PURPOSE: To test the efficacy of naked plasmid that expresses human kringle 5 of plasminogen (K5) in suppressing experimental corneal neovascularization in a rat model. METHODS: A eukaryotic expression plasmid encoding human K5 (pSecK5) was constructed. COS cells were transiently transfected with pSecK5 using a lipid-based transfection reagent. K5 secretion was confirmed by Western blot analysis. The effect of the secreted K5 on the proliferation of human umbilical vein endothelial cells (HUVECs) was investigated colorimetrically. Forty-three Sprague-Dawley rats were used for a corneal neovascularization suppression experiment. Corneal injury was induced by placing a disk of filter paper (immersed in 1 mol/l NaOH, 3.0 mm in diameter) on the corneal surface for 2 min. The cornea was immediately washed with saline. pSecK5 and empty plasmids were injected subconjunctivally, and square-wave electric pulses were immediately applied to the eyes. The expression of K5 was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The extent of corneal neovascularization was evaluated by scores. RESULTS: The constructed plasmid could express itself in COS cells. Conditioned medium from K5-transfected COS cells apparently inhibited HUVEC proliferation, compared with conditioned medium from COS cells transfected with empty plasmid or nontransfected cells. RT-PCR and immunohistochemistry confirmed the expression of K5 in the conjunctiva and cornea. Corneal neovascularization was significantly suppressed by K5 gene transfer in rats' eyes. CONCLUSION: In a rat model, K5 gene transfer by subconjunctival injection and electroporation can effectively inhibit corneal neovascularization induced by an alkali burn. Copyright 2003 S. Karger AG, Basel
PURPOSE: To test the efficacy of naked plasmid that expresses human kringle 5 of plasminogen (K5) in suppressing experimental corneal neovascularization in a rat model. METHODS: A eukaryotic expression plasmid encoding human K5 (pSecK5) was constructed. COS cells were transiently transfected with pSecK5 using a lipid-based transfection reagent. K5 secretion was confirmed by Western blot analysis. The effect of the secreted K5 on the proliferation of human umbilical vein endothelial cells (HUVECs) was investigated colorimetrically. Forty-three Sprague-Dawley rats were used for a corneal neovascularization suppression experiment. Corneal injury was induced by placing a disk of filter paper (immersed in 1 mol/l NaOH, 3.0 mm in diameter) on the corneal surface for 2 min. The cornea was immediately washed with saline. pSecK5 and empty plasmids were injected subconjunctivally, and square-wave electric pulses were immediately applied to the eyes. The expression of K5 was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The extent of corneal neovascularization was evaluated by scores. RESULTS: The constructed plasmid could express itself in COS cells. Conditioned medium from K5-transfected COS cells apparently inhibited HUVEC proliferation, compared with conditioned medium from COS cells transfected with empty plasmid or nontransfected cells. RT-PCR and immunohistochemistry confirmed the expression of K5 in the conjunctiva and cornea. Corneal neovascularization was significantly suppressed by K5 gene transfer in rats' eyes. CONCLUSION: In a rat model, K5 gene transfer by subconjunctival injection and electroporation can effectively inhibit corneal neovascularization induced by an alkali burn. Copyright 2003 S. Karger AG, Basel
Authors: John M Nickerson; Shannon E Getz; Jana T Sellers; Micah A Chrenek; Penny Goodman; Christiana J Bernal; Jeffrey H Boatright Journal: Methods Mol Biol Date: 2014