ZO-1 REORGANIZATION AND MYOFIBROBLAST TRANSFORMATION OF CORNEAL ENDOTHELIAL-CELLS AFTER FREEZE INJURY IN THE CAT

Corneal endothelial wound healing following scrape injury in the rabbi t and cat is characterized by cell spreading and maintenance of a norm al endothelial phenotype consisting of apically-localized, circumferen tial microfilament bands and cell border-associated ZO-1, a tight junc tion protein and marker for endothelial differentiation. In contrast, after freeze injury in the rat and rabbit endothelial cells develop ba sally organized microfilament bundles (stress fibers), and appear to p roliferate and form a multilayered zone at the wound margin. The purpo se of the present study was to determine if similar phenotypic changes are observed after freeze injury in the cat corneal endothelium, whic h like human, normally has limited growth potential. In addition, chan ges in ZO-1 and alpha-smooth muscle actin (a marker for myofibroblast transformation) distribution were evaluated for the first time followi ng freeze injury. In vivo endothelial healing of standard 3 mm diamete r freeze injury was evaluated at 4 hr, 12 hr, 24 hr, 48 hr, 3 days and 5 days after injury in 22 cat eyes. Corneas were stained with phalloi din, propidium iodide, and anti-ZO-1, anti-alpha-smooth muscle-specifi c actin or anti-fibronectin antibodies. Protein organization was then evaluated using immunofluorescence and laser scanning confocal microsc opy. Beginning at 12 hr after injury, endothelial cells appeared to ex tend and elongate over the wound area. By 48 hr after injury, migratin g endothelial cells formed a multilayered activated zone (AZ) at the w ound margin. Endothelial cells immediately adjacent to the AZ maintain ed a normal circumferential organization of f-actin colocalized with c ell border-associated anti-ZO-1 staining at all time points observed, However, within the AZ there was an abrupt increase in phalloidin stai ning and development of prominent microfilament bundles (stress fibers ), as well as a loss of normal anti-ZO-1 staining. The AZ also stained positively for anti-alpha-smooth muscle actin and anti-fibronectin an tibodies. Changes in the distribution of ZO-1 were observed as early a s 4 hr after injury, and appeared to precede f-actin reorganization. T hese data indicate that endothelial healing after freeze injury in the cat involves a loss of normal endothelial differentiation and cell co nnectivity, and transformation to a myofibroblastic phenotype. (C) 199 7 Academic Press Limited.