Tissue plasminogen activator (tPA) is a protein that is active in the process of wound healing and angiogenesis. Recombinant human tPA has been produced and packaged into a retroviral vector that can be used to transduce endothelial cells. This opens the possibility of using endothelial cells that over-express tPA in vivo wound healing and angiogenesis experiments. An initial step in this process is the careful in vitro characterization of transduced cells in regards to normal cellular function and invasiveness. Primary cultures of endothelial cells are obtained from sheep, rabbit, and cows. Pure endothelial cell cultures of the same passage are divided into 3 groups. The first group is transduced with the B2NSt viral vector, which contains the human tPA cDNA and a neomycin resistant gene. The second group is transduced with LBgSN viral vector, which contains the beta-galactosidase gene and a neomycin resistant gene. The second group serves as a transduced cell control. The third group is not transduced and serves as a normal control. Transduced cells are selected in G418, a neomycin analogue toxic to nontransduced eukaryotic cells, for 2 weeks. Cellular functions are tested in assays of cell attachment to gelatin-coated dishes, cell proliferation, migration, invasion of gels, and in vitro tube formation. Differences in the three groups are quantitated and tested for statistical significance. An endothelial cell with increased invasiveness while maintaining differentiated cellular functions may be able to augment natural wound healing and blood vessel growth in areas of injury.
