Cellular movement is an essential component of normal developmental and reparative processes including embryogenesis, wound healing, tissue remodelling, and angiogenesis. Recent studies have identified a small group of proteins which appear to be specific regulators of cell mobility. One example is scatter factor (SF), a soluble fibroblast- derived protein activity which causes sheets of normal epithelial cells to scatter (spread and separate into single cells). We showed that bovine and human arterial smooth muscle cells release a factor similar to the human embryonic fibroblast-derived SF. We the utilized two quantitative assays of cell migration (a microcarrier bead assay developed in our laboratory and a modified Boyden chamber assay) to demonstrate that SF stimulates migration of arterial endothelium and epithelium, but not 3T3 or smooth muscle. While SF's in vivo functions are not yet known, these findings suggest that it might be involved in wound healing and vascular repair. Scattering and migration-stimulating activities were both heat sensitive, trypsin labile, and non-dialyzable (14 kd cutoff), and activities co-purified on standard and FPLC columns. Dose-response curves for stimulation of endothelial or epithelial migration were similar whether crude, partially purified or highly purified SF preparations were utilized. We purified the factor 500-1000 fold from serum-free conditioned medium from a high producer line of ras-transformed mouse 3T3 cells using cation exchange chromatography. FPLC purified fractions showed one major band at 62 kd on analysis by SDS-PAGE with silver- staining. We estimate that SF maximally stimulates migration of target cells at concentrations of about 0.5 nM. In this grant, we proposed to develop efficient methods for chromatographic purification of sufficient quantities of electrophoretically homogeneous SF to allow complete biochemical characterization of the protein, including N-terminal microsequencing. We will use the purified factor to generate monoclonals for development of immunoassays; in vivo detection, and further protein characterization. We will investigate SF's biologic activity on microvascular endothelium and its effects on the cytoskeletal organization of endothelial and epithelial cells. These studies will contribute to basic knowledge of cell movement and of blood vessel biology. They may also yield insights which will ultimately be useful in diagnosis and treatment of vascular disorders.
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