Angiogenesis Impairment in Diabetes: Role of Methylglyoxal-Induced Receptor for Advanced Glycation Endproducts, Autophagy and Vascular Endothelial Growth Factor Receptor 2
Figure 3
Inhibition of autophagy, but not proteasome or caspase, abolishes the reduction of VEGFR2 protein levels and angiogenesis by MGO.
A–E: Suppression of autophagy, but not proteasome and caspase, prevented VEGFR2 reduction induced by MGO. (A–C and E) One hour prior to MGO challenge (25 µM for 16 h), BAEC were pre-incubated respectively with chloroquine (CQ: 100 µM), pepstatin A (Pep A: 10 µM), bafilomycin A1 (Baf A1: 5 nM), MG132 (0.5 µM), epoxomicin (Epo: 0.5 µM), lactacystin (Lact: 1 µM), and z-VAD-fmk (z-VAD: 20 µM); (D) Before MGO treatment (as above), BAEC were transfected either with control siRNA or siRNA targeting Beclin-1, based on instructions from Santa Cruz Biotechnology (Santa Cruz, CA); all 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). F: Administration of autophagy inhibitor rescued MGO-impaired tube formation. One hour before MGO stimulation (25 µM), endothelial cells were incubated respectively with CQ (100 µM), Pep A (10 µM), Baf A1 (5 nM) and subjected to tube formation assay. G: Knockdown of Beclin 1 by siRNA prevented MGO-reduced tube formation. All images presented are representative of three independent experiments. *P<0.05 vs control (n = 3). NS: not significant vs control.