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Sachidananda Kenchegowda, Azucena H. Kakazu, Haydee E. Bazan; Activation of Myofibroblasts by Platelet-Activating Factor (PAF) Induces Fibronectin (FN) Degradation Blocked by Metalloproteinases (MMPs) Inhibitors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2527.
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Corneal myofibrobasts play a role in reconstituting the collagen-rich extracellular matrix (ECM) and promoting wound closure by contraction. Platelet-activating factor (PAF) is a potent inflammatory mediator produced after corneal injury that inhibits wound healing. Corneal myofibroblasts express the PAF-receptor and, when stimulated with PAF, induces MMP-9 and MT1-MMP (Ottino, P. etal., IOVS, 46; 487, 2005). Here we investigate if the action of PAF is through degradation of fibronectin (FN) and if metalloproteinases (MMPs) are involved.
Primary cultures of rabbit corneal myofibroblasts (RCM) and myofibroblasts derived from mouse keratocytes (MCM) were stimulated with PAF (100 nM) for 16, 24, and 36 h. In some experiments, the PAF receptor antagonist LAU- 0901 (10 µM) was added to block the effect of PAF. Some experiments use the MMPs inhibitors GM6001 and Batimastat each at a concentration of 10 µM . Cells and media were collected and analyzed by Western blot using antibodies against FN and α-smooth muscle actin (α-SMA). Immunofluorescence staining was done using anti-FN monoclonal FTIC-conjugated IgG antibody.
RCM and MCM cells stimulated with PAF produced a significant decrease in FN expression, analyzed by Western blot and immunofluorescence. Most of the effect was on FN release to the media. Incubation with the PAF-receptor antagonists LAU-0901 blocked the effect of PAF. When the cells were incubated in the presence of the inhibitors, there was a 44% increase in FN production with respect to controls.
PAF inhibition of corneal wound repair involves the degradation of FN through activation of MMPs, decreasing attachment and migration of myofibroblasts.
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