Autophagy of VEGFR2 Impairs Diabetic Angiogenesis Figure 3. Inhibition of autophagy, but not proteasome or caspase, abolishes the reduction of VEGFR2 protein levels and angiogenesis by MGO. A–E: Suppression of autophagy, but not proteasome and caspase, prevented VEGFR2 reduction induced by MGO. (A–C and E) One hour prior to MGO challenge (25 mM for 16 h), BAEC were pre-incubated respectively with chloroquine (CQ: 100 mM), pepstatin A (Pep A: 10 mM), bafilomycin A1 (Baf A1: 5 nM), MG132 (0.5 mM), epoxomicin (Epo: 0.5 mM), lactacystin (Lact: 1 mM), and z-VAD-fmk (z-VAD: 20 mM); (D) Before MGO treatment (as above), BAEC were transfected either with control siRNA or siRNA targeting Beclin-1, based on instructions from Santa Cruz Biotechnology (Santa Cruz, CA); all cell lysates were subjected to Western blot with indicated antibodies. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). F: Administration of autophagy inhibitor rescued MGO-impaired tube formation. One hour before MGO stimulation (25 mM), endothelial cells were incubated respectively with CQ (100 mM), Pep A (10 mM), Baf A1 (5 nM) and subjected to tube formation assay. G: Knockdown of Beclin 1 by siRNA prevented MGO-reduced tube formation. All images presented are representative of three independent experiments. *P,0.05 vs control (n = 3). NS: not significant vs control. doi:10.1371/journal.pone.0046720.g003 [49,50]. As noted in the confocal imaging of the GFP-LC3 transfected cells (Fig. 4B), the MGO-treated cell presented a significant increase in number of LC3 punctae compared to untreated control (Fig. 4B), which was accompanied by an increase in autophagy markers (Fig. 2A). Furthermore, in VEGFR2- immuno-coprecipitates, the protein staining of LC3B-II was stronger in MGO challenged cells than the control (Fig. 4C), suggesting VEGFR2 as a potential target of autophagy. In agreement with this, genetic overexpression of Beclin 1 by plasmid infection reduced VEGFR2 protein levels without MGO (Fig. 4D), and the reduction was further enhanced with MGO (Fig. 4D). Finally, we explored whether pharmacological induction of autophagy exerted the same effect as MGO. We treated the cell with rapamycin, a well documented autophagy inducer [49]. As shown in Fig. 4E, this autophagy inducer decreased both VEGFR2 protein levels (Fig. 4E) and endothelial tube formation (Fig. 4F) in the absence of MGO. Taken together, these data indicated that autophagy was involved in VEGFR2 reduction induced by MGO. Suppression of Peroxynitrite Generation Prevents MGO- increased Markers of Autophagy in Endothelial Cells Emerging data suggest that generation of reactive oxidative species (ROS), an important etiological factor for diabetic vascular complications [51],_ENREF_10 might trigger autop- hagy [52]. We wondered if the MGO-mediated effects involved peroxynitrite (ONOO2), one of the most important components of ROS [53]. ONOO2 is formed during simultaneous generation of superoxide (O2–.) and NO in a cell; inhibition of O2– or NO abolishes ONOO2 formation [54]. As such, we incubated BAEC either with mito-TEMPO-H (mTempol) (a mitochondria-targeted antioxidant with O2–. scavenging prop- erties [55]), L-NAME (a non-selective inhibitor of eNOS), or uric acid (UA) (a potent scavenger of ONOO2), before MGO stimulation. As shown in Fig. 5, mTempol treatment blocked the increase of Beclin-1 (Fig. 5A). Similarly, the increase was abolished either with L-NAME (Fig. 5B) or UA (Fig. 5C), an approach that suppresses ONOO2 formation. To further demonstrate the mediation by ONOO2, we monitored the expression of autophagy markers in ONOO–treated cells. As PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46720