Abstract 143: Macropinocytosis Mediates Enzymatically Modified LDL (ELDL)-Induced Murine Smooth Muscle Cell Formation: A Role for RAGE In ELDL Endocytosis and Upregulation of Scavenger Receptor LOX1

Abstract

Background: ELDL is present in human atherosclerotic lesions and promotes foam cell formation in cultured macrophages and vascular smooth muscle cells (SMC). Here we study mechanism of ELDL uptake and its effects on SMCs.

Methods and Results: Incubation of wild type murine aortic SMCs with 10 μg/ml ELDL (trypsin, cholesterol esterase modified) results in enhanced 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 oxidized LDL (200 μg/ml; -75%, p<0.01). Inhibitors of macropinocytosis (50 μM LY294006, 2 μM wortmannin, and 3 mM amiloride) attenuated ELDL uptake (-50%, -50%, -100% respectively). In contrast, 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) had no effect on ELDL uptake in SMCs. Moreover, ELDL incubation led to increased expression of scavenger receptor LOX1 (+ 3 fold, p<0.01) in wild type SMC’s, but not in SMC deficient in Receptor for AGE (RAGE-/-), while CD36 and SRA1 remained unchanged in both the SMCs. Importantly, RAGE-/- SMCs upon pretreatment with PI3K inhibitors that only partially inhibited macropinocytosis of ELDL in wild type SMCs, completely prevented ELDL uptake in RAGE-/- SMCs. Mechanistically, ELDL upregulates ROS (detected using H2DCFDA) and down regulates PIP3 (detected by pAkt immunoblotting) in wild type, but not in RAGE-/- SMCs. Since ROS is known to regulate macropinocytosis via increased Ca2+ levels, we tested Ca2+ channel inhibitor lacidipine (30 μM), and found complete inhibition of ELDL uptake in both, wild type and RAGE-/- SMCs. Lastly, we speculate that the fused structure of LDL in the ELDL preparation is preferentially activating RAGE, since oligomerization of ligands are known to increase RAGE signaling, and FPLC analysis demonstrated that ELDL consists mostly of fused LDL particles.

Conclusions: ELDL is highly potent in inducing foam cells in aortic SMCs. ELDL endocytosis is mediated by RAGE-regulated, Ca2+ dependent macropinocytosis.