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. 2017 Jan 1;58(1):9-20.
doi: 10.1167/iovs.16-20009.

Galectin-3 Inhibition by a Small-Molecule Inhibitor Reduces Both Pathological Corneal Neovascularization and Fibrosis

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Free PMC article

Galectin-3 Inhibition by a Small-Molecule Inhibitor Reduces Both Pathological Corneal Neovascularization and Fibrosis

Wei-Sheng Chen et al. Invest Ophthalmol Vis Sci. .
Free PMC article

Abstract

Purpose: Corneal neovascularization and scarring commonly lead to significant vision loss. This study was designed to determine whether a small-molecule inhibitor of galectin-3 can inhibit both corneal angiogenesis and fibrosis in experimental mouse models.

Methods: Animal models of silver nitrate cautery and alkaline burn were used to induce mouse corneal angiogenesis and fibrosis, respectively. Corneas were treated with the galectin-3 inhibitor, 33DFTG, or vehicle alone and were processed for whole-mount immunofluorescence staining and Western blot analysis to quantify the density of blood vessels and markers of fibrosis. In addition, human umbilical vein endothelial cells (HUVECs) and primary human corneal fibroblasts were used to analyze the role of galectin-3 in the process of angiogenesis and fibrosis in vitro.

Results: Robust angiogenesis was observed in silver nitrate-cauterized corneas on day 5 post injury, and markedly increased corneal opacification was demonstrated in alkaline burn-injured corneas on days 7 and 14 post injury. Treatment with the inhibitor substantially reduced corneal angiogenesis and opacification with a concomitant decrease in α-smooth muscle actin (α-SMA) expression and distribution. In vitro studies revealed that 33DFTG inhibited VEGF-A-induced HUVEC migration and sprouting without cytotoxic effects. The addition of exogenous galectin-3 to corneal fibroblasts in culture induced the expression of fibrosis-related proteins, including α-SMA and connective tissue growth factor.

Conclusions: Our data provide proof of concept that targeting galectin-3 by the novel, small-molecule inhibitor, 33DFTG, ameliorates pathological corneal angiogenesis as well as fibrosis. These findings suggest a potential new therapeutic strategy for treating ocular disorders related to pathological angiogenesis and fibrosis.

Figures

Figure 1
Figure 1
Galectin-3 inhibition by 33DFTG reduces corneal angiogenesis in vivo. Neovascularization was induced in mouse corneas by silver nitrate cautery as described in Methods. (A) Ten microliters of 33DFTG (325 ng) in PBS containing 0.5% DMSO or vehicle alone was administered by subconjunctival injections every other day. After 5 days, mice were killed, and flat mounts of corneas were stained with anti-CD31 to visualize blood vessels. Representative corneal flat mounts stained with anti-CD31. (i) Untreated normal cornea; (ii) control eye treated with vehicle alone; (iii) 33DFTG-treated cornea. (B) The density of blood vessels covering the whole cornea was quantified by ImageJ. Blood vessels cover ∼40% and 28% of cornea in vehicle- and 33DFTG-treated mice, respectively. n = 13. Data from three independent experiments were plotted and analyzed with Student's t-test. Scale bar: 1 mm.
Figure 2
Figure 2
Galectin-3 inhibition by 33DFTG abolishes VEGF-A–induced endothelial cell migration and attenuates VEGF-A–induced endothelial cell sprouting. (A) HUVECs were serum starved overnight, detached with Accutase, resuspended in 1% FBS/M199, and added in the upper chamber. The bottom chamber was filled with VEGF-A in the presence or absence of varying concentrations of 33DFTG in 1% FBS/M199. After a 3-hour incubation, HUVECs that migrated to lower side of the membrane were counted. The inhibitor at as low as 1 nM concentration significantly reduced VEGF-A–induced chemotaxis. A value of 1.0 was assigned to vehicle-treated cells. The value of cell migration in response to VEGF-A and 33DFTG is expressed as fold change in migration with respect to the vehicle-treated cells. (B) HUVEC spheroids were prepared as described in Methods. Spheroids were seeded into type I collagen gels in the presence or absence of 33DFTG. After 6-hour pretreatment, spheroids were treated with VEGF-A (50 ng/mL) in the presence or absence of 33DFTG. After 24 hours, spheroids were stained with calcein AM and fluorescent images were acquired using the EVOS FL cell imaging system. Cumulative sprout lengths were quantified by ImageJ. (i) A value of 1.0 was assigned to the sprout length of VEGF-A–treated spheroids. The value of sprouting in response to vehicle alone or VEGF-A with varying doses of 33DFTG is expressed as fold change in sprouting with respect to the VEGF-A–treated spheroids. Each condition had 11 to 15 spheroids. (ii) Representative fluorescence images are shown. Data are plotted as mean ± SEM and analyzed with 1-way ANOVA. *P < 0.05 versus control; ***P < 0.001 versus control; ###P < 0.001 versus VEGF-A. (B) Representative images of sprouts are shown. Ctrl: 0.05% DMSO; VEGF-A: 50 ng/mL; +33DFTG: VEGF-A (50 ng/mL) + 33DFTG (5 μM). Scale bar: 100 μm.
Figure 3
Figure 3
Galectin-3 inhibition by 33DFTG ameliorates corneal fibrosis. (A) Treatment with the inhibitor reduces corneal opacification. Mouse corneas were injured by alkali burn as described in Methods. One day post alkali burn, mice were equally divided into two groups. One group of mice was treated with 33DFTG (50 μM in 10 μL) by local subconjunctival injections on alternate days from day 1 until day 13. Control mice were injected with 10 μL PBS containing 0.5% DMSO (vehicle). Opacity score was recorded on days 1, 7, and 14. (i) Two representative photomicrographs of day 14 post injury of each group are shown. (ii) Opacity scores of three independent experiments are shown. n = 28 for vehicle (0.5% DMSO/PBS)-treated group; n = 29 for 33DFTG-treated group. (B) Expression of α-SMA is reduced in the corneas of 33DFTG-treated eyes. On day 14 post injury, corneal lysates (containing 30 μg protein) from injured eyes along with untreated normal eyes were subjected to electrophoresis in 4% to 15% SDS-PAGE gels. Protein blots of the gels were probed with anti-α-SMA, anti-galectin-3, and anti-β-actin antibodies. (i) Representative immunoblots are shown. (ii) Relative band intensity was quantified by Image Studio. Expression value of α-SMA and galectin-3 was normalized to β-actin, a value of 1.0 was given to vehicle (0.5% DMSO/PBS)-treated corneas, and the expression values of α-SMA and galectin-3 in the 33DFTG (50 μM)-treated corneas were calculated as fold changes. Four corneas were pooled and considered one biological replica. n = 3. Data are plotted as mean ± SEM and analyzed using 1-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle.
Figure 4
Figure 4
Treatment with the galectin-3 inhibitor decreases α-SMA immunoreactivity, inhibits CD45+ cell infiltration, and affects galectin-3 expression and distribution in alkaline-burned corneas. (A) Frozen tissue sections of vehicle (0.5% DMSO in PBS)- and 33DFTG (50 μM)-treated NaOH-cauterized eyes collected on day 14 post injury were immunostained with anti-α-SMA (green), followed by counterstaining with DAPI (blue). (i) No immunoreactivity was detected in corneas that were stained with isotype control IgG (top left). In normal control cornea, no α-SMA immunoreactivity was detected (top right). In contrast, α-SMA immunoreactivity was observed in subepithelial stroma in alkali-burned corneas (middle). Markedly reduced α-SMA immunoreactivity was detected in the 33DFTG-treated corneas (bottom). (ii) Fluorescence intensity (arbitrary unit, AU) of α-SMA within corneal stroma was quantified by ImageJ software. (B) Frozen tissue sections of vehicle- and 33DFTG-treated eyes collected on day 14 post injury were immunostained with anti-galectin-3 (green), anti-CD45 (red), and DAPI (blue). (i) Compared to vehicle-treated controls, 33DFTG-treated corneas exhibited reduced infiltration of CD45+ cells, increased galectin-3 immunoreactivity in corneal epithelium, and decreased galectin-3 immunoreactivity in corneal stroma. (ii) Cell numbers and fluorescence intensity were quantified. Immunostaining processing and fluorescence exposure time of all images are the same. Two representative images of vehicle- and 33DFTG-treated corneas are shown. For both (A) and (B), vehicle control, n = 4; 33DFTG, n = 6. Data are plotted as mean ± SEM and analyzed with Student's t-test. **P < 0.01; ***P < 0.001. Scale bar: 20 μm.
Figure 5
Figure 5
Galectin-3 treatment increases expression of fibrosis-related proteins. Primary human corneal fibrocytes were treated overnight with TGF-β1 (0.5 ng/mL in MEM, a positive control) and varying doses of recombinant galectin-3 ranging from 0.2 to 2 μM. Cell lysates containing 30 μg protein were subjected to electrophoresis in 4% to 15% SDS-PAGE gels. Protein blots of the gels were probed using anti-α-SMA, anti-CTGF, and anti-β-actin as described in Methods. (A) Representative immunoblots from three independent experiments with the same conclusion are shown. (B) Relative band intensity quantified by Image Studio. Expression value of α-SMA (red line) and CTGF (blue line) was normalized to β-actin. A value of 1.0 was assigned to the untreated control cells, and values of cells treated with galectin-3 are expressed as fold change with respect to the untreated control cells. Data from three independent experiments are plotted as mean ± SEM and analyzed using Student's t-test. *P < 0.05, **P < 0.01 versus untreated control cells.
Figure 6
Figure 6
Eye drop formulation of 33DFTG reduces cautery-induced corneal angiogenesis. Corneal neovascularization was induced by silver nitrate cautery as described in Methods. Ten microliters of varying doses of 33DFTG in 1.65% HEC or vehicle (1.65% HEC) alone was applied twice daily. After 7 days, mice were killed, and flat mounts of corneas were stained with anti-CD31 to visualize blood vessels. (A) Representative corneal flat mounts stained with anti-CD31. (i) Untreated normal cornea; (ii) vehicle control eye drop–treated cornea; (iii) 33DFTG (0.01%) eye drop–treated cornea. (B) The density of blood vessels covering the whole cornea was quantified by ImageJ. Blood vessels cover 45% and 19% of cornea in vehicle- and 33DFTG-treated mice, respectively. n ≥ 10 per group. Data are plotted and analyzed with 1-way ANOVA. **P < 0.01, ***P < 0.001. Scale bar: 1 mm.

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