- Email: [email protected]
Plasma levels of granzyme B are increased in patients with lipid-rich carotid plaques as determined by echogenicity
Atherosclerosis 195 (2007) e142–e146
Plasma levels of granzyme B are increased in patients with lipid-rich carotid plaques as determined by echogenicity Mona Skjelland a,∗ , Annika E. Michelsen b , Kirsten Krohg-Sørensen c , Bjørn Tennøe d , Arve Dahl a , Søren Bakke d , Frank Brosstad b , Jan K. Dam˚as b , David Russell a , Bente Halvorsen b , P˚al Aukrust b a Department of Neurology, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Oslo, Norway Research Institute for Internal Medicine, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Oslo, Norway Department of Thoracic and Cardiovascular Surgery, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Oslo, Norway d Department of Radiology, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Oslo, Norway b
c
Received 7 February 2007; received in revised form 30 April 2007; accepted 2 May 2007 Available online 12 June 2007
Abstract Increased echolucency of carotid plaques is associated with an increased risk of ischemic stroke. Inflammation and apoptosis of vascular smooth muscle cells in the arterial wall are involved in the atherosclerotic process and destabilization of the plaque. Granzyme B (GrB) is a key mediator of T cell-mediated cytotoxicity, and we therefore hypothesized that this protease could distinguish echolucent from other plaques. Ultrasound-determined echolucency of atherosclerotic plaques was assessed prior to carotid endarterectomy/angioplasty in 57 consecutively recruited patients with high-grade internal carotid stenosis. Plasma levels of GrB were measured by enzyme immunoassay prior to surgery. Patients with carotid atherosclerosis had significantly higher plasma levels of GrB compared to healthy controls (n = 16) (p < 0.01), with particularly high levels in those with an echolucent lesion. While there were no differences in traditional cardiovascular risk factors or CRP between those with echolucent (n = 16) and those with echogenic/heterogeneous (n = 41) plaques, the echolucent group had markedly raised plasma levels of GrB (p < 0.01). Patients with high levels of circulating granzyme B also had more ischemic lesions on cerebral MRI prior to surgery. Raised plasma levels of GrB in echolucent carotid plaques with increased frequency of cerebrovascular events suggest that GrB may be a marker of plaque instability. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Carotid arteries; Inflammation; Cerebral ischemia; Ultrasonography
1. Introduction The composition of an atherosclerotic plaque is considered more important than the degree of stenosis for defining cardiovascular risk [1,2]. Ultrasound plaque appearance or echogenicity can in principle be classified into plaques with low-level echoes with often a thin incomplete fibrous cap ∗ Corresponding author at: Department of Neurology, RikshospitaletRadiumhospitalet Medical Center, 0027 Oslo, Norway. Tel.: +47 23072764; fax: +47 23074891. E-mail address: [email protected] (M. Skjelland).
0021-9150/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2007.05.001
on the luminal surface (echolucent plaque), and plaque with medium and high level echoes (echogenic plaques), characterized by a higher content of fibrous tissue and calcification. Echolucent carotid plaques, independent of the degree of stenosis, have been found to be associated with symptomatic disease and a higher risk for future ischemic cerebrovascular events as compared with echogenic plaques [1–3]. The mechanisms by which echolucent plaques are related to plaque instability and cerebrovascular events are not fully understood, but some studies have suggested an association between echolucency and inflammation [4,5]. Increased apoptosis of vascular smooth muscle cells (VSMC) and other
M. Skjelland et al. / Atherosclerosis 195 (2007) e142–e146
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stabilizing cells could also contribute to plaque destabilization in echolucent lesions. The serine protease granzyme B (GrB), mainly derived from cytotoxic T cells, is a main regulator of apoptosis through its ability to activate the caspase cascade [6]. T cells play an important regulatory role in the inflammatory and apoptotic processes within the atherosclerotic lesions, and these regulatory functions could potentially involve GrB-related mechanisms. The presence of GrB in advanced atherosclerotic lesions, associated with increasing disease severity and plaque instability [7] provide further support to a hypothesis suggesting that GrB could be involved in this process. The measurements of soluble GrB may be useful to assess clinical disorders associated with enhanced T cell activation and apoptosis such as rheumatoid arthritis [8]. Based on these properties, we hypothesized that plasma levels of GrB could be related to the degree of plaque instability. In the present study, we investigated this hypothesis by analyzing the relationship between plasma levels of GrB and plaque morphology, as determined by ultrasound of the carotid artery in patients who were referred for carotid endarterectomy (CEA) or carotid artery stenting (CAS).
were therefore grouped together (n = 41) and compared to the pure echolucent (n = 16) plaques (Table 1). Patients with concomitant inflammatory diseases such as infections and autoimmune disorders, malignancies, and liver or kidney disease were excluded from the study. Cerebral magnetic resonance imaging (MRI) and a clinical neurological examination were performed within 2 days before and 3 days after CEA/CAS. Previous cerebral ischemia included all signs of ischemic tissue impairment in all vessel territories before to CEA/CAS. New lesions on MRI included new ischemic lesions after CEA/CAS ipsilaterally to the operated/stented carotid artery. The MRIs were reviewed by two experienced neuroradiologists who were blinded for plaque morphology and GrB levels, but not for clinical features. Perioperative cerebrovascular symptoms included stroke, transitory ischemic attacks (TIA) or amaurosis fugax during or within the first 3 days after the CEA/CAS. Blood samples were collected within 2 days prior to CEA/CAS. GrB in patients were compared with values in blood samples collected from 16 sex- and age-matched healthy controls. The regional ethics committee approved the protocol and all patients signed an informed consent form prior to participation in the study.
2. Material and methods
2.1. Ultrasound of the precerebral arteries
Fifty-seven consecutive patients with internal carotid stenosis (≥70%) were treated with CEA (n = 48) or CAS (n = 9). The study included 17 (29.8%) women and 40 (70.2%) men, aged 65.5 years (S.D.: 9.1, range: 44–83 years). The carotid stenoses were diagnosed and classified within 2 days prior to CEA/CAS by precerebral color duplex ultrasound and CT angiography according to consensus criteria [9,10]. The plaques were divided into three groups using ultrasound depending on plaque echogenicity (echolucent, heterogeneous and echogenic) as previously described [1,3]. Since previous studies have shown an increased risk of stroke in echolucent plaques, we compared this group to the more echogenic plaques. The heterogeneous (non-uniform appearance) and the echogenic plaques
The degree of stenosis and morphology of the plaques were determined using Color Duplex examinations (HDI 5000, Philips). Plaque morphology in terms of echogenicity was assessed as previously described, with the vessel lumen as the reference structure for defining echolucency and the bright echo zone produced by the media–adventitia interface in the far wall as the reference for defining echogenicity [1,3]. 2.2. Blood sampling protocol Venipuncture of a forearm vein was performed within 2 days before the CEA/CAS with minimal stasis. Peripheral venous blood was drawn into pyrogen-free tubes with EDTA as anticoagulant. The tubes were immediately immersed in
Table 1 Patient characteristics
Age (years) Male sex Degree of stenosis (%) Previous cerebrovascular symptoms* Previous MRI ischemic lesions Diabetes mellitus Antihypertensive treatment Statin treatment Angiotensin-converting enzyme inhibitors Angiotensin receptor blockers Current smoking
Echolucent (n = 16)
Echogenic/heterogenous (n = 41)
p
67 (9.2) 63 (10) 78 (7.9) 44 (7) 94 (15) 25 (4) 69 (11) 75 (12) 13 (2) 19 (3) 63 (10)
65 (8.9) 73 (30) 86 (7.4) 46 (19) 61 (25) 17 (7) 73 (30) 88 (36) 22 (9) 20 (8) 51 (21)
.164 .431 .019 .860 .029 .496 .990 .243 .417 .948 .505
Numbers are given as percentages (numbers) of patients, except age and degree of stenosis given as mean (S.D.). * Previous cerebrovascular symptoms include ipsilateral ischemic stroke, transitory ischemic attacks or amaurosis fugax within the last 6 months.
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melting ice and centrifuged within 30 min at 2500 × g for 25 min at 4 ◦ C to obtain platelet-poor plasma. All samples were stored at −80 ◦ C and thawed only once. 2.3. Enzyme immunoassay (EIA) Plasma levels of GrB were determined in duplicates by EIA according to the manufacturer’s description (Bender Medsystems, Vienna, Austria). We have no exact data on the proportion of false positive and negative GrB values in the present study population. However, the EIA showed no crossreactivity with different human proteases such as proteinase 2, tryptase, cathepsisn G, granzyme A, human neutrophils elastase, trypsin or chymotrypsin, and the recovery of different concentrations of spiked GrB in human sera was 117%. The intra- and inter-assay coefficients of variations were 8.5% and 10.4%, respectively.
Fig. 1. Box plots of plasma GrB levels in patients with echolucent (EL, n = 16) and heterogeneous/echogenic (EG/het, n = 41) carotid plaques and in healthy controls (ctrls, n = 16). *p < 0.01 vs. controls; # p < 0.02 vs. EG/het.
categorical data. Probability values (two-sided) were considered significant for values <0.05.
2.4. Miscellaneous C-reactive protein (CRP) levels were determined by a high-sensitivity particle enhanced immunoturbidimetric assay on a Modular platform (Roche Diagnostic, Basel, Switzerland). Concentrations of lipid parameters were measured by routine laboratory methods as previously described [11]. 2.5. Statistical methods The study was primarily designed to detect differences between those with echolucent and those with echogenic/heterogeneous plaques. We were looking for large differences (i.e., >2-fold increase or >50% decrease), and we did not perform any sample size calculations. However, on the basis of previous experiments in our laboratory, a sample size of 15–25 individuals in each group was assessed to be sufficient to detect large and biologically relevant differences in plasma levels of mediators related to inflammatory processes in patients with atherosclerotic disorders. Two-sample t-tests were used for comparison of normally distributed data and Mann–Whitney for data with a non-normal distribution. Pearson chi-square was used to assess correlation between
3. Results Fifty-seven patients with carotid stenosis were included in the study and classified into two groups according to their plaque morphology. The inter-observer agreement between the two neurologists who evaluated the plaques was good with κ = 0.70 (p < 0.001) based on data from 34 plaques. Patient characteristics were similar in those who had echolucent (n = 16) and those with echogenic/heterogeneous (n = 41) plaques, except for a less pronounced degree of stenosis and more ischemic MRI lesions previous to operation/stenting in echolucent plaque patients (Table 1). In contrast, there were no differences in previous cerebrovascular symptoms between those with echolucent and those with echogenic/heterogeneous plaques. This suggests that many of the MRI lesions were asymptomatic [12]. There were no differences in preoperative characteristics or postoperative data between patients treated with CEA and CAS. As shown in Fig. 1, the patients with carotid atherosclerosis had significantly raised plasma levels of GrB when compared with sex- and age-matched healthy controls. Partic-
Table 2 Blood parameters according to plaque echogenicity
Granzyme B (pg/mL) Cholesterol (mmol/L)* HDL cholesterol (mmol/L) LDL cholesterol (mmol/L)* Triglycerides (mmol/L)* CRP (mg/L)* Leukocyte count (109 /L)* Fibrinogen (g/L) Platelet count (109 /L)* HbA1c (%)*
Echolucent (n = 16)
Echogenic/heterogenous (n = 41)
p
492.0 (0.7–956.4) 4.23 (0.47) 1.20 (0.8–1.9) 2.68 (0.57) 1.40 (0.74) 6.0 (7.5) 7.4 (1.9) 4.2 (3.1–4.8) 264 (84.0) 6.1 (0.9)
143.8 (0.7–981.1) 4.42 (0.91) 1.30 (0.7–2.5) 2.70 (0.80) 1.40 (0.80) 5.7 (5.1) 7.3 (2.3) 3.8 (2.6–6.9) 254 (70.1) 5.8 (1.2)
.010 .414 .523 .921 .860 .839 .905 .090 .642 .390
Numbers are medians (range) or * unadjusted means (S.D.).
M. Skjelland et al. / Atherosclerosis 195 (2007) e142–e146
ularly high levels of GrB were found in those with echolucent plaques. Although there were no differences in traditional cardiovascular risk factors (i.e., lipid parameters, fibrinogen, and HbA1c) or CRP between those with echolucent and those with echogenic/heterogeneous plaques, the former group had markedly raised plasma levels of GrB as compared to the other patients (Table 2 and Fig. 1). Forty patients had ischemic lesions on cerebral MRI before CEA/CAS (15 patients [94%] versus 25 patients [61%], echolucent and echogenic/heterogeneous plaques, respectively, p = 0.029) Table 2. The highest GrB levels were seen in those with previous ischemic MRI lesions (p = 0.042). Patients with echolucent plaques also had more perioperative ipsilateral cerebrovascular events (three patients [19%] versus no patients, p = 0.003), and more new ischemic lesions on cerebral MRI (four patients [25%] versus one patient [2%], p < 0.001) after CEA/CAS compared to patients with echogenic/heterogeneous plaque, but there were no correlations between GrB levels and these variables.
4. Discussion Echolucent carotid plaques have been consistently associated with symptomatic disease in cross-sectional studies and an increased incidence of cerebrovascular events in large cohorts [1]. The results of the present study with increased perioperative cerebrovascular events and more postoperative MRI ischemic lesions in echolucent plaques, indicate increased instability in echolucent compared to more echogenic plaques, which is in accordance with previous studies [1–3]. More importantly, we report that patients with echolucent plaques had markedly raised plasma levels of GrB compared to those with echogenic/heterogeneous lesions. In contrast, there were no differences in traditional cardiovascular risk factors or CRP values between these two groups of patients. These findings suggest that GrB may be a plasma marker of plaque echolucency and instability in patients with carotid atherosclerosis. The association between high levels of GrB and more cerebral MRI lesions prior to operation further supports this possibility. There was no correlation between GrB levels and perioperative cerebrovascular events or postoperative new MRI lesions, but this may be due to the small number of patients with these pre- or postoperative cerebrovascular complications. T cell-mediated apoptosis has previously been linked to plaque destabilization [7,13], and it has been suggested that GrB may be a key mediator in this process [6]. Indeed, Choy et al. found more GrB in advanced atherosclerotic plaques than in mildly diseased vessels [7], and these authors have also demonstrated the ability of soluble GrB to induce VSMC apoptosis [6]. Based on GrB’s role in T cell-mediated cytotoxicity, it is tempting to hypothesize that GrB is not only is an indicator, but also a mediator of plaque instability. CRP has been shown to be an independent predictor of stroke/TIA in apparently healthy individuals as well as in
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patients with established atherosclerosis [14–16]. There is also one report of raised CRP levels in persons with echolucent carotid plaques suggesting a higher degree of systemic inflammation in these patients [17]. However, although CRP is a stable and reliable marker of inflammation, the inflammatory processes that underlie atherogeneis are mediated by a multitude of cytokines and are unlikely to be reflected by CRP levels alone [18]. We found that while there was no difference in CRP levels between those with echolucent and those with echogenic/heterogeneous plaques, the former group had markedly increased GrB levels in plasma. T cell activation plays an important role in atherogenesis and plaque destabilization [19], and as GrB is mainly released from activated cytotoxic T cells [6], it is tempting to hypothesize that the raised plasma levels of GrB in patients with echolucent plaques may reflect the degree of T cell activation in these patients, a process that may not necessarily be mirrored by CRP. Plasma levels of GrB have previously been suggested as a marker of disease activity in rheumatoid arthritis [8]. Our findings suggest that GrB may also be a suitable plasma marker of plaque instability and echolucency in carotid plaques, potentially reflecting important pathogenic processes in these patients. However, although those with echolucent plaques had significantly raised GrB levels compared with patients in the echogenic/heterogeneous group, there was some overlap, suggesting that the raised GrB levels are not restricted to those with echolucent lesions, possibly reflecting a contributing rather than a quite decisive role for GrB in the development of echolucent plaques. These issues should be further elucidated in forthcoming studies, examining a larger study population.
Conflicts of interest None.
Acknowledgement Supported by a grant from The Norwegian Foundation for Health and Rehabilitation.
References [1] Mathiesen EB, Bonaa KH, Joakimsen O. Echolucent plaques are associated with high risk of ischemic cerebrovascular events in carotid stenosis: the Tromsø study. Circulation 2001;103:2171–5. [2] Nordestgaard BG. Echolucent rupture-prone plaques. Curr Opin Lipidol 2003;14:505–12. [3] European Carotid Plaque Study G. Carotid artery plaque composition—relationship to clinical presentation and ultrasound B-mode imaging. Eur J Vasc Endovasc Surg 1995;10:23–30. [4] Yamagami H, Kitagawa K, Nagai Y, et al. Higher levels of Interleukin-6 are associated with lower echogenicity of carotid artery plaques. Stroke 2004;35:677–81.
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[5] Gronholdt ML, Nordestgaard BG, Bentzon J, et al. Macrophages are associated with lipid-rich carotid artery plaques, echolucency on B-mode imaging, and elevated plasma lipid levels. J Vasc Surg 2002;35:137–45. [6] Choy JC, Hung VHY, Hunter AL, et al. Granzyme B induces smooth muscle cell apoptosis in the absence of perforin: involvement of extracellular matrix degradation. Arterioscler Thromb Vasc Biol 2004;24:2245–50. [7] Choy J, McDonald P, Suarez AC, et al. Granzyme B in atherosclerosis and transplant vascular disease: association with cell death and atherosclerotic disease severity. Modern Pathol 2003;16:460–70. [8] Tak PP, Spaeny-Dekking L, Kraan MC, et al. The levels of soluble granzyme A and B are elevated in plasma and synovial fluid of patients with rheumatoid arthritis (RA). Clin Exp Immunol 1999;116:366–70. [9] Anderson GB, Ashforth R, Steinke DE, Ferdinandy R, Findlay JM. CT Angiography for the detection and characterization of carotid artery bifurcation disease. Stroke 2000;31:2168–74. [10] Grant EG, Benson CB, Moneta GL, et al. Carotid artery stenosis: grayscale and doppler US diagnosis—Society of Radiologists in Ultrasound Consensus Conference. Radiology 2003;229:340–6. [11] Holven KB, Scholz H, Halvorsen B, et al. Hyperhomocysteinemic subjects have enhanced expression of lectin-like oxidized LDL receptor-1 in mononuclear cells. J Nutr 2003;133:3588–91.
[12] Coutts SB, Hill MD, Simon JE, et al. Silent ischemia in minor stroke and TIA patients identified on MR imaging. Neurology 2005;65:513– 7. [13] Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005;352:1685–95. [14] Tsimikas S, Willerson JT, Ridker PM. C-reactive protein and other emerging blood biomarkers to optimize risk stratification of vulnerable patients. J Am Coll Cardiol 2006;47:C19–31. [15] Ridker PM. Clinical application of C-reactive protein for cardiovascular disease detection and prevention. Circulation 2003;107:363– 9. [16] Everett BM, Kurth T, Buring JE, Ridker PM. The relative strength of C-reactive protein and lipid levels as determinants of ischemic stroke compared with coronary heart disease in women. J Am Coll Cardiol 2006;48:2235–42. [17] With Not0 AA-TBgMEEA, Endothelial dysfunction and systemic inflammation in persons with echolucent carotid plaques. Thromb Haemost 2006;96:53–9. [18] Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 2000;342:836–43. [19] Robertson. T cells in atherogenesis: for better or for worse? Arterioscler Thromb Vasc Biol 2006;26:2421.
Plasma levels of granzyme B are increased in patients with lipid-rich carotid plaques as determined by echogenicity Mona Skjelland a,∗ , Annika E. Michelsen b , Kirsten Krohg-Sørensen c , Bjørn Tennøe d , Arve Dahl a , Søren Bakke d , Frank Brosstad b , Jan K. Dam˚as b , David Russell a , Bente Halvorsen b , P˚al Aukrust b a Department of Neurology, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Oslo, Norway Research Institute for Internal Medicine, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Oslo, Norway Department of Thoracic and Cardiovascular Surgery, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Oslo, Norway d Department of Radiology, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Oslo, Norway b
c
Received 7 February 2007; received in revised form 30 April 2007; accepted 2 May 2007 Available online 12 June 2007
Abstract Increased echolucency of carotid plaques is associated with an increased risk of ischemic stroke. Inflammation and apoptosis of vascular smooth muscle cells in the arterial wall are involved in the atherosclerotic process and destabilization of the plaque. Granzyme B (GrB) is a key mediator of T cell-mediated cytotoxicity, and we therefore hypothesized that this protease could distinguish echolucent from other plaques. Ultrasound-determined echolucency of atherosclerotic plaques was assessed prior to carotid endarterectomy/angioplasty in 57 consecutively recruited patients with high-grade internal carotid stenosis. Plasma levels of GrB were measured by enzyme immunoassay prior to surgery. Patients with carotid atherosclerosis had significantly higher plasma levels of GrB compared to healthy controls (n = 16) (p < 0.01), with particularly high levels in those with an echolucent lesion. While there were no differences in traditional cardiovascular risk factors or CRP between those with echolucent (n = 16) and those with echogenic/heterogeneous (n = 41) plaques, the echolucent group had markedly raised plasma levels of GrB (p < 0.01). Patients with high levels of circulating granzyme B also had more ischemic lesions on cerebral MRI prior to surgery. Raised plasma levels of GrB in echolucent carotid plaques with increased frequency of cerebrovascular events suggest that GrB may be a marker of plaque instability. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Carotid arteries; Inflammation; Cerebral ischemia; Ultrasonography
1. Introduction The composition of an atherosclerotic plaque is considered more important than the degree of stenosis for defining cardiovascular risk [1,2]. Ultrasound plaque appearance or echogenicity can in principle be classified into plaques with low-level echoes with often a thin incomplete fibrous cap ∗ Corresponding author at: Department of Neurology, RikshospitaletRadiumhospitalet Medical Center, 0027 Oslo, Norway. Tel.: +47 23072764; fax: +47 23074891. E-mail address: [email protected] (M. Skjelland).
0021-9150/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2007.05.001
on the luminal surface (echolucent plaque), and plaque with medium and high level echoes (echogenic plaques), characterized by a higher content of fibrous tissue and calcification. Echolucent carotid plaques, independent of the degree of stenosis, have been found to be associated with symptomatic disease and a higher risk for future ischemic cerebrovascular events as compared with echogenic plaques [1–3]. The mechanisms by which echolucent plaques are related to plaque instability and cerebrovascular events are not fully understood, but some studies have suggested an association between echolucency and inflammation [4,5]. Increased apoptosis of vascular smooth muscle cells (VSMC) and other
M. Skjelland et al. / Atherosclerosis 195 (2007) e142–e146
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stabilizing cells could also contribute to plaque destabilization in echolucent lesions. The serine protease granzyme B (GrB), mainly derived from cytotoxic T cells, is a main regulator of apoptosis through its ability to activate the caspase cascade [6]. T cells play an important regulatory role in the inflammatory and apoptotic processes within the atherosclerotic lesions, and these regulatory functions could potentially involve GrB-related mechanisms. The presence of GrB in advanced atherosclerotic lesions, associated with increasing disease severity and plaque instability [7] provide further support to a hypothesis suggesting that GrB could be involved in this process. The measurements of soluble GrB may be useful to assess clinical disorders associated with enhanced T cell activation and apoptosis such as rheumatoid arthritis [8]. Based on these properties, we hypothesized that plasma levels of GrB could be related to the degree of plaque instability. In the present study, we investigated this hypothesis by analyzing the relationship between plasma levels of GrB and plaque morphology, as determined by ultrasound of the carotid artery in patients who were referred for carotid endarterectomy (CEA) or carotid artery stenting (CAS).
were therefore grouped together (n = 41) and compared to the pure echolucent (n = 16) plaques (Table 1). Patients with concomitant inflammatory diseases such as infections and autoimmune disorders, malignancies, and liver or kidney disease were excluded from the study. Cerebral magnetic resonance imaging (MRI) and a clinical neurological examination were performed within 2 days before and 3 days after CEA/CAS. Previous cerebral ischemia included all signs of ischemic tissue impairment in all vessel territories before to CEA/CAS. New lesions on MRI included new ischemic lesions after CEA/CAS ipsilaterally to the operated/stented carotid artery. The MRIs were reviewed by two experienced neuroradiologists who were blinded for plaque morphology and GrB levels, but not for clinical features. Perioperative cerebrovascular symptoms included stroke, transitory ischemic attacks (TIA) or amaurosis fugax during or within the first 3 days after the CEA/CAS. Blood samples were collected within 2 days prior to CEA/CAS. GrB in patients were compared with values in blood samples collected from 16 sex- and age-matched healthy controls. The regional ethics committee approved the protocol and all patients signed an informed consent form prior to participation in the study.
2. Material and methods
2.1. Ultrasound of the precerebral arteries
Fifty-seven consecutive patients with internal carotid stenosis (≥70%) were treated with CEA (n = 48) or CAS (n = 9). The study included 17 (29.8%) women and 40 (70.2%) men, aged 65.5 years (S.D.: 9.1, range: 44–83 years). The carotid stenoses were diagnosed and classified within 2 days prior to CEA/CAS by precerebral color duplex ultrasound and CT angiography according to consensus criteria [9,10]. The plaques were divided into three groups using ultrasound depending on plaque echogenicity (echolucent, heterogeneous and echogenic) as previously described [1,3]. Since previous studies have shown an increased risk of stroke in echolucent plaques, we compared this group to the more echogenic plaques. The heterogeneous (non-uniform appearance) and the echogenic plaques
The degree of stenosis and morphology of the plaques were determined using Color Duplex examinations (HDI 5000, Philips). Plaque morphology in terms of echogenicity was assessed as previously described, with the vessel lumen as the reference structure for defining echolucency and the bright echo zone produced by the media–adventitia interface in the far wall as the reference for defining echogenicity [1,3]. 2.2. Blood sampling protocol Venipuncture of a forearm vein was performed within 2 days before the CEA/CAS with minimal stasis. Peripheral venous blood was drawn into pyrogen-free tubes with EDTA as anticoagulant. The tubes were immediately immersed in
Table 1 Patient characteristics
Age (years) Male sex Degree of stenosis (%) Previous cerebrovascular symptoms* Previous MRI ischemic lesions Diabetes mellitus Antihypertensive treatment Statin treatment Angiotensin-converting enzyme inhibitors Angiotensin receptor blockers Current smoking
Echolucent (n = 16)
Echogenic/heterogenous (n = 41)
p
67 (9.2) 63 (10) 78 (7.9) 44 (7) 94 (15) 25 (4) 69 (11) 75 (12) 13 (2) 19 (3) 63 (10)
65 (8.9) 73 (30) 86 (7.4) 46 (19) 61 (25) 17 (7) 73 (30) 88 (36) 22 (9) 20 (8) 51 (21)
.164 .431 .019 .860 .029 .496 .990 .243 .417 .948 .505
Numbers are given as percentages (numbers) of patients, except age and degree of stenosis given as mean (S.D.). * Previous cerebrovascular symptoms include ipsilateral ischemic stroke, transitory ischemic attacks or amaurosis fugax within the last 6 months.
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melting ice and centrifuged within 30 min at 2500 × g for 25 min at 4 ◦ C to obtain platelet-poor plasma. All samples were stored at −80 ◦ C and thawed only once. 2.3. Enzyme immunoassay (EIA) Plasma levels of GrB were determined in duplicates by EIA according to the manufacturer’s description (Bender Medsystems, Vienna, Austria). We have no exact data on the proportion of false positive and negative GrB values in the present study population. However, the EIA showed no crossreactivity with different human proteases such as proteinase 2, tryptase, cathepsisn G, granzyme A, human neutrophils elastase, trypsin or chymotrypsin, and the recovery of different concentrations of spiked GrB in human sera was 117%. The intra- and inter-assay coefficients of variations were 8.5% and 10.4%, respectively.
Fig. 1. Box plots of plasma GrB levels in patients with echolucent (EL, n = 16) and heterogeneous/echogenic (EG/het, n = 41) carotid plaques and in healthy controls (ctrls, n = 16). *p < 0.01 vs. controls; # p < 0.02 vs. EG/het.
categorical data. Probability values (two-sided) were considered significant for values <0.05.
2.4. Miscellaneous C-reactive protein (CRP) levels were determined by a high-sensitivity particle enhanced immunoturbidimetric assay on a Modular platform (Roche Diagnostic, Basel, Switzerland). Concentrations of lipid parameters were measured by routine laboratory methods as previously described [11]. 2.5. Statistical methods The study was primarily designed to detect differences between those with echolucent and those with echogenic/heterogeneous plaques. We were looking for large differences (i.e., >2-fold increase or >50% decrease), and we did not perform any sample size calculations. However, on the basis of previous experiments in our laboratory, a sample size of 15–25 individuals in each group was assessed to be sufficient to detect large and biologically relevant differences in plasma levels of mediators related to inflammatory processes in patients with atherosclerotic disorders. Two-sample t-tests were used for comparison of normally distributed data and Mann–Whitney for data with a non-normal distribution. Pearson chi-square was used to assess correlation between
3. Results Fifty-seven patients with carotid stenosis were included in the study and classified into two groups according to their plaque morphology. The inter-observer agreement between the two neurologists who evaluated the plaques was good with κ = 0.70 (p < 0.001) based on data from 34 plaques. Patient characteristics were similar in those who had echolucent (n = 16) and those with echogenic/heterogeneous (n = 41) plaques, except for a less pronounced degree of stenosis and more ischemic MRI lesions previous to operation/stenting in echolucent plaque patients (Table 1). In contrast, there were no differences in previous cerebrovascular symptoms between those with echolucent and those with echogenic/heterogeneous plaques. This suggests that many of the MRI lesions were asymptomatic [12]. There were no differences in preoperative characteristics or postoperative data between patients treated with CEA and CAS. As shown in Fig. 1, the patients with carotid atherosclerosis had significantly raised plasma levels of GrB when compared with sex- and age-matched healthy controls. Partic-
Table 2 Blood parameters according to plaque echogenicity
Granzyme B (pg/mL) Cholesterol (mmol/L)* HDL cholesterol (mmol/L) LDL cholesterol (mmol/L)* Triglycerides (mmol/L)* CRP (mg/L)* Leukocyte count (109 /L)* Fibrinogen (g/L) Platelet count (109 /L)* HbA1c (%)*
Echolucent (n = 16)
Echogenic/heterogenous (n = 41)
p
492.0 (0.7–956.4) 4.23 (0.47) 1.20 (0.8–1.9) 2.68 (0.57) 1.40 (0.74) 6.0 (7.5) 7.4 (1.9) 4.2 (3.1–4.8) 264 (84.0) 6.1 (0.9)
143.8 (0.7–981.1) 4.42 (0.91) 1.30 (0.7–2.5) 2.70 (0.80) 1.40 (0.80) 5.7 (5.1) 7.3 (2.3) 3.8 (2.6–6.9) 254 (70.1) 5.8 (1.2)
.010 .414 .523 .921 .860 .839 .905 .090 .642 .390
Numbers are medians (range) or * unadjusted means (S.D.).
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ularly high levels of GrB were found in those with echolucent plaques. Although there were no differences in traditional cardiovascular risk factors (i.e., lipid parameters, fibrinogen, and HbA1c) or CRP between those with echolucent and those with echogenic/heterogeneous plaques, the former group had markedly raised plasma levels of GrB as compared to the other patients (Table 2 and Fig. 1). Forty patients had ischemic lesions on cerebral MRI before CEA/CAS (15 patients [94%] versus 25 patients [61%], echolucent and echogenic/heterogeneous plaques, respectively, p = 0.029) Table 2. The highest GrB levels were seen in those with previous ischemic MRI lesions (p = 0.042). Patients with echolucent plaques also had more perioperative ipsilateral cerebrovascular events (three patients [19%] versus no patients, p = 0.003), and more new ischemic lesions on cerebral MRI (four patients [25%] versus one patient [2%], p < 0.001) after CEA/CAS compared to patients with echogenic/heterogeneous plaque, but there were no correlations between GrB levels and these variables.
4. Discussion Echolucent carotid plaques have been consistently associated with symptomatic disease in cross-sectional studies and an increased incidence of cerebrovascular events in large cohorts [1]. The results of the present study with increased perioperative cerebrovascular events and more postoperative MRI ischemic lesions in echolucent plaques, indicate increased instability in echolucent compared to more echogenic plaques, which is in accordance with previous studies [1–3]. More importantly, we report that patients with echolucent plaques had markedly raised plasma levels of GrB compared to those with echogenic/heterogeneous lesions. In contrast, there were no differences in traditional cardiovascular risk factors or CRP values between these two groups of patients. These findings suggest that GrB may be a plasma marker of plaque echolucency and instability in patients with carotid atherosclerosis. The association between high levels of GrB and more cerebral MRI lesions prior to operation further supports this possibility. There was no correlation between GrB levels and perioperative cerebrovascular events or postoperative new MRI lesions, but this may be due to the small number of patients with these pre- or postoperative cerebrovascular complications. T cell-mediated apoptosis has previously been linked to plaque destabilization [7,13], and it has been suggested that GrB may be a key mediator in this process [6]. Indeed, Choy et al. found more GrB in advanced atherosclerotic plaques than in mildly diseased vessels [7], and these authors have also demonstrated the ability of soluble GrB to induce VSMC apoptosis [6]. Based on GrB’s role in T cell-mediated cytotoxicity, it is tempting to hypothesize that GrB is not only is an indicator, but also a mediator of plaque instability. CRP has been shown to be an independent predictor of stroke/TIA in apparently healthy individuals as well as in
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patients with established atherosclerosis [14–16]. There is also one report of raised CRP levels in persons with echolucent carotid plaques suggesting a higher degree of systemic inflammation in these patients [17]. However, although CRP is a stable and reliable marker of inflammation, the inflammatory processes that underlie atherogeneis are mediated by a multitude of cytokines and are unlikely to be reflected by CRP levels alone [18]. We found that while there was no difference in CRP levels between those with echolucent and those with echogenic/heterogeneous plaques, the former group had markedly increased GrB levels in plasma. T cell activation plays an important role in atherogenesis and plaque destabilization [19], and as GrB is mainly released from activated cytotoxic T cells [6], it is tempting to hypothesize that the raised plasma levels of GrB in patients with echolucent plaques may reflect the degree of T cell activation in these patients, a process that may not necessarily be mirrored by CRP. Plasma levels of GrB have previously been suggested as a marker of disease activity in rheumatoid arthritis [8]. Our findings suggest that GrB may also be a suitable plasma marker of plaque instability and echolucency in carotid plaques, potentially reflecting important pathogenic processes in these patients. However, although those with echolucent plaques had significantly raised GrB levels compared with patients in the echogenic/heterogeneous group, there was some overlap, suggesting that the raised GrB levels are not restricted to those with echolucent lesions, possibly reflecting a contributing rather than a quite decisive role for GrB in the development of echolucent plaques. These issues should be further elucidated in forthcoming studies, examining a larger study population.
Conflicts of interest None.
Acknowledgement Supported by a grant from The Norwegian Foundation for Health and Rehabilitation.
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