Heparin Binding Protein Affects Endothelial Cell Permeability Through Transforming Growth Factor Beta Receptor 2

Objective:At the early stage of inflammation,polymorphonuclear neutrophils（PMNs）are the first cells recruited to the inflammatory region of tissue.The activation,chemotaxis and migration of PMN cause vascular endothelial barrier damage,which is closely related to some proteins secreted by PMN.Heparin binding protein（HBP）,as an important granule protein secreted by PMN,participates in the pathophysiological process of diseases.It can affect endothelial cells（EC）by increasing vascular permeability,leading to vascular leakage.Under serious circumstances,it may cause tissue edema,even hypotension and organ dysfunction without proper clinical intervention.In recent years,it has been found that it is related to the severity of septic shock and can be used as a biomarker of severe sepsis.Heparin binding protein（HBP）,also known as azurocidin.It is also named as cationic antimicrobial protein 37 or CAP37 because of its relative molecular weight of 37000 and bactericidal activity.HBP has various functions,which can not only induce vascular leakage,edema formation and chemotaxis of leukocytes,but also promote the repairing process of tissues and cells.A few hours before the development of hypotension or organ dysfunction in patients with sepsis,the increase of plasma HBP level can be observed and HBP can be used as a powerful predictor of disease progression to severe sepsis within 72 hours.HBP changes vascular permeability mainly by affecting endothelial barrier.Vascular EC is the target cell after HBP is released.HBP directly or indirectly destroys endothelial barrier components,such as intercellular junctions and extracellular glycocalyx,affecting signal transduction in endothelial cells,such as endothelial cytoskeleton remodeling and eventually endothelial barrier dysfunction.Vascular barrier dysfunction and inflammatory response promote excessive leakage of macromolecular substances such as proteins out of circulation,resulting in circulatory dysfunction and organ dysfunction.Because there are many heparin-like substances within the glycocalyx structure on the surface of endothelial cells,it is generally believed that polysaccharide coating is the binding site of HBP on the surface of endothelial cells.However,whether there are receptors on the surface of endothelial cells remains unknown.Transforming growth factor-β（TGF-β）is an evolutionarily conserved polypeptide family,which regulates embryogenesis and homeostasis of tissues.TGF-β family members are also involved in the pathophysiological mechanism of many diseases.It is reported that the elevated level of TGF beta is associated with the pathogenesis of infectious diseases,and its receptors play a very important role.TGF-β binds to receptor kinases and transmits phosphorylation signals to downstream mediators（mainly SMADs protein）,and oligomerization-dependent signals to ubiquitin ligase and intracellular protein kinases.The interaction between SMADs and other signaling proteins mediates regulatory signals that control expression of target genes,RNA processing at multiple levels,mRNA translation and protein regulation.Upon ligand binding with TGF beta receptor Ⅱ,on one hand,through the classical pathway,activated SMAD2/3 forms a complex with the SMAD4 and then translocate to the nucleus where it binds to high-affinity DNA binding transcription factors（TF）and chromatin remodeling（CR）in order to positively or negatively regulate the transcription of target genes so as to play the role of damage repair and promote epithelial mesenchymal transition（EMT）;on the other hand,through SMAD-independent pathways,receptor Ⅱ can directly regulate Rho-ROCK pathway,eventually results in actin cytoskeleton reorganization and formation of stress fibers.It also affects the cellular junction,which affects the permeability of endothelial cells.Our studies showed that the phosphorylation level of SMAD2/3(SMAD2-Ser^465/467,SMAD3-Ser^423/425)is increased under the stimulation of HBP.Therefore,it is speculated that TGF beta receptor Ⅱ may be a potential protein receptor of HBP on the surface of endothelial cells.Methods:1.In vitro experiment:Human Umbilical Vein Endothelial Cells（HUVECs）were used as the experimental objects in this study.HUVEC was cultured and stimulated by PBS and HBP（10ug/ml）for 0h,24h,48h,and 72h.Cell viability was observed by CCK-8.2.In vitro experiment:HUVECs was cultured and stimulated by HBP（10ug/ml）for 0h,3h,6h,and 9h.SMAD2/3 and phosph-SMAD2/3 protein levels（SMAD2-Ser465/467,SMAD3-Ser423/425）were detected by West blot.3.In vitro experiment:HUVECs were selected and divided into 4 groups:the control group,the HBP group,the TGFBR2siRNA group and the TGFBR2siRNA+HBP group.We then identified the most efficacious TGFBR2siRNA fragment from three fragments.We transfected HUVECs with control siRNA and siRNA fragment and then stimulated the HUVECs with HBP.SMAD2/3 and phosph-SMAD2/3 protein levels were detected by West blot.4.In vitro experiment:HUVECs were selected and divided into 4 groups:the control group,the HBP group,the halofuginone group and the halofuginone+HBP group.SMAD2/3 and phosph-SMAD2/3 protein levels were detected by West blot.5.In vitro experiment:Co-IP experiment,confocal immunofluorescence microscopy,and GST pull-down test to observe the binding of HBP to TGFBR2 and its binding region.6.In vitro experiment:HUVECs were selected and divided into 4 groups:the control group,the HBP group,the TGFBR2siRNA group and the TGFBR2siRNA+HBP group.Nucleocytoplasmic Shuttling of SMADs were detected by West blot and immunofluorescence.7.In vitro experiment:HUVECs were grown on 0.4um pore transwell filters until confluent.FITC-dextran was applied apically at 1 mg/ml.One hour later,a sample of the medium was removed from the lower chamber to measure permeability.Monolayers were either left untreated（control）,treatment with TGFBR2siRNA,stimulated with 10 ug/ml HBP,or treatment with TGFBR2siRNA and then HBP.Samples were taken from the lower chamber for fluorescence measurements and compared to the control monolayers.All groups are treated in triplicate.8.In vitro experiment:HUVECs were selected and divided into 4 groups:the control group,the HBP group,the TGFBR2siRNA group and the TGFBR2siRNA+HBP group.TJP1,OCLN,and Rho protein levels were detected by West blot.The relative mRNA levels of the RhoA and RhoB genes were determined by reverse transcription qPCR.Actin cytoskeleton was stained using Rhodamin-phalloidin and was observed using fluorescence microscopy.TJP1 was observed using fluorescence microscopy.9.In vivo experiment:12 male C57BL/6 mice were randomly divided into 4 groups,which were the control group,the HBP group,the TGFBR2siRNA group and the TGFBR2siRNA+HBP group,and each group had 3 mice.Weight 25±g.The HBP group model was established by bolus injection of 150ug HBP intravenously,and the TGFBR2siRNA group was established by bolus injection of 20 nmol TGFBR2siRNA intravenously,and the samples of plasma and lung were collected.The pulmonary wet/dry ratio（W/D）were calculated.TJP1,OCLN,Rho,SMAD2/3,and phosphor-SMAD2/3（SMAD2-Ser465/467,SMAD3-Ser423/425）protein levels were detected by West blot.The relative mRNA levels of the RhoA and RhoB genes were determined by reverse transcription qPCR.Lung histology was observed,and lung injury was scored.Blood-gas barrier was observed by transmission electron microscope and scanning electron microscope.Results:1.Viability of HUVECs cells increased with the extension of the incubation time of the HBP stimulation.2.Under the stimulation of HBP（10ug/ml）,we found that the expression of phosph-SMAD2/3 increased in HUVECs.In contrast,HBP stimulation did not affect SMAD2/3 expression.3.We treated HUVECs with halofuginone（10ng/ml）and then stimulated the HUVECs with HBP.Western blot showed that HBP could induce SMAD2/3 phosphorylation.However,halofuginone treatment abolished the phosphorylation of endogenous SMAD2/3 in HUVECs.4.We transfected HUVECs with control siRNA and siRNA fragment 2 and then stimulated the HUVECs with HBP.The expression of TGFBR2 was decreased in TGFBR2 knockdown cells.Western blot showed that HBP could induce SMAD2/3 phosphorylation.However,TGFBR2 knockdown abolished the phosphorylation of endogenous SMAD2/3 in HUVECs.5.We confirmed by endogenous coimmunoprecipitation and confocal immunofluorescence microscopy that TGFBR2 is a novel receptor that interacts with HBP before the activation of TGF-β pathway.At the same time,we determine the combination area between TGFBR2 and HBP.6.Western blot showed and immunofluorescence showed Nuclear translocation of SMAD complexes under the stimulation of HBP.7.HBP treatment for 1 hour increased the permeability of HUVECs and TGFBR2siRNA treatment could partly block the effect.8.HBP treatment induced the expression of Rho protein and stress fiber formation but had no effect on ZO-1 expression.TGFBR2siRNA treatment can partly block TGF-β signaling,compromised the effect of HBP on actin cytoskeleton reorganization and Rho protein expression.The relative mRNA levels of the RhoA and RhoB genes were also increased by HBP treatment.9.In vivo experiment:HBP treatment increased the expression level of Rho,phosph-SMAD2/3 protein.However,HBP strongly downregulated the expression of occludin.Besides,the relative mRNA levels of the RhoA and RhoB genes are also increased by HBP treatment.TGFBR2siRNA treatment can partly block the effect of HBP.In HBP group,lung W/D ratio of lung weight increased significantly than the control group（5.1833±0.23214 vs.4.0224±0.18561,p<0.01）.In TGFBR2siRNA+HBP group,W/D ratio of lung weight was lower than that of HBP group（4.1976±0.04377 vs.5.1833±0.23214,p<0.01）,but still higher than the control group（4.1976±0.04377 vs.8.8798±0.04787,p<0.01）.Lung histopathology showed that the alveoli in control group were opened uniformly,alveolar wall and septa were thin and clear.In the HBP group,the alveolar collapsed.The alveolar wall and septa are damaged significantly,with congestion and inflammatory cell infiltration.In TGFBR2siRNA+HBP group,the alveolar septa swelling,and inflammatory cells infiltration were attenuated compared with HBP group.Scanning electron microscopy showed that HBP caused protein deposited and disappearance of alveoli.The area of failure of the blood-gas barrier could be found.Transmitting electron microscopy showed that the alveolar epithelial and microvascular endothelial cells in control group had no swelling or dissolution,furthermore,there were no large gap between cells.However,in HBP group,the alveolar epithelial membrane was dissolved,cell boundary was not clear.Basal membrane was disrupted and swollen endothelial cells could be found.The alveolar epithelial injury and vascular endothelial cell swelling were attenuated in TGFBR2siRNA+HBP group.Conclusion:1.HBP can activate TGF-β pathway by binding to TGFBR2,upregulate Rho expression level,induce actin cytoskeleton reorganization and formation of stress fibers.Besides,ZO-1 was partially redistributed under the treatment of HBP.2.HBP can increase endothelial permeability by binding to TGFBR2.In vivo experiment,HBP induces acute lung injury and TGFBR2siRNA treatment could partly block the effect.