The PKC?-P120ctn-NF-?B Signaling Pathway Is Involved In The Study Of The Mechanism Of Vascular Endothelial Injury Under The Action Of Fluid Shear Force

Background and Objective:Intracranial aneurysm is an acute cerebrovascular disease that poses a serious threat to human life and health.The pathogenesis of intracranial aneurysms has not been fully elucidated.A large number of studies have shown that vascular endothelial cell injury is the initial link in the formation of intracranial aneurysms.The potential mechanisms of vascular endothelial cell injury include hemodynamic stress,decreased nitric oxide synthase activity,oxidative stress,estrogen imbalance,and endothelial cell-endothelial cell connection damage.However,how hemodynamics causes vascular endothelial injury needs to be elaborated.P120 ctn plays an important role in vascular endothelial connectivity.The highly conserved juxta-membrane domain of P120 ctn binds to VE-cadherin to maintain the stability of endothelial cell adhesion.P120 ctn is one of the natural immune response regulation factor closely associated with inflammation factor NF-B activation.PKC alpha is closely related to vascular diseases and can regulate the expression and phosphorylation of P120 ctn.The purpose of this subject is to address whether PKC alpha-P120ctn-NF-B signal pathways are involved in the vascular endothelial damage caused by fluid shear stress or not.Methods:We designed a new “T” chamber hydrodynamics system to generate a stable fluid shear stress for human umbilical vein endothelial cells(HUVECs).The shear stress and shear stress gradient in the “T” chamber hydrodynamics system were calculated by hydrodynamics simulation software.According to the different shear stress and shear stress gradient on the slide,the slide is divided into three regions: ?zone: stagnation zone,which is the fluid shear stress as low as normal;?zone: acceleration zone,high fluid shear force and high shear stress gradient;? zone:Zone of the high fluid shear stress and high shear stress gradient from negative to zero.Then the human umbilical vein endothelial cells in ?zone were taken for the research.Human umbilical vein endothelial cells were cultured in vitro and transfected with RNAiPKC? recombinant lentivirus or P120 ctn serine mutation plasmid at 879,respectively.The endothelial cells were successively implanted on a glass slide and placed the “T” chamber hydrodynamics system.The 500ml/min flow was loaded to impact the endothelial cells on the slide for 0 h and 6 h.The density,morphology and PKC??P120ctn ?P-P120ctnS879?VE-cadherin?NF-B expression of vascular endothelial cells were detected by inverted microscope and Western Blot.Result:1.Effects of fluid shear stress on the morphology and density of endothelial cells: ? zone: significantly lower endothelial cells density,increased gap between endothelial cells and endothelial cell diversification were observed by inverted microscope;? zone: the density of endothelial cells and morphology were unchanged;however,slightly higher endothelial cell density was found in ? zone.2.Effects of fluid shear stress on the expression of phosphorylated PKC?,P120 ctn,phosphorylated P120ctnS879,VE-cadherin and NF-B in endothelial cells: Compared with controlled cells,the phosphorylation of PKC? expression increased significantly(P < 0.05),the expression of P120 ctn and VE-cadherin decreased significantly(P<0.01),Phosphorylation of P120ctnS879 increased significantly(P < 0.05),the expression of NF-B increased significantly(P<0.01)in endothelial cells under fluid shear stress at 6 hours.3.Effects of PKC? on the expression of endothelial cells P120 ctn,phosphorylated P120ctnS879,VE-cadherin and NF-B: Under the fluid shear stress,the expression of P120 ctn and VE-cadherin in the PKC? knockdown endothelial cells did not change significantly(P>0.05);Phosphorylated P120ctnS879 expression was almost undetectable;NF-B expression was significantly reduced(P<0.01).4.Effects of P120 ctn S879 on the expression of NF-B in endothelial cells: In vascular endothelial cells mutated at P120 ctn serine 879,the phosphorylation of PKC? expression increased significantly in endothelial cells under fluid shear stress at 6 hours(P<0.05);the expression of P120 ctn and VE-cadherin decreased significantly(P<0.01);There was no significant change in NF-B expression(P>0.05),however,compared with normal endothelial cells,NF-B expression was significantly decreased(P<0.05).Conclusion:The PKC alpha-P120ctn-NF-B signaling pathways involved in vascular endothelial damage under the fluid shear stress,the involved mechanisms may include:1.PKC alpha inhibited the expression of P120 ctn and VE-cadherin,leading to the damage of vascular endothelial intercelluar junctions;2.PKC alpha induced phosphorylation P120ctnS879 increased NF-B expression and accerelate the inflammatory response.