Inhibitory Effect Of CCR3-antagonist Signal In Alkali-induced Corneal Neovascularization

Background and AimAngiogenesis is a basic biological process, which is broadly participating in several different physiological of development and has important significance in health and disease. Neovascularization inlvoved in more than twenty ocular diseases, among them corneal neovascularization is still difficult to treatment. Until now, the pathogenesis of corneal neovascularization has not been feen understood and there is no effect treatment. Alkali-induced corneal neovascularization is a sensitive, repeatable, consistent inflammatory animal models, which provides an ideal platform for the research of corneal neovascularization because of its simple establish, convenient observation and so on.CCR3is the CC chemokine receptor-3, which can combine with selective ligands such as Eotaxin-1/CCL11, Eotaxin-2/CCL24, Eotaxin-3/CCL26, and nonselective ligands such as MCP-2, MCP-3, MCP-4, MIP-1α, RANTES and so on. CCR3is mainly expressed on the surface of eosinophils and basophils, and also expressed on the surface of mast cell, TH2lymphocyte, macrophages and so on. Recently studies demonstrate that CCR3and its ligands play pivotal roles in choroidal neovascularization. However, less is known about the role in CRNV.In this study, alkali induced corneal neovascularization is used to explore the effect of CCR3on the process of corneal neovascularization. Firstly, CCR3mRNA expression at different time point after alkali injury during corneal neovascularization was detected by RT-PCR. Secondly, the effect of CCR3-antagonist on alkali induced CRNV was observed by using SB328437after alkali injury. Thirdly, the expressions of MCP-1, MCP-3mRNA between the experimental group and control group were assessed and compared by RT-PCR, and the infiltration of macrophages were observe and compared by immunohistochemistry. Materials and methods1.30BALB/c mice of alkali-burn induced corneal neovascularization, and collect the corneal tissue at0,2,4,7,14days after alkali injury randomly, and use RT-PCR to detect the CCR3mRNA expression at different time point respectively.2. We designed two different time periods. In the early phase treatment group, the mice were treated with CCR3-atagonist after alkali injury immediately, while in the later stage treatment group, the mice were treated from7days after alkali injury. In each group the injured mice were divided into4groups randomly forward, and used different concentrations of CCR3-antagonist (125μg/ml,250μg/ml,500μg/ml) to intervene the development of CRNV, control group use0.2%HA, respectively, three times one day, for7days or14days. At the indicated time intervals, the eyes were photographed and then the mice were killed and the corneas were removed and fixed for whole mount CD31staining.3.36BALB/c mice after alkali injury were randomly divided into experimental group (500μg/ml CCR3-atagonist group) and control group (0.2%HA group).The method of intervention was the same as the early phase treatment group. Corneas at2,4and7days after alkali injury were collected for mRNA extraction followed by RT-PCR to detect and compare the expression of MCP-1, MCP-3mRNA between experimental group and control group.4.28BALB/c mice after alkali injury were randomly divided into experimental group (500μg/ml CCR3-atagonist group) and control group (0.2%HA group).The method of intervention was the same as the early phase treatment group. Eyes at2,4days after alkali injury were enucleated for frozen section, slides were stained with rat anti-mouse F4/80to evaluate and compare the number of macrophage in the corneal tissue between experimental group and control group.Results1. CCR3mRNA were expressed in the process of alkali induced corneal neovascularization, in addition the expressing level of CCR3mRNA at2day,4day,7day were higher than0day (P<0.05), which suggest the involvement of CCR3in corneal neovascularization.2. Repetitive result proved that500μg/ml CCR3-atagonist could inhibit the development of corneal neovascularization at the early phase treated mice (P<0.05), but not the125 μg/ml and250μg/ml CCR3-atagonist (P>0.05). In late phase treated mice, there was no difference between CCR3-antagonist treated groups and control group (P>0.05).3. RT-PCR results demonstrate that500μg/ml CCR3-antagonist treated mice exhibited decreased intra-corneal MCP-1and MCP-3mRNA (P<0.05) expression in the early phase (day2, day4) after alkali injury compared with control group. The result revealed that topical administration of CCR3-antagonist could efficiently decrease the MCP-1and MCP-3mRNA expression in the corneal after alkali injury.4. Immunohistochemistry analysis demonstrated that CCR3-antagonist treatment significantly inhibited F4/80-positive macrophages infiltration into the corneas at day4(P<0.05), but not day2(P>0.05).Conclusion1. CCR3mRNA was expressed in the process of alkali induced corneal neovascularization,500μg/ml CCR3-atagonist could inhibit the development of corneal neovascularization at the early phase.2.500μg/ml CCR3-atagonist could decrease the expression of MCP-1, MCP-3mRNA and also inhibit macrophages infiltration into the corneas in the early phase, which may be suppressing the development of CRNV.3. CCR3-atagonist may be a new clinical treatment for corneal neovascularization.