Screening System of Blocking Agents of the Receptor for Advanced Glycation Endproducts in Cells Using Fluorescence

Abstract

Activation of the receptor for advanced glycation endproducts (RAGE) triggers cellular responses implicated in the pathogenesis of diabetic complications; blockade of RAGE has been shown to inhibit the development of diabetic complications. To develop a screening system to identify novel disruptors of advanced glycation endproducts (AGE)-RAGE binding, we used an AGE-RAGE binding system in RAGE-overexpressing cells; test compounds were screened using this system. To construct human RAGE-overexpressing cells, mouse mesangial cells (MMCs) were stably transfected with the pcDNA-human RAGE (hRAGE) vector and selected under 1 mg/mL gentamicin (G418). RAGE expression in hRAGE-overexpressing MMCs was analyzed by Western blotting with specific RAGE antibody. To identify novel disruptors of AGE-RAGE binding, 50 single compounds and AGE-bovine serum albumin (BSA)-Alexa 488 (AGE-BSA labeled with Alexa 488) were treated to the hRAGE-overexpressing MMCs. Nonbinding AGE-BSA-Alexa 488 was washed and fluorescence measured by microtiter plate reader (excitation wavelength, 485 nm; emission wavelength, 528 nm). In hRAGE-overexpressing cells, only treatment with AGE-BSA-Alexa 488 significantly increased fluorescence intensity in a dose-dependent manner. Of 50 compounds tested, genistein disrupted AGE-RAGE binding in a dose-dependent manner. This AGE-RAGE binding system using AGE-BSA-Alexa 488 in hRAGE-overexpressing cells was suitable for screening of agents that disrupt AGE-hRAGE binding. Key words advanced glycation endproduct; receptor for advanced glycation endproduct; screeningChronic hyperglycemia causes increased formation of advanced glycation endproducts (AGEs) in tissues of individuals with diabetes. The formation and accumulation of AGEs can accelerate the development of diabetic vascular complications.1) Approaches for the development of drugs either to prevent or treat diabetic complications have focused on inhibition of AGE formation, suppression of AGE receptor (RAGE) expression or its downstream pathways, and blockade of the AGE-RAGE interaction.2-4) Inhibitors of AGE formation such as aminoguanidine (Pimagedine) and pyridoxamine (Pyridorin) prevent diabetic nephropathy by inhibiting albuminuria, glomerular hypertrophy, and mesangial expansion in patients with type 1 diabetes and in animal models. 2,5,6) The AGE-protein crosslinker breaker ALT-711 (Alagebrium) improves endothelial dysfunction in patients with isolated systolic hypertension and decreases left ventricular mass in patients with diastolic heart failure. [7][8][9] In our previous study, 700 plant extracts and phytochemicals were tested for inhibition of AGE formation by incubating glucose and bovine serum albumin (BSA) for 14 d; several extracts were shown to prevent diabetic complications, specifically nephropathy, cataract formation, and retinopathy in experimental animal models. [10][11][12]

Effects of Allium victorialis leaf extracts and its single compounds on aldose reductase, advanced glycation end products and TGF-β1 expression in mesangial cells

“…Although, IC 50 level of EtOAc- soluble fraction was higher than TMG (0.94±0.01 μg/ml), among the extracts, it has the inhibitory effects on AGEs formation (IC 50 =30.13±1.68 μg/ml; AG, IC 50 = 76.47±4.81 μg/ml). Previous research indicated that genistein has inhibitory effects of AR activity in vitro, AGEs formation, and AGE-RAGE binding in hRAGE-overexpressing cells [9,23]. Next, we examined the inhibitory effects of compounds and extracts on AGEs formation (Table 1).…”

“…AGEs formation assay was performed as previously described [23,24]. Bovine serum albumin (BSA, 10 mg/ml, Sigma-Aldrich) in 50mM phosphate buffer (pH 7.4) with containing 0.02% sodium azide to prevent bacterial growth was added to 0.2 M fructose and glucose.…”

“…AGE-BSA/RAGE binding in the cells was determined as described previously [23]. Briefly, Alexa 488 labeling of AGE-BSA was performed using the Alexa Fluor® 488 protein labeling kit (Molecular Probes, Eugene, OR).…”

A branched arabinoglucan from Angelica sinensis ameliorates diabetic renal damage in rats

“…The formation of AGEs occurs as a result of the Maillard reaction, a nonenzymatic modification of proteins, lipids, and nucleic acids by reducing sugars. It has been reported that low‐molecular weight heparin (LMWH; Myint et al, ), a β‐(1,2)‐fructan with branches at O‐6 and terminating with a glucosyl residue (Xiao et al, ), quercitrin (Kim et al, ), and genistein (Jung et al, ) antagonize AGE action on RAGE and have preventive effects on DN. LMWH binds RAGE with an affinity approximately six times higher than AGEs.…”

“…A human RAGE (hRAGE)-overexpressing plasmid was constructed as previously described (Jung, Kim, & Kim, 2012). Briefly, full-length human RAGE cDNA (GenBank® accession no.…”