Angiogenesis Impairment in Diabetes: Role of Methylglyoxal-Induced Receptor for Advanced Glycation Endproducts, Autophagy and Vascular Endothelial Growth Factor Receptor 2 Hongtao Liu, Shujie Yu, Hua Zhang, Jian Xu* Section of Endocrinology, Department of Medicine, Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America Abstract Diabetes impairs physiological angiogenesis by molecular mechanisms that are not fully understood. Methylglyoxal (MGO), a metabolite of glycolysis, is increased in patients with diabetes. This study defined the role of MGO in angiogenesis impairment and tested the mechanism in diabetic animals. Endothelial cells and mouse aortas were subjected to Western blot analysis of vascular endothelial growth factor receptor 2 (VEGFR2) protein levels and angiogenesis evaluation by endothelial cell tube formation/migration and aortic ring assays. Incubation with MGO reduced VEGFR2 protein, but not mRNA, levels in a time and dose dependent manner. Genetic knockdown of the receptor for advanced glycation endproducts (RAGE) attenuated the reduction of VEGFR2. Overexpression of Glyoxalase 1, the enzyme that detoxifies MGO, reduced the MGO-protein adducts and prevented VEGFR2 reduction. The VEGFR2 reduction was associated with impaired angiogenesis. Suppression of autophagy either by inhibitors or siRNA, but not of the proteasome and caspase, normalized both the VEGFR2 protein levels and angiogenesis. Conversely, induction of autophagy either by rapamycin or overexpression of LC3 and Beclin-1 reduced VEGFR2 and angiogenesis. MGO increased endothelial LC3B and Beclin-1, markers of autophagy, which were accompanied by an increase of both autophagic flux (LC3 punctae) and co- 2 immunoprecipitation of VEGFR2 with LC3. Pharmacological or genetic suppression of peroxynitrite (ONOO ) generation not only blocked the autophagy but also reversed the reduction of VEGFR2 and angiogenesis. Like MGO-treated aortas from normglycemic C57BL/6J mice, aortas from diabetic db/db and Akita mice presented reductions of angiogenesis or VEGFR2. Administration of either autophagy inhibitor ex vivo or superoxide scavenger in vivo abolished the reductions. Taken together, MGO reduces endothelial angiogenesis through RAGE-mediated, ONOO–dependent and autophagy-induced VEGFR2 degradation, which may represent a new mechanism for diabetic angiogenesis impairment. Citation: Liu H, Yu S, Zhang H, Xu J (2012) Angiogenesis Impairment in Diabetes: Role of Methylglyoxal-Induced Receptor for Advanced Glycation Endproducts, Autophagy and Vascular Endothelial Growth Factor Receptor 2. PLoS ONE 7(10): e46720. doi:10.1371/journal.pone.0046720 Editor: Masuko Ushio-Fukai, University of Illinois at Chicago, United States of America Received April 12, 2012; Accepted September 7, 2012; Published October 3, 2012 Copyright: 2012 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an NIH Grant (P20 RR 024215-05) from the COBRE Program of the National Center for Research Resources, a National Scientist Development Grant (10SDG2600164) from the American Heart Association, a Junior Faculty Award (1-12-JF-58) from the American Diabetes Association, and a Research Award (HR11-200) from the Oklahoma Center for Advancement of Science and Technology (all to J.X.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jian-xu@ouhsc.edu Introduction Angiogenesis, defined as the formation of new blood vessels out of preexisting capillaries, plays a crucial role in maintaining vascular health [1]. Angiogenesis impairment operates in the peripheral vasculature contributing to delayed wound healing, exacerbated peripheral limb ischemia, and even cardiac mortality via reduction of collateral vessel development [2]. Mechanisms underlying diabetic angiogenesis impairment are complex. Mul- tiple molecular mechanisms have been proposed, including oxidative stress/reactive oxygen species, endothelial derange- ments, loss of endothelium-derived nitric oxide (NO) bioactivity, and micro RNA alterations [3]. However, it has not been completely elucidated how diabetes impairs physiological angio- genesis. VEGF receptor 2 (VEGFR2, or kinase-insert domain receptor, KDR/fetal liver kinase, Flk-1) first identified in 1991 [4], is produced within the cell and expressed on the cell surface as a matured 230 kD form (between 200–250 kD) of protein pending on levels of glycosylation [5]. In an intact cell, VEGFR2 binds certain members of the VEGF family [6] through dimerization and strong ligand-dependent tyrosine phosphorylation, which results in a mitogenic, chemotactic, and pro-survival signal [7]. VEGFR2 serves as the principal receptor for VEGF signaling [8] that leads to vasodilatation, endothelial cell migration, and proliferation [9]. VEGFR2-KO mice are embryonic lethal (at E8.5–9.5) with defective blood-island formation and vasculogen- esis suggesting that VEGFR2 signaling is required for cardiovas- cular development [10]. Cell migration signals are recently shown to use, at least partly, a pathway dependent on an adaptor region of VEGFR2 [11]. Endothelial cells respond to VEGF to produce PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46720