A Role for Galectin-3 in Angiogenesis

Certain physiological processes, such as embryonic development, re­productive function, and wound healing, require the formation of new blood vessels. The cellular mechanism by which new blood vessels are formed from the existing vasculature is termed angiogenesis. Angiogenesis is a multi-step process that involves the concerted action of several cytokines and growth factors that exert chemotactic, mito­genic, and modulatory effects on endothelial cells.¹

It has been demonstrated that carbohydrate-binding proteins and their respective glycoconjugate ligands also play an essential role in angiogenesis.^{2, 3} Specifically, recent research has shown that Galectin-3 promotes angiogenesis.^{4, 5} Galectin-3 is one member of the Galectin family of lectins that specifically bind to N-acetyl-lactosamine-containing glycoproteins.⁶ All galectins contain at least one carbohydrate recognition domain (CRD) in their C-termini. However, Galectin-3 is the sole member of the family that contains one CRD linked to a proline, glycine, and tyrosine-rich repeat N-terminal domain. The N-terminal half of Galectin-3 mediates oligomerization upon ligand binding.⁶ Galectin-3 is mainly found in the cytosol but can be secreted to function extra­cellularly, where it binds to cell surface glycoconjugates containing beta-galactosides.⁶

A role for Galectin-3 in angiogenesis was first revealed after research demonstrated that it influences chemotaxis and differentiation of human umbilical vein endothelial cells (HUVECs). In vitro, Galectin-3 is both chemotactic and induces capillary tubule formation in HUVECs.⁴ Galectin-3 also promotes angiogenesis in vivo.^{4, 5} These processes are dependent upon both carbohydrate recognition and oligo­merization by Galectin-3.^{4, 5}

A recent paper by Markowska et al. elucidated a potential mechanism by which Galectin-3 mediates angiogenesis.⁵ These authors discovered that Galectin-3 participates in angiogenesis induced by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Decreasing Galectin-3 expression in HUVECs by small interfering RNA reduced VEGF- and bFGF-mediated migration and capillary tubule formation.⁵ Additionally, both growth factors failed to induce neovascularization in Galectin-3-deficient mice.⁵ Mass spectrometry analysis of HUVEC lysate proteins isolated with a Galectin-3 affinity column, identified Integrin alpha V beta 3 as a binding partner with putative involvement in Galectin-3-mediated effects on angiogenesis.⁵ In general, integrin clustering triggers the activation of intracellular signaling molecules, such as focal adhesion kinase (FAK).⁷ Specifically, Integrin alpha V beta 3 has been shown to be involved in growth factor-mediated angiogenesis.^{7, 8, 9, 10} Markowska and colleagues demon­strated that treating HUVECs with Galectin-3 promoted Integrin alpha V beta 3 clustering and FAK activation.⁵ Moreover, pretreatment with antibodies against Integrin alpha V beta 3 inhibited Galectin-3-induced HUVEC migration and capillary tubule formation.⁵

It was hypothesized that Galectin-3 interacts with complex N-glycans present on Integrin alpha V beta 3, activating signaling pathways that influence VEGF- and bFGF-mediated angiogenesis. The authors generated HUVECs expressing Integrin alpha V beta 3 with reduced expression of the high affinity Galectin-3 ligands, beta 1,6-N-acetylglucosamine (GlcNAc)-branched N-glycans. This was accomplished by transfecting HUVECs with lentivirus carrying small hairpin RNA (shRNA) constructs directed against human N-acetylglucosaminyl-transferase V (GnTV), the enzyme that synthesizes the beta 1,6GlcNAc-branched arm in N-glycans.^{5, 11, 12} Disrupting GnTV expres­sion in HUVECs attenuated binding of Galectin-3 to cell surface glycoproteins.⁵ These GnTV shRNA-transfected cells did not migrate or form tubule structures when exposed to either Galectin-3, VEGF, or bFGF.⁵ In addition, Galectin-3 and both growth factors failed to promote angiogenesis in vivo in GnTV^–/– mice.⁵

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Galectin-3 Activation of Integrin alpha V beta 3 in Growth Factor-mediated Angiogenesis. Galectin-3, a beta-galactoside binding protein, has been previously implicated as participating in angiogenesis; however, the mechanism Galectin-3 employs was still unknown. Markowska and colleagues demonstrated that Galectin-3 binds to complex N-glycans on Integrin alpha V beta 3, triggering integrin clustering and FAK activation.5 Activated Integrin alpha V beta 3 subsequently influences VEGF- and bFGF-induced angiogenesis. This study complements a wealth of research that has implicated angiogenic activity to be dependent upon the coordinated actions of integrins and growth factor receptors. Integrin alpha V beta 3, specifically the beta 3 subunit, complexes with VEGF R2 and FGF R1 upon ligand binding.9, 10, 14 The interaction between Integrin alpha V beta 3 and VEGF R2 is critical for VEGF-induced VEGF R2 phosphorylation and activation of VEGF R2 signaling pathways.7, 9, 10 Integrin alpha V beta 3 is also required for the sustained ERK1/2 activation observed during bFGF-induced angiogenesis.15 In a similar manner, growth factors have also been shown to modulate integrin functioning. VEGF and FGF receptor activation increases the affinity of Integrin alpha V beta 3 for its ligands.8, 9

References

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14. Yan, W. et al. (2008) Mol. Biol. Cell 19:2278.
15. Eliceiri, B.P. et al. (1998) J. Cell Biol. 140:1255.

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