Lysophosphatidylcholine induces apoptotic and non-apoptotic death in vascular smooth muscle cells: in comparison with oxidized LDL

Oxidized low-density lipoprotein (oxLDL) plays a key role in the development of atherogenesis, partly by causing injury to vascular cells. However, different preparations of LDL, methods of oxidation, and/or active components often produce cellular effects of various degrees. To explore the quantitative relationship between dose and level of oxidation of the oxLDL utilized, we employed combinations of different levels of oxidation and concentrations of oxLDL to induce cell death in cultured vascular smooth muscle cells (VSMC). We also examined the effect of lysophosphatidylcholine (lysoPC), a putative active component of oxLDL, on VSMCs by determining, in parallel with a cytotoxicity test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay), DNA fragmentation ([3H]thymidine release), and flow cytometric analyses. We found that oxLDL caused cytotoxicity in an oxidative level- and dose-dependent manner, lysoPC also caused dose-dependent cytotoxicity with or without serum. Fragmentation of DNA was observed in both oxLDL- and lysoPC-treated VSMCs. Furthermore, lysoPC-induced DNA ladder was also demonstrated by gel electrophoresis at a concentration of 25 μmol/l or higher. Flow cytometric analysis yielded similar results for oxLDL- and lysoPC-treated VSMC; namely, an accumulation in the fraction of cells in G0/G1 phase with a reciprocal change in S-phase fraction. Membrane phosphatidylserine exposure, detected by annexin V staining, provided additional evidence that lysoPC induced significant apoptosis in VSMC. Taken together, the degree of oxLDL-induced cytotoxicity/apoptosis of VSMC depended on combined effects of oxLDL concentration and oxidative level. Moreover, lysoPC also elicited a dose-dependent apoptosis in addition to cytotoxicity.