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.
In apoE(-/-) mice, MMP expression is greater than apoptosis as the disease progresses and MPI may be a better imaging agent for more advanced disease.
BackgroundReceptor for advanced glycation endproducts (RAGE) expression contributes to the impaired angiogenic response to limb ischemia in diabetes. The aim of this study was to detect the effect of increased expression of RAGE on the angiogenic response to limb ischemia in diabetes by targeting αvβ3 integrin with 99mTc-labeled Arg-Gly-Asp (RGD).MethodsMale wild-type (WT) C57BL/6 mice were either made diabetic or left as control for 2 months when they underwent femoral artery ligation. Four groups were studied at days 3 to 7 after ligation: WT without diabetes (NDM) (n = 14), WT with diabetes (DM) (n = 14), RAGE-/- NDM (n = 16), and RAGE-/- DM (n = 14). Mice were injected with 99mTc-HYNIC-RGD and imaged. Count ratios for ischemic/non-ischemic limbs were measured. Muscle was stained for RAGE, αvβ3, and lectins.ResultsThere was no difference in count ratio between RAGE-/- and WT NDM groups. Mean count ratio was lower for WT DM (1.38 ± 0.26) vs. WT NDM (1.91 ± 0.34) (P<0.001). Mean count ratio was lower for the RAGE-/- DM group than for RAGE-/- NDM group (1.75 ± 0.22 vs. 2.02 ± 0.29) (P<0.001) and higher than for the WT DM group (P<0.001). Immunohistopathology supported the scan findings.ConclusionsIn vivo imaging of αvβ3 integrin can detect the effect of RAGE on the angiogenic response to limb ischemia in diabetes.
OBJECTIVES The aim of this study was to image expression of receptor for advanced glycation end products (RAGE) in a mouse model of myocardial reperfusion injury. BACKGROUND RAGE and its ligands are implicated in the pathogenesis of ischemia/reperfusion injury and infarction. We hypothesized that RAGE-directed quantitative imaging of myocardial uptake of technetium-99m (99mTc)-anti-RAGE F(ab′)2 in a mouse model of myocardial ischemic injury can detect RAGE expression and show quantitative differences between early (18 to 20 h) and later times (48 h) after reperfusion. METHODS Twenty-five wild-type (WT) mice underwent left anterior descending coronary artery occlusion for 30 min. Mice were injected with 19.98 ± 1.78 MBq of 99mTc anti-RAGE F(ab′)2 at 2 time points after reperfusion (at 18 to 20 h [n = 8] and at 48 h [n = 12]) and 5 h later with 6.14 ± 2.0 MBq of thallium-201 (201Tl). Five WT mice were injected with nonspecific F(ab′)2 and 201Tl 18 to 20 h after reperfusion. Six WT mice underwent sham operation without coronary intervention. After injection with 201Tl, all mice immediately underwent dual isotope single-photon emission computed tomography/computed tomography. At completion of imaging, hearts were counted and sectioned. RESULTS The uptake of 99mTc-anti-RAGE F(ab′)2 in the ischemic zone from the scans as mean percentage injected dose was significantly greater at 18 to 20 h (5.7 ± 2.1 × 10−3%) as compared with at 48 h (1.4 ± 1.1 × 10−3%; p < 0.001) after reperfusion. Disease and antibody controls showed no focal uptake in the infarct. Gamma well counting of the myocardium supported the quantitative scan data. By immunohistochemical staining there was greater caspase-3 and RAGE staining at 18 to 20 h versus at 48 h (p = 0.04 and p = 0.01, respectively). On dual immunofluorescence, RAGE colocalized mainly with injured cardiomyocytes undergoing apoptosis. CONCLUSIONS RAGE expression in myocardial ischemic injury can be imaged in vivo using a novel 99mTc-anti-RAGE F(ab′)2. RAGE plays a role in several cardiovascular diseases and is a potential target for clinical imaging.
Optical agents targeting α(v)β₃ are potential tools to image the angiogenic response to limb ischemia. The left (L) femoral artery was ligated in 17 mice and sham surgery performed on the contralateral right (R) hindlimb. Seven days later, IntegriSense (2 nmol) was injected into 11 mice and 6 were probe controls. Six hours later, mice underwent optical imaging. Ratios of photon flux in the L/R limbs were calculated. Tissue was stained for α(v) , CD31, and lectin. The signal was increased in the ischemic limbs compared to contralateral legs and ratio of photon flux in L/R limb averaged 2.37. Control probe showed no hindlimb signal. IntegriSense colocalized with CD31 by dual fluorescent staining. Ratios for L/R hindlimbs correlated with quantitative lectin staining (r = 0.88, p = 0.003). Optical imaging can identify and quantify angiogenic response to hindlimb ischemia.
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