Study On The Role Of EMC3 And LMBR1L In Retinal Angiogenesis

A sophisticated vessel network gradually forms to provide oxygen and nutrients to meet the high metabolic demands of retina during the eye development.Angiogenesis is a sophisticated process that is precisely regulated by the interaction between various cell populations in the retina,and disorders of these processes may lead to defects in vascular development.Retinal angiogenesis defects lead to disruption of the oxygen and nutrients transportation in the retina,resulting in metabolic imbalance,which causes a series of human retinal vascular disease.Patients with retinal vascular ophthalmopathy exhibit varying degrees of phenotypes,including non-cascular area around the retina,leakage,neovascularization,retinal stretching and fold,and even progress to blindness in severe cases.There is no effective treatment for these diseases at present.It is of great clinical significance to identify new pathogenic genes and clarify mechanisms under these genes to develop prenatal screening and treatment methods for these diseases.In this dissertation,the role of EMC3 and LMBR1 L in retina angiogenesis was investigated.In vivo and in vitro experiments were applied to clarify vascular defect phenotypes in loss of EMC3 and LMBR1 L.The mechanisms underlining retina angiogenesis defects caused by EMC3 and LMBR1 L defecency were studied at both transcriptional and protein level,which providing theoretical basis for the diagnosis and treatment of retinal vascular ophthalmopathy.The main research contents of this dissertation are as follows:1.The role of EMC3 in retinal angiogenesis.Endoplasmic reticulum membrane protein complex(EMC)is involved in the biogenesis of membrane protein and various cellular processes.Studies have shown that EMC1 regulates the development of neural crest in Xenopus by affecting Frizzled expression,which inhibiting Wnt signaling pathway.As a core component of the EMC,EMC3 may also be involved in the regulation of the Wnt signaling pathway.However,the role of EMC3 in retinal angiogenesis has not been reported.In this study,an endothelial cell-specific Emc3 knockout mouse model was generated to explore the role of EMC3 in retinal angiogenesis.Emc3 knockout mice exhibited delayed retinal vascular progression,leakage,decreased tip cell and filopodia numbers,and impaired proliferation ability of endothelial cells.Mechanically,the expression of multiple Wnt related genes were both down-regulated in EMC3 knockdown Human retinal microvascular endothelial cells(HRECs)at both transcriptional level and protein level.Meanwhile,in vitro luciferase assay also showed that the knockdown of EMC3 inhibited the activity of Wnt signaling pathway.Li Cl treatment can rescue the phenotypes caused by EMC3 deletion to some extent.In summary,the experimental results suggested that the deletion of EMC3 inhibit the activity of Wnt signaling pathway,which ultimately leads to defects in retinal angiogenesis.2.The role of LMBR1 L in retinal angiogenesis.Previous study has shown that loss of LMBR1L(Limb Development Membrane Protein 1 Like)overactivates Wnt signaling pathway,leading to impaired lymphogenesis in mice.Previous studies have pointed out that abnormal activation of Wnt signaling pathway can also lead to retinal angiogenesis defects.Therefore,Lmbr1 l globally knockout mouse model was constructed to further explore the effect of Wnt pathway on retinal angiogenesis.The results showed that deletion of Lmbr1 l lead to delayed vascular progression,increased vessel density and delayed regression of hyloaid.And luciferase assay showed that the activity of Wnt signaling pathway was actived in loss of LMBR1 L,which was consistent with previous study that LMBR1 L is a negative regulator of Wnt signaling pathway.Mechanically,in vivo and in vitro studies indicated that LMBR1 L depletion activates Wnt signaling pathway by stabilizing Wnt receptors(FZD4 and LRP5)and by disrupting the degradation complex,which resulting in the accumulation of ?-catenin in the cytoplasm.In addition,loss of LMBR1 L also lead to the enhancement of the adherin junctions between vascular endothelial cells,which may be the reason of delayed vascular progression with increased density.Treatment with AR-A014418,the inhibitor of the Wnt signaling pathway,can rescue the phenotype caused by LMBR1 L deletion to a certain extent.The sum of these experimental results suggested that deletion of LMBR1 L overactivates Wnt signaling pathway,which eventually lead to excessive proliferation and migration defects of retinal endothelial cells.In conclusion,this dissertation elucidated the important role of EMC3 and LMBR1 L in retinal angiogenesis and further confirmed that overactivation or inactivation of the Wnt signaling pathway can lead to defects in retinal vascular development.It provides a theoretical basis for the diagnosis of retinal vascular ophthalmopathy and the development of new therapeutic methods.