The Role And Mechanism Of IQGAP1 On Hyperpermeability Of Pulmonary Microvascular Endothelial Cells Injury Induced By LPS

BackgroundPulmonary microvascular endothelial cells(PMVECs)lined to the endovascular surface are important to the vascular barrier,which can selectively regulate the exchange of substances,such as intracellular and extracellular cytokines,and inflammatory mediators;PMVECs also play a key role in maintaining the dynamic balance of the internal environment;furthermore,the changes of PMVECs functions caused by intracellular and extracellular signal stimulation are the key factors affecting the changes of microvascular permeability.Vascular leakage caused by increased permeability,because of the structure damage of microvascular endothelial cells,is still one of the the main mechanism contributing to many inflammatory diseases.Acute respiratory distress syndrome(ARDS),with a mortality rate of nearly 45% in severe patients,is characterized by increased permeability of pulmonary microvascular endothelial cells caused by over activation of inflammation.Vascular endothelial cells are connected by various means such as tight junction,adhesive junction,and gap junction.Adhesive junction is the main connection mode of vascular endothelial cells,which is closely related to vascular endothelial permeability.Adhesive junction is also involved in establishing and maintaining cells Intercellular adhesion,skeletal remodeling,intracellular signal transduction,and post-transcriptional regulation.The IQGAPs(IQ-domain GTPase-activating proteins,IQGAPs)is a family of eukaryotic proteins containing multiple domains.IQGAPs,as cytoskeleton-binding proteins,plays important roles in regulating biological processes such as cytokinesis,cell migration,tissue remodeling,cell adhesion,and cell signaling.The IQGAPs protein family includes three members: IQGAP1,IQGAP2,and IQGAP3.IQGAP1 is widely distributed,with the exception of brain tissue,which is distributed in all tissues and organs,and has a high expression level in the lungs.IQGAP1 is an actin-regulated protein composed of 1657 amino acids,which plays an important role in maintaining the normal adhesive function of the cytoskeleton and connective function between cells.Among the many factors affecting microvascular endothelial cells,IQGAP1 is considered to be one of important factors in maintaining the permeability and integrity of microvascular endothelial cells.In recent years,the absence of IQGAP1 has been confirmed to affect the permeability of vascular endothelial cells and lead to vascular leakage,but the specific mechanism,and whether it also exists in the change of the permeability of pulmonary microvascular endothelial cells under the induction of inflammation are not clear.Nuclear factor kappa B(NF-?B)is a group of inducible transcription factors composed of polypeptide members of the Rel / NF-?B family.Under the stimulation of trauma,stress,microbial infection and other external substances,its activity can increase abnormally,resulting in the excessive release of a large number of proinflammatory cytokines.In ARDS,the NF-?B signal transduction pathway is activated,and it also participates in the expression of a variety of inflammatory factors,which regulates the occurrence,development and prognosis of ARDS.Lipopolysaccharide(LPS),the main component of the cell wall of Gram-negative bacteria,can not only increase the permeability of vascular endothelial cells,but also activates some intracellular signal transduction pathways and destroys cells connections,thereby increasing cell permeability.A large number of clinical data also confirmed that LPS is involved in the development of ARDS.However,NF-?B,as a control point for the large-scale expression of inflammatory response genes,is activated in LPS induced pulmonary microvascular endothelial cell permeability injury,but whether it can activate the expression of IQGAP1 and its mechanism of action has not been interpreted.ObjectiveWe focused on IQGAP1 expressed in RPMVECs.We wanted to analyzed the molecular regulatory mechanism and the signal pathway of the the permeability of microvascular endothelial cells,under the influence of different interference factors;In the process of the LPS-induced increase in monolayer permeability of RPMVECs,we wanted to study its mechanism and how LPS regulates IQGAP1 through the NF-?B signaling pathway;and our aim is to provide molecular biological experimental evidence for the study of the mechanism of pulmonary microvascular endothelial cell monolayer permeability change diseases such as ARDS.Methods1.Primary PMVECs were isolated,cultured and identified in vitro.2.The expression and distribution of IQGAPs subtypes on PMVECs wereidentified.3.IQGAP1 expression in RPMVECs induced by LPS was detected.4.Low IQGAP1 and over expressed,stably infected RPMVECs were constructedin vitro and identified.5.We successfully establish RPMVECs monolayer cell model in vitro usingtranswell chamber;we also used different concentrations of LPS in different periodsto induce intervention in RPMVECs monolayer fusion cells,monitor changes intrans-endothelial cell resistance(TER),and analyze LPS and its resistance Changesin PMVECs single-layer permeability.6.Under the condition of LPS intervention,we observed and analyzed the changes of skeletal protein F-actin on RPMVECs with normal,low and over expression of IQGAP1.7.With RNA-Sequencing,we analyzed the effect of IQGAP1 deletion on mRNA expression changes in RPMVECs,and screened for meaningful differentiate expressed genes(DEGs).Through mRNA function enrichment analysis such as GO and KEGG,we explored the correlation with IQGAP1 expression Key genes and signaling pathways involved in the biological processes of PMVECs.8.With Western blot,expression of IQGAP1 and NF-?B P65 after si RNAmediated inhibition of NF-?B interference,and under the induction of LPS and NF-?B inhibitor CAPE,we studied if the expression of NF-?B P65 and IQGAP1 correlated with each other under low,normal and over expression condition.9.A monolayer cell model of RPMVECs with normal expression,low expression and over-expression of IQGAP1 was constructed in vitro with a transwell chamber.According to the experimental design,some experimental groups were given pretreatment with NF-?B inhibitor CPAE,followed by LPS-induced intervention,different TER changes were monitored,and the effect on PMVECs monolayer permeability was analyzed.Results 1.The primary RPMVECs were successfully isolated and cultured in vitro and confirmed by morphology,VIII and CD34 immunostaining.2.There were three subtypes of IQGAPs on RPMVECs,which had been confirmed by immunofluorescence,Western blot,q RT-PCR.IQGAP1 had the highest expression content,mainly located at the plasma membrane and cell junctions.3.10 ?g/ml LPS induced RPMVECs for 12 hours,and IQGAP1 expression was confirmed to be the highest.4.The lentiviral infection was used to construct stable IQGAP1 low-and overexpressing RPMVECs,and the construction was successfully confirmed by GFP observation,Western blot,and q RT-PCR.Based on the above results and analysis of infection efficiency,the appropriate stable transformation Strains were subjected to subsequent experiments.5.The TER value of PMVECs detected by 10 ?g/ml LPS concentration was the smallest,and the permeability of PMVECs monolayer was the largest.The PMVECs of rat were treated with 10 ?g/ml LPS at 9 different time points.Permeability was the largest;in summary,PMVECs treated with 10 ?g/ml LPS for 12 hours in rats had the lowest TER value,but the largest single-layer PMVECs permeability.6.In the PMVECs of normal control group,F-actin was mainly distributed on the inner side of the cell membrane,around the cells,and was filamentous.It was close to the cell junction and was arranged in an orderly manner on the inside of the cell membrane and formed a nuclear skeleton.When 10 ?g / ml LPS was induced for 12 hours,F-actin aggregation and remodeling in RPMVECs could be observed,and some F-actin crude stress fibers were formed.F-actin expression levels and morphological changes of RPMVECs in the lentivirus infection control group and the down-regulated IQGAP1 expression group were not significant,and there was no significant difference to the normal control group.However,when LPS was induced to stimulate the low-expression IQGAP1 group,it was found that F-actin formation was significantly reduced,and no significant stress fiber formation was observed.7.We used RNA-Sequencing to analyze the mRNAs related to the low expression of IQGAP1 on PMVECs.The results were as follows:(1)A total of 1,216 DEGs were identified(p-value ?0.05,fold change(29)1.5 and the average FPKM in the group ? 0.5),including 665 up-regulated mRNAs and 551 down-regulated mRNAs,and verified by q RT-PCR.The two results were consistent.Compared with the control,knockdown of IQGAP1 resulted in the upregulation of 33 pathways,while a total of 73 pathways were downregulated.(2)GO function enrichment analysis: Up-regulation of key genes was mostly related to DNA replication and cell cycle,such as: Mcm3,Cdc7;and downregulation of DEGs was mostly related to cytoskeleton,cell connection,and extracellular matrix,such as ICAM-1,IL-6.(3)The up-regulated pathways were mainly involved in apoptosis,protein synthesis,and certain endothelial repair processes,such as p53 and AMPK signal pathways.However,low expression of IQGAP1 resulted in down-regulated cell proliferation and vascular development.The inhibition of IQGAP1 expression affected the integrity of the microvascular endothelial cell barrier,indicating that IQGAP1 may contributed to endothelial cell barrier repair or injury.KEGG enrichment analysis: However,the most significant differential signal down-regulation pathways caused by IQGAP1 knockdown were mostly related to inflammation: such as NF-?B,TNF and IL-17 signal pathways.8.After si NF-?B interference,lentivirus infection induced low and overexpression of IQGAP1,and with the induction of LPS and CAPE,Western blot and double luciferase gene detection confirmed that LPS activates the NF-?B signal pathway to up-regulate the expression of IQGAP1,and IQGAP1 can feedback regulate NF-?B and reduce the damage to PMVECs by stimulation..9.Each group of PMVECs with different expressions of IQGAP1 stimulated with LPS stimulated at 10 ?g / ml showed that TER began to decrease significantly at 30 minutes and decreased to a maximum value at 12 hours.Inhibition of IQGAP1 expression could reduce the decrease of TER induced by LPS.In the IQGAP 1 overexpression group,the decrease of TER was more significant under LPS induction.However,the resistance of the experimental group treated with NF-?B inhibitor CAPE before LPS stimulation was significantly higher than that of the LPS group treated without CAPE.In summary,CAPE could reduce the decrease of transcellular monolayer resistance caused by LPS,and the inhibition of IQGAP1 expression can be further reduced on this basis but still lower than the normal group.Conclusion 1.Three subtypes of IQGAPs family,IQGAP1,IQGAP2,and IQGAP3,exist on PMVECs.But IQGAP1 has the highest expression content and is mainly distributed at the plasma membrane and cell junctions.2.When PMVECs were treated with 10?g/ml LPS for 12 hours,the IQGAP1 expression was most significantly increased,the TER value was the smallest,and the PMVECs single-layer permeability was the largest in a concentration-and timedependent manner.3.LPS may induce an increase in PMVECs skeletal reorganization,which partially depend on the presence of IQGAP1.4.RNA-Sequencing analysis showed that inhibition of IQGAP1 expression would affect the integrity of the microvascular endothelial cell barrier.IQGAP1 may play a key role in endothelial cell barrier repair and injury.5.LPS activates the NF-?B signal pathway to up-regulate the expression of IQGAP1;IQGAP1 can feedback regulate NF-?B and reduce the damage to PMVECs by stimulation.6.Inhibition of IQGAP1 expression and inhibition of NF-?B signaling pathway can alleviate the increase in monolayer permeability of PMVECs induced by LPS.