title: Localization of thrombospondin-1 and myofibroblasts during corneal wound repair.
abstract: Thrombospondin-1 (TSP-1) is a multifunctional matrix protein that has recently been examined in various wound processes, primarily for its ability to activate the latent complex of transforming growth factor-beta (TGF-β). TGF-β has been shown to play a major role in stimulating mesenchymal cells to synthesize extracellular matrix. After injury, corneal keratocytes become activated and transform into fibroblasts and myofibroblasts. Our hypothesis is that TSP-1 regulates the transformation of keratocytes into myofibroblasts (MF) via TGF-β. In the current study, we examined the expression of TSP-1 and α-smooth muscle actin (SMA), a marker of MF, during rat corneal wound healing. Three-mm keratectomy or debridement wounds were made in the central rat cornea and allowed to heal from 8 hours to 8 weeks in vivo. Unwounded rat corneas served as controls. Expression of TSP-1, SMA and Ki67, a marker of proliferating cells, were examined by indirect-immunofluorescence microscopy. In unwounded corneas, TSP-1 expression was observed primarily in the endothelium. No expression was seen in the stroma, and only low levels were detected in the epithelium. Ki67 was localized in the epithelial basal cells and no SMA was present in the central cornea of unwounded eyes. After keratectomy wounds, TSP-1 expression was seen 24 h after wounding in the stroma immediately subjacent to the wound-healing epithelium. The expression of TSP-1 increased daily and peaked 7-8 days after wounding. SMA expression, however, was not observed until 3-4 days after wounding. Interestingly, SMA-positive cells were almost exclusively seen in the stromal zone expressing TSP-1. Peak expression of SMA-positive cells was observed 7-8 days after wounding. Ki67-expressing cells were seen both in the area expressing TSP-1 and the adjacent area. In the debridement wounds, no SMA expressing cells were observed at any time point. TSP-1 was localized in the basement membrane zone from 2 to 5 days after wounding, and the localization did not appear to penetrate into the stroma. These data are in agreement with our hypothesis that TSP-1 localization in the stromal matrix is involved in the transformation of keratocytes into myofibroblasts.
authors.affiliation: The Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA.
authors.forename: Mariko
authors.initials: M
authors.lastname: Matsuba
authors.forename: Audrey E K
authors.initials: AE
authors.lastname: Hutcheon
authors.forename: James D
authors.initials: JD
authors.lastname: Zieske
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D054338:Cell Transdifferentiation; D003315:Cornea / Q000502:physiology*; D065306:Corneal Injuries; D060527:Corneal Keratocytes / Q000166:cytology; D003646:Debridement; D004195:Disease Models, Animal; D005131:Eye Injuries / Q000378:metabolism* / Q000473:pathology; D019084:Fluorescent Antibody Technique, Indirect; D019394:Ki-67 Antigen / Q000378:metabolism; D008856:Microscopy, Fluorescence; D058628:Myofibroblasts / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019700:Thrombospondin 1 / Q000378:metabolism*; D014945:Wound Healing / Q000502:physiology*
chemical_list: D000199:Actins; D015415:Biomarkers; D019394:Ki-67 Antigen; D019700:Thrombospondin 1; C498862:Acta2 protein, rat