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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 VEGF, PEDF, NF-KB p65, PMN and ED1(CD68) expression after corneal alkali burns.(A) Western blot analysis results showed that VEGF was expressed at low levels in normal rat cornea, while there was a dramatic increase at day 14 (D14) after alkali burns in the control group. In the late stage treatment experiment, there was a decrease in VEGF on day 24 (D24). In the netrin-4 treatment group, there was a dramatic down regulation of VEGF on day 14 and day 24. PEDF was expressed in normal corneas and was dramatically decreased on days 14 and 24 after alkali burns, whereas it was restored after netrin-4 treatment. Densitometry of protein expression compared with β-actin showed significant differences between the control group and the netrin-4 treatment group in (B) VEGF and (C) PEDF on days 14 and 24 (** p < 0.01). (E) NK-KB p65 expression detected by western blot. (F) PMN and ED1(CD68) immunostaining after alkali burn day 5, 7.
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pone.0122951.g007: Effect of netrin-4 on VEGF, PEDF, NF-KB p65, PMN and ED1(CD68) expression after corneal alkali burns.(A) Western blot analysis results showed that VEGF was expressed at low levels in normal rat cornea, while there was a dramatic increase at day 14 (D14) after alkali burns in the control group. In the late stage treatment experiment, there was a decrease in VEGF on day 24 (D24). In the netrin-4 treatment group, there was a dramatic down regulation of VEGF on day 14 and day 24. PEDF was expressed in normal corneas and was dramatically decreased on days 14 and 24 after alkali burns, whereas it was restored after netrin-4 treatment. Densitometry of protein expression compared with β-actin showed significant differences between the control group and the netrin-4 treatment group in (B) VEGF and (C) PEDF on days 14 and 24 (** p < 0.01). (E) NK-KB p65 expression detected by western blot. (F) PMN and ED1(CD68) immunostaining after alkali burn day 5, 7.

Mentions: It is well established that corneal neovascularization is tightly regulated by a dynamic, natural equilibrium between local proangiogenic and antiangiogenic molecules [28–31]. Among them, VEGF and PEDF are considered to play major roles. To investigate the mechanism hownetrin-4 inhibits and reverses corneal neovascularization after alkali burns application, we used Western blot analysis to assess VEGF and PEDF protein levels. Our results show that VEGF is expressed at low levels in normal rat corneas, but there was a dramatic increase in the control group on day 14 after alkali burns. In the later stage, there was a decrease in VEGF expression on day 24. After netrin-4 treatment, VEGF expression was lower than that of the control group on days 14 and 24 (Fig 7A and 7B). PEDF was expressed in the normal corneas and dramatically decreased on days 14 and 24 after alkali burns. However, PEDF was restored in the netrin-4 treatment group, although its expression was still lower than that observed in the normal corneas (Fig 7A and 7D). To further illustrate the function of netrin-4 in corneal inflammation, we conducted western blot NF-KB p65 and immunostaining of PMN and ED1(CD-68) antibodies to detect inflammation, neutrophil and macrophage infiltration after alkali burns. In the normal rat cornea, NK-KB p65 expressed lower than after alkali-burn. However, netrin-4 significantly decreased NK-KB p65 expression at day 1, 4, 7 (Fig 7E). Meanwhile, we detected netrin-4 inhibits neutrophil and macrophage infiltration by immunostaining (Fig 7F).


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 VEGF, PEDF, NF-KB p65, PMN and ED1(CD68) expression after corneal alkali burns.(A) Western blot analysis results showed that VEGF was expressed at low levels in normal rat cornea, while there was a dramatic increase at day 14 (D14) after alkali burns in the control group. In the late stage treatment experiment, there was a decrease in VEGF on day 24 (D24). In the netrin-4 treatment group, there was a dramatic down regulation of VEGF on day 14 and day 24. PEDF was expressed in normal corneas and was dramatically decreased on days 14 and 24 after alkali burns, whereas it was restored after netrin-4 treatment. Densitometry of protein expression compared with β-actin showed significant differences between the control group and the netrin-4 treatment group in (B) VEGF and (C) PEDF on days 14 and 24 (** p < 0.01). (E) NK-KB p65 expression detected by western blot. (F) PMN and ED1(CD68) immunostaining after alkali burn day 5, 7.
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pone.0122951.g007: Effect of netrin-4 on VEGF, PEDF, NF-KB p65, PMN and ED1(CD68) expression after corneal alkali burns.(A) Western blot analysis results showed that VEGF was expressed at low levels in normal rat cornea, while there was a dramatic increase at day 14 (D14) after alkali burns in the control group. In the late stage treatment experiment, there was a decrease in VEGF on day 24 (D24). In the netrin-4 treatment group, there was a dramatic down regulation of VEGF on day 14 and day 24. PEDF was expressed in normal corneas and was dramatically decreased on days 14 and 24 after alkali burns, whereas it was restored after netrin-4 treatment. Densitometry of protein expression compared with β-actin showed significant differences between the control group and the netrin-4 treatment group in (B) VEGF and (C) PEDF on days 14 and 24 (** p < 0.01). (E) NK-KB p65 expression detected by western blot. (F) PMN and ED1(CD68) immunostaining after alkali burn day 5, 7.
Mentions: It is well established that corneal neovascularization is tightly regulated by a dynamic, natural equilibrium between local proangiogenic and antiangiogenic molecules [28–31]. Among them, VEGF and PEDF are considered to play major roles. To investigate the mechanism hownetrin-4 inhibits and reverses corneal neovascularization after alkali burns application, we used Western blot analysis to assess VEGF and PEDF protein levels. Our results show that VEGF is expressed at low levels in normal rat corneas, but there was a dramatic increase in the control group on day 14 after alkali burns. In the later stage, there was a decrease in VEGF expression on day 24. After netrin-4 treatment, VEGF expression was lower than that of the control group on days 14 and 24 (Fig 7A and 7B). PEDF was expressed in the normal corneas and dramatically decreased on days 14 and 24 after alkali burns. However, PEDF was restored in the netrin-4 treatment group, although its expression was still lower than that observed in the normal corneas (Fig 7A and 7D). To further illustrate the function of netrin-4 in corneal inflammation, we conducted western blot NF-KB p65 and immunostaining of PMN and ED1(CD-68) antibodies to detect inflammation, neutrophil and macrophage infiltration after alkali burns. In the normal rat cornea, NK-KB p65 expressed lower than after alkali-burn. However, netrin-4 significantly decreased NK-KB p65 expression at day 1, 4, 7 (Fig 7E). Meanwhile, we detected netrin-4 inhibits neutrophil and macrophage infiltration by immunostaining (Fig 7F).

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