SHP2 Regulates VE-cadherin Trafficking And Endothelial Inteerity

Rationale:The endothelium of blood vessels forms a selective barrier,regulating the extravasation of plasma components and cells.Endothelial cell junctions,especially adherens junctions of the utmost importance,contribute fundamentally to the regulation and maintenance of this barrier function and endothelial integrity.Vascular endothelial(VE)-cadherin trafficking is known to play a central role in vascular development,endothelial integrity and homeostasis.VE-cadherin membrane localization and stability is finely tuned by reversible phosphorylation.Protein tyrosine phosphatase SHP2 has been shown to be involved in regulating endothelial barrier function in vitro;however,the physiological mechanisms remain greatly unknown in vivo.Objective and Methods:Our research explored the underlying molecular mechanisms of endothelial SHP2 utilizing human umbilical vein endothelial cells(HUVECs)in vitro.In HUVECs,we constructed lenti virus to knockdown SHP2,performing further approaches including immunofluorescence assay,immunoblotting,immunoprecipitation,permeability assay and biotin-labeling to clarify VE-cadherin membrane localization and the stability of VE-cadherin complex.Also,we utilized HE-staining,immunofluorescence assays,whole-mount immunohistochemistry staining,X-gal staining after paraffin methods,together with electron microscopy to explore in vivo functions of SHP2 on endothelium-specific Shp2 KO mice.Results:SHP2 knockdown or catalytic inhibition in HUVECs disrupted the junctional localization of VE-cadherin and increased endothelial barrier permeability.Loss of SHP2 augmented the GTPase activity of ARF1,thereby promoting VE-cadherin internalization.Either knockdown ARF1 or using SecinH3 to inhibit cytohesins,which are guanine nucleotide-exchange factors for ARF1,markedly attenuated VE-cadherin endocytosis in SHP2-deficient endothelial cells.SHP2 knockdown increased MET(HGFR,Hepatocyte Growth Factor Receptor)expression and phosphorylation level.Knockdown MET or inhibition of MET markedly prevented SHP2 deficiency-induced ARF1 activation and VE-cadherin endocytosis in endothelial cells,suggesting MET activation is required for ARF1 activation and VE-cadherin internalization induced by SHP2 deficiency.Mice(Shp2f/f:Tie2-Cre,or Shp2f/f:Cdh5-Cre)with selective depletion of Shp2 in the endothelia showed embryonic lethality characterized by massive hemorrhage and discontinuous junctional VE-cadherin localization.Inhibitors for ARF1 or MET used in pregnant mice rescued the vascular leakage in endothelial Shp2 knockout embryos,extending embryonic survival.Conclusions:1.SHP2 deficiency leads to increase of endothelial permeability.2.Loss of SHP2 potentiates VE-cadherin internalization.3.ARF1 mediates SHP2 depletion-induced endothelial barrier disruption.4.MET mediates SHP2 depletion-induced ARF1 activation.5.Mice with selective depletion of Shp2 in the endothelia showed embryonic lethality characterized by massive hemorrhage and discontinuous junctional VE-cadherin localization.6.Inhibition of either ARF1 or MET rescues vascular leakage in endothelial Shp2 KO embryos and extend embryonic survival.