Role Of Tie1/Tie2 Regulated By Neutrophils In The Development Of Acute Lung Injury Induced By Dual-insults In Mice

[Objectives]Loss of endothelial cell barrier function plays a significant role in the development of acute respiratory distress syndrome(ARDS),directly correlated with injuried endothelium in lung.Endothelial cell(EC)growth factors,angiopoietin(Ang)-1 & 2,maintain vascular homeostasis via tightly regulated competitive interaction with tyrosine kinase receptor,Tie2 which is expressed on ECs.Ang-1/Tie2 binding leads to Tie2 phosphorylation(p Tie2)and signaling for downstream anti-inflammatory and pro-survival phenotype,whereas Ang-2,stored pre-formed and released from activated ECs,can act as an apparent antagonist.Our previous studies showed Ang-2 significantly elevated in our murine ALI/ARDS model of hemorrhage(priming)followed by secondary septic challenge,and additionally,neutrophil(PMN)interactions with resident pulmonary vascular ECs contribute significantly to Ang-2 releasing and mediate Ang-2-associated pulmonary EC dysfunction central to the development of indirect ARDS.The orphan receptor,Tie1,has recently been described as contributing additional mediation of Ang/Tie2 signialing.While neither Ang-1 nor Ang-2 binds Tie1,Tie1 has been shown to complex with Tie2,inhibiting Ang-1/Tie2 interactions,thus promoting an activated,pro-inflammatory EC phenotype.Whether hemorrhage alters Tie1/Tie2 expression and interactions leading to the primed/activated EC phenotype,and the contribution of hemorrhage primed PMN to this altered expression is not known.To begin to investigate this,we tested the hypothesis that Tie1/Tie2 expression is altered following hemorrhage,and this change is associated with EC interaction with activated(shock primed)PMN.A murine model of hemorrhagic shock–induced priming for the development of i ARDS after subsequent septic challenge was used in this study.Our study will determine Tie1/Tie2 expressions following hemorrhage 6h,24 h challenged with sepsis.To better understand the significance of Tie1 in mediating EC activation leading to loss of barrier function and the development of ALI in our model,we delivered liposomal-encapsulated short interference(si)RNA to target Tie1 via tail vein at 1h post hemorrhage,in order to assess effects of Tie1 knock down on lung injury.To assess the contribution of hemorrhage-primed neutrophil to changes in Tie1/Tie2 expression,mice were depleted of peripheral blood neutrophils via intra-peritoneal(i.p.)injection of rat anti-mouse neutrophil antibody at 48 h before hemorrhage.Tie1,Tie2 and phosphorylated Tie2 expressions in lung homogenates were assessed using commercial ELISA kits.our aim was to verify our hypothesis neutrophil following hemorrhage alters Tie1/Tie2 expressions thereby decreasing Tie2 availability to Ang-1 binding,which will provide a theoretical basis for clinical prevention and treatment of ALI or ARDS and seek a potential therapeutic target.[Methods]1.Tie1/Tie2 expressions in ALI induced by dual insults of hemorrhagic shock and sepsisNa?ve male C57BL/6 mice(8-10 weeks old)were randomly divided into groups: na?ve group,sham hemorrhage/cecal ligation and puncture(CLP)group(Shem/C),H/C group.Mice in H/C group will be induced CLP at 6h and 24 h after hemorrhage,while mice in Shem/C group are subjected to CLP at 24 h following sham hemorrhage.Tie1 and Tie2 expression will be assessed in lung homogenates using commercial ELISA kits and Western blot,respectively.2.The role of Tie1 in the development of ARDS in our modelwe delivered liposomal-encapsulated short interference(si)RNA to target Tie1 via tail vein injection at 1h post hemorrhage,CLP will be induced at 24 h post hemorrhage.Plasma and lung tissue were collected at 24 h post CLP.Lung hematoxylin-eosin(H&E)staining was used to assess lung histopathology.Tie1 protein expression in plasma and lung tissue was detected with ELISA.Lung injury including neutrophils infiltration and proteins levels in BLA was assessed by MPO and BCA assays,respectively.Additionally,cytokines(IL-6,TNF-ɑ,IL-10)and MIP2 protein levels were examined by ELISA kits.3.The contribution of neutrophils to Tie1/Tie2 expression altering in ALI induced by dual insults of hemorrhagic shock and sepsisNa?ve male C57BL/6 mice were randomly divided into groups: Na?ve,H/C,H/C+ anti-Gr-1 and H/C+isotype groups(6 mice per group).Anti-Gr-1 and isotype antibody were intraperitoneally administrated at 48 h before hemorrhage while mice in H/C group were injected the same volume of saline at the same time.Tie1,Tie2 and phosphorylated Tie2 expressions in lung homogenates were assessed using commercial ELISA kits at 24 h after CLP.Lung hematoxylin-eosin(H&E)staining was used to assess lung histopathology.cytokines(IL-6,TNF-ɑ,IL-10)and MIP2 protein levels were examined by ELISA kits[Results]1.Tie1/Tie2 expressions in ALI induced by dual insults of hemorrhagic shock and sepsisIn our “two-hit” model,p-Tie2 was decreased at 6h and restored at 24 h after hemorrhagic shock,while Tie1 expression was elevated at both 6h and 24 h after hemorrhagic shock.2.The role of Tie1 in the development of ARDS in our modelSilencing of Tie1 receptor expression on lung endothelial cells using intra-venous tail vein injection of Tie1 targeted si RNA significantly decreased Tie2,pro-inflammatory mediator IL-6 and neutrophil chemotactic protein MIP-2.Neutrophil influx to the lung(MPO)and lung leak(BAL: plasma protein)were significantly decreased in mice that received Tie1 si RNA following hemorrhagic shock.3.The contribution of neutrophils to Tie1/Tie2 expression altering in ALI induced by dual insults of hemorrhagic shock and sepsisDepletion of neutrophil significantly inhibited the inflammatory response in lung tissue in ALI and improved lung injury,including decreased inflammatory cytokines and protein level in BALF.Importantly,neutrophil depletion markedly upregulated p-Tie2 and downregulated Tie1 expression.There was no difference in anti-Gr-1 and isotype groups.[Conclusion]These findings support our hypothesis and suggest 1)that Tie1 plays a role in mediating EC dysfunction potentially by prolonging EC activation by decreasing Tie2 availability to Ang-1 binding,2)that hemorrhage-primed neutrophil contributes to the altered Tie2/Tie1 expression in acute lung injury,3)that Tie1 contributes to the development of i ARDS and is a potential therapeutic target or biomarker of treatment efficacy/ARDS resolution.