Marika Villa1, Mariacristina Parravano2, Arianna Micheli3, Lucia Gaddini4, Andrea Matteucci4, Cinzia Mallozzi3, Francesco Facchiano5, Fiorella Malchiodi-Albedi4, Flavia Pricci6. 1. Dept of Cardiovascular, Dysmetabolic and Aging-associated diseases. 2. Dept of Ophthalmology, G.B. Bietti Eye Foundation-IRCCS, Rome, Italy. 3. Dept of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy. 4. National Center for Drug Research and Evaluation. 5. Dept of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy. 6. Dept of Cardiovascular, Dysmetabolic and Aging-associated diseases. Electronic address: flavia.pricci@iss.it.
Abstract
OBJECTIVE: Advanced glycation end-products (AGEs) constitute a highly heterogeneous family of compounds, relevant in the pathogenesis of diabetic complications, which could represent efficient biomarkers of disease progression and drug response. Unfortunately, due to their chemical heterogeneity, no method has been validated to faithfully monitor their levels in the course of the disease. In this study, we refine a procedure to quantitatively analyze fluorescent AGEs (fAGEs), a subset considered remarkably representative of the entire AGE family, and measure them in in vitro glycated BSA (gBSA) and in plasma and vitreous of diabetic rats, for testing its use to possibly quantify circulating AGEs in patients, as markers of metabolic control. METHODS: fAGE levels were evaluated by spectrofluorimetric analysis in in vitro and in vivo experimental models. BSA was glycated in vitro with increasing D-glucose concentrations for a fixed time or with a fixed D-glucose concentration for increasing time. In in vivo experiments, streptozotocin-induced diabetic rats were studied at 1, 3, 6 and 12weeks to analyze plasma and vitreous. To confirm the presence of AGEs in our models, non-diabetic rat retinal explants were exposed to high glucose (HG), to reproduce short-term effects, or in vitro gBSA, to reproduce long-term effects of elevated glucose concentrations. Rat retinal explants and diabetic retinal tissues were evaluated for the receptor for advanced glycation end-product (RAGE) by Western blot analysis. RESULTS: In in vitro experiments, fluorescence emission showed glucose concentration- and time-dependent increase of fAGEs in gBSA (p≤0.05). In streptozotocin-induced diabetic rats, fAGE in plasma and vitrei showed an increase at 6 (p≤0.005) and 12 (p≤0.05) weeks of diabetes, with respect to control. RAGE was time-dependently upregulated in retinas incubated with gBSA, but not with HG, and in diabetic retinal tissue, substantiating exposure to AGEs. CONCLUSIONS: Applying the proposed technique, we could show that fAGEs levels increase with glucose concentration and time of exposure in vitro. Furthermore, in diabetic rats, it showed that circulating fAGEs are similarly upregulated as those in vitreous, suggesting a correlation between circulating and tissue AGEs. These results support the use of this method as a simple and reliable test to measure circulating fAGEs and monitor diabetes progression.
OBJECTIVE: Advanced glycation end-products (AGEs) constitute a highly heterogeneous family of compounds, relevant in the pathogenesis of diabetic complications, which could represent efficient biomarkers of disease progression and drug response. Unfortunately, due to their chemical heterogeneity, no method has been validated to faithfully monitor their levels in the course of the disease. In this study, we refine a procedure to quantitatively analyze fluorescent AGEs (fAGEs), a subset considered remarkably representative of the entire AGE family, and measure them in in vitro glycated BSA (gBSA) and in plasma and vitreous of diabeticrats, for testing its use to possibly quantify circulating AGEs in patients, as markers of metabolic control. METHODS:fAGE levels were evaluated by spectrofluorimetric analysis in in vitro and in vivo experimental models. BSA was glycated in vitro with increasing D-glucose concentrations for a fixed time or with a fixed D-glucose concentration for increasing time. In in vivo experiments, streptozotocin-induced diabeticrats were studied at 1, 3, 6 and 12weeks to analyze plasma and vitreous. To confirm the presence of AGEs in our models, non-diabeticrat retinal explants were exposed to high glucose (HG), to reproduce short-term effects, or in vitro gBSA, to reproduce long-term effects of elevated glucose concentrations. Rat retinal explants and diabetic retinal tissues were evaluated for the receptor for advanced glycation end-product (RAGE) by Western blot analysis. RESULTS: In in vitro experiments, fluorescence emission showed glucose concentration- and time-dependent increase of fAGEs in gBSA (p≤0.05). In streptozotocin-induced diabeticrats, fAGE in plasma and vitrei showed an increase at 6 (p≤0.005) and 12 (p≤0.05) weeks of diabetes, with respect to control. RAGE was time-dependently upregulated in retinas incubated with gBSA, but not with HG, and in diabetic retinal tissue, substantiating exposure to AGEs. CONCLUSIONS: Applying the proposed technique, we could show that fAGEs levels increase with glucose concentration and time of exposure in vitro. Furthermore, in diabeticrats, it showed that circulating fAGEs are similarly upregulated as those in vitreous, suggesting a correlation between circulating and tissue AGEs. These results support the use of this method as a simple and reliable test to measure circulating fAGEs and monitor diabetes progression.
Authors: Maria Ferraro; Serena Di Vincenzo; Claudia Sangiorgi; Stefania Leto Barone; Sebastiano Gangemi; Luigi Lanata; Elisabetta Pace Journal: Pharmaceuticals (Basel) Date: 2022-02-11