Autophagy of VEGFR2 Impairs Diabetic Angiogenesis Figure 6. Suppression of ONOO2 generation prevents reduction of VEGFR2 protein and endothelial cell angiogenesis by MGO. A–E: Suppression of ONOO2 generation prevented reduction of VEGFR2 protein. BAEC were incubated respectively with vehicle (controls), mTempol (1 mM for 1 h), L-NAME (1 mM for 1 h), and UA (100 mM for 1 h), adenoviral infection of GFP (control), or SOD1 and SOD2, prior to MGO incubation (25 mM, 16 h). The treated cells were subjected to Western blot for VEGFR2 protein. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). F: Inhibition of ONOO2 generation normalized the tube formation. Endothelial cells were incubated respectively with vehicle (controls), mTempol (1 mM for 1 h), L-NAME (1 mM for 1 h), and UA (100 mM for 1 h) prior to MGO challenge (25 mM). The treated cells were subjected to tube formation assay. The images shown are representative of three independent experiments. *P,0.05 vs control (n = 3). NS: not significant vs control. doi:10.1371/journal.pone.0046720.g006 treated aortas manifested decreased capillary-like endothelial outgrowth (Fig. 8A); however, in the presence of either Pep A or Baf A1, the endothelial outgrowth was significantly restored (Fig. 8A), suggesting that autophagy mediated the MGO-reduced angiogenic response in aorta. Aortas of Genetic Diabetic Mice Present Autophagy- mediated Lower Angiogenic Response Compared to the Genetic Controls We next examined whether aortas from the genetic diabetic db/ db and Akita mice had lower angiogenic capacity and whether that could be reversed through autophagy inhibition. To this end, we assessed angiogenesis of the aortas from db/db and the genetic control mice with or without autophagy suppression. As depicted in Fig. 8, aortas from db/db vs the control mice presented reduced aortic endothelial outgrowth (Fig. 8B); similar blunted angiogenic responses were also observed in another type of genetic diabetic Akita mice (Fig. 8C). However, pre-treatment either with Pep A or Baf A1 normalized the endothelial outgrowth (Fig. 8B), indicating the involvement of autophagy in angiogenesis impairment in diabetes. Administration of mTempol in vivo Prevents the Reduction of VEGFR2 Protein Levels and Restores Aortic Angiogenic Response in Akita Mice We took one step further to determine if administration of mTempol in vivo rescued aortic angiogenic capacity through VEGFR2. To achieve this, we treated the Akita mice either with mTempol (0.1 mM, in drinking water) or vehicle (normal drinking water) for 4 weeks and assessed the angiogenic response and VEGFR2 protein levels of the aortas. As shown in Fig. 8C, mTempol treatment in vivo improved aortic endothelial outgrowth (Fig. 8C). With a VEGFR2 enrichment approach, we were able to detect aortic VEGFR2 proteins of non-diabetic genetic control mice (Fig. 8D), which were reduced in Akita mice (Fig. 8D). However, administration of mTempol, but not of the vehicle, restored the aortic VEGFR2 protein levels (Fig. 8D). In summary, diabetes impaired endothelial angiogenic capacity appeared to be associated with VEGFR2 protein levels, both of which could be normalized by administration of mTempol, a scavenger of O2–. PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e46720