Lysophosphatidylcholine and reactive oxygen species mediate the synergistic effect of mildly oxidized LDL with serotonin on vascular smooth muscle cell proliferation

Background-Mild oxidation of LDL enhances its atherogenic potential and induces a synergistic interaction with serotonin (5HT) on vascular smooth muscle cell (VSMC) proliferation. Because of its complex chemical nature, the mitogenic components of mildly oxidized LDL (moxLDL) remain unclear. Methods and Results-We examined both the effects of lysophosphatidylcholine (LPC) and hydrogen peroxide (H2O2), a donor of reactive oxygen species, as major components of moxLDL and their interactions with 5HT on VSMC proliferation. Growth-arrested VSMCs were incubated with different concentrations of moxLDL, LPC, H2O2, or LPC with H2O2 in the absence or presence of 5HT. DNA synthesis in VSMCs was examined by [H-3]thymidine incorporation. MoxLDL, LPC, H2O2, and 5HT stimulated DNA synthesis in a dose-dependent manner. MoxLDL had a maximal stimulatory effect at a concentration of 5 mug/mL (211%), LPC at 15 mu mol/L (156%), H2O2 at 5 mu mol/L (179%), and 5HT at 50 mu mol/L (205%). Added together, moxLDL (50 ng/mL) and 5HT (50 mu mol/L) synergistically increased DNA synthesis (443%). Coincubation of LPC (1 mu mol/L) with H2O2 (0.5 mu mol/L) and 5HT (5 mu mol/L) resulted in a synergistic increase in DNA synthesis (439%), which was nearly equal to that of moxLDL with 5HT (443%), The combined effects of LPC, H2O2, and 5HT on DNA synthesis were completely reversed by the combined use of an antioxidant, N-acetylcysteine (400 mu mol/L) or butylated hydroxytoluene (20 mu mol/L), with a 5HT(2) receptor antagonist, LY281067 (10 mug/mL). Conclusions-Our results suggest that both LPC and reactive oxygen species may contribute to the mitogenic effect of moxLDL on VSMCs and its synergistic effect with 5HT.