Annexin II is a phospholipid-binding calcium-regulated protein expressed on the surface of endothelial cells. It possesses the to bind tissue plasminogen activator (t-PA), an endothelial cell secretory product, as well as plasmin, a fibrinolytic serine protease of broad substrate specificity, by 60-fold. We recently created an annexin II null deletion mouse which displays cardiomyopathy with reduced capillary density. Additional preliminary data suggest that annexin II is required for post- natal angiogenesis in selected in vivo settings such as microvascular invasion of Matrigel implants and development of oxygen-induced retinal neovascularization. In this proposal, we plan to examine the scope of the angiogenic defect in annexin II deficiency, and to determine its molecular mechanism. We will employ a variety of genetic and molecular approaches to address this question.
Under Specific Aim I, we will study the role of annexin II in vivo in the Matrigel invasion assay, in oxygen- induced retinopathy, in the corneal pocket assay, in a carotid artery ligation model, and in an auricular translation assay using annexin II """"""""knockout"""""""" mice. In addition, we will determine whether the annexin II promoter is up-regulated in these settings of neovascularization using AnnII-LacZ transgenic mice. Finally, we will examine the behavior of mice deficient in other elements of the fibrinolytic system (plasminogen, t-PA, urokinase) in these assays to determine whether the role of annexin II in angiogenesis is dependent upon plasmin generation.
Under Aim II, we will determine the proliferative capacity, survival profile, proteolytic activity, and migratory potential of microvascular endothelial cells isolated from annexin II-deficient animals, and study their response to known angiogenic modulators.
Under Aim III, we will examine the influence of homocysteine (HC) on annexin II function in vivo. HC is a thiol-containing amino acid that specifically blocks the t-PA binding domain within the """"""""tail"""""""" region of annexin II. We will determine the effect of HC-induced annexin II blockade on angiogenesis in mice that are hyperhomocysteinemic due to cystathionine beta-synthase deficiency, and whether this defect reflects impaired plasmin generation. Taken together, these experiments should provide a detailed, mechanistic understanding of the role of annexin II in post-natal neovascular development, and will likely provide new strategies for therapeutic interventions in angiogenesis in the future.
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