Pharmacological inhibition of Axl affects smooth muscle cell functions under oxidative stress

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Reactive oxygen species (ROS) activate Axl receptor tyrosine kinase (RTK), promoting survival in rat aortic smooth muscle cells (RASMs).
• Axl knockout mice exhibit reduced vascular pathologies, suggesting Axl's role in disease.
• Targeting Axl with pharmacological inhibitors is a potential therapeutic strategy for oxidative stress-related vascular diseases.

Purpose of the Study:

• To investigate the efficacy of novel Axl-specific compounds, R428 and R572, in inhibiting ligand-independent Axl activation.
• To evaluate the effects of R428 and R572 on cell survival, migration, and ROS production in RASMs under oxidative stress.

Main Methods:

• Stimulation of RASMs with hydrogen peroxide (H2O2) to induce oxidative stress.
• Assessment of Akt phosphorylation (p-Akt) to determine Axl signaling inhibition.
• Flow cytometry using Annexin V to measure apoptosis and cleaved caspase-3 levels.
• Evaluation of cell migration in response to serum stimulation.
• Measurement of intracellular ROS production.

Main Results:

• R428 and R572 demonstrated potent inhibition of p-Akt at significantly lower concentrations (100 nM and 10 nM, respectively) compared to Fc-Axl (2 μg/mL).
• Both compounds induced a 2-fold increase in apoptosis and elevated cleaved caspase-3 levels, indicating enhanced cell death.
• R428 and R572 reduced cell migration by approximately 50% and decreased intracellular ROS production.
• These compounds proved more potent than Fc-Axl in inhibiting ligand-independent Axl signaling under oxidative stress.

Conclusions:

• R428 and R572 are potent inhibitors of ligand-independent Axl signaling in RASMs subjected to oxidative stress.
• These novel compounds effectively reduce Axl-mediated cell survival and migration.
• R428 and R572 show promise as therapeutic agents for vascular diseases linked to oxidative stress.