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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

Cell viability, HUVEC proliferation and apoptosis on different doses of netrin-4 was detected by using the CCK-8 assay and flow cytometry.(A) Cultured HUVECs were serum-starved for 24 h followed by 72 h culture in serum free media with or without dosage netrin-4 protein. The cells were then harvested, stained with propidium iodide (PI), and analyzed by flow cytometry. The cell proliferation rate was expressed as the percentage of cells in the S phase and as the G2 + S/G1 ratio (** p < 0.01). (B) Cell viability detected by CCK-8. (C) S phase percentage of cells. (D) G2 + S/G1 ratio. (E) Apoptosis assay by flow cytometry, analysis with annexin V-FITC / PI double staining. The numbers in the upper left, upper right, lower left and lower right quadrants represent the percentage of necrotic cells (annexin V positive, PI positive), advanced apoptotic cells (annexin V negative, PI positive), viable cells (annexin V negative, PI negative) and early apoptotic cells (annexin V positive, PI negative), respectively. The values represent the mean percentages of apoptotic cells in different groups (* p < 0.05). (F) Quantitative results of the apoptotic cells. Each value represents the mean ± SD, n = 3. * p < 0.05; ** p < 0.01; compared with the control group.
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pone.0122951.g002: Cell viability, HUVEC proliferation and apoptosis on different doses of netrin-4 was detected by using the CCK-8 assay and flow cytometry.(A) Cultured HUVECs were serum-starved for 24 h followed by 72 h culture in serum free media with or without dosage netrin-4 protein. The cells were then harvested, stained with propidium iodide (PI), and analyzed by flow cytometry. The cell proliferation rate was expressed as the percentage of cells in the S phase and as the G2 + S/G1 ratio (** p < 0.01). (B) Cell viability detected by CCK-8. (C) S phase percentage of cells. (D) G2 + S/G1 ratio. (E) Apoptosis assay by flow cytometry, analysis with annexin V-FITC / PI double staining. The numbers in the upper left, upper right, lower left and lower right quadrants represent the percentage of necrotic cells (annexin V positive, PI positive), advanced apoptotic cells (annexin V negative, PI positive), viable cells (annexin V negative, PI negative) and early apoptotic cells (annexin V positive, PI negative), respectively. The values represent the mean percentages of apoptotic cells in different groups (* p < 0.05). (F) Quantitative results of the apoptotic cells. Each value represents the mean ± SD, n = 3. * p < 0.05; ** p < 0.01; compared with the control group.

Mentions: We first measured the cell viability with CCK-8 assays in the presence of different concentrations of netrin-4 (100, 500, 1000, 5000 ng/mL), which reduced HUVEC viability at 1000 and 5000 ng/mL compared with the control (Fig 2B). The effects of netrin-4 on cell cycle were assessed using flow cytometry. After treating for 72 hours, cells treated with 100, 500, 1000, and 5000 ng/mL netrin-4, 5000ng/mL netrin-4 decreases the percentage of cells in S phase as well as the ratio of cells in G2+S phase compared with G1 phase. However, the opposite effects were observed at the concentration of 100 ng/mL (Fig 2A, 2C and 2D). In addtion, HUVECs were starved overnight and then treated with 100, 500, 1000, and 5000 ng/mL netrin-4, and apoptosis was analyzed using flow cytometry. Representative dot plots of annexin V/PI staining are shown (Fig 2E). The lower left quadrant shows the vital population, the lower right quadrant shows the apoptotic population (annexin V+/PI-), and the upper right quadrant shows the late apoptotic/necrotic population (annexin V+/PI+). The total number of apoptotic HUVECs increased after treatment with 5000 ng/mL netrin-4, and accordingly the total percentage of apoptotic cells increased from 12.12 ± 0.27% to 20.45 ± 0.51% at the high dosage (Fig 2E). The results demonstrate that netrin-4 promotes HUVECs apoptosis at the concentration of 5000 ng/mL but decreases HUVEC apoptosis (Fig 2F).


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)

Cell viability, HUVEC proliferation and apoptosis on different doses of netrin-4 was detected by using the CCK-8 assay and flow cytometry.(A) Cultured HUVECs were serum-starved for 24 h followed by 72 h culture in serum free media with or without dosage netrin-4 protein. The cells were then harvested, stained with propidium iodide (PI), and analyzed by flow cytometry. The cell proliferation rate was expressed as the percentage of cells in the S phase and as the G2 + S/G1 ratio (** p < 0.01). (B) Cell viability detected by CCK-8. (C) S phase percentage of cells. (D) G2 + S/G1 ratio. (E) Apoptosis assay by flow cytometry, analysis with annexin V-FITC / PI double staining. The numbers in the upper left, upper right, lower left and lower right quadrants represent the percentage of necrotic cells (annexin V positive, PI positive), advanced apoptotic cells (annexin V negative, PI positive), viable cells (annexin V negative, PI negative) and early apoptotic cells (annexin V positive, PI negative), respectively. The values represent the mean percentages of apoptotic cells in different groups (* p < 0.05). (F) Quantitative results of the apoptotic cells. Each value represents the mean ± SD, n = 3. * p < 0.05; ** p < 0.01; compared with the control group.
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pone.0122951.g002: Cell viability, HUVEC proliferation and apoptosis on different doses of netrin-4 was detected by using the CCK-8 assay and flow cytometry.(A) Cultured HUVECs were serum-starved for 24 h followed by 72 h culture in serum free media with or without dosage netrin-4 protein. The cells were then harvested, stained with propidium iodide (PI), and analyzed by flow cytometry. The cell proliferation rate was expressed as the percentage of cells in the S phase and as the G2 + S/G1 ratio (** p < 0.01). (B) Cell viability detected by CCK-8. (C) S phase percentage of cells. (D) G2 + S/G1 ratio. (E) Apoptosis assay by flow cytometry, analysis with annexin V-FITC / PI double staining. The numbers in the upper left, upper right, lower left and lower right quadrants represent the percentage of necrotic cells (annexin V positive, PI positive), advanced apoptotic cells (annexin V negative, PI positive), viable cells (annexin V negative, PI negative) and early apoptotic cells (annexin V positive, PI negative), respectively. The values represent the mean percentages of apoptotic cells in different groups (* p < 0.05). (F) Quantitative results of the apoptotic cells. Each value represents the mean ± SD, n = 3. * p < 0.05; ** p < 0.01; compared with the control group.
Mentions: We first measured the cell viability with CCK-8 assays in the presence of different concentrations of netrin-4 (100, 500, 1000, 5000 ng/mL), which reduced HUVEC viability at 1000 and 5000 ng/mL compared with the control (Fig 2B). The effects of netrin-4 on cell cycle were assessed using flow cytometry. After treating for 72 hours, cells treated with 100, 500, 1000, and 5000 ng/mL netrin-4, 5000ng/mL netrin-4 decreases the percentage of cells in S phase as well as the ratio of cells in G2+S phase compared with G1 phase. However, the opposite effects were observed at the concentration of 100 ng/mL (Fig 2A, 2C and 2D). In addtion, HUVECs were starved overnight and then treated with 100, 500, 1000, and 5000 ng/mL netrin-4, and apoptosis was analyzed using flow cytometry. Representative dot plots of annexin V/PI staining are shown (Fig 2E). The lower left quadrant shows the vital population, the lower right quadrant shows the apoptotic population (annexin V+/PI-), and the upper right quadrant shows the late apoptotic/necrotic population (annexin V+/PI+). The total number of apoptotic HUVECs increased after treatment with 5000 ng/mL netrin-4, and accordingly the total percentage of apoptotic cells increased from 12.12 ± 0.27% to 20.45 ± 0.51% at the high dosage (Fig 2E). The results demonstrate that netrin-4 promotes HUVECs apoptosis at the concentration of 5000 ng/mL but decreases HUVEC apoptosis (Fig 2F).

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