Abstract
Abstract: :
Purpose: Keratocytes of the corneal stroma respond to wounding by expression of a myofibroblastic phenotype characterized by alpha smooth muscle actin cytoskeletal stress fibers. Fibrotic extracellular matrix deposited in wounds includes collagens I and III, cellular fibronectin, biglycan and dermatan sulfate of increased size and sulfation. We recently demonstrated (J Biol Chem 276, 44173) that cultured keratocytes assume a myofibroblastic phenotype and secrete fibrotic matrix components in response to transforming growth factor beta (TGFß). The current study seeks to determine if the myofibroblastic phenotype and fibrotic matrix expression in these cells is linked. Methods: Primary bovine keratocytes were embedded in collagen gels or plated on collagen-coated surfaces and treated 5-6 days with TGFß1. Cells and matrix molecules were recovered from gels after solubilization with collagenase or urea. Smooth muscle actin, biglycan, and cellular fibronectin protein and mRNA were detected using western blotting and real-time RT-PCR. Dermatan sulfate from culture matrix was analyzed by polyacrylamide gel electrophoresis and fluorophore assisted carbohydrate electrophoresis (FACE). Results: TGFß treatment caused contraction of both floating and anchored collagen gels. Smooth muscle actin was upregulated in keratocytes exposed to TGFß cultured as monolayers but not keratocytes embedded in anchored or floating or collagen gels. Keratocytes secreted cellular fibronectin, biglycan, and dermatan sulfate with increased abundance, size, and sulfation in response to TGFß in all conditions. Conclusions: Altered matrix deposition can occur independently of alpha smooth muscle actin stress fiber development and thus myofibroblasts in healing wounds are not the sole source of fibrotic tissue.
Keywords: cornea: stroma and keratocytes • proteoglycans/glycosaminoglycans • wound healing