Expression of Tissue Factor in High-Grade Carotid Artery Stenosis. Association With Plaque Destabilization

Transcription

1 Expression of Tissue Factor in High-Grade Carotid Artery Stenosis Association With Plaque Destabilization Sebastian Jander, MD; Matthias Sitzer, MD; Angélique Wendt, MS; Michael Schroeter, MD; Martin Buchkremer, MD; Mario Siebler, MD; Wolfram Müller, MD; Wilhelm Sandmann, MD; Guido Stoll, MD Background and Purpose The procoagulant protein tissue factor (TF) has been implicated in thromboembolic complications associated with advanced atherosclerosis. In this study, we investigated whether TF expression in high-grade stenoses of the internal carotid artery (ICA) is associated with clinical features of plaque destabilization and addressed the relationship between TF expression and plaque inflammation. Methods In 36 consecutive patients undergoing surgery for high-grade ICA stenosis, clinical evidence of plaque instability was provided by the recent occurrence of ischemic symptoms attributable to the stenosis and the detection of cerebral microembolism by means of transcranial Doppler ultrasound monitoring of the ipsilateral middle cerebral artery. Endarterectomy specimens were stained immunocytochemically for TF expression as well as macrophage (CD68) and T cell (CD3) infiltration. Results Morphologically, TF immunoreactivity was codistributed with plaque inflammation and predominantly localized to CD68 macrophages. Accordingly, statistical analysis revealed a significant association of TF expression with plaque infiltration by macrophages (P ) and T cells (P 0.013). Plaques extensively stained for TF (median of TF total section area 40% in semiquantitative assessment) were more frequent in symptomatic (12/27) than in asymptomatic patients (1/9). Conversely, plaques exhibiting little TF expression (median of TF section area 20%) were more frequent in asymptomatic (3/9) than in symptomatic (1/27) patients (P 0.016). Likewise, we found a highly significant association of TF expression with the occurrence of cerebral microembolism (P 0.008). Conclusions Induction of TF at sites of plaque inflammation may play an important role in the destabilization of high-grade ICA stenosis. (Stroke. 2001;32: ) Key Words: atherosclerosis carotid arteries inflammation leukocytes procoagulant Arterio-arterial thromboembolism from extracranial stenoses of the internal carotid artery (ICA) is an important pathomechanism of ischemic stroke. 1,2 However, even highgrade ICA stenoses ( 70% luminal narrowing) carry a highly variable annual risk of stroke that can be as high as 13% after the recent occurrence of transient cerebral or retinal ischemia or as low as 1% to 2% in clinically asymptomatic patients. 3 5 The cellular and molecular mechanisms converting a stable plaque into unstable ICA disease are incompletely understood. In many patients with high-grade ICA stenosis, longterm transcranial Doppler ultrasonography (TCD) can reveal clinically silent formed-element microemboli passing through the ipsilateral middle cerebral artery. 6 The rate of microemboli is higher in recently symptomatic than asymptomatic patients, 7 predicts the occurrence of future ischemic symptoms, 8,9 and declines after carotid endarterectomy. 10 Thus, cerebral microembolism provides reliable paraclinical evidence of plaque destabilization. Inflammatory mechanisms are considered to play a key role in the pathogenesis of atherosclerosis. 11 Plaqueinfiltrating T cells and macrophages are potential sources of matrix-degrading enzymes and thrombogenic substances that have been implicated in the key events of plaque destabilization, ie, rupture of the fibrous cap and subsequent luminal thrombosis, finally leading to the manifestation of acute ischemic syndromes such as stroke or myocardial infarction. 16,17 In patients with high-grade ICA stenosis, several groups have recently found a significant correlation between the extent of inflammatory pathology and the development of plaque-related ischemic complications (see also Received August 23, 2000; final revision received December 1, 2000; accepted December 14, From the Departments of Neurology (S.J., A.W., M.Sch., M.Sie., G.S.), Pathology (W.M.), and Vascular Surgery and Renal Transplantation (W.S.), Heinrich-Heine-University, Düsseldorf, Germany, and the Department of Neurology (M.Sit., M.B.), Johann Wolfgang Goethe-University, Frankfurt am Main, Germany. Correspondence to Dr Sebastian Jander, Department of Neurology, Heinrich-Heine-University, Moorenstr 5, D Düsseldorf, Germany. jander@uni-duesseldorf.de 2001 American Heart Association, Inc. Stroke is available at 850

2 Jander et al Tissue Factor in Unstable ICA Disease 851 TABLE 1. Clinical Features of Symptomatic and Asymptomatic Patients With High-Grade ICA Stenosis Age (mean SD), y Sex, % Male Luminal Narrowing, mean (%) Microemboli Count 0h 1 0h 1 Asymptomatic (n 9) (84.6) 7 2 Symptomatic (n 27) (84.3) 6 21 Golledge et al 22 for a recent review). Tissue factor (TF) is a glycoprotein that is strongly induced in activated inflammatory macrophages and T cells By its ability to bind factor VIIa, TF directly activates the coagulation cascade. Therefore, TF is a candidate molecule linking plaque inflammation with arterial thromboembolism. 26,27 In the present study, we performed an immunocytochemical analysis of TF expression in endarterectomy specimens from 36 consecutive patients undergoing surgery for high-grade ICA stenosis and addressed the relationship of TF expression to clinical features of plaque destabilization and inflammatory plaque infiltration by macrophages and T cells. Subjects and Methods Patients This study prospectively included 36 consecutive surgical inpatients enlisted to undergo carotid endarterectomy for extracranial highgrade ICA stenosis ( 70% luminal narrowing). The study was approved by the local ethics review committee and performed in accordance with institutional guidelines. Informed consent was obtained from all patients before each examination. Baseline characteristics of the study population are provided in Table 1. The degree of luminal narrowing was determined by intra-arterial cerebral angiography, using the criteria of the North American Symptomatic Carotid Endarterectomy Trial (NASCET). 28 Antiplatelet medication (n 30 patients) or oral anticoagulants (n 1) were routinely stopped at least 6 days before operation. At the day of operation, routine coagulation parameters were normal in all patients. During surgery, no anticoagulation was performed. Routine cardiological and medical assessment performed preoperatively did not provide evidence of cardiac embolism or systemic infectious disease. According to NASCET criteria, 28 patients were defined as symptomatic if they had recently ( 121 days before) experienced transient retinal or cerebral symptoms or minor ischemic strokes attributable to the high-grade ICA stenosis. For the assessment of cerebral microembolism, all patients received long-term transcranial Doppler (TCD) signal recording of the middle cerebral artery ipsilateral to the high-grade ICA stenosis for at least 1 hour, as described in detail elsewhere. 10 The patients were monitored 1 to 21 days before endarterectomy (median 4.5 days). Histological Procedures and Immunocytochemistry After longitudinal arteriotomy, the carotid atherosclerotic plaque was excised en bloc (routine endarterectomy), fixed immediately in 4% paraformaldehyde, decalcified, and transversely sectioned at 2-mm intervals. 16 Each 2-mm tissue bloc was embedded separately into paraffin. The quantitative analysis was based on all blocs derived from each individual plaque. The number of blocs examined per plaque was (mean SD). The total number of blocs examined was 403. For immunocytochemistry, 10- m sections were mounted onto gelatin-coated slides. After deparaffinization, sections were incubated with a monoclonal antibody (mab) against human tissue factor (No. 4509, American Diagnostica Inc) at 1:100 dilution, followed by biotinylated horse anti-mouse IgG (Vector Laboratories) and the ABC ELITE kit reagents (Vector) with diaminobenzidine as substrate. In parallel experiments, substitution of the TF-specific primary mab with an irrelevant, isotype-matched control antibody yielded negative results. Furthermore, preadsorption of the TF mab with an excess of human recombinant TF (American Diagnostica) led to a complete disappearance of staining (Figures 1D and 1E). To study the relationship between TF expression and inflammation, serial sections from all blocs were stained with mab KP1 against the macrophage marker CD68 (1:100 dilution) or rabbit polyclonal IgG against human CD3 (1:100) as a T cell marker (both primary antibodies from DAKO). For antigen retrieval, sections were microwaved in 10 mmol/l sodium citrate buffer, ph 6.0, for 10 minutes before staining. Quantification Because TF-immunoreactivity (TF-IR) was distributed both diffusely in the extracellular matrix and in large confluent infiltrates, quantification by cell counting was not possible. In addition, attempts to quantify TF section area by computer-aided planimetry were unsuccessful because of the diffuse appearance of the staining that caused large interrater variability. Therefore, TF expression was determined semiquantitatively by 2 independent observers who were blinded as to the identity and clinical status of the patients. For each section, TF expression was rated as either weak ( 20% of section area staining positively for TF), moderate (20% to 40% TF section area), or abundant ( 40% TF section area). Based on these values, the median of TF section area was calculated for the entire plaque. For the semiquantitative rating procedure, statistics revealed excellent interrater agreement ( 0.96). T-cell counting (number of cells per mm 2 total section area) and macrophage planimetry (% total section area occupied by CD68 cells) were performed as described previously. 21 Statistical Analysis To analyze the relationship between the expression of TF and inflammation, we performed Pearson correlation analyses, including TF section area, the percentage of macrophage-rich areas, and the number of T cells per mm 2 section area determined for each section. To analyze the relationship between the extent of TF expression (median TF total section area of each individual plaque) and the occurrence of ischemic symptoms and cerebral microembolism, respectively, we used the nonparametric Mann-Whitney U test. Because we performed 2 consecutive statistical tests, values of P (0.05/2) were considered indicative of statistically significant findings ( adjustment). Results TF Expression in the Fibrous Cap: Codistribution With Macrophages and T Cells TF immunoreactivity was found in all plaques, although its extent showed large interindividual variation. The median TF section area was 20% in 4 plaques, 20% to 40% in 19 plaques, and 40% in 13 plaques. TF expression was overall strongly accentuated within the atheromatous core and its immediate surroundings (Figure). In the lesion core, TF immunostaining was predominantly diffuse in the extracellular matrix. In contrast, strong cellular immunostaining was found at the transition between atheromatous core and fibrous cap, where large confluent infiltrates stained positively for TF

3 852 Stroke April 2001 TABLE 2. Relationship Between TF Expression and Ischemic Symptoms Ipsilateral Ischemic Symptoms Within Past 120 Days Absent TF Section Area, median Present n % n % 20% % % TF expression was determined semiquantitatively in each plaque section and for each entire plaque the median of TF section area was calculated. Mann-Whitney U test revealed a significant association between TF expression and the occurrence of ischemic symptoms (P 0.016). (Figure, panel A). In addition, TF was localized in cells immediately lining the luminal surface of the plaque (arrowheads). Staining of serial sections for macrophages (B) and T cells (C) revealed that TF immunoreactivity was overall codistributed with inflammatory infiltration and predominantly localized to CD68 macrophages (A, B, and D). To corroborate the relationship between inflammation and TF expression, we quantified the extent of macrophage and T cell infiltration in each plaque section as described previously21 and studied their relationship to TF expression by linear regression analysis. In line with the morphological observations, we found a significant association between TF expression and the percentage of macrophage-rich areas (r 0.335, P ), and the number of T cells per mm2 section area (r 0.124, P 0.013), respectively. Increased TF Expression Is Associated With Plaque Destabilization To clarify the clinical significance of TF expression in carotid artery plaques, we first studied the relationship between TF immunoreactivity and the occurrence of ischemic symptoms attributable to the stenosis during the last 120 days before inclusion into the study (Table 2). Plaques extensively stained for TF (median of TF total section area 40%) were more frequent in symptomatic (12/27 patients; 44%) than in asymptomatic (1/9; 11%) patients. Conversely, plaques exhibiting little TF immunoreactivity (median of TF total section area 20%) were more frequent in asymptomatic (3/9; 33%) than in symptomatic (1/27; 4%) patients. Plaques with an intermediate degree of TF section area (20% to 40%) displayed a similar frequency in both symptomatic and asymptomatic patients. Statistical testing revealed a signifiimmunocytochemical localization of TF (A, D), macrophages (CD68, B), and T cells (CD3, C) in a carotid plaque from a symptomatic patient. TF is expressed both diffusely in the necrotic lesion core (asterisk) and in dense cellular infiltrates at the transition between lesion core and fibrous cap. In addition, some TF cells are localized at the luminal surface of the plaque (arrowheads). Note the similar distribution and morphology of TF and CD68 cells. D, High-power view of TF immunoreactivity. E, TF immunostaining is completely abolished after preadsorption of TF-specific mab with recombinant TF. Scale bars: 100 m in A C, 50 m in D and E.

4 Jander et al Tissue Factor in Unstable ICA Disease 853 TABLE 3. Relationship Between TF Expression and Preoperative Microemboli Count Microemboli Count TF Section 0h 1 0h 1 Area, Median n % n % 20% % % See Table 2 for details concerning TF quantification. Overall, a significant association between TF expression and the occurrence of cerebral microembolism was found (P 0.008). cant association between TF expression and a history of previous ischemic symptoms (P 0.016). To further substantiate the association of TF expression with plaque destabilization, we analyzed the relationship between TF section area and the occurrence of cerebral microembolism in long-term TCD monitoring. Table 3 shows that strongly TF plaques were more frequent in microemboli-positive patients (48% versus 15% in the microemboli-negative group) whereas plaques with little TF immunoreactivity were more frequent in microembolinegative patients (31% versus 0% in microemboli-positive patients). As for ischemic symptoms, statistical testing showed a highly significant association of TF expression with the occurrence of cerebral microembolism (P 0.008). Discussion In our study we have shown that increased expression of TF in high-grade stenoses of the ICA is associated with plaque destabilization evidenced clinically both by a history of previous ischemic symptoms and the detection of microemboli in long-term TCD monitoring of the ipsilateral middle cerebral artery. Morphologically, TF immunoreactivity was codistributed with plaque inflammation and mostly localized to CD68 macrophages. In line with this observation, previous studies showed induction of TF by inflammatory cytokines such as interferon- 23 and CD40 ligand (CD154). 25 Our data therefore strongly suggest that TF induction at sites of plaque inflammation may play an important role in the destabilization of high-grade ICA stenosis. TF has the ability to directly activate the coagulation cascade by the interaction with factors VIIa and X. 24 Accordingly, TF has been implicated in the development of thromboembolism due to advanced atherosclerotic lesions. 26,27 In our present study, most TF immunoreactivity was localized diffusely in the acellular necrotic core and within inflammatory infiltrates in the fibrous cap of the atheroma. However, we also found some TF expression in cells immediately lining the vascular lumen. Thus, exposure of TF activity to coagulation factors may either occur directly at the intimal surface or may result from plaque rupture leading to the release of macrophage-bound or extracellular material from deeper parts of the plaque to the bloodstream. The rupture of complicated plaques has been suggested to be due to the expression of matrix metalloproteinases (MMPs) that degrade extracellular matrix components and thereby weaken the fibrous cap. 13,14 Interestingly, a recent study by Loftus et al 29 indeed indicates a correlation of MMP-9 expression with carotid plaque destabilization. Thus, it is an intriguing hypothesis that the concerted action of MMP-9 and TF may be a key mechanism of plaque destabilization in cerebrovascular disease patients at risk of stroke. A potential limitation of our present study arises from the fact that the sensitive immunohistochemical staining procedure used for the detection of TF antigen does not allow direct conclusions with respect to the actual presence of TF bioactivity. However, in a comparative study, Thiruvikraman et al 15 used both TF-specific antibody and digoxygenin-labeled factors VIIa and X for the in situ detection of TF and observed essentially identical staining patterns with both types of detection reagents. Similar to our immunohistochemical data, the digoxygenin-labeled factors bound to the acellular lipid core and numerous macrophages. It is therefore likely that the TF immunoreactivity detected in our study indeed reflects TF binding activity for its physiologically relevant ligands. On the other hand, TF expression in atherosclerotic plaques has been shown to be paralleled by the induction of an endogenous TF pathway inhibitor (TFPI) 30,31 that may interfere with TF bioactivity in a complex manner. 32,33 Therefore, additional studies using TF as well as TFPI bioassays will be necessary to definitively clarify the role of TF in ICA plaque destabilization. Acknowledgment This work was supported by the Deutsche Forschungsgemeinschaft. Dr Stoll holds a Hermann and Lilly Schilling professorship. References 1. Pessin MS, Hinton RC, Davis KR, Duncan GW, Robertson GH, Ackermann RH, Mohr JP. Mechanisms of acute carotid stroke. Ann Neurol. 1979;6: Fieschi C, Argentino C, Lenzi GL, Sacchetti ML, Toni D, Bozzao L. Clinical and instrumental evaluation of patients with ischemic stroke within the first six hours. J Neurol Sci. 1989;91: Hennerici M, Hülsbömer H-B, Hefter H, Lammerts D, Rautenberg W. Natural history of asymptomatic extracranial arterial disease. Brain. 1987;110: North American Symptomatic Carotid Endarterectomy Trial Collaborators. Beneficial effect of carotid endarterectomy in symptomatic patients with high-grade carotid stenosis. N Engl J Med. 1991;325: European Carotid Surgery Trialists Collaborative Group. MRC European Carotid Surgery Trial: interim results for symptomatic patients with severe (70 99%) or with mild (0 29%) carotid stenosis. Lancet. 1991; 337: Spencer MP, Thomas GI, Nicholls SC, Sauvage LR. Detection of middle cerebral artery emboli during carotid endarterectomy using transcranial Doppler ultrasonography. Stroke. 1990;21: Siebler M, Kleinschmidt A, Sitzer M, Steinmetz H, Freund H-J. Cerebral microembolism in symptomatic and asymptomatic high-grade internal carotid artery stenosis. Neurology. 1994;44: Siebler M, Nachtmann A, Sitzer M, Rose G, Kleinschmidt A, Rademacher J, Steinmetz H. Cerebral microembolism and the risk of ischemia in asymptomatic high-grade internal carotid artery stenosis. Stroke. 1995;26: Molloy J, Markus HS. Asymptomatic embolization predicts stroke and TIA risk in patients with carotid artery stenosis. Stroke. 1999;30: Siebler M, Sitzer M, Rose G, Bendfeldt D, Steinmetz H. Silent cerebral embolism caused by neurologically symptomatic high-grade carotid stenosis: event rates before and after carotid endarterectomy. Brain. 1993; 116:

5 854 Stroke April Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999; 340: Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci U S A. 1989;86: Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994;94: Halpert I, Sires UI, Roby JD, Potter-Perigo S, Wight TN, Shapiro SD, Welgus HG, Wickline SA, Parks WC. Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme. Proc Natl Acad Sci U S A. 1996;93: Thiruvikraman SV, Guha A, Roboz J, Taubman MB, Nemerson Y, Fallon JT. Localization of tissue factor in human atherosclerotic plaques by binding of digoxygenin-labeled factors VIIa and X. Lab Invest. 1996;75: Sitzer M, Müller W, Siebler M, Hort W, Kniemeyer H-W, Jäncke L, Steinmetz H. Plaque ulceration and lumen thrombus are the main sources of cerebral microemboli in high-grade internal carotid artery stenosis. Stroke. 1995;26: Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes (1). N Engl J Med. 1992;326: Bassiouny HS, Sakaguchi Y, Mikucki SA, McKinsey JF, Piano G, Gewertz BL, Glagov S. Juxtalumenal location of plaque necrosis and neoformation in symptomatic carotid stenosis. J Vasc Surg. 1997;26: Carr SC, Farb A, Pearce WH, Virmani R, Yao JST. Activated inflammatory cells are associated with plaque rupture in carotid artery stenosis. Surgery. 1997;122: DeGraba TJ, Siren AL, Penix L, McCarron RM, Hargraves R, Sood S, Pettigrew KD, Hallenbeck JM. Increased endothelial expression of intercellular adhesion molecule-1 in symptomatic versus asymptomatic human carotid atherosclerotic plaque. Stroke. 1998;29: Jander S, Sitzer M, Schumann R, Schroeter M, Siebler M, Steinmetz H, Stoll G. Inflammation in high-grade carotid stenosis: a possible role for macrophages and T cells in plaque destabilization. Stroke. 1998;29: Golledge J, Greenhalgh RM, Davies AH. The symptomatic carotid plaque. Stroke. 2000;31: Del Prete G, De Carli M, Lammel RM, D Elios MM, Daniel KC, Giusti B, Abbate R, Romagnani S. Th1 and Th2 T-helper cells exert opposite regulatory effects on procoagulant activity and tissue factor production by human monocytes. Blood. 1995;86: Camerer E, Kolsto A-B, Prydz H. Cell biology of tissue factor, the principal initiator of blood coagulation. Thromb Res. 1996;81: Mach F, Schönbeck U, Bonnefoy J-Y, Pober JS, Libby P. Activation of monocyte/macrophage functions related to acute atheroma complication by ligation of CD40: induction of collagenase, stromelysin, and tissue factor. Circulation. 1997;96: Moreno PR, Bernardi VH, López-Cuëllar J, Muria AM, Palacios IF, Gold HK, Mehran R, Sharma SK, Nemerson Y, Fuster V, Fallon JT. Macrophages, smooth muscle cells, and tissue factor in unstable angina: implications for cell-mediated thrombogenicity in acute coronary syndromes. Circulation. 1996;94: Toschi V, Gallo R, Lettino M, Fallon JT, Gertz SD, Fernández-Ortiz A, Chesebro JH, Badimon L, Nemerson Y, Fuster V, Badimon JJ. Tissue factor modulates the thrombogenicity of human atherosclerotic plaques. Circulation. 1997;95: North American Symptomatic Carotid Endarterectomy Trial (NASCET) Steering Committee. North American Symptomatic Carotid Endarterectomy Trial: methods, patient characteristics, and progress. Stroke. 1991; 22: Loftus IM, Naylor AR, Goodall S, Crowther M, Jones L, Bell PR, Thompson MM. Increased matrix metalloproteinase-9 activity in unstable carotid plaques: a potential role in acute plaque disruption. Stroke. 2000; 31: Drew AF, Davenport P, Apostolopoulos J, Tipping PG. Tissue factor pathway inhibitor expression in atherosclerosis. Lab Invest. 1997;77: Kaikita K, Takeya M, Ogawa H, Suefuji H, Yasue H, Takahashi K. Co-localization of tissue factor and tissue factor pathway inhibitor in coronary atherosclerosis. J Pathol. 1999;188: Crawley J, Lupu F, Westmuckett AD, Severs NJ, Kakkar VV, Lupu C, Expression, localization, and activity of tissue factor pathway inhibitor in normal and atherosclerotic human vessels. Arterioscler Thromb Vasc Biol. 2000;20: Caplice NM, Mueske CS, Kleppe LS, Simari RD. Presence of tissue factor pathway inhibitor in human atherosclerotic plaques is associated with reduced tissue factor activity. Circulation. 1998;98: