The Influence Of Mechanical Stretch On The Proliferation,Migration And Extracellular Matrix Metabolism Of Cultured Corneal Fibroblasts In Vitro

Corneal ectasia is one of the most serious complications after Laser in situ keratomileusis (LASIK). In some cases, patients have to receive corneal transplantation. At present, the etiology of the corneal ectasia is still unclear. The survival environment of the keratocytes alters because the stress state of the cornea redistributes after LASIK, and many kinds of cytokines and growth factors are released after corneal injury. In this study, cultured corneal fibroblasts were subjected to cyclic mechanical stretch. We focused on how mechanical stretch and cytokines regulate the proliferation, migration and extracellular matrix metabolism of the corneal fibroblasts in vitro. This study will enhance our understanding on the process of corneal wound healing and the mechanism of corneal ectasia after LASIK. The main work and findings are as follows:(1) Cyclic stretch was applied to mimics the alteration of the cornea LASIK in vitro. Our goal was to explore the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the proliferation and migration of the cultured corneal fibroblasts with5%cyclic stretching. Our results showed that a5%stretching promoted the proliferation of the corneal fibroblasts, but inhibited the cell migration behavior. This inhibitory effect can be attenuated by bFGF or EGF, and is associated with the applying manner of the mechanical force.(2) The effects of the mechanical stretch on the expression of MMPs, TIMPs, and Collagen I were investigated. Cyclic stretching had a biphasic effect in regulating the expression of MMPs, TIMPs, and Collagen I. A small-magnitude stretch (5%) inhibited the expression of MMP-2, and had no obvious influence on MT1-MMP. However, it increased the expression of TIMPs, and Collagen I. Whereas a large-magnitude stretching (15%) increased the expression of MMP-2and MT1-MMP, but inhibited the expression of TIMPs, and Collagen I. This indicated that mechanical stress played an important role in regulating corneal fibroblasts matrix metabolism.(3) Inflammation often occurred in the corneal wound healing after LASIK or keratoconus. The cornea is exposed to a complicated environment if the stress state changes. How the mechanical stress influences the matrix metabolism in the presence of inflammatory cytokine is still unclear. In this study, we evaluated the combined effects of IL-1β and injury mechanical stretch on the expression of MMPs, TIMPs, and Collagen I in corneal fibroblasts in vitro.Our results showed that15%mechanical stretch and IL-1β had a synergistic effect on up-regulating the expression of MMP-1, MMP-3, and MMP-9, but had no significant influence on the expression of MMP-2. It also down-regulated the expression of TIMP-1and further down-regulated the expression of Collagen Ial, but had no significant influence on the expression of TIMP-2. This indicated that inflammatory cytokine might initiate some MMPs expression, and injury mechanical stretch amplified these effects.(4) ERK activation seemed to be directly involved in the stretch-induced MMP-2secretion in corneal fibroblasts.We first examined which members of the MAPKs family were involved in the injury stretch-induced MMP-2. The present results demonstrated that15%cyclic stretch not only activated ERK1/2phosphorylation but also increased MMP-2secretion. As PD098059, an inhibitor of MEK, inhibited both stretch-induced ERK phosphorylation and MMP-2secretion. However, inhibition of p38and JNK using SB203850and SP600125did not suppress stretch-induced pro-MMP-2increase, implying that it is not p38and JNK, but ERK1/2activation that seems to be required for overload-induced pro-MMP-2production in rabbit corneal fibroblasts.In summary, our results suggested that the corneal fibroblasts could response to mechanical stress by regulating the proliferation, migration, and matrix metabolism of the corneal fibroblasts in vitro. A small-magnitude stretching (5%) might promote corneal matrix synthetic events; whereas a large-magnitude stretching (15%) might facilitate the corneal tissue destruction, especially in the presence of inflammatory cytokine.