This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.It has been well established that diabetes leads to microvascular complications, and accelerates macrovascular diseases. One feature of diabetes is excessive oxidant stress, which impairs nitric oxide (NO) bioactivity, increase adhesion molecule expression, and promote atherosclerotic lesion formation.Preliminary studies demonstrate that hyperglycemia and free fatty acids (FFA) impart an oxidant stress in endothelial cells, resulting in lipid peroxidation, tyrosine nitration of prostacyclin synthase (PGIS), and endothelial dysfunction; Treatment of cells with either AICAR or with adenoviruse overexpressing constitutively active AMPK prevents the increased O2.-, inactivation of NO, PGIS nitration and endothelial dysfunction; Activation of AMPK by ischemic preconditioning (IPC) effectively blocked the markers of oxidant stress, likely via over expression of UCP-2. The most conclusive evidence that AMPK reduced oxidant stress is that (IPC) failed to alter both the markers of oxidant stress and UCP-2 expression in the AMPK-KO mice.We hypothesize that AMPK activation could protect the endothelial cell against the adverse effects of hyperglycemia and FFA by increasing antioxidant potentials (UCP-2 and/or superoxide dismutase) that lead to a decrease in oxidant stress and increase in NO bioactivity. This hypothesis will be pursued in two specific aims. 1) Determine if activation of AMPK reduces oxidant stress and endothelial dysfunction induced byhyperglycemia and FFA, and evaluate how it works. HAEC will be incubated with glucose and FFA, the markers of oxidant stress such as (O2.-), peroxynitrite, UCP2 expression, NO bioactivity and expression of adhesion molecules will be monitored under conditions in which AMPK activity is altered by using AICAR oradenoviral constructs. 2) Determine if AMPK-dependent reduction in oxidant stress and endothelial dysfunction is operating in vivo. Wild type and AMPK alpha 1 and alpha 2 KO mice will be used to make diabetic animal model. Aortic rings will be assayed or assayed after being incubated for various time in hyperglycemia/FFA media by measurements of functional parameters UCP-2 expression and selected parameters of ONOOprostacyclin synthase pathway. The proposed studies will provide insight into the mechanism by which diabetes leads to vascular diseases and may lead to use activation of AMPK as an important target for the prevention and treatment of diabetic vascular complications.
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