Critical Role Of SDF-1α/CXCR4 Signaling In Proliferation,Migration And Capillary Tube Formation Of HRECs In Vitro

Background and Objective Retinal pathological neovascularization,a leading cause of refractory blindness worldwide,is seen in age-related macular degeneration,diabetic retinopathy,retinopathy of prematurity,and retinal vein occlusion.Stromal cell derived factor 1α(SDF-1α)is a representative factor of the chemokine family,which specifically binds to C-X-C receptor 4(CXCR4).The SDF-1α/CXCR4 axis regulates numerous activities,including chemotaxis,adhesion,proliferation,and survival.Moreover,CXCR4 was also detectable in endothelial cells,which points to a possible role for SDF-1α/CXCR4 cell signaling in angiogenesis.Considerable evidence suggests that SDF-1α/CXCR4 signaling is involved in the progression of pathological neovascularization.However,the precise mechanism of its effects in retinal neovascularization still needs further exploration.The purpose of this study is to address the critical effect and mechanism of SDF-1α/CXCR4 signaling in human retinal vascular endothelial cells(HRECs)behavior of tube formation,proliferation and migration,providing a basis for further development of SDF-1α/CXCR4 signaling as a promising therapeutic target for the treatment of ocular neovascularization.Methods The HRECs were divided into several groups,as follows:PBS treated control group;SDF-1α groups,to which specific concentrations of 10 ng/ml,50 ng/ml,100 ng/ml and 200 ng/ml of recombinant human SDF-1α protein were added;and CXCR4 antagonist groups,to which SDF-1α protein(200 ng/ml)combined with CXCR4 antagonist were added(1 μmol/ml).The expression of CXCR4 in HRECs was quantified by reverse transcriptase polymerase chain reaction(RT-PCR)and western blot.Cell proliferation of HRECs was examined using cell counting kit(CCK)-8 assay in the presence of different concentrations of SDF-1α protein.The effect of SDF-1α/CXCR4 signaling in migration and capillary tube formation of HRECs was examined using wound scratching assay and three-dimensional matrigel assay respectively in vitro.The effect of SDF-1α/CXCR4 signaling in HREC expression of angiogenic associated factors such as VEGF,bFGF,IL-8 and ICAM-1 was examined using Real-time PCR and western blot.The level of phosphorylated ERK1/2 and PI3K expression in HRECs was evaluated by western bolt.Results RT-PCR and western blot detection revealed CXCR4 was expressed in HRECs.CCK-8 assay showed that the proliferation of HRECs in the presence of SDF-1αwas promoted in a dosage-dependent manner when compared with control group after cultured for 24 h.However,it was reduced while treated with CXCR4 antagonist.Wound scratching assay showed a significant increase in migration distance of HRECs under SDF-1α stimulation at 24h after injury,and it was reduced while treated with CXCR4 antagonist.As shown in tube formation assay,the number of intact capillary tubes formed by HRECs in the presence of SDF-1α was remarkably more than that in PBS treated control group after incubation for 12 h.It was also reduced while treated with CXCR4 antagonist.The result of Realtime-PCR and western blot showed that the expression of VEGF,bFGF,IL-8 and ICAM-1 was much more augmented in SDF-1α treated HRECs than control group.Furthermore,the level of pERK1/2 and pPI3K protein expression was also markedly enhanced in SDF-1α treated HRECs than control group as shown by western blot analysis.Conclusion Our present results indicate that SDF-1α/CXCR4 signaling has direct effect in HRECs bio-functions by promoting cell proliferation,cell migration and tube formation capability.The effects may through enhancing pro-angiogenic factors expression,such as VEGF,bFGF,IL-8 and ICAM-1 and the promoting property via activating PI3K/Akt and ERK1/2 signaling.