The Effect Of Rgma On Angiogenesis In Vitro And Related Mechanism

Objective:Acute ischemic stroke is a leading cause of sever cognitive disorder,physical disability and mortality worldwide.The therapeutic methods for ischemic stroke have made enormously progress in revascularization,such as intravenous thrombolysis,intravascular intervention therapy,whereas most patients are not suitable for these effective treatments in terms of the narrow therapeutic time window.Thus,rehabilitation after stroke is more important during the cause of the disease.The concept of neurovascular unit emphasizes that vascular play a critical role in maintaining the structure and function of neurons.Regarding this,novel effective approaches are needed for stroke therapy.Angiogenesis is an important strategy for post-stroke rehabilitation,because enough perfusion has access to generate more nutrients for neuronal regeneration as well as take away the metabolic waste.Repulsive guidance molecule?RGM?was originally identified as a membrane-bound protein that functions as an axon repellent guidance molecule in chick nervous system.We previously found that RGMa was significantly increased and induced the growth cone collapse in MCAO/reperfusion rat model.Moreover,we found that down-regulating the expression of RGMa can promote the neuronal functional recovery after acute cerebral ischemic.The nervous and vascular system have a remarkably parallel way in organization,axon guidance and the pattern of blood vessels share the same cues and receptors together.Neural guidance factors,such as netrins,semaphorins,ephrins and slits,play a critical role in regulating angiogenesis.We thus speculate that RGMa may be a regulator during angiogenesis.Neugenin,the receptor of RGMa,is also the receptor of netrin-1,netrin-4,which were involved in anti-angiogenesis via binding to neogenin.The axon attractant induced by netrin1 can be converted by the expression of Unc5b on neurons.Unc5b was as an essential co-receptor of neogenin during the inhibition of angiogenesis and phosphorylation of FAK induced by netrin-1 and netrin-4.Unc5b was confirmed as a repulsive factor in endothelial cells during angiogenesis.Therefore,we speculate that the RGMa as a neural guidance factor may also be a regulator in angiogenesis,the involvement of Unc5b may be essential for the function of RGMa in angiogenesis.However,the function of RGMa on angiogenesis remain unclear.In our present study,we aim to explore the role of RGMa in angiogenesis and its mechanism.Methods:Part?:1.Endothelial cells?HUAEC\HUVEC\RBMEC?were treated with recombinant human vascular endothelial growth factor?rVEGF?.Then,western blot was used to evaluate the expression of RGMa protein.qrt-PCR was used to evaluate the level of RGMa mRNA.VEGFA protein level of cell culture supertanant were detected by ELISA kit after endothelial cells were treated with recombinant repulsive guidance molecule a?rRGMa?.2.To determine the optimal concentration of RGMa in this study,scratch assay was used to explore the effect of different concentration of RGMa?500ng/ml,1ug/ml,1.5ug/ml,2ug/ml,3ug/ml?on HUAECs migration.3.CCK-8?Cell counting kit 8?and EdU kit?5-Ethynyl-2'-deoxyuridine kit?were used to evaluate the effect of RGMa on cell proliferation of normal vascular endothelial cells and VEGF-stimulated vascular endothelial cells.4.Scratch assay and transwell assay were used to investigate the effect of RGMa on the tube formation ability of normal vascular endothelial cells and VEGF-stimulated vascular endothelial cells.5.Using Matrigel tube formation to investigate the effect of RGMa on cell migration of normal vascular endothelial cells and VEGF-stimulated vascular endothelial cells.6.Western blot was used to detect the effect of RGMa on phosphorylation of focal adhesion kinase?p-FAK?in normal vascular endothelial cells and VEGF-stimulated vascular endothelial cells.7.FITC conjugated phalloidin and DAPI were used to stain F-actin and nuclear DNA,respectively,to reveal the location of the cytoplasm and microvilli in the HUAECs.To detect the intensity and location of FAK?pY397?,primary antibody of FAK?p Y397?and its second antibody were used to label FAK.Part?:1.Neogenin knockout for HUAECs was done by using CRISPR/Case9knockout kit.HUAECs transferred with the plasmid were sub cultured for7-8 passages,then HUAEC^neo-was established after purifying with puromycin and identifying with western blot.2.Scratch assay and transwell assay were used to investigate the role of neogenin in RGMa inhibition on endothelial cell migration.3.Using Matrigel tube formation to investigate the role of neogenin in the RGMa inhibition on tube formation of endothelial cell.4.Western blot was used to detect the role of neogenin in RGMa dephosphorylation of focal adhesion kinase?p-FAK?in endothelial cells.5.FITC conjugated phalloidin and DAPI were used to stain F-actin and nuclear DNA,respectively,to reveal the reassemble of cytoskeleton and microvilli in the endothelial cells.Part?:1.Unc5b knockout for HUAECs was done by using CRISPR/Case9knockout kit.HUAECs transferred with the plasmid were sub cultured for7-8 passages,then HUAEC^Unc5b-was established after purifying with puromycin and identifying with western blot.2.Scratch assay and transwell assay were used to investigate the role of Unc5b in RGMa inhibition on endothelial cell migration.3.Using Matrigel tube formation to investigate the role of Unc5b in the RGMa inhibition on tube formation of endothelial cell.4.Western blot was used to detect the role of Unc5b in RGMa dephosphorylation of focal adhesion kinase?p-FAK?in endothelial cells.5.FITC conjugated phalloidin and DAPI were used to stain F-actin and nuclear DNA,respectively,to reveal the reassemble of cytoskeleton and microvilli in the endothelial cells.Results:Part?1.Compared with control group,the addition of RGMa significantly suppressed the expression of VEGF in endothelial cells?P<0.01?and cell culture supernatant?P<0.01?.In contrast,the expression of RGMa was significantly increased in endothelial cells treated with VEGF?P<0.05?.RGMa mRNA level was significantly increased in entothelial cells treated with VEGF?P<0.01?.2.Cells migration distance was measured to determine the most effective suppressive concentration of RGMa.HUAEC migration was suppressed by the addition of different concentration of RGMa?500ng/ml,1ug/ml,1.5ug/ml,2ug/ml,2.5ug/ml,3ug/ml?,2ug/ml was considered to be the optimum concentration in this experiment.3.RGMa?2ug/ml?suppressed the proliferation of normal EC?P<0.05?or EC stimulated with VEGF?P<0.001?significantly in both CCK-8 test?P<0.05?and EdU test.4.RGMa?2ug/ml?significantly inhibites cell migration in normal EC?P<0.05?or EC stimulated with VEGF?P<0.001?in both scratch assay and transwell assay.5.The results of Matrigel tube formation showed that RGMa?2ug/ml?significantly inhibite the ability of tube formation?number of tubes,branch number,tube area,tube length?in normal normal EC?P<0.001?or EC stimulated with VEGF?P<0.001?.6.RGMa significantly reduced the level of FAK?pY397?in both normal EC?P<0.05?or EC stimulated with VEGF?P<0.001?,with no difference in total FAK.7.The intensity and distribution of phosphorylated FAK on cell edges in control or VEGF group were both down-regulated by the addition of RGMa,the formation of filopodia and lamellipodium were also attenuated at the same time.Part?:1.The results of immunoblotting showed that neogenin was not expressed in cells transferred with CRISPR/case9 neogenin knockout plasmid,which suggest that stable HUAEC^neo-strain was established.2.The scratch assays and Transwell assays showed that migration distance and number of migration cells have no difference between HUAEC^neo-treated with RGMa?2ug/ml?and HUAEC^neo-without any treatment?P?29?0.05?.But the migration distance and number of migration cells of Sg RNA group treated with RGMa was significantly decreased than SgRNA group?P<0.001?.3.The matrigel tube formation assays showed that cells ability of tube formation?number of tubes,tube area,tube length?has no differences between HUAEC^neo-treated with RGMa?2ug/ml?and HUAEC ^neo-without any treatment?P?29?0.05?.But the tube formation of Sg RNA group treated with RGMa was significantly decreased compare with SgRNA group?P<0.001?.4.Western blot analysis show that the level of FAK?p Y397?has no difference between RGMa?2ug/ml?treated HUAEC^neo-and HUAEC^neo-without treatment?P?29?0.05?.Compare with SgRNA group,the level of FAK?pY397?is ignificantly decreased in Sg RNA group treated with RGMa?P<0.05?.5.Images from cyto-immunofluorescence showed that the inhibition of RGMa on the formation of filopodia and lamellipodium were conversed by neogenin knockout in HUAEC and HUAEC stimulated with VEGF.Part?:1.The results of immunoblotting showed that Unc5b was not expressed in cells transferred with CRISPR/case9 Unc5b knockout plasmid,which suggest that stable HUAEC^Unc5b-strain was established.2.The scratch assays and Transwell assays showed that migration distance and number of migration cells have no difference between HUAEC^Unc5b-treated with RGMa?2ug/ml?and HUAEC^Unc5b-without any treatment?P?29?0.05?.But the migration distance and number of migration cells of Sg RNA group treated with RGMa was significantly decreased than SgRNA group?P<0.001?.3.The matrigel tube formation assays showed that cells ability of tube formation?number of tubes,tube area,tube length?has no differences between HUAEC^Unc5b--treated with RGMa?2ug/ml?and HUAEC^Unc5b-without any treatment?P?29?0.05?.But the tube formation of SgRNA group treated with RGMa was significantly decreased compare with SgRNA group?P<0.001?.4.Western blot analysis show that the level of FAK?p Y397?has no difference between RGMa?2ug/ml?treated HUAEC^Unc5b-and HUAEC^Unc5b-without treatment?P?29?0.05?.Compare with SgRNA group,the level of FAK?pY397?is significantly decreased in SgRNA group treated with RGMa?P<0.05?.5.Images from cyto-immunofluorescence showed that the inhibition of RGMa on the formation of filopodia and lamellipodium were conversed by Unc5b knockout in HUAEC and HUAEC stimulated with VEGF.Conclusions:1.RGMa may be a negative regulator of angiogenesis through suppressing the expression of VEGF;2.RGMa inhibition on the proliferation,migration and tube foration of normal EC and EC stimulated with VEGF;3.RGMa inhibiton on angiogenesis may via the Tyr-397dephosphorylationofFAK throughthe interactionbetween neogenin-RGMa complex and Unc5b.