Influence of Modified Phospholipids on Vascular Cell Function in Restenosis

Fiona Greig1, Corinne Spickett2, Simon Kennedy1. 1University of Glasgow, Glasgow, UK, 2Aston University, Birmingham, UK.

Atherosclerosis is now widely accepted as a chronic inflammatory disease. Modification of phospholipids is thought to be a primary risk factor in the progression of this disease. The phagocytic enzyme, myeloperoxidase, is released from inflammatory cells found inside atherosclerotic lesions and can lead to the production of both oxidized and chlorinated phospholipids. Restenosis is characterised by the migration and proliferation of vascular smooth muscle cells (VSMCs) causing the luminal area of the atherosclerotic artery to re-narrow after treatments to improve blood flow such as balloon angioplasty or stenting. Modified phospholipids could play a critical role in this process and to investigate this, the effect of chlorinated and oxidized phospholipids on vascular cell function such as proliferation, migration and to induce cytotoxicity was measured. Rabbit aortic VSMCs and human saphenous vein endothelial cells (HSVECs) were incubated with either chlorinated lipids, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine chlorohydrins (SOPC ClOH) or 2-chlorohexadecanal (2-ClHDA) or oxidized lipids, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) or 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) for 2 or 6 hours prior to or for 24 hours simultaneously with FCS stimulation. Statistical analysis was performed using a one-way ANOVA followed by Dunnett’s post hoc test. Data are expressed as mean±SEM. Chlorinated lipids had only a very limited effect on proliferation, viability and migration in both VSMCs and HSVECs. However, following 6 hour incubation with 100µM SOPC ClOH, there was a significant reduction in cell viability (54.1±5.0% of cells viable vs control, P<0.01, n = 4). Incubation with oxidized lipids for 2 hours prior to stimulation with FCS caused a large decrease in VSMC proliferation and viability with both 100µM PGPC (0.2±1.0%, 0.5±3.1% vs control, P<0.001, n = 6) and POVPC (58.2±11.3%, 50.2±9.3% vs control, P<0.01, n = 6). Western blot analysis indicated an increase in caspase-3 activity following treatment with both oxidised lipids. Similar results were seen for HSVECs, at a lower concentration range of 1-50 µM, with incubation of chlorinated and oxidized phospholipids. The apoptotic action of the oxidized phospholipids could be abolished by the presence of 10% FCS. 100μM concentrations of both PGPC and POVPC also caused a substantial decrease in VSMC migration (PGPC: 17.1±13.6% vs control, P<0.01, n = 4; POVPC: 54.3±2.6% vs control, P<0.01, n = 4). These data suggest the potential importance of the modification of phospholipids in the atherosclerotic process and also in neointima formation in injured vessels. Oxidised lipids appear to be have more potent effects than equivalent concentrations of chlorinated lipids.