Xiaopei Gao

Phosphatidylinositol 3-kinase γ signaling through protein kinase Cζ induces NADPH oxidase-mediated oxidant generation and NF-κB activation in endothelial cells

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kγ in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kγ-mediated oxidant signaling in the mechanism of NF-κB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110γ catalytic subunit of PI3Kγ. Tumor necrosis factor (TNF) α challenge of wild type ECs caused p110γ translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110γ–/– ECs. ROS production was the result of TNFα activation of Ser phosphorylation of NADPH oxidase subunit p47phox and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110γ–/– ECs. Additionally, the TNFα-activated NF-κB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kγ. TNFα challenge of p110γ–/– ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCζ. Thus, PI3Kγ lies upstream of PKCζ in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-κB activation and ICAM-1 expression.

Endothelial cell–restricted disruption of FoxM1 impairs endothelial repair following LPS-induced vascular injury

Recovery of endothelial integrity after vascular injury is vital for endothelial barrier function and vascular homeostasis. However, little is known about the molecular mechanisms of endothelial barrier repair following injury. To investigate the functional role of forkhead box M1 (FoxM1) in the mechanism of endothelial repair, we generated endothelial cell-restricted FoxM1-deficient mice (FoxM1 CKO mice). These mutant mice were viable and exhibited no overt phenotype. However, in response to the inflammatory mediator LPS, FoxM1 CKO mice displayed significantly protracted increase in lung vascular permeability and markedly increased mortality. Following LPS-induced vascular injury, FoxM1 CKO lungs demonstrated impaired cell proliferation in association with sustained expression of p27(Kip1) and decreased expression of cyclin B1 and Cdc25C. Endothelial cells isolated from FoxM1 CKO lungs failed to proliferate, and siRNA-mediated suppression of FoxM1 expression in human endothelial cells resulted in defective cell cycle progression. Deletion of FoxM1 in endothelial cells induced decreased expression of cyclins, Cdc2, and Cdc25C, increased p27(Kip1) expression, and decreased Cdk activities. Thus, FoxM1 plays a critical role in the mechanism of the restoration of endothelial barrier function following vascular injury. These data suggest that impairment in FoxM1 activation may be an important determinant of the persistent vascular barrier leakiness and edema formation associated with inflammatory diseases.

Cdc42 Regulates Adherens Junction Stability and Endothelial Permeability by Inducing α-Catenin Interaction With the Vascular Endothelial Cadherin Complex

The endothelial adherens junctions (AJs) consist of trans-oligomers of membrane spanning vascular endothelial (VE)-cadherin proteins, which bind β-catenin through their cytoplasmic domain. β-Catenin in turn binds α-catenin and connects the AJ complex with the actin cytoskeleton. We addressed the in vivo effects of loss of VE-cadherin interactions on lung vascular endothelial permeability and the role of specific Rho GTPase effectors in regulating the increase in permeability induced by AJ destabilization. We used cationic liposomes encapsulating the mutant of VE-cadherin lacking the extracellular domain (&Dgr;EXD) to interfere with AJ assembly in mouse lung endothelial cells. We observed that lung vascular permeability (quantified as microvessel filtration coefficient [Kf,c]) was increased 5-fold in lungs expressing &Dgr;EXD. This did not occur to the same degree on expression of the VE-cadherin mutant, &Dgr;EXD&Dgr;β, lacking the β-catenin–binding site. The increased vascular permeability was the result of destabilization of VE-cadherin homotypic interaction induced by a shift in the binding of β-catenin from wild-type VE-cadherin to the expressed &Dgr;EXD mutant. Because &Dgr;EXD expression in endothelial cells activated the Rho GTPase Cdc42, we addressed its role in the mechanism of increased endothelial permeability induced by AJ destabilization. Coexpression of dominant-negative Cdc42 (N17Cdc42) prevented the increase in Kf,c induced by &Dgr;EXD. This was attributed to inhibition of the association of α-catenin with the &Dgr;EXD–β-catenin complex. The results demonstrate that Cdc42 regulates AJ permeability by controlling the binding of α-catenin with β-catenin and the consequent interaction of the VE-cadherin/catenin complex with the actin cytoskeleton.

Differential Role of CD18 Integrins in Mediating Lung Neutrophil Sequestration and Increased Microvascular Permeability Induced by Escherichia coli in Mice

The in vivo contributions of CD18 integrin-dependent and -independent mechanisms in mediating the increases in lung neutrophil (polymorphonuclear leukocyte; PMN) sequestration and microvascular permeability are not well understood. We determined the time course of these responses to Gram-negative sepsis in the mouse lung and addressed the specific contributions of CD18 integrins and ICAM-1. PMN sequestration in the lung was assessed by morphometric analysis, and transalveolar PMN migration was assessed by bronchoalveolar lavage. Lung tissue PMN number increased by 6-fold within 1 h after i.p. Escherichia coli challenge; this value peaked at 3 h (7-fold above control) and decreased at 12 h (3.5-fold above control). PMN migration into the airspace was delayed; the value peaked at 6 h and remained elevated up to 12 h. Saturating concentrations of anti-CD18 and anti-ICAM-1 mAbs reduced lung tissue PMN sequestration and migration; however, peak responses at 3 and 6 h were inhibited by 40%, indicating that only a small component of PMN sequestration and migration was CD18 dependent at these times. In contrast to the time-dependent decreased role of CD18 integrins in mediating PMN sequestration and migration, CD18 and ICAM-1 blockade prevented the increase in lung microvascular permeability and edema formation at all times after E. coli challenge. Thus, Gram-negative sepsis engages CD18/ICAM-1-independent mechanisms capable of the time-dependent amplification of lung PMN sequestration and migration. The increased pulmonary microvascular permeability induced by E. coli is solely the result of engagement of CD18 integrins even when PMN accumulation and migration responses are significantly CD18 independent.

Caveolin-1-eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability.

Caveolin-1–mediated inhibition of endothelial nitric oxide synthase signaling is essential for adherens junction integrity and endothelial permeability homeostasis.

E3 ubiquitin ligase Cblb regulates the acute inflammatory response underlying lung injury.

The E3 ubiquitin ligase Cblb has a crucial role in the prevention of chronic inflammation and autoimmunity. Here we show that Cblb also has an unexpected function in acute lung inflammation. Cblb attenuates the sequestration of inflammatory cells in the lungs after administration of lipopolysaccharide (LPS). In a model of polymicrobial sepsis in which acute lung inflammation depends on the LPS receptor (Toll-like receptor 4, TLR-4), the loss of Cblb expression accentuates acute lung inflammation and reduces survival. Loss of Cblb significantly increases sepsis-induced release of inflammatory cytokines and chemokines. Cblb controls the association between TLR4 and the intracellular adaptor MyD88. Expression of wild-type Cblb, but not expression of a Cblb mutant that lacks E3 ubiquitin ligase function, prevents the activity of a reporter gene for the transcription factor nuclear factor-κB (NF-κB) in monocytes that have been challenged with LPS. The downregulation of TLR4 expression on the cell surface of neutrophils is impaired in the absence of Cblb. Our data reveal that Cblb regulates the TLR4-mediated acute inflammatory response that is induced by sepsis.

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 &agr; (TNF&agr;)–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—TNF&agr; exposure of mouse lungs for 5 minutes produced a 3-fold increase in 125I-anti-ICAM-1 monoclonal antibody (mAb) binding and 111In 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, TNF&agr; increased the Bmax 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. TNF&agr; 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.

Evidence of a common mechanism of disassembly of adherens junctions through Gα13 targeting of VE-cadherin

Disruption of endothelial adherens junctions in response to inflammatory signals is mediated by the heterotrimeric G protein Gα13, which binds to VE-cadherin and induces VE-cadherin internalization through Src-dependent signaling pathway.

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-&agr; 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-&agr;–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-&agr; 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.

Sphingosine Kinase 1 Mediation of Expression of the Anaphylatoxin Receptor C5L2 Dampens the Inflammatory Response to Endotoxin

The complement anaphylatoxin C5a has a pathogenetic role in endotoxin-induced lung inflammatory injury by regulating phagocytic cell migration and activation. Endotoxin and C5a activate the enzyme sphingosine kinase (Sphk) 1 to generate the signaling lipid sphingosine-1-phosphate (S1P), a critical regulator of phagocyte function. We assessed the function of Sphk1 and S1P in experimental lung inflammatory injury and determined their roles in anaphylatoxin receptor signaling and on the expression of the two C5a receptors, C5aR (CD88) and C5L2, on phagocytes. We report that Sphk1 gene deficient (Sphk1−/−) mice had augmented lung inflammatory response to endotoxin compared to wild type mice. Sphk1 was required for C5a-mediated reduction in cytokine and chemokine production by macrophages. Moreover, neutrophils from Sphk1−/− mice failed to upregulate the anaphylatoxin receptor C5L2 in response to LPS. Exogenous S1P restored C5L2 cell surface expression of Sphk1−/− mouse neutrophils to wild type levels but had no effect on cell surface expression of the other anaphylatoxin receptor, CD88. These results provide the first genetic evidence of the crucial role of Sphk1 in regulating the balance between expression of CD88 and C5L2 in phagocytes. S1P-mediated up-regulation of C5L2 is a novel therapeutic target for mitigating endotoxin-induced lung inflammatory injury.