Abstract 646: Redox Control of Macropinocytosis; An Unexplored Target in Atherosclerosis

Aims: Early studies established the paradigm that oxidation of low density lipoprotein (LDL) is necessary for scavenger receptor-mediated LDL uptake and lipid accumulation in macrophages. In addition to this “classical” mode of lipid internalization, scavenger receptor-independent uptake of native, non-oxidized LDL (nLDL) via macropinocytosis has been demonstrated to contribute to lipid uptake by macrophages. Despite this previous information the precise signaling mechanisms regulating macropinocytosis of nLDL and the relative contribution of lipid macropinocytosis to atherosclerosis remain unknown. This study was designed to examine the role of phagocyte NADPH oxidase (a.k.a. Nox2) in macropinocytosis and to investigate macropinocytotic uptake of lipids in hypercholesterolemic ApoE -/- mice in vivo . Results: Phorbol myristate acetate (4β-PMA) activation of human and murine macrophages stimulated membrane ruffling, macropinosome formation, and subsequent uptake of nLDL by macropinocytosis. FACS data indicated that 4β-PMA stimulates lipid accumulation following nLDL treatment in macrophages lacking scavenger receptor CD36. Mechanistically, we found that pharmacological blockade of protein kinase C (PKC), inhibition of flavoenzymes by diphenyleneiodonium, and scavenging intracellular superoxide anion abolished phorbol ester-induced macropinocytosis. Transcriptional knockdown of Nox2 using siRNA inhibited 4β-PMA-induced macropinocytosis in THP-1 macrophages. Delving further into the mechanism, we found that Nox2 via redox inactivation of PTEN and activation of the PI3K/Akt pathway dephosphorylates the actin-binding protein cofilin, stimulates membrane ruffling, and induces macropinocytosis. Finally, peritoneal chimera experiments indicate that macropinocytotic uptake of lipids in hypercholesterolemic ApoE -/- mice was attenuated in Nox2 y/- macrophages compared to wild type controls. Innovation and Conclusion: These findings suggest a previously undescribed redox-sensitive signaling pathway leading to internalization of nLDL by macropinocytosis. The signaling mechanism described herein may identify new targets in atherosclerosis and other disease conditions involving macropinocytosis.