(18)F-FDG imaging of human atherosclerotic carotid plaques reflects gene expression of the key hypoxia marker HIF-1α

Abstract

To investigate the association between gene expression of key molecular markers of hypoxia and inflammation in atherosclerotic carotid lesions with 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)F-FDG) uptake as determined clinically by positron emission tomography (PET). Studies using PET have demonstrated (18)F-FDG-uptake in patients with confirmed plaques of the carotid artery. Inflammatory active or "vulnerable" plaques progressively increase in bulk, develop necrotic cores, poor vessel-wall vascularization and become prone to hypoxia. We used quantitative polymerase-chain reaction (qPCR) to determine gene expression of hypoxia-inducible factor 1α (HIF-1α) and cluster of differentiation 68 (CD68) on plaques recovered by carotid endarterectomy (CEA) in 18 patients. Gene expression was compared with (18)F-FDG-uptake quantified as the maximum standardized uptake value (SUVmax) on co-registered PET/computed tomography (CT) scans performed the day before CEA. Immunohistochemistry was used to validate target-gene protein expression. In univariate linear regression analysis HIF-1α was significantly correlated with (18)F-FDG-uptake (SUVmax) as was CD68. A two-tailed Pearson regression model demonstrated that HIF-1α and CD68 gene expression co-variated and accordingly when entering the variables into multivariate linear regression models with SUV-values as dependent variables, HIF-1α was eliminated in the final models. (18)F-FDG-uptake (SUVmax) is correlated with HIF-1α gene expression indicating an association between hypoxia and glucose metabolism in vivo. The marker of inflammation CD68 is also associated with (18)F-FDG-uptake (SUVmax). As CD68 and HIF-1α gene expression co-variate their information is overlapping.

Keywords: 18F-FDG PET/CT imaging; HIF-1α; Hypoxia; carotid atherosclerosis; gene expression.

Figure 1

Coronal contrast enhanced CT-image of the right carotid with enlarged PET/CT insert. Carotis communis and carotis internae dxt: white arrows pointing right, carotis externae dxt: single white arrow pointing down. Inserted and enlarged is the fused ¹⁸FDG-PET/CT modality; yellow to white coloration depicts glucose-uptake intensity. Green lines indicate where the excised plaque is physically split upon recovery. The distance between two green lines is 3 mm and the total number of lines corresponds to the total size of the excised plaque from this particular patient. The dotted line indicates the level of the bifurcation. ROIs are drawn on transaxial images, corresponding to each line on the coronal image presented here. This creates a segmented cylinder of voxels encompassing lumen, plaque and vessel wall from where the SUV values are noted. Importantly the size of the excised plaque determines the number of slices and thus corresponding ROIs on the PET/CT data. Note the calcified plaque in the bifurcation ranging from the distal common carotid artery, past the bulb and protruding into the internal carotid artery. CT = computed tomography; ¹⁸FDG-PET = ¹⁸F-flurodeoxyglucose-positron emission tomography; ROI = region of interest; SUV = standardized uptake value.

Figure 2

Contrast enhanced CT; diagnostic CTA performed with intravenous injection of contrast with bolus tracking of the ascending aorta and a cutoff value of 80 HU. In green are ROIs encircling the left and right internal carotid artery, white arrows point to the jugular veins. CT = computed tomography, CTA = CT angiography, HU = Hounsfield units, ROI = region of interest.

Figure 3

Gene expression; correlation to SUV_max. Univariate linear regression analysis of gene expression of HIF-1α relative to SUV_max as an expression of ¹⁸F-FDG-uptake for all patients and all lesion slices (n = 126). Note log-transformation of gene expression data. The 95% confidence interval is indicated by the broken line. ¹⁸F-FDG = 2-[¹⁸F] fluoro-2-deoxy-D-glucose; HIF-1α = hypoxia inducible factor 1α; SUV_max = maximum standardized uptake value.

Figure 4

Correlation between CD68 and HIF-1α. Scatterplot of a two-tailed Pearson correlation analysis of gene expression of CD68 and HIF-1α for all patients and all lesion slices (n = 126). Note log-transformation of gene expression data. The 95% confidence interval is indicated by the broken line. CD68 = cluster of differentiation 68; HIF1α = hypoxia inducible factor 1α.

Figure 5

Immunohistochemical stainings. Immunohistochemical stains of CD68 and HIF1α: Negative control stains (left panel x 100 magnification, panel A and D). Immunohistochemical stain as indicated in each sub-panel lower left corner (middle panel x 100 magnification, panel B and E). Insert boxes indicate magnified areas that are shown in (right panel x 400 magnification, panel C and F). Scale bars: 200 μm two left columns and 50 μm right columns. (*) arterial lumen. CD68 = cluster of differentiation 68; HIF-1α = hypoxia inducible factor 1α.