Development of Receptor for Advanced Glycation End Products–Directed Imaging of Atherosclerotic Plaque in a Murine Model of Spontaneous Atherosclerosis

Abstract

Background— The receptor for advanced glycation end products (RAGE) is implicated in the development and progression of atherosclerosis. We tested the hypothesis that 99m Tc-labeled anti-RAGE F(ab′) 2 can be used as a noninvasive tool to image atherosclerotic lesions in apolipoprotein E–deficient (apoE −/− ) mice. Methods and Results— A sequence in the V-type Ig extracellular domain of RAGE was used to develop a peptide injected into rabbits; serum was retrieved, IgG prepared and affinity-purified, and pepsin-digested into F(ab′) 2 . Thirteen 6-week apoE −/− mice were fed a Western diet. At 20 weeks, 6 were injected with 15.2�1.9 MBq (350 to 411 μCi) 99m Tc-labeled anti-RAGE F(ab′) 2 , 6 were injected with 99m Tc-labeled control nonspecific IgG F(ab′) 2 , and 1 was injected with dual-labeled 99m Tc and rhodamine anti-RAGE F(ab′) 2 . Four 20-week C57BL/6 mice were injected with 99m Tc-labeled anti-RAGE F(ab′) 2 . All mice were imaged on a high resolution mini-γ camera 4 hours after injection and euthanized. The aortic tree was dissected and photographed, and the proximal aorta was sectioned for staining after γ scintillation counting. All 6 apoE −/− mice injected with 99m Tc-labeled anti-RAGE F(ab′) 2 fragments showed focal tracer uptake in the proximal aorta (mean %ID/g, 1.98%). Disease and antibody controls showed no focal tracer uptake in the thorax (%ID/g, <1.0%). Histological sections of the proximal aorta showed American Heart Association class III lesions with lipid laden macrophages, smooth muscle cells, and positive staining for RAGE. On immunofluorescence, RAGE colocalized with macrophages. Conclusion— These data show the feasibility of noninvasively imaging RAGE in atherosclerotic lesions in a murine model and confirm levels of RAGE expression sufficient to allow detection on in vivo imaging.

Simvastatin Suppresses Apoptosis in Vulnerable Atherosclerotic Plaques Through Regulating the Expression of p53, Bcl-2 and Bcl-xL

“…Sections from BCA were used for immunohistochemistry staining to identify macrophages, smooth muscle cells (SMC) by a method reported previously [15]. Briefly, the sections were incubated with polyclonal antibodies at 37°C for 60 min or at 4°C overnight and then labeled with HRPconjugated anti-rabbit IgG at 37°C for 60 min.…”

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

“…Preparation of F(ab9) 2 Fragments and Radiolabeling F(ab9) 2 fragments of the purified anti-RAGE antibody were prepared as previously described (10). These fragments have more antigen binding sites available than Fab and faster blood-pool and renal clearance than whole antibody.…”

“…These fragments have more antigen binding sites available than Fab and faster blood-pool and renal clearance than whole antibody. Direct coupling of anti-RAGE F(ab9) 2 antibodies to DTPA (Sigma) for 99m Tc labeling was performed as previously described (10,14). The mean specific activity was 6.58 6 0.68 MBq/mg of protein, and the mean radiopurity was 98% 6 0.54% by instant thin-layer chromatography.…”

“…For cellular characterization of atherosclerotic lesions, immunohistochemical staining was performed as previously described (10). Briefly, tissue sections were deparaffinized in xylene, and endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide.…”

“…In a proof-of-concept paper, we showed the ability to visualize uptake of an F(ab9) 2 fragment of a 99m Tc-labeled polyclonal antibody in atherosclerotic plaque in the prox-imal aortae of 20-wk-old nondiabetic ApoE 2/2 mice using planar imaging (10). The generation of monoclonal antibodies is now a standard and increasingly routine procedure.…”