EphrinB2 Signaling Mediates PTH-induced Endothelial-to-mesenchymal Transition (EndMT) In Aortic Endothelial Cells

Background: Experimental studies found that over-physiological dose of PTH could induce End MT,but its underlying mechanisms are still largely unknown.Recent studies have shown that PTH up-regulated the expression of Ephrin B2.And activated Ephrin B2/FAK pathway mediated epithelial-mesenchymal transition,but whether Ephrin B2/FAK pathway is involved in mediating End MT has not yet been devised.Methods: In the first part: Effect of Ephrin B2 on End MT induced by PTH in HAECs.(1)HAECs were divided into four groups: control group with PTH concentration of 0 mol/L,experimental group with PTH concentration of 10-9mol/L,experimental group with PTH concentration of 10-8mol/L,experimental group with PTH concentration of 10-7mol/L.HAECs incubated with different concentration of PTH for 48 h.The m RNA and protein of Ephrin B2 and End MT makers were measured by real-time PCR,WB and immunofluorescence.The phosphorylation level of Ephrin B2 was tested by WB.(2)HAECs were divided into two groups:PTH+ nontargeting si RNA(vehicle,vehi)as control group,PTH+Ephrin B2 si RNA as experimental group.The m RNA and protein of End MT-related makers were measured by real-time PCR,WB.In the second part :whether Ephrin B2 mediates End MT induced by PTH by activating FAK signal.(1)HAECs were divided into four groups: control group with PTH concentration of 0 mol/L,experimental group with PTH concentration of 10-7mol/L,PTH+ nontargeting si RNA(vehicle,vehi)as experimental group,PTH+Ephrin B2 si RNA as experimental group.FAK was tested by WB.The phosphorylation level of FAK was tested by WB and immunofluorescence after incubation of 48 h.(2)HAECs were divided into two groups: PTH+ nontargeting si RNA(vehicle,vehi)as control group,PTH+FAK si RNA as experimental group.The m RNA and protein of End MT-related makers were measured by real-time PCR,WB.Results:In the first part,our data showed that PTH down-regulated expressions of CD31 and VE-Cadherin and up-regulated expression of a-SMA,FSP1 and ephrin B2 in a dose-dependent manner.In contrast to PTH +Vehi group,the treatment of Ephrin B2 si RNA resulted in a down-regulation of MSC-related markers and a up-regulation of EC-related markers.In the second part,we found that PTH increased the expression phosphorylated-FAK.The treatment of Ephrin B2 si RNA abolished the expression of phosphorylated-FAK.Compared with PTH +Vehi group,the changes above were attenuated by the treatment of FAK si RNA.Conclusion: Ephrin B2/FAK pathway was involved in End MT induced by PTH.Inhibition of this pathway by Ephrin B2 si RNA or FAK si RNA could partially block the expression of End MT related markers.