EXPRESSION OF ALPHA-SMOOTH MUSCLE (ALPHA-SM) ACTIN DURING CORNEAL STROMAL WOUND-HEALING

Purpose. The purpose of this study was to correlate the temporal expre ssion of alpha-smooth muscle specific actin (alpha-SM actin), a molecu lar marker for myofibroblast transformation, with corneal wound contra ction. Methods. After full-thickness, central corneal injury in rabbit eyes, the anterior width of the wound (wound gape) was measured in th e same animals using in vivo confocal microscopy. In addition, animals were sacrificed at various times after injury for the determination o f alpha-SM actin expression by immunofluorescent microscopy using a mo use monoclonal antibody specific for human alpha-actin. Antibody speci ficity was confirmed by Western blot analysis of normal and wound fibr oblasts. Expression of alpha-SM actin also was related spatially to f- actin and the wound margin by co-localization with phalloidin and DTAF (5{[4,6-dichlorotriazin-2yl] amino)fluorescein), a fluorescent marker bound to the wound margin. Results. Wound contraction was most eviden t from days 7 to 42, when wound gape progressively decreased from 574 +/- 120 mu m to 250 +/- 61 mu m. Thereafter, the wound remained stable to day 84 (304 +/- 58 mu m). Expression of alpha-SM actin directly co rrelated with wound contraction-appearing across the wound at day 7, t he full thickness of the wound at day 14, and the posterior wound at d ay 28. alpha-SM actin was localized exclusively to phalloidin-stained, f-actin microfilament bundles or stress fibers within wound healing f ibroblasts, and the disappearance of alpha-SM actin correlated with th e concomitant disappearance of stress fibers at days 28 to 42. Stainin g of the wound margin with DTAF confirmed that the expression of alpha -SM actin was limited to fibroblasts within the wound. Conclusions. Th e expression of alpha-SM actin was directly correlated to corneal woun d contraction, appearing at the initiation of and disappearing at the completion of the contraction process. Furthermore, the exclusive expr ession of alpha-SM actin by fibroblasts present only within the wound suggests that local environmental factors unique to the wound may play an important role in myofibroblast transformation.