Angiogenesis depends upon proper collagen biosynthesis and crosslinking, moreover, type I collagen is an ideal scaffold for angiogenesis in vitro. Despite this, mechanisms of type I collagen-mediated angiogenesis remain poorly understood. We propose to define the features of the type / collagen fibril, endothelial cell surface, and intracellular signaling pathways that act together to mediate type I collagen-induced angiogenesis. We have developed a unique model for studying endothelial tube morphogenesis in vitro. Our preliminary data using our system support the hypothesis that engagement between the alpha2beta1 integrin receptor and integrin-binding sequences of type I collagen result in D38MAPK activation, and inactivation of Focal Adhesion Kinase during endothelial tube morphogenesis. We will test this hypothesis in the following aims:1) Define the roles of endothelial cell surface alpha1beta1, alpha2beta1, and alphaVbeta3 integrin receptors, sulfated proteoglycans, and fibronectin by testing the activities of integrin or fibronectin function-blocking antibodies and inhibitors of GAG function on angiogenesis; 2) Synthesize triple helical, type I collagen mimetic peptides (THPs) including putative integrin-binding sites, and study their capacities to inhibit cell collagen attachment, bind integrin receptors, and influence angiogenesis; 3) Study the consequences of integrin function-blocking antibodies, integrin-binding THPs, and chemical and dominant-negative construct inhibitors of signaling pathway function on p38MAPK and FAK activation and angiogenesis; and 4) Examine the role of alpha2beta1 integrin ligation of discrete collagen sequences, and the p38MAPK and FAK pathways, a) in vivo in the chick CAM, and b) in angiogenesis induction by other polymers including heterotypic collagen fibrils and fibrin. Our work will define the type I collagen structural features critical for its morphogenic activity, probe the functional link between alpha2beta1 integrin-collagen ligation, p38 MAPK and FAK activation, and angiogenesis, and contribute to the understanding and treatment of human diseases involving vascular insufficiencies, such as ischemia, or abnormal angiogenesis, as in tumor growth.
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