Manuscript Number: THIJ-09-813R2
Title: Paclitaxel induces thrombomodulin downregulation in human aortic endothelial cells Article Type: Laboratory Investigation
Corresponding Author: Mrs. Huey-Chun Huang, Ph.D.
Corresponding Author's Institution: China Medical University First Author: Huang-Joe Wang, M.D., Ph.D.
Order of Authors: Huang-Joe Wang, M.D., Ph.D.; Te-Ling Lu, Ph.D.; Haimei Huang, Ph.D.; Huey-Chun Huang, Ph.D.
Abstract: Patients with paclitaxel-eluting stents are at risk of developing stent thrombosis on premature discontinuation of dual antiplatelet therapy. In this study, we aimed to clarify whether paclitaxel can modulate thrombomodulin (TM) expression in human aortic endothelial cells (HAECs). HAECs were stimulated with paclitaxal. Methoxyphenyl tetrazolium inner salt cell viability assay, Western blotting, real-time PCR, and immunohistochemistry were done. In HAECs, paclitaxel (10−5 to 10−9 mol/L) treatment for 13 h caused significant cytotoxicity at drug concentrations greater than 10−7 mol/L. Paclitaxel (10−5 to 10−9 mol/L) treatment for 5 h downregulated TM expression dose-dependently, persisting even at 13 h. Thrombin-paclitaxel cotreatment did not alter the effect of paclitaxel on TM downregulation. Paclitaxel caused a 0.63-fold decrease in TM mRNA expression, but thrombin cotreatment did not alter this decrease. In-vivo studies confirmed that paclitaxel (10 mg/kg) caused endothelial TM downregulation in mice. In summary, paclitaxel downregulates TM expression irrespective of thrombin stimulation, which is an important factor for patients receiving PESs.
Therefore, further designs of drug-eluting stents should consider the influence of eluting drugs on endothelial thrombogenicity.
May 8, 2010
James T. Willerson, MD
Editor
Texas Heart Institute Journal
Dear Editor
RE: Manuscript Ms. No. THIJ-09-813, R3; Title: "Paclitaxel induces
thrombomodulin downregulation in human aortic endothelial cells" Enclosed please find the revised manuscript file. Thank you for the
constructive comments. The manuscript was revised point-by-point, based on
the reviewer's suggestion. We hope the revision meets your approval for the
publication of the manuscript.
Sincerely Yours,
Huey-Chun Huang, PhD,
Department of Medical Laboratory Science and Biotechnology, China Medical University
Revisions made in the manuscript
Revisions made in response to reviewer#3’s comments:
Reviewer #3: The authors addressed my comments; however, I think that they should conduct some in vivo experiments to back up the in vitro studies reported here, rather than just add to the discussion.
Response: In-vivo experiments have been done to back up our in-vitro study. The
in-vivo study has been addressed in Abstract (page 2, line 6 and page 2, line 13) ,
Material and Methods (page 6, line 6, page 7, line 13 and page 9, line 6), Results
(page 12, line 1) and Discussion (page 14, line 17), Acknowledgments (page 16, line
8), References (Page 21, line 14), Figure 5 A and 5 B, and Figure legends (Page 25,
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Paclitaxel induces thrombomodulin downregulation in human aortic
endothelial cells
Huang-Joe Wang1, 2, M.D., Ph.D., Te-Ling Lu3, Ph.D.; Haimei Huang1,Ph.D.; Huey-Chun Huang4, Ph.D.
1
Institute of Biotechnology, National Tsing Hua University, Taiwan;
2
Division of Cardiology, Department of Medicine, China Medical University Hospital,
Taiwan;
3
School of Pharmacy, China Medical University, Taiwan.;
4
.Department of Medical Laboratory Science and Biotechnology, China Medical
University, Taiwan.
Address correspondence to:
Huey-Chun Huang, PhD, Department of Medical Laboratory Science and
Biotechnology, China Medical University, No. 91 Hsueh-Shih Road,Taichung 40402,
Taiwan. Tel: 886-4-22053366 ext. 7207; Fax: 886-4-22057414; E-mail:
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Abstract
Patients with paclitaxel-eluting stents are at risk of developing stent thrombosis on premature discontinuation of dual antiplatelet therapy. In this study, we aimed to clarify whether paclitaxel can modulate thrombomodulin (TM) expression in human aortic endothelial cells (HAECs). HAECs were stimulated with paclitaxal. Methoxyphenyl tetrazolium inner salt cell viability assay, Western blotting, real-time PCR, and immunohistochemistry were done. In HAECs, paclitaxel (10−5 to 10−9 mol/L) treatment for 13 h caused significant cytotoxicity at drug concentrations greater than 10−7 mol/L. Paclitaxel (10−5 to 10−9 mol/L) treatment for 5 h downregulated TM expression dose-dependently, persisting even at 13 h. Thrombin–paclitaxel cotreatment did not alter the effect of paclitaxel on TM downregulation. Paclitaxel caused a 0.63-fold decrease in TM mRNA expression, but thrombin cotreatment did not alter this decrease. In-vivo studies confirmed that paclitaxel (10 mg/kg) caused endothelial TM downregulation in mice. In summary, paclitaxel downregulates TM expression irrespective of thrombin stimulation, which is an important factor for patients receiving PESs. Therefore, further designs of drug-eluting stents should consider the influence of eluting drugs on endothelial thrombogenicity.
Key words: Endothelial cells; Drug-eluting stent; Paclitaxel; Thrombin;
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Introduction
Since their introduction, drug-eluting stents (DESs) have revolutionized the field of
interventional cardiology. Compared with bare-metal stents, DESs substantially reduce
restenosis and represent a significant advance in percutaneous coronary interventions
(PCI). Accordingly, DESs have been rapidly adopted in clinical practice and are
currently used in the majority of the PCI procedures worldwide [1]. Despite their rapid
acceptance, the major limitation of DESs is the risk of stent thrombosis (1–2%
incidence), which is often associated with myocardial infarction or sudden death [2].
The most significant predicator of DES thrombosis is premature discontinuation of dual
antiplatelet therapy, especially thienopyridines [3,4]. Unfortunately, prolonged use of
dual antiplatelet therapy can result in bleeding events in up to 32.4% of the patients, of
which 85.7% are just minor bleeding cases but cause 11.1% of these patients to
discontinue clopidogrel (the most commonly used theinopyridine), predisposing to
further catastrophic events [5]. Even with continuation of dual antiplatelet therapy, an
aspirin and clopidogrel nonresponder status may develop, which is associated with a
high risk of DES thrombosis or death [6,7].
Paclitaxel is a microtubule-stabilizing drug causing inhibition of smooth muscle cell
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reduction in the restenosis rate among patients receiving paclitaxel-eluting stents (PESs;
Boston Scientific, Natick, MA). PESs were approved by the US Food and Drug
Administration in 2003, and nearly 5 million PESs have been implanted in patients
worldwide [1]. However, PES implantation to treat coronary artery diseases can result in
significant local inflammation in the coronary vascular wall and apoptosis in the
endothelial cells [9]. A large meta-analysis revealed that the risk of late definite stent
thrombosis (>30 days) is increased with PESs compared with bare-metal stents or
sirolimus-eluting stents [10].
Tissue factor (TF) is an initiator of the extrinsic pathway of blood coagulation, the
predominant clotting pathway for thrombin generation in vivo [11,12]. This
procoagulant cascade is closely controlled. One of the most important anti-coagulation
pathways is thrombomodulin (TM) - protein C - endothelial cell protein C receptor
(EPCR) system [13]. TM, a transmembrane glycoprotein binding with high affinity to
thrombin, is constitutively synthesized by endothelial cells. The procoagulant properties
of thrombin are lost upon binding of functionally important exosite I of thrombin to TM,
and antithrombin can efficiently inhibit TM-bound thrombin [14]. In addition, TM is an
important cofactor for thrombin-mediated activation of protein C, and protein C
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activation can be further enhanced by 20-fold when protein C is bound to EPCRs [15].
Activated protein C, facilitated by protein S, exerts a potent anticoagulation effect by
destroying factors Va and VIIIa and suppressing further thrombin production. Under
normal conditions, anticoagulant forces prevail over the procoagulant forces to maintain
blood fluidity [16].
Stähli et al. [17] and Wang et al. [18] have reported that paclitaxel can enhance
thrombin-induced endothelial TF expression, which can partly explain the
PES-associated thrombotic risks. However, it is unknown whether paclitaxel can
modulate TM expression in endothelial cells. Therefore, in this study, we aimed to
clarify the effect of paclitaxel on TM expression in human aortic endothelial cells
(HAECs).
Materials and methods
Cell culture
HAECs were purchased from Cell Applications, Inc. (San Diego, CA), and cultured
in the endothelial cell growth medium (Cell Applications, Inc.) according to the
manufacturer’s recommendations. The cells were grown to near confluence in
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serum-starved conditions in M-199 medium supplemented with 1% fetal bovine
serum (FBS; HyClone, Logan, UT). Paclitaxel was then added to the dishes for 5 or
13, and 4 h for TM protein analysis, and TM mRNA analysis. For human thrombin (1
unit/mL; Sigma-Aldrich, St. Louis, MO) stimulation assays (thrombin alone or in
combination with paclitaxel), thrombin was added 1 h after paclitaxel treatment.
Animals
Female BALB/c mice (age 8 to 10 weeks) with a mean weight of 24 g were used
in all experiments, The mice were handled and housed according to institutional
guidelines. Mice were anesthetized with intraperitoneal injection of 0.4 g/kg chloral
hydrate. Paclitaxel was diluted with DMSO and then injected into the tail vein in a
volume of 0.14 ml at a concentration of 10 mg/kg with a 27-gauge needle. The same
volume of DMSO was used in control mice. Five hours after paclitaxel treatment,
mice were sacrificed and the aortas were harvested for Western blotting and
immunohistochemistry assay.
Methoxyphenyl tetrazolium inner salt cell viability assay
HAECs were seeded in a 96-well plate to a final concentration of 1 × 104 cells/well. After serum-starvation for 24 h and treatment, 20 µL of methoxyphenyl tetrazolium
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cultivated at 37°C for 2 h. The absorbance was then recorded at 490 nm. During the
initial viability test, we treated human aortic endothelial cells with paclitaxel for 6, 13
and 24 h. The serum starvation design before paclitaxel treatment to HAECs caused
only few endothelial viable at 24 h.
Western blotting
The HAECs 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
(SDS-PAGE). The proteins were transferred onto a poly(vinylidene difluoride) PVDF
membrane by a semidry transfer method at 5 V for 100 min. Antibodies against
thrombomodulin (Santa Cruz Biotechnology, Santa Cruz, CA) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology) were
used at concentrations of 1:2000 and 1:3000, respectively. All the blots were normalized
to the blot for GAPDH expression and probed for the whole cell lysates. The mouse
aorta was homogenized in 100 μL of RIPA buffer (Millipore-Upstate, Billerica, MA) with freshly added complete protease inhibitor cocktail solution (Roche
Diagnostics, Basel, Switzerland) 5 hours after paclitaxel or DMSO vehicle
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and GAPDH (Santa Cruz Biotechnology) were used at a concentration of 1:1000
and 1:3000, respectively.
Real-time polymerase chain reaction analysis
Endothelial cells were harvested by trypsinization and obtained as a pellet. Total
ribonucleic acid (RNA) was extracted from the pellet using a Micro-to-Midi total RNA
purification system (Invitrogen, Carlsbad, CA). Complementary deoxyribonucleic acid
(cDNA) was synthesized from the total RNA using Moloney murine leukemia virus
reverse transcriptase (M-MLV RT, Superscript II; Invitrogen), and polymerase chain
reaction (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 primer sequences were as follows:
TM: 5′-GGCCAAGATGGAGTACAAGTGC-3′ (forward)
and 5′-CGCCAGTTAGCCATGGAATAGA-3′ (reverse)
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and 5′-TGAAGACGCCAGTGGACTCC-3′ (reverse)
Real-time PCR was performed using the following parameters: one cycle at 95°C
for 10 min; and 40 cycles at 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min.
GAPDH mRNA expression served as a loading control and a melting-curve analysis
was performed after amplification to verify the accuracy of the amplicon.
Immunohistochemistry
Immunohistochemistry assay was performed with the Bond-Max autostainer
(Leica Microsystems, Taiwan Branch). Briefly, formalin-fixed and
paraffin-embedded tissue specimens were deparaffinized in xylene, rehydrated
through serial dilutions of alcohol, and washed in phosphate-buffered saline (pH
7.2). Slides were stained with thrombomodulin monoclonal antibodies (Dako North
America, Carpinteria, CA) at 1:100 dilution on the fully automated Bond-Max
system using onboard heat induced antigen retrieval and a VBS Refine polymer
detection system (Leica Microsystems). Diaminobenzidine (Leica Microsystems)
was used as the chromogen in all these immunostainings.
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Statistical analyses were performed using SPSS 12.0 statistical software for Windows
(SPSS Inc., Chicago, IL, USA). All data are presented as the mean ± standard error of
the mean (SEM). The significance was determined by unpaired t-test, and P values less
than 0.05 were considered statistically significant.
Results
Paclitaxel was cytotoxic to the HAECs at 13 h
MTS cell viability assay revealed that the treatments with paclitaxel (10–5 mol/L for 6 h), thrombin (1 unit/mL for 5 h), and paclitaxel (10–5 mol/L for 6 h)/thrombin (1 unit/mL for 5 h) were not toxic to HAEC (figure 1A). These results suggested that
neither paclitaxel nor paclitaxel/thrombin cotreatment could cause significant damage to
HAEC for up to 6 h. However, paclitaxel (10−5 to 10−9 mol/L) treatment for 13 h caused significant cytotoxicity at drug concentrations greater than 10−7 mol/L (Fig. 1B). These results suggested that paclitaxel exerted an antiproliferative effect on the HAECs at 13
h.
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In the Western blotting analysis, paclitaxel (10−5 to 10−9 mol/L for 5 h) downregulated the expression of TM protein in a dose-dependent manner compared
with the control (Fig. 2).
We noted that paclitaxel alone caused down-regulation of TM protein expression at
different time points (6, 9 and 13 hr). The blots of 6 and 9 h were omitted for their
redundancy. We chose 13 h as the longest time to examine the effect of paclitaxel on
TM protein expression, at a time when around 90% HEACs still remained their viability.
Paclitaxel (10−5 mol/L) caused persistent downregulation of TM protein expression at 13 h (Fig. 3), at a time when paclitaxel still caused tissue factor upregulation (data not
shown). The addition of thrombin (1 unit/mL) did not alter the effect of paclitaxel on
TM downregulation at 13 h.
Paclitaxel downregulated TF mRNA expression
In the real-time PCR analysis, paclitaxel (10−5 mol/L for 4 h) caused a 0.63-fold decrease in TM mRNA expression (Fig. 4), but paclitaxel–thrombin (1 unit/mL for 3 h)
cotreatment did not alter the magnitude of TM downregulation induced by paclitaxel
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Paclitaxel downregulated TM expression in-vivo
In the Western blotting analysis, paclitaxel (10 mg/kg for 5 h) caused a decrease
in TM protein expression compared with DMSO vehicle control in mouse aorta
(Fig 5A). In immunohistochemistry assay, TM expression was demonstrated within
the endothelium of DMSO vehicle treated aorta (5B). In contrast, treatment with
paclitaxel (10 mg/kg for 5 h) caused a significant reduction in endothelial TM
expression (Fig 5C).
Discussion
The major findings of this study are as follows: paclitaxel can downregulate the
mRNA and protein expression of TM; the effect of paclitaxel on TM expression is
dose-dependent, and persists for up to 13 h, during which HAEC cell viability decreases
significantly at drug concentrations greater than 10−7 mol/L; and addition of thrombin to paclitaxel does not alter the ability of paclitaxel to downregulate TM expression.
Some mediators have been reported to downregulate TM (e.g., tumor necrosis
factor-α, interleukin 1, endotoxin, homocyteine, hypoxia, glucose modified protein, etc.)
[13]. Our finding revealed that paclitaxel alone causes a significant decrease in TM
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TM downregulation is preceded by a comparable magnitude of mRNA decrease. Both
the endothelial viability and the TM level were decreased at 13 h, implying a
predisposition to thrombotic complications after PES implantation, especially in patients
with inadequately implanted PESs or in aspirin and clopidogrel nonresponders
[7,19,20].
Thrombin causes a controversial effect on TM [13]. Maruyama et al. [21] reported
that thrombin causes a decrease in the availability and function of TM in the
endothelium due to the effect of thrombin causing internalization of the thrombin–TM
complex by human umbilical vein endothelial cells and A549 lung-cancer cells. In
contrast, Beretz et al. [22] did not observed such endocytosis in human saphenous vein
endothelial cells (HSVECs) and endothelial cell line EA.hy 926. Further, thrombin
increased TM mRNA synthesis in the HSVECs but did not alter the surface antigen in
the cells [23]. Archipoff et al. [24] also reported the absence of the effect of thrombin on
TM expression in HSVECs. Utilization of different endothelial cell sources, treatment
protocols, and detection methods clearly explained the conflicting results. Our data are
similar to the report by Archipoff et al., and we did not observe an effect of thrombin on
TM expression in the HAECs.
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stimulation can aggravate TF expression in paclitaxel-treated HAECs [17,18].
Furthermore, paclitaxel can cause downregulation of TM expression in HAECs, as
shown in our data. Such reciprocal regulation of TF and TM in endothelial cells by
paclitaxel is similar to other inflammatory mediators, including tumor necrosis factor-α,
interleukin 1, lipopolysaccharide, and so on [12,13]. In the clinical scenario, as
thrombin is generated in PES coronary plaques, paclitaxel can accentuate TF expression
in the thrombin-producing milieu, although TM expression was decreased by paclitaxel.
These situations cause an imbalance of the procoagulant and anticoagulant, due to
which patients receiving PESs are prone to thrombotic complications. These findings
may explain the observation that premature withdrawal of dual antiplatelet therapy is
particularly hazardous after PES implantation.
Joner et al. reported that TM expression was absent or weak in rabbits receiving
different DESs, including sirolimus, zotarolimus, everolimus and paclitaxel-eluting
stents (25). Their study examined the endothelial TM at 14- and 28-days, at a time when
many inflammatory mediators that cause TM downregulation can be induced by the
eluting drugs. Therefore, it is difficult to determine whether paclitaxel per se can cause
TM downregulation from their study. In contrast, our in-vitro and in-vivo data
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the paclitaxel concentration (10 mg/kg) in our study could achieve a plasma
concentration around 3.0x 10-6 mol/L at 1.5 h and 1.5 x 10-6 mol/L at 3 h (26), at a concentration ranges that also caused TM downregulation in our HAEC data.
Paclitaxel is deposited in the blood vessel at concentration 30- to 40 times bulk
concentration because of its hydrophobic binding properties (27). Particularly, the
endovascular implantation of PES results in the highest tissue concentration in the
intima (28). The TAXUS Express2 stent system coats 50 μg paclitaxel in a 2.25-mm diameter (8-mm length) PES and 282 μg paclitaxel in a 4.0-mm (32-mm length) PES
(29). A porcine coronary artery model using a biodegradable paclitaxel-loaded (170 μg)
polylactide stent caused 3.2 μ g/g tissue (corresponding to 3.7 x 10 -6 mol/L) concentration at 28 days(30). Similarly, a rabbit iliac artery model using a commercial
slow-realease PES (137 μg) caused 1.7 μg/g tissue (2.0 x 10 -6 mol/L) concentration at 1 day and 1.3 μg/g tissue (1.5 x 10 -6 mol/L) concentration at 8 days (31). Our data revealed that paclitaxel significantly downregulated TM protein at drug concentrations
of 10 -5 - 10 -6 mol/L. Thus, the paclitaxel dosages used in our study are relevant to local tissue concentrations after PES deployment.
In summary, paclitaxel can cause TM mRNA and protein downregulation in HAECs.
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In HAECs, the effect of paclitaxel on TM downregulation persists even upon thrombin
stimulation. This finding provides insight into stent thrombosis in the patients receiving
PESs. Further efforts to develop a safe DES should consider the influence of eluting
drugs on the thrombogenicity of the underlying endothelium.
Acknowledgments
This study was supported in part by grants from the National Science Council (NSC
97-2314-B-039-026), China Medical University Hospital (DMR-99-005), and China
Medical University (CMU-95- 088, CMU-95-320). The authors thank Ying-Ti Chen
for her valuable technical assistance.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Figure legends
Fig 1. Paclitaxel is toxic to endothelial cells at drug concentrations greater than 10−7 mol/L at 13 h. A. HAEC cell viability was determined by MTS assay. The HAECs are
serum-starvated for 24 hrs and treat with DMSO (0.1%, 6 hrs), thrombin (1 unit/mL, 5
hrs), paclitaxel (10-5 mol/L, 6 hrs), and paclitaxel (10-5 mol/L, 6 hrs) / thrombin (1 unit/mL, 5 hrs). Treatment with thrombin, paclitaxel, and paclitaxel /thrombin do not
cause significant change of cell viability at this time (all P = not significant). B.
HAECs were treated with paclitaxel (10−5 to 10−9 mol/L) for 13 h. Paclitaxel caused cytotoxicity at drug concentrations greater than 10−7 mol/L. The data shown are the mean ± SEM of three independent experiments with duplication in each experiment. *P
= 0.001 for paclitaxel (10−5 mol/L) vs. the control; **P = 0.003 for paclitaxel (10−6 and 10−7 mol/L) vs. the control.
Fig. 2. Paclitaxel decreases TM expression dose-dependently. TM protein expression
was determined by Western blot analysis. After serum-starvation, HAECs were treated
with paclitaxel (10−5 to 10−9 mol/L) for 5 h. Paclitaxel downregulated the expression of TM in a dose-dependent manner. The blot represents results from three different
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
experiments. All the blots were normalized to the blot for GAPDH. The bars represent
the mean ± SEM of three experiments. *P < 0.001 for paclitaxel (10−5 mol/L) vs. the control; *P < 0.05 for paclitaxel (10−6 mol/L) vs. the control.
Fig. 3. Paclitaxel causes persistent downregulation of TM expression at 13 h. TM
protein expression was determined by Western blot analysis. HAECs were treated with
paclitaxel (10−5 mol/L) for 13 h. Paclitaxel caused persistent TM downregulation at 13 h irrespective of the presence or absence of thrombin. The blot represents results from
three different experiments. *P < 0.05 vs control.
Fig. 4. Paclitaxel induces TM mRNA downregulation. The relative quantity of TF
mRNA was determined by real-time PCR. HAECs were treated with DMSO (0.1% for 4
h), thrombin (1 unit/mL for 3 h), paclitaxel (10−5 mol/L for 4 h), and paclitaxel (10−5 mol/L for 4 h) with thrombin (1 unit/mL for 3 h). Paclitaxel caused a 0.63-fold decrease
in TM mRNA expression. Thrombin cotreatment did not cause a further change in TM
mRNA expression compared with paclitaxel alone. The bars represent the mean ± SEM
of five experiments. *P < 0.01 for paclitaxel vs. the control; **P < 0.05 for paclitaxel
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
Fig 5. Paclitaxel induces TM downregulation in-vivo, A. TM protein expression in
mouse aorta was determined by Western blot analysis. Mouse was treated with
paclitaxel (10 mg/kg) or DMSO vehicle control for 5 h. Paclitaxel caused a
reduction in TM protein expression compared to DMSO vehicle control. The blot
represents results from three different experiments. B. Mouse endothelial TM
expression was determined by immunohistochemistry assay. Mouse was treated
with DMSO vehicle control for 5 h, TM expression was demonstrated within the
endothelium of DMSO vehicle treated aorta. C. In contrast, paclitaxel (10 mg/kg
for 5 h) markedly reduced endothelial TM expression. The photographs represent
results from three different experiments. Shown are x400 magnification
