PROJECT SUMMARY Atherosclerosis, the underlying cause of heart attack, stroke and peripheral arterial disease, is the leading cause of death in the United States. A better understanding of the pathomechanisms of atherosclerosis is critical to identify more effective therapeutic strategies to treat atherosclerosis. Recent studies demonstrated that majority of the circulating monocyte pool in patients with atherosclerosis are lipid-laden foamy monocytes and pharmacological depletion of foamy monocytes in hypercholesterolemic mice attenuates atherosclerosis development. Although these studies suggest that monocyte uptake of plasma LDL is a therapeutic target in atherosclerosis, the mechanism of foamy monocyte formation is currently unknown. Preliminary studies demonstrate for the first time that chemical stimulation of macropinocytosis promotes native LDL uptake, leading to foamy monocyte formation in vitro. Moreover, pharmacological inhibition of macropinocytosis and genetic deletion of the macropinocytosis regulator, NHE1, selectively in myeloid cells attenuates atherosclerosis development in hypercholesterolemic mice. The central hypothesis of this proposal is that stimulation of macropinocytosis promotes monocyte lipid uptake and foamy monocyte formation. Aim 1 will test the hypothesis that physiologically relevant stimulators of macropinocytosis increase foamy monocyte formation in vitro. We will assess the ability of human primary and THP1 monocytes to uptake lipids in the presence or absence of physiologically relevant macropinocytosis stimulators and pharmacological inhibitors. The relative contribution of scavenger receptor-mediated lipid uptake vs. macropinocytosis will be quantified using monocytes from wild type, CD36-/-, SRA-/- and CD36-/-/SRA-/- mice. Finally, we will assess phenotypic and functional changes in foamy monocytes relevant to the pathogenesis of atherosclerosis. Aim 2 will test the hypothesis that myeloid cell- specific deletion of the macropinocytosis regulator, NHE1, inhibits foamy monocyte formation in vivo. Monocytes from NHE1f/f Lysm Cre+ and littermate Cre- mice injected with AAV8-PCSK9 will be isolated and analyzed for lipid content, inflammatory cytokine secretion, inflammatory markers, and adhesion ability. Additionally, we will use wild type and CD36-/-/SRA-/- mice to assess the effect of a repurposed FDA-approved drug that inhibits macropinocytosis in attenuating circulating foamy monocyte levels. This project will allow me to develop new technical skills, gain expertise in conducting rigorous, hypothesis-driven research and improve my independent skills. The project will be conducted under the mentorship of Dr. Gabor Csanyi and Dr. Neal Weintraub in the Vascular Biology Center at the Medical College of Georgia, Augusta University, which has a rich history of successful pre- and post-doctoral training. The proposed project is for 3 years of funding with the aims divided amongst the 3 years of funding, culminatin...