RESUMEN
Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent. Copyright 2000 Academic Press.
Asunto(s)
Bovinos , Embrión de Pollo , Ratones , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/aislamiento & purificación , Inhibidores de la Angiogénesis/genética , Animales , Western Blotting , Movimiento Celular/efectos de los fármacos , Corion/patología , Corion/efectos de los fármacos , Dicroismo Circular , Colágeno/farmacología , Colágeno/aislamiento & purificación , Colágeno/genética , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/citología , Escherichia coli/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/genética , Pliegue de Proteína , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/genética , Solubilidad , Levaduras/genéticaRESUMEN
Angiostatin is a potent angiogenesis inhibitor that is composed of the first four kringles of plasminogen fragment. Angiostatin with one less kringle molecule (kringle 1 to 3) was recently demonstrated to be an effective angiogenic inhibitor. To determine whether recombinant plasminogen kringle 1-3 (rPK1-3) can inhibit the corneal neovascularization induced by potent angiogenic factors; angiogenin, bFGF, or VEGF, hydron polymer discs each containing 2.0 microg of angiogenin, 500 ng of bFGF, or 500 ng of VEGF respectively were implanted into the corneal stroma of 138 rabbit eyes, and then discs each containing 10 microg, 12.5 microg, 20 microg or 30 microg of rPK1-3 were implanted randomly. Discs containing phosphate buffered saline were also implanted as a control. The angiogenesis score on number and length of newly formed vessels on the each of the rabbit's cornea were recorded daily by two observers (blinded). The treated corneas were also examined histologically. Recombinant PK1-3 treated corneas showed less neovascularization induced by all angiogenic factors (p < 0.05). and the extent of inhibition of neovascularization was proportional to the concentration of rPK1-3 (p < 0.05). Histologic examination showed leukocyte infiltration into the corneal stroma on the PBS treated eyes whereas rPK1-3 treated eyes showed only traces of leukocytes. These results of the effective rPK1-3 inhibition of corneal neovascularization induced by angiogenin, bFGF, or VEGF suggest that this angiostatin related fragment, rPK1-3, may be useful in the treatment of various neovascular diseases. Copyright 2000 Academic Press.