Human tissue factor pathway inhibitor-2 suppresses the wound-healing activities of human Tenon’s capsule fibroblasts in vitro

PURPOSE Human tissue Factor Pathway Inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin, which activates metalloproteinases involved in extracellular matrix degradation. Its secretion in the extracellular matrix makes TFPI-2 a potential inhibitor of tumor cell invasion. However, no studies have yet evaluated the wound-healing activities of human Tenon's capsule fibroblasts (hTCFs). The aim of the study is to elucidate the effect of TFPI-2 overexpression on hTCF proliferation and migration, to determine whether TFPI-2 may act as an antiscarring agent in vivo after glaucoma filtration surgery. METHODS Plasmid vector pBos-Cite-neo/TFPI-2 was transfected into hTCFs with Lipofectamine 2000. After selection by G418, transfected, non-transfected, and mock-transfected cells were screened for TFPI-2 mRNA and protein by reverse transcription-PCR and western blot analysis respectively. Cell proliferation and viability were determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Cell migration was studied on restrained collagen gels and with a scratch-wound assay. RESULTS TFPI-2 expression of mRNA and protein was confirmed in transfected cells. The transfected, non-transfected, and mock-transfected cells showed no significant difference in cell proliferation and apoptosis, with TFPI-2 found not to be cytotoxic in hTCFs. Overexpression of TFPI-2 significantly suppressed cell migration three- to four-fold on collagen gel for 2 weeks and in the scratch-wound assay for 2 d (39.27+/-2.40% versus 16.43+/-1.10% at 1 d, and 79.0+/-3.04% versus 30.13+/-2.1% at 2 d). CONCLUSIONS TFPI-2 expression may strongly inhibit the migration ability of hTCFs in vitro, making it a promising candidate for novel therapies to minimize scar development after glaucoma drainage surgery.