Abstract-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 p38 mapk , 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, nϭ3 to 7, PϽ0.01). Activation of RhoA but not p38 mapk 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 -nitro-L-arginine methyl ester (100 mol/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. Key Words: atherosclerosis Ⅲ cell culture/isolation Ⅲ endothelial dysfunction Ⅲ tissue factor Ⅲ lipoproteins T issue factor (TF), a 47-kDa integral membrane protein, appears to be a critical determinant of atherosclerotic plaque thrombogenicity that is important for triggering acute coronary syndromes. 1-4 TF is highly expressed in unstable atherosclerotic plaque, 5-8 whereas its expression is mainly limited to adventitial fibroblasts, and it is sporadically found in the media of normal arteries. 5 Healthy endothelial cells express negligible amounts and have low activity of TF that can be upregulated by various atherogenic factors such as thrombin, tumor necrosis factor-␣ (TNF-␣), and interleukin-1. 9,10 Although intracellular regulatory mechanisms of endothelial TF expression have not been completely elucidated, recent studies demonstrate that endothelial TF expression is positively regulated by the small G-protein RhoA and the serine/threonine protein kinase p38 mapk , but negatively regulated by protein kinase B (Akt). 9,11,12 Akt activation is dependent on the upstream phosphatidylinositol 3-kinase (PI3K) and influences cellular functions by activation of various downstream effectors. 13 In human endothelial cells, Akt phosphorylates endothelial nitric oxide synthase (eNOS) at serine-1177 leading to...

Background: Reconstituted high-density lipoprotein (rHDL) is prepared from apolipoprotein A-I, isolated from human plasma, and soybean-derived phosphatidylcholine and exhibits biochemical and functional characteristics similar to endogenous nascent high-density lipoprotein (HDL). This study tested the hypothesis that pretreatment with rHDL may reduce neuronal damage in 2 experimental rat models of stroke. Methods: In the first model, an excitotoxic lesion was induced by unilateral injection of N-methyl-D-aspartate (NMDA) in the right striatum (excitotoxic lesion model). In the second model, temporary occlusion of the middle cerebral artery (MCA) was attained by inserting a nylon thread through the carotid artery and blood flow was restored 30 min later (MCAo model). In both models, either rHDL (120 mg/kg) or saline (control) were infused over 4 h, starting 2 h before the injection of NMDA or the induction of ischemia, respectively. 24 h after the interventions, the rats were sacrificed and the brains removed for histochemical preparation. The necrotic area was delimited using an image analysis system. In addition, the levels of reactive oxygen species (ROS) in human endothelial (ECV 304) and neuroblastoma (SK-N-BE) cell lines were measured fluorometrically as 2′,7′-dichlorofluorescein fluorescence in the presence and absence of rHDL and under basal and stress-induced conditions. Results: In the excitotoxic lesion and MCAo models, pretreatment with rHDL significantly reduced the brain necrotic area by 61 and 76%, respectively (p < 0.01). Overnight incubation of ECV 304 and SK-N-BE cells with 0.5 mg/ml rHDL decreased basal and stress-induced ROS levels by 73 and 72% (ECV 304) and by 76 and 43% (SK-N-BE), respectively (p < 0.01). Conclusion: These results suggest that rHDL reduces neuronal damage after onset of ischemic stroke, possibly by involving an anti-oxidative mechanism. Thus, rHDL may be a powerful neuroprotective tool for the treatment of cerebrovascular diseases.

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