title: Blockade by phosphorothioate aptamers of advanced glycation end products-induced damage in cultured pericytes and endothelial cells.
abstract: Advanced glycation end products (AGEs) not only inhibit DNA synthesis of retinal pericytes, but also elicit vascular hyperpermeability, pathological angiogenesis, and thrombogenic reactions by inducing vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) through the interaction with the receptor for AGEs (RAGE), thereby being involved in the pathogenesis of diabetic retinopathy. In this study, we screened novel phosphorothioate-modified aptamers directed against AGEs (AGEs-thioaptamers) using a combinatorial chemistry in vitro, and examined whether these aptamers could inhibit the AGE-induced damage in both retinal pericytes and human umbilical vein endothelial cells (HUVECs). We identified 11 AGEs-thioaptamers; among them, clones #4, #7s and #9s aptamers had higher binding affinity to AGEs-human serum albumin (HSA) than the others. Surface plasmon resonance analysis revealed that KD values of #4s, #7s and #9s were 0.63, 0.36, and 0.57nM, respectively. Furthermore, these 3 clones dose-dependently restored the decrease in DNA synthesis in AGE-exposed pericytes. AGEs significantly increased RAGE, VEGF and PAI-1 mRNA levels in HUVEC, all of which were completely blocked by the treatment with 20nM clone #4s aptamer. Quartz crystal microbalance analysis confirmed that #4s aptamer dose-dependently inhibited the binding of AGEs-HSA to RAGE. Our present study demonstrated that AGEs-thioaptamers could inhibit the harmful effects of AGEs in pericytes and HUVEC by suppressing the binding of AGEs to RAGE. Blockade by AGEs-thioaptamers of the AGEs-RAGE axis might be a novel therapeutic strategy for diabetic retinopathy.
authors.affiliation: Department of Chemistry, Kurume University School of Medicine, Kurume, Japan. Electronic address: higashiy@med.kurume-u.ac.jp.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Higashimoto
authors.forename: Takanori
authors.initials: T
authors.lastname: Matsui
authors.forename: Yuri
authors.initials: Y
authors.lastname: Nishino
authors.forename: Junichi
authors.initials: J
authors.lastname: Taira
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Inoue
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Sho-Ichi
authors.initials: S
authors.lastname: Yamagishi
mesh_terms: D052157:Aptamers, Nucleotide / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication; D015723:Gene Library; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D020286:Pericytes / Q000378:metabolism* / Q000473:pathology; D054735:Phosphorothioate Oligonucleotides / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D012171:Retinal Vessels / Q000378:metabolism* / Q000473:pathology; D052156:SELEX Aptamer Technique; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D017127:Glycation End Products, Advanced; D054735:Phosphorothioate Oligonucleotides; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; C051321:SERPINE1 protein, human; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D004247:DNA
keywords: 2′-deoxyadenosine-5′-O-(1-thiotriphosphate); 2′-deoxythymidine-5′-O-(1-thiotriphosphate); AGEs; AGEs–thioaptamers; BSA; EC; ELISA; FBS; HSA; HUVECs; K(D); PAI-1; PCR; QCM; RAGE; RT-PCR; SELEX; SPR; VEGF; advanced glycation end products; bovine serum albumin; dATP(αS); dTTP(αS); dissociation constant; double-stranded DNA; dsDNA; endothelial cells; enzyme-linked immunosorbent assay; fetal bovine serum; human serum albumin; human umbilical vein endothelial cells; phosphorothioate aptamers directed against AGEs; plasminogen activator inhibitor-1; polymerase chain reactions; quartz crystal microbalance; real-time reverse transcription PCR; receptor for AGEs; single-stranded DNA; ssDNA; surface plasmon resonance; systematic evolution of ligands by exponential enrichment; v-domain of RAGE; vRAGE; vascular endothelial growth factor