Investigating a Ligand‐receptor Role for Glutathione in Angiogenesis

Abstract

IntroductionIn addition to its classic role in redox chemistry, glutathione (GSH) also acts as an allosteric regulator of the calcium‐sensing receptor (CaSR). CaSR expression has been found in a variety of cell types including vascular smooth muscle and endothelial cells (ECs), where its activity has been linked to EC adhesion and migration. Although a relationship between GSH and CaSR has been discovered, no link has been established between GSH‐CaSR interaction and regulation of blood vessel development. We hypothesize that a novel mechanism for GSH regulation of blood vessel development occurs through an extracellular interaction with CaSR as a part of normal physiology, with potential extensions to pathological angiogenesis.MethodsMatrigel tube formation and under agarose migration assays were used to measure EC activity in primary human aortic endothelial cells (HAECs). Fura‐2 AM was used with cells from an hTERT immortalized aortic endothelial cell line (tHAECs) to measure changes in intracellular calcium. Cell surface expression assay (CelLISA) and flow cytometry were used to measure surface CaSR expression. Endothelial and endothelial‐mesenchymal transition (EndoMT) markers were measured using real‐time PCR.ResultsExtracellular application of GSH increased endothelial tube length and branching in tube formation assay, while treating cells with selective CaSR inhibitor (NPS‐2143) abrogated these effects, indicating a role for CaSR in vascular modeling. Extracellular GSH influenced EC migration as measured by under agarose migration assay. GSH or a CaSR agonist (R‐568) reduced surface CaSR expression, with little/no change in intracellular calcium. RT‐PCR results suggest that extracellular GSH alters endothelial phenotype along the endothelial‐mesenchymal spectrum, promoting endothelial differentiation.ConclusionIn addition to its classic antioxidant role, GSH appears to act extracellularly to influence EC activity. GSH caused significant increases in EC branching as measured by the tube formation assay. With the use of a selective CaSR inhibitor, we were able to confirm the contribution of CaSR to this effect. Additionally, GSH influenced EC migration in the under agarose assay. These results could be explained by GSH‐CaSR‐mediated alterations in endothelial differentiation. These results suggest a role for GSH in vascular development and angiogenesis, through a ligand interaction with the CaSR G‐protein coupled receptor, which represents a novel mechanism of action for physiological and pathological blood vessel development.