The transcription factor DREAM represses the deubiquitinase A20 and mediates inflammation.

Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.

The redox-sensitive cation channel TRPM2 modulates phagocyte ROS production and inflammation.

The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is critical for host defense, but ROS overproduction can also lead to inflammation and tissue injury. Here we report that TRPM2, a nonselective and redox-sensitive cation channel, inhibited ROS production in phagocytic cells and prevented endotoxin-induced lung inflammation in mice. TRPM2-deficient mice challenged with endotoxin (lipopolysaccharide) had an enhanced inflammatory response and diminished survival relative to that of wild-type mice challenged with endotoxin. TRPM2 functioned by dampening NADPH oxidase-mediated ROS production through depolarization of the plasma membrane in phagocytes. As ROS also activate TRPM2, our findings establish a negative feedback mechanism for the inactivation of ROS production through inhibition of the membrane potential-sensitive NADPH oxidase.

Phospholipase D2 restores endothelial barrier function by promoting PTPN14-mediated VE-cadherin dephosphorylation.

Increased permeability of vascular lung tissues is a hallmark of acute lung injury and is often caused by edemagenic insults resulting in inflammation. Vascular endothelial (VE)-cadherin undergoes internalization in response to inflammatory stimuli and is recycled at cell adhesion junctions during endothelial barrier re-establishment. Here, we hypothesized that phospholipase D (PLD)-generated phosphatidic acid (PA) signaling regulates VE-cadherin recycling and promotes endothelial barrier recovery by dephosphorylating VE-cadherin. Genetic deletion of PLD2 impaired recovery from protease-activated receptor-1-activating peptide (PAR-1-AP)-induced lung vascular permeability and potentiated inflammation in vivo In human lung microvascular endothelial cells (HLMVECs), inhibition or deletion of PLD2, but not of PLD1, delayed endothelial barrier recovery after thrombin stimulation. Thrombin stimulation of HLMVECs increased co-localization of PLD2-generated PA and VE-cadherin at cell-cell adhesion junctions. Inhibition of PLD2 activity resulted in prolonged phosphorylation of Tyr-658 in VE-cadherin during the recovery phase 3 h post-thrombin challenge. Immunoprecipitation experiments revealed that after HLMVECs are thrombin stimulated, PLD2, VE-cadherin, and protein-tyrosine phosphatase nonreceptor type 14 (PTPN14), a PLD2-dependent protein-tyrosine phosphatase, strongly associate with each other. PTPN14 depletion delayed VE-cadherin dephosphorylation, reannealing of adherens junctions, and barrier function recovery. PLD2 inhibition attenuated PTPN14 activity and reversed PTPN14-dependent VE-cadherin dephosphorylation after thrombin stimulation. Our findings indicate that PLD2 promotes PTPN14-mediated dephosphorylation of VE-cadherin and that redistribution of VE-cadherin at adherens junctions is essential for recovery of endothelial barrier function after an edemagenic insult.

Nonmuscle myosin light-chain kinase mediates neutrophil transmigration in sepsis-induced lung inflammation by activating beta2 integrins.

Nonmuscle myosin light-chain kinase (MYLK) mediates increased lung vascular endothelial permeability in lipopolysaccharide-induced lung inflammatory injury, the chief cause of the acute respiratory distress syndrome. In a lung injury model, we demonstrate here that MYLK was also essential for neutrophil transmigration, but that this function was mostly independent of myosin II regulatory light chain, the only known substrate of MYLK. Instead, MYLK in neutrophils was required for the recruitment and activation of the tyrosine kinase Pyk2, which mediated full activation of beta(2) integrins. Our results demonstrate that MYLK-mediated activation of beta(2) integrins through Pyk2 links beta(2) integrin signaling to the actin motile machinery of neutrophils.

Pyk2 phosphorylation of VE-PTP downstream of STIM1-induced Ca2+ entry regulates disassembly of adherens junctions.

Vascular endothelial protein tyrosine phosphatase (VE-PTP) stabilizes endothelial adherens junctions (AJs) through constitutive dephosphorylation of VE-cadherin. Here we investigated the role of stromal interaction molecule 1 (STIM1) activation of store-operated Ca2+ entry (SOCE) in regulating AJ assembly. We observed that SOCE induced by STIM1 activated Pyk2 in human lung microvascular endothelial cells (ECs) and induced tyrosine phosphorylation of VE-PTP at Y1981. Pyk2-induced tyrosine phosphorylation of VE-PTP promoted Src binding to VE-PTP, Src activation, and subsequent VE-cadherin phosphorylation and thereby increased the endothelial permeability response. The increase in permeability was secondary to disassembly of AJs. Pyk2-mediated responses were blocked in EC-restricted Stim1 knockout mice, indicating the requirement for STIM1 in initiating the signaling cascade. A peptide derived from the Pyk2 phosphorylation site on VE-PTP abolished the STIM1/SOCE-activated permeability response. Thus Pyk2 activation secondary to STIM1-induced SOCE causes tyrosine phosphorylation of VE-PTP, and VE-PTP, in turn, binds to and activates Src, thereby phosphorylating VE-cadherin to increase endothelial permeability through disassembly of AJs. Our results thus identify a novel signaling mechanism by which STIM1-induced Ca2+ signaling activates Pyk2 to inhibit the interaction of VE-PTP and VE-cadherin and hence increase endothelial permeability. Therefore, targeting the Pyk2 activation pathway may be a potentially important anti-inflammatory strategy.

p120-catenin expressed in alveolar type II cells is essential for the regulation of lung innate immune response.

The integrity of the lung alveolar epithelial barrier is required for the gas exchange and is important for immune regulation. Alveolar epithelial barrier is composed of flat type I cells, which make up approximately 95% of the gas-exchange surface, and cuboidal type II cells, which secrete surfactants and modulate lung immunity. p120-catenin (p120; gene symbol CTNND1) is an important component of adherens junctions of epithelial cells; however, its function in lung alveolar epithelial barrier has not been addressed in genetic models. Here, we created an inducible type II cell-specific p120-knockout mouse (p120EKO). The mutant lungs showed chronic inflammation, and the alveolar epithelial barrier was leaky to (125)I-albumin tracer compared to wild type. The mutant lungs also demonstrated marked infiltration of inflammatory cells and activation of NF-κB. Intracellular adhesion molecule 1, Toll-like receptor 4, and macrophage inflammatory protein 2 were all up-regulated. p120EKO lungs showed increased expression of the surfactant proteins Sp-B, Sp-C, and Sp-D, and displayed severe inflammation after pneumonia caused by Pseudomonas aeruginosa compared with wild type. In p120-deficient type II cell monolayers, we observed reduced transepithelial resistance compared to control, consistent with formation of defective adherens junctions. Thus, although type II cells constitute only 5% of the alveolar surface area, p120 expressed in these cells plays a critical role in regulating the innate immunity of the entire lung.

G protein-dependent basal and evoked endothelial cell vWF secretion.

von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13(-/-);Gα12(-/-) mice that could be normalized by infusion of human vWF. Blood from Gα12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-Gαq(-/-);Gα11(-/-) and Gα12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.

Cooperative interaction of trp melastatin channel transient receptor potential (TRPM2) with its splice variant TRPM2 short variant is essential for endothelial cell apoptosis.

RATIONALE: Oxidants generated by activated endothelial cells are known to induce apoptosis, a pathogenic feature of vascular injury and inflammation from multiple pathogeneses. The melastatin-family transient receptor potential 2 (TRPM2) channel is an oxidant-sensitive Ca2+ permeable channel implicated in mediating apoptosis; however, the mechanisms of gating of the supranormal Ca2+ influx required for initiating of apoptosis are not understood. OBJECTIVE: Here, we addressed the role of TRPM2 and its interaction with the short splice variant TRPM2 short variant (TRPM2-S) in mediating the Ca2+ entry burst required for induction of endothelial cell apoptosis. METHODS AND RESULTS: We observed that TRPM2-S was basally associated with TRPM2 in the endothelial plasmalemma, and this interaction functioned to suppress TRPM2-dependent Ca2+ gating constitutively. Reactive oxygen species production in endothelial cells or directly applying reactive oxygen species induced protein kinase C-α activation and phosphorylation of TRPM2 at Ser 39. This in turn stimulated a large entry of Ca2+ and activated the apoptosis pathway. A similar TRPM2-dependent endothelial apoptosis mechanism was seen in intact vessels. The protein kinase C-α-activated phosphoswitch opened the TRPM2 channel to allow large Ca2+ influx by releasing TRPM2-S inhibition of TRPM2, which in turn activated caspase-3 and cleaved the caspase substrate poly(ADP-ribose) polymerase. CONCLUSIONS: Here, we describe a fundamental mechanism by which activation of the trp superfamily TRPM2 channel induces apoptosis of endothelial cells. The signaling mechanism involves reactive oxygen species-induced protein kinase C-α activation resulting in phosphorylation of TRPM2-S that allows enhanced TRPM2-mediated gating of Ca2+ and activation of the apoptosis program. Strategies aimed at preventing the uncoupling of TRPM2-S from TRPM2 and subsequent Ca2+ gating during oxidative stress may mitigate endothelial apoptosis and its consequences in mediating vascular injury and inflammation.

Cooperative signaling via transcription factors NF-κB and AP1/c-Fos mediates endothelial cell STIM1 expression and hyperpermeability in response to endotoxin.

Stromal interacting molecule 1 (STIM1) regulates store-operated Ca(2+) entry (SOCE). Here we show that STIM1 expression in endothelial cells (ECs) is increased during sepsis and, therefore, contributes to hyperpermeability. LPS induced STIM1 mRNA and protein expression in human and mouse lung ECs. The induced STIM1 expression was associated with augmented SOCE as well as a permeability increase in both in vitro and in vivo models. Because activation of both the NF-κB and p38 MAPK signaling pathways downstream of TLR4 amplifies vascular inflammation, we studied the influence of these two pathways on LPS-induced STIM1 expression. Inhibition of either NF-κB or p38 MAPK activation by pharmacological agents prevented LPS-induced STIM1 expression. Silencing of the NF-κB proteins (p65/RelA or p50/NF-κB1) or the p38 MAPK isoform p38α prevented LPS-induced STIM1 expression and increased SOCE in ECs. In support of these findings, we found NF-κB and AP1 binding sites in the 5'-regulatory region of human and mouse STIM1 genes. Further, we demonstrated that LPS induced time-dependent binding of the transcription factors NF-κB (p65/RelA) and AP1 (c-Fos/c-Jun) to the STIM1 promoter. Interestingly, silencing of c-Fos, but not c-Jun, markedly reduced LPS-induced STIM1 expression in ECs. We also observed that silencing of p38α prevented c-Fos expression in response to LPS in ECs, suggesting that p38α signaling mediates the expression of c-Fos. These results support the proposal that cooperative signaling of both NF-κB and AP1 (via p38α) amplifies STIM1 expression in ECs and, thereby, contributes to the lung vascular hyperpermeability response during sepsis.

Combinatorial therapy with acetylation and methylation modifiers attenuates lung vascular hyperpermeability in endotoxemia-induced mouse inflammatory lung injury.

Impairment of tissue fluid homeostasis and migration of inflammatory cells across the vascular endothelial barrier are crucial factors in the pathogenesis of acute lung injury (ALI). The goal for treatment of ALI is to target pathways that lead to profound dysregulation of the lung endothelial barrier. Although studies have shown that chemical epigenetic modifiers can limit lung inflammation in experimental ALI models, studies to date have not examined efficacy of a combination of DNA methyl transferase inhibitor 5-Aza 2-deoxycytidine and histone deacetylase inhibitor trichostatin A (herein referred to as Aza+TSA) after endotoxemia-induced mouse lung injury. We tested the hypothesis that treatment with Aza+TSA after lipopolysaccharide induction of ALI through epigenetic modification of lung endothelial cells prevents inflammatory lung injury. Combinatorial treatment with Aza+TSA mitigated the increased endothelial permeability response after lipopolysaccharide challenge. In addition, we observed reduced lung inflammation and lung injury. Aza+TSA also significantly reduced mortality in the ALI model. The protection was ascribed to inhibition of the eNOS-Cav1-MLC2 signaling pathway and enhanced acetylation of histone markers on the vascular endothelial-cadherin promoter. In summary, these data show for the first time the efficacy of combinatorial Aza+TSA therapy in preventing ALI in lipopolysaccharide-induced endotoxemia and raise the possibility of an essential role of DNA methyl transferase and histone deacetylase in the mechanism of ALI.

A critical role for Lyn kinase in strengthening endothelial integrity and barrier function.

The Src family kinases (SFKs) c-Src and Yes mediate vascular leakage in response to various stimuli including lipopolysaccharide (LPS) and vascular endothelial growth factor (VEGF). Here, we define an opposing function of another SFK, Lyn, which in contrast to other SFKs, strengthens endothelial junctions and thereby restrains the increase in vascular permeability. Mice lacking Lyn displayed increased mortality in LPS-induced endotoxemia and increased vascular permeability in response to LPS or VEGF challenge compared with wild-type littermates. Lyn knockout mice repopulated with wild-type bone marrow-derived cells have higher vascular permeability than wild-type mice, suggesting a role of endothelial Lyn in the maintenance of the vascular barrier. Small interfering RNA-mediated down-regulation of Lyn disrupted endothelial barrier integrity, whereas expression of a constitutively active mutant of Lyn enhanced the barrier. However, down-regulation of Lyn did not affect LPS-induced endothelial permeability. We demonstrate that Lyn association with focal adhesion kinase (FAK) and phosphorylation of FAK at tyrosine residues 576/577 and 925 were required for Lyn-dependent stabilization of endothelial adherens junctions. Thus, in contrast to c-Src and Yes, which increase vascular permeability in response to stimuli, Lyn stabilizes endothelial junctions through phosphorylation of FAK. Therefore, therapeutics activating Lyn kinase may strengthen the endothelial barrier junction and hence have anti-inflammatory potential.

Loss of caveolin-1 and adiponectin induces severe inflammatory lung injury following LPS challenge through excessive oxidative/nitrative stress.

Excessive reactive oxygen/nitrogen species have been associated with the onset, progression, and outcome of sepsis, both in preclinical and clinical studies. However, the signaling pathways regulating oxidative/nitrative stress in the pathogenesis of sepsis-induced acute lung injury and acute respiratory distress syndrome are not fully understood. Employing the novel mouse model with genetic deletions of both caveolin-1 (Cav1) and adiponectin (ADPN) [double knockout (DKO) mice], we have demonstrated the critical role of Cav1 and ADPN signaling cross talk in regulating oxidative/nitrative stress and resulting inflammatory lung injury following LPS challenge. In contrast to the inhibited inflammatory lung injury in Cav1(-/-) mice, we observed severe lung inflammation and markedly increased lung vascular permeability in DKO mice in response to LPS challenge. Accordingly, the DKO mice exhibited an 80% mortality rate following a sublethal dose of LPS challenge. At basal state, loss of Cav1 and ADPN resulted in a drastic increase of oxidative stress and resultant nitrative stress in DKO lungs. Scavenging of superoxide by pretreating the DKO mice with MnTMPYP (a superoxide dismutase mimetic) restored the inflammatory responses to LPS challenge including reduced lung myeloperoxidase activity and vascular permeability. Thus oxidative/nitrative stress collectively modulated by Cav1 and ADPN signalings is a critical determinant of inflammatory lung injury in response to LPS challenge.

ICAM-1-activated Src and eNOS signaling increase endothelial cell surface PECAM-1 adhesivity and neutrophil transmigration.

Polymorphonuclear neutrophil (PMN) extravasation requires selectin-mediated tethering, intercellular adhesion molecule-1 (ICAM-1)-dependent firm adhesion, and platelet/endothelial cell adhesion molecule 1 (PECAM-1)-mediated transendothelial migration. An important unanswered question is whether ICAM-1-activated signaling contributes to PMN transmigration mediated by PECAM-1. We tested this concept and the roles of endothelial nitric oxide synthase (eNOS) and Src activated by PMN ligation of ICAM-1 in mediating PECAM-1-dependent PMN transmigration. We observed that lung PMN infiltration in vivo induced in carrageenan-injected WT mice was significantly reduced in ICAM-1(-/-) and eNOS(-/-) mice. Crosslinking WT mouse ICAM-1 expressed in human endothelial cells (ECs), but not the phospho-defective Tyr(518)Phe ICAM-1 mutant, induced SHP-2-dependent Src Tyr530 dephosphorylation that resulted in Src activation. ICAM-1 activation also stimulated phosphorylation of Akt (p-Ser473) and eNOS (p-Ser1177), thereby increasing NO production. PMN migration across EC monolayers was abolished in cells expressing the Tyr(518)Phe ICAM-1 mutant or by pretreatment with either the Src inhibitor PP2 or eNOS inhibitor L-NAME. Importantly, phospho-ICAM-1 induction of Src signaling induced PECAM-1 Tyr686 phosphorylation and increased EC surface anti-PECAM-1 mAb-binding activity. These results collectively show that ICAM-1-activated Src and eNOS signaling sequentially induce PECAM-1-mediated PMN transendothelial migration. Both Src and eNOS inhibition may be important therapeutic targets to prevent or limit vascular inflammation.

A critical role for phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac exchanger 1 in endothelial junction disruption and vascular hyperpermeability.

RATIONALE: The small GTPase Rac is critical to vascular endothelial functions, yet its regulation in endothelial cells remains unclear. Understanding the upstream pathway may delineate Rac activation mechanisms and its role in maintaining vascular endothelial barrier integrity. OBJECTIVE: By investigating phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac exchanger 1 (P-Rex1), one of the Rac-specific guanine nucleotide exchange factors previously known for G protein-coupled receptor signaling, we sought to determine whether Rac-guanine nucleotide exchange factor is nodal for signal integration and potential target for drug intervention. METHODS AND RESULTS: Using gene deletion and small interference RNA silencing approach, we investigated the role of P-Rex1 in human lung microvascular endothelial cells. Tumor necrosis factor α (TNF-α) exposure led to disruption of endothelial junctions, and silencing P-Rex1 protected junction integrity. TNF-α stimulated Rac activation and reactive oxygen species production in a P-Rex1-dependent manner. Removal of P-Rex1 significantly reduced intercellular adhesion molecule-1 expression, polymorphonuclear leukocyte transendothelial migration, and leukocyte sequestration in TNF-α-challenged mouse lungs. The P-Rex1 knockout mice were also refractory to lung vascular hyperpermeability and edema in a lipopolysaccharide-induced sepsis model. CONCLUSIONS: These results demonstrate for the first time that P-Rex1 expressed in endothelial cells is activated downstream of TNF-α, which is not a G protein-coupled receptor agonist. Our data identify P-Rex1 as a critical mediator of vascular barrier disruption. Targeting P-Rex1 may effectively protect against TNF-α- and lipopolysaccharide-induced endothelial junction disruption and vascular hyperpermeability.

PKCα activation of p120-catenin serine 879 phospho-switch disassembles VE-cadherin junctions and disrupts vascular integrity.

RATIONALE: Adherens junctions (AJs) are the primary intercellular junctions in microvessels responsible for endothelial barrier function. Homophilic adhesion of vascular endothelial (VE) cadherin forms AJs, which are stabilized by binding of p120-catenin (p120). p120 dissociation from VE-cadherin results in loss of VE-cadherin homotypic interaction and AJ disassembly; however, the signaling mechanisms regulating p120 dissociation from VE-cadherin are not understood. OBJECTIVE: To address the mechanism of protein kinase C (PKC)-α function in increasing endothelial permeability, we determined the role of PKCα phosphorylation of p120 in mediating disruption of AJ integrity. METHODS AND RESULTS: We showed that PKCα phosphorylation of p120 at serine (S)879 in response to thrombin or lipopolysaccharide challenge reduced p120 binding affinity for VE-cadherin and mediated AJ disassembly secondary to VE-cadherin internalization. In studies in mouse lung vessels, expression of the phosphodeficient S879A-p120 mutant prevented the increase in vascular permeability induced by activation of the thrombin receptor PAR-1. CONCLUSIONS: PKCα phosphorylation of p120 at S879 is a critical phospho-switch mediating disassociation of p120 from VE-cadherin that results in AJ disassembly. Therefore, blocking PKCα-mediated p120 phosphorylation represents a novel targeted anti-inflammatory strategy to prevent disruption of vascular endothelial barrier function.

The Ca(2+) sensor stromal interaction molecule 1 (STIM1) is necessary and sufficient for the store-operated Ca(2+) entry function of transient receptor potential canonical (TRPC) 1 and 4 channels in endothelial cells.

We addressed the requirement for stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+)-sensor, and Orai1, a Ca(2+) selective channel, in regulating Ca(2+) entry through the store-operated channels mouse transient receptor potential canonical (TRPC) 4 or human TRPC1. Studies were made using murine and human lung endothelial cells (ECs) challenged with thrombin known to induce Ca(2+) entry via TRPC1/4. Deletion or knockdown of TRPC4 abolished Ca(2+) entry secondary to depletion of ER Ca(2+) stores, preventing the disruption of the endothelial barrier. Knockdown of STIM1 (but not of Orai1or Orai3) or expression of the dominant-negative STIM1(K684E-K685E) mutant in ECs also suppressed Ca(2+) entry secondary to store depletion. Ectopic expression of WT-STIM1 or WT-Orai1 in TRPC4(-/-)-ECs failed to rescue Ca(2+) entry; however, WT-TRPC4 expression in TRPC4(-/-)-ECs restored Ca(2+) entry indicating the requirement for TRPC4 in mediating store-operated Ca(2+) entry. Moreover, expression of the dominant-negative Orai1(R91W) mutant or Orai3(E81W) mutant in WT-ECs failed to prevent thrombin-induced Ca(2+) entry. In contrast, expression of the dominant-negative TRPC4(EE647-648KK) mutant in WT-ECs markedly reduced thrombin-induced Ca(2+) entry. In ECs expressing YFP-STIM1, ER-store Ca(2+) depletion induced formation of fluorescent membrane puncta in WT but not in TRPC4(-/-) cells, indicating that mobilization of STIM1 and engagement of its Ca(2+) sensing function required TRPC4 expression. Coimmunoprecipitation studies showed coupling of TRPC1 and TRPC4 with STIM1 on depletion of ER Ca(2+) stores. Thus, TRPC1 and TRPC4 can interact with STIM1 to form functional store-operated Ca(2+)-entry channels, which are essential for mediating Ca(2+) entry-dependent disruption of the endothelial barrier.

Epinephrine induces rapid deterioration in pulmonary oxygen exchange in intact, anesthetized rats: a flow and pulmonary capillary pressure-dependent phenomenon.

BACKGROUND: Previous studies indicate epinephrine adversely affects arterial oxygenation when administered in a rat model of local anesthetic overdose. The authors tested whether epinephrine alone exerts similar effects in the intact animal. METHODS: Anesthetized rats received a single intravenous injection of epinephrine (25, 50, or 100 mcg/kg); matched cohorts were pretreated with phentolamine (100 mcg/kg); n = 5 for each of the six treatment groups. Arterial pressure and blood gases were measured at baseline, 1 and 10 min after epinephrine administration. Pulmonary capillary pressures during epinephrine infusion with normal and increased flows were measured in an isolated lung preparation. RESULTS: Epinephrine injection in the intact animal caused hypoxemia, hypercapnia, and acidosis at all doses. Arterial oxygen tension was reduced within 1 min of injection. Hyperlactatemia occurred by 10 min after 50 and 100 mcg/kg. Rate pressure product was decreased by 10 min after 100 mcg/kg epinephrine. Pretreatment with phentolamine attenuated these effects except at 100 mcg/kg epinephrine. In the isolated lung preparation, epinephrine in combination with increased pulmonary flow increased pulmonary capillary pressure and lung water. CONCLUSIONS: Bolus injection of epinephrine in the intact, anesthetized rat impairs pulmonary oxygen exchange within 1 min of treatment. Effects were blunted by α-adrenergic receptor blockade. Edema occurred in the isolated lung above a threshold pulmonary capillary pressure when epinephrine treatment was coupled with an increase in pulmonary flow. These results potentially argue against using traditional doses of epinephrine for resuscitation, particularly in the anesthetized patient.

Src phosphorylation of endothelial cell surface intercellular adhesion molecule-1 mediates neutrophil adhesion and contributes to the mechanism of lung inflammation.

OBJECTIVE: The goal of this study was to determine whether tumor necrosis factor α (TNFα)-induced Src activation and intercellular adhesion molecule-1 (ICAM-1) phosphorylation rapidly increase endothelial cell adhesivity and polymorphonuclear leukocyte (PMN) sequestration independently of de novo ICAM-1 synthesis. METHODS AND RESULTS: TNFα exposure of mouse lungs for 5 minutes produced a 3-fold increase in (125)I-anti-ICAM-1 monoclonal antibody (mAb) binding and (111)In oxine-labeled PMN sequestration, as well as Src activation, ICAM-1 Tyr518 phosphorylation, and phospho- Tyr518-ICAM-1 coimmunoprecipitation with actin. The response was absent in Nox2(-/-) lungs or following Src inhibition. In COS-7 cells transfected with wild-type (WT), phospho-defective (Tyr518Phe), or phospho-mimicking (Tyr518Asp) mouse ICAM-1 cDNA constructs, TNFα increased the B(max) of YN1/1.7.4 anti-ICAM-1 mAb binding to WT-ICAM-1 but not to Tyr518Phe-ICAM-1, indicating increased binding avidity secondary to ICAM-1 phosphorylation. This effect was mimicked by expression of the Tyr518Asp-ICAM-1 mutant. TNFα also increased the staining intensity and cell surface clustering of YN1/1.7.4 mAb-labeled WT-ICAM-1 that colocalized with F-actin, which was not observed with Tyr518Phe-ICAM-1 but was recapitulated with Tyr518Asp-ICAM-1. Finally, overexpression of ICAM-1 in mouse lungs significantly increased lipopolysaccharide-induced transvascular albumin leakage and bronchoalveolar lavage PMN counts at 2 and 24 hours after lipopolysaccharide inhalation compared with lungs expressing the Tyr518Phe ICAM-1 mutant. CONCLUSION: Src-dependent phosphorylation of endothelial cell ICAM-1 Tyr518 induces PMN adhesion by promoting ICAM-1 clustering, which we propose mediates rapid-phase lung vascular accumulation of PMNs during inflammation.

TAK1 is essential for endothelial barrier maintenance and repair after lung vascular injury.

TGF-ß-activated kinase 1 (TAK1) plays crucial roles in innate and adaptive immune responses and is required for embryonic vascular development. However, TAK1's role in regulating vascular barrier integrity is not well defined. Here we show that endothelial TAK1 kinase function is required to maintain and repair the injured lung endothelial barrier. We observed that inhibition of TAK1 with 5Z-7-oxozeaenol markedly reduced expression of ß-catenin (ß-cat) and VE-cadherin at endothelial adherens junctions and augmented protease-activated receptor-1 (PAR-1)- or toll-like receptor-4 (TLR-4)-induced increases in lung vascular permeability. In inducible endothelial cell (EC)-restricted TAK1 knockout (TAK1i∆EC) mice, we observed that the lung endothelial barrier was compromised and in addition, TAK1i∆EC mice exhibited heightened sensitivity to septic shock. Consistent with these findings, we observed dramatically reduced ß-cat expression in lung ECs of TAK1i∆EC mice. Further, either inhibition or knockdown of TAK1 blocked PAR-1- or TLR-4-induced inactivation of glycogen synthase kinase 3ß (GSK3ß), which in turn increased phosphorylation, ubiquitylation, and degradation of ß-cat in ECs to destabilize the endothelial barrier. Importantly, we showed that TAK1 inactivates GSK3ß through AKT activation in ECs. Thus our findings in this study point to the potential of targeting the TAK1-AKT-GSK3ß axis as a therapeutic approach to treat uncontrolled lung vascular leak during sepsis.

A novel function of sphingosine kinase 1 suppression of JNK activity in preventing inflammation and injury.

The mechanism underlying the protective effect of sphingosine kinase 1 (SphK1) in inflammatory injury is not clear. We demonstrated using SphK1-null mice (SphK1(-/-)) the crucial role of SphK1 in suppressing lipopolysaccharide-induced neutrophil oxidant production and sequestration in lungs and mitigating lung inflammatory injury. This effect of SphK1 was independent of the production of sphingosine 1-phosphate, the product of SphK1 activity. The anti-inflammatory effect of SphK1 in the lipopolysaccharide model was mediated through SphK1 interaction with JNK. SphK1 stabilization of JNK in turn inhibited JNK binding to the JNK-interacting protein 3 (JIP3) and thus abrogated the activation of NADPH oxidase and oxidant generation and resultant NF-kappaB activation. Therefore, SphK1-mediated down-regulation of JNK activity serves to dampen inflammation and tissue injury.