Effect And Mechanism Of CYP2J2 And Its Metabolites EETs On Permeability Increase In Lipopolysaccharide Induced Acute Lung Injury

Background and Objective:Acute lung injury is a syndrome caused by alveolar epithelial cells and capilary endothelial cell insult which is pathologically characterized by diffuse pulmonary interstitial and alveolar edema and clinically characterized by acute hypoxic respiratory insufficiency. Although it is generally accepted that sepsis-associated mortality is related to the host response and involves a multitude of inflammatory cell types, inflammatory mediators, and coagulation factors, clinical studies have largely failed to identify an effective therapeutic target and Mechanical ventilation is currently the main support treatment. Future advances in sepsis related lung injury therapy will require a better understanding of how the individual components of the host response interact. Integrity of endothelial barrier is a determination of the prognosis in the acute lung injury caused by sepsis. Vascular endothelial cadherin (VE-cadherin, also known as cadherin 5), the major type of inter-endothelial junctions, is a trans-membrane receptor whose extracellular domain homophilically binds to the extracellular domain of another VE-cadherin from an adjacent cell and whose intracellular domain is anchored to the cell cytoskeleton via a family of actin-binding proteins called catenins (α, β,γ and p120 catenins). Phosphorylation and internalization of VE-cadherin, and reorganization of the actin cytoskeleton into stress fibers, thus applying mechanical forces to AJs that break apart the junctions are responsible for increases in vascular permeability increase. Any agent capable of inhibiting VE cadherin and myosin light chain (MLC) phosphorylation may effectively reduce vascular permeability induced by inflammatory agents. The EETs, metabolites of cytochrome P450 epoxygenase of Arachidonic acid, exhibit protective effect in various pathogenic states, however, whether the EETs play a role in endothelial barrier enhancement and the relative mechanism remains to be investigated. We uses to focus on the properties of inflammation or leukocytes themselves in acute lung injury, however, therer are few studies that sought to explore the mechanism of leukocytes infiltration from the view of endothelial cells. Thus the present study is designed to investigate the effect and mechanism of CYP2J2 on sepsis induced permeability increase.Methods:In the in vivo study, male CYP2J2 transgenic and their Wild-type control littermates were divided into four groups randomly (with each group 10 mice for each tests): the wildtype-saline treated group, the wildtype-LPS treated group, the CYP2J2 transgenic-saline treated group, the CYP2J2 transgenic-LPS treated group. Besides using the transgenic mice, we also induced the sEH inhibitor TPPU to further confirm the protective effect of CYP2J2 on endothelial barrier function. Having been treated by LPS for 6 hours, some mice were tail vein injected with 2mg/100g evans blue and after 1 hour were right ventricle levaged with cold PBS and then the lung were exised to take photos and homogenized to tests the OD value; some other mice were bronchoalveolar lavaged by intratracheal injection of 0.5 mL of PBS solution for three times, followed by gentle aspiration. The recovered fluids were processed for determination of total protein concentration and cytokines assay; some mice were excised the lung to paraffined or frozened in -80 for further tests such as MPO activity assay or western blot.In the in vitro study, we treated the HUVEC with LPS to induce the conformational change between adjacent endothelial cells. In order to investigate the effet of the CYP2J2 sysyem on endothelial barrier and the potential mechanism, we incubated the cell with sEH inhibitor AUDA and the VE-cadherin and MLC phosphorylation and relative indexes were measured. The concreat procegures are as follows.1. To investigate the effect of endothelial specific CYP2J2 overexpression on sepsis induced acute lung injury1) To obvserve the moratility rate after being injected with LPS and draw a survival curve2) HE staining:to observe the infiltration of leukocytes in lung3) Immunohistochemistry of MPO:to observe the nutrophage infiltration in lung4) MPO activity assay:to observe the nutrophage infiltration in lung5) Evans blue staining and protein concerntration of bronchoalveolar leavage fluid measurements:to obseve the transvascular lekeage of protein6) The measurement of the ratio of wet weight versus dry weight of the lung:to assess the fluid accumulation in lungs7) Immunofluorenscence:staing of NG2 and CD31 to asscess the loss of pericytes2. The effect of sEH inhibitor AUDA on constituents of endothelial cell junctions1) FITC-dextran transwell:to test the effect of AUDA on LPS induced permeability increase of endothelial monolayer2) Western blot:to test the exchange of soluble and insoluble ingredient of VE-cadherin and the phosphorylation of VE-cadherin and MLC3) Flow cytometry:to investigate the effect of AUDA on endothelial cell apoptosis3. The potential mechanisms that AUDA regulates endothelial permeability1) The role of Rho-Rock in AUDA regulated endothelial permeability2) The role of oxidative stress in AUDA regulated endothelial permeability that are mediated by LPS activated ROCK3) The role of Src in AUDA regulated endothelial permeability4) The crosstalk between Src and oxidative stressResults:Here, we show that the endothelial specific 2J2 over-expression and sEH inhibitor TPPU reduced LPS induced endothelial permeability increase in vivo, which is also imitated in the in vitro study by sEH inhibitor AUDA and 11,12-EET, an effect mediated by reduced GRP78-Src interaction and ROS production that leaded to reduced RhoA/Rock activation, and eventually reduced VE-cadherin and MLC phosphorylation. In addition, the AUDA reduced LPS induced VE-cadherin endocytosis and stress fiber formation.1. Endothelial specific CYP2J2 overexpression protected against sepsis induced acute lung injury1) Endothelial specific CYP2J2 overexpression reduced sepsis induced mortality2) Endothelial specific CYP2J2 overexpression reduced sepsis induced leukocytes infiltration of lung especially neutrophage infiltration.3) Endothelial specific CYP2J2 overexpression reduced sepsis induced fluid accumulation in lungs4) Endothelial specific CYP2J2 overexpression reduced sepsis induced transvascular lekeage of protein5) Endothelial specific CYP2J2 overexpression reduced sepsis induced pericytes loss6) sEH inhibitor TPPU protected against sepsis induced permeability increase as CYP2J2 overexpression did2. The effect of sEH inhibitor AUDA on constituents of endothelial cell junctions1) AUDA suppressed LPS induced FITC-dextran transendothelial infiltration which indicated that AUDA protected against LPS induced permeability increase of endothelial monolayer2) AUDA suppressed the conversion of VE-cadherin from insoluble to insoluble form3) AUDA suppressed the phosphorylation of VE-cadherin as well as MLC4) The protection of AUDA against LPS induced endothelial barrier dysfuction is independent of apoptosis 3. The potential mechanism that AUDA regulates endothelial permeability1) The role of Rho-Rock in AUDA regelated endothelial permeabilitya) AUDA suppressed the increased Rho activaty that was induced by LPSb) AUDA suppressed the phosphorylation of MYPT that acted downstream of ROCK pathway2) The role of oxidative stress in AUDA regulated endothelial permeability that are mediated by LPS activated ROCKa) Ros eliminator NAC reduced LPS induced Ros generation and mitigated LPS induded endothelial permeability increaseb) NAC remitted LPS induced Rho-rock activationc) AUDA reduced LPS induced ROS generation and NOX2 expressiond) AUDA mitigated H2O2 directly induced permeability increase, which is similar to the effect of NAC3) The role of Src in AUDA regulated endothelial permeabilitya) AUDA inhibited interaction of GRP78 with SRC and subsequent Src activationb) The Src inhibitor PP1 protected against LPS induced permeability increasec) The Src inhibitor PP1 reduced LPS induced VE-cadherin and MLC phosphorylation4) The crosstalk between Src and oxidative stressa) The Src inhibitor PP1 reduced LPS induced ROS generationb) The Src inhibitor PP1 reduced LPS induced NOX2 expressionc) Src inhibitor PP1 reduced LPS induced ROCK activationConclusion:1. CYP2J2 protected against sepsis related acute lung injury, suppressed sepsis induced vascular permeability increase and reduced mortality2. CYP2J2 reduced opening of cell-cell junctions by reducing phosphorylation of VE-cadherin and MLC as well as stress fiber formation3. The effect of CYP2J2 on endothelial barrier maintenance is mediated by suppression of GRP78/src interaction and subsequent src activation, leading to reduced ROS generation and resulting in mitigation of Rho-rock signal pathway activation eventuallyIn words, the CYP2J2-EETs is crucial for RhoA-dependent regulation of cytoskeletal architecture leading to reversible changes in vascular permeability which may contribute to the development of new therapeutic approaches for pulmonary edema and other diseases caused by abnormal vascular permeability. However, the mechanism by which AUDA regulated the interaction between GRP78 and Src need to be further investigated.