SMC macropinocytosis: a novel target in atherosclerotic vascular disease

Project Summary Atherosclerosis is a leading cause of morbidity and mortality globally. Recently, cell lineage tracing, single-cell RNA sequencing and human genomic studies have been integrated to demonstrate that a) majority of plaque foam cells are of vascular smooth muscle cell (SMC) origin and b) SMC can undergo a fate switch to transitional, multipotential cells that can adopt plaque altering phenotypes. Although these results identify SMC as potential therapeutic targets, most current treatments for atherosclerosis have little direct impact on SMC. The endocytic processes by which SMC take up lipids and become foam cells in the arterial wall are not clearly defined. In addition, the mechanisms underlying SMC phenotypic switching in the arterial wall remain largely unknown. Using a combination of high-resolution imaging, 3D cell reconstruction, and LDL immunolabeling, we have recently demonstrated that foam cells (lineage unknown) in human and murine atherosclerotic plaques internalize LDL via macropinocytosis. Novel preliminary data using SMC lineage tracing identifies SMC subsets performing macropinocytosis in the arterial wall in vivo. Stimulation of macropinocytosis of LDL in SMC promotes dedifferentiation and phenotype switching into plaque-promoting phenotypes in vitro. Further, pharmacological blockade of macropinocytosis using the Na+/H+ exchanger 1 (NHE1) blocker, EIPA, inhibits LDL uptake in atherosclerotic arteries and abrogates atherosclerosis development in multiple murine models of atherosclerosis. Preliminary data also show that the matricellular protein thrombospondin-1 (TSP1) stimulates macropinocytosis via CD47 in SMC, leading to foam cell formation and phenotypic switching, and global Cd47-/- mice are protected from atherosclerosis. Based on these observations, we hypothesize that SMC macropinocytosis drives atherosclerosis through foam cell formation and regulation of SMC phenotypic switching. The hypothesis will be tested via the following specific aims: Aim 1: SMC internalize LDL via macropinocytosis in atherosclerotic arteries. Aim 2: SMC-specific inhibition of macropinocytosis attenuates transdifferentiation of SMC into a plaque- promoting phenotype and inhibits atherosclerosis. Aim 3: TSP1 via CD47 stimulates SMC macropinocytosis, promotes SMC phenotypic switching and contributes to atherosclerosis development. The proposal will employ SMC-specific knockout mice (Nhe1-/-, Cd47-/- and Cd36-/-), primary human and murine aortic SMC, a vascular SMC-restricted Cre driver mouse model (Itga8-CreERT2+/-; mTmG+/-), and other genetic tools to test the hypothesis. Multiple complementary techniques will be used to study SMC macropinocytosis in vitro (pharmacological, genetic, fluorescence/SEM imaging) and in vivo (TEM/IEM microscopy, SMC-specific Nhe1 knockout mice). At their conclusion, the proposed studies will define key mechanism(s) promoting SMC foam cell formation and phenotypic switching and potentially identify new targets for therapeutic interventions of atherosclerosis.