Control of angiogenesis dictated by picomolar superoxide levels.

Control of vascular insufficiencies due to various cardiovascular pathologies is important for developing specific and effective treatments. Fluctuations in oxidative stress significantly alter the progression of angiogenesis under physiological and pathological conditions. However, the precise amount of reactive oxygen species (ROS) required to influence subsequent signaling pathways for ischemic angiogenesis remains undefined. Here, we have determined the effect of ROS-mediated molecular mechanisms on angiogenesis in a murine model of peripheral artery disease using Gclm mutant mice (a model of compromised glutathione synthesis and therefore reduced antioxidant capacity). Left femoral artery ligation and excision were performed in Gclm WT (+/+), heterozygous (+/-), and null (-/-) mice. Blood flow (laser Doppler), angiogenic index (CD31/DAPI), and proliferation index (Ki67/DAPI) were significantly increased in Gclm(+/-) mice but not in Gclm(+/+) or Gclm(-/-) mice. Measurements of reactive oxygen species suggest that the amount of superoxide required to stimulate angiogenesis after the induction of ischemia is 9.82 pmol/mg of tissue. Protein carbonyl levels increased in a manner consistent with increasing oxidative stress. Superoxide and protein carbonyl levels were reduced by the addition of the nitroxide tempol, a known superoxide dismutase mimetic. Finally, restoration of blood flow in Gclm(+/-) mice was attenuated by a VEGF164 aptamer, verifying that slightly elevated levels of ROS restore blood flow by stimulating endothelial cell proliferation through a VEGF-dependent pathway. The results of this study reveal new information on the amount of ROS necessary for angiogenic activity and provide the foundation of critical redox parameters for vascular remodeling responses. The information obtained from this study on vascular ischemia, using a model of decreased antioxidant capacity, has provided insight into the control of revascularization and is a step forward in our ability to regulate angiogenic therapies.