Role of Thrombospondin 1 in Oxidative Stress-Mediated Vascular Dysfunction

The matricellular protein thrombospondin 1 (TSP1) is elevated in the vasculature of patients suffering from a variety of cardiovascular disorders, including atherosclerosis, diabetes, restenosis and stroke. In vitro TSP1 inhibits vascular relaxation, stimulates vascular smooth muscle cell proliferation and migration and induces endothelial cell apoptosis. In vivo, TSP1 has been found to limit blood pressure changes to vasodilators via its receptor CD47. All of these processes are known to involve increased reactive oxygen species production. Recently, TSP1 has been reported to stimulate reactive oxygen species production in macrophages and neurons. However, the ability of TSP1 to stimulate reactive oxygen species in vascular cells and its potential role in oxidative stress-mediated vascular dysfunction are not known. We hypothesize that TSP1, through CD47, is a potent inducer of oxidative stress in vascular parenchymal cells, leading to decreased nitric oxide bioavailability and vascular dysfunction. In aim 1 we will investigate whether TSP1, via CD47, increases reactive oxygen species production by induction of NADPH oxidase in endothelial and vascular smooth muscle cells. The effect of TSP1 on superoxide and hydrogen peroxide production will be evaluated in endothelial cells and vascular smooth muscle cells from wild-type, TSP1- and CD47-null mice using luminometric and fluorometric techniques as well as electron paramagnetic resonance. The enzymatic source of TSP1-induced reactive oxygen species will be investigated using a variety of pharmacologic and moleculogenetic approaches. The contribution of NADPH oxidase isoforms Nox1, Nox2 and Nox4 will be tested using Western blot, qPCR, siRNA methodologies and Nox null mice. In aim 2 expression and activity of key antioxidant enzymes, including superoxide dismutase, glutathione peroxidase-1, catalase and thioredoxin 1, will be compared among wild-type, TSP1- and CD47-null mice using Western blot and qPCR. In aim 3 the potential role of reactive oxygen species in TSP1-impaired vascular relaxation will be tested in aortas isolated from wild-type, CD47-, and Nox-null mice. These studies will elucidate a potentially deleterious role of TSP1 in oxidative stress-mediated vascular dysfunction and provide a novel therapeutic target to treat the same. (AHA Program: Postdoctoral Fellowship)