Abstract 489: Glutathione Regulates Endothelial Cell Function And Vegfr2 Signaling

Under physiological and pathological conditions angiogenesis progresses in an idealized redox window. Mice heterozygous for the Glutamate cysteine ligase modifier (GCLm) subunit knockout have augmented ischemic remodeling. During occlusion, the ensuing ischemic milieu and accompanying vascular changes regulate the activity of vascular endothelial receptor 2 (VEGFR2). We hypothesized that the cellular GSH concentration regulates endothelial cell function via altered VEGFR2 signaling. To test this hypothesis, we first performed mouse aortic endothelial cell (MAEC) outgrowth assays on aortic segments dissected from mice homozygous (KO) and heterozygous (Het) for GCLm knockout as well as wildtype control (WT). Outgrowth area in the Het group was significantly higher than WT and KO at day 7 of outgrowth (2.15 fold, n=4, P<0.001). MAECs were isolated and relative migration rates were determined using under-agarose migration assays. Het MAECs were 1.25 fold faster than WT (n=4 p<0.001) while KO MAECs were slower, 0.90 fold (n=3, p<0.05). Cell proliferation assays are currently underway. Concurrently, we inhibited glutathione reductase in primary human aortic endothelial cells using 2-AAPA. 2-AAPA (75 μm) alone didn’t activate VEGFR2 (determined by western blot for pY1175 VEGFR2). 2-AAPA in combination with H 2 O 2 (100 μM) induced a 5.5 fold (p<0.01, n=4) increase in the relative amount of active VEGFR2 compared to vehicle treated HAECs. 2-AAPA with H 2 O 2 induced a significant increase in pY416 proto-oncogene c-Src (Src) 9.5 fold (p<0.01, n=6), This effect was attenuated by the addition of DTT. Furthermore, Src kinase inhibitor 1 (SKI) (200 nM) attenuated the effect of 2-AAPA + H 2 O 2 on VEGFR2. While the VEGFR2 kinase inhibitor SU1948 inhibited activation of VEGFR2, it had no significant effect on the 2-AAPA + H 2 O 2 mediated activation of Src. Combined these data suggest Src mediates the 2-AAPA + H 2 O 2 activation of VEGFR2. Glutathione S-transferase P1 (GSTP1) catalyzes the enzymatic glutathionylation of proteins, The GSTP1 inhibitor TLK199 (50 μM) abrogated the effects of 2-AAPA + H 2 O 2 on VEGFR2 and experiments are underway to determine the glutathionylation status of Src in our system and its effect on the interaction of Src and VEGFR2.