Increased SPARC accumulation during corneal repair.
Keratocytes can become fibroblasts and myofibroblasts during corneal injury and wound healing. We used the in vitro bovine keratocyte repair model system, which involves culturing collagenase-isolated keratocytes in serum-free media and then adding serum or serum plus TGF-beta to the culture media to induce the fibroblast and myofibroblast phenotypes, respectively, to evaluate the synthesis of secreted products by the cells. Serum and serum plus TGF-beta rapidly induced the fibroblast morphology and alpha smooth muscle actin, a marker of myofibroblasts. Keratocytes cultured in serum and serum plus TGF-beta also increased the synthesis of several high molecular weight products (approximately 100kD and larger) and the accumulation of a 43kD protein shown to be osteonectin/SPARC by both sequencing tryptic peptides from the protein and by reaction with antisera to osteonectin/SPARC. Immunohistochemical staining of mouse corneas with antisera to SPARC seven days post-wounding also demonstrated an increased accumulation of SPARC in the regions undergoing repair. These results indicate SPARC accumulation is a marker for stromal repair.
Berryhill
Bridgette L
BL
The Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, FL, USA.
Kane
Bradley
B
Stramer
Brian M
BM
Fini
M Elizabeth
ME
Hassell
John R
JR
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D002417:Cattle; D002478:Cells, Cultured; D003315:Cornea / Q000378:metabolism* / Q000473:pathology; D003320:Corneal Ulcer / Q000378:metabolism* / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix; D007150:Immunohistochemistry / Q000379:methods; D015603:Keratinocytes / Q000737:chemistry; D008233:Lymphotoxin-alpha / Q000494:pharmacology; D051379:Mice; D015676:Osteonectin / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014945:Wound Healing
D015415:Biomarkers; D008233:Lymphotoxin-alpha; D015676:Osteonectin; D012333:RNA, Messenger