Abstract 181: Enhanced Cell Death by Oxidized Phospholipids in Vascular Smooth Muscle Cells via Activation of AMP-Activated Protein Kinase

Abstract

The modification of phospholipids, principally found in low density lipoproteins, is thought to be a primary risk factor in the progression of atherosclerosis. AMP-activated protein kinase (AMPK) is involved in regulating cellular energy balance and believed to be vasculoprotective in many cardiovascular diseases including atherosclerosis. This study investigates the interaction of modified phospholipids on AMPK function in vascular smooth muscle. Aortic smooth muscle cells (SMCs) were isolated from male New Zealand white rabbits and incubated with increasing concentrations of oxidized phospholipids, 1-palmitoyl-2-glutaroyl- sn -glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)- sn -glycero-3-phosphocholine (POVPC) for 2 hours before stimulation with 10% FCS for 24 hours. Cell proliferation and viability was measured for each condition using bromodeoxyuridine (BrdU) and ATP assays respectively. Incubation of POVPC caused a modest reduction in proliferation at the highest concentration (100μM, 58.16±11.3% of viable cells, n=6) while, PGPC dramatically reduced the number of viable cells in a concentration-dependent manner (25μM, 53.78±4.6%, n=6). Pretreatment of SMCs with an AMPK activator, A769662 for 45 mins, enhanced cell death to 10μM of PGPC (79.32±9.6% vs 102.96±5.8% for lipid alone, n=4). Pre-incubation with AMPK inhibitor, compound C for 30 mins, abolished the enhanced cell death at this concentration (88.54±14.9%, n=3). Similarly, activation of AMPK prior to 25μM POVPC treatment caused substantially more cell death than with lipid alone (48.01±13.24% vs 82.89±6.0% respectively, n=3). In contrast to PGPC, pretreatment with compound C had no effect on proliferation (25μM, 49.76±9.3%, n=3). Phosphorylated acetyl-CoA carboxylase, a downstream target of AMPK, showed a significant increase in expression after incubation of A76662 while no change after compound C was observed, analyzed by immunoblotting (4.10±0.95 vs 0.68±0.38, n=3). This novel study reveals that the activation of AMPK enhanced cytotoxicity of oxidized phospholipids, with PGPC and POVPC apparently operating through separate pathways. This contrasts with the protective effect usually observed for AMPK and may be critical in restenosis.