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Original citation: J Clin Invest. 2020;130(7):3684-3698. https://doi.org/10.1172/JCI136908. In the original version of Figure 4D, the representative lung histology sections of control and LPS-exposed animals, specifically the Rolipram (5 mg/kg)/PBS, Rolipram [...]

Unchecked inflammation is a hallmark of inflammatory tissue injury in diseases such as acute respiratory distress syndrome (ARDS). Yet the mechanisms of inflammatory lung injury remain largely unknown. Here we showed that bacterial endotoxin lipopolysaccharide (LPS) and cecal ligation and puncture-induced (CLP-induced) polymicrobial sepsis decreased the expression of transcription factor cAMP response element binding (CREB) in lung endothelial cells. We demonstrated that endothelial CREB was crucial for VE-cadherin transcription and the formation of the normal restrictive endothelial adherens junctions. The inflammatory cytokine IL-1[beta] reduced cAMP generation and CREB-mediated transcription of VEcadherin. Furthermore, endothelial cell-specific deletion of CREB induced lung vascular injury whereas ectopic expression of CREB in the endothelium prevented the injury. We also observed that rolipram, which inhibits type 4 cyclic nucleotide phosphodiesterase-mediated (PDE4-mediated) hydrolysis of cAMP, prevented endotoxemia-induced lung vascular injury since it preserved CREB-mediated VE-cadherin expression. These data demonstrate the fundamental role of the endothelial cAMP-CREB axis in promoting lung vascular integrity and suppressing inflammatory injury. Therefore, strategies aimed at enhancing endothelial CREB-mediated VE-cadherin transcription are potentially useful in preventing sepsis-induced lung vascular injury in ARDS., Introduction Sepsis-induced acute respiratory distress syndrome (ARDS), a syndrome characterized by acute respiratory failure and intractable hypoxemia, is the result of pulmonary vascular injury and edema formation (1, 2). The [...]

Acute lung injury is a leading cause of death in bacterial sepsis due to the wholesale destruction of the lung endothelial barrier, which results in protein-rich lung edema, influx of proinflammatory leukocytes, and intractable hypoxemia. Pyroptosis is a form of programmed lytic cell death that is triggered by inflammatory caspases, but little is known about its role in EC death and acute lung injury. Here, we show that systemic exposure to the bacterial endotoxin lipopolysaccharide (LPS) causes severe endothelial pyroptosis that is mediated by the inflammatory caspases, human caspases 4/5 in human ECs, or the murine homolog caspase-11 in mice in vivo. In caspase-11-deficient mice, BM transplantation with WT hematopoietic cells did not abrogate endotoxemia-induced acute lung injury, indicating a central role for nonhematopoietic caspase-11 in endotoxemia. Additionally, conditional deletion of caspase-11 in ECs reduced endotoxemia- induced lung edema, neutrophil accumulation, and death. These results establish the requisite role of endothelial pyroptosis in endotoxemic tissue injury and suggest that endothelial inflammatory caspases are an important therapeutic target for acute lung injury., Introduction Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury (ALI), is a devastating clinical complication of bacterial sepsis, with a mortality rate of greater than [...]

Cyclooxygenase 2 (COX-2) is induced by microbial products, pro-inflammatory cytokines, growth factors, and oncogenes. The Rho family includes RhoA, Rac1, Rac2, Rac3, and Cdc42 and is involved in regulation of the actin cytoskeleton organization, cell growth, vesicular cell trafficking, and transcriptional regulation. Rac2 binds to NADPH oxidase protein complex, and Rac2 null neutrophils are known to have poor phagocytic activity. We examined whether Rac2, the predominant small GTPase in hematopoietic cells, influences COX-2 expression in bone marrow-derived macrophages (BMDM). We showed that BMDM from [Rac2.sup.-/-] null mice have reduced COX-2 expression in response to treatment with endotoxin. Despite a compensatory increase in Racl, BMDM from [Rac2.sup.-/-] null mice have less biologically active GTP-bound Rac in response to LPS stimulation. Signaling molecules (downstream of Rac2 and Toll-like receptor 4) such as p42/44, p38, and pAKT were also affected in BMDM from [Rac2.sup.-/-] null mouse macrophages. We also observed that BMDM from [Rac2.sup.-/-] null failed to degrade I[kappa]B[alpha] significantly and had less immunoreactive PU.1. We show that both NF-[kappa]B pathway and PU.1 are involved in normal macrophage function and play a role in macrophage COX-2 expression. In summary, these data indicate that Rac2 regulates COX-2 expression in BMDM. small guanosine triphosphatases; Rac1; Cdc42

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-[kappa]B (NF-[kappa]B) 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., Author(s): Kurt Bachmaier (corresponding author) [1, 3]; Sophie Toya [1, 2, 3]; Xiaopei Gao [1]; Thomas Triantafillou [1]; Sean Garrean [1]; Gye Young Park [2]; Randall S Frey [1]; Stephen [...]

Lung inflammatory disease is characterized by increased polymorphonuclear leukocyte (PMN) infiltration and vascular permeability. PMN infiltration into tissue involves signaling between endothelial cells and migrating PMNs, which leads to alterations in the organization of adherens junctions (AJs). We addressed the possible role of the protein constituents of AJs, endothelium-specific vascular-endothelial (VE)-cadherin, in the migration of PMNs. Studies were made using VE-cadherin mutant constructs lacking the extracellular domain ([DELTA]EXD) or, additionally, lacking the COOH-terminus [beta]-catenin-binding domain ([DELTA]EXD[DELTA][beta]). Either construct was transduced in pulmonary microvessel endothelia of mice using cationic liposome-encapuslated cDNA constructs injected intravenously. Optimal expression of constructs was seen by Western blot analysis within 24 h. Vessel wall liquid permeability measured as the lung microvessel capillary filtration coefficient increased threefold in [DELTA]EXD-transduced lungs, indicating patency of interendothelial junctions, whereas the control [DELTA]EXD[DELTA][beta] construct was ineffective. To study lung tissue PMN recruitment, we challenged mice intraperitoneally with LPS (3 mg/kg) for 6 h and measured PMN numbers by bronchoalveolar lavage and their accumulation morphometrically in lung tissue. [DELTA]EXD expression markedly reduced the PMN sequestration and migration seen in nontransfected (control wild type) or [DELTA]EXD[DELTA] [beta]-transfected (negative control) mice challenged with LPS. In addition, [DELTA]EXD transfection suppressed LPS-induced activation of NF-[kappa]B and consequent ICAM-1 expression. These results suggest that disassembly of VE-cadherin junctions serves as a negative signal for limiting transendothelial PMN migration secondary to decreased ICAM-1 expression in the mouse model of LPS-induced sepsis. vascular endothelial cadherin; intercellular adhesion molecule-1; polymorphonuclear leukocyte adhesion; vascular endothelial-cadherin mutants

Gao, Xiaopei, Panos Kouklis, Ning Xu, Richard D. Minshall, Raudel Sandoval, Stephen M. Vogel and Asrar B. Malik. Reversibility of increased microvessel permeability in response to VE-cadherin disassembly. Am J Physiol Lung Cell Mol Physiol 279: L1218-L1225, 2000.--We determined the role of vascular endothelial (VE)-cadherin complex in regulating the permeability of pulmonary microvessels. Studies were made in mouse lungs perfused with albumin-Krebs containing EDTA, a [Ca.sup.2+] chelator, added to study the VE-cadherin junctional disassembly. We then repleted the perfusate with [Ca.sup.2+] to restore VE-cadherin integrity. Confocal microscopy showed a disappearance of VE-cadherin immunostaining in a time- and dose-dependent manner after [Ca.sup.2+] chelation and reassembly of the VE-cadherin complex within 5 min after [Ca.sup.2+] repletion. We determined the [sup.125]I-labeled albumin permeability-surface area product and capillary filtration coefficient ([K.sub.fc]) to quantify alterations in the pulmonary microvessel barrier. The addition of EDTA increased [sup.125]I-albumin permeability-surface area product and [K.sub.fc] in a concentration-dependent manner within 5 min. The permeability response was reversed within 5 min after repletion of [Ca.sup.2+]. All anti-VE-cadherin monoclonal antibody against epitopes responsible for homotypic adhesion augmented the increase in [K.sub.fc] induced by [Ca.sup.2+] chelation and prevented reversal of the response. We conclude that the disassembled VE-cadherins in endothelial cells are mobilized at the junctional plasmalemmal membrane such that VE-cadherins can rapidly form adhesive contact and restore microvessel permeability by reannealing the adherens junctions. vascular endothelial-cadherin; homotypic adhesion; vascular permeability; ethylenediaminetetraacetic acid; isolated lungs; mouse