Inhibited Effect Of CCR3Antagonist In Experimental Mouse Corneal Neovascularization

Background and AimThe transparency of cornea is one of the important factors to maintain normal visualacuity. Pathological causes such as inflammation, corneal trauma, misuse of contact lensesand so on can break the balance of pro-angiogenic and anti-angiogenic molecules. Newvessels were then growing into the transparent cornea from corneal limbus, which lead tosevere impaired vision. Until now, the pathogenesis of corneal neovascularization (CRNV)has not been fully understood and the effects of growth factors, cytokines, chemokines andother inflammatory mediators on CRNV were focused.CC chemokine receptor3(CCR3) is a G protein coupled receptor that expressedmainly in eosinophils, basophils, a subset of Th2lymphocytes, mast cells and macrophage,with the highest levels in eosinophils. It is believed to function in recruiting leukocytes,mainly the Th2cells and eosinophils, to inflammatory sites. In allergic diseases such asasthma, it can also be found expression in vascular endothelial cells, and has been shownto be involved in angiogenesis. Recently studies demonstrate that CCR3signal play pivotalroles in choroidal neovascularization. It also mediates the formation of CRNV in mice, butthe specific mechanism and potential invention role of CCR3signal in CRNV is still notclear.Alkali-induced CRNV is a widely accepted corneal neovascularization animal models.In this study, alkali induced corneal neovascularization is used to explore the effect ofCCR3signal on the process of corneal neovascularization. Firstly, CCR3and eotaxinmRNA time kinetic expression after alkali injury was detected by Real-time PCR.Secondly, CCR3antagonist was locally administrated after alkali injury immediately andthe whole mount CD31staining and flow cytometry was used to examine the formation ofcorneal neovascular after injury. The mRNA and protein expression of VEGF in burnedcorneas was detected by Real-time PCR and Western blot. Thirdly, the effect of CCR3 antagonist on human retinal endothelial cells (HRECs) tube formation and proliferationwere detected in vitro.Materials and Methods1. Corneal neovascularization were induced by alkali injury of60BALB/c mice, thecorneal tissue at0,2,4,7days after alkali injury randomly collected. CCR3andeotaxin mRNA expression at the indicated time were detected by Real-time PCR.2.60BALB/c mice after alkali injury were randomly divided into2groups and eachgroup were administrated topically with concentration of1μmol/ml CCR3-antagonistor0.2%sodium hyaluronate (HA) respectively, three times a day for1week afteralkali injury immediately.2weeks after alkali injury the experimental corneas weremicroscopically observed with slit lamp for the progression of inflammation, cornealperforation and neovascularization. The mice were killed and corneas were enucleatedand CRNV were detected and compared by corneal whole mount CD31staining orflow cytometric analysis of intracorneal CD31positive cell.3.60BALB/c mice after alkali injury were randomly divided into experimental group (1μmol/ml CCR3-antagonist group) and control group (0.2%HA group). Theintracorneal expression of VEGF and other relative cytokines were detected andcompared between stimulated groups and vehicle groups by Real-time PCR andWestern blot.4. To observe the CCR3signal on HREC proliferation, cultured HRECs in96well weredivided into5groups. After24h incubation with different concentrations ofCCR3-antagonist (0.1μmol/ml,0.2μmol/ml,0.5μmol/ml,1μmol/mlCCR3-antagonist) and PBS buffer, CCK8were added into culture cluster and the ODvalue in wave length450nm were measured.5. To observe the CCR3signal on the tube formation of HREC, cultured HRECs in96well coated with Matrigel were divided into2groups (1μmol/ml CCR3-antagonistgroup and control group). After24h incubation, the tube formation were counted.Results1. The time kinetic expression of CCR3and eotaxin mRNA in the process of alkaliinduced corneal neovascularization suggest that CCR3signal was involved in thedevelopment of corneal neovascularization.2. CCR3-antagonist local administration can inhibit the development of CRNV. 3. CCR3-antagonist inhibited the proliferation of HRECs in a certain range ofconcentration with a dose-dependent manner. CCR3antagonist influenced the tubeformation of HRECs.ConclusionAlkali-induced corneal neovascularization was inhibited by CCR3antagonist. CCR3pathway plays an important role in corneal neovascularization by regulating the endothelialcell proliferation and tube formation. CCR3-antagonist may be a new clinical treatment forcorneal neovascularization.