The Critical Role And Mechamism Of SDF-1α/CXCR4in The Corneal Neovascularization

Background and ObjectiveOcular neovascular diseases such as corneal neovascularization induced by infectionor chemical injury, iris and atria angle neovascularization, neovascular glaucoma inducedby atria angle closure with fibrovascular membrane formation, age-related maculardegeneration which leaded by choroidal neovascularization and proliferative retinopathycaused by retinal neovascularization and so on are all included. To date, the exactpathogenesis of ocular neovascular disease is yet unclear, and thus it is still lack of preciseand effective clinical prevention or treatment for ocular neovascular diseases. All clinicaltreatment were not efficacy and resulted that ocular neovascular diseases become one ofthe main reasons of blindness. According to the domestic provinces data from Sichuan,Hubei, Zhejiang and Shandong, it showed that since1990only corneal diseases inducedblindness accounted for1/4in whole blindness diseases. As a result, it is extremelyimportant for exploration on the pathogenesis and prevention methods of ocularneovascular diseases.The chemokine receptor, CXCR4, was initially cloned as an orphan chemokinereceptor and was found to be expressed on many different cell types such as monocytes,lymphocytes, hematopoietic and endothelial progenitor cells. CXCR4is activated by theligand, stromal-derived factor1(SDF-1/CXCL12), and mediates several different activitiessuch as chemotaxis, adhesion, proliferation, survival, and, in some cells, apoptosis.Activation of CXCR4on lymphocytes and monocytes stimulates chemotaxis, resulting inrecruitment to sites of immune and inflammatory reactions. Moreover, CXCR4was alsodetected in endothelial cells, which points to a role for SDF-1a/CXCR4cell signaling invascularization. The signaling pathway of SDF-1α/CXCR4plays a critical role in ocular neovascular diseases such as choroidal neovascularization, diabetic retinopathy andhypoxia-induced retinopathy. However, their function in other diseases such as cornealneovascularization is yet unclear. In order to delineate their possibly specific mechanism incorneal neovascularization, we have prepared alkali-inducing experimental CRNV modelusing NaOH, topically administrated SDF-1α recombinant protein, neutralizing anti-mouseSDF-1α mAbs and CXCR4antagnist, microscopically and immunostaining detected theoccurrence of corneal neovascularization, and examined the target gene expression ininjury corneas. Through these exploration and resultant statistically analysis, theexperiment conclusion will provide us a new thinking about SDF-1α/CXCR4signalingpathway involvement in ocular neovascularization.Materials and MethodsAlkali-induced corneal injury model (CRNV) was prepared using NaOH on left eyesof30BALB/c mice. They were randomly divided into3groups (n=10) and each groupwere administrated topically with concentration of10μg/mL SDF-1α neutralizing antibody,5μg/mL CXCR4antagonist or0.2%sodium hyaluronate respectively three times a day for1week after alkali injury.2weeks after alkali injury the experimental corneas weremicroscopically observed with slit lamp for the progression of inflammation, cornealperforation and neovascularization, then the mice were killed and their alkali injury eyeswere enucleated for immunohistochemical detection of CD31positive area ofneovascularization and they were compared by statistical methods and were analysed forthe effection of SDF-1α neutralizing antibody and CXCR4antagonist on CRNV.Alkali-induced injury model were prepared with the same methods for corneal injuryand group dividing. The corneas were removed for total RNA extraction at indicated timeintervals of day2, day4and day7after injury. The mRNA expression of VEGF in injurycorneas was examined by RT-PCR and the difference level of each group were comparedby statistically analysis. We also concomitantly examined VEGF protein expression tofurther explore the effection of SDF-1α neutralizing antibody and CXCR4antagonist onintra-corneal VEGF expression.In order to verify the animal experiments, we also isolated mouse peritonealmacrophages and cultured them with RPMI-1640medium in in vitro. Then the cells werestimulated by SDF-1α recombinant protein, SDF-1α neutralizing antibody or CXCR4 antagonist at different concentration. The expression of VEGF was detected and comparedbetween stimulated groups and vehicle groups.ResultsRepeated results indicated that the positive CRNV areas in injury cornea of SDF-1αneutralizing antibody and CXCR4antagonist administrated groups was significantlydecreased than control group and this tendency was statistically significant (P<0.05).Concomitantly, the intra-corneal VEGF expression in experimented groups was reduced atsame trend compared with corneas of contral group (P<0.05).We also found that VEGF expression in mouse peritoneal macrophages was increasedafter stimulated with SDF-1α recombinant protein In vitro and controversically, theirexpression of VEGF were decreased significantly after stimulated by CXCR4antagonistand the tendency were statistically significant (P<0.05).ConclusionBlocking SDF-1α/CXCR4signaling reduce the VEGF expression and neovasculari-zation in alkali-induced injuried cornea. SDF-1α/CXCR4may play a critical role in theexperimental corneal neovascularization by affecting VEGF expression in intra-cornealmonocytes/macrophages.