Angiogenesis, the highly regulated process of new capillary formation rarely occurs in normal adults, but is necessary during embryogenesis, corpus luteum formation, and wound healing. Uncontrolled angiogenesis plays an important role in diseases such as rheumatoid arthritis, hemangiomas, tumor growth and metastasis. Development of effective agents which can inhibit angiogenesis has potential value in the treatment of these diseases. Thrombospondin 1 (TS1) and certain peptides derived from TS I block angiogenesis in vivo and inhibit the proliferation and migration of endothelial cells (ECs) in vitro. These investigators have shown that TS I is a major regulator of EC phenotype and its expression is sufficient to restore a normal phenotype and suppress hemangioma formation in Polyoma middle T transformed mouse brain ECs. This is mediated, at least in part, by complete suppression of platelet endothelial cell adhesion molecule- I (PECAM-1) expression, an important regulator of EC adhesion and angiogenesis. The main objective of this proposal is to delineate the expression and adhesive function of different PECAM- I isoforms and to characterize their signaling pathways in ECs. The expression pattern of TS I and PECAM- I isoforms will be examined in ECs of developing murine blood vessels by in situ hybridization and immunohistochemistry. Expression of different PECAM- I isoforms and/or their cytoplasmic chimeras in ECs will determine whether different isoforms have distinct roles in regulation of EC phenotype and require interactions with cytoplasmic proteins. The GST-PECAM- I cytoplasmic fusion proteins phosphorylated on their tyrosine, serine, and threonine residues will be utilized in pull down experiments to identify the signal transducing molecules which interact with PECAM- 1. Expression of PECAM- I isoforms or his-myc tagged cytoplasmic domains in an epithelial cell model (MDCK cells), which forms adherens junctions very similar to ECs, will illustrate whether they affect formation of adherens junctions and influence PECAM- I cellular adhesive and signaling functions. These studies will provide insight into the coordinated expression of TS 1 and PECAM- 1 isoforms and their interactive roles in regulating EC phenotype. Characterization of the intracellular proteins which interact with PECAM- I cytoplasmic domains will provide further knowledge of the signaling pathways which regulate PECAM- I adhesive functions. It is suggested that a therapeutic benefit can be derived by exploiting these signaling pathways to control the hypervascularization characteristic of arthritis.
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