Martin Græbe

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

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=0.71, P=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.

Gene expression and 18FDG uptake in atherosclerotic carotid plaques.

PurposeMetabolic assessment of vascular inflammation by 2-[18F]fluoro-2-deoxy-D-glucose positron emission tomography (FDG)-PET is a promising new approach for the evaluation of the vulnerability of atherosclerotic plaques. Quantitative real-time PCR allows measurement of gene expression of markers of atherosclerotic plaque vulnerability. These techniques were applied in advanced atherosclerotic disease to relate metabolism and inflammatory activity to the gene expression profile of the vulnerable atherosclerotic plaque. MethodsSeventeen patients with clinical symptoms of cerebral vascular events (<3 months) and an additional ipsilateral internal carotid artery stenosis of greater than 60% were recruited. FDG uptake in the carotids was determined by PET/computed tomography and expressed as mean and maximal standardized uptake values (SUVmean and SUVmax). The atherosclerotic plaques were subsequently recovered by carotid endarterectomy. The gene expression of markers of vulnerability – CD68, IL-18, matrix metalloproteinase 9, cathepsin K, GLUT-1, and hexokinase type II (HK2) – were measured in plaques by quantitative PCR. ResultsIn a multivariate linear regression model, GLUT-1, CD68, cathepsin K, and HK2 gene expression remained in the final model as predictive variables of FDG accumulation calculated as SUVmean (R2=0.26, P<0.0001). In addition, a multivariate linear regression model found GLUT-1, CD68, cathepsin K, and HK2 gene expression as independent predictive variables of FDG accumulation calculated as SUVmax (R2=0.30, P<0.0001). ConclusionGLUT-1, HK2, CD68, and cathepsin K remained in both multivariate models and thus provided independent information regarding FDG uptake. We suggest that FDG uptake is a composite indicator of macrophage load, overall inflammatory activity and collagenolytic plaque destabilization. Accordingly, FDG-PET could prove to be an important predictor of cerebrovascular events in patients with carotid plaques.

18FDG PET and Ultrasound Echolucency in Carotid Artery Plaques

OBJECTIVES The objective was to evaluate inflammation in echolucent carotid artery plaques. BACKGROUND Ultrasound echolucency of carotid artery plaques has been proven to differentiate patients at high risk of stroke. On the other hand, positron emission tomography (PET) of plaques with the use of [(18)F]-fluorodeoxyglucose (FDG) identifies highly inflamed plaques, and the combination of molecular imaging and morphology could improve identification of vulnerable plaques. METHODS A total of 33 patients with cerebrovascular symptoms and carotid artery plaques were included prospectively for ultrasound and PET imaging. Plaque standardized gray scale medians (GSM) were measured in longitudinal ultrasound images to quantitate echolucency, and GSM values were compared with FDG PET uptake quantified by maximum standardized uptake values (SUV). Symptomatic plaques were compared with contralateral carotid artery plaques considered asymptomatic, and in 17 symptomatic patients, endarterectomized plaque specimens were analyzed for CD68 expression. RESULTS There was a negative correlation between GSM and FDG SUV (r = -0.56, p < 0.01). Whereas echo-rich plaques tended to show low FDG uptake, echolucent plaques ranged from high to low inflammatory activity, as depicted with PET. Quantitative FDG SUV differentiated asymptomatic from symptomatic plaques, whereas GSM values did not. There was a positive correlation between CD68 expression and FDG uptake (r = 0.50, p = 0.04). CONCLUSIONS Our results substantiate previous findings of an association between plaque FDG uptake and inflammation. Echolucent plaques exhibit a wide range of inflammatory activity, whereas echorich plaques show little inflammation. FDG PET may be useful for further stratification of echolucent plaques being either active (vulnerable) or inactive.

Microvessel Density But Not Neoangiogenesis Is Associated with 18F-FDG Uptake in Human Atherosclerotic Carotid Plaques

IntroductionThe vulnerable atherosclerotic lesion exhibits the proliferation of neovessels and inflammation. The imaging modality 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography (18FDG-PET) is considered for the identification of vulnerable plaques.PurposeThe purpose of this study was to compare the gene expression of neoangiogenesis and vulnerability-associated genes with 18FDG uptake in patients undergoing carotid endarterectomy.ProceduresHuman atherosclerotic carotid artery plaques from symptomatic patients were used for gene expression analysis by quantitative PCR of vascular endothelial growth factor (VEGF) and integrin αV and integrin β3 subunits, genes essential during neoangiogenesis. We also evaluated the gene expression of CD34, a measure of microvessel density (MVD), as well as CD68, MMP-9, and cathepsin K, genes of major importance in plaque vulnerability. Gene expression analysis was compared with 18FDG-PET.ResultsVEGF and integrin αVβ3 gene expression did not correlate with 18FDG uptake, whereas CD34 gene expression exhibited an inverse correlation with 18FDG uptake. Additionally, we established that markers of vulnerability were correlated with 18FDG uptake.ConclusionsNeoangiogenesis is not associated with 18FDG uptake, whereas MVD and markers of vulnerability correlate with 18FDG uptake. The absence of correlation between markers of neoangiogenesis and 18FDG uptake suggests a temporal separation between the process of neoangiogenesis and inflammatory activity.

When to image carotid plaque inflammation with FDG PET/CT.

ObjectiveQuantification of 18-fluorodeoxyglucose (FDG) uptake in inflamed high-risk carotid atherosclerotic plaques is challenged by the spatial resolution of positron emission tomography (PET) and luminal blood activity. Late acquisition protocols have been used to overcome these challenges to enhance the contrast between the plaque and blood-pool FDG activity. However, for prospective studies the late acquisition is inconvenient for the patient and staff, and most retrospective studies of plaque uptake use data from early acquisition protocols. The objective was to evaluate changes in the quantification methods of FDG uptake in carotid artery plaques between early and late PET scans. MethodsFDG uptake 1 and 3 h after tracer injection was compared in 19 carotid artery plaques. The average plaque maximum standardized uptake value (SUVmax) and a target to background ratio (TBR), using venous blood-pool activity as background, were evaluated at the two time points. These methods have been shown earlier to quantitate the degree of inflammation in late hour scans. ResultsA good individual plaque FDG uptake consistency was found between the two time points for SUVmax, r2=0.86. In contrast, the ratio method did not conserve the results between the two time points: TBR r2=0.34. For both methods, absolute values changed over time. TBR values generally increased as blood pool activity decreased, whereas the individual plaque SUVmax values showed both increases and decreases over time. ConclusionIdentification of carotid plaque inflammation with PET can be performed 1 h after FDG injection using SUVmax for plaque FDG uptake quantification.

Reproducibility of Two 3-D Ultrasound Carotid Plaque Quantification Methods

Compared with single 2-D images, emerging 3-D ultrasound technologies hold the promise of reducing variability and increasing sensitivity in the quantification of carotid plaques for individual cardiovascular risk stratification. Inter- and intra-observer agreement between a manual, cross-sectional, 2-D freehand sweep and a mechanical 3-D ultrasound investigation of 62 carotid artery plaques is reported with intra-class correlation coefficients (with 95% confidence intervals). Inter-observer agreement was 0.60 (0.29-0.77) for the freehand method and 0.89 (0.83-0.93) for the mechanical 3-D acquisition. The use of semi-automated computerized planimetric measurements of plaque burden has high intra-observer repeatability, but is vulnerable to systematic inter-observer differences. For the 2-D freehand sweep, a considerable contribution to variation is introduced by the scanning procedure itself, that is, the lack of controlled motion along the third dimension. Future implementation of 3-D ultrasound quantification in large-scale studies of inter-individual cardiovascular risk assessment seems justified using the methods described.

Congestive heart failure in rats is associated with increased collecting duct vasopressin sensitivity and vasopressin type 2 receptor reexternalization.

A number of studies have shown that rats with congestive heart failure (CHF) have increased protein levels of the vasopressin (AVP)-regulated water channel aquaporin-2 (AQP2) even during conditions with unchanged circulating levels of AVP, suggesting an increase in the sensitivity of the AVP type 2 (V2) receptor in experimental CHF. The present study was aimed at investigating AVP signaling in rats with moderate CHF (left ventricular end diastolic pressure >10 mmHg; normal plasma AVP levels) induced by ligation of the left anterior descending coronary artery. Sham-operated rats were used as controls. Western blotting analyses revealed an increased abundance of AQP2 in renal cortex (+33 ± 9% of sham; P < 0.05) and in inner medulla (IM) (+54 ± 15% of sham; P < 0.05) in CHF rats compared with sham-operated controls. Dose-response studies on isolated collecting ducts (CDs) showed an increased accumulation of cAMP in response to AVP in CHF rats compared with controls. V2 receptor surface-binding studies in isolated IMCDs showed a marked and comparable AVP-induced V2 receptor internalization in response to AVP in both CHF and control rats. As expected V2 receptor surface binding remained low after AVP challenge in control rats. In contrast to this, V2 receptor surface binding returned to pre-AVP levels within 30 min in the CHF rats, indicating an obtained recycling ability of the V2 receptor in CHF. Together the results indicate the presence of an increased AVP sensitivity in the CDs from CHF rats, associated with an acquired recycling ability of the V2 receptor.

The Significance of Echolucent Plaques: Past and Future Perspectives

Like most other scientific discoveries of significance, assessment of plaque pathohistological features using ultrasonographic morphology was first described in scattered and scarce single observations. In the beginning of the 1980s, visual assessment of echolucency was linked to the histology of symptomatic carotid plaques (at that time mainly considered to be intraplaque hemorrhage) and the first prospective studies considering risk of stroke within different gradings of echolucency were published.