Iron overload damages many organs. Unfortunately, therapeutic iron chelators also have undesired toxicity and may deliver iron to microbes. Here we show that a mutant form (K3Cys) of endogenous lipocalin 2 (LCN2) is filtered by the kidney but can bypass sites of megalin-dependent recapture, resulting in urinary excretion. Because K3Cys maintains recognition of its cognate ligand, the iron siderophore enterochelin, this protein can capture and transport iron even in the acidic conditions of urine. Mutant LCN2 strips iron from transferrin and citrate, and delivers it into the urine. In addition, it removes iron from iron overloaded mice, including models of acquired (iron-dextran or stored red blood cells) and primary (Hfe−/−) iron overload. In each case, the mutants reduce redox activity typical of non-transferrin-bound iron. In summary, we present a non-toxic strategy for iron chelation and urinary elimination, based on manipulating an endogenous protein:siderophore:iron clearance pathway.
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.
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. Methods: A monoclonal murine antibody was developed against the V-domain of RAGE, fragmented into F(ab9) 2 and labeled with 99m Tc, and injected at a dose of 15.14 6 1.23 MBq into 24-wk-old male apolipoprotein E null (ApoE 2/2 ) mice (n 5 22), including mice with streptozotocin-induced diabetes mellitus (n 5 8), nondiabetic mice (n 5 8), and control ApoE 2/2 /RAGE 2/2 double-knock-out mice (n 5 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. Results: Radiotracer uptake in the aortic lesions was clearly visualized noninvasively by SPECT. RAGE uptake as percentage injected dose in diabetic ApoE 2/2 mice (1.39 6 0.16 · 10 22 ) was significantly higher than that in nondiabetic ApoE 2/2 mice (0.48 6 0.27 · 10 22 ) (P , 0.0001). The radiotracer uptake was highly correlated with RAGE expression by quantitative immunohistomorphometry (r 5 0.82, P 5 0.002) and with percentage of macrophages (r 5 0.86, P , 0.0001). Conclusion: In this study, 99m Tc-labeled anti-RAGE F(ab9) 2 SPECT successfully identified early accelerated disease in diabetes mellitus for age-matched ApoE 2/2 mice and quantified RAGE expression over a range of lesion severities.
BackgroundThe purpose of this study is to image the effect of diabetes on expression of receptor for advanced glycation endproducts (RAGE) in limb ischemia in live animals.MethodsMale wild-type C57BL/6 mice were either made diabetic or left as control. Two months later, diabetic and non-diabetic mice underwent left femoral artery ligation. The right leg served as lesion control. Five days later, mice were injected with 15.1 ± 4.4 MBq 99mTc-anti-RAGE F(ab’)2 and 4 to 5 h later (blood pool clearance) underwent SPECT/CT imaging. At the completion of imaging, mice were euthanized, hind limbs counted and sectioned, and scans reconstructed. Regions of interest were drawn on serial transverse sections comprising the hind limbs and activity in millicuries summed and divided by the injected dose (ID). Quantitative histology was performed for RAGE staining and angiogenesis.ResultsUptake of 99mTc-anti-RAGE F(ab')2 as %ID × 10−3 was higher in the left (ischemic) limbs for the diabetic mice (n = 8) compared to non-diabetic mice (n = 8) (1.20 ± 0.44% vs. 0.49 ± 0.40%; P = 0.0007) and corresponded to less angiogenesis in the diabetic mice. Uptake was also higher in the right limbs of diabetic compared to non-diabetic animals (0.82 ± 0.33% vs. 0.40 ± 0.14%; P = 0.0004).ConclusionsThese data show the feasibility of imaging and quantifying the effect of diabetes on RAGE expression in limb ischemia.
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