Characterization of avascular corneal wound healing fibroblasts. New insights into the myofibroblast.

The characteristics and derivation of corneal wound healing fibroblasts (myofibroblasts) were evaluated by studying the temporal changes in the cellular actin distribution of corneal fibrocytes following full thickness 3-mm diameter central corneal wounds in the rabbit. Under certain conditions these wounds heal without neovascularization, allowing for the detailed analysis of invading fibroblasts with minimal contamination by other cell types. The authors employed transmission electron microscopy to localize microfilaments, fluorescent microscopy using NBD-phallacidin, a mushroom toxin which binds specifically to f-actin and oligomeres of g-actin, to localize actin filaments, and isoelectric focusing gels to characterize actin isotypes. During the early stages of wound healing (1-7 days) there is a gradual change in the corneal fibrocytes adjacent to the wound margin characterized by the development of extensive rough endoplasmic reticulum, microtubules, a prominent Golgi apparatus, and a cortical microfilament network. This is in contrast to the normal fibrocyte, which, for the most part, lacks these structures. The development of microfilaments correlated with increased NBD-phallacidin fluorescence of fibrocytes adjacent to the wound as compared with fibrocytes farther removed from the site of injury. Fibroblasts appearing within the wound from 7 days to 2 months after injury had ultrastructural characteristics similar to those of myofibroblasts, including parallel arrays of microfilaments, stress fibers and cell-cell, cell-matrix attachments. Furthermore, these cells stained intensely with NBD-phallacidin, supporting the ultrastructural findings. At 1 month after injury, cells contained within the wound possessed predominantly nonmuscle isoactins (gamma) as seen by silver staining of isoelectric focusing gels, but little or no (smooth muscle) isoactins could be detected. Moreover, no significant differences could be detected between electrophoretic profiles obtained from wounded versus normal corneas. These morphologic and biochemical data suggest that the corneal fibrocyte may develop into a fibroblastlike cell similar to the myofibroblast, and is characterized by a marked increase in filamentous actin.