Autophagy of VEGFR2 Impairs Diabetic Angiogenesis Figure 1. MGO reduces VEGFR2 protein levels and impairs endothelial cell angiogenesis. A and B: MGO reduced VEGFR2 protein levels in a time and dose dependent fashion. BAEC were incubated with MGO at indicated concentrations for up to 24 h. Then cells were subjected to Western blot respectively with a rabbit derived VEGFR2 antibody and a mouse derived b-actin antibody. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). C and D: MGO reduced endothelial cell angiogenesis in a dose dependent manner. Cells incubated with MGO (10–100 mM) were subjected to angiogenesis assessment by endothelial cell tube formation (upper panel in C) and migration (bottom panel in C). *P,0.05 vs control (n = 3). doi:10.1371/journal.pone.0046720.g001 reduced VEGFR2 protein levels (Fig. 2C). The reduction was further enhanced when MGO was present (Fig. 2C). To further confirm that the effect was MGO-specific, we quantified the levels of MGO-protein adduct in the treated cells by ELISA. As depicted, MGO vs control increased intracellular MGO-protein adducts (Fig. 2D). Knockdown of Glo1 alone elevated MGO- protein adducts (Fig. 2D); such an effect was dramatically strengthened in MGO-treated cells (Fig. 2D). Next, we infected the cells with a Glo1-containing plasmid, using the cells infected with an empty plasmid as control. In line with the previous results (Fig. 1A/B, 2C and Fig. S1), MGO reduced VEGFR2 protein levels in the control cells (Fig. 2E); however, the reduction was absent in Glo1-overexpressing cells (Fig. 2E). Similarly, MGO augmented MGO-protein adduct levels in the control-plasmid but not the Glo1-overexpressing cells (Fig. 2F). Finally, we asked whether the effect of MGO required RAGE, the receptor for AGE. We choose to check VEGFR2 protein levels after the RAGE was knockdown. As expected, the RAGE protein staining was reduced in RAGE- but not control-siRNA treated cells (Fig. 2G). Importantly, the otherwise reduction of VEGFR2 was markedly prevented (around 80%) when RAGE was knockdown (Fig. 2G), suggesting a dominant role of RAGE in the studied effects of MGO. In sum, these data suggest that the MGO-generated effects were indeed physiologically relevant. Inhibition of Autophagy, but not Proteasome or Caspase, Abolishes MGO-induced VEGFR2 Protein Reduction To dissect the potential mechanism underlying VEGFR2 protein reduction, we measured VEGFR2 protein levels in the presence of proteolytic inhibitors. To this end, we pre-incubated BAEC with respective inhibitors of lysosome or autophagy, proteasome and caspase, prior to MGO challenge. As shown in Fig. 3A, pre-incubation with chloroquine (CQ), a widely recog- nized inhibitor for autophagy and lysosome, prevented VEGFR2 protein reduction. Other lysosome/autophagy inhibitors, such as pepstatin A (Pep A) (Fig. 3B) and bafilomycin A1 (Baf A1) (Fig. 3C) showed similar protective effects. To confirm the role of autophagy, we examined the effects of MGO in BAEC where important autophagy effectors, such as Beclin-1 and LC 3B [47], were knocked down. As presented in Fig. 3, MGO reduced VEGFR2 protein levels in the control-siRNA-infecting BAEC, as anticipated (Fig. 3D); however, Beclin-1 siRNA infection de- creased Beclin-1 protein levels (Fig. 3D) and prevented VEGFR2 protein reduction by MGO (Fig. 3D). Similar VEGFR2-rescuring effects were observed when LC 3B was knocked down (Fig. S2). However, respective pre-incubation with MG132, epoxomicin, and lactacystin, the structurally unrelated inhibitors of protea- some, could not block the reduction (Fig. 3E), although VEGFR2 can be a proteasome target [48]; pre-incubation with z-VAD-fmk (z-VAD), a broad caspase inhibitor, also did not prevent the PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e46720