Hydrogen peroxide activates the Gas6-Axl pathway in vascular smooth muscle cells

Abstract

Axl, a receptor tyrosine kinase, is involved in cell survival, proliferation, and migration. We have shown that Axl expression increases in the neointima of balloon-injured rat carotids. Because oxidative stress is known to play a major role in remodeling of injured vessels, we hypothesized that H 2O2 might activate Axl by promoting autophosphorylation. H2O2 rapidly stimulated Axl tyrosine phosphorylation in rat vascular smooth muscle cells within 1 min that was maximal at 5 min (6-fold). The response to H2O2 was concentration-dependent with EC50 of ∼500 μM. Axl phosphorylation was partly dependent on production of its endogenous ligand, growth arrest gene 6 (Gas6), because Axl-Fc, a fragment of Axl extracellular domain that neutralizes Gas6, inhibited H2O2-induced Axl phosphorylation by 50%. Axl phosphorylation by H2O2 was also attenuated by warfarin, which inhibits Gas6 activity by preventing post-translational modification. In intact vessels Axl was phosphorylated by H2O2, and Axl phosphorylation was inhibited by warfarin treatment in balloon-injured carotids. Akt, a downstream target of Axl, was phosphorylated by H2O 2 in Axl+/+ mouse aorta but significantly inhibited in Axl-/- aorta. Intimal proliferation was decreased significantly in a cuff injury model in Axl-/- mice compared with Axl+/+ mice. In summary, Axl is an important signaling mediator for oxidative stress in cultured vascular smooth muscle cells and intact vessels and may represent an important therapeutic target for vascular remodeling and response to injury.