Abstract
Purpose: :
Corneal neovascularization (NV) is associated with a number of corneal conditions that result in blindness for example trachoma and hepatic keratitis. Furthermore, vascularization of the corneal bed in corneal transplant recipients significantly increases the risk of graft rejection and failure. The purpose of this study is to determine the role of a carbohydrate-binding protein, galectin-3, and its inhibitors in the process of NV.
Methods: :
Capillary tubule formation and migration assays were performed using HUVEC to determine whether galectin-3 promotes angiogenesis in vitro . Mouse corneal micropocket assay was performed to determine whether galectin-3 promotes angiogenesis in vivo . To determine the role of the carbohydrate-based recognition system the experiments were performed in presence of saccharide inhibitors of galectins and also using Magt5 deficient (Mgat5-/-) HUVEC. Mgat5 is a glycosyltransferase that enhances the synthesis of galectin-3 ligands. Mgat5-/- HUVEC were generated using shRNA lentiviral transfection.
Results: :
Galectin-3 promoted capillary tubule formation and migration of HUVEC in vitro , as well as, the formation of vessels in vivo in the corneal micropocket assay in a dose-dependent manner. Galectin-3-mediated capillary tubule formation and migration were inhibited by competing saccharides: (i) 0.1M β-lactose, (ii) 0.005M Galα1-3[Fucα1-2]Galβ1-4GlcNAcβ, (iii) 0.005M Neu5Acα2-3[Galβ1-4GlcNAcβ1-3)3β, and (iv) 0.1% modified citrus pectin, but not by noncompeting saccharides: (i) sucrose, (ii) Galα1-4Galβ1-4Glcβ, (iii) Neu5Acα2-6GalNAcβ1-4GlcNAcβ, or (iv) citrus pectin. Also, galectin-3 induced angiogenesis was markedly reduced in corneal pocket assay in the presence of the saccharide inhibitors of galectin-3. Galectin-3 failed to promote capillary tubule formation and migration of Mgat5-/- HUVEC. Of particular significance are our findings that VEGF- and bFGF-mediated migration and capillary tubule formation were also inhibited by specific inhibitors of galectin-3 and that VEGF and bFGF failed to promote angiogenesis in vitro of Mgat5-/- HUVEC.
Conclusions: :
Galectin-3 promotes angiogenesis in vitro and in vivo via a carbohydrate-based recognition system. The carbohydrate-based recognition, most likely, also plays a role in VEGF- and bFGF-mediated angiogenesis.
Keywords: neovascularization • cornea: basic science • glycoconjugates/glycoproteins