| Background:Retinal astrocytes construct special structure for stalk endothelial cells?EC?to proliferate or cling and for tip EC sprout out in early developmental stages.EC secret important factors to regulate the maturation and development of astrocytes.In addition,it is essential for the stability of retinal vascular endothelium-glial unit,which is an important part of the development of the whole retinal network.As an important nuclear transcription factor downstream of HIPPO pathway,Yes-associated protein?YAP?has been proved to be an important regulator of retinal vascular development in early stage,and the absence of endothelial-specific YAP will lead to the stagnation of vascular development.Current studies of our group show that retinal blood vessel-origin YAP deficiency not only slowed the stagnation of endothelial cells,but also affected the maturation of astrocyte network.Due to the importance of leukaemia inhibitor factor and its receptor?LIF-LIFR?in endothelium-glial units,that is,endothelial cells secrete LIF and act on the LIFR of astrocytes,which affects the maturation and differentiation process of astrocytes,but the specific mechanism is still unclear.When YAP was deleted in EC,LIF was correspondingly declined,thus,we speculate YAP may play an important role in this process.By generating a Tek promotor driven YAP conditional knock out mice(Tek-Cre;YAPf/f)and a endothelial cells-astrocytes co-culture system,we investigated the molecular mechanism of YAP governing astrocyte maturation by regulating endothelial LIF secretion.Besides,the significance of this study can also be applied to some retinal vascular model such as OIR,which is similar to retinopathy of prematurity?ROP?.The unstable development of astrocyte was subject to the neovascular in the OIR disease model.However,rescue of YAP provides the correction of a series of retina problems such as bleeding and leakage caused by the excessive proliferation of endothelial cells.In addition,it benefits retinal astrocyte maturation and the stability of endothelium-glia networks.Our study may shed light on retinal neovascularization and provide a new therapeutic target for further clinical usage.Purpose:1.To testify the location and expression of YAP in the retina at the development stage;Endothelial cell-specific YAP knockout mice have stagnation of vascularization accompany by astrocyte development inhibition.2.Confirm that the astrocyte development retardation caused by YAP deficiency of endothelial cells is due to the increase of immature astrocytes,and the reason for the imbalance between immature and mature astrocytes is the decreased secretion of LIF from EC.3.Through the co-culture system of endothelial cells and astrocytes in vitro,it is confirmed that the increase or decrease of YAP in endothelial cells change the secretion of LIF,thus affecting the expression of astrocytes.4.To establish the OIR model to confirm that the deficiency of YAP in endothelial cells increase the neovascular which is associated with the abnormal morphology of astrocytes and the increased immature astrocytes.5.OIR mice are treated with YAP agonist?Y27632?,which provide a normal morphology of astrocytes and the increase number of the mature cells accompany with decreased neovascular.6.To testify that the changes in astrocyte maturation and differentiation caused by YAP knockout mice in OIR model are also related to the LIF secretion of endothelial cells.7.The in vitro co-culture model confirm the correlation of YAP-LIF-LIFR axle.Method:1.Mice bearing floxed alleles of YAP were crossed with mice expressing a Cre recombinase driven by the endothelial Tek promoter?Tek-Cre?to finally obtain the following three genotypes:YAPf/f,Tek-Cre;YAPf/w and Tek-Cre;and YAPf/f.In all experiments,control animals(YAPf/f mice)were littermates not expressing Cre.Male and female mice were used for the analysis.2.E16,P0,P3,P7,P14 and P21 were selected as important point of early development,and the retinas were measured for fluorescence immunostaining in wholemount retinas,immunofluorescence staining for frozen section of retinas,and retinal protein expression were detected by Westernblot.3.OIR model is useful for investigating the dynamic pathological process of OIR,which includes initial microvascular degeneration?P7–P12?,vascular regrowth?P12–P17?,and neovessel formation?P14–P17?.Briefly,P7 mice pups were kept with nursing mothers exposed to 75%oxygen until P12,followed by a return to room air?21%oxygen?to induce retinal NV until P17.Age-matched YAPf/f mice were kept in room air throughout postnatal development to serve as the normoxic controls.The experimental contents included:immunofluorescence staining of the whole retinas,immunofluorescence staining of frozen retinas,and Westernblot detection of protein expression.4.Co-cultured astrocytes and microvascular endothelial cells in vitro?hmec-1?.Astrocytes in the cerebral cortex were extracted from mice within 24 hours after birth and cultured on 24-well plates for 3-5 days.Microvascular endothelial cell lines were cultured in6-well plates.When the fusion degree of astrocytes reached about 40%-50%,the glia cells were placed into 6-well plates containing endothelial cell lines to achieve co-culture.5.In vitro co-culture experiments were conducted to detect the expression of specific markers in endothelial cells and astrocytes by immunofluorescence staining and Westernblot.The relationship between YAP and the LIF-LIFR axis was verified by two different sets of experiments:endothelial cells knock down YAP and add LIF protein,and astrocyte LIFR receptor knock down after endothelial cells overexpress YAP.Result:1.The results of WB and co-localization by immunofluorescence at each time point of retinal development showed that YAP was located in the endothelial cells of retinal RGC layer,and a small number of astrocytes expressed YAP at the same time.Meanwhile,the results of fluorescence immunostaining in wholemount retinas showed that endothelial cells and astrocytes in the retina were indeed codependent.2.Compared with WT mice,the endothelial cells specificity knockout mice homozygous and heterozygous appear different degree of endothelial cells:growth stagnation,decreasing tip cells,lumen sparse,accompany with developmental delay of mature astrocytes,disorder structure,and the mature and immature astrocytes cells ratio changed.3.Co-culture of astrocytes and endothelial cells in vitro showed that the increased or decreased expression of YAP in endothelial cells could change the expression of astrocyte specific markers at the same time.In addition,LIF,the intermediate molecule secreted from the endothelial cells that regulates the maturation of astrocytes,was also closely related to YAP at the developmental stage.4.OIR model was established,and it was found that compared with WT mice,YAP specific knockout mice showed significantly increased central avascular area at P12 and increased neovascularization at P17,accompanied by astrocyte fibrosis.In vitro mice were injected with YAP agonist which could effectively increase central blood vessels and decreased the neovascularization.At the same time,the ratio of immature astrocytes to mature astrocytes was reduced and the structure of astrocytes network became order.5.The morphological changes in the retina of mice in the OIR model were also related to the regulation of LIF secretion by YAP in endothelial cells.After injection of YAP agonist,LIF expression were increased,and the mature astrocytes increased while immature astrocytes decreased.6.Co-culture of astrocytes and endothelial cells in vitro revealed the correlation between the YAP-LIF-LIFR axis by changing the YAP expression of endothelial cells and then exogenous addition of LIF or blocking LIFR to detect the expression of astrocytes specific maker.Conclusion:This study not only revealed that YAP in endothelial cells plays an important role in retinal vascular development,but also plays a regulatory role in the stability of vascular-neural units by regulating the LIF-LIFR axis.This is an important finding in the development of retinal blood vessels and astrocytes,also can be applied to the retina vascular disease model.YAP maintained the stable development of mature astrocytes in OIR and changed the abnormal proliferation of neovascularization,reduced blood vessel leakage at the same time.Different from the inhibition of vascular growth factors point as previous reported,the treatment of regulating vascular-neural units provided a new theoretical basis in retinal neovascular diseases. |
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