Autophagy of VEGFR2 Impairs Diabetic Angiogenesis Figure 4. MGO induces autophagy markers and autophagic flux, whereas genetic or pharmacological induction of autophagy mimics MGO-elicited effects. A: MGO increased autophagy markers Beclin 1 and LC3B. B: MGO induced LC3 punctae formation (autophagic flux) (Confocal microscopy imaging). C: MGO increased immunoprecipitation of VEGFR2 with LC3B, an autophagy marker and a component of the autophagosome. D: overexpression of Beclin 1 decreased VEGFR2 protein levels, and the reduction was accelerated when MGO was present. E: Rapamycin (1 mM for 1 h) decreased VEGFR2 protein levels. F: Rapamycin (1 mM) reduced endothelial tube formation. BAEC were incubated with MGO (25 mM) for 1 h (A–C) and 16 h (D). Cells were collected for Western blotting with a rabbit derived Beclin-1 or LC 3B antibody and a mouse derived b-actin antibody. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). doi:10.1371/journal.pone.0046720.g004 expected, ONOO2 incubation in the absence of MGO increased both Beclin 1 and LC3B protein levels peaking at 2 h (Fig. 5D). These data suggested that the MGO-induced autophagy was ONOO2 dependent and inhibition of ONOO2 generation inhibited autophagy induced by MGO. Blocking ONOO2 Formation Abrogates MGO-mediated Reduction of VEGFR2 Protein Levels and Endothelial Cell Tube Formation We wondered if inhibition of ONOO2 mimicked the effects of autophagy suppression which was shown to restore VEGFR2 protein levels (Fig. 3A/B/C) and endothelial angiogenesis (Fig. 3F/G). In this regard, we monitored VEGFR2 protein levels in the presence of ONOO2 suppression which was achieved with pharmacological or genetic means. We found that pre-incubation with mTempol restored VEGFR2 protein levels (Fig. 6A) in BAEC. Similarly, the reduction was abolished either by L-NAME (Fig. 6B) or UA (Fig. 6C). However, administra- tion of mTempol, L-NAME, or UA alone did not affect endothelial tube formation (Fig. S4). Adenoviral overexpression of either SOD1 (Fig. 6D) or SOD2 (Fig. 6E), but not the control (GFP) (Fig. 6D/E), also blocked VEGFR2 reduction (Fig. 6D/E). Furthermore, the impaired endothelial angiogenesis appeared to be normalized, since a restored tube formation was evident when ONOO2 suppression through different mechan- isms was present (Fig. 6F). Hydrogen peroxide is an important member of the reactive oxygen species and a regulator of endothelial function [56]. To decide whether hydrogen peroxide mediated the effects exerted by MGO, we compared these effects in cells overexpressing either a control (GFP) or an enzyme called catalase that catalyzes hydrogen peroxide degradation. As shown in Fig. S5, MGO reproducibly decreased both VEGFR2 (Fig. S5A) and endothelial tube formation (Fig. S5B). However, overexpression of catalase (Fig. S5A) could not prevent the reductions (Fig. S5A and S5B). MGO Induces VEGFR2 Reduction in Endothelial Cell Cytosol which can be Blocked by Administration of Either O2–. Scavenger or Autophagy Inhibitor VEGFR2 trafficking has recently been found to be associated with its function and stability [14,57,58]. We sought to detect VEGFR2 distributions in BAEC. As depicted in Fig. 7A/B (low/ high power field), immune-fluorescent staining of VEGFR2 was PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e46720