A Research Into The Effects And Mechanisms Of Basement Membrane Regulating The Nfκb Signaling In Endothelial Cells

Background and Objectives:NF-кB signaling in endothelial cells(EC)has been increasingly recognized as a critical player in the pathogenesis of many inflammation-relevant diseases such as sepsis and atherosclerosis.EC sit atop of a layer of basement membrane(BM)comprised of multiple distinct macromolecules.Although individual component of BM can impact various EC functions,the studies of whether and how BM as a whole regulates EC functions,including NFкB signaling,are still lacking.Based on the in vivo observations that non-inflammatory EC have intact BM while inflammationengaging capillary EC lose BM integrity,we hypothesize that BM as a whole may regulate EC NFкB signaling.Indeed,using ex vivo BM model that preserves BM integrity and authenticity,we have revealed an inhibitory effect of BM on EC NFкB signaling by regulating VE-Cadherin in our preliminary studies.This study aimed to reveal the effects and molecular mechanisms of BM regulating the NFκB signaling in endothelial cells.Method:(1)A peritoneum was obtained from a living rat to prepare a basement membrane model that maintains the integrity and authenticity of the basement membrane.(2)Western blot,RT-PCR,NFкB luciferase reporter gene and other techniques were used to study the differences in the activation of NF κB signaling and related protein expression.(3 The effects of basement membrane integrity on endothelial cell NF κB signaling were investigated by disrupting the integrity of the basement membrane by high-salt elution and activation of leukocyte incubation.(4)The effect of VE-Cadherin-mediated intercellular adhesion on endothelial cell NF κB signaling was investigated by recombinant lentiviral-mediated shRNA silencing of VE-Cadherin expression and different cell culture densities.(5)The luciferase reporter gene and Western Blot were used to study the expression of VE-Cadherin and regulate β-Catenin transcriptional activity and β-Trcp1 expression,thereby regulating I κB expression and NF κB signaling.(6)The effect of VE-Cadherin on the activity of RhoA small GTPase was studied by using GST fusion protein pulldown technique,and then the effect of RhoA activity on NFкB activity was studied by gene point mutation technique.(7)VE-Cadherin-Cre/LoxP technology was used to establish a vascular basement membrane tissue-specific Laminin gamma knockout mouse strain,providing an animal model for studying the effects of vascular basement membrane integrity on NF κB signaling.Result:(1)The omental basement membrane isolated from the body inhibits endothelial cell NF κB signaling;(2)The integrity of the basement membrane is critical for the inhibition of endothelial cell NF κB signaling;(3)The intact basement membrane inhibits NF κB signaling by enhancing endothelial cell VE-Cadherin-mediated intercellular adhesion;(4)VE-Cadherin can regulate I κB protein stability and NF κB signaling by regulating β-catenin transcriptional activity and β-TRCP expression;(5)VE-Cadherin regulates NF κB signaling by regulating RhoA activity.Conclusion:The intact basement membrane inhibits NF κB signaling in endothelial cells in vitro,and this inhibition is achieved by VE-Cadherin-mediated intercellular adhesion,which in turn regulates β-catenin transcriptional activity and RhoA activity.