Résumé
To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound-healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.
Sujets)
Animaux , Humains , Mâle , Souris , Mouvement cellulaire , Cellules cultivées , Dependovirus/génétique , Cellules endothéliales/métabolisme , Techniques de transfert de gènes , Vecteurs génétiques , Souris de lignée C57BL , Néovascularisation physiologique , Pancreatic ribonuclease/biosynthèse , Veines ombilicales/cytologie , Facteur de croissance endothéliale vasculaire de type A/biosynthèseRésumé
PURPOSE: The purpose of this study was to determine whether immunosuppressant, rapamycin could inhibit corneal angiogenesis induced by angiogenin and to evalutate its role by micropocket assay. METHODS: The rabbit's eye was implanted intrastromally into the superior cornea with pellet for the control group, pellet containing of angiogenin for the angiogenin group, and pellet containing of angiogenin and rapamycin for the angiogenin+rapamycin group. Biomicrographically, corneal angiogenesis was evaluated for 14 days after pellet implantation, based on the number and the length of new vessels. The neovascularized cornea also was examined histologically. RESULTS: We could observe that the angiogenin inducing corneal angiogenesis was inhibited by rapamycin. The score of neovascularization was significantly decreased in the angiogenin+rapamycin group than in the angiogenin group at 3, 7 and 10 days after pellet implantation (p<0.05). Histologically, the cornea of angiogenin+rapamycin group also showed much less new vessels than that of angiogenin group, in which inflammatory cells and edema was observed. CONCLUSIONS: Rapamycin appears to inhibit angiogenin induced angiogenesis in a rabbit corneal micropocket assay and may have therapeutic potential as an antiangiogenic agent.
Sujets)
Lapins , Cornée , Néovascularisation cornéenne , Oedème , SirolimusRésumé
The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50)= 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.
Sujets)
Animaux , Bovins , Cricetinae , Humains , Techniques de biocapteur , Division cellulaire/effets des médicaments et des substances chimiques , Mouvement cellulaire/effets des médicaments et des substances chimiques , Cellules cultivées , Poulets , Cellules endothéliales/cytologie , Cinétique , Kringles , Ligands , Fragments peptidiques/composition chimique , Liaison aux protéines , Récepteurs de surface cellulaire/métabolisme , Récepteurs à l'activateur du plasminogène de type urokinase , Activateur du plasminogène de type urokinase/composition chimique , Facteur de croissance endothéliale vasculaire de type A/pharmacologieRésumé
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.
Sujets)
Embryon de poulet , Lapins , Inhibiteurs de l'angiogenèse/pharmacologie , Inhibiteurs de l'angiogenèse/génétique , Animaux , Chorion/effets des médicaments et des substances chimiques , Chorion/vascularisation , Cornée/anatomopathologie , Cornée/effets des médicaments et des substances chimiques , Cornée/vascularisation , Facteurs de croissance endothéliale/pharmacologie , Facteur de croissance fibroblastique de type 2/pharmacologie , Kringles/génétique , Lymphokines/pharmacologie , Microscopie/méthodes , Néovascularisation pathologique/traitement médicamenteux , Plasminogène/pharmacologie , Plasminogène/génétique , Protéines recombinantes/pharmacologie , Protéines recombinantes/génétique , Pancreatic ribonuclease/pharmacologieRésumé
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.