HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Oxidized phospholipids in oxidized low-density lipoprotein down-regulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RAR -RXR heterodimers and Sp1 and Sp3 to their binding sequences in the TM promoter

The present work investigated the mechanism for down-regulation of thrombomodulin (TM), an anticoagulant glycoprotein, on cultured umbilical vein endothelial cells (HUVECs) exposed to lipid extracts from oxidized low-density lipoprotein (oxLDL). HUVECs exposed to phospholipid extracts, but not to free cholesterol, triglyceride, or cholesterol ester, isolated from ox-LDL reduced TM mRNA levels to nearly the same extent as native ox-LDL. Oxidized 1-palmitoyl-2-arachidonyl-snglycero-3-phosphocholine (ox-PAPC), but not native PAPC or a reduced form of ox-PAPC, markedly decreased TM mRNA levels. The apparent half-life (t 1/2 2.7 hours) of TM mRNA in control cells was not significantly different from that in cells exposed to ox-LDL or ox-PAPC. TM mRNA levels were regulated by transcriptional activation via a retinoid receptor (RAR ). The binding activities of nuclear proteins from HUVECs treated with oxLDL or ox-PAPC to the DR4 or stimulatory protein 1 (Sp1) sequence in the TM promoter were significantly reduced with decreased expression of RAR , retinoid X receptor (RXR ), Sp1, and Sp3 in the nuclei. The promoter activity in HUVECs transfected with a reporter plasmid expressing the TM promoter with targeted deletions in the DR4 and Sp1 binding elements was decreased to about 20% of that with the wild-type construct. Treatment of the cells with ox-PAPC had no additional effect on the promoter activity. These results suggest that oxidized phospholipids in ox-LDL inhibit transcription of the TM gene in HUVECs by inhibiting the binding of RAR -RXR heterodimer and Sp, including Sp1 and Sp3, to the DR4 element and Sp1 binding element, respectively, in the TM promoter with reduced expression of RAR , RXR , and Sp1 and Sp3 in the nuclei. (Blood. 2003; 101:4765-4774)