The Interleukin (IL)-17A Promotes Angiogenesis In An Experimental Corneal Neovascularization Model

Background Ocular neovascular diseases,including neovascular glaucoma induced by atria angle closure with fibrovascular membrane formation,age-related macular degeneration and corneal neovascularization,are refractory eye diseases which can lead to blindness.To date,the exact pathogenesis of ocular neovascular disease is yet unclear,and thus it is still lack of precise and effective clinical prevention or treatment for ocular neovascular diseases.As a result,it is extremely important for exploration on exact pathogenesis and prevention methods on ocular neovascular diseases.Tissue avascularity is maintained by the balance between angiogenic and anti-angiogenic factor expression.In the process of inflammation and tumor growth,many kinds of cells are induced to express angiogenic factors,such as VEGF and other cytokines.These factors are expressed by various cells,including infiltrated macrophages and neutrophils.The cornea is transparent and is an immunoprivileged site,in part,because of its avascular nature.Corneal neovascularization(CRNV)can arise from various causes,including corneal infections,misuse of contact lens,chemical burn and inflammation and frequently leads to impaired vision.The physiologic regulation of angiogenesis is dependent on a delicate temporal and spatial balance of activators and inhibitors present within the vascular microenvironment.Based upon these findings,identification of endogenous angiogenesis stimulators or inhibitors is an area of great interest.The interleukin(IL)-17 cytokine family consists of 6 members(IL-17 A to IL-17F)and at least 5 receptors(IL-17 RA to IL-17RE;reviewed in Moseley et al.).It has been shown that IL-17 A has several biologic activities,including induction of IL-6,IL-8,prostaglandin E2(PGE2),and metalloprotease 1(MMP-1),as well as enhancement of monocyte chemotactic protein 1(MCP-1)and granulocyte colony-stimulating factor (G-CSF)expression in fibroblasts and keratinocytes.In addition,IL-17 A induces the secretion of tumor necrosis factor(TNF)-? and stromelysin by macrophages to inhibit tumor growth.Although the proinflammatory function and intracellular signaling pathways of IL-17 A were strikingly similar to those of IL-1 and Toll receptors,IL-17 RA has no homology with other known receptor sequences,thus,IL-17 A,homologous proteins,and its viral homologue constitute a novel cytokine family.It was reported that IL-17 A contributed to tumor angiogenesis by causing the proliferation and migration of vascular endothelial cells into tissues.However,the role of IL-17 A in mediating CRNV subsequent to severe injury remains unclear.To further address the role of IL-17A-mediated signals in ocular neovascularization,alkali-induced CRNV was analyzed in mouse recombinant IL-17A-or in neutralizing anti-mouse IL-17 A antibody(Ab)-treated mice compared with vehicle-treated mice.Here,we describe the complicated role of IL-17A-induced progenitor cell recruitment,neutrophil and macrophage infiltration,and vascular endothelial growth factor(VEGF)and IL-6 production,involving corneal fibroblasts and infiltrated macrophages in experimental CRNV.Part ? Intracorneal expression of IL-17 A and its effects on experimental corneal neovascularizationObjective: To explore intracorneal expression of IL-17 A and its receptor and the role of IL-17 A in alkali-induced corneal neovascularization(CRNV).Methods: Induction of CRNV by alkali injury was performed and compared in recombinant mouse IL-17 A protein or anti-mouse IL-17 A neutralizing antibody(Ab)-treated mice.The expression of IL-17 A,IL-17 A receptor(IL-17RA),vascular endothelial growth factor(VEGF),IL-6,IL-8 and monocyte chemoattractant protein-1(MCP-1)in burned corneas or other cells was examined by RT-PCR and western blot.The infiltration of macrophages,neutrophils and c-Kit positive progenitor cells into burned corneas was detected by flow cytometry(FCM).The Raw264.7 cell line and mouse corneal fibroblasts were stimulated by mouse recombinant IL-17 A or anti-mouse IL-17 A neutralizing Ab in vitro,and the expression of IL-6 and VEGF in culture supernatant was determined by ELISA assay.Results: The alkali-induced CRNV peaked 2 weeks after the alkali burn.Compared to the control group,mouse recombinant IL-17 A protein administration significantly increased the amount of corneal neovascularization,and it decreased in mice treated with neutralizing anti-mouse IL-17 A Ab.RT-PCR and western blotting confirmed that the IL-17 A protein up-regulated intracorneal VEGF,IL-6 and phosphorylated focal adhesion kinase(p FAK)expression.FCM revealed that the numbers of intracorneal infiltrated progenitor cells and macrophages were also augmented in IL-17A-treated mice.The ELISA assay showed that IL-17 A markedly induced VEGF and IL-6 expression in the RAW264.7 murine macrophage cell line and in the mouse corneal fibroblasts.Conclusions: IL-17A-treated mice exhibited enhanced alkali-induced CRNV through enhanced intracorneal progenitor cell and inflammatory cell infiltration,and increased VEGF and IL-6 expression by fibroblasts and macrophages.Part ? The effects of IL-17 A on capillary tube formation of human retinal vascular endothelial cellsObjective: To address the effects and mechanism of IL-17 A in capillary tube formation of human retinal vascular endothelial cells(HRECs).Methods: IL-17 A receptor(IL-17RA)expression in human retinal vascular endothelial cells(HRECs)was quantified by reverse transcriptase polymerase chain reaction(RT-PCR)and western blot.The effect of IL-17 A in capillary tube formation and migration of HRECs was detected using three-dimensional matrigel assay and transwell assay respectively in vitro.Cell proliferation of HRECs was examined using cell counting kit(CCK)-8 assay in the presence of different concentrations of IL-17 A.The effect of IL-17 A in HRECs expression of IL-6,VEGF,IL-8 and intercellular cell adhesion molecule(ICAM)-1 was examined using real-time PCR and western blot.Results: RT-PCR and western blot detection revealed IL-17 A receptor(IL-17RA)was expressed in HRECs.The number of intact capillary tubes formed by HRECs in the presence of IL-17 A was remarkably more than that in blank control group.Transwell assay showed a significant increase in the number of migrated HRECs under 100 ng/ml or 500 ng/ml IL-17 A stimulation.Real-time PCR and western blot detection showed that IL-17 A significantly promoted IL-6,VEGF,IL-8 and ICAM-1 expression in HRECs.The proliferation of HRECs in the presence of IL-17 A was promoted in a dosage-dependent manner.Conclusions: IL-17 A can increase HRECs capillary tube formation through promoting HRECs migration,proliferation and expression of IL-6,IL-8,VEGF and ICAM-1.