Lysophosphatidylcholine is a major contributor to the synergistic effect of mildly oxidized low-density lipoprotein with endothelin-1 on vascular smooth muscle cell proliferation

Endothelin-1 (ET-1) and oxidized low-density lipoprotein (ox-LDL) are associated with atherosclerosis and essential hypertension. We assessed the effect of mildly oxidized LDL (mox-LDL) and ox-LDL and their major oxidative components, i.e., reactive oxygen species (ROS), lysophosphatidylcholine (LPC) and 4-hydroxy-2-nonenal (HNE) and their interaction with ET-1 on vascular smooth muscle cell (VSMC) proliferation. Growth-arrested VSMCs isolated from the rabbit aorta were incubated with different concentrations of LDL, mox-LDL, ox-LDL, hydrogen peroxide (H2O2) (a donor of ROS), LPC, or HNE with or without ET-1. DNA synthesis in VSMCs was measured by [3 H] thymidine incorporation. Mox-LDL, ox-LDL, H2O2, LPC, HNE, or ET-1 stimulated DNA synthesis in a dose-dependent manner. Maximal effect was observed at 5 mug/ml for mox-LDL (162%) or ox-LDL (154%), 15 muM LPC (156%), 5 muM H2O2 (177%), 1 muM HNE (144%), and 0.1 muM ET-1 (195%). By contrast, LDL was without any significant effect. When added together, there was no synergistic effect of LDL, H2O2, or HNE with ET-1 on DNA synthesis. However, the effect of mox-LDL (0.1 mug/ml), ox-LDL (0.5 mug/ml), or LPC (10 muM was potentiated by ET-1 (114%-338%, 133%-425%, 118%-333%, respectively). The mitogenic effect of mox-LDL, ox-LDL, or LPC and their interaction with ET-1 were inhibited by defatted albumin (10 mug/ml), antioxidant N-acetylcysteine (400 muM), the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (1 muM). The ETA/B receptor antagonist TAK044 (1 muM) or the MAPK kinase inhibitor PD098059 (10 muM) inhibited the mitogenic effect of ET-1 and its interaction with mox-LDL, ox-LDL, or LPC. The synergistic interaction of mox-LDL, ox-LDL, or LPC with ET-1 was completely reversed by the combined use of N-acetylcysteine and TAK044. Our results suggest that mox-LDL, ox-LDL, and their major phospholipid component LPC act synergistically with ET-1 in inducing VSMC proliferation by way of the activation of redox-sensitive and MAPK pathways.