(Circulation. 2000;102:602.)
© 2000 American Heart Association, Inc.
Brief Rapid Communication |
Smoking Increases Tissue Factor Expression in Atherosclerotic Plaques
Implications for Plaque Thrombogenicity
From the Atherosclerosis Research Center, Division of Cardiology and Department of Medicine, Cedars-Sinai Medical Center and University of California School of Medicine, Los Angeles.
Correspondence to Bojan Cercek, MD, Division of Cardiology, Cedars-Sinai Medical Center, Room 5314, 8700 Beverly Blvd, Los Angeles, CA 90048. E-mail cercek{at}cshs.org
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—Smoking increases the risk of atherothrombotic events. To determine whether smoking influences plaque thrombogenicity, we examined the effect of cigarette smoking and aspirin use on tissue factor (TF) expression in atherosclerotic plaques.
Methods and Results—A total of 23 apoE-/- mice were exposed to cigarette smoke with (n=9) or without (n=14) aspirin treatment. Eleven mice who were exposed to filtered room air served as controls. Aortic root plaques of mice exposed to smoke had higher immunoreactivity for TF (14±4% versus 6.4±3%; P=0.0005), vascular cell adhesion molecule-1 (15±4% versus 5±2%; P=0.002), and macrophages (16±5% versus 6±2%; P=0.002) compared with nonsmoking controls. Aspirin treatment attenuated smoking-induced changes in plaque composition. In human plaques obtained by carotid endarterectomy, TF immunoreactivity (8±5% versus 2±2%; P=0.0002) and activity (P=0.03) were higher in the plaques from smokers (n=28) than those from nonsmokers (n=28). Aspirin use was associated with reduced TF expression in smokers (9±8% versus 3±4%; P=0.0017).
Conclusions—Our results suggest increased plaque TF expression and thrombogenicity as a novel mechanism for the increased risk of atherothrombotic events in smokers. Treatment with aspirin may reduce TF expression.
Key Words: smoking • thromboplastin • aspirin • atherosclerosis
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Smoking doubles the risk of myocardial infarction and ischemic stroke.1 The mechanisms by which smoking increases the risk of these atherothrombotic events are not fully understood. Cigarette smoking confers a hypercoagulable state,2 3 but its effect on plaque thrombogenicity is unknown. Tissue factor (TF) plays a key role in thrombus formation after plaque disruption.4 TF forms a high-affinity complex with factors VII and VIIa; this leads to thrombin formation, which in turn activates the clotting cascade and platelets.5 Smoking increases the number of tissue macrophages,6 which are a predominant source of TF in atherosclerotic plaques. We tested the hypothesis that smoking increases plaque TF expression. We further hypothesized that aspirin, an inhibitor of lipopolysaccharide-induced TF upregulation,7 would attenuate smoking-induced TF upregulation.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
ApoE-/- Mice
Six-week-old male apoE-/- mice were fed high cholesterol chow throughout the study period. At 20 weeks of age, 14 mice (smokers) were exposed to the smoke of half of a research, nonfiltered cigarette each day, 5 days a week, for 8 weeks in a specially designed apparatus, which has been described previously.8 An additional 9 mice who were equally exposed to cigarette smoke were treated with aspirin (0.5 mg/kg SC per day). Blood salicylate levels (determined in 3 mice) were maximal (16.8±2 mg/dL) at 60 minutes after injection. The control group consisted of 11 mice (nonsmokers) exposed to filtered air in a similar apparatus. The study was approved by the Institutional Animal Care and Use Committee.
Immunohistochemistry
After euthanasia at 28 weeks of age, the base of the heart and
the first 3 mm of the ascending aorta were embedded in OCT (Tissue
Tek, Allegiance). In each mouse, 3 nonconsecutive, 10-µm-thick
sections of the aortic root were stained for TF (polyclonal sheep
anti-rabbit tissue factor, American Diagnostica). Sections
from 5 mice in each group were stained for the expression of vascular
adhesion molecule (VCAM-1; rat anti-mouse CD 106, PharMingen) and
monocyte/macrophages (rat anti-mouse monocyte/macrophages,
Serotec).
Western Blot
A total of 50 µg of protein from the aortas of 5 mice in each
group were loaded on 12% SDS-PAGE gels, incubated with the anti-TF
antibody and then the HRP-conjugated anti-sheep antibody, and detected
according to the enhanced chemiluminescence protocol
(Amersham).
Aortic TF Expression and Activity
TF procoagulant activity was measured by 1-stage recalcification
clotting time, as described previously.9 A total of 25
µL of mouse plasma (Sigma) was mixed with 25 µL of the aortic
extracts and 25 µL of 25 mmol/L calcium chloride. Clotting time
was measured by a clot timer. For each experiment, a standard curve
with lipidated recombinant TF (American Diagnostica)
was constructed.
Patient Population
A total of 166 consecutive patients underwent carotid
endarterectomy, 92 (55%) for
symptomatic carotid disease (cerebrovascular accident,
transitory ischemic event, or amaurosis fugax) and 74 (44%)
for severe (>80%) asymptomatic carotid artery
stenosis.
Twenty-eight patients (17%) were current smokers, 87 (52%) were former smokers, and 51 (31%) had never smoked (nonsmokers). The plaques of 28 smokers and 28 nonsmokers matched for age, sex, and indications for the carotid endarterectomy were studied for TF expression (n=23 in each group) or activity (n=5 in each group).
TF Expression and Activity in Carotid Plaques
TF expression was determined using computer-assisted morphometry
(Optima 5.1 bioscan) of immunohistochemical staining with murine
anti-human monoclonal antibodies against TF (American
Diagnostica) and was expressed as percent of plaque
area.
Frozen carotid plaque samples were homogenized using CAPS buffer. Protein concentration was determined using the Coomassie blue protein assay (Pierce). TF activity was determined using a commercially available kit (Actichrom TF activity assay, American Diagnostica), which detected TF based on its ability to form TF/VII complex and to cleave Stectrozyme-factor VII, resulting in the release of the pNA chromophore. TF activity is presented as lipidized TF concentration (nmol/L) per 100 µg of protein.
Statistical Analysis
Normally distributed continuous variables are
presented as mean±SD and compared by t test.
Otherwise, the variables are compared by Wilcoxon test.
Parametric variables are presented as percentage
and compared using a 2-tailed 
2 or Fisher
exact test.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
ApoE-/- Mice
The body weight (33±6, 32±4, and 30±3 g; P=0.3) and cholesterol levels (1383±245, 1419±197, and 1339±306 g/dL; P=0.8) were similar in the smoker, nonsmoker, and smoker mice treated with aspirin, respectively.
TF Expression
Plaques from untreated smoker mice had a significantly
larger TF immunoreactive area compared with plaques from nonsmoker mice
(14±4% versus 6.4±3%; P=0.0005) and smoker mice treated
with aspirin (14±4% versus 6.5±4.5%; P=0.002; Figures 1A
through 1C). TF was largely located in
the lipid-rich core and in the shoulders of the plaques.
|
By Western blotting, smokers had a 2.3±0.7-fold greater TF
content compared with nonsmoker mice (P=0.004). Aspirin
treatment reduced TF content to only 1.3±0.17-fold of that of
nonsmoker mice (P=0.07; Figure 1D
).
Smoker mice also had a larger VCAM-1 immunoreactive area compared with nonsmoker mice (15±4% versus 5±2%; P=0.002) and smoker mice treated with aspirin (15±4% versus 5±3%; P=0.02). Increased VCAM-1 expression was associated with greater macrophage immunoreactivity in smoker compared with nonsmoker mice (16±5% versus 6±2%; P=0.002) and with smoker mice treated with aspirin (16±5% versus 7±5%; P=0.002).
Aortic TF Procoagulant Activity
Exposure to smoke was associated with a 2- to 3-fold
increase in TF procoagulant activity compared with nonsmoker mice
(1.96±1.4 compared with 0.75±0.7 nmol/L TF per 100 µg of protein;
n=3 in each group)
Human Carotid Plaque
TF Expression
Carotid plaques obtained from patients undergoing carotid
endarterectomy for symptomatic carotid
disease had significantly higher TF immunoreactivity compared with
plaques from asymptomatic patients (7.4±7% versus
2.2±3%; P<0.04).
Smokers (n=23) and the matched nonsmokers (n=23) had similar
clinical characteristics
(Table). TF
immunoreactivity was detected in 22 of the 23 plaques from smokers
(96%) compared with 14 of the 23 plaques from nonsmokers (61%;
P=0.0098). TF immunoreactive area was significantly larger
in plaques from smokers compared with those from nonsmokers (8±6%
versus 2.2±2%; P=0.0002; Figure 2A). TF colocalized with
macrophage immunoreactivity (Figure 2B).
|
|
TF Activity
TF activity was significantly higher in the plaques from
smokers (n=5) compared with those from nonsmokers (n=5; 14.5±2.3
versus 7±2.6 nmol/L lipidated TF per 100 µg of protein;
P=0.03).
Aspirin and TF Expression
A total of 29 of the patients (63%) were using aspirin
(average dose, 208 mg/d) before surgery. The prevalence of smoking
among patients treated and untreated with aspirin was similar (48%
versus 53%). Aspirin use was associated with a reduced TF-stained area
compared with no aspirin treatment (3.6±4% versus 7.5±5%;
P=0.0053). Although aspirin use was associated with a
significant reduction in TF among smokers (14.5±9% versus 4.4±4%;
P=0.0017), the reduction among nonsmokers was not
statistically significant (3.4±2% versus 2.0±2%;
P=0.4).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The key findings of this study are that (1) exposure to cigarette smoke increases immunoreactivity for TF, VCAM-1, and macrophages in the atherosclerotic plaques of apoE-/- mice and that aspirin attenuates these effects and (2) smoking is associated with increased TF immunoreactivity and activity in human carotid plaques, with attenuation of this effect by prior aspirin use.
Smoking enhances systemic coagulability, as evidenced by increased circulating thrombin generation and activity,3 fibrinogen levels,3 4 and platelet activation.4 Our findings suggest yet another mechanism, enhanced plaque TF content and procoagulant activity, by which smoking may predispose to acute arterial thrombosis. Our finding that aspirin use is associated with reduced plaque TF content suggests another mechanism for the protective effects of aspirin in vascular disease. The clinical significance of these findings is supported by the finding that plaques from patients with asymptomatic severe carotid artery stenosis had lower TF expression compared with those with symptomatic disease, which is similar to findings in coronary atherectomy samples from patients with unstable and stable angina.10
In apoE-/- mice, TF content paralleled VCAM-1 and macrophage immunoreactivity. These findings suggest that smoking may increase plaque macrophage content through increased VCAM-1 expression. A similar mechanism was described in increased monocyte adhesion to endothelial cells exposed to cigarette smoke condensate.11 Whether smoking increases TF expression by enhanced macrophage recruitment alone or if it also upregulates macrophage TF gene expression requires further assessment. Aortic TF procoagulant activity was also higher (2-fold) in mice exposed to smoke. When compared with human plaques, TF activity in mice aortas was several-fold lower. At least in part, this difference can be explained by the dilution of aortic plaque TF procoagulant activity, because only 15% to 20% of the total aortic surface was covered by atheromas.
The effect of aspirin on plaque TF expression observed in our study is similar to that of cholesterol lowering reported in cholesterol-fed rabbits.13 Cholesterol lowering was implied as a potential mechanism for the powerful preventive effect of statins in patients surviving acute myocardial infarction. Our analogous findings suggest an interesting implication for the beneficial effect of aspirin in patients with occlusive arterial disease.
We conclude that exposure to cigarette smoke is associated with a significant increase in atherosclerotic plaque TF expression and activity, which may in part explain the increased atherothrombotic risk associated with smoking.
|
Acknowledgments |
|---|
S. Matetzky was supported by Save A Heart Foundation of Los Angeles, Calif.
Received March 24, 2000; revision received June 16, 2000; accepted June 19, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Ockene IS, Miller NH. Cigarette smoking, cardiovascular disease, and stroke: a statement for healthcare professionals from the American Heart Association. Circulation. 1997;96:3243–3247.
2. Miller GJ, Bauer KA, Cooper JA, et al. Activation of the coagulant pathway in cigarette smokers. Thromb Haemost. 1998;79:549–553.[Medline] [Order article via Infotrieve]
3. Benowitz NL, Fitzgerald GA, Wilson M, et al. Nicotine effects on eicosanoid formation and hemostatic function: comparison of transdermal nicotine and cigarette smoking. J Am Coll Cardiol. 1993;22:1159–1167.[Abstract]
4.
Toschi V, Gallo R, Lettino M, et al. Tissue
factor modulates the thrombogenicity of human atherosclerotic plaques.
Circulation. 1997;95:594–599.
5.
Nemerson Y. Tissue factor and hemostasis.
Blood. 1988;71:1–8.
6. Botti TP, Amin H, Hiltscher L, et al, for the PDAY Research Group. A comparison of the quantitation of macrophage foam cell populations and the extent of apolipoprotein E deposition in developing atherosclerotic lesions in young people: high and low serum thiocyanate groups as an indication of smoking. Atherosclerosis. 1996;124:191–202.[Medline] [Order article via Infotrieve]
7. Mackman N. Regulation of the tissue factor gene. Thromb Haemost. 1997;78:747–754.[Medline] [Order article via Infotrieve]
8.
Hautamaki RD, Kobayashi DK, Senior RM, et al.
Requirement for macrophage elastase for
cigarette-smoke-induced emphysema in mice. Science. 1997;277:2002–2004.
9. Meisel SR, Shimon I, Edgington T, et al. Leukemia inhibitory factor enhances tissue factor expression in human monocyte-derived macrophages: a gp-130-mediated mechanism. Br J Haematol.. 1999;107:747–755.[Medline] [Order article via Infotrieve]
10. Ardissino D, Merlini PA, Arlens R, et al. Tissue-factor antigen and activity in human coronary atherosclerotic plaques. Lancet. 1997;349:769–771.[Medline] [Order article via Infotrieve]
11.
Shen Y, Rattan V, Sultana C, et al. Cigarette
smoke condensate-induced adhesion molecule expression and
transendothelial migration of monocytes. Am
J Physiol. 1996;270:H1624–H1633.
12.
Parry GCN, Mackman N. Transcriptional regulation
of tissue factor expression in human endothelial cells.
Arteriosler Thromb Vasc Biol. 1995;15:612–621.
13.
Aikawa M, Voglic SJ, Sugiyama S, et al. Dietary
lipid lowering reduces tissue factor expression in rabbit
atheroma. Circulation. 1999;100:1215–1222.
This article has been cited by other articles:
 |
|
 |
|
  A. Churg, X. Wang, R. D. Wang, S. C. Meixner, E. L. G. Pryzdial, and J. L. Wright {alpha}1-Antitrypsin Suppresses TNF-{alpha} and MMP-12 Production by Cigarette Smoke-Stimulated Macrophages Am. J. Respir. Cell Mol. Biol., August 1, 2007; 37(2): 144 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Steffel, T. F. Luscher, and F. C. Tanner Tissue Factor in Cardiovascular Diseases: Molecular Mechanisms and Clinical Implications Circulation, February 7, 2006; 113(5): 722 - 731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Raupach, K. Schafer, S. Konstantinides, and S. Andreas Secondhand smoke as an acute threat for the cardiovascular system: a change in paradigm Eur. Heart J., February 2, 2006; 27(4): 386 - 392. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. K. MacCallum Markers of Hemostasis and Systemic Inflammation in Heart Disease and Atherosclerosis in Smokers Proceedings of the ATS, April 1, 2005; 2(1): 34 - 43. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Johnson, L. Mureebe, and D. Silver Hypercoagulable States: A Review Vascular and Endovascular Surgery, March 1, 2005; 39(2): 123 - 133. [Abstract] [PDF]
|
|
|
|
 |
|
 S. Kangavari, S. Matetzky, P. K. Shah, J. Yano, K.-Y. Chyu, M. C. Fishbein, and B. Cercek Smoking Increases Inflammation and Metalloproteinase Expression in Human Carotid Atherosclerotic Plaques Journal of Cardiovascular Pharmacology and Therapeutics, October 1, 2004; 9(4): 291 - 298. [Abstract] [PDF]
|
|
|
|
 |
|
 J. A. Ambrose and R. S. Barua The pathophysiology of cigarette smoking and cardiovascular disease: An update J. Am. Coll. Cardiol., May 19, 2004; 43(10): 1731 - 1737. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. O. Aalami, T. D. Fang, H. M. Song, and R. P. Nacamuli Physiological Features of Aging Persons Arch Surg, October 1, 2003; 138(10): 1068 - 1076. [Full Text] [PDF]
|
|
|
|
 |
|
 N. L. Kobrinsky, M. G. Klug, P. J. Hokanson, D. E. Sjolander, and L. Burd Impact of Smoking on Cancer Stage at Diagnosis J. Clin. Oncol., March 1, 2003; 21(5): 907 - 913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. K. Shah Mechanisms of plaque vulnerability and rupture J. Am. Coll. Cardiol., February 19, 2003; 41(4_Suppl_S): 15S - 22S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. L Ruberg and J. Loscalzo Prothrombotic determinants of coronary atherothrombosis Vascular Medicine, November 1, 2002; 7(4): 289 - 299. [Abstract] [PDF]
|
|
|
|
 |
|
 S. Kiechl, P. Werner, G. Egger, F. Oberhollenzer, M. Mayr, Q. Xu, W. Poewe, and J. Willeit Active and Passive Smoking, Chronic Infections, and the Risk of Carotid Atherosclerosis: Prospective Results From the Bruneck Study Stroke, September 1, 2002; 33(9): 2170 - 2176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Barua, J. A. Ambrose, D. C. Saha, and L.-J. Eales-Reynolds Smoking Is Associated With Altered Endothelial-Derived Fibrinolytic and Antithrombotic Factors: An In Vitro Demonstration Circulation, August 20, 2002; 106(8): 905 - 908. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Eaton, H. L. Byers, N. Leeds, M. A. Ward, and M. J. Shattock Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-Thiolated during Ischemia and Reperfusion J. Biol. Chem., March 15, 2002; 277(12): 9806 - 9811. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. H.M Moons, M. Levi, and R. J.G Peters Tissue factor and coronary artery disease Cardiovasc Res, February 1, 2002; 53(2): 313 - 325. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Undas, K. Brummel, J. Musial, K. G. Mann, and A. Szczeklik Blood coagulation at the site of microvascular injury: effects of low-dose aspirin Blood, October 15, 2001; 98(8): 2423 - 2431. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||