Angiogenesis is the sprouting of new vascular structures from preexisting vascular beds and occurs due to various stimuli. A common mediator of angiogenesis is vascular endothelial growth factor (VEGF). VEGF after association with its receptor (VEGFR2) acts through a reactive oxygen species (ROS) dependent mechanism allowing it to initiate a signaling process necessary for angiogenesis. Glutathione (GSH) acts as an initial defense against oxidative stress within the endothelial cells. De novo synthesis of GSH depends on a rate limiting holoenzyme, glutamate cysteine ligase (GCL), consisting of a modulatory (GCL-M) and catalytic subunit (GCL-C). Due to increases in ROS levels, GSH is oxidized to neutralize any associated stresses. As reduced GSH levels are altered other anti-oxidant defense mechanisms are activated, among these are E2-related factor-2 (Nrf-2) which binds to DNA antioxidant response elements (ARE) on several genes including GCL-M. Our laboratory has recently discovered that GCL-M mice display enhanced angiogenic activity suggesting that endothelial cell glutathione levels critically regulate blood vessel growth. Previous observations in the literature and preliminary data provided herein led to the hypothesis that GCL- M mediates angiogenesis by controlling tissue ROS levels induced by VEGF through activation of NRF-2. To test this hypothesis the specific aims of this project are: (1) Determine the migratory and proliferative phenotype of GCL-M KO mouse endothelial cells in response to increased ROS levels through VEGF stimulation. For this purpose a group of the GCL-M KO mice will be implanted with a VEGF containing diffusion disk to determine vascular recruitment, and another group will have their femoral artery ligated and angiogenesis measured over a period of 21 days. Tissues from various models will be examined for GSH levels, endothelial cell density and proliferation, and ROS generation. (2) Determine the molecular and cellular mechanisms by which GCL-M mediates angiogenesis through the activation of Nrf-2. For this aim endothelial cells will be harvested from mice and used in Dunn cell migration chambers in the presence of VEGF to determine mechanisms of motility as well as ROS production. The gene proposed for study (GCL-M) has been linked to many vascular abnormalities. Enhancing our knowledge of antioxidant regulation of angiogenesis will lead to a better understanding of many vascular abnormalities associated with increased levels of ROS.
