Autophagy of VEGFR2 Impairs Diabetic Angiogenesis Figure 2. Knockdown of Glo1 enhances VEGFR2 reduction induced by MGO, whereas Glo1 overexpression prevents the reduction. A–B: MGO did not affect cell viability or apoptotic markers. BAEC were incubated with MGO at the indicated concentrations (for 24 h) or time course (up to 24 h) followed either by viability measurement (MTT assay) or Western blot staining for markers of apoptosis with respective antibodies against Bcl-2, Bax and Caspase 3. C: Knockdown of Glo1 by siRNA partly mimicked the MGO effect and enhanced MGO-induced VEGFR2 reduction. Transfection of control or Glo1 siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA). D: MGO increased levels of MGO-protein adduct which were further enhanced by siRNA knockdown of Glo1. E: Overexpression of Glo1 blocked MGO-induced VEGFR2 reduction. Infection of either control or Glo1 containing plasmid DNA was performed according to the protocol provided by OriGene (Rockville, MD). F: Overexpression of Glo1 blocked MGO-increased MGO-protein adducts. G: Knockdown of RAGE by siRNA prevented MGO-induced VEGFR2 reduction. Transfection of control or RAGE siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA). Western blot was performed with indicated antibodies. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). NS: not significant vs control (at 0 mM of MGO). Casp-3: Caspase-3; c-Casp-3: cleaved Caspase-3. doi:10.1371/journal.pone.0046720.g002 reduction (Fig. 3E). Next we wondered whether MGO affected VEGFR2 at the transcriptional level. To answer this question, we performed RT-PCR to quantify the mRNA levels of VEGFR2 in the presence of MGO. As shown in the Fig. S3, MGO at indicated time and dose did not change VEGFR2 mRNA levels; similar results were obtained in other settings where Glo1 was manipu- lated (Fig. S3). The data suggested that the effect of MGO mainly occurred at a protein level and that autophagy played a key role in MGO-mediated VEGFR2 reduction. Autophagy Suppression Rescues MGO-impaired Endothelial Cell Tube Formation To determine the physiological consequence of VEGFR2 protein restoration, we monitored endothelial cell tube formation with and without the presence of autophagy inhibitors. As demonstrated in Fig. 3, respective administration of CQ, Pep A and Baf A1 reversed the impaired tube formation (Fig. 3F), which was associated with restored VEGFR2 protein levels (Fig. 3A/B/ C). However, application of the inhibitors alone did not affect endothelial tube formation (Fig. S4). Furthermore, the potential off-target effect of the inhibitors could be excluded, since the siRNA knockdown of Beclin 1 also rescued MGO-impaired endothelial tube formation (Fig. 3G), which was accompanied with restored VEGFR2 protein levels (Fig. 3D). All these data indicated the essential contribution of autophagy to the MGO-elicited effects. MGO Increases Markers of Autophagy and Autophagic Flux, whereas Autophagy Induction Mimics the MGO- effects in Endothelial Cells To further confirm the involvement of autophagy and exploit the underlying mechanism, we first measured protein levels of Beclin-1 and LC 3B, two common markers of autophagy [47], in MGO treated BAEC. As shown in Fig. 4, MGO increased both Beclin-1 and LC3B (mainly the LC 3B-II) protein levels (Fig. 4A). Of note, the increase in LC3B-II protein levels implicated an augmented conversion of LC3B-I to LC3B-II, a hallmark of autophagy in mammalian cells [49]. Next, we assayed the autophagic flux by counting the ‘‘LC3-dots or punctae’’ [50], a marker of autophagy initiation or autophagosome formation PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e46720