Unfractionated Heparin Ameliorates Pulmonary Microvascular Endothelial Barrier Dysfunction Via Microtubule Stabilization In Acute Lung Injury

IntroductionSepsis-associated acute lung injury(ALI)is a common and severe consequence of infection,which contributes to significant morbidity and mortality in critically-ill patients.A large number of studies show that pulmonary endothelial cells are key modulators and orchestrators of ALI.As the central pathophysiological process,the aberrant and dysfunctional endothelial barrier induces capillary leakage,causing ALI.Lipopolysaccharide(LPS),the main component of the outer membrane of Gram-negative bacteria,always induces sepsis-associated ALI and endothelial barrier dysfunction.As we known,endothelial cell permeability is basically controlled by dynamic cytoskeletal remodeling and inter-cell junction protein complexes,which are also tightly linked to the cytoskeleton.Numerous researches demonstrated that the actin dynamics are critical for the regulation of endothelial barrier stability and vascular permeability.Rho GTPases are considered as the foremost mediators that control actin remodeling related to endothelial barrier function in response to different stimuli.The Rho guanine nucleotide exchange factor GEF-H1,acts as a microtubule-associated Rho specific guanosine nucleotide(GDP/GTP)exchange factor and couples microtubules to the Rho GTPase-dependent actin cytoskeleton.An increasing body of evidence corroborates the role of microtubule dynamics in regulation of endothelial permeability.Unfractionated heparin(UFH),a widely used anticoagulant drug,participates in the regulation of multifarious biological functions.Beyond anticoagulation,UFH has been shown other effects,such as anti-inflammation,anti-adhesion,anti-metastatic properties.We previously demonstrated that LPS induced the remodeling of F-actin cytoskeleton and formation of stress fibers,which increased human umbilical vascular endothelial cell(HUVEC)permeability,in contrast,UFH protected cells from endothelial hyperpermeability.Furthermore,we also showed that UFH attenuated pulmonary vascular hyperpermeability in mouse model of sepsis via the RhoA/Rho kinase(ROCK)pathway.However,the mechanism of action was not clear.Therefore,we investigated whether UFH ameliorates LPS-induced endothelial barrier dysfunction by inhibiting MT disassembly,and regulating GEF-H1 expression and F-actin remodeling.The p38 MAPK pathway is involved in various biological functions,including endothelial barrier function in response to exogenous and endogenous stimuli.We observed that UFH regulated anti-inflammatory activity by inhibiting p38 MAPK activation.Hence,we investigated whether the p38 MAPK pathway was involved in UFH-mediated attenuation of LPS-induced MT disassembly and endothelial barrier dysfunction.Objective:To investigate whether UFH ameliorates pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization in acute lung injury.Methods:Methods 1: C57BL/6J mice were randomized into four groups: Vehicle,UFH,LPS and LPS + UFH.Briefly,mice were anesthetized and given a 30 mg/kg body weight LPS(in 100 ?L of saline)intraperitoneally.Subcutaneous injection of 8 units UFH diluted in sterile saline(LPS + UFH group)or equal volume sterile saline(LPS group)was administered 30 min prior to the LPS injection.Then we assessed the effect of UFH on endothelial permeability in mice of LPS-induced sepsis associated ALI through histologic examination,lung wet/dry(W/D)weight ratio and assessment of lung capillary leakage by Evans Blue extravasation.We also evaluated the expression of acetylized-?-tubulin,GEF-H1 and the phosphorylation of MYPT1.Methods 2: HPMECs were plated on 96-well plates and the cell viability was determined by methyl thiazoyltetrazolium(MTT)assay.HPMECs were randomly divided into the Vehicle,UFH,LPS and LPS plus UFH groups.HPMECs was exposed to LPS alone or pretreatment with UFH.Then we assessed the protective effect of UFH on LPS-induced endothelial hyperpermeability through measurement of transendothelial electrical resistance(TEER),the permeability to FITC-dextran,and the actin cytoskeleton examined by immunofluorescence staining with TRITC-phalloidin.Then we evaluated the expression of GEF-H1 and the phosphorylation of MYPT1.Furthermore,we observed tubulin and acetylized-?-tubulin by immunofluorescence staining,as well as the expression of acetylized-?-tubulin,monomeric tubulin and polymeric tubulin.In the end,we examined the protective effect of UFH against Nocodazole-induced endothelial barrier dysfunction.Methods 3: HPMECs were randomly divided into the Vehicle,LPS,LPS plus UFH groups and LPS plus SB203580.We evaluated the effect of UFH on p38 phosphorylation and activation.Then we assessed the effect of UFH and SB203580 on endothelial permeability through measurement of transendothelial electrical resistance(TEER)and the permeability to FITC-dextran,as well as immunoprecipitation of F-actin.Then we evaluated the expression of GEF-H1 and the phosphorylation of MYPT1.In the end,we observed tubulin and acetylized-?-tubulin by immunoprecipitation,as well as the expression of acetylized-?-tubulin,monomeric tubulin and polymeric tubulin.Results:Results 1: Pulmonary vascular endothelial permeability of mice with sepsis associated ALI model by intraperitoneal injection of LPS was significantly increased.UFH attenuated pulmonary vascular endothelial hyperpermeability,also increased the degree of microtubule acetylation,decreased the expression of GEF-H1 and the phosphorylation of MYPT1 in mice with sepsis associated ALI.Results 2: The transendothelial electrical resistance(TEER)across HPMECs were decreased and the influx of FITC-conjugated dextran were increased after 6 hours treated with LPS(10?g/ml),UFH(10U/ml)ameliorated LPS-induced endothelial barrier dysfunction.After LPS stimulation,the remodeling of F-actin and the formation of stress fibers were also observed,which led to the increase of intercellular space.The pretreatment of UFH improved the change,also reduced the increase of GEF-H1 expression and MYPT1 phosphorylation level induced by LPS.Furthermore,UFH elevated the decrease of acetylated tubulin and improved MT depolymerization stimulated by LPS.We also demonstrated that Nocodazole increased the permeability of HPMECs through causing MTs depolymerization,increasing GEF-H1 expression and MYPT1 phosphorylation and F-actin remodeling,which were all remarkably ameliorated by UFH pretreatment.Results 3: UFH significantly deacreased p38 phosphorylation and activation stimulated by LPS.UFH and p38 inhibitor SB203580(10?M)ameliorated LPS-induced endothelial barrier dysfunction by increasing TEER and decreasing the influx of FITC-conjugated dextran across HPMECs.UFH and SB203580 also improved the remodeling of F-actin and the formation of stress fibers,reduced the increase of GEF-H1 expression and MYPT1 phosphorylation.Moreover,UFH and SB203580 elevated the decrease of acetylated tubulin and improved MT depolymerization stimulated by LPS.Conclusion: 1.UFH ameliorates pulmonary endothelial barrier dysfunction in a mice model of sepsis associated acute lung injury.2.LPS induces F-actin remodeling and hyperpermeability of HPMECs via causing MT depolymerization and motivating GEF-H1.3.UFH ameliorates LPS-induced endothelial barrier dysfunction through inhibiting MT depolymerization,motivating GEF-H1 and causing F-actin remodeling.4.The inhibition of p38 MAPK represents one of the mechanisms by which UFH ameliorates LPS-induced endothelial barrier dysfunction through inhibiting MT depolymerization,motivating GEF-H1 and causing F-actin remodeling.