64Cu-labeled divalent cystine knot peptide for imaging carotid atherosclerotic plaques

Abstract

The rupture of vulnerable atherosclerotic plaques that lead to stroke and myocardial infarction may be induced by macrophage infiltration and augmented by the expression of integrin αvβ;3. Indeed, atherosclerotic angiogenesis may be a promising marker of inflammation. In this study, an engineered integrin αvβ;3-targeting PET probe, 64Cu-NOTA-3-4A, derived from a divalent knottin miniprotein was evaluated in a mouse model for carotid atherosclerotic plaques. Methods: Atherosclerotic plaques in BALB/C mice, maintained on a high-fat diet, were induced with streptozotocin injection and carotid artery ligation and verified by MR imaging. Knottin 3-4A was synthesized by solid-phase peptide synthesis chemistry and coupled to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) before radiolabeling with 64Cu. PET probe stability in mouse serum was evaluated. Mice with carotid atherosclerotic plaques were injected via the tail vein with 64Cu-NOTA-3-4A or 18F-FDG, followed by small-animal PET/ CT imaging at different time points. Receptor targeting specificity of the probe was verified by coinjection of c(RGDyK) administered in molar excess. Subsequently, carotid artery dissection and immunofluorescence staining were performed to evaluate target expression. Results: 64Cu-NOTA-3-4A was synthesized in high radiochemical purity and yield and demonstrated molecular stability in both phosphate-buffered saline and mouse serum at 4 h. Smallanimal PET/CT showed that 64Cu-NOTA-3-4A accumulated at significantly higher levels in the neovasculature of carotid atherosclerotic plaques (7.41 ± 1.44 vs. 0.67 ± 0.23 percentage injected dose/gram, P , 0.05) than healthy or normal vessels at 1 h after injection. 18FFDG also accumulated in atherosclerotic lesions at 0.5 and 1 h after injection but at lower plaque-to-normal tissue ratios than 64Cu-NOTA-3-4A. For example, plaque-to-normal carotid artery ratios for 18F-FDG and 64Cu-NOTA-3-4A at 1 h after injection were 3.75 and 14.71 (P , 0.05), respectively. Furthermore, uptake of 64Cu-NOTA-3-4A in atherosclerotic plaques was effectively blocked (∼90% at 1 h after injection) by coinjection of c(RGDyK). Immunostaining confirmed integrin αvβ;3 expression in both the infiltrating macrophages and the neovasculature of atherosclerotic plaques. Conclusion: 64Cu-NOTA-3-4A demonstrates specific accumulation in carotid atherosclerotic plaques in which macrophage infiltration and angiogenesis are responsible for elevated integrin αvβ;3 levels. Therefore, 64Cu-NOTA-3-4A may demonstrate clinical utility as a PET probe for atherosclerosis imaging or for the evaluation of therapies used to treat atherosclerosis.