The Effect Of Connexin43 On Corneal Neovascularization And Its Mechanism

Background:Corneal neovascularization(CNV)is a concomitant process of corneal injury healing.Neovascularization grows into the clear cornea,bringing nutrients to the cornea as well as a large number of inflammatory cells,and the prolonged presence of neovascularization can lead to loss of corneal transparency and thus affect vision.Currently,the treatment of CNV is based on corticosteroids,but these drugs have many side effects,such as persistent corneal epithelial healing,corneal collagen lysis and increased intraocular pressure.The researchers have used anti-retinal neovascularization drugs(Ranibizumab,Compazepine)in the treatment of corneal neovascularization and obtained some efficacy,but the results of the study showed that these drugs have some damage to the corneal nerve,and their are no clear clinical guidelines to include these drugs in the treatment of corneal neovascularization.Therefore,there has been a search for a safe and effective anti-corneal neovascularization drug.Gap junction protein 43(Cx43)is a transmembrane protein highly expressed in vascular endothelial cells.The classical function of gap junction proteins is to mediate communication between adjacent cells to promote tissue and organ homeostasis.Researchers have identified a variety of gap junction protein inhibitors and called them gap junction protein mimetic peptides,such as gap19,gap26,gap27,P5,L2,etc.These gap junction protein mimetic peptides inhibit gap junction protein function by specifically binding to the intracellular or extracellular loops of gap junction proteins.Studies have shown that the use of these inhibitors is effective in reducing the growth of neovascularization in various organs in vivo,but the mechanism of action has not been fully elucidated.Purpose:1.To clarify the changes of Cx43 expression during the growth of corneal neovascularization and to explore the effects of Cx43 inhibition on corneal neovascularization and mechanisms.2.To identify the changes of Cx43 expression in human umbilical vein endothelial cells(HUVEC)after VEGFA stimulation and to investigate the effects of Cx43 inhibition on HUVEC proliferation,tube formation,migration and mechanism.3.To clarify the role of VE-cadherin and β-catenin in Cx43-mediated angiogenesis and to explore the specific mechanisms involved.Methods:1.This study performed animal experiments using C57BL/6J mice to induce corneal neovascularization by sutures and subconjunctival injection of Cx43 inhibitor gap26 to observe corneal neovascularization growth in mice.Corneal specimens from the experiments were subjected to whole cornea fluorescence staining using CD31 antibody to observe angiogenesis further.Corneal sections were prepared for HE staining to observe vascular and inflammatory cell infiltration in corneal tissue.Immunofluorescence staining of corneal sections was performed to observe the protein expression of Cx43,VEGFR2,VEGFR1,and CD31 in corneal tissues.Protein detection of Cx43,VEGFR1,VEGFR2,p VEGFR2,ERK,and p ERK in corneal tissue was performed using western blot(WB).Cx43,VEGFR1 and VEGFR2 gene expression in corneal tissues was examined using realtime fluorescence quantitative PCR(RT-q PCR).Primary human umbilical vein endothelial cells(HUVEC)were extracted and characterized.The effect of Cx43 inhibitor gap26 on HUVEC function under VEGFA stimulation was observed by cell proliferation assay,scratch assay and tube formation assay.The effect of gap26 on neovascular sprouts was observed by mouse aortic ring germination assay.The expression of Cx43,VEGFR1,VEGFR2,p VEGFR2,ERK and p ERK in HUVEC was analyzed by cellular immunofluorescence,WB,RT-q PCR.2.WB and RT-q PCR were performed to detect changes in VE-cadherin and β-catenin expression in the cornea and HUVEC following the use of the Cx43 inhibitor gap26.WB and RT-q PCR examined the effect of si RNA silencing of VE-cadherin and β-catenin in HUVEC.The effects of silencing VE-cadherin or β-catenin on HUVEC were observed by proliferation assay,scratch assay and tube formation assay in the presence of VEGFA and gap26.WB,RT-q PCR and cellular immunofluorescence assays were performed to detect the effects of silencing VE-cadherin or β-catenin in HUVEC in the presence of VEGFA and gap26 on alterations in the expression of VE-cadherin,β-catenin,VEGFR2,p VEGFR2,ERK and p ERK in HUVEC.Results:1.Cx43 expression is increased during corneal neovascularization growth.According to the mouse corneal neovascularization model experiments results,corneal neovascularization was significantly reduced after subconjunctival administration of gap26 compared to the control group.CD31 whole cornea fluorescence staining also verified this result.The results of HE staining of corneal sections showed that both vascular lumen and inflammatory cell infiltration in the corneal tissue were reduced after administration of the inhibitor compared with the control group.Immunofluorescence staining of corneal tissue sections showed that CD31,VEGFR1 and VEGFR2 expression were reduced in the inhibitor group.Immunoblotting experiments showed decreased expression of VEGFR1,VEGFR2,p VEGFR2 and p ERK.The real-time fluorescence quantitative PCR assay results showed decreased expression of VEGFR1 and VEGFR2.2.Cx43 gene and protein expression were increased in HUVEC under VEGFA stimulation.Inhibition of Cx43 in vitro assays reduced the migration,tube-forming and proliferation ability of VEGFA-stimulated HUVEC.Cellular immunofluorescence and immunoblotting assays showed no change in VEGFR2 expression after using gap26.p VEGFR2,VEGFR1 and p ERK expression were decreased.The real-time fluorescence quantitative PCR assay results showed no change in VEGFR2 expression,but VEGFR1 expression was decreased.3.VE-cadherin and β-catenin expression was reduced under VEGFA-stimulated conditions,and their expression was restored after using gap26.Knockdown of VE-cadherin or β-catenin blocked the inhibitory effects of gap26 on vascular endothelial cell proliferation,tube formation and migration.And knockdown of both genes blocked the inhibitory effect of gap26 on VEGFR2 and ERK phosphorylation.In addition,β-catenin inhibited the VEGFR2-ERK signaling pathway by regulating VE-cadherin,thereby affecting the angiogenic process.Conclusion:1.1.Cx43 expression was increased during corneal neovascularization growth.Cx43 inhibitor gap26 effectively reduced corneal neovascularization and inhibited VEGFR2-ERK signaling pathway in the cornea.Cx43 expression was increased in HUVEC under VEGFA stimulation.2.Cx43 expression was increased in HUVEC under VEGFA stimulation.Cx43 inhibitor gap26 reduced the proliferation,migration and tube-forming function of HUVEC under VEGFA stimulation,inhibited the growth of neovascular sprouts in mouse aortic rings,and suppressed VEGFR2-ERK signaling in HUVEC.3.Cx43 inhibitor gap26 inhibited angiogenesis via the β-catenin-VE-cadherin-VEGFR2-ERK signaling pathway under VEGFA-stimulated conditions.