Molecular Pathology in Vulnerable Carotid Plaques: Correlation with [18]-Fluorodeoxyglucose Positron Emission Tomography (FDG-PET)

Eur J Vasc Endovasc Surg (2009) 37, 714e721

Molecular Pathology in Vulnerable Carotid Plaques: Correlation with [18]-Fluorodeoxyglucose Positron Emission Tomography (FDG-PET) M. Græbe a,*, S.F. Pedersen b, L. Borgwardt c, L. Højgaard c, H. Sillesen a, A. Kjær b,c a

Department of Vascular Surgery, Rigshospitalet RK-3111, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen E, Denmark b Cluster for Molecular Imaging, University of Copenhagen, Copenhagen, Denmark c Department of Clinical Physiology, Nuclear Medicine & PET, Rigshospitalet. University of Copenhagen, Copenhagen, Denmark Submitted 21 August 2008; accepted 9 November 2008 Available online 27 December 2008

KEYWORDS Carotid artery plaque; Stroke; Positron emission tomography (PET); Gene expression; [18]-Fluorodeoxyglucose (FDG)

Abstract Objectives: Atherosclerosis is recognised as an inflammatory disease, and new diagnostic tools are warranted to evaluate plaque inflammatory activity and risk of cardiovascular events. We investigated [18]-fluorodeoxyglucose (FDG) uptake in vulnerable carotid plaques visualised by positron emission tomography (PET). Uptake was correlated to quantitative gene expression of known markers of inflammation and plaque vulnerability. Methods: Ten patients with recent transient ischaemic attack and carotid artery stenosis (>50%) underwent combined FDG-PET and computed tomography angiography (CTA) the day before carotid endarterectomy. Plaque mRNA expression of the inflammatory cytokine interleukin 18 (IL-18), the macrophage-specific marker CD68 and the two proteinases, Cathepsin K and matrix metalloproteinase 9 (MMP-9), were quantified using real-time quantitative polymerase chain reaction. Results: Consistent up-regulation of CD68 (3.8-fold  0.9; mean  standard error), Cathepsin K (2.1-fold  0.5), MMP-9 (122-fold  65) and IL-18 (3.4-fold  0.7) were found in the plaques, compared to reference-artery specimens. The FDG uptake by plaques was strongly correlated with CD68 gene expression (r Z 0.71, P Z 0.02). Any correlations with Cathepsin K, MMP-9 or IL-18 gene expression were weaker. Conclusions: FDG-PET uptake in carotid plaques is correlated to gene expression of CD68 and other molecular markers of inflammation and vulnerability. ª 2008 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: þ45 3545 8082. E-mail address: [email protected] (M. Græbe). 1078-5884/$34 ª 2008 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejvs.2008.11.018

FDG-PET and Molecular Pathology in Plaques The risk of recurrent stroke within 90 days is 15e20% in patients presenting with symptoms of transient ischaemic attack (TIA) or minor stroke.1 Those with severe arterial diseasedthat is, carotid artery atherosclerotic plaquesdhave a conceivable higher risk of recurrence, typically reported to be 35e40%.2e4 Large, randomised trials conducted in the 1990s documented that carotid endarterectomy (CEA) significantly reduced the risk in patients with at least 50% stenosis of the relevant (ipsilateral) carotid artery.5,6 Although the risk of recurrent stroke is, thus, evident in those having symptomatic carotid artery stenosis, some of the patients with stenosis do not get a second event; and asymptomatic patients have only a small benefit of CEA. A better stratifying mechanism for patients with TIA or minor stroke being at risk of a second stroke would prove to be a major improvement in patient selection. During the last decade, plaque molecular and structural composition, rather than degree of stenosis, have proved a far more important trigger of thromboembolic events.7e9 Evidence of inflammatory processes being pivotal in the development of vulnerability is growing, and several of the underlying molecular processes leading to plaque rupture have been elucidated.10,11 Circulating biomarkers are currently being evaluated as markers of plaque vulnerability to unmask which patients might benefit from intensified medical treatment and, eventually, endarterectomy.12,13 In addition, several imaging techniques trying to depict the vulnerable plaque have emerged.14,15 Positron emission tomography (PET) is one such promising imaging technology currently under investigation. Using the glucose analogue radiotracer [18F]2-fluoro-2-deoxy-Dglucose (FDG), it has been shown in both, animal and human, studies that atherosclerotic lesions tends to show a high FDG uptake. It has been shown, that FDG-PET uptake in arteries shows good correlation to other known factors of atherosclerosis (e.g., age),16e19 and that the FDG uptake in carotid plaques in TIA patients correlates well to macrophage quantities within the plaque.20,21 The aim of the present study was to assess to what extent FDG-PET reflects key pathological molecular processes in carotid plaques to permit non-invasive imaging of these processes. To do so, we combined real-time quantitative polymerase chain reaction (qPCR) of excised endarterectomised plaques with preoperative FDG-PET imaging. We investigated gene expression of the mainly constitutively expressed macrophage-surface-protein CD68 and of known mediators of vulnerabilitydinterleukin 18 (IL-18),11,22 matrix metalloproteinase 9 (MMP-9)10 and the cathepsin cysteine proteinase Cathepsin K (CatK), which degrades collagen.23e25

Methods Ethics The study was approved by the Danish National Committee on Biomedical Research Ethics (Jr. no.: 0120065513), and all participants gave written informed consent on inclusion.

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Patients Ten patients with a carotid artery stenosis of 50% or more, shown by duplex ultrasound,26 and symptoms of an ipsilateral cerebral event within 3 months prior to operation were included. The day before carotid endarterectomy, all patients had an FDG-PET/computed tomography (CT) and a physical exam, and blood samples were drawn.

FDG-PET/computed tomography angiography (CTA) After an overnight fast (6 h), a combined PET/CTA (Siemens Biograph 16, Siemens AG Healthcare Sector, Erlangen, Germany) was performed over the neck region, 3 h after intravenous administration of 400 MBq FDG. Two trained investigators (MG and LB), blinded to molecular data, used the advanced, open-source PACS station DICOM viewer OsiriX v. 2.7.5 (http://www.osirix-viewer.com) to place regions of interest (ROIs) around the common carotid artery and the internal carotid artery. ROIs placed within the lumen of the jugular veins were used for blood-pool readings. Mean and maximum standardised uptake values (SUVmean and SUVmax) were noted, which represent counts within the region, corrected for patient weight and decaycorrected injected FDG dose. Alignment of excised plaques to corresponding transverse image sections was secured by measurements of bidirectional distance of plaque from the bifurcation, as noted preoperatively. An average of SUVmax ROIs corresponding to the excised plaque was divided by an average of SUVmean in blood-pool, which yields the targetto-background ratio (TBR).20,21,27,28

Plaque Preparation Carotid endarterectomy was performed under local anaesthesia and plaques were removed gently, photographed and sliced transversely in 3-mm thick slices. Careful attention was given to orientation and slice origin (common carotid artery, bifurcation and internal carotid artery). The slices were immediately conserved in RNALater (Applied Biosystems, CA, USA) and stored at 4  C for 24 h before they were dried and frozen at 80  C until further analysis. A piece of the superior thyroid artery was conserved in the same way from all patients, referred to as the ‘reference’. No visual (macroscopic) pathology was present in these specimens, but to diminish possible individual non-visual pathology revealed in qPCR analysis, gene-expression analysis from all reference arteries were pooled in a mathematical manner to ensure a baseline reference for the entire population.

RNA Extraction and Gene-Expression Analysis The following molecular analyses on the extracted plaque slices were done by a team of specialists (SFP and AK) blinded to the image data. Samples of 10e35 mg of tissue were dissected from each slice sample and the TRIzol reagents (Invitrogen Corporation Carlsbad, CA, USA) were used for total RNA extraction.

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The RNA was quantified and assigned RNA-integrity values using the Agilent 2100 Bioanalyzer (Agilent Technologies Inc. Santa Clara, CA, USA). Due to partially degraded RNA, we performed a baseline correction on all assays; effectively excluding degraded RNA to produce a measure of intact RNA for subsequent input into the reverse transcriptase procedure. Reverse transcriptase polymerase chain reaction (RTPCR) was performed using the AffinityScript QPCR cDNA Synthesis Kit (Stratagene, La Jolla, CA, USA). Samples were run on a Mastercycler gradient RT-PCR machine (Eppendorf Hamburg, Germany) and cDNA was stored at 20  C until quantification analysis. The quantitative PCR (qPCR) analyses were performed using the Mx3000P and Mx3005 Real-Time PCR Systems (Stratagene La Jolla, CA, USA). We used SYBR Green for optimising primer concentrations and for housekeeping gene-stability measures, whereas the TaqMan assay was used for multiplex measurements of genes of interest (GOIs). Reference-tissue data were pooled for all patients as a ‘universal’ reference to diminish possible non-macroscopic individual pathology in the reference arteries. Initial sequence data were obtained from the National Center for Biotechnology Information website, and primer and probe sets were designed using Beacon Designer 7.0 software (Premier Biosoft International, Palo Alto, CA, USA). All primers and probes were purchased from Sigma-Aldrich (St. Louis, MO, USA) and designed with different reporters and quenchers at the 50 -end and 30 -end, respectively, allowing multiplex analysis as desired. Two triplex analyses were performed. One with housekeeping gene combined with CD68 and IL-18 and another with housekeeping gene combined with MMP-9 and Cathepsin K.

passed the test. MMP-9 data were log-transformed to obtain normal distribution. The P-values of the Kolmogorove Smirnov tests were: 0.84 (CD68), 0.81 (IL-18), 0.99 (Cathepsin K), 0.93 (log MMP-9) and 0.52 (TBR), in accordance with previous testing of larger test materials also demonstrating normal distribution of these variables. A one-sample t-test was used to test for overall increase in expression, compared to the reference (all set to 1, except for MMP-9, where we use the fact that log 1 Z 0). To test for correlation between gene overexpression and FDG uptake, an average of both values were calculated for each patient to reduce uncertainty of co-registering of image and plaque slices. Correlation coefficients and P-values were computed using the Pearson method. Where an extreme value (outlier) was present, statistical testing was repeated without this value to test robustness of the correlations. The P-values are all two-tailed and P < 0.05 is considered as significant. Statistical analysis and graphing were done using the software programs SPSS v.16 (SPSS Inc.) and Prism v.5a (Graphpad Software Inc.), respectively.

Statistical Analysis

Data from all slices (n Z 68), independent of patient origin, are shown in Fig. 1A. Fig. 1B shows, in the same manner, distribution of gene expression when slices were averaged for each of the 10 patients. Real-time qPCR average for individuals (n Z 10, see Fig. 1B) revealed that CD68 was overexpressed by 3.78  0.92 (P Z 0.014), IL-18 was

Average plaque-slice expressions of examined genes are presented as mean  standard error of mean (SEM). Data were tested for deviation from Gaussian distribution by using the KolmogoroveSmirnov test. All data, but MMP-9,

Results Single-patient data, with emphasis on known risk factors of stroke, are outlined in Table 1. Notably, all patients had normal low-density lipoprotein (LDL) levels, compared to American guidelines by NCEP,29 and no correlations between the shown parameters and plaque TBR were found (data not shown). All patients received both oral antiplatelet therapy and cholesterol-lowering drugs (statins) by the time of the PET scan. All patients were previous smokers.

Quantitative Gene Expression

Table 1

Individual patient data

Patient #

Gender

Age (years)

Time (days)a

Glucose (mmol/l)

HgbA1c (%)

LDL (mg/dL)

Blood pressure (mmHg)

Degree of stenosis (%)b

Plaque TBRc

1 2 3 4 5 6 7 8 9 10

F M M F M F M M M M

81 76 74 77 67 71 54 66 66 78

27 33 74 41 22 28 70 51 74 21

6.20 5.70 7.10 6.00 5.70 7.40 3.80 4.60 4.50 5.90

5.50 6.00 6.50 6.50 5.50 5.90 5.80 5.30 6.00 5.70

101 66 93 104 101 143 46 89 77 107

120/60 120/70 160/100 140/70 180/90 180/80 140/80 140/80 105/70 120/60

75 55 85 75 60 80 65 70 70 95

1.56 1.66 1.20 1.28 1.50 1.31 1.90 1.40 2.71 1.61

All blood samples were drawn before FDG injection after an overnight fast. No correlation was found between TBR and the tabled data. a Time: time from last symptom till PET in days. b Degree of stenosis: carotid artery stenosis determined by duplex flow analysis. c TBR: target-to-background ratio of PET FDG uptake.

FDG-PET and Molecular Pathology in Plaques

717 respectively, on transverse PET/CT image slices corresponding to the extension of the excised plaque was used for quantification of individual plaque uptake.

Correlation of Gene Expression with FDG Uptake Figs. 3 and 4 show correlation plots for each of the four genes investigated, compared to FDG uptake of the plaques. Using data from all 10 patients, significant correlations were found between TBR and CD68 (r Z 0.71, P Z 0.02), log MMP-9 (r Z 0.69, P Z 0.04), IL-18 (r Z 0.76, P Z 0.01) and Cathepsin K (r Z 0.70, P Z 0.02) as shown in Fig. 3. There was one high TBR ‘outlier’. When this was excluded, for the remaining nine patients, CD68 correlation to TBR strengthened (r Z 0.88, P Z 0.002), whereas correlation coefficients of IL-18 (r Z 0.56, P Z 0.12), log MMP-9 (r Z 0.62, P Z 0.07) and Cathepsin K weakened and were no longer significantly correlated to TBR. Apart from MMP-9, significant correlations were observed between average plaque SUVmax and gene expression (CD68: r Z 0.64, P Z 0.04; log MMP-9 r Z 0.45, P Z 0.22; IL-18 r Z 0.77, P Z 0.01; Cathepsin K: r Z 0.67, P Z 0.03) as shown in Fig. 4. In contrast, poor correlations were found between SUVmean and gene expression (CD68: r Z 0.36, P Z 0.30; log MMP-9 r Z 0.59, P Z 0.07; IL-18 r Z 0.65, P Z 0.04; Cathepsin K: r Z 0.69, P Z 0.03). Figure 1 Plaque mRNA expression relative to reference (Z1, dotted line). (A) Every point represents a slice, 4e9 slices per patient, 68 slices in all. (B) Average of slices for each patient. Mean  SEM. Note log scale.

similarly overexpressed 3.43  0.74 (P Z 0.009), MMP-9 was highly overexpressed by an average of 122  65 (P Z 0.001) whereas Cathepsin K averaged in expression by 2.12  0.46fold (P Z 0.039), all in comparison to gene expression in pooled, reference arterial tissue. Correlates between expression of CD68 and the other genes are shown in Table 2.

PET/CT Imaging Fig. 2 shows examples of FDG uptake in plaques in different subjects, all with symptomatic right-sided carotid plaques. Averaging of SUVmax, SUVmean and calculation of TBR,

Table 2

Correlation between gene expressions

Correlates (n Z 10)

Pearson correlation coefficient (r)

P-value (two-sided)

CD68 vs. IL-18 CD68 vs. log MMP-9 CD68 vs. Cathepsin K IL-18 vs. log MMP-9 IL-18 vs. Cathepsin K

0.80* 0.48* 0.36 0.75* 0.63

0.005 0.033 0.312 0.012 0.052

MMP-9 data were log-transformed to obtain Gaussian distribution as tested by the KolmogoroveSmirnov test. *P < 0.05.

Discussion We found up-regulation of CD68, IL-18, MMP-9 and Cathepsin K gene expression in endarterectomised carotid plaques from patients with recent TIA. This is in accordance with previous studies concerning CD68, IL-18 and MMP9,10,11 whereas Cathepsin K gene expression has not previously been studied in endarterectomised plaques. CD68 (which is mainly constitutively expressed) is a recognised marker of macrophages30 and we found that both TBR and SUVmax were correlated with CD68 gene expression, whereas SUVmean showed a weaker correlation. This is in agreement with previous reports about the use of TBR.20,21,27 Hence, our results suggest that TBR is the optimal parameter to investigate associations between FDG uptake and plaque gene expression. The 3.8-fold overexpression of CD68 observed (Fig. 1) is most probably due to an increased number of macrophages in the regions of study. We suspect that the increased uptake of glucose, as demonstrated with FDG-PET, was largely due to the abundance of macrophages yielding a high metabolic rate. Macrophages are thought to be the major contributor of harmful inflammatory actions in atherosclerosis. In addition, several cytokines, metalloproteinases and cathepsins expressed in macrophages have been identified as contributors in the development of atherosclerotic lesions. IL-18 is a cytokine identified as having a role in the late stages of atherosclerotic lesion development, when thinning of the extracellular matrix (ECM) renders plaques prone to rupture.7,11 IL-18 is a known mediator of MMP-9 activation22 and as plasma IL-18 concentrations are elevated in patients with atherosclerosis, its potential as a biomarker of future cardiovascular

Figure 2 Examples of qualitative assessments of FDG uptake in right-side carotid artery plaques from three patients. (A) Transverse (and (B) sagittal) slice of same patient with high plaque uptake. (C) Intermediate uptake and (D) no or normal uptake. Both CTA (left), FDG-PET (right) and fused image (middle) are shown.

FDG-PET and Molecular Pathology in Plaques

Figure 3 Correlation plot of A: CD68, B: IL-18, C: log MMP-9 and D: Cathepsin K vs. FDG uptake (TBR). Dotted line represents reference (‘normal’ artery gene expression).

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Figure 4 Correlation plot of (A) CD68, (B) IL-18, (C) log MMP-9 and (D) Cathepsin K vs. FDG uptake (SUVmax). Dotted line represents reference.

720 events has been investigated.31e33 IL-18 was up-regulated 3.2-fold in our study (Fig. 1), but the correlation between IL-18 and TBR was less robust as significance was lost when the outlier was excluded. The degradation of ECM is a pivotal step in the process of destabilising plaques, that is, making them vulnerable, and the effect is propagated by the release of ECM degrading proteolytic enzymes including matrix metalloproteinases and cathepsins.34,35 MMP-9 expression is induced in activated macrophages36 and evidence is growing for a key role for MMP-9 in late steps of plaque remodelling.9,37e39 Levels of circulating MMP-9 are increased in patients with acute coronary events40 and our group has shown that baseline plasma levels are associated with the risk of stroke among patients with carotid artery plaques.41 The extremely high levels of MMP-9 expression found in the present study (w1000-fold up-regulation in single specimens) corroborate our previous findings. Like IL-18, the correlation between MMP-9 expression and FDG uptake was less robust than that for CD68 expression. Recent emphasis on cathepsin cysteine proteases in plaque development and rupture led us to include Cathepsin K in our study.10,25,34 In comparison to other cathepsins, Cathepsin K exhibits collagenolytic properties, and it is expressed by several of the cell types involved in plaque remodelling.23,24 Although, overall, there was a 2.1-fold increase in the expression of Cathepsin K in endarterectomised carotid plaques, any correlation to FDG uptake was weak. A limitation of our study was the low number of patients enrolled. We were able to clearly link gene expression of CD68 expression with FDG uptake, but an increased number of patients may be necessary to confirm possible associations between FDG uptake and IL-18, MMP-9 and Cathepsin K. FDG uptake appears to be a promising method for noninvasive plaque characterisation and, possibly, risk stratification of patients with TIAs: this requires elucidation in prospective studies. In conclusion, our study reveals that, in a rather homogenous population of high-risk patients, a grading of the overall increase in macrophage gene expression of CD68 in carotid plaques can be visualised non-invasively by means of FDG-PET.

Conflict of Interest

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Acknowledgements The study was funded in part by The Danish Heart Foundation, The Research Fund of Rigshospitalet, The Danish Medical Research Council and The John and Birthe Meyer Foundation. The PET/CT scanner was donated by the John and Birthe Meyer Foundation.

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