Inhibition of VEGF expression and corneal neovascularization by shRNA targeting HIF-1α in a mouse model of closed eye contact lens wear

摘要

Purpose: Inappropriate contact lens (CL) use and care often lead to corneal neovascularization (corneal NV). We used mouse eyes which wore CL as the animal model to assess the reason for corneal NV with CL wear. The similar and overlapping activity of vascular endothelial growth factor (VEGF) and the potent angiogenic hypoxia-inducible factor 1α (HIF-1α) called for a study of the temporal relationship in the expression of these two autocoids. We determined the time dependent expression of HIF-1α and correlated it to that of VEGF expression in the mouse model of closed eye with CL wear. Methods: Mouse eyes were fitted with CL followed by a silk suture tarsorrhaphy. The anterior surface was analyzed at 4, 7, and 10 days after tarsorrhaphy by slit lamp and corneal NV. HIF-1α and VEGF levels were measured by reverse transcription PCR, western blotting and immunofluorescence with specific primers and antibodies. We used shRNA targeting HIF-1α to substantiate the link between HIF-1α, VEGF expression, and angiogenesis in the CL wear model. Results: Corneal NV scores increased in a time dependent manner in the model of closed eye CL induced hypoxic injury. Corneal epithelial HIF-1α and VEGF expression increased in a time dependent manner. The prolonged hypoxic state brought by closed eye CL wear induced a time dependent neovascular response which was significantly attenuated by HIF-1α specific shRNA but not by nonspecific shRNA. Both HIF-1α and VEGF levels were reduced significantly in corneal homogenates from eyes treated with the HIF-1α specific shRNA. Conclusions: The present study documented the increased expression of HIF-1α in the corneal epithelium during CL wear. It also demonstrated the presence of VEGF in the corneal epithelium and its increased expression in this model. Altogether, the results of this study raised the possibility of interaction between HIF-1α and VEGF, in mediating the neovascularization response induced by the prolonged hypoxic state brought about by closed eye CL wear. The results strongly implicated corneal HIF-1α as a component of the inflammatory and neovascular cascade initiated by hypoxic and further suggested that HIF-1α was a proximal regulator of VEGF expression in this model.