Abstract 16342: Rage Dependent Macropinocytosis Mediates Endocytosis of Enzyme Modified Non Oxidized LDL and Foam Cell Formation in Mouse Aortic Smooth Muscle Cells
Background: Vascular smooth muscle cell (SMC) derived foam cells contribute to human atherosclerosis. Enzymatically modified LDL (E LDL) induces SMC foam cells. Mechanism of E LDL uptake and the relative contribution of E LDL compared with other modified forms of LDL such as oxidized LDL in SMC foam cell formation are unknown.
Methods and Results: Electrophoresis and FPLC analysis suggests E LDL (trypsin,cholesterol esterase modified) consists of fused LDL particles. Incubation of murine aortic SMCs with 10 μg/ml E LDL results in significant foam cell formation (analyzed by Oil Red O, lipid measurement) compared to SMCs incubated with acetylated LDL (500 μg/ml; -50%, p<0.01) and copper oxidized LDL (200 μg/ml; -75%, p<0.01). E LDL incubation led to increased mRNA of scavenger receptor LOX 1(+ 6 fold, p<0.01), while CD36 and SRA1 remained unchanged; moreover ABCA1 and LDLR mRNA levels were reduced in E LDL treated SMCs (-25%, p<0.05). Inhibitors of receptor mediated endocytosis (100 μM dynasore, 0.1 M Sucrose), and inhibitors of caveolae /lipid raft mediated endocytosis (5mM MBCD, 5 μM filipin) failed to prevent E LDL uptake in SMCs, while inhibitors of macropinocytosis including LY294006 (50 μM) and wortmannin (2 μM) partially (-50%) and amiloride (3 μM) completely inhibited E LDL uptake in wild type SMCs. Importantly, SMC deficient for receptor for advanced glycation end products (RAGE) were completely protected from E LDL uptake and foam cell formation when pretreated with the same concentration of the PI3 K inhibitors that only partially inhibited macropinocytosis in wild type SMCs. RAGE is known to activate Src K that activates PI3 K which plays crucial roles in macropinocytosis. Src K inhibitor PP2 (2 μM) in RAGE-/- SMCs, unlike in wild type SMCs, also completely blocked E LDL uptake. Oligomerization of ligands is known to increase RAGE signaling, and we speculate that the fused nature of LDL in E LDL may activate RAGE.
Conclusions: E LDL is highly potent in inducing foam cells in mouse aortic SMCs. This is mediated by Src, PI3K and RAGE regulated endocytosis of E LDL into SMCs. Inhibitors for macropinocytosis together with genetic ablation of RAGE completely prevents uptake of E LDL and foam cell formation in murine SMC.
Author Disclosures: B. Chellan: None. L. Yan: None. C.A. Reardon: None. M.H. Bowman: None.
- © 2014 by American Heart Association, Inc.