Quantification of MGO-modified Proteins (Adducts) The quantification of MGO-modified proteins (MGO-protein adducts) was determined with an enzyme immunoassay kit (OxiSelectTM Methylglyoxal ELISA Kit) purchased from Cell BioLabs Inc. (San Diego, CA). This was accomplished by comparing the absorbance with that of a known MGO-bovine serum albumin standard curve according to the product manual provided by the manufacture. RNA Isolation, Reverse Transcription, and Real-time PCR Total cellular RNA was isolated from endothelial cells, reverse transcribed into cDNA, and the resulting cDNA was subjected to quantitative polymerase chain reaction (PCR) as previously described [40]. Forward and reverse specific primer sequences were: VEGFR2 [40], forward: 59- TGTGGGTTTGCCTAGTGTTTCT; reverse: 59-CACT- CAGTCACCTCCACCCTT. b-actin (75 bp, Human) primer: Forward: 59-ACGGCATCGTCACCAACTG; reverse: 59- GAGCCACACGCAGCTCATT. Total RNA Kit I (#R683401) was purchased from Omega Bio-Tek (Norcross, GA), iScriptTM cDNA Synthesis Kit (170-8891), iQTM SYBR Green Supermix (170-8880), and the Real Time Detection System were obtained from Bio-Rad (Hercules, CA). PCR specific primers were synthesized by Sigma (St. Louis, MO). Autophagy Assays (Confocal Microscopy of the Autophagosome) Autophagy was visualized in endothelial cells by transfection of the plasmid pGFP-LC3 followed by analysis with confocal microscopy (Olympus FV500: Japan). The plasmid vector pGFP-LC3 was kindly provided by Dr. Toren Finkel through Addgene (Cambridge, MA: Addgene plasmid 24920). The autophagy assays were performed as previously described [41]. The number of GFP-LC3 punctae was assessed from .6 random high-power fields, and a minimum of 30 cells per sample were counted. An LC3 punctae was regarded as an isolated GFP- positive structure .1 mm diameter. Mouse Aortic Ring Assay The assay was performed with aortic ring as previously described [42]. Briefly, thoracic aortas were excised from the mice, and peri-adventitial fibro-adipose tissues were removed. Aortas were then cut into 1-mm rings, washed in culture medium and transferred to 48-well tissue culture plates coated with Matrigel (100 mL per well), which was then overlaid with an additional 100 mL of Matrigel. The plates were incubated at 37uC, and media were changed every 2 days. MGO or other reagents were incubated with cell culture medium. Aortic rings were examined daily, and digital images were taken on day 6 for quantitative analysis of vascular endothelial outgrowth (sprouts) by the NIH ImageJ Program and expressed as a percentage of the total slide area per section. Mice Male C57BL/6J mice as well as genetic diabetic Akita and db/ db (and control lean) mice, 8 weeks of age, 20–30 g, were obtained from the Jackson Laboratory (Bar Harbor, ME). Some groups of Akita mice were treated with mTempol (0.1 mM in the drinking water) or vehicle (normal drinking water) for 4 weeks. The animal protocols were reviewed and approved by the University of Oklahoma Institute Animal Care and Use Committee (IACUC). The approved IACUC protocol numbers are: 10-153-H, 11-072- H, and 11-045. Autophagy of VEGFR2 Impairs Diabetic Angiogenesis Statistical Analysis Values are expressed as mean 6 SEM. All data were analyzed with 1-way ANOVA followed by multiple t-tests, except for those obtained by studies of time course and dose effects which were analyzed by repeated-measures ANOVA. Significance was accepted at P,0.05. Results MGO Reduces VEGFR2 Protein Levels and Impairs Endothelial Cell Migration and Tube Formation We sought to investigate if MGO affected the protein levels of VEGFR2 in BAEC. First, we tested this in a range of MGO concentrations which have been widely reported. The results showed that incubation of MGO with endothelial cell reduced VEGFR2 protein levels in a time (Fig. 1A) and dose (Fig. 1B) dependent manner. The same dose and time effects of MGO were also observed in HUVEC (data not shown). Therefore, the lowest effective MGO concentration on VEGFR2 protein levels was defined as 25 mM overnight (16 h) under normal cell culture condition. To test the physiological outcome of the VEGFR2 reduction, we assessed angiogenesis, which was evaluated by assays of tube formation and cell migration in BAEC. In parallel with VEGFR2 protein reduction (Fig. 1A/B), the presence of MGO reduced tube formation (Fig. 1C, upper panel) and cell migration (Fig. 1C, bottom panel) in a dose-dependent fashion (Fig. 1D). MGO at the Studied Concentrations do not Affect Cell Viability and Markers of Apoptosis MGO has been estimated to be produced to a level as high as 400 mM per cell per day [43], and similar concentrations have been applied in many physiological studies in literature. However, it is also well known that MGO at high concentra- tions induces cell death [44]. To decide whether MGO at the studied concentrations either altered the viability or induced cell apoptosis, we performed an MTT assay and examined apoptotic markers. As depicted in Fig. 2, there was no significant difference of viability between the control and MGO-treated cells at the studied concentrations of MGO (Fig. 2A). MGO at the indicated time course did not induce apoptosis of the treated cells, evidenced by no significant changes in Western blot staining for Bcl-2, Bax, and caspase 3, the classic markers of apoptosis (Fig. 2B). Inhibition of Glo1 Enhances VEGFR2 Reduction Induced by MGO, whereas Overexpression of Glo1 Prevents the Reduction To confirm that MGO generated its effect through a physi- ological pathway and that it was MGO specific, we monitored the effect of MGO in intact cells with either loss-of-function or gain-of-function approach that modulates Glo1, the MGO detoxifying enzyme [44]. In this regard, we first pre-incubated the cells with MTX, a Glo1 inhibitor [45] that increases MGO levels [46], before MGO-challenging. As shown in Fig. S1, the presence of MTX alone decreased VEGFR2 protein levels, mimicking the effect of MGO (Fig. S1); such an effect was enhanced in the presence of exogenous MGO where VEGFR2 protein levels were further decreased (Fig. S1). To exclude potential off-target effects of MTX, we employed a genetic approach with siRNA to knockdown Glo1 protein levels (Fig. 2C). Like MTX, Glo1 siRNA, but not the control, PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46720