Galectin-1 and -3 in high amounts inhibit angiogenic properties of human retinal microvascular endothelial cells in vitro

Abstract

Purpose: Galectin-1 and -3 are β-galactoside binding lectins with varying effects on angiogenesis and apoptosis. Since in retinal pigment epithelial cells high amounts of human recombinant galectin (hr-GAL)1 and 3 inhibit cell adhesion, migration and proliferation, we investigated if hr-GAL1 and 3 have homologous effects on human retinal microvascular endothelial cells (HRMEC) in vitro.

Methods: To investigate the effect of galectin-1 and -3 on HRMEC, proliferation, apoptosis and viability were analyzed after incubation with 30, 60 and 120 μg/ml hr-GAL1 or 3 by BrdU-ELISA, histone-DNA complex ELISA, live/dead staining and the WST-1 assay, respectively. Further on, a cell adhesion as well as tube formation assay were performed on galectin-treated HRMEC. Migration was investigated by the scratch migration assay and time-lapse microscopy. In addition, immunohistochemical staining on HRMEC for β-catenin, galectin-1 and -3 were performed and β-catenin expression was investigated by western blot analysis.

Results: Incubation with hr-GAL1 or 3 lead to a decrease in proliferation, migration, adhesion and tube formation of HRMEC compared to the untreated controls. No toxic effects of hr-GAL1 and 3 on HRMEC were detected. Intriguingly, after treatment of HRMEC with hr-GAL1 or 3, an activation of the proangiogenic Wnt/β-catenin signaling pathway was observed. However, incubation of HRMEC with hr-GAL1 or 3 drew intracellular galectin-1 and -3 out of the cells, respectively.

Conclusion: Exogenously added hr-GAL1 or 3 inhibit angiogenic properties of HRMEC in vitro, an effect that might be mediated via a loss of intracellular endogenous galectins.

Fig 1. Localization of galectin-1 and -3 following incubation of HRMEC with hr-GAL1 and 3.

Immunostaining of HRMEC for galectin-1 (A) and -3 (B) after incubation with 120 μg/ml hr-GAL1 and 3 for 2 as well as 24 h. C. Detection of biotinylated hr-GAL1 and 3 (green) after incubation of HRMEC with 30 μg/ml recombinant protein for 2 h and immunostaining for MCT-1 (red) as a marker for the cell membrane (C). Membrane, focal plane of the cell membrane; cytoplasm, focal plane of the cytoplasm; magnification bar in A–C, 20 μm; blue, Hoechst staining. D. The intracellular fluorescent signal for galectin-1 or -3 of the control group and hr-GAL1 or 3 treated HRMEC was quantified, normalized against the control group and plotted as relative intracellular fluorescent signal. **p<0.01; ***p<0.001; n = 30 of 3 independent experiments.

Fig 2. Hr-GAL1 and 3 decreases intracellular galectin-1 and -3 level via exocytosis and proteasomal degradation.

Immunostaining of HRMEC for galectin-1 and -3 after incubation with 120 μg/ml hr-GAL1 or 3 with or without 5 μM epoxomicin, an inhibitor of proteasomal degradation, or 10 μM brefeldin A, an inhibitor of exocytosis, for 12 h. Magnification bar, 20 μm; blue, Hoechst staining.

Fig 3. hr-GAL1 and 3 block cell proliferation of HRMEC in vitro.

BrdU ELISA (A) and WST-1 (B) assay after treatment of HRMEC with 30, 60, and 120 μg/ml hr-GAL1 and 3 for 48 h (BrdU) as well as 72 h (WST-1). *co vs all hr-GAL1 and 3 concentrations, ***p<0.001; BrdU n = 40 of 5 independent experiments; WST-1, n = 32 of 4 independent experiments.

Fig 4. hr-GAL1 and 3 inhibit migration of HRMEC in vitro.

Scratch migration assay (A) and quantification (B) of HRMEC after incubation with 30, 60 and 120 μg/ml hr-GAL1 or 3 for 24 h. The left half of all images shows the wounded area immediately after scratching (0 h), the right half the same region after incubation for 24 h. *positive control (pos co) vs treated cells or negative control (neg co), ***p<0.001; n = 20 of 4 independent experiments.

Fig 5. hr-GAL1 and 3 slow down cell velocity of HRMEC in vitro.

Time lapse microscopy documentation of mean cell migration velocity at the documented time intervals (A), the overall mean velocity after incubation (B) and the single-cell superimposed cell tracks (C) during 24 h of incubation with 30, 60 and 120 μg/ml hr-GAL1 or 3. Each color in (C) represents one superimposed cell track. *co vs all hr-GAL for velocity, ***p<0.001, n = 60 of 3 independent experiments.

Fig 6. hr-GAL1 and 3 inhibit attachment of HRMEC in vitro.

Cell adhesion of HRMEC in the presence of 30, 60 and 120 μg/ml of hr-GAL1 and 3. Adherent cells were quantified after 15, 30, 60 and 120 min. *positive control (pos co) vs hr-GAL1 or 3; *p<0.05, (vs hr-GAL1 60 μg/ml: p = 0.021, vs hr-GAL1 120 μg/ml: p = 0.036) **p<0.01(vs hr-GAL3 120 μg/ml: p = 0.01); n = 9 of 3 independent experiments.

Fig 7. Hr-GAL1 and 3 inhibit formation of capillary-like structures in vitro.

A. Formation of capillary-like structures after incubation of HRMEC in basal (neg co) or cell supplemented culture medium without (pos co) or with 120 μg/ml rh-GAL1 or 3 for 4h. B. The area, which was covered by capillary-like structures with a width of more than 30 μm, was quantified after incubation with hr-GAL1 or 3 for 4 h and plotted as relative area of capillary-like structures. *positive control (pos co) vs hr-GAL1 and 3; *** p<0.001; n = 24 of 4 independent experiments; magnification bar, 200 μm.

Fig 8. Hr-GAL1 and 3 have no toxic effects on HRMEC.

A. Relative level of histone-DNA complexes after incubation of HRMEC in cell culture medium without (neg co) or with supplements (pos co) and hr-GAL1 or 3 for 72 h. B. Live/dead staining of HRMEC with Hoechst 33342 (blue) and propidium iodide (red) after 72 h treatment with 120 μg/ml hr-GAL1 or 3. *pos co vs neg co; ***p<0.001; n = 12 of 3 independent experiments; magnification bar, 75 μm.

Fig 9. Hr-GAL1 and 3 activate Wnt/β-catenin signaling in HRMEC in vitro.

A, B. Western blot analysis (B) and densitometry (A) for β-catenin on proteins from HRMEC after incubation with 120 μg/ml hr-GAL1 or 3 for 24 h (*co vs hr-GAL1: p = 0.019, *Co vs hr-GAL3: p = 0.021) *p<0.5; n = 3 of 3 independent experiments. C. Immunofluorescent staining for β-catenin (red) after incubation with 120 μg/ml hr-GAL1 or 3 for 24 h. Magnification bars, 20 μm; blue, Hoechst staining.