Cells of F-P and F-TFPI-2 were harvested by trypsinization, resuspended in a culture medium without serum at a density of 1106cells/ml, seeded onto the polymerized gels, and allowed to form cell aggregates for 2-3 h at 37 C
Cells of F-P and F-TFPI-2 were harvested by trypsinization, resuspended in a culture medium without serum at a density of 1106cells/ml, seeded onto the polymerized gels, and allowed to form cell aggregates for 2-3 h at 37 C. 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 (39.272.40% versus 16.431.10% at 1 d, and 79.03.04% versus 30.132.1% 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. == Introduction == Trabeculectomy is the most frequently used surgical method to reduce intraocular pressure in patients with glaucoma unresponsive to medical therapy. However, excessive scarring of the filtering bleb after glaucoma filtration surgery can lead to an increase in intraocular pressure, and is the most important cause of treatment failure. A variety of antimetabolites, such as 5-fluorouracil and mitomycin C, has been shown to be clinically effective at preventing bleb failure after filtration surgery [1,2]. Their antifibrotic effect has been shown to derive mostly from the inhibition of human Tenons capsule fibroblasts (hTCFs) proliferation, as well as from apoptotic cell death [3]. However, these agents are associated with significant adverse side effects and postoperative complications, such as ocular hypotony, following choroidal detachment and hypotonic maculopathy, progressive thinning of the filtering bleb following bleb infection, and endophthalmitis [4,5]. Most studies on the filtering bleb healing process and its modulation have concentrated on fibroblast proliferation. However, in some high-risk patients, even after antiproliferative treatment, surgery still fails, in part due to residual activity of the growth-arrested cells and their interaction with surrounding untreated fibroblasts. Therefore, alternative targets to prevent scar formation after trabeculectomy are needed. The wound-healing processes include proliferation, migration, synthesis of extracellular matrix (ECM) components, and collagen contraction by hTCFs (the key cells involved in the subconjunctival wound-healing response). After injury, quiescent fibroblasts in the surrounding matrix are activated; they proliferate, and migrate into the wound site to deposit and remodel a new matrix, resulting in tissue fibrosis and scar formation. This process involves a family of enzymes capable of cleaving components of the ECM, such as matrix metalloproteinases (MMPs). Human tissue factor pathway inhibitor-2 (TFPI-2), an inhibitor of MMPs, may inhibit Rabbit polyclonal to Caspase 3 scarring APR-246 after glaucoma filtration surgery. TFPI-2 is a member of the same Kunitz-type serine protease inhibitor family as tissue factor pathway inhibitor-1 (TFP-1). TFPI-2 contains three Kunitz domains arranged in tandem with a high degree of conservation. APR-246 The basic carboxy terminus of TFPI-2 mediates ionic interactions that associate this protein with glycosaminoglycans in ECMs [6,7]. Studies have demonstrated that TFPI-2 is a strong serine protease inhibitor with broad inhibitory spectra, whose expression can decrease the invasion capacity of various tumor cells [8-12]. Its effect on the wound-healing activities of hTCFs is unknown. APR-246 Therefore, the aim of the present study was to elucidate the effect of TFPI-2 on hTCF proliferation and migration to determine its suitability as an antiscarring agent for in vivo use APR-246 after glaucoma filtration surgery. == Methods == == Cell culture == Cultures of human Tenon’s capsule were established from patients undergoing routine cataract surgery. The tenets of the Declaration of Helsinki were followed in the collection of human material, and patients consent was APR-246 obtained. Excised specimens were dissected into 1-2 mm cubes and maintained in Dulbecco’s modified Eagle’s medium (Gibco BRL, Gaithersburg, MD), supplemented with 20 mM HEPES,.