Research On Related Genes Of Eye Disease With Abnormal Retinal Angiogenesis

Angiogenesis is the process of forming new blood vessels through the sprouting of existing blood vessels,in which both physiological and pathological blood vessels are produced after embryonic development has been completed.Physiological angiogenesis plays a critical role in wound healing,tissue growth,and regeneration.The abnormal development of physiological blood vessels and the formation of pathological blood vessels are often related to the occurrence of many diseases.The abnormal development of retinal blood vessels,which serve as the channels for oxygen and nutrient supply in the retina,will lead to many eye diseases,such as familial exudative vitreoretinopathy,diabetic retinopathy,retinopathy of prematurity,and so on.However,there are few effective diagnosis and treatment methods for these diseases.Finding genes that affect vascular development and clarifying the mechanisms of physiological vascular development and pathological angiogenesis are of great significance for the diagnosis and treatment of these diseases.In this dissertation,the role of three genes in retinal angiogenesis and their regulatory mechanism were studied,which provided a reference for the treatment of vascular blinding eye disease.The main contents of this dissertation are as follows:1.The role and mechanism of TMEM30 A in retinal angiogenesis were explored for the first time.Experiment data indicate that knockdown of TMEM30 A in primary human retinal endothelial cells led to abnormal tube formation.The specific knockout of TMEM30 A in mice vascular endothelial cells resulted in retinal surface vascular development retardation,decreased density,vascular terminal enlargement,decreased number of apical endothelial cells,and decreased number of filopodia.Besides,knockout of TMEM30 A in mice bodies also resulted in damage to the integrity of the blood vessel walls.However,knocking out Tmem30 a after the development of the retinal blood vessels in mice will not affect the maintenance of the vascular network.Furthermore,analysis at the transcriptome level revealed that the expression of genes related to the cell cycle was reduced when TMEM30 A was knockout in primary human retinal endothelial cells and mice,while western blot analysis found that the phosphorylation level of related proteins involved in VEGF-induced signaling was also significantly reduced.In summary,these results above suggest that TMEM30 A plays a role in angiogenesis and maintenance of vascular barrier integrity by regulating VEGF signaling to control angiogenesis.2.The role of TWIST1 gene in pathological neovascularization of the retina was explored.Experiments showed that overexpression of Twist in vascular endothelial cells led to the growth of superficial vessels delayed,abnormal proliferation of the front end,and vascular leakage occurred.Further immunofluorescence staining found that the mice vascular endothelial cell proliferation rate was significantly accelerated,the expression of the glial fibrillary acidic protein in the mice retina increased,and the vascular endothelial growth factor secreted abnormally.The staining of vascular endothelial cell nucleus showed that the vascular endothelial cell nucleus was mostly spherical and did not point to the avascular area.The above results indicate that Twist1 overexpression leads to abnormal activation of astrocytes,and the secretion of vascular endothelial growth factor increases,which leads to abnormal blood vessel proliferation.Meanwhile,the loss of vascular endothelial cell polarity caused by Twist1 overexpression will lead to vascular development delay.Overexpression of Twist1 did not affect the maintenance of vascular network after the development of retinal vessels in mice.However,when the retinal vascular was mature,overexpression of Twist1 does not affect the maintenance of the vascular network.Besides,in the global Twist1 knockin mouse,the eyeballs of mice became smaller,and the blood vessels in the retinal surface developed retinal retardation,decreased density,and local leakage,which were different from the phenotypes of Twist1 overexpression in vascular endothelial cells,indicating that Twist1 also plays a role in other cells.Mechanistically,in vitro luciferase assay showed that overexpression of Twist1 resulted in increased activity of the Norrin/?-catenin signaling pathway.In summary,these results suggest that Twist1 may cause pathologic neovascularization of the retina through abnormal activation of the Norrin/?-catenin signal.3.A new candidate gene DLG1 for familial exudative vitreoretinopathy was identified and was explored.For the first time,DLG1 was associated with FEVR,further clarifying the mechanism of DLG1 affecting angiogenesis.Results showed that the missense mutation S598 G of DLG1 prevents DLG1 from activating ?-catenin signal transmission normally.Knockdown of DLG1 reduced the tube formation of human retinal vascular endothelial cells in vitro.Western blot experiment proved that the phosphorylation level of VEGFR2 protein decreased after DLG1 deletion.In conclusion,these studies above elucidate the role of TMEM30 a in retinal angiogenesis and its molecular mechanism,confirm the role of TWIST1 in pathological angiogenesis,and identify a new candidate gene DLG1 for familial exudative vitreoretinopathy,which provides a new therapeutic target for the treatment of vascular ophthalmopathy and other pathological angiogenic diseases.