Role of tissue factor pathway inhibitor-2 in the expressions of matrix metallopro- teinases in keratocytes in vitro

Abstract

AIM:To elucidate the relation between tissue factor pathway inhibitor-2(TFPI-2)expression and the expression of matrix metalloproteinases(MMPs)in keratocytes. METHODS: Primary culture and subculture of rabbit keratocytes were established in vitro. Plasmid vector pBos-Cite-neo/TFPI-2 was transfected into keratocytes with Lipofectamine 2000. After being selected by G418, three groups of cells including TFPI-2 gene transfected cells K-TFPI-2, empty vector transfected cells K-V and non-transfected cells K-P were screened for TFPI-2 mRNA and protein by reverse transcription-polymerase chain reaction and Western blot analysis, respectively. The activity of MMPs in the three groups of cells was detected by substrate zymography and compared by ANOVA. RESULTS: Expression of mRNA and protein of TFPI-2 was more in the cells of K-TFPI-2 than in the other cells of K-P and K-V with a significant difference(mRNA:0.79±0.02 vs 0.51±0.03 and 0.48±0.02, P=0.000 and P=0.000; Protein:24.5±0.8 vs 15.5±0.5 and 14.9±0.9,P=0.000 and P=0.000). Compared with the two groups of K-P and K-V, the cells of K-TFPI-2 had a significant decreased activity of MMP1(12.3±0.7 vs 16.7±1.2 and 15.9±0.7, P=0.001 and P=0.003)and MMP2(15.4±1.3 vs 18.2±1.1 and 17.8±1.1, P=0.027 and P=0.046). CONCLUSION: It is suggested that the expression of TFPI-2 may strongly inhibit the activity of MMPs in keratocytes in vitro, which provides an experimental basis for curing CNV with gene therapy.

Keywords

• keratocytes
• tissue factor pathway inhibitor-2
• matrix metalloproteinases
• extracellular matrix