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SL Chen; Expression of a Fusion Gene Containing Angiostatin and Endostatin Motifs via Lentiviral Transduction - a Novel, Potentially Antiangiogenic Reagent . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1757.
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Purpose: Angiostatin, a 38 kD protein derived from an internal fragment of plasminogen, and Endostatin, a 20kD protein product consisting of the COOH terminal of collagen 18, are both inhibitors of endothelial cell proliferation. We wished to develop a lentiviral vector able to introduce a fusion construct containing both endostatin and angiostatin motifs into ocular cells. In addition we wished to test the efficacy of this reagent to inhibit angiogenesis in vivo in a rabbit model of corneal neovascularization. Methods: An Endostatin-Angiostatin (Endo-Ang) fusion cDNA was cloned into the lentiviral vector pHR'-IRES-eGFP under the control of the EF1α/HTLV promoter. Replication-defective Endo-Ang lentivirus was prepared. Recombinant viral particles were tested for in vitro expression of the fusion-gene by RT-PCR of RNA from transduced human cells, utilizing primers that spanned the 8 aa linker region. A nylon mesh soaked with the Endo-Ang lentivirus was inserted into a corneal stromal pocket in one eye of New Zealand White rabbits. The virus soaked mesh was removed after 24 hours. Controls included eyes treated with nylon mesh soaked with pHR'-IRES-eGFP virus and without virus. An alkali burn of the corneal surface was employed to induce neovascularization. Corneal neovascularization is being graded in an ongoing fashion by a single blinded observer. Results: A fusion cDNA coding for a 20 aa secretion signal sequence, human Endostatin (derived from the 184 aa at the COOH end of collagen 18), a 8 amino acid linker sequence and 365 aa of human plasminogen (corresponding to the Angiostatin portion) was successfully cloned into a pHR'-EF1α/HTLV-IRES-eGFP plasmid. Positive RT-PCR of transduced human microvascular endothelial cells indicated efficient expression and effective production of lentiviral supernatants containing the Endo-Ang fusion product. Confocal microscopy analysis of OCT cryosections indicated that eGFP expression was localized to the corneal pocket area. Conclusion: A replication defective lentiviral system has been used to express an Endo-Ang fusion gene. This product is a potentially useful tool for studying the combined anti-angiogenic properties of Endostatin-18 and Angiostatin. The rabbit model of corneal neovascularization is being used to test whether the fusion product Endo-Ang has an enhanced inhibitory effect on angiogenesis. Supported by the Clayton Foundation for Research and Research to Prevent Blindness
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