Inhibited corneal neovascularization in rabbits following corneal alkali burn by double-target interference for VEGF and HIF-1α

Abstract

Expression of hypoxia-inducible factor (HIF) 1α has been observed in corneal neovascularization (CNV). Vascular endothelial growth factor (VEGF), one of the most well-known angiogenic factors in CNV, is under the regulation of HIF-1. The present study aims to investigate the synergistic effects of VEGF and HIF-1α gene silencing on alkali burn-induced CNV in rabbits. The models of rabbits in corneal alkali burn were established. SiRNA recombinant adenovirus was used to explore the synergistic effects of VEGF and HIF-1α gene silencing on alkali burn-induced CNV. CNV area and ultrastructure of cornea were observed. The expression of VEGF and HIF-1α was detected. CNV was observed in rabbits following alkali burn. In addition, overexpressed VEGF and HIF-1α was also observed in rabbits following alkali burn. Then, silencing HIF-1α or silencing VEGF decreased area of CNV, inhibited neovascularization and improved pathological changes, while double-target interference for VEGF and HIF-1α decreased area of CNV inhibited neovascularization, and improved pathological changes to a greater extent. Our study provides evidences emphasizing the distinct notion that VEGF and HIF-1α play the contributory role in alkali burn-induced CNV as a result of double-target interference for VEGF and HIF-1α inhibiting CNV in rabbits following corneal alkali burn.

Keywords: Alkali burn; Cornea neovascularization; Gene silencing; HIF-1α; Rabbit model; Synergistic effect; VEGF.

Figure 1. Under the slit lamp microscope, double-target interference for VEGF and HIF-1α significantly inhibits neovascularization in corneas

(A) The experimental results on the 14th day after alkali burn under slit lamp microscope; (B) the area of CNV of rabbits after alkali burn in each group. *, P<0.05 compared with the normal group; ^#, P<0.05 compared with the HIF-1α-VEGF siRNA group; ^&, P<0.05 compared with the HIF-1α siRNA group.

Figure 2. Under the confocal microscopy (×800), double-target interference for VEGF and HIF-1α promotes the healing of corneal epithelial cells and inhibits inflammatory cell infiltration in rabbits after alkali burn

The results of the corneas on the 1st, 7th, and 14th days after alkali burn observed under the confocal microscopy with the field of vision of 400 μm × 400 μm and the resolving power of 1 μm.

Figure 3. HE staining reveals that double-target interference for VEGF and HIF-1α significantly improves pathological changes of corneal tissues on the 14th day after alkali burn

The neovascularization containing erythrocyte in the corneal stroma was expressed by black arrowhead, and the corneal stroma presented as edema and mononuclear inflammatory reaction.

Figure 4. Double-target interference for VEGF and HIF-1α decreases the positive expression of VEGF and HIF-1α in alkali-burned corneal tissue

(A) The positive staining in each group; (B) the positive expression of VEGF and HIF-1α in corneas were examined by immunohistochemistry. *P<0.05 compared with the normal group; ^#P<0.05 compared with the HIF-1α-VEGF siRNA group; ^&P<0.05 compared with the HIF-1α siRNA group.

Figure 5. RT-qPCR reveals that double-target interference for VEGF and HIF-1α reduces mRNA expression of VEGF and HIF-1α in corneal tissues after alkali burn

(A) mRNA expression of VEGF; (B) mRNA expression of HIF-1α. *P<0.05 compared with the normal group; ^#P<0.05 compared with the HIF-1α-VEGF siRNA group; ^&P<0.05 compared with the HIF-1α siRNA group.

Figure 6. Western blot analysis demonstrates that double-target interference for VEGF and HIF-1α reduces the protein level of VEGF and HIF-1α in corneal tissues after alkali burn

(A) Protein bands and protein level of VEGF in corneal tissues after alkali burn; (B) protein bands and protein level of HIF-1α in corneal tissues after alkali burn. *P<0.05 compared with the normal group; ^#P<0.05 compared with the HIF-1α-VEGF siRNA group; ^&P<0.05 compared with the HIF-1α siRNA group.