Decreases in Glutathione Levels Augment VEGFR2 Activation
Authors: Prasai Priya K , Pattillo Christopher B 
Published: November 2023

Ischemia is a key factor contributing to tissue injury in most cardiovascular pathologies. Although tissue hypoxia is a potent stimulus for angiogenesis, new blood vessel formation in cardiovascular diseases is severely impaired. New vessel formation may in turn salvage ischemic tissue by increasing blood flow. Previously, our laboratory showed that a small decrease in glutathione (GSH) in ischemic tissue enhanced angiogenesis thereby protecting the tissue. Therefore, we hypothesized that a decrease in glutathione positively modulates angiogenesis through the vascular endothelial growth factor‐A (VEGF‐A)/VEGF receptor 2 (VEGFR2) pathway. To test this hypothesis, we used human aortic endothelial cells (HAEC) and oxidized GSH using diamide, a thiol‐specific oxidant that oxidizes GSH to GSSG. We confirmed the decrease in GSH and a concomitant increase in GSSG in HAEC after 15 minutes exposure to diamide using high performance liquid chromatography (HPLC). To study the effect of diamide on VEGFR2 activation, HAEC cells were subjected to diamide for 15 minutes and a subsequent activation of VEGFR2 was triggered by exogenous addition of VEGF‐A. Receptor activation was assessed using western blotting for phosphorylated VEGFR2. Diamide caused an amplification of VEGF dependent VEGFR2 activation in a dose‐dependent manner. Surprisingly, we also observed receptor activation in high concentration (150μM, 250μM and 375μM) diamide treated groups that were not treated with VEGF‐A. To test whether receptor activation was due to increased reactive oxygen species (ROS), we first measured the total cellular ROS using CM‐H2DCFDA, chloromethyl derivative of fluorescein diacetate. Simultaneously, we subjected HAEC cells to different concentrations of H2O2 and probed for phosphorylated VEGFR2 after western blotting. The total cellular ROS in diamide treated cells increased linearly with increasing diamide concentration, however, did not exceed the ROS generated by highest H2O2 dose (200μM). Interestingly, we did not observe any receptor activation in H2O2 treatment group alone suggesting that the ligand‐independent activation of VEGFR2 could be a GSSG driven mechanism. To test whether cysteine sulfinic acid (cys‐SOH) formation is essential for VEGFR2 ligand‐independent activation, we pretreated the cells with dimedone (a cys‐SOH trapping agent) and then repeated the diamide experiment. The results show a marked reduction in receptor activation after pretreatment with dimedone. This data suggests that cys‐SOH formation is only partially responsible for the ligand‐independent activation of VEGFR2 and the remaining is contributed by GSSG. Increased protein S‐glutathiolation after diamide treatment is evident from literature. Hence, we tested whether diamide increased S‐glutathiolation of proteins using western blotting and found tremendous increase in protein S‐glutathiolation in samples treated with high concentrations (150μM, 250μM and 375μM) of diamide. It is important to note that those concentrations also elicited ligand‐independent receptor activation. Taken together, our results suggest potential S‐glutathiolation of VEGFR2 could lead to ligand‐independent activation of the angiogenic pathway.

Support or Funding Information

American Heart Association 15SDG25710038