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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

Netrin-4 promotes the regression of corneal neovascularization and inhibit apoptosis after alkali burns.(A) Ten days after the injury, dense neovascularization reached the central cornea. In this experiment, netrin-4 treatment began on day 10. By day 24, the new blood vessels regressed from the central cornea to the peripheral cornea in the control group. In contrast, almost all the new blood vessels had regressed to the limbal area in the netrin-4 treatment group by day 24. (B) The inflammatory index continuously decreased from day 10 to day 24 in both groups, while the index was significantly lower in the netrin-4 treatment group on days 17, 20, and 24 (* p < 0.05). (C) The NV area gradually reduced from day 10 to day 24 in both groups. There was a dramatic decrease of NV area in the netrin-4 treatment group on day 20 and day 24, and there was a significant difference between the two groups (* p < 0.05 and ** p < 0.01). (D) The average NV length was continuously reduced from day 10 to day 24 in both groups, and the length was shorter in the netrin-4 treatment group on days 17, 20, and 24 than in the other group (* p < 0.05 and ** p < 0.01). (E) Netrin-4 reduced alkali burn-induced apoptosis of corneal cells. (F) A statistical analysis of the apoptotic cells on day 7 between the two groups showed significant difference (*** p < 0.001).
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pone.0122951.g006: Netrin-4 promotes the regression of corneal neovascularization and inhibit apoptosis after alkali burns.(A) Ten days after the injury, dense neovascularization reached the central cornea. In this experiment, netrin-4 treatment began on day 10. By day 24, the new blood vessels regressed from the central cornea to the peripheral cornea in the control group. In contrast, almost all the new blood vessels had regressed to the limbal area in the netrin-4 treatment group by day 24. (B) The inflammatory index continuously decreased from day 10 to day 24 in both groups, while the index was significantly lower in the netrin-4 treatment group on days 17, 20, and 24 (* p < 0.05). (C) The NV area gradually reduced from day 10 to day 24 in both groups. There was a dramatic decrease of NV area in the netrin-4 treatment group on day 20 and day 24, and there was a significant difference between the two groups (* p < 0.05 and ** p < 0.01). (D) The average NV length was continuously reduced from day 10 to day 24 in both groups, and the length was shorter in the netrin-4 treatment group on days 17, 20, and 24 than in the other group (* p < 0.05 and ** p < 0.01). (E) Netrin-4 reduced alkali burn-induced apoptosis of corneal cells. (F) A statistical analysis of the apoptotic cells on day 7 between the two groups showed significant difference (*** p < 0.001).

Mentions: To determine if netrin-4 induces the regression of well-formed corneal neovascularization, netrin-4 treatment was started on day 10 after applying alkali burns. In the rat alkali-burn model, corneal neovascularization peaked 10 days after injury (Fig 6C). Thereafter, gradual blood vessel regression was noted in the control group (Fig 6C). By 24 days post injury, the blood vessels regressed from the central cornea to the peripheral cornea in the control group. On the other hand, the rapid regression of corneal neovascularization was noticed when the eyes were treated with netrin-4, and almost all of the blood vessels regressed to the limbal area 24 days after treatment (Fig 6A). The inflammatory index showed gradual decrease in the PBS group, while it was further decreased in the netrin-4 group at days 17, 20, and 24 (Fig 3B). The area of corneal neovascularization (Fig 6C) and length (Fig 6D) on day 24 were significantly lower in the netrin-4 group in comparison with the control group. Meanwhile, to determine the effect of netrin-4 on alkali-burn induced apoptosis, we performed TUNEL assay on corneas treated with PBS or netrin-4 post alkali injury. As expected, there were no apoptotic cells present in normal rat corneas (Fig 6E). After alkali-burn, there were still many apoptotic cells in the basal epithelia, stromal, and endothelia at day 7. However, when the corneas were treated with netrin-4, there were much fewer apoptotic cells compared with the PBS control, and the majority of the apoptotic cells resided in the endothelia (Fig 6E). Statistical analysis showed a significant difference of apoptotic cells between the two groups (Fig 6F)


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)

Netrin-4 promotes the regression of corneal neovascularization and inhibit apoptosis after alkali burns.(A) Ten days after the injury, dense neovascularization reached the central cornea. In this experiment, netrin-4 treatment began on day 10. By day 24, the new blood vessels regressed from the central cornea to the peripheral cornea in the control group. In contrast, almost all the new blood vessels had regressed to the limbal area in the netrin-4 treatment group by day 24. (B) The inflammatory index continuously decreased from day 10 to day 24 in both groups, while the index was significantly lower in the netrin-4 treatment group on days 17, 20, and 24 (* p < 0.05). (C) The NV area gradually reduced from day 10 to day 24 in both groups. There was a dramatic decrease of NV area in the netrin-4 treatment group on day 20 and day 24, and there was a significant difference between the two groups (* p < 0.05 and ** p < 0.01). (D) The average NV length was continuously reduced from day 10 to day 24 in both groups, and the length was shorter in the netrin-4 treatment group on days 17, 20, and 24 than in the other group (* p < 0.05 and ** p < 0.01). (E) Netrin-4 reduced alkali burn-induced apoptosis of corneal cells. (F) A statistical analysis of the apoptotic cells on day 7 between the two groups showed significant difference (*** p < 0.001).
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pone.0122951.g006: Netrin-4 promotes the regression of corneal neovascularization and inhibit apoptosis after alkali burns.(A) Ten days after the injury, dense neovascularization reached the central cornea. In this experiment, netrin-4 treatment began on day 10. By day 24, the new blood vessels regressed from the central cornea to the peripheral cornea in the control group. In contrast, almost all the new blood vessels had regressed to the limbal area in the netrin-4 treatment group by day 24. (B) The inflammatory index continuously decreased from day 10 to day 24 in both groups, while the index was significantly lower in the netrin-4 treatment group on days 17, 20, and 24 (* p < 0.05). (C) The NV area gradually reduced from day 10 to day 24 in both groups. There was a dramatic decrease of NV area in the netrin-4 treatment group on day 20 and day 24, and there was a significant difference between the two groups (* p < 0.05 and ** p < 0.01). (D) The average NV length was continuously reduced from day 10 to day 24 in both groups, and the length was shorter in the netrin-4 treatment group on days 17, 20, and 24 than in the other group (* p < 0.05 and ** p < 0.01). (E) Netrin-4 reduced alkali burn-induced apoptosis of corneal cells. (F) A statistical analysis of the apoptotic cells on day 7 between the two groups showed significant difference (*** p < 0.001).
Mentions: To determine if netrin-4 induces the regression of well-formed corneal neovascularization, netrin-4 treatment was started on day 10 after applying alkali burns. In the rat alkali-burn model, corneal neovascularization peaked 10 days after injury (Fig 6C). Thereafter, gradual blood vessel regression was noted in the control group (Fig 6C). By 24 days post injury, the blood vessels regressed from the central cornea to the peripheral cornea in the control group. On the other hand, the rapid regression of corneal neovascularization was noticed when the eyes were treated with netrin-4, and almost all of the blood vessels regressed to the limbal area 24 days after treatment (Fig 6A). The inflammatory index showed gradual decrease in the PBS group, while it was further decreased in the netrin-4 group at days 17, 20, and 24 (Fig 3B). The area of corneal neovascularization (Fig 6C) and length (Fig 6D) on day 24 were significantly lower in the netrin-4 group in comparison with the control group. Meanwhile, to determine the effect of netrin-4 on alkali-burn induced apoptosis, we performed TUNEL assay on corneas treated with PBS or netrin-4 post alkali injury. As expected, there were no apoptotic cells present in normal rat corneas (Fig 6E). After alkali-burn, there were still many apoptotic cells in the basal epithelia, stromal, and endothelia at day 7. However, when the corneas were treated with netrin-4, there were much fewer apoptotic cells compared with the PBS control, and the majority of the apoptotic cells resided in the endothelia (Fig 6E). Statistical analysis showed a significant difference of apoptotic cells between the two groups (Fig 6F)

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