Pharmacological inhibition of Axl affects smooth muscle cell functions under oxidative stress.
We previously demonstrated that reactive oxygen species (ROS) activate Axl, a receptor tyrosine kinase, resulting in increased survival of rat aortic smooth muscle cells (RASMs). Our experiments in Axl knockout mice showed significant reduction in vascular pathologies. We hypothesize that selective pharmacological inhibitors of Axl could prove beneficial in treating vascular diseases associated with oxidative stress. We investigated a role for two novel compounds specific for Axl (R428 and R572) on ligand independent activation of Axl mediated cell survival and migration. Stimulation of RASMs with H(2)O(2) for 5 min significantly increased Akt phosphorylation (p-Akt). Inhibition at 50% (IC(50)) of p-Akt was calculated at lower concentrations in R428 (100 nM) and R572 (10 nM) compared to Fc-Axl (2 microg/mL). Flow cytometry staining with Annexin V showed a 2-fold increase in apoptosis with R428 and R572 compared to Fc-Axl after H(2)O(2), which was validated by concomitant increases in cleaved caspase-3. Pretreatment with R428 and R572 decreased cell migration by approximately 50% in response to 20% serum (similar to that after Fc-Axl). R428 and R572 decreased intracellular production of ROS in comparison to Fc-Axl. In conclusion, R428 and R572 are more potent inhibitors of ligand independent mediated Axl signaling compared to Fc-Axl in RASMs under oxidative stress.
Smolock
E M
EM
Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Korshunov
V A
VA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001567:Benzocycloheptenes / Q000494:pharmacology*; D053148:Caspase 3 / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D006861:Hydrogen Peroxide / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000502:physiology; D018384:Oxidative Stress; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D014230:Triazoles / Q000494:pharmacology*
D001567:Benzocycloheptenes; D017382:Reactive Oxygen Species; D014230:Triazoles; C548378:bemcentinib; D006861:Hydrogen Peroxide; D020794:Receptor Protein-Tyrosine Kinases; D051057:Proto-Oncogene Proteins c-akt; D053148:Caspase 3