Abstract
Diabetes impairs physiological angiogenesis by molecular mechanisms that are not fully understood. Methylglyoxal (MGO), a metabolite of glycolysis, is increased in patients with diabetes. This study defined the role of MGO in angiogenesis impairment and tested the mechanism in diabetic animals. Endothelial cells and mouse aortas were subjected to Western blot analysis of vascular endothelial growth factor receptor 2 (VEGFR2) protein levels and angiogenesis evaluation by endothelial cell tube formation/migration and aortic ring assays. Incubation with MGO reduced VEGFR2 protein, but not mRNA, levels in a time and dose dependent manner. Genetic knockdown of the receptor for advanced glycation endproducts (RAGE) attenuated the reduction of VEGFR2. Overexpression of Glyoxalase 1, the enzyme that detoxifies MGO, reduced the MGO-protein adducts and prevented VEGFR2 reduction. The VEGFR2 reduction was associated with impaired angiogenesis. Suppression of autophagy either by inhibitors or siRNA, but not of the proteasome and caspase, normalized both the VEGFR2 protein levels and angiogenesis. Conversely, induction of autophagy either by rapamycin or overexpression of LC3 and Beclin-1 reduced VEGFR2 and angiogenesis. MGO increased endothelial LC3B and Beclin-1, markers of autophagy, which were accompanied by an increase of both autophagic flux (LC3 punctae) and co-immunoprecipitation of VEGFR2 with LC3. Pharmacological or genetic suppression of peroxynitrite (ONOO−) generation not only blocked the autophagy but also reversed the reduction of VEGFR2 and angiogenesis. Like MGO-treated aortas from normglycemic C57BL/6J mice, aortas from diabetic db/db and Akita mice presented reductions of angiogenesis or VEGFR2. Administration of either autophagy inhibitor ex vivo or superoxide scavenger in vivo abolished the reductions. Taken together, MGO reduces endothelial angiogenesis through RAGE-mediated, ONOO–dependent and autophagy-induced VEGFR2 degradation, which may represent a new mechanism for diabetic angiogenesis impairment.
Highlights
Angiogenesis, defined as the formation of new blood vessels out of preexisting capillaries, plays a crucial role in maintaining vascular health [1]
The results showed that incubation of MGO with endothelial cell reduced vascular endothelial growth factor receptor 2 (VEGFR2) protein levels in a time (Fig. 1A) and dose (Fig. 1B) dependent manner
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
Summary
Angiogenesis, defined as the formation of new blood vessels out of preexisting capillaries, plays a crucial role in maintaining vascular health [1]. Multiple molecular mechanisms have been proposed, including oxidative stress/reactive oxygen species, endothelial derangements, loss of endothelium-derived nitric oxide (NO) bioactivity, and micro RNA alterations [3]. It has not been completely elucidated how diabetes impairs physiological angiogenesis. Endothelial cells respond to VEGF to produce new blood vessels. This angiogenic process makes a critical contribution during embryogenesis and in the response to ischemia in adult tissues [12,13,14,15]. The mechanism underlying VEGFR2 reduction and the contributions to angiogenesis impairment in diabetes are not known
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