Reconstituted high-density lipoprotein inhibits thrombin-induced endothelial tissue factor expression through inhibition of RhoA and stimulation of phosphatidylinositol 3-kinase but not Akt/endothelial nitric oxide synthase.
Endothelial cells express negligible amounts of tissue factor (TF) that can be induced by thrombin, which is important for acute coronary syndromes. Recent research suggests that endothelial TF expression is positively regulated by RhoA and p38mapk, but negatively by Akt/endothelial nitric oxide synthase (eNOS) pathway. High-density lipoprotein (HDL) is atheroprotective and exerts antiatherothrombotic effect. This study investigated the effect of a reconstituted HDL (rHDL) on endothelial TF expression induced by thrombin and the underlying mechanisms. In cultured human umbilical vein and aortic endothelial cells, thrombin (4 U/mL, 4 hours) increased TF protein level, which was reduced by rHDL (0.1 mg/mL, 43% inhibition, n=3 to 7, P<0.01). Activation of RhoA but not p38mapk by thrombin was prevented by rHDL. rHDL stimulated Akt/eNOS pathway. The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002 abolished the activation of Akt/eNOS and reversed the inhibitory effect of rHDL on TF expression. Adenoviral expression of the active PI3K mutant (p110) reduced TF expression stimulated by thrombin without inhibiting RhoA activation, whereas expression of the active Akt mutant (m/p) further facilitated TF upregulation by thrombin. Moreover, a dominant-negative Akt mutant (KA) reduced thrombin's effect and did not reverse the rHDL's inhibitory effect on TF expression. Inhibition of eNOS by N(omega)-nitro-L-arginine methyl ester (100 micromol/L) did not affect the rHDL's effect. In conclusion, rHDL inhibits thrombin-induced human endothelial TF expression through inhibition of RhoA and activation of PI3K but not Akt/eNOS. These findings implicate a novel mechanism of antiatherothrombotic effects of HDL.
Department of Medicine, Divisions of Physiology, University of Fribourg, Fribourg, Switzerland.
Hema
H
Viswambharan
Xiu-Fen
XF
Ming
Shengsi
S
Zhu
Alphonse
A
Hubsch
Peter
P
Lerch
Guy
G
Vergères
Sandro
S
Rusconi
Zhihong
Z
Yang
D000256:Adenoviridae / Q000235:genetics; D000730:Androstadienes / Q000494:pharmacology; D001011:Aorta; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008075:Lipoproteins, HDL / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000502:physiology; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D013917:Thrombin / Q000037:antagonists & inhibitors*; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics; D014161:Transduction, Genetic; D014471:Umbilical Veins; D000077191:Wortmannin; D048051:p38 Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000502:physiology*
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D008075:Lipoproteins, HDL; D020928:Mitogen-Activated Protein Kinases; D009025:Morpholines; D019331:NG-Nitroarginine Methyl Ester; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins; D013917:Thrombin; D013925:Thromboplastin; D000077191:Wortmannin; D048051:p38 Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C496314:NOS3 protein, human; C494918:AKT1 protein, human