Angiogenesis is the growth of blood cells from preexisting vessels. The broad, long-term objectives of this proposal are to identify cis-acting DNA elements that regulate transient expression of tissue factor (TF) in endothelial cells during angiogenesis, and to determine how TF expression contributes to various forms of angiogenesis. Our hypothesis is that TF expression by endothelial cells initiates limited activation of the clotting cascade to generate thrombin, which subsequently stimulates its receptor PAR-1 on vascular cells to permit the maturation of blood vessels. There are three specific aims: 1/ To elucidate the role of TF in angiogenesis using in vitro and in vivo models of angiogenesis. 2/ To investigate how TF contributes to yolk sac vessel development during angiogenesis. 3/ To identify an angiogenic response element (ARE) that regulates TF gene expression in endothelial cells during blood vessel development.
These aims will be accomplished using established in vitro and in vivo models of angiogenesis. We will employ a genetic approach to determine the role of the intracellular domain of TF and the binding of factor VII to the extracellular domain of TF in E9.5 yolk sac vessel formation by attempting to rescue mTF null embryos with mutated versions of human TF protein. Identification of the ARE in the TF promoter will be achieved by analyzing the in vivo expression during angiogenesis of transgene containing various lengths of 5' flanking sequence with or without intron 1. Detailed mapping of the ARE will use EMSA and antibody supershift experiments. Angiogenesis is a key component in human diseases such as cancer and heart disease. Currently, anti-angiogenic therapy is being used to treat cancer and pro-angiogenic therapy is being used to treat cancer and pro- angiogenic agents are being used to enhance collateral vessel development in ischemic myocardium.
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