Protective Effects And Mechanism Of Targeting Endothelial Cells With Anticoagulants On Acute Lung Injury Of Mice

BackgroundAcute lung injury(ALI)is a clinical syndrome characterized with diffuse interstitial and non-cardiogenic pulmonary edema resulting in severe hypoxemia and difficulty breathing,even further developing into acute respiratory distress syndrome(ARDS).A great part of pathogenesis of ALI is on basis of endothelial injury which leads to increased vascular permeability and inflammatory pulmonary infiltrates.As ALI is one of the most frequent causes of morbidity and mortality in the critically ill patients,researchers are constitute searching for novel therapeutic targets.In ALI or ARDS,there are mutual amplifying effects between inflammation and coagulation.Abundant evidence has proved that coagulopathy is a significant event in systemic inflammation,ALI or ARDS.In some previous studies,researchers infused endotoxin or bacteria into the vascular space to activate inflammation.In succession,intravascular coagulation was activated by increasing tissue factor(TF)activity.In patients with ARDS,elevated markers of coagulating proteins,including thrombin,soluble TF,Ⅶa,and TF-dependent factor Ⅹ(FⅩ)were detected in bronchoalveolar lavage fluid(BALF),and the TF-FⅦa pathway mediated the coagulation disturbance.TF binds and activates FⅦ,by which downstream coagulation cascades are activated.Under normal states,there are just small amounts of TF in circulating blood,but in pathophysiologic conditions,TF can be expressed on the surface of monocytes and endothelial cells.The TF expression can be stimulated by endotoxin and proinflammatory cytokines,for instance tumor necrosis factor(TNF),interleukin-1(IL-1),and IL-6.Activation of coagulation is a physiologic response for inflammation and contributes to contain inflammation.However,lung injury may be aggravated by excessive pulmonary inflammation-induced coagulopathy,which thus contributes to ALI.Coagulation brings up proteases which activates intracellular signaling pathways by binding with specific cell receptors.Coagulation proteases(such as TF-FVIIa complex,FXa,and thrombin)may generate a powerful proinflammatory effect by activating corresponding receptors on the surface of endothelial cells,monocytes and leukocytes,of which the most important receptors are protease-activated receptors(PARs).By these mechanisms,coagulation products promote the generation of proinflammatory cytokines,for example IL-6,chemokines,and vascular endothelial growth factor(VEGF),which enhance vascular permeability,extravasation of leukocytes,and lung injury.Meanwhile,the increased coagulation activity cannot be sufficiently balanced by the natural anticoagulants.Compared with those in plasma from patients with ARDS,activated protein C(APC)levels were reduced and soluble levels of thrombomodulin are higher in pulmonary edema fluid.Antithrombin(AT)levels were reduced in BALF of healthy volunteers who accepted locally challenged with either lipoteichoic acid(LTA)or lipopolysaccharide(LPS)as well as in patients with inhalation trauma because of burn.Similarly,the TF procoagulant activity was unable to be sufficiently blocked by intra-alveolar TFPI because of truncation and inactivation.Therapeutic interventions aimed at coagulation blockade are considered to be theoretically useful for ALI/ARDS by dampening the inflammatory response.In rats,LPS-induced lung injury was attenuated by intratracheal administration of APC through inhibiting accumulation of leukocytes.In a rat model of Streptococcus pneumoniae pneumonia,AT treatment generated significant lung-protective effects.TFPI reduced the mortality of Escherichia coli septic shock in baboons.But in all of current preclinical studies,agents that aimed at initiation of coagulation by TF and natural inhibitors of proteases were applied through intravein,intratrachea or nebulization.The disadvantages of these administration strategies involve in bioavailability,sufficiency of local effect in injury sites,and systemic side effects like bleeding.Therefore,we attempt to explore the effects of targeted therapy of anticoagulation on mouse model of acute lung injury.We used two strains of transgenic mice,CD31-TFPI-Tg(XCON6)and CD31-Hir-Tg(XCON7)expressing respectively a membrane tethered human tissue factor pathway inhibitor(hTFPI)and hirudin fusion proteins on CD31-positive cells,including vascular endothelial cells,monocytes and platelets.Part Ⅰ Increased expression levels of correlative proteins of TF-PARspathway on mouse lung vascular endothelial cells induced by LPSObjective:To identify the expression levels of correlative proteins of TF-PARs pathway on mouse lung microvascular endothelial cells induced by LPS.Methods:Primary mouse lung microvascular endothelial cells(MLECs)were isolated and identified from neonatal wildtype(WT)mice and routinely grown in culture medium(Vasculife EnGS-Mv complete kit,10%FCS).Cell cultures were maintained in the exponential growth phase and used between the passages 1 and 2.Cells(>80%confluent in culture dishes)were intervened with 50ug/ml LPS from Pseudomonas aeruginosa or with isometric PBS as the control.Cells were cultured for 24 hours before assaying for tissue factor(TF),protease activated receptor 1(PAR1)and PAR2.In vivo experiments,WT mice were randomly treated as follows:intratracheally instilled with lipopolysaccharide(LPS)from Pseudomonas aeruginosa(10mg/Kg)to induce ALI(WT-LPS group)and intratracheally instilled with normal saline as a control(WT-CON).24 hours later,cell suspension were obtained from Ⅳ-type collagenase digested lung tissues to test the expression levels of tissue factor(TF),protease activated receptor 1(PAR1)and PAR2.Results:The analysis of Flow Cytometry showed that the expression levels of TF,PAR1 and PAR2 were significantly up-regulated on MLECs intervened with LPS and on microvascular endothelial cells of lung tissues of LPS-induced ALI mice.Conclusion:LPS induces the increased expression levels of correlative proteins of TF-PARs pathway on mouse lung microvascular endothelial cells.Part Ⅱ Targeted Anticoagulant Fusion Proteins protect LPS-induced Acute Lung InjuryObjective:To explore whether targeted anticoagulant fusion proteins can protect LPS-induced ALI.Methods:We used two strains of transgenic mice,CD31-TFPI-Tg(XCON6)and CD31-Hir-Tg(XCON7)expressing respectively a membranetetheredhuman tissue factor pathway inhibitor(hTFPI)and hirudin fusion proteins on CD31-positive cells,including vascular endothelial cells,monocytes and platelets.Some transgenic strains were reconstituted with WT bone marrow and some XCON6 mice were injected with an anti-hTFPI antibody through tail vein.All mice were intratracheally instilled with LPS from Pseudomonas aeruginosa(10mg/Kg)or normal saline(NS)for 24 hours.The mice were sacrificed for analyzing lung inflammation,blood-gas barrier injury,lung injury and survival rate.Results:Both hTFPI and hirudin,the membrane-tethered anticoagulant fusion proteins and highly specifically expressed in endothelial cells,inhibit significantly inflammation by reducing leukocyte infiltrates,decreasing the level of proinflammatory cytokines(KC,MIP-2 and IL-6),and preventing significantly blood-gas barrier injury and lung injury,and increase the survival rate in LPS-induced mouse ALI.Conclusion:Targeted anticoagulant fusion proteins in microvascular endothelial cells of lung tissues protect LPS-induced ALI by suppressing cell inflammation.PAR III Targeted Anticoagulant Fusion Proteins protect LPS-induced Acute Lung Injury through Decreasing CCL2Objective:To study the potential mechanism of the protective effects of targeted anticoagulant fusion proteins on LPS-induced ALI.Methods:Two strains of transgenic mice,CD31-TFPI-Tg and CD31-Hir-Tg,and WT mice were employed in this study.Some transgenic strains were reconstituted with WT bone marrow and some XCON6 mice were injected with an anti-hTFPI antibody through tail vein.All mice were intratracheally instilled with LPS from Pseudomonas aeruginosa(10mg/Kg)or normal saline(NS)for 24 hours.The mice were sacrificed to detect the chemokine(C-C motif)ligand 2(CCL2)and the macrophage migratory inhibitory factor(MIF)in lung tissues.Results:The expression level of CCL2 in both XCON6 and XCON7 mice was decreased by RT-PCR and immunofluorescence analysis but no change of MIF level by PCR analysis compared to WT mice.Conclusion:Targeted anticoagulant fusion proteins protect LPS-induced acute lung injury through decreasing CCL2.