Thrombomodulin and atherosclerosis

• Project/Area Number: 13671180
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Metabolomics
• Research Institution: 福井医科大学
• Principal Investigator: OIDA Koji University of Fukui Hospital, Lecturer, 医学部附属病院, 講師 (90203687)
• Co-Investigator(Kenkyū-buntansha): ISHII Hidemi Showa Pharmaceutical University, Professor, 薬学部, 教授 (50102710)
• Project Period (FY): 2001 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥3,400,000 (Direct Cost: ¥3,400,000); Fiscal Year 2003: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000); Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
• Keywords: Thrombomodulin / Endothelial Cells / Arteriosclerosis / Oxidized LDL / Monocyte & Macrophage / PPARγ / Statin (HMG-CoA reductase inhibitor) / 単球マクロファージ / マクロファージ / マイクロアレイ

Research Abstract

A. The mechanism for down-regulation of thrombomodulin (TM), an anticoagulant glycoprotein, on cultured umbilical vein endothelial c ells (HUVECs) exposed to lipid extracts from oxidized low-density lipoprotein (ox-LDL) was investigated. HUVECs exposed to phospho lipid extracts isolated from ox-LDL reduced TM mRNA levels to nearly the same extent as native ox-LDL. Oxidized 1-palmitoyl-2-arac hidonyl-sn-glycero-3-phosphocholine (ox-PAPC) markedly decreased TM mRNA levels. The binding activities of nuclear proteins from H UVECs treated with ox-LDL or ox-PAPC to the DR4 or stimulatory protein 1 (Sp1) sequence in the TM promoter were significantly r educed with decreased expression of RARbeta, retinoid X receptor alpha (RXRalpha), 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. These results s uggest that oxidized phospholipids in ox-LDL inhibit transcription of the TM gene in HUVECs by inhibiting the binding of RARbeta-RXRalpha heterodimer and Sp, including Sp1 and Sp3, to the DR4 element and Sp1 binding element, respectively, in the TM promoter with reduced expression of RARbeta, RXRalpha, and Sp1 amd Sp3 in the nuclei.
B. Since the expression of thrombomodulin (TM) by monocytes is upregulated during differentiation into macrophages, we investigated the effect of pioglitazone, a thiazolidinedione (TZD) that is a synthetic ligand of PPARgamma, on the expression of TM by a human monocyte/macrophage cell line ; human acute monocytic leukemia (TEP-1) cells. Pioglitazone dose-dependently upregulated TM antigen expressian by THP-1 cells accompanied by an upregulation of TM cofactor activity for thrombin-dependent protein C activation. Thrombomodulin mRNA expression in THP-1 cells was also upregulated by pioglitazone. Treatment cells with a natural PPARgamma ligand, 15-deoxy-delta12,14-prostaglandin J(2) (PGJ2), also enhanced TM protein expression. PGF(2alpha) an agent known to inactivate PPARgamma, diminished the stimulatory effect of pioglitazone and PGJ2 on TM protein expression. These results suggest that PPARgamma activation in macrophages may counteract potentially prothrombotic and putative inflammatory properties in activated macrophages.
C. The effect of statins on the expression of TM and TF by endothelial cells was investigated. The incubation of endothelial cells with statins led to a concentration-and time-dependent increase in cellular TM antigen and mRNA levels. In contrast, the expression of TF mRNA was not induced under the same conditions. The stimulation of TM expression by statins was prevented by either mevalonate or geranylgeranylpyrophosphate. Specific inhibition of geranylgeranyltransferase-I and Rac/Cdc42 by GGTI-286 and Clostridium sordellii lethal toxin, respectively, enhanced TM expression, whereas inactivation of Rho by Clostridium botulinum C3 exoenzyme was ineffective. These results suggest that statins regulate TM expression via inhibition of small G proteins of the Rho family ; Rac/Cdc42. A statin-mediated increase in TM expression by endothelial cells may contribute to the beneficial effects of statins on endothelial function.

Research Products

• [Publications] Ishii H, Tezuka T, Ishikawa H, Takada K, Oida K, Horie S.: "Oxidized phospholipids in oxidized low-density lipoprotein downregulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RAR{beta}-RXR{alpha} heterodimers and Sp1 and 3 to their binding sequences in the TM promoter."Blood. 101(6). 4765-4774 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Kanehara H, Tohda G, Oida K, Suzuki J, Ishii H, Miyamori I.: "Thrombomodulin expression by THP-1 but not by vascular endothelial cells is upregulated by pioglitazone."Thrombosis Research. 108(4). 227-234 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Masamura K, Oida K, Kanehara H, Suzuki J, Horie S, Ishii H, Miyamori I.: "Pitavastatin-induced thrombomodulin expression by endothelial cells acts via inhibition of small G proteins of the Rho family."Arteriosclerosis Thrombosis and Vascular Biology. 23(3). 512-517 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Masamura K, Oida K, Kanehara H, Suzuki J, Horie S, Ishii H, Miyamori I.: "Pitavastatin-induced thrombomodulin expression by endothelial cells acts via inhibition of small G proteins of the Rho family."Arterioscler Thromb Vasc Biol.. 23(3). 512-517 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Ishii H, Tezuka T, Ishikawa H, Takada K, Oida K, Horie S.: "Oxidized phospholipids in oxidized low-density lipoprotein downregulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RAR{alpha} heterodimers and Sp1 and 3 to their binding sequences in the TM promoter."Blood. 101(12). 4765-4674 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Kanehara H, Tohda G, Oida K, Suzuki J, Ishii H, Miyamori I.: "Thrombo modulin expression by THP-1 but not by vascular endothelial cells is up regulated by pioglitazone."Thromb Res.. 108(4). 227-234 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Ishii H, Tezuka T, Ishikawa H, Takada K, Oida K, Horie S.: "Oxidized phospholipids in oxidized low-density lipoprotein downregulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RAR{beta}-RXR{alpha}heterodimers and Sp1 and 3 to their binding sequences in the TM promoter"Blood. 101(6). 4765-4774 (2003)
• [Publications] Kanehara H, Tohda G, Oida K, Suzuki J, Ishii H, Miyamori I.: "Thrombomodulin expression by THP-1 but not by vascular endothelial cells is upregulated by pioglitazone"Thrombosis Research. 108(4). 227-234 (2003)
• [Publications] Masamura K, Oida K, Kanehara H, Suzuki J, Horie S, Ishii H, Miyamori I.: "Pitavastatin-induced thrombomodulin expression by endothelial cells acts via inhibition of small G proteins of the Rho famil"Arteriosclerosis Thrombosis and Vascular Biology. 23(3). 512-517 (2003)
• [Publications] Kanehara H, Tohda G, Oida K.Suzuki J, Ishii H, Miyamori I: "Thrombomodulin expression by THP-1 but not by vascular endothelial cells is upregulated by pioglitazone"Thromb Res. 108(4). 227-234 (2002)
• [Publications] Masamura K, Oida K, Kanehara H, Suzuki J, Horie S, Ishii H, Miyamori I.: "Pitavastatin-induced thrombomodulin expression by endothelial cells acts via inhibition of small g proteins of the rho family"Arterioscler Thromb Vasc Biol.. 23(3). 512-517 (2002)
• [Publications] Ishii H, Tezuka T, Ishikawa H, Takada K, Oida K, Horie S.: "Oxidized phospholipids in oxidized low-density lipoprotein downregulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RAR{beta}-RXR{alpha} heterodimers and Sp1 and 3 to their binding sequences in the TM promoter"Blood. (印刷中). (2003)