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(Circulation Research. 2004;94:918.)
© 2004 American Heart Association, Inc.

Molecular Medicine

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

Hema Viswambharan, Xiu-Fen Ming, Shengsi Zhu, Alphonse Hubsch, Peter Lerch, Guy Vergères, Sandro Rusconi, Zhihong Yang

From the Department of Medicine, Divisions of Physiology (H.V., X.-F.M., S.Z., Z.Y.) and Biochemistry (S.R.), University of Fribourg, Fribourg, Switzerland; and ZLB Bioplasma AG (A.H., P.L., G.V.), Bern, Switzerland.

Correspondence to Prof Dr Zhihong Yang, Vascular Biology, Department of Medicine, Division of Physiology, University of Fribourg, Rue du Musée 5, CH-1700, Fribourg, Switzerland. E-mail Zhihong.Yang{at}unifr.ch

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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, n=3 to 7, P<0.01). 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 µmol/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 • cell culture/isolation • endothelial dysfunction • tissue factor • lipoproteins

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tissue 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 activation of the enzyme and production of the important vascular protective factor, NO.^14,15 NO is a potent vasodilator and inhibitor of smooth muscle cell proliferation and platelet aggregation.¹⁶ It is also implicated in inhibition of TF expression in macrophages and endothelial cells.^17–19

The protective effects of HDL against ischemic heart disease are well established.^20,21 However, the underlying mechanisms have not been completely understood. Besides reverse-cholesterol transport, the cholesterol-independent (ie, pleiotropic) effects of HDL have been proposed to play an important role in cardiovascular protection.²² Experimental and clinical studies demonstrated that HDL or reconstituted HDL (rHDL) improves endothelial functions, which may be attributed to activation of eNOS through stimulation of Akt and p42/44mapk.^23–28 Indeed, intravenous infusion of rHDL restores plasma HDL concentration and improves endothelial function in ABCA1 heterozygote subjects and hypercholesterolemic patients.^26,27 Direct inhibition of platelet aggregation by HDL or rHDL have also been shown.^29–33 Moreover, anticoagulative and profibrinolytic effects of HDL were reported, but no conclusive results are available.^25,34 For example, HDL augments anticoagulant activities of protein S and activated protein C.³⁵ Inhibitory effects of HDL on secretion of plasminogen activator inhibitor (PAI) as well as tissue-type plasminogen activator (tPA) by endothelial cells were reported.^36,37 Similarly, intravenous infusion of rHDL reduces endotoxin-induced activation of coagulation (measured by decreased plasma levels of prothrombin fragment F1+2) as well as fibrinolysis (measured by increased plasma levels of tissue-type plasminogen activator, tPA) in human volunteers.³³ In an isolated in vitro reaction assay, HDL has been shown to inhibit activation of coagulation factor X by factor VIIa and TF purified from placenta.³⁸ These studies suggest a modulatory role of HDL or rHDL in hemostasis. The present study was aimed to investigate whether rHDL inhibits TF expression in response to thrombin by interfering with RhoA, p38^mapk, and/or PI3K/Akt/eNOS pathways in cultured human endothelial cells.

	Materials and Methods

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Chemicals
rHDL was produced as described.³⁹ For other chemicals, see the expanded Materials and Methods in the online data supplement available at http://circres.ahajournals.org.

Endothelial Cell Cultivation
Endothelial cells were isolated from human umbilical veins and characterized as described.⁴⁰ Human aortic endothelial cells were purchased from PromoCell (see expanded Materials and Methods for details).

Generation of Recombinant Adenoviruses (rAd)
Expression plasmid encoding an active PI3K mutant (pEF-BOS-rCD2-p110) was generously provided by D.A. Cantrell (Imperial Cancer Research Fund, London, UK), the plasmids encoding an active RhoA (pcDNA3.1-Rho63L) and a dominant-negative RhoA (pcDNA3.1-Rho19L) were kindly provided by J.J. Baldassare (Saint Louis University School of Medicine, St. Louis Mo), and pCMV constructs encoding a hemagglutinin (HA) epitope–tagged active Akt (pCMV-m/p-HA-Akt) and negative Akt (pCMV-HA-Akt-KA) were from B. Hemmings (FMI Institute, Basel, Switzerland). Generation of recombinant adenovirus expressing the mutants was carried out as described⁴⁰ (see expanded Materials and Methods for details).

Adenoviral Infection and Transgene Expression
Adenoviral infection of human umbilical vein endothelial cells (HUVECs) was performed as previously described.⁴⁰ The transgene expression of LacZ, Rho63, Rho19, m/p-Akt, Akt-KA, and rCD2-p110 was analyzed by immunoblotting using either anti-HA or anti-CD2 antibodies.

TF Expression
To study whether rHDL inhibits thrombin-induced TF expression involving activation of PI3K/Akt/eNOS pathway, quiescent cells were preincubated with wortmannin, LY294002, or L-NAME at the indicated concentrations for 60 minutes before treatments with rHDL followed by thrombin stimulation. Appropriate vehicles were added to the unstimulated control cells. In another series of experiments, cells expressing the active PI3K mutant (rCD2-p110) or the active (m/p) and negative (KA) Akt mutant were prepared as described in the previous section, and stimulated with thrombin (4 U/mL, 4 hours) in the presence or absence of rHDL to analyze the effect of PI3K and Akt on TF expression. Cell extracts were prepared by lysing cells in extraction buffer as previously described.⁴⁰ Extracts (30 µg) were subjected to SDS-PAGE, and immunoblotting for detection of TF expression was then performed. Quantification of the signals was performed using NIH Image 1.62 software. Ponceau S staining or tubulin protein levels were used to ensure equal protein loading.

Pull-Down Assay of GTP-RhoA
Activation of RhoA was assessed by a pull-down assay in the cells stimulated with thrombin as described previously.⁴⁰ To analyze the effect of rHDL on RhoA activation, the cells were preincubated with rHDL (0.1 mg/mL) for 1 hour followed by stimulation with thrombin (4 U/mL, 15 minutes). To study whether PI3K interferes with RhoA activation, cells overexpressing the active PI3K (rCD2-p110) or control gene (LacZ) were stimulated with thrombin (4 U/mL, 15 minutes), and RhoA activation was then measured.

Activation of Akt
As previously described,⁴⁰ Akt activation was detected by immunoblotting of Akt phosphorylation at Ser-473 and/or by nonradioactive immunoprecipitation-kinase assay using the Akt kinase assay kit from Cell Signaling Technology.

p38^mapk Activation
p38^mapk activation was analyzed by immunoblotting of p38^mapk phosphorylation at Thr180/Tyr185. Cell extracts (30 µg) were subjected to 10% SDS-PAGE, and phosphorylated p38^mapk was detected with anti-phospho-p38^mapk (T180/Y185) antibody. Activation of p38^mapk was calculated as ratio of phospho-p38^mapk against total p38^mapk.

Activation of eNOS
eNOS activation was measured by phosphorylation of the enzyme at Ser1177 and L-citrulline production as previously described.⁴⁰ (see details in the expanded Materials and Methods).

Statistical Analysis
TF expression stimulated by thrombin served as 100%. The effects of interventions with rHDL, substances, or mutants were calculated as percentage changes of the TF level stimulated with thrombin. Data are given as mean±SEM. The ANOVA with Bonferroni’s posttest was used for statistical analysis. A two-tailed value of P<0.05 was considered to indicate a statistically significant difference.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
rHDL Inhibits Thrombin-Induced TF Expression
Stimulation of HUVECs with thrombin (4 U/mL) for 4 hours markedly increased TF protein level (Figure 1A, top panel, n=7) confirming the results of previous study by Eto et al.⁹ The induction of endothelial TF level by thrombin was significantly inhibited by rHDL (0.1 mg/mL, 43% inhibition; Figure 1A, bottom panel, n=7; P<0.001), whereas rHDL alone had no significant effect on the basal TF level in the cells. Similarly, HDL isolated from human plasma also inhibits thrombin-induced TF expression (data not shown). Similar to HUVECs, in cultured human aortic endothelial cells, upregulation of TF protein level by thrombin (4 U/mL, 4 hours) was also reduced by rHDL (0.1 mg/mL, 47% inhibition; Figure 1B, n=3, P<0.001). Due to the greater availability of the material, the subsequent experiments were performed in HUVECs with rHDL.

View larger version (23K): [in this window] [in a new window]   Figure 1. rHDL inhibits endothelial tissue factor (TF) expression by thrombin. In HUVECs (A, n=7) as well as human aortic endothelial cells (B, HAECs, n=3) thrombin (Thr, 4 U/mL; 4 hours) upregulated TF protein level that was significantly inhibited by rHDL (0.1 mg/mL), whereas rHDL alone had no significant effect on the basal level of TF expression. *P<0.001 vs Thr.

Effect of rHDL on Thrombin-Induced Activation of RhoA
Because RhoA has been demonstrated to be involved in thrombin-mediated induction of TF expression in endothelial cells,^9,11 we analyzed whether rHDL interferes with RhoA activation. Activation of RhoA by thrombin (4 U/mL, 15 minutes) was inhibited by rHDL (0.1 mg/mL, n=5, P<0.01; Figure 2A). The role of RhoA in the induction of TF was further demonstrated by the experiment showing that adenoviral expression of the constitutively active RhoA mutant Rho63 (24 hours after infection) enhanced endothelial TF expression, whereas Rho19, the negative mutant has no effect on the basal TF expression (n=5, Figure 2B). The expression of the transgenes LacZ, Rho63, and Rho19 in the cells was demonstrated by immunoblotting using anti-HA antibody (Figure 2C).

View larger version (40K): [in this window] [in a new window]   Figure 2. rHDL inhibits endothelial tissue factor (TF) expression via inhibition of RhoA. A, Pull-down assay shows that thrombin (Thr, 4 U/mL; 15 minutes) activated RhoA (Rho-GTP), which was blocked by preincubation of the cells with rHDL (0.1 mg/mL, n=5). B, Adenoviral expression of active RhoA mutant Rho63 enhanced endothelial TF expression (n=5). C, Expression of transgenes of LacZ, Rho63, and Rho19 was shown by Western blot using anti-HA antibody. *P<0.001 vs control; P<0.01 vs Thr.

Effect of rHDL on Thrombin-Induced Activation of p38^mapk
We further analyzed whether rHDL reduces endothelial TF protein level by interfering with p38^mapk, which has been shown to be a positive regulatory mechanism of TF expression in endothelial cells.^9,41 Preincubation of the cells with rHDL (0.1 mg/mL) for 60 minutes did not significantly affect activation of p38^mapk (phosphorylation at T-180/Y-185) in response to thrombin (4 U/mL, 15 minutes, n=5; Figure 3), although rHDL significantly inhibited thrombin-induced TF expression under this condition (see Figure 1A).

View larger version (29K): [in this window] [in a new window]   Figure 3. rHDL did not inhibit activation of p38mapk. Phosphorylation of p38mapk at T180/Y185 residues was significantly stimulated by thrombin (Thr, 4 U/mL, 15 minutes), which was not affected by rHDL (0.1 mg/mL, n=5). *P<0.001 vs control.

rHDL Activates PI3K/Akt/eNOS Pathway
Initial experiments with different concentrations of rHDL (0.01 to 1.0 mg/mL) demonstrated that rHDL at the concentration of 0.1 mg/mL gives the maximal activation of Akt and eNOS (data not shown). Stimulation of the cells with rHDL (0.1 mg/mL) over 60 minutes induced a 1.7-fold increase in Akt phosphorylation at serine-473 (Figure 4A, n=6; P<0.05). Activation of Akt by rHDL (0.1 mg/mL, 60 minutes) was abolished by the PI3K inhibitor, wortmannin (0.1 µmol/L; Figure 4A, n=6; P<0.01).

View larger version (20K): [in this window] [in a new window]   Figure 4. rHDL activates PI3K/Akt/eNOS pathway. A, rHDL (0.1 mg/mL) phosphorylated Akt at serine-473, which was inhibited by the PI3K inhibitor wortmannin (WM, 0.1 µmol/L, P<0.05; n=6). B, rHDL also stimulated phosphorylation of eNOS at serine-1177 and L-citrulline production, which was blocked by wortmannin (WM, 0.1 µmol/L, P<0.01; n=6). *P<0.05 vs control; **P<0.01 vs control; P<0.05 vs rHDL; P<0.01 vs rHDL.

In parallel with Akt activation, rHDL stimulated eNOS phosphorylation at serine-1177 (1.5-fold increase) reaching the maximum at 30 minutes (Figure 4B). eNOS phosphorylation at serine-1177 was correlated with an increased L-citrulline production (1.9-fold, n=6; P<0.05), which was inhibited by wortmannin (0.1 µmol/L; Figure 4B, n=6; P<0.01).

rHDL Prevents Thrombin-Induced TF Expression Involving PI3K but not Akt/eNOS
The inhibitory effect of rHDL on TF expression induced by thrombin (4 U/mL, 4 hours) was fully reversed by the PI3K inhibitors wortmannin (0.1 µmol/L; Figure 5A, n=3; P<0.01) or LY294002 (1 µmol/L, n=5, P<0.01; Figure 5B). Moreover, adenoviral expression of a constitutively active PI3K (rCD2-p110) significantly reduced TF protein level stimulated by thrombin (Figure 5C, n=4; P<0.05). The p110 transgene expression was demonstrated by immunoblotting using anti-CD2 antibody (Figure 5D, lane 2; lane 1 are cells expressing HA-tagged LacZ, which is therefore not detectable with anti-CD2 antibody). In contrast to the active PI3K mutant, adenoviral expression of a constitutively active Akt mutant (m/p-Akt) enhanced the upregulation of TF expression stimulated by thrombin (Figure 6A, n=3; P<0.05). The transgene expression of m/p-Akt and LacZ was demonstrated by immunoblotting using monoclonal anti-HA antibody (Figure 6B). The role of Akt on TF expression was further investigated with the negative Akt mutant Akt-KA whose expression was demonstrated by immunoblotting on Figure 6C. An in vitro Akt kinase assay revealed that Akt was inhibited by ectopic expression of Akt-KA (Figures 6Da and 6Dc), indicating that Akt-KA functions as dominant-negative mutant in the cells. It is also important to notice that thrombin (4 U/mL, 4 hours) reduced Akt activity, which was reversed in the presence of rHDL (0.1 mg/mL), and this recovering effect of rHDL on Akt activity was abolished by the dominant negative Akt-KA mutant (Figures 6Da and 6Dc). In line with the effect of the active Akt-m/p mutant, which enhanced TF upregulation by thrombin (Figure 6A), Akt-KA reduced TF upregulation by thrombin and did not reverse the inhibitory effect of rHDL on TF expression, although neither active nor negative Akt mutants alone exerted any effect on basal TF expression in the cells (Figures 6A, 6Db, and 6Dc). Moreover, inhibition of eNOS by L-NAME (100 µmol/L) did not affect rHDL’s effect, and L-NAME alone did not modify basal level of TF expression in the cells (Figure 6E, n=3). It is important to notice that adenoviral expression of the active PI3K mutant p110 in the cells did not inhibit activation of RhoA stimulated by thrombin (4 U/mL, 15 minutes, n=6; Figure 7).

View larger version (38K): [in this window] [in a new window]   Figure 5. rHDL inhibits tissue factor (TF) expression via PI3K. Inhibition of TF expression in response to thrombin (4 U/mL, 4 hours) by rHDL (0.1 mg/mL) was abolished by the PI3K inhibitors wortmannin (0.1 µmol/L, n=3; A) and LY294002 (1 µmol/L, n=5; B). Adenoviral expression of the PI3K active mutant rCD2-p110 reduced thrombin-induced TF upregulation (n=4, C). Transgene expressions of CD2-tagged p110 (rCD2-p110) was demonstrated by immunoblotting using anti-CD2 antibody (D, lane 2; lane 1 is the HA-tagged LacZ-expressing cell that is not detectable under this condition. p110 appears larger on the SDS-PAGE gel due to the tagged CD2). *P<0.05 vs thrombin; P<0.01 vs Thr+rHDL.

View larger version (46K): [in this window] [in a new window]   Figure 6. rHDL inhibits tissue factor (TF) expression independent of Akt/eNOS. A, Adenoviral expression of the active Akt mutant (m/p) further enhanced the stimulation of TF level by thrombin. B and C, Expression of transgenes of HA-tagged LacZ (B and C, lane 1), the active m/p-Akt mutant (B, lane 2), and the negative Akt mutant (KA; C, lane 2) was demonstrated by immunoblotting using anti-HA antibody. Da, In HUVECs expressing LacZ control gene, basal Akt activity (lane 1) measured by phosphorylation of GSK-3 (p-GSK) as substrate was suppressed by thrombin (Thr 4 U/mL, 4 hours, lane 2), which was reversed in the presence of rHDL (0.1 mg/mL, lane 3). Expression of the negative Akt-KA mutant inhibited Akt activity (lane 4), which remained low by thrombin treatment (lane 5) and could not be reversed by rHDL (lane 6). Db, In HUVECs expressing LacZ control gene, no significant basal TF expression was observed (lane 1), but it was markedly upregulated by thrombin (4 U/mL, 4 hours, lane 2) reduced by rHDL (0.1 mg/mL, lane 3). Negative Akt-KA mutant alone had no effect on basal TF expression (lane 4) but reduced thrombin’s effect (lane 5) and did not reverse rHDL’s inhibitory effect on TF expression (lane 6). Tubulin expression was used to ensure equal protein loading. Quantification of the data from Da and Db is shown in Dc. E, eNOS inhibitor L-NAME, (100 µmol/L, n=3) did not affect thrombin’s effect on TF expression. *P<0.05 vs Thr alone in A and E; *P<0.05, **P<0.01, and ***P<0.001 between the groups in D.

View larger version (39K): [in this window] [in a new window]   Figure 7. Adenoviral expression of the active PI3K (rCD2-p110) did not inhibit RhoA activation by thrombin. Expression of the active PI3K (rCD2-p110) had no effect on RhoA activation stimulated by thrombin (4 U/mL, 15 minutes). n=6, *P<0.05 vs LacZ.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
There is compelling evidence showing that HDL is cardioprotective and low HDL level is an independent risk factor of coronary artery disease.²⁸ The cardioprotective mechanisms of HDL are complex and attributed to multiple biological functions beyond reverse-cholesterol transport.⁴² Among other mechanisms, protective effects of HDL on endothelial function seem to play an important role.^25,28,43 The vascular endothelial cells exert a wide spectrum of biological functions as diverse as the control of smooth muscle contraction, platelet aggregation, inflammation, as well as hemostasis.¹⁶ Alterations of one or more of the above-mentioned physiological roles constitute of endothelial dysfunction that is important in triggering cardiovascular events. Among other factors, TF is importantly involved in atherothrombosis.^1–8 TF is upregulated in atherosclerotic plaque and the plasma concentration of TF is increased in patients with unstable angina and myocardial infarction.^2–4 Previous studies showed that thrombin upregulates TF expression, which is associated with an increased cell surface TF activity.^9,10 Our current study further confirmed the stimulating effect of thrombin on TF expression. Most interestingly, the induction of endothelial TF expression by thrombin was significantly inhibited by rHDL. It is important to note that rHDL exerts similar biological functions as plasma HDL and intravenous infusion of rHDL restores HDL plasma concentration and improves endothelial function in ABCA1 heterozygote subjects with isolated low HDL level or in hypercholesterolemic patients.^26,27 The results of our present study not only implicate a novel cardiovascular protective function of HDL, but also the therapeutic potential of rHDL in patients with coronary heart disease.^26,27 Indeed, our unpublished results showed that HDL isolated from human plasma, similar to rHDL, was also able to inhibit thrombin-induced endothelial TF expression.

Although the regulatory mechanisms of endothelial TF expression have not been fully elucidated yet, recent research provides compelling evidence that RhoA and p38^mapk are important positive regulators of TF expression in endothelial cells.^9,11 In the present study, we demonstrated that adenoviral expression of an active RhoA mutant increased endothelial TF protein level, which further confirms the role of RhoA in upregulating TF expression in endothelial cells.^9,11 Most interestingly, rHDL was able to inhibit thrombin-induced RhoA activation, indicating that rHDL reduces endothelial TF expression by blocking RhoA activation. The mechanisms of RhoA inhibition by rHDL are unclear at this stage. Several possibilities could be postulated. First, rHDL may inhibit upstream mechanisms of RhoA activation, such as guanine nucleotide exchange factors (GEFs) that transform the inactive RhoA.GDP into the active RhoA.GTP form, or inhibit GTPase-activating proteins (GAP) that return RhoA.GTP to the RhoA.GDP form, or rHDL may enhance activity of GDP-dissociation inhibitor that prevents GDP dissociation from the GDP-bound form and keeps the small G-protein inactive. Secondly, rHDL may inhibit HMG-CoA reductase and thereby the production of HMG-CoA/mevalonate intermediates that are required for activation of RhoA.⁴⁴ Those hypotheses warrant further investigation.

The fact that TF upregulation by thrombin was only partially inhibited by rHDL in spite of a full inhibition of RhoA suggests that the other mechanism that upregulates TF expression may not be affected by rHDL. Several studies demonstrated the crucial role of p38^mapk in TF upregulation in endothelial cells and monocytes.^9,12,41,45 In our present study, we showed that p38^mapk activation by thrombin remained unaffected by rHDL, suggesting that rHDL inhibits endothelial TF expression via interfering with RhoA but not p38^mapk.

Whereas RhoA and p38^mapk positively regulate TF expression, PI3K/Akt pathway was shown to inhibit TF expression in human endothelial cells.^9,12 In the present study, we showed that rHDL activates Akt, which is in line with the results of several other studies using plasma HDL.^24,46 The effects of PI3K/Akt on thrombin-induced TF upregulation in human endothelial cells were further investigated in our present study. Two lines of evidences support a role of PI3K in the rHDL-mediated suppression of TF upregulation by thrombin. Firstly, inhibition of PI3K by two different inhibitors wortmannin or LY294002 reversed the effect of rHDL, suggesting that rHDL inhibits TF upregulation by thrombin via PI3K. It is important to notice that wortmannin alone, but not LY294002, enhanced TF expression, indicating that wortmannin may have certain nonspecific effects not related to PI3K. Secondly, expression of an active PI3K mutant (rCD2-p110) reduced thrombin-stimulated TF expression (Figure 5C). The function of the active PI3K in the cells was confirmed by the activation of Akt (phosphorylation of Akt at serine-473; data not shown). To our surprise, adenoviral expression of the active Akt mutant (m/p) enhanced thrombin-induced TF expression. Moreover, expression of a dominant negative Akt mutant (KA) reduced thrombin’s effect and did not reverse the rHDL’s inhibitory effect on TF expression (Figures 6Db and 6Dc). These data suggest that PI3K reduces TF expression stimulated by thrombin not via Akt under the experimental condition. Activation of other downstream effectors of PI3K, which is independent of Akt, such as PKC, p70s6k, protein tyrosin kinases (Tec family), and Rac⁴⁷ or even some other yet to be identified effector(s) must play a dominant role in the inhibition of TF expression. Which of these downstream effector(s), that is dominant to Akt, accounts for the negative regulation of PI3K on TF expression, remains to be identified. Our results contrast the observation by a recent study showing that expression of a constitutively active Akt mutant reduces VEGF-induced TF expression in human endothelial cells.¹² The discrepancy between this study and ours remains obscure. It might be due to the different agents used for the stimulation of TF expression. Whereas VEGF was used in that study, we used thrombin as a stimulator of TF expression. It is noteworthy that neither the constitutively active Akt mutant nor the dominant-negative mutant alone has any effect on basal TF expression, but they influence thrombin’s effect, ie, the active Akt mutant facilitated and the negative Akt mutant reduced thrombin-induced endothelial TF upregulation, suggesting that activation of Akt alone is not sufficient to modify TF expression and an interaction between Akt and thrombin-induced signaling mechanisms related to TF expression must exist, which may be different from that of Akt with VEGF. Hence, differential interaction between Akt and the signal transduction pathways stimulated by VEGF or thrombin may explain the different effect of active Akt on TF regulation. This issue is currently under investigation.

Activation of Akt affects cellular functions involving several downstream targets.¹³ In human endothelial cells, Akt phosphorylates eNOS at serine-1177 and enhances eNOS enzyme activity.^14,15,40 In line with the observation with plasma HDL,²⁴ we also showed in the present study that rHDL stimulates Akt and eNOS. The activation of Akt/eNOS could be abolished by PI3K inhibitor wortmannin. However, inhibition of eNOS by L-NAME did not affect the inhibitory effect of rHDL on thrombin-induced up-regulation of TF expression implicating that eNOS is not involved in the inhibition of endothelial TF expression by rHDL. This result further supports our conclusion that rHDL inhibits thrombin-induced endothelial TF expression through stimulation of PI3K but not Akt/eNOS. Similar observations were obtained in human endothelial cells stimulated with VEGF, a well-known eNOS stimulator.¹² Indeed, VEGF-induced TF expression in the cells was not modified by L-NAME.¹² Our results contrast with the observations in macrophages and endothelial cells when stimulated with endotoxin or IL-1ß,^17–19 where NO is implicated in suppression of TF expression. The discrepancy between our present study and those studies may be explained by the fact that large amounts of NO are released from iNOS in macrophages or endothelial cells stimulated with endotoxin, whereas stimulation of eNOS by rHDL is relatively weak that is not sufficient to inhibit TF expression under our experimental condition. Moreover, in the experiments where L-arginine was supplemented to enhance NO production, an inhibition of TF production was observed.^18,19 However, a non–NO-mediated role for L-arginine in modulating TF expression cannot be excluded with complete certainty.⁴⁸

A recent study by Gratton et al⁴⁹ demonstrated that adenoviral expression of a constitutively active Akt in endothelial cells causes inactivating phosphorylation of MEKK3, which leads to subsequent inhibition of MKK3/MKK6-p38^mapk pathway, and conversely, dominant-negative Akt mutant decreases inactivating phosphorylation of MEKK3 and therefore enhances MKK-p38^mapk activation in response to VEGF, suggesting a negative regulation of p38^mapk pathway by PI3K/Akt pathway. This mechanism, however, does not account for the inhibition of thrombin-induced TF expression by rHDL, because p38^mapk activation by thrombin was not affected by rHDL (see Figure 3). The possibility that rHDL reduces thrombin-induced endothelial TF expression via interfering RhoA activation by PI3K can also be excluded, because adenoviral expression of a constitutively active PI3K mutant did not inhibit thrombin-induced RhoA activation in the cells (Figure 7). Conversely, active RhoA did not interfere with PI3K activity as reported in our previous study.⁴⁰ Therefore, both inhibition of RhoA and activation of PI3K contribute to the suppression of thrombin-induced TF expression by rHDL. The inhibition of RhoA by rHDL can also explain the result presented on Figure 6 showing that rHDL reversed thrombin’s inhibitory effect on Akt activation, because RhoA activation induced by thrombin inactivates Akt in the cells as demonstrated by our previous study.⁴⁰

In conclusion, rHDL inhibits thrombin-induced TF upregulation via inhibition of RhoA and stimulation of PI3K but not Akt/eNOS (Figure 8). This finding may represent a novel atheroprotective mechanism of HDL in clinical settings and implicate the therapeutic potential of rHDL in coronary artery disease.

View larger version (13K): [in this window] [in a new window]   Figure 8. Schematic diagram showing mechanisms of inhibition of endothelial tissue factor expression by HDL. Interaction between RhoA and Akt was not shown.

	Acknowledgments

 
This study was supported by the Swiss National Science Foundation (31-63811.00), Swiss Heart Foundation, Roche Research Foundation, and a ZLB Bioplasma Research grant. Shengsi Zhu was a recipient of Swiss Federal Scholarship and Hema Viswambharan was supported by the Swiss University Conference Program (SUK). We would like to thank the Maternity Unit of Clinique Sainte Anne of Fribourg for providing human umbilical cords for isolation of endothelial cells.

	Footnotes

 
A. Hubsch, P. Lerch, and G. Vergères are employees of ZLB Bioplasma AG. Z. Yang received a research grant from ZLB Bioplasma AG.

Original received July 18, 2003; resubmission received December 8, 2003; revised resubmission received February 13, 2004; accepted February 16, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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