Effect Of Tissue Culture Irradiation On Orientation Of Cytoskeletal Fibers In Migrating Sheets Of Endothelial Cells

Abstract

Irradiation of endothelial cells (EC) in tissue culture has been used to markedly reduce cell proliferation in order to study the effects of substances on the regulation of EC migration. Since irradiated EC (IRR-EC) migrate the same distance as do non-irradiated EC (NIRR-EC) over periods of up to six days, it is assumed that EC migration is not effected by irradiation. The purpose of this study was to examine the effect of irradiation on the cytoskeleton of migrating EC. The in-vitro experimental wound technique was used as a model system. A linear wound was made in confluent cultures of porcine thoracic aortic EC. EC were observed to migrate into the wound as a cohesive sheet of cells with only a few free unattached cells being present along the front edge of the wound. Cultures were irradiated one hour before wounding with 1500 rads. NIRR-EC migrated the same distance as did the IRR-EC although the latter were much flatter and each IRR-EC covered a larger surface than did the NIRR-EC. The orientation of cytoskeletal fiber bundles localized by immunofluorescence microscopy using antisera produced against electrophoretically purified porcine uterine myosin and chicken gizzard tropomyosin were different when comparing migrating IRR and NIRR-EC in the first row of the endothelial sheet. At 44 hours after wounding myosin localization showed that the main myosin stained fibers in 89% of the IRR-EC were roughly parallel to the wound edge while 11% were roughtly perpendicular to the wound edge. In NIRR-EC the figures were 54% and 46% respectively. Tropomyosin localization showed a similar diffence, 73% and 27% in IRR-EC and 45% and 55% in NIRR-EC. The data shows that there are differences in the extent of spreading of and in the orientation of cytoskeletal fiber bundles in migrating IRR-EC. These differences may reflect different cytoskeletal processes involved during migration of IRR and NIRR-EC.