocytes promotes nLDL and ox-LDL internalization in vitro , leading to foamy monocyte formation. • Circulating monocytes utilize macropinocytosis and CD36 to internalize plasma lipoproteins in hypercholesterolemic mice in vivo. • Genetic blockade of macropinocytosis inhibits foamy monocyte formation and their adhesion to atherosclerotic arteries. • Activation of NOX2 stimulates macropinocytosis in monocytes.