한빛사논문
WonMo Ahn 1,#, Faith N. Burnett 1,#, Kamila Wojnar-Lason 1, Jaser Doja 1, Amritha Sreekumar 1, Pushpankur Ghoshal 1,$, Bhupesh Singla 1,†, Graydon Gonsalvez 3, Ryan A. Harris 4, Xiaoling Wang 4, Joseph Miano 1, Gábor Csányi 1,2
1Vascular Biology Center, Augusta University, Medical College of Georgia, Augusta, GA, 30912, USA
2Department of Pharmacology and Toxicology, Augusta University, Medical College of Georgia, Augusta, GA, 30912, USA
3Department of Cellular Biology & Anatomy, Augusta University, Medical College of Georgia, Augusta, GA, 30912, USA
4Georgia Prevention Institute, Augusta University, Medical College of Georgia, Augusta, GA, 30912, USA
$Present address: Maryland Cannabis Administration, Linthicum, MD, 21090, USA.
†Present address: Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN, 38163, USA.
#Authors contributed to the manuscript equally.
Corresponding Author: Gábor Csányi
Abstract
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+ Nhe1f/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 (O2⋅−) 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.
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