Reactive oxygen species stimulate VEGF production from C2C12 skeletal myotubes through a PI3K/Akt pathway

Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulus, the expression of which increases in skeletal muscle after exercise. Becaus e exercise is also accompanied by increased intramuscular reactive oxygen s pecies (ROS) generation, we tested the hypothesis that ROS stimulate VEGF p roduction from skeletal myotubes. Differentiated C2C12 skeletal myotubes ex posed to ROS-producing agents exhibited a concentration-dependent increase in VEGF production, whereas undifferentiated myoblasts did not respond to o xidants. Moreover, conditioned medium from ROS-treated myotubes increased t he bovine lung microvascular cell proliferation rate. To study the mechanis m(s) involved in the stimulation of VEGF production by ROS, myotubes were p retreated with a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, LY-294002, before being exposed to hydrogen peroxide or pyrogallol. LY-2940 02 attenuated both Akt phosphorylation and VEGF production. In addition, ox idants increased nuclear factor-kappaB-dependent promoter activity in trans iently transfected myotubes; however, pretreatment with the pharmacological inhibitor of nuclear factor-kappaB, diethyldithiocarbamate, did not affect the oxidant-stimulated VEGF release. We conclude that ROS induce VEGF rele ase from myotubes via a PI3K/Akt-dependent pathway.