Evaluation of translocator protein quantification as a tool for characterising macrophage burden in human carotid atherosclerosis

Abstract

Macrophage presence within atherosclerotic plaque is a feature of instability and a risk factor for plaque rupture and clinical events. Activated macrophages express high levels of the translocator protein/peripheral benzodiazepine receptor (TSPO/PBR). In this study, we investigated the potential for quantifying plaque inflammation by targeting this receptor. TSPO expression and distribution in the plaque were quantified using radioligand binding assays and autoradiography. We show that cultured human macrophages expressed 20 times more TSPO than cultured human vascular smooth muscle cells (VSMCs), the other abundant cell type in plaque. The TSPO ligands [3H](R)-1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H](R)-PK11195) and [3H]N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide ([3H]-DAA1106) bound to the same sites in human carotid atherosclerotic plaques in vitro, and demonstrated significant correlation with macrophage-rich regions. In conclusion, our data indicate that radioisotope-labelled DAA1106 has the potential to quantify the macrophage content of atherosclerotic plaque.

Fig. 1

Quantification of translocator protein (TSPO) in cultured cells, with co-localisation of TSPO and macrophage presence in a carotid endarterectomy tissue section from a single patient. Saturation and Scatchard plots of [³H](R)-PK11195 binding in cultured, blood-derived monocytes (A) and vascular smooth muscle cells (B). Specific binding of [³H](R)-PK11195 (C) and [³H]-DAA1106 (D) in atherosclerotic vessel. Total and non-specific binding were obtained by autoradiography and specific binding was obtained by subtracting non-specific binding from total binding. Immunohistochemical staining of macrophages with antibody to CD68 (E). Microscopy images were digitized, co-registered to autoradiography images and quantified. Control sections (isotype-specific IgG antibody) showed negligible staining (F). Bar = 1 mm.