Galectin-3 induces endothelial cell morphogenesis and angiogenesis

Abstract

Increasing evidence suggests that carbohydrate-binding proteins play an essential role in tumor growth and metastasis. However, conflicting results on their function in the regulation of cell proliferation and differentiation during angiogenesis have been reported. We have examined the role of galectin-3 in the regulation of human umbilical vein endothelial cell proliferation, differentiation, migration, and neovascularization. Galectin-3, a carbohydrate-binding protein, with specificity for type 1 and 11 ABH blood group epitopes and polylactosamine glycan containing cell surface glycoproteins, is the major nonintegrin cellular laminin-binding protein. Because galectin-3 expression was shown to be associated in some tumor systems with metastasis, we questioned whether it induces endothelial cell morphogenesis. Here we show that galectin-3 affects chemotaxis and morphology and stimulates capillary tube formation of HUV-EC-C in vitro and angiogenesis in vivo. Endothelial cell morphogenesis is a carbohydrate-dependent process, as it is neutralized by specific sugars and antibodies. These findings demonstrate that endothelial cell surface carbohydrate recognition event(s) can induce a signaling cascade leading to the differentiation and angiogenesis of endothelial cells.

Figure 1.

Endothelial cell organization on 4.5 mg/ml diluted Matrigel in the presence of exogenous galectin-3 (A: control; B: 1 μg/ml; C: 10 μg/ml; D: 20 μg/ml) or in the presence of conditioned medium from the galectin-3-secreting cell line 11-9-1-4 (E) and the null galectin-3 cell line BT-549 (F). Inset: Western blot of 100 μl conditioned medium. Lane 1: 11-9-1-4; lane 2: BT-549. Scale bar, 100 μm.

Figure 2.

Capillary tube formation on Matrigel. A: Control; B: polyclonal antibody; C: preimmune serum. Scale bar, 100 μm.

Figure 3.

Inhibition of capillary tube formation on Matrigel. A: Control; B: 0.1% modified citrus pectin; C: 50 mmol/L lactose; D: 50 mmol/L sucrose. Scale bar, 50 μm.

Figure 4.

Binding assay of various concentrations of iodinated galectin-3 to HUV-EC-C in the presence (○) or absence (•) of 50 mmol/L lactose.

Figure 5.

Scatchard plot analysis of ¹²⁵I-labeled galectin-3 binding to the endothelial cell surface. Experiments were performed as described in Materials and Methods. Each value is represented as a mean of four determinations.

Figure 6.

Chemotaxis of HUV-EC-C, by Boyden chamber assay. The bottom chamber contained 0–20 μg/ml galectin-3 in F12K, conditioned medium from galectin-3-secreting clone 11-9-1-4, or the conditioned medium from the null galectin-3 parental breast cancer cell line BT-549. An arbitrary unit of 1 was given to cells that migrated in 0 μg/ml gal-3. The rest of the values were calculated accordingly. Each point represents an average of eight readings.

Figure 7.

In vivo angiogenesis using BT-549 (A and B) and its galectin-3-transfected clone, 11-9-1-4 (C and D). Cells (1 × 10⁶) were injected with 300 μl Matrigel subcutaneously into nude mice. The tumors were removed after 10 days, fixed with 10% buffered formaldehyde, sectioned, and stained (brown) with anti-von Willebrand factor (A and C) and anti-galectin-3 antibodies (B and D). Scale bar, 8 μm.

Figure 8.

Microvessel density of Matrigel plugs injected with galectin-3-expressing and nonexpressing cells. The P value was 0.0038 as calculated by the Mann-Whitney test.

Figure 9.

Factor VIII staining of neovessels. Four hundred microliters of Matrigel containing 5 μg/ml bFGF or 10 μg/ml galectin-3 was injected dorsolaterally into nude mice. Gels recovered after 7 days were sectioned and stained with von Willebrand factor antibody. A: Matrigel alone; B: with galectin-3; C: with bFGF. Arrows indicate blood vessels. M, Matrigel. Scale bar, 40 μm.