The Mechanism Of Endoglin Regulating The Vasculogenesis Of Zebrafish

Hereditary hemorrhagic telangiectasia(HHT),also known as Lang-Owe syndrome,is a rare autosomal dominant hereditary disease characterized by abnormal angiogenesis in skin,mucosa and organs.HHT can be divided into five main types according to the different mutation genes.Endoglin is the pathogenic gene of type I HHT,accounting for about 50%of the disease.In this study,zebrafish model and HHT patient samples were used to illustrate the mechanism of Endoglin regulating vascular development and the downstream effect gene.The experimental methods used in the research include,1.Zebrafish was selected as in vivo model in this study.Morpholino was injected to inhibit the expression of Endoglin.Real-time PCR(RT-PCR),in situ hybridization,and fluorescence activate sorting(FACS)were used to study the mechanism of Endoglin regulating vascular development 2.Endothelial cells(ECs)derived from a HHT patient induced pluripotent stem cells(iPSCs)were used as in vitro model.Western Blot and Tube formation were used to analyze the possibility of Endoglin downstream effector gene as a therapeutic target of HHT.The research is mainly divided into six parts:1.Conservative Analysis of Different Vertebrates Endoglin:By amino acid sequence alignment of Endoglin between zebrafish and vertebrate orthologs,we found that the sequence of the transmembrane region(TM)is similar in different vertebrates.Moreover,Phylogenetic analysis provided evidence that vertebrate Endoglin originates from a common ancestor.2.Spatial and temporal expression pattern of Endoglin during the zebrafish early embryonic development:The total expression and the specific location of Endoglin at different dvelopmental stages was detected by RT-PCR and in situ hybridization respectively.The results showed that Endoglin was highly expressed in the zygote stage and decreased gradually.In the gastrointestinal stage,the expression of Endoglin increased gradually,and finally Endoglin was highly expressed in the vascular region.3.Endoglin knockdown affects the vascular development:Tg(fli1a:EGFP)yl transgenic line was used to show the vasculature through the different developmental stages by detecting the endothelial cells(ECs)expressing green fluorescence.The blood vessels developed abnormally with some IS Vs breaking in the 24 hpf eng-MO group.Flil is a well known endothelial cell marker in zebrafish and FACS analysis showed that the ratio of GFP+ECs was decreased.4.The effect of Endoglin knockdown on the expression of endothelial markers:RT-PCR and in situ hybridization were used to detect the expression of endothelial markers(kdrl cdh5,hey2,lmo2 and pu.1).We found that the expression of Endoglin decreased significantly after Endoglin knockdown.5.Potential downstream effective factors of Endoglin:Bmper was selected as a potential downstream modulator of Endoglin using RNA sequencing.We found that the expression pattern of Bmper and Endoglin were highly similar during embryogenesis,and the overexpression of Bmper rescued the defective blood vessels in the Endoglin knockdown group.6.The possibility of Bmper as a therapeutic target for HHT therapy:Western Blot and Tube formation experiments were used to detect the expression of ID1 and angiogenesis in ECs from HHT patients respectively.It was found that the expression of ID1 and angiogenesis were significantly increased after BMPER stimulation.In conclusion,the research summarizes the pattern of Endoglin regulating early vascular development which would help to understand the pathogenesis of HHT in future work.In addition,the research identified the potential downstream effector-Bmper and analyzed the possibility of Bmper as a potential target for HHT therapy which brings the hope for the treatment of HHT.