Abstract
Abstract: :
Purpose: Previous studies have shown that differentiation of corneal fibroblasts to myofibroblasts occurs during corneal wound healing and that these cells are important in wound contraction. We, have recently shown that unlike fibroblasts, myofibroblasts express the PAF receptor (He J and Bazan H, Arvo 2003). PAF, a potent inflammatory mediator which accumulates rapidly in the cornea after an injury, stimulates the expression of selective MMPs in corneal epithelium. Since these enzymes are key components of the remodeling process that occurs during wound healing, we investigate the effects of PAF on the expression of MMP-1, -2, -9, TIMP-2 and membrane type 1 MMP (MT1-MMP) in rabbit corneal myofibroblasts. Methods:Corneas from rabbit eyes (Pel-freeze Biologicals, AR) were dissected and epithelium, endothelium and descements membrane were removed. The stroma was then cut into 1 mm2 pieces and incubated in DMEM containing 1 mg/ml collagenase for 3 hours. Keratocytes were pelleted and resuspended in DMEM-F12 medium containing 0.5 % fetal bovine serum (FBS). Corneal myofibroblasts were prepared by seeding keratocytes at low density (5 cells per mm2) and cultured in DMEM-F12 medium containing 0.5% FBS for 5 days. For gene expression studies, cells were starved overnight in serum free medium, followed by treatment with 100 nM cPAF. mRNA was extracted from myofibroblast cells and the levels of gene expression for MMP2, MMP1, MT-MMP-1, TIMP2, were determined by RT-PCR. Values were normalized to the 18s rRNA (endogenous control) and smooth muscle-α-actin (SM-α-actin). Results:Corneal myofibroblasts, defined by their expression of SM-α-actin, express basal levels of MMP-1, -2, TIMP-2 and MT1-MMP mRNA, while MMP-9 was undetected in these cells. Moreover, the mRNA levels of MT1-MMP, TIMP-2 and MMP-2 were markedly higher than MMP-1 in control cultures. Stimulation with 100 nM cPAF produced a marked increase in the expression of MT1-MMP, TIMP-2 and MMP-1 at 8 hours in corneal myofibroblasts, while MMP-2 levels where unchanged when compared to untreated controls. Conclusions: Although the function of myofibroblasts in corneal wound healing remains unclear, evidence suggests a role for these cells in mediating extracellular matrix organization and wound closure. Given that a tightly regulated release of MMPs is essential for extracellular matrix remodeling during the wound healing process, the induction of MMP-1, TIMP-2 and MT1-MMP by PAF highlights an important process that eventually could lead to corneal scaring and loss of visual function.
Keywords: gene/expression • lipids • wound healing