Abstract
Abstract: :
Purpose:To develop a lentiviral vector capable of promoting the expression of a potential angiogenesis inhibitor composed of the five kringle domains of human plasminogen (K1-5). Methods:A cDNA coding for the 452 amino acid protein K1-5 was cloned into the lentiviral vector pHR'-IRES-eGFP under the control of the EF-1α/HTLV promoter. Replication-defective K1-5 lentivirus was prepared by three plasmid co-transfection into 293T cells. Transcription of this gene produces a bicistronic message, capable of the independent translation of K1-5 and eGFP proteins. Expression of K1-5 in human microvascular endothelial cells transduced with this virus was analyzed by RT-PCR analysis. To assay the angiogenic potential of this reagent, nylon mesh soaked with either the pHR'-EF1α/HTLV-K1-5-IRES-eGFP virus or pHR'-IRES-eGFP (a K1-5-deficient control) virus was inserted into a corneal stromal pocket in right and left eyes, respectively, of New Zealand White rabbits by a single blinded surgeon and removed after 24 hours. Corneas were insulted with standardized alkali burns 14 days after transduction. Analysis of the development of corneal vascularization by a single blinded observer is underway. Results:Expression of K1-5 in human microvascular endothelial cells transduced with this vector was proven by RT-PCR. On the third post-insult day an average of 0.3 mm2 of neovascularization was evident in K1-5-virus treated eyes, compared with 5.5 mm2 of neovascularization in control eyes. On day 5 this trend was reversed, with an average of 1.5-fold more vessels in the K1-5-treated eyes compared with controls. Immunohistologic correlation of the development of corneal neovascularization with the temporal and spatial characteristics of K1-5 expression is underway. Conclusion:We have developed a lentiviral reagent capable of transferring an expressible gene containing the five kringle domains of human plasminogen. This reagent may prove useful as an inhibitor of ocular angiogenesis.
Keywords: 419 gene transfer/gene therapy • 483 neovascularization • 370 cornea: basic science