Imaging receptor for advanced glycation end product expression in mouse model of hind limb ischemia

Abstract

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.

Treatment effect with anti-RAGE F(ab′)2 antibody improves hind limb angiogenesis and blood flow in Type 1 diabetic mice with left femoral artery ligation

“…6,7 We developed an antibody that binds a unique peptide on the extracellular domain of RAGE for radiolabeling as a tool to localize and quantify RAGE expression in live animals. [8][9][10] We demonstrated increased RAGE expression in the ischemic hind limbs of diabetic mice compared to control legs and to ischemic hind limbs in non-diabetic mice. [8][9][10] In a human pathology paper, Ritthaler et al in 1995 documented prominent enhancement of endothelial RAGE expression in small and medium size arteries in patients with occlusive peripheral vascular disease both with and without diabetes.…”

“…[8][9][10] We demonstrated increased RAGE expression in the ischemic hind limbs of diabetic mice compared to control legs and to ischemic hind limbs in non-diabetic mice. [8][9][10] In a human pathology paper, Ritthaler et al in 1995 documented prominent enhancement of endothelial RAGE expression in small and medium size arteries in patients with occlusive peripheral vascular disease both with and without diabetes. 11 For imaging, microgram quantities of antibody are linked to the radionuclide and this amount has no biological effect.…”

“…[8][9][10] The extent of RAGE expression and its contribution to the progression of limb ischemia through inhibition of angiogenesis, indicates that blocking RAGE is a potential therapeutic approach to restore blood flow and reduce symptoms. We tested the blocking properties of the antibody against VSMCs in cell culture and the promising results of these experiments prompted us to design the present placebo-controlled treatment study.…”

“…8,9 Briefly, mice were anesthetized via intraperitoneal injection of ketamine (100 mg/ml) and xylazine (5 mg/kg). Under sterile conditions, a skin incision was made on the upper thigh of the mouse.…”

AGE-RAGE Axis in the Pathophysiology of Chronic Lower Limb Ischemia and a Novel Strategy for Its Treatment

“…AGE, RAGE, and sRAGE may play an important role in the development of atherosclerosis and hence CLLI. As described earlier in this article, plasma and skin [25][26][27][28][29][30][31][32][33][34] levels of AGE and RAGE expression in tissue [33][34][35] are elevated while the serum levels of sRAGE 37,38 are reduced in patients with CLLI. In addition, AGE is present in the atherosclerotic plaque of diabetic patients.…”