Shear Stress Activates Akt in Cultured A7r5 Aortic Smooth Muscle Cell Line:

BACKGROUND: Vascular intervention procedures such as balloon angioplastyand stent implantation lead to denudation of the vessel endothelium, exposing the underlying smooth muscle layer directly to effects of blood flow, especially shear stress. Smooth muscle cells are shown to proliferate in areas of damaged endothelium. However, the effect of shear stress on smooth muscle cell signaling has not been studied in depth. Constitutive Akt activity is required for cell survival and drastic mechanical stimuli may be expected to regulate Akt signaling. PURPOSE: In this study, we examined time dependent changes in activation of Akt, MAPK and p70S6k related signaling molecules in response to orbital shear stress on cultured A7r5 cells. METHODS: A7r5 cells cultured to sub-confluence on 150mm culture dishes were subjected to orbital shear for zero, 5min, 15min, 30min, 1h, 4h and 24h at 210rpm. SDS-PAGE and immunoblot analyses were performed using antibodies towards phosphorylated and unphosphorylated forms of various molecules of the Akt signaling pathway. RESULTS: Akt phosphorylation was found to increase to 165% of control at 15min then staying nearly constant up to 1h with a maximum phosphorylation at 4h (185% of control). Upon exposure to shear stress, GSK-3β phosphorylation was markedly decreased at 5min (50% of control) and stayed low up to 4h (40% of control), followed by recovery to 70% of control at 24h. The phosphorylation levels of mTOR, p70S6k and PTEN were also found to be in keeping with the activation of Akt in response to shear stress. CONCLUSION: Akt signaling may be responsible for proliferative response of vascular smooth muscle cells when exposed to shear stress upon endothelial denudation due to vascular interventional procedures such as angioplasty and vessel grafting, leading to increased restenosis. These data may provide insights into possible pharmacological interventions to prevent or retard the development of restenosis. (Supported by funds from NSF Grant 0314742)