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A.H. Dahlmann, M. Bailly, P.T. Khaw; The Role of PDGF-Induced Cell Ruffling During Matrix Contraction in Corneal Wound Healing . Invest. Ophthalmol. Vis. Sci. 2003;44(13):896.
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Background: After wounding of the corneal stroma, dormant keratocytes differentiate into fibroblasts which are able to migrate and to contract, synthesize and remodel extracellular matrix. This transformation is stimulated by various growth factors released shortly after injury. Clinical investigations have shown platelet-derived growth factor (PDGF) to play a prominent role in corneal wound repair following refractive surgery. Purpose: To determine mechanisms involved in PDGF-modulated matrix contraction Methods: Primary human corneal fibroblasts (HCF) in monolayers and in three-dimensional fibroblast-populated collagen matrix (FPCL) were stimulated with PDGF-BB. Cells were microinjected with molecules that block Arp2/3-complex mediated actin assembly (function blocking antibodies against the p34 subunit of the Arp2/3 complex, inhibitory peptides from the C-terminal part of WASp family proteins), or pre-incubated with an inhibitor of myosin II contractile activity (myosin light chain kinase inhibitor ML9). They were then studied with time-lapse phase, epifluorescence and confocal microscopy. Fixed cells were labelled for actin and proteins of the actin-assembling pathway. Results: More than 80% of HCF in monolayers show concentric ruffling of the dorsal cell membrane (DMR) after exposure to PDGF, with a maximum within 3 minutes post-stimulation. Stimulation with other growth factors does not induce this phenomenon. DMR is associated with visible contraction of at least one side of the cell in 60% of cells; twenty percent of cells show simultaneous lamellipod extension on the opposite side. DMR appear as F-actin enriched dorsal structures, and the amount of F-actin bound in stress fibres is reduced. Proteins of the actin-assembling complex Arp 2/3 are present in DMR. However, blocking Arp2/3 mediated actin assembly through injection of inhibitory antibodies or peptides does not significantly alter PDGF-induced DMR, nor does the blocking of the myosin II contraction pathway. In FPCLs, PDGF stimulates matrix contraction, particularly within the first 4 hours after stimulation. HCFs display strong actin stress fibres and the same DMR as observed in monolayers. Conclusions: These results suggest that PDGF-stimulated DMR might initiate the strong contractile response by directly promoting both cell contraction and protrusive activity in HCF. This fast response might play an important role in the generation of forces during corneal wound healing.
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