We studied the association between the production of reactive oxygen specie
s, actin organization, and cellular motility. We have used an endothelial c
ell monolayer-wounding assay to demonstrate that the cells at the margin of
the wound thus created produced significantly more free radicals than did
cells in distant rows. The rate of incorporation of actin monomers into fil
aments was fastest at the wound margin, where heightened production of free
radicals was detected. We have tested the effect of decreasing reactive ox
ygen species production on the migration of endothelial cells and on actin
polymerization. The NADPH inhibitor diphenylene iodonium and the superoxide
dismutase mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (M
nTMPyP) virtually abolished cytochalasin D-inhibitable actin monomer incorp
oration at the fast-growing barbed ends of filaments. Moreover, endothelial
cell migration within the wound was significantly retarded in the presence
of both diphenylene iodonium and MnTMPyP. We conclude that migration of en
dothelial cells in response to loss of confluence includes the intracellula
r production of reactive oxygen species, which contribute to the actin cyto
skeleton reorganization required for the migratory behavior of endothelial
cells.