Gene transfer of kringle 5 of plasminogen by electroporation inhibits corneal neovascularization.
OBJECTIVE
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.


CONCLUSIONS
In a rat model, K5 gene transfer by subconjunctival injection and electroporation can effectively inhibit corneal neovascularization induced by an alkali burn.
Yu
Wen-Zhen
WZ
Department of Ophthalmology, Peking University People's Hospital, Beijing, China.
Li
Xiao-Xin
XX
She
Hai-Cheng
HC
He
Pei-Ying
PY
Dong
Jian-Qiang
JQ
Rui
Min
M
Ma
Da-Long
DL
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002455:Cell Division / Q000187:drug effects; D003228:Conjunctiva / Q000378:metabolism; D003315:Cornea / Q000378:metabolism; D016510:Corneal Neovascularization / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D004195:Disease Models, Animal; D018274:Electroporation; D004730:Endothelium, Vascular / Q000473:pathology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006801:Humans; D007124:Immunoenzyme Techniques; D007267:Injections; D018082:Kringles / Q000235:genetics*; D008297:Male; D010957:Plasmids; D010958:Plasminogen / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
D010958:Plasminogen