A Novel Monoclonal Antibody for RAGE-Directed Imaging Identifies Accelerated Atherosclerosis in Diabetes

Abstract

Receptor for advanced glycation end products (RAGE) binds advanced glycation end products and other inflammatory ligands and is expressed in atherosclerotic plaques in diabetic and nondiabetic subjects. The higher expression in diabetes mellitus corresponds to the accelerated course of the atherosclerosis. This study was designed to test the hypothesis that the level of RAGE expression in atherosclerosis can be detected by quantitative in vivo SPECT and that counts in the target will correlate with the strength of the biologic signal. A monoclonal murine antibody was developed against the V-domain of RAGE, fragmented into F(ab')(2) and labeled with (99m)Tc, and injected at a dose of 15.14 +/- 1.23 MBq into 24-wk-old male apolipoprotein E null (ApoE(-/-)) mice (n = 22), including mice with streptozotocin-induced diabetes mellitus (n = 8), nondiabetic mice (n = 8), and control ApoE(-/-)/RAGE(-/-) double-knock-out mice (n = 6). Four hours later (allowing for blood-pool clearance), the mice were imaged and sacrificed, and the proximal aorta was removed and counted to calculate the percentage injected dose of RAGE per gram of tissue, followed by histologic and immunohistochemical characterization. Radiotracer uptake in the aortic lesions was clearly visualized noninvasively by SPECT. RAGE uptake as percentage injected dose in diabetic ApoE(-/-) mice (1.39 +/- 0.16 x 10(-2)) was significantly higher than that in nondiabetic ApoE(-/-) mice (0.48 +/- 0.27 x 10(-2)) (P < 0.0001). The radiotracer uptake was highly correlated with RAGE expression by quantitative immunohistomorphometry (r = 0.82, P = 0.002) and with percentage of macrophages (r = 0.86, P < 0.0001). In this study, (99m)Tc-labeled anti-RAGE F(ab')(2) SPECT successfully identified early accelerated disease in diabetes mellitus for age-matched ApoE(-/-) mice and quantified RAGE expression over a range of lesion severities.