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.Caspase-11-mediated endothelial pyroptosis underlies endotoxemia-induced lung injury The Journal of Clinical Investigation R E S E A R C H A R T I C L E4 1 2 5 jci.org Volume 127 Number 11 November 2017the complex process of inflammation involves multiple programmed cell death pathways, depending on the type and magnitude of the inciting stimulus and cell type. From an evolutionary perspective, pyroptosis of cells harboring intracellular bacteria or in which LPS has breached the plasma membrane is an effective means of eliminating an intracellular bacterial niche and activating the host through release of inflammatory mediators such as IL-1β, while sparing uninfected neighboring cells (13,(28)(29)(30). Thus, pyroptosis induced by inflammatory caspases 1/4/5/11 is an innate immune response distinct from the canonical inflammasome activation pathway via cell-surface TLR4 (11,(31)(32)(33)(34). However, caspase-11 can also be immunopathologic in sepsis (35). Caspase-11-deficient mice were protected in endotoxemic shock (10,11), suggesting that in the setting of an overwhelming inflammatory response, the potentially protective pyroptotic mechanism activates an exaggerated pathologic response due to overwhelming cell lysis. The role of the inflammatory caspases 4/5/11 in mediating cytoplasmic LPS signaling and pyroptosis has until now been primarily studied in macrophages or dendritic cells (11,12,14,28,32,36). Their role in destroying the endothelial barrier through widespread endothelial death and pathogenesis of ALI remains unknown. Here, we tested the hypothesis that lung ECs are a primary target for pyroptosis via intracellular sensing of LPS by the inflammatory caspases 4/5/11 and that endothelial pyroptosis is required for the induction of ALI. Results LPS in ECs induces pyroptotic cell death via activation of inflammatory cas...

We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein Gi, and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial 125I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11–amino acid Gαi carboxyl-terminal peptide inhibited endothelial 125I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of Gαi with the caveolin-1, whereas dn-Src inhibited Gαi binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60 with caveolin-1, followed by the activation of the downstream Gi-coupled Src kinase signaling pathway.

Abstract-We investigated the possibility that the TRPC gene family of putative store-operated Ca 2ϩ entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca 2ϩ signaling. Studies were made in wild-type (wt) and TRPC4 knockout (TRPC4 Ϫ/Ϫ ) mice and lung vascular endothelial cells (LECs) isolated from these animals. RT-PCR showed expression of TRPC1, TRPC3, TRPC4, and TRPC6 mRNA in wt LECs, but TRPC4 mRNA expression was not detected in TRPC4

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Caveolin‐1 (Cav‐1), the principal structural protein of caveolae, is implicated in normal endothelial barrier function, buts its role in lung inflammation is not well understood. Using caveolin‐1 knockout (Cav‐1 −/−) mice, we addressed the role of Cav‐1 in sepsis‐induced lung injury. We assessed lung inflammation in Cav‐1 −/− mice following i.p. injection of LPS. Neutrophil (PMN) binding was measured by an adhesion assay performed on primary cultured mouse endothelial cells. PMN sequestration was assessed by myeloperoxidase activity in the whole lung. Lung microvascular permeability was determined using iodine‐125 radio‐labeled albumin. When compared to wild type (WT) mice, Cav‐1 −/− mice exhibited significantly impaired PMN binding and sequestration after LPS challenge. LPS‐induced increases in lung microvascular permeability and edema formation were also markedly reduced in Cav‐1 −/− mice relative to WT. To address the basis of the reduced lung inflammation, we examined the specific role of nitric oxide (NO). As Cav‐1 is known to sequester eNOS rendering it inactive, we postulated that in Cav‐1 −/− mice there should be increased eNOS activity and eNOS‐derived NO levels in response to LPS challenge. We observed a marked increase in eNOS activity and NO production in Cav‐1 −/− lung relative to WT. Corresponding to the known role of NO in modulating NF‐κB activity, we also noted a time‐dependent suppression of NF‐κB activity in Cav‐1 −/− mice. Thus, Cav‐1 expression and its ability to regulate eNOS‐derived NO production is a crucial determinant of the lung inflammatory response to sepsis. This abstract is funded by: NIH(T32HL007829, HL60678, HL77806)

Abstract-The lipid mediator sphingosine-1-phosphate (S1P), the product of sphingosine kinase (SPHK)-induced phosphorylation of sphingosine, is known to stabilize interendothelial junctions and prevent microvessel leakiness. Here, we investigated the role of SPHK1 activation in regulating the increase in pulmonary microvessel permeability induced by challenge of mice with lipopolysaccharide or thrombin ligation of protease-activating receptor (PAR)-1. Both lipopolysaccharide and thrombin increased mouse lung microvascular permeability and resulted in a delayed activation of SPHK1 that was coupled to the onset of restoration of permeability. In contrast to wild-type mice, Sphk1 Ϫ/Ϫ mice showed markedly enhanced pulmonary edema formation in response to lipopolysaccharide and PAR-1 activation. Using endothelial cells challenged with thrombin concentration (50 nmol/L) that elicited a transient but reversible increase in endothelial permeability, we observed that increased SPHK1 activity and decreased intracellular S1P concentration preceded the onset of barrier recovery. Thus, we tested the hypothesis that released S1P in a paracrine manner activates its receptor S1P1 to restore the endothelial barrier. Knockdown of SPHK1 decreased basal S1P production and Rac1 activity but increased basal endothelial permeability. In SPHK1-depleted cells, PAR-1 activation failed to induce Rac1 activation but augmented RhoA activation and endothelial hyperpermeability response. Knockdown of S1P1 receptor in endothelial cells also enhanced the increase in endothelial permeability following PAR-1 activation. S1P treatment of Sphk1 Ϫ/Ϫ lungs or SPHK1-deficient endothelial cells restored endothelial barrier function. Our results suggest the crucial role of activation of the SPHK13 S1P3 S1P1 signaling pathway in response to inflammatory mediators in endothelial cells in regulating endothelial barrier homeostasis. Key Words: sphingosine kinase Ⅲ lung vascular permeability Ⅲ thrombin Ⅲ PAR-1 Ⅲ RhoGTPases Ⅲ S1P1 Ⅲ S1P T he vascular endothelium forms a semipermeable barrier separating intravascular and tissue compartments. Disruption of endothelial barrier is a crucial factor in the pathogenesis of tissue inflammation, the hallmark of inflammatory diseases such as the acute respiratory distress syndrome. 1 Increased microvessel endothelial permeability leads to protein-rich alveolar edema that severely impairs oxygenation. 2 Thrombin, a serine protease, generated during sepsis and intravascular coagulation, ligates the endothelial cell surface receptor protease activating receptor 1 (PAR-1) and increases endothelial permeability. 1,[3][4][5][6] This increase in endothelial permeability is typically followed by a recovery period of Ϸ2 hours, during which barrier integrity is restored. 7,8 It has been surmised that PAR-1 signaling stimulates intrinsic repair mechanisms that restore barrier function. 7-9 Sphingosine-1-phosphate (S1P), a lipid mediator, was shown to be 1 such factor promoting endothelial barrier function. 10 -13 S1P binds to S...

Increased endothelial permeability is the hallmark of inflammatory vascular edema. Inflammatory mediators that bind to heptahelical G protein-coupled receptors trigger increased endothelial permeability by increasing the intracellular Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i activates key signaling pathways that mediate cytoskeletal reorganization (through myosin-light-chain-dependent contraction) and the disassembly of VE-cadherin at the adherens junctions. The Ca2+-dependent protein kinase C (PKC) isoform PKCalpha plays a crucial role in initiating endothelial cell contraction and disassembly of VE-cadherin junctions. The increase in [Ca2+]i induced by inflammatory agonists such as thrombin and histamine is achieved by the generation of inositol 1,4,5-trisphosphate (IP3), activation of IP3-receptors, release of stored intracellular Ca2+, and Ca2+ entry through plasma membrane channels. IP3-sensitive Ca2+-store depletion activates plasma membrane cation channels (i.e., store-operated cation channels [SOCs] or Ca2+ release-activated channels [CRACs]) to cause Ca2+ influx into endothelial cells. Recent studies have identified members of Drosophila transient receptor potential (TRP) gene family of channels that encode functional SOCs in endothelial cells. These studies also suggest that the canonical TRPC homologue TRPC1 is the predominant isoform expressed in human vascular endothelial cells, and is the essential component of the SOC in this cell type. Further, evidence suggests that the inflammatory cytokine tumor necrosis factor-alpha can induce the expression of TRPC1 in human vascular endothelial cells signaling via the nuclear factor-kappaB pathway. Increased expression of TRPC1 augments Ca2+ influx via SOCs and potentiates the thrombin-induced increase in permeability in human vascular endothelial cells. Deletion of the canonical TRPC homologue in mouse, TRPC4, caused impairment in store-operated Ca2+ current and Ca2+-store release-activated Ca2+ influx in aortic and lung endothelial cells. In TRPC4 knockout (TRPC4-/-) mice, acetylcholine-induced endothelium-dependent smooth muscle relaxation was drastically reduced. In addition, TRPC4-/- mouse-lung endothelial cells exhibited lack of actin-stress fiber formation and cell retraction in response to thrombin activation of protease-activated receptor-1 (PAR-1) in endothelial cells. The increase in lung microvascular permeability in response to PAR-1 activation was inhibited in TRPC4-/- mice. These results indicate that endothelial TRP channels such as TRPC1 and TRPC4 play an important role in signaling agonist-induced increases in endothelial permeability.

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