new blood vessels. This angiogenic process makes a critical contribution during embryogenesis and in the response to ischemia in adult tissues [12,13,14,15]. VEGF resistance has been recently observed in diabetic angiogenesis which is attribute to monocytic VEGFR1 down-regulation [16]. The observation suggests that components or downstream targets of VEGF signaling, such as VEGFR2, could be missing or dysfunctional in diabetes. In fact, VEGFR2 protein reduction has been observed in patients with diabetes [17,18] and in experimental diabetic animals [19]. The mechanism underlying VEGFR2 reduction and the contributions to angiogenesis impairment in diabetes are not known. Methylglyoxal (MGO) is the major source of intracellular advanced glycation end-products (AGEs) [20]. It is a highly reactive a-oxoaldehyde being formed primarily from the intermediates of glycolysis in cells [21]. MGO has been implicated in the pathogenesis of diabetic complications [22]. Consistent with the finding that high glucose increases MGO production in cell culture in vitro [23], hyperglycemia enhances MGO production in patients with diabetes [24]. MGO can be detoxified efficiently by Glyoxalase (Glo) 1 [25]. While over- expression of Glo1 inhibits AGEs formation in cultured endothelial cells [26] and in diabetic animals [27], Glo1 deficiency is associated with increased intracellular AGEs [28]. Moreover, it has been reported that AGEs attenuate the angiogenic response in vitro [29]. In contrast, overexpression of Glo1 reverses high glucose-impaired angiogenesis in cultured endothelial cells [30]; blockade of AGEs formation by aminoguanidine restores ischemia-induced angiogenesis in pe- ripheral limbs of diabetic mice in vivo [31]. Provided the crucial role of VEGFR2 in endothelial angiogenesis [32] and the implications of MGO in diabetic complications [22], this study investigated the impacts of MGO on VEGFR2 protein levels and endothelial angiogenesis in cell culture and mouse models to define a potentially new mechanism underlying angiogenesis impairment in diabetes. Materials and Methods Materials The antibodies used in the present study included: VEGFR2 (55B11), b-actin, Beclin-1, LC3B, SOD1, and peroxidase conju- gated secondary antibodies (Cell Signaling, Danvers, MA); VEGFR2 (Flk-1:A-3), Bcl-2, Bax, Caspase 3, and Glo1 (Santa Cruz Biotechnology, Santa Cruz, CA); SOD2 (Fisher scientific, Pittsburgh, PA). The reagents included: MGO (Santa Cruz Biotechnology, Santa Cruz, CA); MG132 and mito-TEMPO-H (Enzo Life Sciences, Farmingdale, NY) (mTempol); protease inhibitor cocktail (EMD Chemicals, San Diego, CA); chloroquine, bafilomycin A1, pepstatin A, L-NAME, methotrexate (MTX), MTT, peroxynitrite, rapamycin, and uric acid (Fisher scientific, Pittsburgh, PA); ProLongH Gold and SlowFadeH Gold Antifade Reagents and goat anti-rabbit IgG conjugates labeled with fluorescent dyes (Invitrogen, Carlsbad CA); epoxomicin, lactacys- tin, and z-VAD-fmk (Sigma, St. Louis, MO); SiRNA of human LC3B, Beclin-1,Receptor for AGE (RAGE), or control (Santa Cruz Biotechnology, Santa Cruz, CA); Adenoviral vectors expressing GFP, SOD1 or SOD2 (Vectors BioLabs, Philadelphia, PA); transfection-ready plasmids pCMV-Glo1 and pCMV-cata- lase, as well as their control DNA plasmids pCMV-GFP (OriGene, Rockville, MD). Autophagy of VEGFR2 Impairs Diabetic Angiogenesis Endothelial Cells and Infection with Adenovirus, Plasmid, and siRNA The endothelial cells: bovine aortic endothelial cells (BAEC) and human umbilicus vessel endothelial cells (HUVEC) cells and mediums were from ATCC (Manassas, VA). The cells were grown at 70–80% confluent and used between passages 3 and 8 as previously reported [33,34,35]. Adenoviral infection of GFP, SOD1, and SOD2 were performed as previously reported [35]. Confluent endothelial cells were infected with transfection-ready plasmid encoding Glo1 (pCMV-Glo1) or catalase (pCMV- catalase), as well as their control DNA plasmids (pCMV-GFP) according to instructions provided by OriGene (Rockville, MD). Transfection of pCDNA3 control or Beclin-1 was performed as previously described [36]. The plasmid vector pCDNA3-Beclin 1 was kindly provided by Dr. Junying Yuan through Addgene (Cambridge, MA: Addgene plasmid 21150). Transfection of control or target (LC3B, Beclin-1, and Glo1) siRNA was performed based on protocols provided by Santa Cruz Bio- technology (Santa Cruz, CA). All cells were incubated in a humidified atmosphere of 5% CO2+95% O2 at 37uC. Western Blot Analysis Cultured cells or aortas were homogenized on ice in cell-lysis buffer. Aortic VEGFR2 was detected through an immunoprecip- itation-combined Western blot, where VEGFR2-co-immunopre- cipitates were detected by Western blot with an antibody recognizing another VEGFR2 epitope. Western blotting and band densitometry were performed as previously reported [33,34]. Cell Viability Measurement Endothelial cells were seeded at a density of 56104 cells/ml in 96-well plates. After being treated with MGO at studied concentrations, cell viability was measured using the MTT assay as previously reported [37]. Briefly, the cells after MGO or vehicle treatment were incubated with MTT (5 mg/ml, culture medium) and followed by absorption measurement at 490 nm. The cell viability was presented as a percentage of the DTT counts of the vehicle-treated cells. Cell Migration Assay Cell migration was measured as previously reported [38]. Briefly, BAEC were plated at confluence and the monolayer artificially wounded by scraping with a pipette tip, followed by wash with medium and incubation with the studying reagents. Phase contrast cell images were taken after healing overnight. The wound healing was quantified by measuring the wounding area using the NIH ImageJ Program and expressed as percentage of the section area. Tube Formation Assay The tube or vascular-like structure formation by endothelial cells was assessed on growth factor-reduced Matrigel (BD Biosciences), as described [39]. Briefly, endothelial cells were seeded on Matrigel-coated 24-well plates at 16105 cells per well in 1% FBS medium containing MGO or other reagents and incubated at 37uC for 18 hours. Tube formation images were observed using an inverted phase contrast microscope, and the degree of tube formation was quantified by measuring the number of tubes in 30 randomly selected fields quantified by the NIH ImageJ Program and expressed as a percentage of the total number of sprouts in control. PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46720