Effect Of LCN10 On The Endothelial Barrier Integrity In Septic Mice And Potential Mechanism Behind This Effect

BackgroundSepsis is defined as life-threatening multiple organ failure caused by dysregulated immune response to infection.Sepsis continues to be a major challenge for the worldwide healthcare systems due to its high mortality and high therapeutic cost.Despite recent advances in antibiotic therapy and supportive critical care result in the decrease in the mortality,sepsis remains a leading cause of death in intensive care units,and the total number of both incidences and deaths are still increased Notably,patients who survive from sepsis often suffer from long-term physical,mental,and cognitive dysfunctions which significantly and seriously decreased the quality of life.Therefore,up to now,sepsis remains a widely major healthcare problem worldwide.However,its underlying mechanisms are extremely complicated It is regarded as related to several sequential events including systemic inflammation,immune dysfunction,multi-organ damages,and aberrant immune response to pathogens.The exact pathogenesis mechanism of sepsis is still unclear.Therefore,it will be of great clinical interest to better understand the molecular mechanism behind of sepsis.Clinically,we have found that septic patients often develop progressive accumulation of edema fluid in the interstitial spaces,subcutaneous tissue,and body cavities,which suggests the disruption of endothelial integrity and increased vascular permeability in sepsis.Sepsis is well recognized to induce vascular hyperpermeability,which subsequently causes hypotension,heart failure,acute respiratory distress syndrome(ARDS),acute kidney injury(AKI),intestinal dysfunction,etc.The evolution toward multi-organ failure(MOF),which is known as a crucial predictor of survival,directly results from vascular hyperpermeability and microcirculatory dysfunction.Therefore,the vascular endothelial barrier disruption is a main pathophysiological process in sepsis,which resulted multi-organ dysfunctions and lead to high mortality.Given that all blood vessels are lined with endothelial cells,the loss of endothelial integrity accounts for the vascular leakage and tissue edema in sepsis Dysregulation of transendothelial paracellular permeability is the main determinant of sepsis-induced vascular extravasation.This results from the disruption of interendothelial junctions(IEJs)and the disorganization of actin cytoskeleton.The interendothelial junctions(IEJs)play an important role in maintaining the endothelial integrity.IEJs include adheren junctions(AJs)and tight junctions(TJs),which are both composed of multiprotein complexes.AJs are composed of VE-cadherin and catenin while TJs are composed of occludins,claudins,zona occludins(ZO),and junctional adhesion molecules(JAMs).The IEJs molecules can be altered by various factors such as vascular endothelial growth factor(VEGF),oxidative stress,renin-angiotensin system,oxidized low-density lipoprotein,homocysteine,etc.At present,many studies have focused on the mechanism of vascular permeability in sepsis,especially on paracellular pathway.However,due to its complex process and regulators,the specific mechanism remains unclear.Therefore,it is important to understand both cellular and molecular mechanisms behind the endothelial barrier dysfunction.Such studies might lead to new therapies for sepsisLipocalins(LCNs)are a family of evolutionarily conserved small proteins(18-40 kDa).They share an eight-stranded antiparallel ?-sheet structure that possesses a binding pocket,which could bind small hydrophobic ligands(i.e.retinoids,fatty acids,steroids,and odorants)and interact with cell surface receptors.In fact,accumulating evidences have shown that lipocalins play important roles in the regulation of inflammatory responses,cell proliferation/differentiation,and animal behavior.In humans,several LCN family proteins(i.e.,LCN1,LCN2,and H-FABP)have been extensively studied as biomarkers for the diagnosis of various diseases.Regarding the protective effects on inflammation and permeability,most prior studies have focused on increased circulating level of lipocalin-2(LCN2),also referred to as neutrophil gelatinase-associated lipocalin(NGAL).One recent study by Tsalik showed that LCN2 expression was significantly increased in septic patients compared to healthy donors and its expression level was highest in non-survival patients.However,conversely,the plasma level of LCN10 was significantly higher in survival septic patients when comparing to the non-survival group and healthy donors.These pilot clinical data suggested that LCN10 may play a protective role in sepsis.UP to now,only a few studies showed that LCN10 might be related to inflammatory bowel diseases.However,there are no research on the role of LCN10 in sepsis.Therefore,the role of Lipocalin-10(LCN10)in sepsis,especially its effects on vascular permeability,are unknown.ObjectivesThe purpose of this study was to explore whether circulating LCN10 could protect endothelial barrier integrity in sepsis and the underlying mechanismsMethods1.LCN 10 protein expression levels in different tissues especially in total heart tissue under different stimulation were determined by Western Blot.2.LCN 10 mRNA expression levels in total heart tissue,different types of cardiac cells,and mouse cardiac endothelial cells(MCECs)were determined by qPCR.3.MCECs were transfected with siRNA to knock down LCN 10,and,then,treated with lipopolysaccharide(LPS,1 ?g/ml)or tumor necrosis factor ?(TNF-?,10ng/ml).Endothelial permeability was monitored by determining values of trans-endothelial electrical resistance(TEER)and fluorescein isothiocyanate-dextran(FITC-dextran)/Evans blue dye(EB)-albumin flux.4.In vivo,sepsis model was induced in both wild-type(WT)and LCN 10-knock out(KO)mice by intraperitoneal(IP)injection of LPS(10mg/kg body weight)or cecal ligation and puncture(CLP).After 24 h,the mice were intravenously administered 200 ?l of 0.5%Evans blue dye in PBS.One hour later,organs were harvested.Evans blue dye was quantified by measuring the optical density at 620nm.Frozen sections(7?m in thickness)were observed under a confocal microscope LSM 710.Vascular leakage corresponding to the amount of dye in the extravascular compartment was analyzed with Image J software5.Using WT and KO sepsis models induced by LPS,both animal survival and organ damage were assessed at indicated time points.6.MCECs were transfected with Ad.LCN10,and.then,treated with LPS or TNF-?.The monolayer permeability was monitored at different time points.7.Treating MCECs with recombinant LCN10(rLCN10),then the monolayer permeability was measured by the three methods mentioned above8.Mechanistically,RNA-seq was used to identify the genes in MCECs which were significantly changed by the overexpression of LCN10.Among these genes,the vascular permeability related genes were further confirmed by qPCR.9.The target genes(SSH1 and LRP2)were selected out and further confirmed by Western Blot.These genes were silenced by siRNA transfection to further confirm the protective effects of LCN10 on endothelial integrity.10.Using immunofluorescence staining technique,the location and distribution of F-actin and ZO-1 protein were observed under confocal microscopy.11.GraphPad Prism 7.0(GraphPad Software,San Diego,CA,USA)was utilized for statistical analysis.Data were presented as means ± SEM/SD.Significance was determined by Student's t test and 1-or 2-way ANOVA to determine differences within groups where appropriate.Survival was presented as Kaplan-Meier curves and differences were analyzed by the log-rank test in GraphPad Prism software.p<0.05 was considered statistically significant.Results1.According to our western blot results,LCN10 presented higher expression level in heart,spleen and blood.Considering that this study focused on the vascular permeability,we chose heart as our target organ.The LCN10 protein level in heart was firstly elevated,and then decreased in both LPS and CLP models.2.Similarly,the mRNA was also firstly increased,and then decreased in the heart after LPS injection or CLP surgery.Compared to other cells isolated from heart(cardiomyocytes,fibroblasts),only endothelial cells were found to be able to express more LCN10 after LPS or CLP stimulation.Similarly,LCN10 mRNA peaked at 6h in MCECs after LPS or TNF-? stimulation.3.In vitro,MCECs were transfected with siRNA to knock down LCN10.Three different methods including TEER,FITC-dextran and EB-BSA flux were used to determine the permeability of MCECs upon LPS or TNF-? challenge.The data showed that the loss of LCN10 aggravated the endotoxin induced hyperpermeability of MCEC monolayer.4.In vitro,compared with WT mice,LCN10-KO mice showed increased extravasation of the EB dye in heart,aorta,lung,intestine,and stomach in response to LPS treatment and CLP surgery.Furthermore,our confocal microscope observation confirmed the significant increases in EB fluorescence intensity in heart,aorta,intestine,and stomach.5.In vivo,LCN 10 deficiency significantly increased multiple organ damages and mice mortality during sepsis.Moreover,compared to WT mice,LCN10-KO mice exhibited more severe edema in lung and intestine and more protein exudation in BALF,associated with worse myocardial contractile function(lower EF%and FS%)20h after LPS injection.6.In vivo,LCN 10 gain of function by transfecting MCECs with Ad.LCN 10 significantly attenuated endotoxin-induced MCEC monolayer damage without affecting the viability of MCECs,which indicated the protective role LCN 10 in MCEC monolayer integrity.7.In vitro experiment also showed that administration of recombinant LCN 10(rLCN10)protected the endothelial barrier integrity of MCECs upon endotoxin challenge using three different methods mentioned above.8.Mechanically,using RNA-Seq method,we found SSH1,LRP2,and several other genes that involved in the cytoskeleton and cell junction pathways were significantly upregulated,which were also confirmed by qPCR.9.Western Blot analysis showed that the increase of LCN 10 was accompanied by increased SSH1 and decreased P-Cofilin(increased dephosphorylation)whereas no change in total Cofilin protein,which resulted in the decrease of P-Cofilin/Cofilin ratio.When SSH1 was silenced,there was no change in P-Cofilin and P-Cofilin/Cofilin ratio with the overexpression of LCN10.These findings indicated that LCN10 might protect endothelial integrity through SSH1.To further confirm,the protective effects of LCN10 on endothelial integrity disappeared when SSH1 was silenced.To investigate how LCN10 upregulated the expression of SSH1,we assumed that LCN10 upregulated the expression of SSH1 through binding with LRP2 according to our RNA-Seq result.Consistently,LCN10-induced SSH1 upregulation disappeared when LRP2 was knocked down by transfected MCECs with siLRP2.Meanwhile,the protective effects of LCN10 on endothelial integrity disappeared when LRP2 was silenced.All of these data support our hypothesis that LCN10-induced SSH1 upregulation is dependent on the LRP2 signaling pathway.10.Confocal microscope images showed that LCN 10 overexpression reorganized F-actin from stress fibers to peripheral cortical actin(cytoskeleton)after LPS stimulation,which,in turn,stabilized tight junction molecules(ZO-1)compared to contol group.The protective role of LCN10 disappeared when SSH1 was silencedConclusionLCN10 activates SSH1/Cofilin signaling pathway via binding to LRP2 receptor,reorganizes F-actin and stabilized ZO-1 which,in turn,protects vascular integrity during sepsis.Our study uncovers the protective role of LCN 10 in maintaining the vascular integrity and presents a novel molecular mechanism in the pathogenesis of sepsis,which suggests that LCN10 may regarded as a potential future agent for protection against sepsis.