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Therapeutic effects of topical netrin-4 inhibits corneal neovascularization in alkali-burn rats.

Han Y, Shao Y, Liu T, Qu YL, Li W, Liu Z - PLoS ONE (2015)

Bottom Line: Netrin-4 and netrin-1 have been found to be either pro- or antiangiogenic factors.We found that netrin-4 functions similarly as netrin-1 in angiogenesis.These results indicate that netrin-4 shed new light on its potential roles in treatmenting for angiogenic diseases that affect the ocular surface, as well as other tissues.

View Article: PubMed Central - PubMed

Affiliation: Eye Institute of Xiamen University, Xiamen, Fujian, China; Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.

ABSTRACT
Netrins are secreted molecules involved in axon guidance and angiogenesis. However, the role of netrins in the vasculature remains unclear. Netrin-4 and netrin-1 have been found to be either pro- or antiangiogenic factors. Previously, we found that netrin-1 acts as an anti-angiogenic factor in rats by inhibiting alkali burn-induced corneal neovascularization. Here, we further investigate the effects of netrin-4, another member of the same netrin family, on neovascularization in vitro and in vivo. We found that netrin-4 functions similarly as netrin-1 in angiogenesis. In vitro angiogenesis assay shows that netrin-4 affected human umbilical vein endothelial cell (HUVEC) tube formation, viability and proliferation, apoptosis, migration, and invasion in a dose-dependent manner. Netrin-4 was topically applied in vivo to alkali-burned rat corneas on day 0 (immediately after injury) and/or day 10 post-injury. Netrin-4 subsequently suppressed and reversed corneal neovascularization. Netrin-4 inhibited corneal epithelial and stromal cell apoptosis, inhibited vascular endothelial growth factor (VEGF), but promoted pigment epithelium-derived factor (PEDF) expression, decreased NK-KB p65 expression, and inhibits neutrophil and macrophage infiltration. These results indicate that netrin-4 shed new light on its potential roles in treatmenting for angiogenic diseases that affect the ocular surface, as well as other tissues.

No MeSH data available.


Related in: MedlinePlus

Effect of netrin-4 on HUVECs migration and invasion.HUVEC were treated during 24 h in medium without serum, in the presence of EBM2 alone or EBM2 with different concentrations of netrin-4. (A)Pictures were taken at 0 h and 24 h after insert removing. (B) Results are expressed as percentage of wound closure (percentage closure) (mean ± SD). (C) Netrin-4 affects HUVEC migration in a transwell assay. HUVEC cells were allowed to migrate for 24 h at 37°C in a 5% CO2 humidified incubator. After 24 h, the cells that migrated across the membrane were stained with DAPI and the fluorescence intensity was measured. (D)The % invasion is compared to the fluorescence intensity of migrated cells in the absence of netrin-4 (** p < 0.01). Each experiment was performed three times and a representative picture of each condition is shown. Data are expressed as mean ± SD.
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pone.0122951.g004: Effect of netrin-4 on HUVECs migration and invasion.HUVEC were treated during 24 h in medium without serum, in the presence of EBM2 alone or EBM2 with different concentrations of netrin-4. (A)Pictures were taken at 0 h and 24 h after insert removing. (B) Results are expressed as percentage of wound closure (percentage closure) (mean ± SD). (C) Netrin-4 affects HUVEC migration in a transwell assay. HUVEC cells were allowed to migrate for 24 h at 37°C in a 5% CO2 humidified incubator. After 24 h, the cells that migrated across the membrane were stained with DAPI and the fluorescence intensity was measured. (D)The % invasion is compared to the fluorescence intensity of migrated cells in the absence of netrin-4 (** p < 0.01). Each experiment was performed three times and a representative picture of each condition is shown. Data are expressed as mean ± SD.

Mentions: After verifying the effects of netrin-4 on cell proliferation, we assessed its effects on HUVEC migration using a scratch-wound model. After the scratch wound was created, the wounds were allowed to heal with or without netrin-4. The control wounds closed within approximately 24 hours, while the group treated with 5000 ng/mL netrin-4 did not heal within 24 hours. There was still 980 μm blank in the netrin-4-treated group at 24 h. After 24 hours treatment, the migration rates were 42.6 and 26.1 μm/hour in the control and netrin-4–treated groups, respectively (Fig 4A and 4B). Based on the results of the transwell invasion assays, the 5000 ng/mL netrin-4–treated group demonstrated lower invasive abilities than the controls, but 100ng/mL netrin-4 increases HUVEC invasive abilities (Fig 4C and 4D). These results suggest that netrin-4 exhibits a dose-dependent effects on HUVEC proliferation, migration, apoptosis, and invasion. At 5000ng/mL, netrin-4 can inhibit HUVEC proliferation, migration, invasion, but increase apoptosis. However, at lower dosage 100ng/mL, the role of netrin-4 is opposite.


Therapeutic effects of topical netrin-4 inhibits corneal neovascularization in alkali-burn rats.

Han Y, Shao Y, Liu T, Qu YL, Li W, Liu Z - PLoS ONE (2015)

Effect of netrin-4 on HUVECs migration and invasion.HUVEC were treated during 24 h in medium without serum, in the presence of EBM2 alone or EBM2 with different concentrations of netrin-4. (A)Pictures were taken at 0 h and 24 h after insert removing. (B) Results are expressed as percentage of wound closure (percentage closure) (mean ± SD). (C) Netrin-4 affects HUVEC migration in a transwell assay. HUVEC cells were allowed to migrate for 24 h at 37°C in a 5% CO2 humidified incubator. After 24 h, the cells that migrated across the membrane were stained with DAPI and the fluorescence intensity was measured. (D)The % invasion is compared to the fluorescence intensity of migrated cells in the absence of netrin-4 (** p < 0.01). Each experiment was performed three times and a representative picture of each condition is shown. Data are expressed as mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390284&req=5

pone.0122951.g004: Effect of netrin-4 on HUVECs migration and invasion.HUVEC were treated during 24 h in medium without serum, in the presence of EBM2 alone or EBM2 with different concentrations of netrin-4. (A)Pictures were taken at 0 h and 24 h after insert removing. (B) Results are expressed as percentage of wound closure (percentage closure) (mean ± SD). (C) Netrin-4 affects HUVEC migration in a transwell assay. HUVEC cells were allowed to migrate for 24 h at 37°C in a 5% CO2 humidified incubator. After 24 h, the cells that migrated across the membrane were stained with DAPI and the fluorescence intensity was measured. (D)The % invasion is compared to the fluorescence intensity of migrated cells in the absence of netrin-4 (** p < 0.01). Each experiment was performed three times and a representative picture of each condition is shown. Data are expressed as mean ± SD.
Mentions: After verifying the effects of netrin-4 on cell proliferation, we assessed its effects on HUVEC migration using a scratch-wound model. After the scratch wound was created, the wounds were allowed to heal with or without netrin-4. The control wounds closed within approximately 24 hours, while the group treated with 5000 ng/mL netrin-4 did not heal within 24 hours. There was still 980 μm blank in the netrin-4-treated group at 24 h. After 24 hours treatment, the migration rates were 42.6 and 26.1 μm/hour in the control and netrin-4–treated groups, respectively (Fig 4A and 4B). Based on the results of the transwell invasion assays, the 5000 ng/mL netrin-4–treated group demonstrated lower invasive abilities than the controls, but 100ng/mL netrin-4 increases HUVEC invasive abilities (Fig 4C and 4D). These results suggest that netrin-4 exhibits a dose-dependent effects on HUVEC proliferation, migration, apoptosis, and invasion. At 5000ng/mL, netrin-4 can inhibit HUVEC proliferation, migration, invasion, but increase apoptosis. However, at lower dosage 100ng/mL, the role of netrin-4 is opposite.

Bottom Line: Netrin-4 and netrin-1 have been found to be either pro- or antiangiogenic factors.We found that netrin-4 functions similarly as netrin-1 in angiogenesis.These results indicate that netrin-4 shed new light on its potential roles in treatmenting for angiogenic diseases that affect the ocular surface, as well as other tissues.

View Article: PubMed Central - PubMed

Affiliation: Eye Institute of Xiamen University, Xiamen, Fujian, China; Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.

ABSTRACT
Netrins are secreted molecules involved in axon guidance and angiogenesis. However, the role of netrins in the vasculature remains unclear. Netrin-4 and netrin-1 have been found to be either pro- or antiangiogenic factors. Previously, we found that netrin-1 acts as an anti-angiogenic factor in rats by inhibiting alkali burn-induced corneal neovascularization. Here, we further investigate the effects of netrin-4, another member of the same netrin family, on neovascularization in vitro and in vivo. We found that netrin-4 functions similarly as netrin-1 in angiogenesis. In vitro angiogenesis assay shows that netrin-4 affected human umbilical vein endothelial cell (HUVEC) tube formation, viability and proliferation, apoptosis, migration, and invasion in a dose-dependent manner. Netrin-4 was topically applied in vivo to alkali-burned rat corneas on day 0 (immediately after injury) and/or day 10 post-injury. Netrin-4 subsequently suppressed and reversed corneal neovascularization. Netrin-4 inhibited corneal epithelial and stromal cell apoptosis, inhibited vascular endothelial growth factor (VEGF), but promoted pigment epithelium-derived factor (PEDF) expression, decreased NK-KB p65 expression, and inhibits neutrophil and macrophage infiltration. These results indicate that netrin-4 shed new light on its potential roles in treatmenting for angiogenic diseases that affect the ocular surface, as well as other tissues.

No MeSH data available.


Related in: MedlinePlus