Pathological Lymphangiogenesis Is Regulated by Galectin-8-Dependent Crosstalk among VEGF-C, Podoplanin and Integrin Pathways.
Abstract: Galectin expression patterns: Although members of the galectin family of carbohydrate-binding proteins are thought to play a role in the immune response and regulation of allograft survival, little is known about the galectin expression signature in diseased corneas. In this study, we compare the galectin expression pattern in normal, chemically injured, Pseudomonas aeruginosa-infected, ... read moreand allograft rejected or accepted mouse corneas. In normal corneas, galectins-1, -3, -7, -8 and -9 were expressed in normal corneas. Galectin-1 was distributed mainly in the stroma, galectin-3 was localized mainly in epithelium, and galectins-7, -8 and -9 were detected in both corneal epithelium and stroma. Expression levels of the five galectins were drastically altered under pathological conditions. In both infected and cauterized corneas, overall galectin-3 expression was downregulated, whereas overall galectins-8 and -9 were upregulated. Changes in the expression level of galectins-7, -8 and -9 were distinct in the epithelium of infected and cauterized corneas. Expression of these three galectins was upregulated in corneal epithelium of infected corneas but not in cauterized corneas. Consistent with the changes in protein expression: (i) galectins-7, -8 and -9 mRNA expression was upregulated in cauterized corneas and (ii) galectin-3 mRNA was downregulated and galectin-9 mRNA expression was upregulated in infected corneas. Although in both accepted and rejected grafts, expression levels of the five galectins were upregulated compared to normal corneas, there were distinct differences in the expression levels of galectins-8 and -9 between accepted and rejected grafts, as both Western blot and immunofluorescence staining revealed galectin-8 is upregulated, whereas galectin-9 is downregulated in rejected grafts compared to accepted grafts. Our data demonstrate differential regulation of various members of the galectin family in the course of corneal infection, neovascularization and allograft acceptance/rejection. The emerging functionality of the sugar code of cell surface receptors via endogenous galectins reflect to the pertinent roles of the five tested galectins in the diseases of cornea. Role of galectin-8 in lymphangiogenesis: Lymphangiogenesis is associated with diverse pathological conditions including metastatic dissemination, graft rejection, type 2 diabetes, obesity, hypertension, lymphedema and glaucoma. Despite recent studies have demonstrated that the members of the galectin family play a critical role in hemangiogenesis, the role of galectins in lymphangiogenesis has not been elucidated. In vitro studies have shown that galectin-8 binds podoplanin and that the lectin promotes haptotaxis of lymphatic endothelial cells (LECs). However, the evidence that galectin-8 exerts its biological functions through podoplanin is lacking. Here, we demonstrate for the first time that a carbohydrate-binding protein, galectin-8, is a potent lymphangiogenic factor. Galectin-8 was markedly upregulated in inflamed human and mouse corneas, and inhibitors of galectin-8 reduced inflammatory lymphangiogenesis. In corneal micropocket assays and 3D sprouting assays, galectin-8 promoted lymphangiogenesis in a carbohydrate-dependent manner. Galectin-8 was identified as a key mediator of integrin-dependent crosstalk between VEGF-C (vascular endothelial growth factor-C) and podoplanin lymphangiogenic pathways. Galectin-8 inhibitors reduced VEGF-C-induced lymphangiogenesis. Conversely, exogenous galectin-8 markedly enhanced VEGF-C-induced lymphangiogenesis in a carbohydrate-dependent manner. Knockdown of podoplanin attenuated not only galectin-8 but also VEGF-C-mediated LEC sprouting. Also, in corneal micropocket assays, VEGF-C-induced lymphangiogenesis was significantly reduced in the galectin-8-/- and podoplanin-/- mice; likewise, galectin-8-induced lymphangiogenesis was reduced in podoplanin-/- mice. Interestingly, knockdown of VEGFR-3 did not affect galectin-8-mediated LEC sprouting. Instead, inhibiting integrins α1β1 and α5β1 curtailed both galectin-8- and VEGF-C-mediated LEC sprouting. Additionally, podoplanin knockdown in LECs interfered with integrin activation. Immunoprecipitation assays further confirmed galectin-8-dependent interactions between podoplanin and integrins α5 and β1. In summary, this study has uncovered a unique lymphangiogenic pathway in which galectin-8-mediated interactions between podoplanin and integrins α1β1/α5β1 play a key role.
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisors: Noorjahan Panjwani, and Peter Brooks.
Committee: Lucy Liaw, John Castellot, Pedram Hamrah, and Pablo Argueso.
Keywords: Cellular biology, and Biochemistry.read less