Refractive surgery not only leads to tissue injury but also evokes mechanical stress increase of the cornea. How the mechanical stress affects the corneal matrix remodeling, specifically, matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases; TIMPs) is not well understood. In this study, cultured rabbit corneal fibroblasts in vitro were subjected to regimen of 5%, 10%, or 15% equibiaxial stretch at 0.1 Hz for 3 or 24 h. MMP-2 protein level was measured by gelatin zymography and Western blotting. MMP-2, membrane type 1 MMP (MT1-MMP), and TIMP-2 mRNA levels were quantified by real-time quantitative PCR. Extracellular regulated protein kinase (ERK) phosphorylation protein levels were assessed by Western blotting. Our results showed that a 15% stretch resulted in increases in MMP-2 protein, MMP-2 mRNA, and MT1-MMP mRNA levels, but a decrease in TIMP-2 mRNA level. However, a 5% stretch caused decreases in MMP-2 protein and mRNA level, but an increase in TIMP-2 mRNA level, and no change in MT1-MMP mRNA level. A 15% stretch also caused a significant increase in ERK1/2 phosphorylation. Inhibition of the mitogenactivated protein kinase (MEK) pathway with PD98059 attenuated stretch-induced increase in MMP-2 production and ERK activity. These results suggest that small-magnitude stretching may promote corneal matrix synthetic events, whereas large-magnitude stretching promotes corneal matrix degradation by changing the balance between MMPs and TIMPs in corneal fibroblasts. Large-magnitude stretch-induced increase in pro-MMP-2 production was in an ERK-dependent manner.
Inflammatory molecules and matrix metalloproteinase (MMPs) have been found over-expressed in the tear film of patients with keratoconus. However, the mechanistic link between inflammatory molecules and MMPs in the pathogenesis of keratoconus remains still elusive. Therefore, we investigated the effect of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) on MMP-1 expression and used IL-6 antibody (IL-6 Ab) to examine the role of IL-6 on TNF-α mediated regulation of MMP-1 in fibroblasts of normal cornea and keratoconus. Real-time polymerase chain reaction, Enzyme-linked immunosorbent assay, and Western blot data demonstrated that MMP-1 and IL-6 were expressed in fibroblasts of normal cornea and keratoconus. Levels of MMP-1 and IL-6 were significantly higher in keratoconus than normal cornea. TNF-α treatment led to a significant increase in IL-6 levels. IL-6 treatment induced MMP-1 synthesis in normal cornea and keratoconus. TNF-α increased MMP-1 expression in a dose- and time-dependent manner and this response was completely inhibited by the IL-6 Ab. In conclusion, these results indicate that fibroblasts of keratoconus shows increased levels of IL-6 and MMP-1 gene and protein expression and IL-6 mediates the TNF-α-induced MMP-1 expression.
BackgroundCorneal tensile strain increases if the cornea becomes thin or if intraocular pressure increases. However, the effects of mechanical stress on extracellular matrix (ECM) remodelling in the corneal repair process and the corneal anomalies are unknown.MethodsIn this study, the combined effects of interleukin-1β (IL-1β) on matrix metalloproteinases (MMPs) in corneal fibroblasts under cyclic stretching were investigated in vitro. Cultured rabbit corneal fibroblasts were subjected to 5, 10 or 15 % cyclic equibiaxial stretching at 0.1 Hz for 36 h in the presence of IL-1β. Conditioned medium was harvested for the analysis of MMP2 and MMP9 protein production using the gelatin zymography and western blot techniques.Results and conclusionsCyclic equibiaxial stretching changed the cell morphology by increasing the contractility of F-actin fibres. IL-1β alone induced the expression of MMP9 and increased the production of MMP2, and 5 % stretching alone decreased the production of MMP2, which indicates that a low stretching magnitude can reduce ECM degradation. In the presence of IL-1β, 5 and 10 % stretching increased the production of MMP2, whereas 15 % stretching increased the production of MMP9. These results indicate that MMP expression is enhanced by cyclic mechanical stimulation in the presence of IL-1β, which is expected to contribute to corneal ECM degradation, leading to the development of post-refractive surgery keratectasia.
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