In Vivo and In Vitro Inhibitory Effect of Amniotic Extraction on Neovascularization

Abstract

Purpose: 

To prepare amniotic extraction (AE) and to test its antiangiogenic effect in vivo and in vitro.

Methods: 

AE was prepared and diluted to 50, 100, and 200 μg/mL concentrations. Alkali burn-induced corneal neovascularization (NV) was produced and topically treated with different concentrations of AE or 0.1% dexamethasone for 7 days. Normal saline was used as a control. Corneal NV was visualized by heart perfusion of Chinese ink and quantified as the percentage of corneal NV area to the whole corneal area. Human umbilical vein endothelial cells (HUVECs) were primarily cultured. The effects of AE on proliferation and tube formation of HUVECs were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and in vitro angiogenesis assay. Pigment epithelium-derived factor (PEDF) in AE was detected by Western blot.

Results: 

Relative corneal NV area in the control group was 56.6% ± 9.9%, which was significantly reduced by 50 μg/mL AE (47.6% ± 6.9%; P = 0.043) and 200 μg/mL AE (34.3% ± 7.8%; P < 0.001) and by 0.1% dexamethasone (21.1% ± 1.8%; P < 0.001). HUVEC cell proliferation was significantly decreased after treatment with AE at concentrations of 50 and 100 μg/mL compared with control (P = 0.036 and 0.001, respectively). The tube formation was significantly suppressed by 100 μg/mL AE (70.03% ± 4.35%) compared with control (100% ± 4.84%; P = 0.002). No expression of PEDF was detected in AE.

Conclusion: 

AE inhibits NV induced by alkali burn. This effect may be elicited at least in part through the inhibiting activity of blood vessel endothelial cells and is not associated with PEDF.