Native low-density lipoprotein (nLDL) was one of the modifiable risk factors contributed directly to cardiovascular diseases development. We investigated that nLDL stimulation induced NADPH oxidase activation and superoxide production that was an important factor on human aortic smooth muscle cells (hAoSMC) proliferation.

Superoxide generation was recorded with fluorescent-staining of dihydroethidine or by measuring lucigenin-induced chemiluminescence for 5 minutes. We examined cell proliferation with 4[-3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) reagent and analyzed the change of gene expression by northern blot analysis.

nLDL stimulation increased superoxide anion production in hAoSMC that confirmed through dihydroethidine staining and lucigenin-induced chemiluminescence methods. nLDL-induced proliferation abolished with preincubation of superoxide scavengers or NADPH oxidase inhibitor. NADPH as a substrate of NADPH oxidase increased superoxide generation in both nLDL-stimulated and unstimulated cell homogenate, which was completely blocked at the diphenylene iodinium (DPI)- or apocynin-pretreated hAoSMC homogenates. Furthermore, superoxide generation was only observed at the fraction of cellular precipitate, but not in soluble fraction. Expression of p22phox in mRNA level increased with nLDL treatment as early as 30 minutes and transfection of anti-sense oligonucleotide of p22phox completely abolished nLDL-induced proliferation of hAoSMC.

The above results have shown that nLDL-induced proliferation in hAoSMC depends on superoxide production through NADPH oxidase activation.