Autophagy of VEGFR2 Impairs Diabetic Angiogenesis Figure 8. VEGFR2 protein levels and the aortic endothelial angiogenesis are reduced in diabetic mice, which can be rescued either by autophagy suppression or O2–. scavenging. A: Aortic angiogenic response was reduced in MGO-challenged aortas of C57BL/6J mice, which could be restored by autophagy inhibitors. Aortic rings removed from normoglycemic C57BL/6J mice were incubated with MGO (25 mM) with or without the presence of autophagy inhibitors (Pep A: 10 mM, Baf A1: 5 mM) and subjected to aortic ring assay; *P,0.05 vs the vehicle treated (n = 5/ group). B: Aortic endothelial outgrowth was impaired in db/db vs genetic control mice that could be rescued by autophagy inhibitors. Aortic rings prepared from the genetic control and db/db mice were treated with vehicle or autophagy inhibitors (Pep A: 10 mM, Baf A1: 5 mM) and followed by aortic ring assay. *P,0.05 vs the vehicle treated (n = 5/group). C–D: Aortic angiogenesis and VEGFR2 protein were reduced in diabetic Akita vs genetic control mice, which could be improved by mTempol administration in vivo. Aortas prepared from the genetic control and the treated Akita mice (mTempol: 0.1 mM in the drinking water, vehicle: normal drinking water, for 4 weeks) were subjected to aortic ring assay and aortic VEGFR2 protein detection by immune-precipitation (VEGFR2 enrichment)-combined Western blot. # indicates antibody recognizing different epitope of the VEGFR2 molecule. The Western blot of b-actin in aortic tissue homogenates with a mouse derived antibody is used as an Input for the loading control. *P,0.05 vs the vehicle treated (n = 5/group). E: the proposed mechanism of diabetic angiogenesis impairment: MGO, a glycolytic metabolite which is found to be increased in patients with diabetes, impairs endothelial angiogenesis through ONOO–dependent autophagy-mediated VEGFR2 degradation. NS: not significant vs control. doi:10.1371/journal.pone.0046720.g008 endosome-to-plasma membrane recycling [73]. The MGO-in- duced VEGFR2 reduction identified in the present study occurred predominantly in cytosol (and may be in plasma membrane as well). The observation implicates that MGO-mediated VEGFR2 degradation could be a detour from its normal trafficking. Such a deviation might represent an unidentified cellular mechanism by which diabetes reduces VEGR2 protein levels. Although the role of trafficking in MGO-mediated VEGFR2 reduction remains to be established, the alteration of VEGFR2 protein immune- fluorescent-staining in the presence of MGO and the involvement of both ONOO2 and autophagy (Fig. 7A/B/C) were confirmed with other approaches (Fig. 1–6 and Fig. 8A/B/C). Furthermore, aortic rings from diabetic (Akita and db/db) vs genetic control mice demonstrated lower angiogenic capacities (Fig. 8C/D), which were associated with VEGFR2 protein reductions (Fig. 8D). More importantly, the clinical relevance of VEGFR2 reduction in diabetes is apparent in that diabetes-associated VEGFR2 reduc- tions have been observed both in humans [17,18] and experi- mental animals [19]. It is believed that such reductions impair collateral formation in the myocardium of patients with diabetes [17,18] or causes erectile dysfunction in the penile of diabetic rats [19]. To the best of our knowledge, this is the first report that links autophagy-mediated VEGFR2 degradation to angiogenesis im- pairment in diabetes. The supporting evidence not only includes the induction of autophagy markers (Fig. 4A and 5A/B/C: LC3B and/or Beclin-1) but also the pharmacological (Fig. 3A/B/C and Fig. 4E/F: experiments with autophagy/lysosome inhibitors or inducer) and genetic (Fig. 3 D/G and Fig. S2/Fig. 4D: experi- ments with siRNA or overexpression) connections of autophagy to the reductions of VEGFR2 (Fig. 3A/B/C/D, Fig. 4D, and Fig. S2) and angiogenesis (Fig. 3F/G and Fig. 4F). The key evidence included the demonstration of MGO-enhanced autophagy initi- ation (increase in the number of LC3 punctae) (Fig. 4B) and PLOS ONE | www.plosone.org 11 October 2012 | Volume 7 | Issue 10 | e46720