Function And Mechanism Of Receptor Tyrosine Kinase TIE In Vascular Development And Maintenance

The arteriovenous differentiation is a key event in the development of cardiovascular system in mammals.Vascular endothelial growth factor receptor-2(VEGFR)mediated signaling pathway plays an important role in angiogenesis and arterial differentiation.In contrast,knowledge on venogenesis is still limited.The TIE(Tyrosine kinase with immunoglobulin-like and EGF-like domains)receptors include two members Tie1 and Tie2.While it is well recognized that they are important regulators of vascular maturation and remodeling,mechanisms underlying their functions in vascular development require further investigation.In addition,lymphatic vessels originate from cardinal veins during embryonic development in mammals.Previous studies from our research group and others showed that Tiel was necessary for the formation and remodeling of lymphatic vessel network.However,it is not clear whether Tie2 is directly involved in regulating lymphatic vessel development.Objective:To explore the role of Tiel and Tie2 during postnatal vascular development,and the role of Tie2 in lymphatic vessel growth.Methods:In this study,conditional knockout mice targeting Tiel and Tie2,in combination with transgenic mice with ubiquitous and endothelial cell specific expression of CreERT2 recombinase were used to analyze their effects on angiogenesis and lymphatic vessel growth at embryonic and postnatal stages.Results:1.We have found that induced deletion of Tie1 from the 1st to 4th day after birth led to the reduction of retinal vascular area at P7(the first layer)and also the retinal angiogenic sprouting at deep layers(P9-P11).At the same time,Tiel deficiency induced abnormal vascular proliferation at the venous side at P9-P11.However,the phenotype was partly compensated at 3 weeks after birth.2.The previous work of our research group showed that Tie2 knockout inhibited the angiogenesis process of retina after birth.In this study,we induced Tie2 knockout on the 1st to 4th days after birth and characterized the vascular phenotypes in details.(1)It was found that Tie2 knockout mice had abnormal vein morphology of retinal vascular network at P9-P11.Tie2 deletion led to vein degeneration and haemangioma-like vascular tufts formation at 3 weeks of age.The vascular abnormality was more severe than that of Tiel knockout mice.The venous marker EphB4 and pericyte cell marker NG2 were used to find out that the vascular tufts had lumens and they were covered by pericyte cells.Induction of Tie2 knockout at various postnatal stages(including days 5-8 and 11-16)also led to venous defects.(2)In adult mice,Tie2 knockout resulted in venous tortuosity.This suggests that Tie2 is not only required for vein development,but also is crucial for the maintenance of veins.(3)Subsequent analysis revealed that Tie2 deletion led to the decrease of the mRNA levels of APJ and EphB4,two vein specific markers in lung and retina.Interestingly,although there was no significant change of the transcript level of COUP-TFII,the key transcription factor for vein differentiation,its protein level was dramatically suppressed.Biochemical studies by other researchers in our group showed that Tie2 was essential for the specification and maintenance of vein via the Akt mediated regulation of COUP-TFII protein stability.3.Using double knockout mouse models,we showed that Tie1 deficiency(from the 1st to 4th day after birth)combined with Tie2 heterozygous mutation resulted in the reduction of retinal vascular area.At the age of 3 weeks after birth,we found that vein degeneration was accompanied by haemangioma-like angiogenesis.These results indicate that Tie2 heterozygous mutation worsened the vascular phenotype caused by Tiel knockout,thus implying the synergistic effect of Tiel and Tie2 on postnatal vascular growth.4.In this study,Tie2 deficiency was induced in lymphatic endothelial cells using tamoxifen in Tie2-/iLECKO mouse model at embryonic stage(E10.5).Tie2 deficiency led to the hemorrhage and edema in the back skin of the embryo.However,Tie2 deletion at embryonic stage(E12.5)produced little if any effect on lymphangiogenesis.We further showed that Proxl-CreERT2 mediates Tie2 knockout on veins in Tie2-/iLECKO mouse models.Immunostaining for red blood cell marker Terl 19 showed that Tie2-/iKECKO mice had vascular leakage,suggesting that lymphatic abnormality in the mutant mice may be due to the blood vascular defects.Conclusion:1.Tiel deficiency leads to the delayed retinal angiogenesis after birth,and the vascular defects was partly recovered at 3 weeks after birth2.Tie2 plays an important role in promoting postnatal angiogenesis,venous formation and maintenance.3.Tiel and Tie2 interact synergistically on postnatal vascular growth.4.Abnormal blood vascular development caused by Tie2 deficiency may account for the defects with lymphangiogenesis in mutant mice with Tie2 deletion in lymphatic endothelial cells.