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Abstract
The oxidative modification of low-density lipoprotein (LDL) and subsequent alteration of endothelial cell function are generally accepted as an important early event in the pathogenesis of atherosclerosis. To understand the mechanism by which oxidized LDL (oxLDL) causes dysfunction in endothelial cells, human umbilical vein endothelial cells (HUVEC) were exposed to oxLDL at a concentration that induces cellular dysfunction, and proteomic analysis was carried out, together with the analysis of cellular lipid peroxidation products. Time-dependent accumulation of 7-ketocholesterol and the progression of oxidative modification of peroxiredoxin 2 were observed, together with the suppression of cell proliferation. Proteomic analysis using two-dimensional gel electrophoresis (2-D gel) revealed that nucleophosmin, stathmin, and nucleolin were differentially expressed after exposure to oxLDL. Both 2-D gel and western blot analyses revealed that (1) nucleophosmin was dephosphorylated in a time-dependent manner; (2) stathmin was transiently phosphorylated at 6 h, and the unphosphorylated form was continuously down-regulated; and (3) nucleolin was identified as a 20-kDa fragment and a 76-kDa form, which were down-regulated. These observations suggest that the exposure of HUVEC to oxLDL results in the suppression of cell proliferation, which is ascribed to protein modification and/or altered expression of nucleophosmin, stathmin, and nucleolin under these oxidative stress conditions.
| Abbreviations | ||
| AAPH | = | 2,2′-azobis(2-amidinopropane) dihydrochloride |
| AIPH | = | 2,2′-azobis(2-[2-imidazolin-2-yl]propane) dihydrochloride |
| CE | = | cholesteryl ester |
| CEOH | = | cholesteryl ester hydroxide |
| CEOOH | = | cholesteryl ester hydroperoxide |
| CEO(O)H | = | cholesteryl ester hydroxide and hydroperoxide |
| CHAPS | = | 3-([3-cholamidopropyl] dimethylammonio)-1-propane sulfonate |
| 2-D gel | = | two-dimensional gel electrophoresis |
| DTT | = | dithiothreitol |
| EDTA | = | ethylenediaminetetraacetic acid |
| Ch | = | free cholesterol |
| 7-KCh | = | 7-ketocholesterol |
| 7-OHCh | = | 7-hydroxycholesterol |
| HNE | = | 4-hydroxynonenal |
| 7-OOHCh | = | cholesterol 7-hydroperoxide |
| HUVEC | = | human umbilical vein endothelial cells |
| HPLC | = | high-performance liquid chromatography |
| LDL | = | low-density lipoprotein |
| nano-ESI | = | nano-electrospray ionization |
| nonospray HPLC-MS/MS | = | nanospray interface capillary HPLC-electrospray ionization/tandem mass spectrometry |
| MS/MS | = | tandem mass spectrometry |
| MTT | = | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
| oxLDL | = | oxidized low-density lipoprotein |
| oxPrx | = | oxidatively modified peroxiredoxin |
| PBS | = | phosphate-buffered saline |
| PC | = | phosphatidylcholine |
| PCOH | = | phosphatidylcholine hydroxide |
| PCOOH | = | phosphatidylcholine hydroperoxide |
| PCO(O)H | = | phosphatidylcholine hydroxide and hydroperoxide |
| pI | = | isoelectric point |
| Prx | = | peroxiredoxin |
| SDS | = | sodium dodecyl sulfate |
| SDS-PAGE | = | sodium dodecyl sulfate-polyacrylamide gel electrophoresis |
| TFA | = | trifluoroacetic acid |
| Abbreviations | ||
| AAPH | = | 2,2′-azobis(2-amidinopropane) dihydrochloride |
| AIPH | = | 2,2′-azobis(2-[2-imidazolin-2-yl]propane) dihydrochloride |
| CE | = | cholesteryl ester |
| CEOH | = | cholesteryl ester hydroxide |
| CEOOH | = | cholesteryl ester hydroperoxide |
| CEO(O)H | = | cholesteryl ester hydroxide and hydroperoxide |
| CHAPS | = | 3-([3-cholamidopropyl] dimethylammonio)-1-propane sulfonate |
| 2-D gel | = | two-dimensional gel electrophoresis |
| DTT | = | dithiothreitol |
| EDTA | = | ethylenediaminetetraacetic acid |
| Ch | = | free cholesterol |
| 7-KCh | = | 7-ketocholesterol |
| 7-OHCh | = | 7-hydroxycholesterol |
| HNE | = | 4-hydroxynonenal |
| 7-OOHCh | = | cholesterol 7-hydroperoxide |
| HUVEC | = | human umbilical vein endothelial cells |
| HPLC | = | high-performance liquid chromatography |
| LDL | = | low-density lipoprotein |
| nano-ESI | = | nano-electrospray ionization |
| nonospray HPLC-MS/MS | = | nanospray interface capillary HPLC-electrospray ionization/tandem mass spectrometry |
| MS/MS | = | tandem mass spectrometry |
| MTT | = | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
| oxLDL | = | oxidized low-density lipoprotein |
| oxPrx | = | oxidatively modified peroxiredoxin |
| PBS | = | phosphate-buffered saline |
| PC | = | phosphatidylcholine |
| PCOH | = | phosphatidylcholine hydroxide |
| PCOOH | = | phosphatidylcholine hydroperoxide |
| PCO(O)H | = | phosphatidylcholine hydroxide and hydroperoxide |
| pI | = | isoelectric point |
| Prx | = | peroxiredoxin |
| SDS | = | sodium dodecyl sulfate |
| SDS-PAGE | = | sodium dodecyl sulfate-polyacrylamide gel electrophoresis |
| TFA | = | trifluoroacetic acid |