immune-coprecipitation of LC3B with VEGFR2 (Fig. 4C). Importantly, induction of autophagy either mimicked (Fig. 4D) or enhanced the MGO-generated effects (Fig. 4D/E/F). These data suggest that VEGFR2 is a potential target of autophagy. Autophagy is a lysosomal degradation pathway and essential for survival, differentiation, development, and homeostasis [47]. The pathway refers to a regulated catabolic cellular process for the lysosomal-dependent turnover of organelles and proteins [74]. Autophagy principally plays an adaptive role to protect organisms against diverse pathologies, including infections, cancer, aging, heart disease, and neurodegeneration. However, in certain disease settings, self-eating or even the pro-survival functions of autophagy could be deleterious [47]. Although emerging evidence supports the role of autophagy in the pathophysiology of diabetes, especially in the maintenance of pancreatic function, it remains largely undecided whether autophagy plays a protective or harmful role in other organ-tissue systems [75,76]. Interestingly, several angio- genesis inhibitors employed in anti-angiogenesis therapy induce autophagy activation [77,78], linking autophagy initiation to angiogenesis suppression [79]._ENREF_41 Consistently, suppres- sion of autophagy apparently promotes angiogenesis [79]. Indeed, mice deficient in the autophagic protein Beclin-1 display a pro- angiogenic phenotype associated with hypoxia [80]. Therefore, an autophagy-mediated reduction of VEGFR2 may contribute to diabetic angiogenesis impairment. Evidence in this paper also supports an ONOO2 dependent mechanism regulating autop- hagy and its effects, consistent with the recognized role of oxidative stress in diabetic complications [24]. However, it merits further investigations on how autophagy is triggered to reduce VEGFR2. Due to the heterogeneity of angiogenic dysregulation in diabetes [3] and the fact that angiogenesis is determined by a range of factors beyond vascular cell [81], it remains to be elucidated if VEGFR2-associated proteins or other angiogenic-related factors in vascular cell and beyond [82] would be affected by similar mechanism under diabetic conditions. Provided the important role of VEGFR2 in angiogenesis [67,68,69,70] and the association of AGEs/precursors (including MGO) with the pathogenesis of diabetic complications [22,83,84,85], we propose that impaired physiological angiogen- esis in diabetes results, at least in part, from downregulation of VEGFR2. Mechanistically, MGO reduces endothelial cell angio- genesis through ONOO2 dependent and autophagy-mediated VEGFR2 protein degradation, which may represent a potentially new mechanism for angiogenesis impairment in diabetes. Supporting Information Figure S1 Administration of Glo1 inhibitor mimics the effect of MGO and accelerates MGO-induced VEGFR2 reduction. BAEC were pre-incubated with Glo1 inhibitor MTX (100 mM for 1 h) followed by MGO administration at 25 mM for 16 h. Cell homogenates were subjected to Western blot 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). (JPG) Figure S2 tion in LC3B knockdown prevents VEGFR2 reduc- MGO-treated endothelial cells. BAEC were References 1. Carmeliet P (2005) Angiogenesis in life, disease and medicine. Nature 438: 932– 936. 2. Duh E, Aiello LP (1999) Vascular endothelial growth factor and diabetes: the agonist versus antagonist paradox. Diabetes 48: 1899–1906. Autophagy of VEGFR2 Impairs Diabetic Angiogenesis transfected either with control siRNA or siRNA targeting LC3B, according to instructions from Santa Cruz Biotechnology (Santa Cruz, CA). Then the cells were challenged by MGO (25 mM for 16 h) and the 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). NS: not significant vs control. (JPG) Figure S3 MGO does not change mRNA levels of VEGFR2. A: MGO did not alter mRNA levels of VEGFR2. BAEC were incubated with MGO at indicated representative (1X and 2X) concentrations (1625 mM, which was used through the present study; 2625 mM) for up to 8 h. B: Manipulation of Glo1 by administration of Glo1 inhibitor did not change VEGFR2 mRNA levels. BAEC were pre-incubated with of Glo1 inhibitor MTX (100 mM for 1 h) followed by MGO administration at 25 mM for 16 h. All cells from A and B were subjected to total cellular RNA isolation (Total RNA Kit I: #R683401, Omega Bio- Tek), reverse transcription into cDNA (iScriptTM cDNA Synthesis Kit, #170-8891, Bio-Rad), and quantitative polymerase chain reaction (PCR) (iQTM SYBR Green Supermix, 170-8880 and Real Time Detection System, Bio-Rad). The mRNA levels of VEGFR2 were normalized with those of the actin mRNA. NS: not significant vs control (n = 3). (JPG) Figure S4 The studded reagents do not alter endothelial tube formation. BAEC were pre-incubated respectively with chloroquine (CQ: 100 mM), pepstatin A (Pep A: 10 mM), bafilomycin A1 (Baf A1: 5 nM), mTempol (1 mM), L-NAME (1 mM), and UA (100 mM) for 1 h, and then all were subjected to assay of endothelial tube formation. All images presented are representative of three independent experiments. NS: not significant vs control (n = 3). (JPG) Figure S5 Overexpression of catalase does not prevent MGO-induced reduction of VEGFR2 protein levels and endothelial cell tube formation. HUVEC were transfected with pCMV control (GFP) or catalase (CAT)-expressing plasmid, according to instructions from OriGene (Rockville, MD). The transfected BAEC were incubated with MGO (25 mM for 16 h) and then subjected to A: Western blot. B: tube formation assay. All images or blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). CAT: catalase. NS: not significant vs control (n = 3). (JPG) Acknowledgments A portion of this study has been presented in the 72nd Scientific Sessions of American Diabetes Association on June 8–12, 2012, Philadelphia, PA, USA. Author Contributions Conceived and designed the experiments: HL JX. Performed the experiments: HL SY HZ JX. Analyzed the data: HL SY HZ JX. Wrote the paper: HL JX. 3. Leeper NJ, Cooke JP (2011) MicroRNA and mechanisms of impaired angiogenesis in diabetes mellitus. Circulation 123: 236–238. PLOS ONE | www.plosone.org 12 October 2012 | Volume 7 | Issue 10 | e46720