Abstract
Abstract: :
Purpose: In response to stromal damage, keratocyte acquisition of a myofibroblast phenotype can be involved in a fibrotic response leading to scar formation. Myofibroblast transformation in other organs such as skin, is controlled in part by platelet release of TGF-ßs from damaged blood vessels. However, normal corneal repair is avascular and other resident corneal cell types are hypothesized to substitute for platelets in controlling the myofibroblast transition. The aim of this study was to investigate epithelial/stromal interactions controlling the myofibroblast transition in cornea. Methods: Epithelial and stromal cells were isolated from normal rabbit corneas and used for in vitro culture. For in vivo fibrotic repair, a 1mm button of tissue was removed from central mouse cornea and the wounds were allowed to heal for 5, 7, 10, and 14 days prior to analysis. Results: Stromal cells with a myofibroblast phenotype were observed transiently during corneal repair in the mouse model. Removal of epithelial cells daily inhibited the appearance of myofibroblasts. Medium conditioned by cultured corneal epithelial cells induced myofibroblast transformation in cultures of corneal stromal cells. Western analysis revealed that cultured epithelial cells, unlike stromal cells, spontaneously produced the TGF-ß2 isoform. Immunolocalization of TGF-ßs in vivo during wound repair confirmed the in vitro data with TGF-ß2 expressed in normal epithelium as well as strongly localized directly beneath the wounded epithelium. In contrast, TGF-ß1 was never detected in the epithelium and localized mainly to the posterior region of the wounded corneal stroma within the endothelial region. When epithelial cells were plated on matrigel, a stratum of basement membrane extracellular matrix, TGF-ß2 levels in conditioned media was reduced. The return of a basement membrane after wounding in vivo, as examined by immunolocalization of the basement membrane protein entactin, correlated with the disappearance of myofibroblasts. Conclusion: These data are consistent with the hypothesis that the epithelium controls stromal cell myofibroblast transformation during corneal repair. They further suggest that the basement membrane indirectly controls the myofibroblast transition by regulating the extent of TGF-ß release into the corneal stroma. Therefore the epithelial substratum may determine the extent of corneal fibrosis that develops after wounding.
Keywords: 374 cornea: stroma and keratocytes • 631 wound healing • 372 cornea: epithelium