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Paclitaxel induces up-regulation of tissue factor in human aortic endothelial cells
International Immunopharmacology 9 (2009) 144–147
Contents lists available at ScienceDirect
International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p
Preliminary report
Paclitaxel induces up-regulation of tissue factor in human aortic endothelial cells Huang-Joe Wang a,b, Haimei Huang b, Yi-Ching Chuang c, Huey-Chun Huang c,⁎ a b c
Division of Cardiology, Department of Medicine, China Medical University Hospital, Taiwan Institute of Biotechnology, National Tsing Hua University, Taiwan Department of Medical Laboratory Science and Biotechnology, China Medical University, Taiwan
a r t i c l e
i n f o
Article history: Received 21 September 2008 Received in revised form 10 October 2008 Accepted 10 October 2008 Keywords: Endothelial cells Drug-eluting stent Stent thrombosis Paclitaxel Tissue factor
a b s t r a c t Patients who underwent paclitaxel-eluting stent implantation are at a risk of developing late stent thrombosis. However, it is unclear whether paclitaxel alone can modulate tissue factor (TF) expression in human aortic endothelial cells (HAEC). HAEC were stimulated with paclitaxel. Western blotting, real-time PCR, and a chromogenic TF activity assay were done. In HAEC, while paclitaxel (10 − 5 mol/L to 10 − 9 mol/L) treatment for 5 h up-regulated the expression of TF in a dose-dependent manner, paclitaxel cotreatment with thrombin further enhanced it. While paclitaxel (10 − 5 mol/L) itself induced a 3.7-fold enhancement in TF activity, its cotreatment along with thrombin elicited a 7.6-fold increase in TF activity. Paclitaxel also caused an 8.1-fold increase in TF mRNA expression, and paclitaxel cotreatment with thrombin caused a 13.6-fold enhancement in TF mRNA expression. In summary, paclitaxel alone can up-regulate endothelial TF expression. These findings are significant for the patients receiving paclitaxel-eluting stents, and they may provide opportunities to develop novel therapeutic strategies for DES thrombosis. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Atherosclerosis is a gradual process that starts early in childhood. It is a systemic disease that affects the intima of large and medium-sized arteries (e.g., the aorta and the coronary, carotid, renal, and peripheral arteries). It may clinically manifest as coronary artery disease, ischemic stroke, and peripheral arterial occlusive disease [1]. In the later stages of life, atherothrombosis, which is defined as an atherosclerotic plaque disruption with superimposed thrombosis, is the leading cause of morbidity and mortality in the developed countries [2]. Percutaneous intervention using coronary metallic stents has been commonly used in the treatment of atherothrombotic lesions in the coronary arteries since the mid-1990s. However, angiographic in-stent restenosis (stenosis with diameter greater than 50%) still occurs in 20–30% of the patients [3]. In order to prevent in-stent restenosis, drug-eluting stents (DES) capable of providing prolonged local delivery of antiproliferative agents (e.g., paclitaxel, sirolimus, zotarolimus, and everolimus) were developed in the early 2000s [4]. However, late DES thrombosis has raised serious concerns due to the significant mortality rate in patients reporting with stent thrombosis [5]. Despite the controversy over the actual risk, recent data suggests a 0.5% higher long-term thrombotic risk associated with DES [6]. However, significantly higher DES thrombosis rates (up to 4.3–6.3%) were reported when DES implantation was performed for off-label ⁎ Corresponding author. Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91 Hsueh-Shih Road, Taichung, 40402 Taiwan. Tel.: +886 4 22053366x7207; fax: +886 4 22057414. E-mail address: [email protected] (H.-C. Huang). 1567-5769/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2008.10.003
conditions, including bifurcation lesions or residual coronary dissections developing after DES implantation [7,8]. Tissue factor (TF) is a key initiator of the extrinsic clotting cascade, which is the predominant coagulation pathway in vivo [9]. The magnitude of the thrombotic processes that are triggered on the ruptured plaques or stented arteries depends on the TF activity [10]. Atherosclerotic plaques contain significant amounts of TF mRNA and antigen, which are associated with macrophage and smooth muscle cells within the plaque [11]. TF in the plaques can bind to factor VIIa, and the TF:VIIa complex can activate factor IX or factor X [12]. Further, factor Xa catalyzes the formation of thrombin to initiate fibrin formation. This chain reaction of coagulation results in thrombus formation in the ruptured plaques. In the stented arteries, DES can delay the healing of the vessel wall, delay endothelization, and cause persistent fibrin deposition [13]. Such effects arising from the use of DES may result in the loss of the antithrombotic endothelial barrier and cause TF-induced coagulation cascades and late stent thrombosis [14,15]. One of the first-generation DES was coated with paclitaxel. Paclitaxel is a microtubule-stabilizing drug that disrupts the cell cycle in the G2/M phase. It is used in DES because of its ability to reduce in-stent restenosis by inhibiting vascular smooth-muscle cell proliferation and migration [16]. Several randomized clinical trials have demonstrated that paclitaxel-eluting stents decrease intimal hyperplasia and in-stent restenosis and reduce revascularization as compared to bare-metal stents [17]. However, the implantation of paclitaxel-coated stents to treat coronary artery diseases can result in significant local inflammation and apoptotic death in the coronary vascular wall and the endothelial cells, respectively [18]. The ensuing in-stent thrombosis can cause significant
H.-J. Wang et al. / International Immunopharmacology 9 (2009) 144–147
morbidity or mortality in combination with the paclitaxel-eluting stent [5,19,20]. Paclitaxel was reported to enhance TF expression in the thrombinstimulated endothelial cells by up-regulating JNK activation [21]. Nonetheless, it is not known if paclitaxel per se can sufficiently stimulate TF expression in the endothelial cells. The current study aims to examine the effect of paclitaxel on TF expression in human aortic endothelial cells (HAEC). 2. Materials and methods 2.1. Cell culture HAEC were purchased from Cell Applications, Inc. (San Diego, CA); they were cultured in the endothelial cell growth medium (Cell Applications, Inc.) according to the manufacturer's recommendations. The cells were grown to near confluence in 10-cm culture dishes before the experiment. The HAEC were grown for 24 h under serum-starved conditions in an M-199 medium supplemented with 1% fetal bovine serum (HyClone, Logan, Utah). Paclitaxel was then added to the dishes for 5 or 6, 6, and 4 h for the TF protein analysis, the TF activity analysis, and the TF mRNA analysis, respectively. For the human thrombin (SigmaAldrich,1 unit/mL) stimulation assays (thrombin alone or in combination with paclitaxel), thrombin was added 1 h after the paclitaxel treatment. 2.2. Methoxyphenyl tetrazolium inner salt (MTS) cell viability assay HAEC were seeded in a 96-well plate to a final concentration of 1 × 104 cells/well. After serum starvation for 24 h, HAEC were treated with DMSO (0.1%, vehicle for 10− 5 mol/L paclitaxel; treatment for 6 h), thrombin (1 unit/mL; 5 h), paclitaxel (10− 5 mol/L; 6 h), and paclitaxel (10− 5 mol/L; 6 h) + thrombin (1 unit/mL; 5 h). Twenty microliters of MTS (0.5 mg/mL, Promega, Madison, WI) were then added to each well, and the cells were cultivated at 37 °C for 2 h. The absorbance was then recorded at 490 nm. 2.3. Western blotting for TF The cells were lysed in 50 mM Tris buffer, and 35 μg of the samples were loaded and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred onto a PVDF membrane by a semidry transfer method at 5 V for 100 min. Antibodies against TF (American Diagnostica, Stamford, CT) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) were used at concentrations of 1:2000 and 1:3000, respectively. All the blots were normalized to the blot for GAPDH expression and were probed for the whole cell lysates.
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2.5. Real-time polymerase chain reaction analysis Endothelial cells were harvested by trypsinization and obtained as a pellet. Total RNA was extracted from the pellet using a Micro-to-Midi total RNA purification system (Invitrogen, Carlsbad, CA). cDNA was synthesized from the total RNA using M-MLV reverse transcriptase (Superscript II, Invitrogen), and PCR was performed using cDNA as the template in a reaction mixture (25 μL) containing a specific primer pair of each cDNA. The cDNA pool obtained by using the reverse transcriptase served as a template for subsequent PCR amplification. PCR amplification was performed in the ABI PRISM 7900 HT sequence detection system using the SYBR PCR kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocols. The primers were designed using Primer Express 2.0 (Applied Biosystems) and were synthesized by Blossom Biotechnologies (Taiwan). The primer sequences were as follows: TF forward primer: 5′-TCCCGAACAGTTAACCGGAA-3′ reverse primer: 5′-GACCACAAATACCACAGCTCCA-3′ GAPDH forward primer: 5′-ATCCCTCCAAAATCAAGTGGG-3′ reverse primer: 5′-TGAAGACGCCAGTGGACTCC-3′ Real-time PCR was performed using the following parameters: 95 °C for 10 min for 1 cycle; 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min for 40 cycles. The GAPDH mRNA expression served as a loading control and a melting-curve analysis was performed after amplification to verify the accuracy of the amplicon. 2.6. Statistical analysis All data are presented as mean± SEM. The significance was determined by the unpaired t test. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Paclitaxel was non-toxic to endothelial cells HAEC were first grown under serum-starved conditions in the M-199 medium containing 1% fetal bovine serum for 24 h before treatment and
2.4. TF activity assay The TF activity in HAEC was analyzed with an Actichrome® TF assay (American Diagnostica) according to the manufacturer's recommendations. Briefly, HAEC were grown, starved, and stimulated in 6-well plates according to the treatment protocols. After the stimulation, the cells were washed twice with phosphate-buffered saline (PBS) and lysed by sonicating them in a buffer (50 mM Tris–HCl, 100 mM NaCl, 0.1% Triton X-100, pH 7.4) for 30 min at 37 °C. The extracted TF was placed in a 96-well microplate and incubated with human Factor VIIa and human Factor X for 15 min at 37 °C. The FVIIa/FXa complex thus formed could cleave a chromogenic substrate Spectrozyme FXa, which was added to each well and incubated for 20 min at 37 °C. This chromogenic reaction was stopped by adding glacial acetic acid after the 20-min incubation period. The data were recorded as the absorbance of the reaction mixtures at 405 nm. A standard curve of different concentrations of lipidated human TF was prepared to ensure that the measurements were recorded in the linear range of detection.
Fig. 1. Paclitaxel enhances the expression of TF in a dose-dependent manner. The expression of TF protein was determined by western blot analysis. After serum starvation, HAEC were treated with paclitaxel (10 − 5 mol/L to 10 − 9 mol/L) for 5 h. Paclitaxel upregulates the expression of TF in a dose-dependent manner. The blot represents results from 3 different experiments. All the blots were normalized to the blot for GAPDH. Bars represent the means ± SEM from 3 experiments. ⁎P b 0.05 vs. the control.
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Fig. 2. Effect of Paclitaxel on the expression of TF is further enhanced by thrombin. HAEC were treated with paclitaxel (10− 5 mol/L) for 6 h. Thrombin (1 unit/mL) was added 1 h after the paclitaxel treatment. The synergic effect of paclitaxel/thrombin cotreatment on the expression of TF is more obvious than that of the paclitaxel treatment alone. The blot represents results from 5 different experiments. All the blots were normalized to the blot for GAPDH. The bars represent the means ± SEM from 5 experiments. ⁎P b 0.01 vs. the control.
then treated according to the treatment protocols. MTS cell viability assay revealed that the treatments with paclitaxel (10− 5 mol/L, 6 h), thrombin (1 unit/mL, 5 h), and paclitaxel (10− 5 mol/L, 6 h)/thrombin (1 unit/mL, 5 h) were not toxic to HAEC (data not shown). These results suggested that neither paclitaxel nor paclitaxel/thrombin cotreatment could cause significant damage to HAEC for up to 6 h. 3.2. Paclitaxel up-regulated the expression of TF in a dose-dependent manner HAEC were treated with paclitaxel (10− 5 mol/L to 10− 9 mol/L) for 5 h. The TF protein expression was determined by western blot analysis. Compared with the control, paclitaxel up-regulated the expression of TF in a dose-dependent manner (Fig. 1). 3.3. Effect of paclitaxel on the expression of TF was further enhanced by thrombin HAEC were treated with paclitaxel (10− 5 mol/L) for 6 h. Thrombin (1 unit/mL) was added 1 h after paclitaxel treatment. The synergic effect of paclitaxel/thrombin cotreatment on the expression of TF was more obvious than the effect of paclitaxel alone (Fig. 2).
Fig. 3. Paclitaxel induces up-regulation of TF activity. TF activity was determined by a colorimetric assay. HAEC were treated with DMSO (0.1%, 6 h), thrombin (1 unit/mL, 5 h), paclitaxel (10− 5 mol/L, 6 h), and paclitaxel (10− 5 mol/L, 6 h)/thrombin (1 unit/mL, 5 h). Paclitaxel caused a 3.7-fold increase in TF activity. Paclitaxel cotreatment with thrombin caused a 7.6-fold enhancement in TF activity. Bars represent the means ± SEM from 6 experiments. ⁎P b 0.05 for paclitaxel vs. control, ⁎⁎P b 0.01 for thrombin vs. control, and † P b 0.005 for paclitaxel/thrombin vs. control.
Fig. 4. Paclitaxel induces up-regulation of TF mRNA production. The relative quantity of TF mRNA was determined by real-time PCR. HAEC were treated with DMSO (0.1%, 4 h), thrombin (1 unit/mL, 3 h), paclitaxel (10− 5 mol/L, 4 h), and paclitaxel (10− 5 mol/L, 4 h)/ thrombin (1 unit/mL, 3 h). Paclitaxel causes an 8.1-fold increase in TF mRNA expression. Thrombin cotreatment with paclitaxel enhances TF mRNA expression by 13.6-fold. Bars represent the means ± SEM from 6 experiments. ⁎P b 0.05 for paclitaxel vs. control, ⁎⁎P b 0.01 for thrombin vs. control, and †P b 0.001 for paclitaxel/thrombin vs. control.
3.4. Paclitaxel induced up-regulation of TF activity HAEC were treated with paclitaxel (10− 5 mol/L) for 6 h. The TF activity was determined by a colorimetric assay. Consistent with the above experiments, while paclitaxel itself caused a 3.7-fold enhancement in TF activity (Fig. 3), its cotreatment with thrombin (1 unit/mL, 5 h) caused a 7.6-fold enhancement in TF activity. 3.5. Paclitaxel up-regulated TF mRNA expression HAEC were treated with paclitaxel (10− 5 mol/L) for 4 h. The relative quantity of TF mRNA was determined by real-time PCR. While paclitaxel caused an 8.1-fold enhancement in TF mRNA expression (Fig. 4), paclitaxel cotreatment with thrombin (1 unit/mL, 3 h) caused a 13.6-fold enhancement in TF mRNA expression. 4. Discussion The major findings of the present study are presented as follows: Paclitaxel per se can sufficiently stimulate enhancement in the mRNA expression and activity of TF protein. The effect of paclitaxel on the expression of TF is dose-dependent. The up-regulated TF protein is functional and can increase the TF activity. Furthermore, increased mRNA production was noted before the increase in TF protein and activity. This phenomenon suggests that TF up-regulation by paclitaxel is under some form of transcriptional control. To our knowledge, this is the first report showing that TF can be up-regulated by paclitaxel alone. Stähli BE et al. reported that paclitaxel could enhance thrombininduced endothelial TF expression, which is consistent with our findings [21]. Interestingly, paclitaxel was not found to alter the expression of basal TF expression in their report. These paradoxical results can be explained by the differences in the 2 study designs. First, while we used an M-199 medium with 1% fetal bovine serum as our starvation medium, Stähli BE et al. utilized 0.5% calf serum. The basal TF level in our western blot did not disappear completely even after serum starvation for 24 h. However, even Stähli's group, which utilized 0.5% calf serum as the starvation medium, did not always show a complete quiescent basal TF level; this was elucidated in another paper by the same author [22]. This basal TF expression was frequently noted even under non-stimulated conditions [23–25]. These findings could be explained by taking into account the stress exerted on the cells during cell culture [26]. Despite the minor basal TF expression, thrombin, paclitaxel, and the combination of both stimulants could cause TF up-regulation in HAEC in our study.
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Second, while Stähli BE et al. pre-treated HAEC with paclitaxel 1 h before thrombin stimulation without disclosing whether the paclitaxel was washed out from medium before the thrombin stimulation, we did not wash out the paclitaxel, irrespective of the thrombin stimulation. After the paclitaxel-eluting DES is deployed, the localized milieu can generate thrombin due to plaque disruption by the DES. During this period, the endothelial cells are continuously exposed to paclitaxel. Therefore, our cotreatment design was more similar to the actual pathological condition. Third, despite the fact that we used the same commercial HAEC, the heterogeneity of the endothelial cells may lead to completely different results [27]. Some patients such as those suffering from diabetes and uremia are prone to atherothrombosis [28,29]. The endothelial cells of diabetic and uremic patients are continuously exposed to hyperglycemic and uremic toxin stresses. Such stresses inevitably cause minor endothelial inflammation, which is similar to our HAEC model. Our data suggested that a stressed endothelium may induce significant amounts of TF by paclitaxel stimulation alone, which explained the higher DES thrombosis rate in diabetic and uremic patients in actual clinical cases [5]. Various agonists and inflammatory cytokines, including tumor necrosis factor-α, interleukin 1, lipopolysaccharide, histamine, vascular endothelial growth factor, and thrombin can up-regulate TF expression in the endothelial cells [10]. Such induction of TF gene expression in the endothelial cells is dependent on 2 binding sites, namely, activator protein 1 (AP1) binding site (proximal 5′-TGAGTCA-3′ and distal 5′TGAATCA-3′) and a non-consensus NF-κB-like binding site (5′CGGAGTTTCC-3′); their transcription factors are c-Fos/c-Jun and c-Rel/ p65, respectively [30–32]. Paclitaxel was reported to activate both the NFκB pathway and AP-1 pathway in murine macrophages and certain human ovarian cancer cell lines [33–35]. The IL-8 promoter activation by paclitaxel was mediated by the AP-1 and NF-κB pathways [33]. Interestingly, IL-8 shares the same non-consensus κB-like site that binds cRel/P65 heterodimers such as the TF promoter [36]. Therefore, it is not surprising that paclitaxel can activate NF-κB and AP-1 pathways in endothelial cells, with resultant TF up-regulation, as shown by our data. Our data pointed out that paclitaxel caused up-regulation of TF in endothelial cells, resulting in an increase in the procoagulant force. These findings may explain the observation that the premature withdrawal of dual antiplatelets is particularly hazardous after the deployment of paclitaxel-eluting stents [5,6]. In summary, paclitaxel per se is sufficient to induce TF expression in HAEC. The transcriptional control exerted by paclitaxel causes an increase in the production of TF mRNA. This finding provides an insight into the stent thrombosis in the patients receiving paclitaxel-eluting DES. Further studies to elucidate the underlying mechanisms of this observation will provide opportunities to develop new therapeutic strategies to prevent paclitaxel-eluting stent thrombosis. Acknowledgements This study was supported in part by grants from the National Science Council (NSC 96-2314-B-039-024), China Medical University Hospital (DMR-96-012, DMR-97-011), and China Medical University (CMU-96-184). References [1] Corti R, Hutter R, Badimon JJ, Fuster V. Evolving concepts in the triad of atherosclerosis, inflammation and thrombosis. J Thromb Thrombolys 2004;17:35–44. [2] Viles-Gonzalez JF, Fuster V, Badimon JJ. Atherothrombosis, a widespread disease with unpredictable and life-threatening consequences. Eur Heart J 2004;25:1197–207. [3] Cutlip DE, Chauhan MS, Baim DS, Ho KK, Popma JJ, Carrozza JP, et al. Clinical restenosis after coronary stenting: perspectives from multicenter clinical trials. J Am Coll Cardiol 2002;40:2082–9. [4] Popma JJ, Baim DS, Resnic FS. Percutaneous coronary and valvular intervention. In: Libby P, Bonow RO, Mann DL, Zipes DP, editors. Heart disease: a textbook of cardiovascular medicine. eighth ed. Saunders Elsevier; 2008. p. 1429–36.
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Contents lists available at ScienceDirect
International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p
Preliminary report
Paclitaxel induces up-regulation of tissue factor in human aortic endothelial cells Huang-Joe Wang a,b, Haimei Huang b, Yi-Ching Chuang c, Huey-Chun Huang c,⁎ a b c
Division of Cardiology, Department of Medicine, China Medical University Hospital, Taiwan Institute of Biotechnology, National Tsing Hua University, Taiwan Department of Medical Laboratory Science and Biotechnology, China Medical University, Taiwan
a r t i c l e
i n f o
Article history: Received 21 September 2008 Received in revised form 10 October 2008 Accepted 10 October 2008 Keywords: Endothelial cells Drug-eluting stent Stent thrombosis Paclitaxel Tissue factor
a b s t r a c t Patients who underwent paclitaxel-eluting stent implantation are at a risk of developing late stent thrombosis. However, it is unclear whether paclitaxel alone can modulate tissue factor (TF) expression in human aortic endothelial cells (HAEC). HAEC were stimulated with paclitaxel. Western blotting, real-time PCR, and a chromogenic TF activity assay were done. In HAEC, while paclitaxel (10 − 5 mol/L to 10 − 9 mol/L) treatment for 5 h up-regulated the expression of TF in a dose-dependent manner, paclitaxel cotreatment with thrombin further enhanced it. While paclitaxel (10 − 5 mol/L) itself induced a 3.7-fold enhancement in TF activity, its cotreatment along with thrombin elicited a 7.6-fold increase in TF activity. Paclitaxel also caused an 8.1-fold increase in TF mRNA expression, and paclitaxel cotreatment with thrombin caused a 13.6-fold enhancement in TF mRNA expression. In summary, paclitaxel alone can up-regulate endothelial TF expression. These findings are significant for the patients receiving paclitaxel-eluting stents, and they may provide opportunities to develop novel therapeutic strategies for DES thrombosis. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Atherosclerosis is a gradual process that starts early in childhood. It is a systemic disease that affects the intima of large and medium-sized arteries (e.g., the aorta and the coronary, carotid, renal, and peripheral arteries). It may clinically manifest as coronary artery disease, ischemic stroke, and peripheral arterial occlusive disease [1]. In the later stages of life, atherothrombosis, which is defined as an atherosclerotic plaque disruption with superimposed thrombosis, is the leading cause of morbidity and mortality in the developed countries [2]. Percutaneous intervention using coronary metallic stents has been commonly used in the treatment of atherothrombotic lesions in the coronary arteries since the mid-1990s. However, angiographic in-stent restenosis (stenosis with diameter greater than 50%) still occurs in 20–30% of the patients [3]. In order to prevent in-stent restenosis, drug-eluting stents (DES) capable of providing prolonged local delivery of antiproliferative agents (e.g., paclitaxel, sirolimus, zotarolimus, and everolimus) were developed in the early 2000s [4]. However, late DES thrombosis has raised serious concerns due to the significant mortality rate in patients reporting with stent thrombosis [5]. Despite the controversy over the actual risk, recent data suggests a 0.5% higher long-term thrombotic risk associated with DES [6]. However, significantly higher DES thrombosis rates (up to 4.3–6.3%) were reported when DES implantation was performed for off-label ⁎ Corresponding author. Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91 Hsueh-Shih Road, Taichung, 40402 Taiwan. Tel.: +886 4 22053366x7207; fax: +886 4 22057414. E-mail address: [email protected] (H.-C. Huang). 1567-5769/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2008.10.003
conditions, including bifurcation lesions or residual coronary dissections developing after DES implantation [7,8]. Tissue factor (TF) is a key initiator of the extrinsic clotting cascade, which is the predominant coagulation pathway in vivo [9]. The magnitude of the thrombotic processes that are triggered on the ruptured plaques or stented arteries depends on the TF activity [10]. Atherosclerotic plaques contain significant amounts of TF mRNA and antigen, which are associated with macrophage and smooth muscle cells within the plaque [11]. TF in the plaques can bind to factor VIIa, and the TF:VIIa complex can activate factor IX or factor X [12]. Further, factor Xa catalyzes the formation of thrombin to initiate fibrin formation. This chain reaction of coagulation results in thrombus formation in the ruptured plaques. In the stented arteries, DES can delay the healing of the vessel wall, delay endothelization, and cause persistent fibrin deposition [13]. Such effects arising from the use of DES may result in the loss of the antithrombotic endothelial barrier and cause TF-induced coagulation cascades and late stent thrombosis [14,15]. One of the first-generation DES was coated with paclitaxel. Paclitaxel is a microtubule-stabilizing drug that disrupts the cell cycle in the G2/M phase. It is used in DES because of its ability to reduce in-stent restenosis by inhibiting vascular smooth-muscle cell proliferation and migration [16]. Several randomized clinical trials have demonstrated that paclitaxel-eluting stents decrease intimal hyperplasia and in-stent restenosis and reduce revascularization as compared to bare-metal stents [17]. However, the implantation of paclitaxel-coated stents to treat coronary artery diseases can result in significant local inflammation and apoptotic death in the coronary vascular wall and the endothelial cells, respectively [18]. The ensuing in-stent thrombosis can cause significant
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morbidity or mortality in combination with the paclitaxel-eluting stent [5,19,20]. Paclitaxel was reported to enhance TF expression in the thrombinstimulated endothelial cells by up-regulating JNK activation [21]. Nonetheless, it is not known if paclitaxel per se can sufficiently stimulate TF expression in the endothelial cells. The current study aims to examine the effect of paclitaxel on TF expression in human aortic endothelial cells (HAEC). 2. Materials and methods 2.1. Cell culture HAEC were purchased from Cell Applications, Inc. (San Diego, CA); they were cultured in the endothelial cell growth medium (Cell Applications, Inc.) according to the manufacturer's recommendations. The cells were grown to near confluence in 10-cm culture dishes before the experiment. The HAEC were grown for 24 h under serum-starved conditions in an M-199 medium supplemented with 1% fetal bovine serum (HyClone, Logan, Utah). Paclitaxel was then added to the dishes for 5 or 6, 6, and 4 h for the TF protein analysis, the TF activity analysis, and the TF mRNA analysis, respectively. For the human thrombin (SigmaAldrich,1 unit/mL) stimulation assays (thrombin alone or in combination with paclitaxel), thrombin was added 1 h after the paclitaxel treatment. 2.2. Methoxyphenyl tetrazolium inner salt (MTS) cell viability assay HAEC were seeded in a 96-well plate to a final concentration of 1 × 104 cells/well. After serum starvation for 24 h, HAEC were treated with DMSO (0.1%, vehicle for 10− 5 mol/L paclitaxel; treatment for 6 h), thrombin (1 unit/mL; 5 h), paclitaxel (10− 5 mol/L; 6 h), and paclitaxel (10− 5 mol/L; 6 h) + thrombin (1 unit/mL; 5 h). Twenty microliters of MTS (0.5 mg/mL, Promega, Madison, WI) were then added to each well, and the cells were cultivated at 37 °C for 2 h. The absorbance was then recorded at 490 nm. 2.3. Western blotting for TF The cells were lysed in 50 mM Tris buffer, and 35 μg of the samples were loaded and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred onto a PVDF membrane by a semidry transfer method at 5 V for 100 min. Antibodies against TF (American Diagnostica, Stamford, CT) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) were used at concentrations of 1:2000 and 1:3000, respectively. All the blots were normalized to the blot for GAPDH expression and were probed for the whole cell lysates.
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2.5. Real-time polymerase chain reaction analysis Endothelial cells were harvested by trypsinization and obtained as a pellet. Total RNA was extracted from the pellet using a Micro-to-Midi total RNA purification system (Invitrogen, Carlsbad, CA). cDNA was synthesized from the total RNA using M-MLV reverse transcriptase (Superscript II, Invitrogen), and PCR was performed using cDNA as the template in a reaction mixture (25 μL) containing a specific primer pair of each cDNA. The cDNA pool obtained by using the reverse transcriptase served as a template for subsequent PCR amplification. PCR amplification was performed in the ABI PRISM 7900 HT sequence detection system using the SYBR PCR kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocols. The primers were designed using Primer Express 2.0 (Applied Biosystems) and were synthesized by Blossom Biotechnologies (Taiwan). The primer sequences were as follows: TF forward primer: 5′-TCCCGAACAGTTAACCGGAA-3′ reverse primer: 5′-GACCACAAATACCACAGCTCCA-3′ GAPDH forward primer: 5′-ATCCCTCCAAAATCAAGTGGG-3′ reverse primer: 5′-TGAAGACGCCAGTGGACTCC-3′ Real-time PCR was performed using the following parameters: 95 °C for 10 min for 1 cycle; 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min for 40 cycles. The GAPDH mRNA expression served as a loading control and a melting-curve analysis was performed after amplification to verify the accuracy of the amplicon. 2.6. Statistical analysis All data are presented as mean± SEM. The significance was determined by the unpaired t test. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Paclitaxel was non-toxic to endothelial cells HAEC were first grown under serum-starved conditions in the M-199 medium containing 1% fetal bovine serum for 24 h before treatment and
2.4. TF activity assay The TF activity in HAEC was analyzed with an Actichrome® TF assay (American Diagnostica) according to the manufacturer's recommendations. Briefly, HAEC were grown, starved, and stimulated in 6-well plates according to the treatment protocols. After the stimulation, the cells were washed twice with phosphate-buffered saline (PBS) and lysed by sonicating them in a buffer (50 mM Tris–HCl, 100 mM NaCl, 0.1% Triton X-100, pH 7.4) for 30 min at 37 °C. The extracted TF was placed in a 96-well microplate and incubated with human Factor VIIa and human Factor X for 15 min at 37 °C. The FVIIa/FXa complex thus formed could cleave a chromogenic substrate Spectrozyme FXa, which was added to each well and incubated for 20 min at 37 °C. This chromogenic reaction was stopped by adding glacial acetic acid after the 20-min incubation period. The data were recorded as the absorbance of the reaction mixtures at 405 nm. A standard curve of different concentrations of lipidated human TF was prepared to ensure that the measurements were recorded in the linear range of detection.
Fig. 1. Paclitaxel enhances the expression of TF in a dose-dependent manner. The expression of TF protein was determined by western blot analysis. After serum starvation, HAEC were treated with paclitaxel (10 − 5 mol/L to 10 − 9 mol/L) for 5 h. Paclitaxel upregulates the expression of TF in a dose-dependent manner. The blot represents results from 3 different experiments. All the blots were normalized to the blot for GAPDH. Bars represent the means ± SEM from 3 experiments. ⁎P b 0.05 vs. the control.
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Fig. 2. Effect of Paclitaxel on the expression of TF is further enhanced by thrombin. HAEC were treated with paclitaxel (10− 5 mol/L) for 6 h. Thrombin (1 unit/mL) was added 1 h after the paclitaxel treatment. The synergic effect of paclitaxel/thrombin cotreatment on the expression of TF is more obvious than that of the paclitaxel treatment alone. The blot represents results from 5 different experiments. All the blots were normalized to the blot for GAPDH. The bars represent the means ± SEM from 5 experiments. ⁎P b 0.01 vs. the control.
then treated according to the treatment protocols. MTS cell viability assay revealed that the treatments with paclitaxel (10− 5 mol/L, 6 h), thrombin (1 unit/mL, 5 h), and paclitaxel (10− 5 mol/L, 6 h)/thrombin (1 unit/mL, 5 h) were not toxic to HAEC (data not shown). These results suggested that neither paclitaxel nor paclitaxel/thrombin cotreatment could cause significant damage to HAEC for up to 6 h. 3.2. Paclitaxel up-regulated the expression of TF in a dose-dependent manner HAEC were treated with paclitaxel (10− 5 mol/L to 10− 9 mol/L) for 5 h. The TF protein expression was determined by western blot analysis. Compared with the control, paclitaxel up-regulated the expression of TF in a dose-dependent manner (Fig. 1). 3.3. Effect of paclitaxel on the expression of TF was further enhanced by thrombin HAEC were treated with paclitaxel (10− 5 mol/L) for 6 h. Thrombin (1 unit/mL) was added 1 h after paclitaxel treatment. The synergic effect of paclitaxel/thrombin cotreatment on the expression of TF was more obvious than the effect of paclitaxel alone (Fig. 2).
Fig. 3. Paclitaxel induces up-regulation of TF activity. TF activity was determined by a colorimetric assay. HAEC were treated with DMSO (0.1%, 6 h), thrombin (1 unit/mL, 5 h), paclitaxel (10− 5 mol/L, 6 h), and paclitaxel (10− 5 mol/L, 6 h)/thrombin (1 unit/mL, 5 h). Paclitaxel caused a 3.7-fold increase in TF activity. Paclitaxel cotreatment with thrombin caused a 7.6-fold enhancement in TF activity. Bars represent the means ± SEM from 6 experiments. ⁎P b 0.05 for paclitaxel vs. control, ⁎⁎P b 0.01 for thrombin vs. control, and † P b 0.005 for paclitaxel/thrombin vs. control.
Fig. 4. Paclitaxel induces up-regulation of TF mRNA production. The relative quantity of TF mRNA was determined by real-time PCR. HAEC were treated with DMSO (0.1%, 4 h), thrombin (1 unit/mL, 3 h), paclitaxel (10− 5 mol/L, 4 h), and paclitaxel (10− 5 mol/L, 4 h)/ thrombin (1 unit/mL, 3 h). Paclitaxel causes an 8.1-fold increase in TF mRNA expression. Thrombin cotreatment with paclitaxel enhances TF mRNA expression by 13.6-fold. Bars represent the means ± SEM from 6 experiments. ⁎P b 0.05 for paclitaxel vs. control, ⁎⁎P b 0.01 for thrombin vs. control, and †P b 0.001 for paclitaxel/thrombin vs. control.
3.4. Paclitaxel induced up-regulation of TF activity HAEC were treated with paclitaxel (10− 5 mol/L) for 6 h. The TF activity was determined by a colorimetric assay. Consistent with the above experiments, while paclitaxel itself caused a 3.7-fold enhancement in TF activity (Fig. 3), its cotreatment with thrombin (1 unit/mL, 5 h) caused a 7.6-fold enhancement in TF activity. 3.5. Paclitaxel up-regulated TF mRNA expression HAEC were treated with paclitaxel (10− 5 mol/L) for 4 h. The relative quantity of TF mRNA was determined by real-time PCR. While paclitaxel caused an 8.1-fold enhancement in TF mRNA expression (Fig. 4), paclitaxel cotreatment with thrombin (1 unit/mL, 3 h) caused a 13.6-fold enhancement in TF mRNA expression. 4. Discussion The major findings of the present study are presented as follows: Paclitaxel per se can sufficiently stimulate enhancement in the mRNA expression and activity of TF protein. The effect of paclitaxel on the expression of TF is dose-dependent. The up-regulated TF protein is functional and can increase the TF activity. Furthermore, increased mRNA production was noted before the increase in TF protein and activity. This phenomenon suggests that TF up-regulation by paclitaxel is under some form of transcriptional control. To our knowledge, this is the first report showing that TF can be up-regulated by paclitaxel alone. Stähli BE et al. reported that paclitaxel could enhance thrombininduced endothelial TF expression, which is consistent with our findings [21]. Interestingly, paclitaxel was not found to alter the expression of basal TF expression in their report. These paradoxical results can be explained by the differences in the 2 study designs. First, while we used an M-199 medium with 1% fetal bovine serum as our starvation medium, Stähli BE et al. utilized 0.5% calf serum. The basal TF level in our western blot did not disappear completely even after serum starvation for 24 h. However, even Stähli's group, which utilized 0.5% calf serum as the starvation medium, did not always show a complete quiescent basal TF level; this was elucidated in another paper by the same author [22]. This basal TF expression was frequently noted even under non-stimulated conditions [23–25]. These findings could be explained by taking into account the stress exerted on the cells during cell culture [26]. Despite the minor basal TF expression, thrombin, paclitaxel, and the combination of both stimulants could cause TF up-regulation in HAEC in our study.
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Second, while Stähli BE et al. pre-treated HAEC with paclitaxel 1 h before thrombin stimulation without disclosing whether the paclitaxel was washed out from medium before the thrombin stimulation, we did not wash out the paclitaxel, irrespective of the thrombin stimulation. After the paclitaxel-eluting DES is deployed, the localized milieu can generate thrombin due to plaque disruption by the DES. During this period, the endothelial cells are continuously exposed to paclitaxel. Therefore, our cotreatment design was more similar to the actual pathological condition. Third, despite the fact that we used the same commercial HAEC, the heterogeneity of the endothelial cells may lead to completely different results [27]. Some patients such as those suffering from diabetes and uremia are prone to atherothrombosis [28,29]. The endothelial cells of diabetic and uremic patients are continuously exposed to hyperglycemic and uremic toxin stresses. Such stresses inevitably cause minor endothelial inflammation, which is similar to our HAEC model. Our data suggested that a stressed endothelium may induce significant amounts of TF by paclitaxel stimulation alone, which explained the higher DES thrombosis rate in diabetic and uremic patients in actual clinical cases [5]. Various agonists and inflammatory cytokines, including tumor necrosis factor-α, interleukin 1, lipopolysaccharide, histamine, vascular endothelial growth factor, and thrombin can up-regulate TF expression in the endothelial cells [10]. Such induction of TF gene expression in the endothelial cells is dependent on 2 binding sites, namely, activator protein 1 (AP1) binding site (proximal 5′-TGAGTCA-3′ and distal 5′TGAATCA-3′) and a non-consensus NF-κB-like binding site (5′CGGAGTTTCC-3′); their transcription factors are c-Fos/c-Jun and c-Rel/ p65, respectively [30–32]. Paclitaxel was reported to activate both the NFκB pathway and AP-1 pathway in murine macrophages and certain human ovarian cancer cell lines [33–35]. The IL-8 promoter activation by paclitaxel was mediated by the AP-1 and NF-κB pathways [33]. Interestingly, IL-8 shares the same non-consensus κB-like site that binds cRel/P65 heterodimers such as the TF promoter [36]. Therefore, it is not surprising that paclitaxel can activate NF-κB and AP-1 pathways in endothelial cells, with resultant TF up-regulation, as shown by our data. Our data pointed out that paclitaxel caused up-regulation of TF in endothelial cells, resulting in an increase in the procoagulant force. These findings may explain the observation that the premature withdrawal of dual antiplatelets is particularly hazardous after the deployment of paclitaxel-eluting stents [5,6]. In summary, paclitaxel per se is sufficient to induce TF expression in HAEC. The transcriptional control exerted by paclitaxel causes an increase in the production of TF mRNA. This finding provides an insight into the stent thrombosis in the patients receiving paclitaxel-eluting DES. Further studies to elucidate the underlying mechanisms of this observation will provide opportunities to develop new therapeutic strategies to prevent paclitaxel-eluting stent thrombosis. Acknowledgements This study was supported in part by grants from the National Science Council (NSC 96-2314-B-039-024), China Medical University Hospital (DMR-96-012, DMR-97-011), and China Medical University (CMU-96-184). References [1] Corti R, Hutter R, Badimon JJ, Fuster V. Evolving concepts in the triad of atherosclerosis, inflammation and thrombosis. J Thromb Thrombolys 2004;17:35–44. [2] Viles-Gonzalez JF, Fuster V, Badimon JJ. Atherothrombosis, a widespread disease with unpredictable and life-threatening consequences. Eur Heart J 2004;25:1197–207. [3] Cutlip DE, Chauhan MS, Baim DS, Ho KK, Popma JJ, Carrozza JP, et al. Clinical restenosis after coronary stenting: perspectives from multicenter clinical trials. J Am Coll Cardiol 2002;40:2082–9. [4] Popma JJ, Baim DS, Resnic FS. Percutaneous coronary and valvular intervention. In: Libby P, Bonow RO, Mann DL, Zipes DP, editors. Heart disease: a textbook of cardiovascular medicine. eighth ed. Saunders Elsevier; 2008. p. 1429–36.
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