Fig 1.
Levels of netrin-4 in endothelial cells, and in cornea before and after alkali-burn were detected by ELISA, IHC and Western blot.
Netrin-4 expression in normal (A) and after alkali-burn rat cornea (B) by immunohistochemical staining. Netrin-4 concentrations in rat cornea and HUVEC (C). Netrin-4 concentration in normal rat cornea and different time after alkali-burn treatment.
Fig 2.
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.
Fig 3.
Effect of netrin-4 on HUVECs tube formation.
Tube formation was assessed in tubulogenesis assay in vitro model, HUVECs were serum-starved with EBM2 medium (0.1% FBS, no growth factor) (Lonza) for 12 hours. HUVECs (1.5×104) were then cultured in a 24-well plate (Gibco) coated with 150 mL Matrigel Basement Membrane Matrix GFR (BD Biosciences). Different concentrations of netrin-4 (100, 500, 1000, and 5000 ng/mL) or PBS (control) were added, and the results were quantified 6 h later using Image J. (B) Representative images out of three independent experiments are shown. * p < 0.05; ** p < 0.01. Each experiment was performed three times and representative pictures are shown. Data are expressed as mean ± SD.
Fig 4.
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.
Fig 5.
Netrin-4 prevents corneal neovascularization after alkali burns.
(A) One day after the alkali burns, the central corneal stroma of the rats appeared opaque and edematous in both groups. On day 14 after the injury, new blood vessels reached the central corneas in the control group, and there was a remarkable decrease in corneal transparency. In contrast, there was only slight new blood vessel formation in the limbal areas in the netrin-4 treatment group, and the corneas remained transparent on day 14. (B) The inflammatory index of the ocular surface declined from day 1 to day 14 in both groups. However, it was significantly lower in eyes treated with netrin-4 on day 7 and day 14 (* p < 0.05 and ** p < 0.01). (C) New blood vessel formation area (NV area) in the control group increased from day 1 to day 7, and there was a mild decrease on day 14 after the alkali burns. In contrast, corneas treated with netrin-4 showed only a mild increase in NV area on day 7. There was a significant difference between the two groups on day 7 and day 14 (** p < 0.01). (D) The average new blood vessel length (NV length) increased from day 1 to day 7 and decreased on day 14 in the PBS group, while the NV length continued to be very short in the netrin-4 treatment group. There were significant differences between the two groups on day 7 and day 14 (** p < 0.01). (E) H&E staining showed prominent new blood vessel formation from the limbal areas to the central corneas in the control group on day 14, which was well-indicated by the red blood cells that remained in the blood vessels. However, the netrin-4 treatment group only showed a few blood vessels in the limbal areas and none in the peripheral and central corneas.
Fig 6.
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).
Fig 7.
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.