Autophagy of VEGFR2 Impairs Diabetic Angiogenesis Figure 7. MGO induces endothelial VEGFR2 reduction predominantly in cytosol, which can be prevented by administration of O2–. scavenger or autophagy inhibitor. A–B (low and high-power fields of the same slides): MGO reduced VEGFR2 immune-staining in cytosol or cytoplasm membrane, without affecting staining in the nucleus. BAEC were incubated with MGO (25 mM, 16 h) and subjected to cell immunofluorescent staining with a commercial immune-staining kit including ProLongH Gold and SlowFadeH Gold Antifade, by using a rabbit derived VEGFR2 antibody or stained with DAPI, and a goat anti-rabbit IgG conjugate labeled with fluorescent dyes. All images shown are representative of three independent experiments. C: The quantification results of B. *P,0.05 vs control (n = 3). NS: not significant vs control. doi:10.1371/journal.pone.0046720.g007 Discussion The present study provided evidence to demonstrate that MGO, a glycolysis metabolite found increased in patients with diabetes, impairs endothelial angiogenesis both in cell culture and aortic tissues. This is operative through ONOO–dependent and autophagy-mediated VEGFR2 degradation, because pharmaceu- tical and genetic approaches that inhibit either autophagy or ONOO2 formation reverse the MGO-mediated impacts. Impor- tantly, the same approaches both ex vivo and in vivo restored the angiogenic response or VEGFR2 protein levels in diabetic aortas, suggesting a previously unidentified mechanism for angiogenesis impairment in diabetes (Fig. 8E). Angiogenesis impairment in diabetic peripheral vasculature contributes to delayed wound healing, increased risk of rejection of transplanted organs, exacerbated peripheral limb ischemia, and even cardiac mortality [2,60]. However, the mechanisms un- derlying diabetes-impaired angiogenesis are not completely un- derstood. A growing body of evidence support that AGEs, as well as MGO, the major precursor of AGEs, and the receptor of AGEs (RAGE) are implicated in the pathogenesis of diabetic vascular complication [61]. MGO is believed to contribute to diabetic complications either as a direct toxin or as a precursor for advanced glycation end products. Consistent with this notion, the present study has implicated an important role of RAGE (Fig. 2G) and the MGO-detoxifying system (Fig. 2E) for MGO-generated effects. Indeed, it has been demonstrated that AGE restriction may improve angiogenesis and wound healing in diabetic animal model [62]. The relevance of AGEs-altered angiogenesis can also be seen in MGO-impaired gastric ulcer healing [63]. Recently, it has been found that O-GlcNAc modification mediated-Akt inhibition reduces angiogenesis [64] and that the Akt-pathway also mediates the AGEs-effects on angiogenesis [65]. However, the dependency of Akt-pathway is lost pending serum presence, and it is yet to establish what serum factor contributes to the observations [65]. Nevertheless, these data imply that other factors essential in the angiogenic pathway are involved. For instance, the alterations of VEGFR1 have been shown to contribute to the impaired angiogenesis after hindlimb ischemia in a type 2 diabetic mouse model [66]. In current study, we have not been able to detect as significant changes of the VEGFR1 as those of the VEGFR2 (data not shown). Given the crucial role of VEGFR2 in endothelial angiogenesis [67,68,69,70], downregulation of endothelial VEGFR2 protein demonstrated in the present study could be a new mechanism. Surprisingly, quantitative data of VEGFR2 protein in aortic tissues are few in literature, in contrast to those reported either in cultural endothelial cells or in the circulation. One plausible reason could be that endothelial VEGFR2 expression in normal vascular tissue is low [71]. Indeed, only by immunoprecipitation enrichment could we detect VEGFR2 protein in control aortic tissues and its reduction in diabetes (Fig. 8D). In endothelia cells, VEGFR2 is found localized in endothelial caveolae, associated with caveolin-1, and this complex is rapidly dissociated upon stimulation with VEGF [72]. A recent study showed that a significant amount of VEGFR2 exists in endosome [15] and undergoes constitutive PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e46720