Barrier-protective Response Research Articles

Treatment of peanut allergy and colitis in mice via the intestinal release of butyrate from polymeric micelles

The microbiome modulates host immunity and aids the maintenance of tolerance in the gut, where microbial and food-derived antigens are abundant. Yet modern dietary factors and the excessive use of antibiotics have contributed to the rising incidence of food allergies, inflammatory bowel disease and other non-communicable chronic diseases associated with the depletion of beneficial taxa, including butyrate-producing Clostridia. Here we show that intragastrically delivered neutral and negatively charged polymeric micelles releasing butyrate in different regions of the intestinal tract restore barrier-protective responses in mouse models of colitis and of peanut allergy. Treatment with the butyrate-releasing micelles increased the abundance of butyrate-producing taxa in Clostridium cluster XIVa, protected mice from an anaphylactic reaction to a peanut challenge and reduced disease severity in a T-cell-transfer model of colitis. By restoring microbial and mucosal homoeostasis, butyrate-releasing micelles may function as an antigen-agnostic approach for the treatment of allergic and inflammatory diseases.

Flagella and indole produced by commensal bacteria protect the intestinal barrier to prevent food allergy

Abstract Lifestyle-induced changes to the diversity of the commensal microbiota have been causally linked to the increasing prevalence of food allergies and other non-communicable chronic diseases. We have shown that bacteria from the Clostridia class prevent an allergic response to food by eliciting an IL-22 dependent barrier protective response that limits allergen access to the systemic circulation. We have now examined the mechanisms by which commensal Clostridia induce this allergy-protective effect. We identified taxa in a consortium of Clostridia that possess flagella and produce indole, which are ligands for TLR5 and AhR, respectively. Lysates and flagella isolated from this consortium induced IL-22 in mouse intestinal explants. IL-22 was not induced in explants from mice in which TLR5 or MyD88 was knocked out globally or conditionally in CD11c+ cells. Treatment with the commensal flagellar isolate also reduced detection of intragastrically administered FITC dextran in the serum of antibiotic-treated mice. Similarly, indole exposure induced IL-22 in intestinal explants and reduced intestinal permeability to FITC dextran. Importantly, AhR signaling in RORγt+ cells was necessary for IL-22 induction by flagella. These results suggest that flagella and indole act synergistically to prevent an allergic response. Finally, we have isolated and characterized two Clostridial taxa which bear flagella and produce indole. We hypothesize that germ-free mice colonized with these two taxa will exhibit improved IL-22 dependent barrier function and be protected against an allergic response. Our work reveals novel features of Clostridia key to their allergy-protective capability which may be further exploited to develop therapeutics. Supported by NIH AI106302 and the Bunning Professorship Endowment Fund

Factor VIIa suppresses inflammation and barrier disruption through the release of EEVs and transfer of microRNA 10a

AbstractCoagulation protease, factor VIIa (FVIIa), binds to endothelial cell protein C receptor (EPCR) and induces anti-inflammatory and endothelial barrier protective responses via protease-activated receptor-1 (PAR1)–mediated, biased signaling. Our recent studies had shown that the FVIIa-EPCR-PAR1 axis induces the release of extracellular vesicles (EVs) from endothelial cells. In the present study, we investigated the mechanism of FVIIa release of endothelial EVs (EEVs) and the contribution of FVIIa-released EEVs to anti-inflammatory and vascular barrier protective effects, in both in vitro and in vivo models. Multiple signaling pathways regulated FVIIa release of EVs from endothelial cells, but the ROCK-dependent pathway appeared to be a major mechanism. FVIIa-released EEVs were enriched with anti-inflammatory microRNAs (miRs), mostly miR10a. FVIIa-released EEVs were taken up readily by monocytes/macrophages and endothelial cells. The uptake of FVIIa-released EEVs by monocytes conferred anti-inflammatory phenotype to monocytes, whereas EEV uptake by endothelial cells resulted in barrier protection. In additional experiments, EEV-mediated delivery of miR10a to monocytes downregulated the expression of TAK1 and activation of the NF-κB–mediated inflammatory pathway. In in vivo experiments, administration of FVIIa-released EEVs to wild-type mice attenuated LPS-induced increased inflammatory cytokines in plasma and vascular leakage into vital tissues. The incorporation of anti-miR10a into FVIIa-released EEVs diminished the ability of FVIIa-released EEVs to confer cytoprotective effects. Administration of the ROCK inhibitor Y27632, which significantly inhibits FVIIa release of EEVs into the circulation, to mice attenuated the cytoprotective effects of FVIIa. Overall, our study revealed novel insights into how FVIIa induces cytoprotective effects and communicates with various cell types.

The role of the commensal microbiota in the regulation of tolerance to dietary allergens.

We review the evidence that environmental stimuli that perturb naturally selected host-microbe interactions are driving the increasing prevalence of food allergy and examine the mechanisms by which commensal bacteria regulate tolerance to dietary allergens. Antibiotic use and the consumption of a high-fat/low-fiber diet have a major and rapid impact on gut bacterial populations, with long-term consequences for both overall microbial community structure and the regulation of host immunity. Recent work emphasizes the role of mucosa-associated commensal bacteria in eliciting a barrier-protective response critical to preventing allergic sensitization to food. Murine model studies are informing the development of novel live biotherapeutic approaches as an adjunctive therapy to enhance antigen-specific oral desensitization and to promote lasting tolerance in patients with food allergy. Strategies based on modulating the composition and/or functionality of the gut microbiome hold promise for the treatment of food allergy.

GRP78 is a novel receptor initiating a vascular barrier protective response to oxidized phospholipids.

Vascular integrity and the maintenance of blood vessel continuity are fundamental features of the circulatory system maintained through endothelial cell-cell junctions. Defects in the endothelial barrier become an initiating factor in several pathologies, including ischemia/reperfusion, tumor angiogenesis, pulmonary edema, sepsis, and acute lung injury. Better understanding of mechanisms stimulating endothelial barrier enhancement may provide novel therapeutic strategies. We previously reported that oxidized phospholipids (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine [OxPAPC]) promote endothelial cell (EC) barrier enhancement both in vitro and in vivo. This study examines the initiating mechanistic events triggered by OxPAPC to increase vascular integrity. Our data demonstrate that OxPAPC directly binds the cell membrane-localized chaperone protein, GRP78, associated with its cofactor, HTJ-1. OxPAPC binding to plasma membrane-localized GRP78 leads to GRP78 trafficking to caveolin-enriched microdomains (CEMs) on the cell surface and consequent activation of sphingosine 1-phosphate receptor 1, Src and Fyn tyrosine kinases, and Rac1 GTPase, processes essential for cytoskeletal reorganization and EC barrier enhancement. Using animal models of acute lung injury with vascular hyperpermeability, we observed that HTJ-1 knockdown blocked OxPAPC protection from interleukin-6 and ventilator-induced lung injury. Our data indicate for the first time an essential role of GRP78 and HTJ-1 in OxPAPC-mediated CEM dynamics and enhancement of vascular integrity.

Commensal bacteria induce a barrier protective response to prevent sensitization to food allergens (P6037)

Abstract Environmentally-induced alterations in the commensal microbiome have been implicated in the increasing prevalence of food allergy. We found that antibiotic treatment of neonatal mice leads to reduced proportions of regulatory T cells(Tregs) in the colonic lamina propria(LP), impaired production of intestinal IgA, and elevated peanut (PN) specific IgE/IgG1 in response to sensitization. Selective colonization of gnotobiotic mice linked the Treg/IgA inducing capacity to a consortium of bacteria within the Clostridia class. Introduction of either a conventional SPF microbiota or a mixture of Clostridia strains to antibiotic-treated mice restored the Treg and IgA compartments and blocked the induction of a food allergic response. Clostridia colonized gnotobiotic mice displayed increased expression of IL-23 and IL-22 in the colonic LP compartment and the induction of anti-microbial REG3β expression in the epithelium. Collectively, these results suggest that the maintenance of oral tolerance to dietary antigens relies on bacterial populations that induce a barrier protective response, which includes activation of the IL-23/IL22 axis and the expansion of intestinal Tregs and IgA secreting B cells. Our findings hold promise for the development of approaches to prevent or treat food allergy based on modulation of the composition of intestinal microbiota.

Barrier-Protective Effects of Activated Protein C in Human Alveolar Epithelial Cells

Acute lung injury (ALI) is a clinical manifestation of respiratory failure, caused by lung inflammation and the disruption of the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importance to prevent alveolar edema. Barrier integrity depends largely on the balance between physical forces on cell-cell and cell-matrix contacts, and this balance might be affected by alterations in the coagulation cascade in patients with ALI. We aimed to study the effects of activated protein C (APC) on mechanical tension and barrier integrity in human alveolar epithelial cells (A549) exposed to thrombin. Cells were pretreated for 3 h with APC (50 µg/ml) or vehicle (control). Subsequently, thrombin (50 nM) or medium was added to the cell culture. APC significantly reduced thrombin-induced cell monolayer permeability, cell stiffening, and cell contraction, measured by electrical impedance, optical magnetic twisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resulted in more elongated ZO-1 aggregates at cell-cell interface areas and induced an increase in ZO-1 membrane protein content. APC attenuated the length of these ZO-1 aggregates and reduced the ZO-1 membrane protein levels induced by thrombin. In conclusion, pretreatment with APC reduced the disruption of barrier integrity induced by thrombin, thus contributing to alveolar epithelial barrier protection.

Activated protein C promotes endothelial barrier protection through biased PAR1 signaling mediated by β‐arrestin and dishevelled‐2 scaffolds

Thrombin, a coagulant protease contributes to inflammatory responses associated with vascular injury and thrombosis. Activated protein C (APC), an anti‐coagulant protease reduces thrombin generation and promotes anti‐inflammatory signaling in endothelial cells. Thrombin and APC signal through the G‐protein coupled protease‐activated receptor1 (PAR1) but elicit opposite effects on endothelial barrier function. The mechanism of APC‐activated PAR1 signaling in endothelial barrier protection is poorly understood. In human endothelial cells, APC activates caveolar PAR1 populations to promote barrier protective responses independent of Gαi signaling. PAR1 is precoupled to β‐arrestins and co‐segregate in caveolin‐1 enriched fractions. APC‐activated PAR1 promotes β‐arrestin‐dependent recruitment and activation of dishevelled‐2 (Dvl‐2), a key mediator of the Wnt/Frizzled signaling pathways. Depletion of β‐arrestins or Dvl‐2 resulted in the loss of APC‐induced Rac1 activation and protection against thrombin‐activated endothelial barrier permeability but not thrombin‐induced RhoA signaling. These findings indicate that PAR1 utilizes β‐arrestins to promote biased signaling and unveils a distinct pathway where β‐arrestins and Dvl‐2 scaffolds promote APC effects on endothelial barrier protection. Funded by R01 HL073328, UC TRDRP 20FT‐0074.

A Role for VEGFR2 Activation in Endothelial Responses Caused by Barrier Disruptive OxPAPC Concentrations

IntroductionOxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) differentially modulate endothelial cell (EC) barrier function in a dose-dependent fashion. Vascular endothelial growth factor receptor-2 (VEGFR2) is involved in the OxPAPC-induced EC inflammatory activation. This study examined a role of VEGFR2 in barrier dysfunction caused by high concentrations of OxPAPC and evaluated downstream signaling mechanisms resulting from the effect of OxPAPC in EC from pulmonary and systemic circulation.MethodsEC monolayer permeability in human pulmonary artery endothelial cells (HPAEC) and human aortic endothelial cells (HAEC) was monitored by changes in transendothelial electrical resistance (TER) across EC monolayers. Actin cytoskeleton was examined by immunostaining with Texas Red labeled phalloidin. Phosphorylation of myosin light chains (MLC) and VE-Cadherin was examined by Western blot and immunofluorescence techniques. The role of VEGFR2 in OxPAPC-induced permeability and cytoskeletal arrangement were determined using siRNA-induced VEGFR2 knockdown.ResultsLow OxPAPC concentrations (5–20 µg/ml) induced a barrier protective response in both HPAEC and HAEC, while high OxPAPC concentrations (50–100 µg/ml) caused a rapid increase in permeability ; actin stress fiber formation and increased MLC phosphorylation were observed as early as 30 min after treatment. VEGFR2 knockdown dramatically decreased the amount of MLC phosphorylation and stress fiber formation caused by high OxPAPC concentrations with modest effects on the amount of VE-cadherin phosphorylation at Y731. We present evidence that activation of Rho is involved in the OxPAPC/VEGFR2 mechanism of EC permeability induced by high OxPAPC concentrations. Knockdown of VEGFR2 did not rescue the early drop in TER but prevented further development of OxPAPC-induced barrier dysfunction.ConclusionsThis study shows that VEGFR2 is involved in the delayed phase of EC barrier dysfunction caused by high OxPAPC concentrations and contributes to stress fiber formation and increased MLC phosphorylation.

Melatonin inhibits IL-1β-induced monolayer permeability of human umbilical vein endothelial cells via Rac activation

Melatonin is a key factor in the coordination of circadian rhythms and seasonal reproduction. Melatonin and its metabolites directly scavenge free radicals, increase the expression of antioxidant enzymes, and play a role in the anti-inflammatory phase of defense responses. At present, there are no direct data available as to melatonin's possible influence on endothelial cell monolayer permeability, which is a major biological process responsible for vascular diseases. The aim of this study was to investigate the effect of melatonin on IL-1β-induced human umbilical vein endothelial cells (HUVECs) monolayer permeability and then to test the involvement of small GTPase Rac in the melatonin-induced endothelial barrier-protective effects as well as cell contact reorganization. It was found that IL-1β treatment increased the permeability of HUVECs monolayer, disrupted adherens junctions, and down-regulated the expression of VE-cadherin which is the main functional protein of adherens junctions. Melatonin, however, decreased dextran permeability and increased intercellular adherens junction areas reflecting an endothelial cell barrier-protective response. Furthermore, melatonin dramatically improved IL-1β-induced Rac inactivation. Our results show that the barrier-protective effects of melatonin on endothelial cells are mediated by Rac activation and leads to enhancement of adherens junctions.

Factor X/Xa Elicits Protective Signaling Responses In Endothelial Cells Directly Via PAR-2 and Indirectly Via EPCR-Dependent Recruitment of PAR-1

AbstractAbstract 2115We recently demonstrated that the Gla-domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier protective response if endothelial cells were pre-incubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells, also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a crosstalk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla-domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells. Disclosures:No relevant conflicts of interest to declare.

Factor X/Xa Elicits Protective Signaling Responses in Endothelial Cells Directly via PAR-2 and Indirectly via Endothelial Protein C Receptor-dependent Recruitment of PAR-1

We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.

Akt-Mediated Transactivation of the S1P 1 Receptor in Caveolin-Enriched Microdomains Regulates Endothelial Barrier Enhancement by Oxidized Phospholipids

Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, leading to increased mass transport across the vessel wall and leukocyte extravasation, the key mechanisms in pathogenesis of tissue inflammation and edema. We have previously demonstrated that OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) significantly enhances vascular endothelial barrier properties in vitro and in vivo and attenuates endothelial hyperpermeability induced by inflammatory and edemagenic agents via Rac and Cdc42 GTPase dependent mechanisms. These findings suggested potential important therapeutic value of barrier-protective oxidized phospholipids. In this study, we examined involvement of signaling complexes associated with caveolin-enriched microdomains (CEMs) in barrier-protective responses of human pulmonary ECs to OxPAPC. Immunoblotting from OxPAPC-treated ECs revealed OxPAPC-mediated rapid recruitment (5 minutes) to CEMs of the sphingosine 1-phosphate receptor (S1P(1)), the serine/threonine kinase Akt, and the Rac1 guanine nucleotide exchange factor Tiam1 and phosphorylation of caveolin-1, indicative of signaling activation in CEMs. Abolishing CEM formation (methyl-beta-cyclodextrin) blocked OxPAPC-mediated Rac1 activation, cytoskeletal reorganization, and EC barrier enhancement. Silencing (small interfering RNA) Akt expression blocked OxPAPC-mediated S1P(1) activation (threonine phosphorylation), whereas silencing S1P(1) receptor expression blocked OxPAPC-mediated Tiam1 recruitment to CEMs, Rac1 activation, and EC barrier enhancement. To confirm our in vitro results in an in vivo murine model of acute lung injury with pulmonary vascular hyperpermeability, we observed that selective lung silencing of caveolin-1 or S1P(1) receptor expression blocked OxPAPC-mediated protection from ventilator-induced lung injury. Taken together, these results suggest Akt-dependent transactivation of S1P(1) within CEMs is important for OxPAPC-mediated cortical actin rearrangement and EC barrier protection.

Thrombin inhibits nuclear factor κB and RhoA pathways in cytokine-stimulated vascular endothelial cells when EPCR is occupied by protein C

The occupancy of endothelial protein C receptor (EPCR) by protein C switches the protease activated receptor 1 (PAR-1)-dependent signalling specificity of thrombin from a permeability enhancing to a barrier protective response in vascular endothelial cells. In this study, the modulatory effects of thrombin and thrombin receptor agonist peptides (TRAP) on tumour necrosis factor (TNF)-alpha-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A were evaluated by monitoring the expression of cell surface adhesion molecules (VCAM-1, ICAM-1 and E-selectin), adhesion of freshly isolated neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factor-kappaB (NF-kappaB) pathway. The analysis of results indicate that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither PAR-1 agonist influenced the proinflammatory effects of TNF-alpha in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin, thus leading to thrombin inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNF-alpha-activated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-kappaB pathways in response to TNF-alpha in cells pretreated with protein C-S195A. Based on these results we conclude that when EPCR is ligated by protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses, thus leading to activation of Rac1 and inhibition of RhoA and NF-kappaB signalling cascades in vascular endothelial cells.

Thrombin Inhibits NF-κb and RhoA Pathways in Cytokine-Stimulated Vascular Endothelial Cells When EPCR Is Occupied by Protein C.

We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by protein C switches the PAR-1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells. To determine whether the occupancy of EPCR by protein C renders thrombin a protective enzyme, thus up-regulating the expression of signaling molecules in the antiinflammatory pathways, we investigated the effects of thrombin and thrombin receptor agonist peptides (TRAP) on TNF-a-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A by monitoring the expression of cell surface adhesion molecules (VCAM- 1, ICAM-1 and E-selectin), adhesion of neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factorkB (NF-kB) pathway. Analysis of the results indicates that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither thrombin nor TRAP influenced the proinflammatory effects of TNF-a in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin and TRAP, thus leading to inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNFa-stimulated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-kB pathways in response to TNF-a in cells pretreated with protein C-S195A. Based on these results we conclude that when EPCR is bound by its natural ligand protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses in vascular endothelial cells.

Cross talk between paxillin and Rac is critical for mediation of barrier-protective effects by oxidized phospholipids

We previously reported that the barrier-protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cells (ECs) delineate the role of Rac- and Cdc42-dependent mechanisms and described the involvement of the focal adhesion (FA) protein paxillin in enhancement of the EC barrier upon OxPAPC challenge. This study examined a potential role of paxillin in the feedback mechanism of Rac regulation by FAs in OxPAPC-stimulated ECs. Our results demonstrate that OxPAPC induced Rac-dependent, Rho-independent peripheral accumulation of paxillin-containing FAs and time-dependent paxillin phosphorylation. Molecular inhibition of Rac decreased association of paxillin with the Rac-specific guanine nucleotide exchange factor beta-PIX. Molecular inhibition of paxillin also attenuated OxPAPC-induced enhancement of adherens junctions critical for the EC barrier-protective response, accumulation of vascular endothelial cadherin in the membrane fractions, and decreased activation of Rac and its effector p21-activated kinase (PAK1). Expression of paxillin with a mutated PAK1-dependent phosphorylation site (S273A) attenuated OxPAPC-induced PAK1 activation and the EC barrier-protective response. These results suggest that PAK1-specific paxillin phosphorylation at Ser(273) is critically involved in the positive-feedback regulation of the Rac-PAK1 pathway and may contribute to sustained enhancement of the EC barrier caused by oxidized phospholipids.

Activated protein C–cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin

Activated protein C (APC) signals in endothelial cells ex vivo through protease activated receptor-1 (PAR1). However, it is controversial whether PAR1 can mediate APC's protective effects in sepsis because the inflammatory response results in thrombin generation and thrombin proteolytically activates PAR1 much more efficiently than APC. Here we show that APC can induce powerful barrier protective responses in an endothelial cell monolayer in the presence of thrombin. Using cell surface immunoassays with conformation sensitive monoclonal anti-PAR1 antibodies we analyzed cleavage of endogenous PAR1 on the endothelial cell surface by APC in the absence and presence of thrombin. Incubation with APC caused efficient PAR1 cleavage and upon coincubation with thrombin APC supported additional PAR1 cleavage. Thrombin-cleaved PAR1 rapidly disappeared from the cell surface whereas, unexpectedly, the APC-cleaved PAR1 remained and could be detected on the cell surface, even when thrombin at concentrations of up to 1 nM was also present. Our findings demonstrate for the first time directly that APC can generate a distinct PAR1 population on endothelial cells in the presence of thrombin. The data suggest that different trafficking of activated PAR1 might explain how PAR1 signaling by APC can be relevant when thrombin is present.

Epac/Rap and PKA are novel mechanisms of ANP‐induced Rac‐mediated pulmonary endothelial barrier protection

Acute lung injury, sepsis, lung inflammation, and ventilator-induced lung injury are life-threatening conditions associated with lung vascular barrier dysfunction, which may lead to pulmonary edema. Increased levels of atrial natriuretic peptide (ANP) in lung circulation reported in these pathologies suggest its potential role in the modulation of lung injury. Besides well recognized physiological effects on vascular tone, plasma volume, and renal function, ANP may exhibit protective effects in models of lung vascular endothelial cell (EC) barrier dysfunction. However, the molecular mechanisms of ANP protective effects are not well understood. The recently described cAMP-dependent guanine nucleotide exchange factor (GEF) Epac activates small GTPase Rap1, which results in activation of small GTPase Rac-specific GEFs Tiam1 and Vav2 and Rac-mediated EC barrier protective responses. Our results show that ANP stimulated protein kinase A and the Epac/Rap1/Tiam/Vav/Rac cascade dramatically attenuated thrombin-induced pulmonary EC permeability and the disruption of EC monolayer integrity. Using pharmacological and molecular activation and inhibition of cAMP-and cGMP-dependent protein kinases (PKA and PKG), Epac, Rap1, Tiam1, Vav2, and Rac we linked ANP-mediated protective effects to the activation of Epac/Rap and PKA signaling cascades, which dramatically inhibited the Rho pathway of thrombin-induced EC hyper-permeability. These results suggest a novel mechanism of ANP protective effects against agonist-induced pulmonary EC barrier dysfunction via inhibition of Rho signaling by Epac/Rap1-Rac and PKA signaling cascades.

The ligand occupancy of endothelial protein C receptor switches the protease-activated receptor 1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells

AbstractRecent studies have indicated that activated protein C (APC) may exert its cytoprotective and anti-inflammatory activities through the endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on vascular endothelial cells. Noting that (1) the activation of protein C on endothelial cells requires thrombin, (2) relative to APC, thrombin cleaves PAR-1 with approximately 3 to 4 orders of magnitude higher catalytic efficiency, and (3) PAR-1 is a target for the proinflammatory activity of thrombin, it is not understood how APC can elicit a protective signaling response through the cleavage of PAR-1 when thrombin is present. In this study, we demonstrate that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that its occupancy by the γ-carboxyglutamic acid (Gla) domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through coupling of PAR-1 to the pertussis toxin–sensitive Gi-protein. Thus, when EPCR is bound by protein C, the PAR-1 cleavage-dependent protective signaling responses in endothelial cells can be mediated by either thrombin or APC. These results provide a new paradigm for understanding how PAR-1 and EPCR participate in protective signaling events in endothelial cells.

Paxillin-β-catenin interactions are involved in Rac/Cdc42-mediated endothelial barrier-protective response to oxidized phospholipids

Oxidized phospholipids may appear in the pulmonary circulation as a result of acute lung injury or inflammation. We have previously described barrier-protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on human pulmonary endothelial cells (EC) mediated by small GTPases Rac and Cdc42. This work examined OxPAPC-induced focal adhesion (FA) and adherens junction (AJ) remodeling and potential interactions between FA and AJ protein complexes involved in OxPAPC-induced EC barrier enhancement. Immunofluorescence analysis, subcellular fractionation, and coimmunoprecipitation assays have shown that OxPAPC induced translocation and peripheral accumulation of FA complexes containing paxillin, focal adhesion kinase, vinculin, GIT1, and GIT2, increased association of AJ proteins vascular endothelial-cadherin, p120-catenin, alpha-, beta-, and gamma-catenins, and dramatically enhanced cell junction areas covered by AJ. Coimmunoprecipitation, pulldown assays, and confocal microscopy studies have demonstrated that OxPAPC promoted novel interactions between FA and AJ complexes via paxillin and beta-catenin association, which was critically dependent on Rac and Cdc42 activities and was abolished by pharmacological or small interfering RNA (siRNA)-mediated inhibition of Rac and Cdc42. Depletion of beta-catenin using the siRNA approach attenuated OxPAPC-induced paxillin translocation to the cell periphery, but also significantly decreased interaction of paxillin with AJ protein complex. In turn, paxillin knockdown by specific siRNA attenuated AJ enhancement in response to OxPAPC. These results show for the first time the novel interactions between FA and AJ protein complexes critical for EC barrier regulation by OxPAPC.