sphingosine-1-phosphate and Pulmonary-Edema

In the lungs, increased vascular permeability can lead to acute lung injury. Because vascular permeability is regulated primarily by endothelial cells, many researchers have studied endothelial cell monolayers in culture, in order to understand the pathomechanisms of pulmonary edema. Such studies are based on the assumption that endothelial cells in culture behave like endothelial cells in situ. Here we show that this assumption is largely unfounded. Cultured endothelial cells show profound differences compared to their physiological counterparts, including a dysregulated calcium homeostasis. They fail to reproduce the pulmonary responses to agents such as platelet-activating factor. In contrast, they respond in a Rho-kinase depend fashion to thrombin, LPS or TNF. This is a striking finding for three reasons: (i) in the lungs, none of these agents increases vascular permeability by a direct interaction with endothelial cells; (ii) The endothelial Rho-kinase pathway seems to play little role in the development of pulmonary edema; (iii) This response pattern is similar for many endothelial cells in culture irrespective of their origin, which is in contrast to the stark heterogeneity of endothelial cells in situ. It appears that most endothelial in culture tend to develop a similar phenotyp that is not representative of any of the known endothelial cells of the lungs. We conclude that at present cultured endothelial cells are not useful to study the pathomechanisms of pulmonary edema.

Topics: Animals; Capillary Permeability; Endothelial Cells; Humans; Lung; Lysophospholipids; Platelet Activating Factor; Pulmonary Edema; rho-Associated Kinases; Signal Transduction; Sphingosine; Thrombin

Acute ethanol intoxication increases the risk of sepsis and aggravates the symptoms of sepsis and lung injury. Therefore, this study aimed to explore whether sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/S1P receptor 1 (S1PR1) signaling pathway functions in lung injury caused by acute ethanol intoxication-enhanced sepsis, as well as its underlying mechanism. The acute ethanol intoxication model was simulated by intraperitoneally administering mice with 32% ethanol solution, and cecal ligation and puncture (CLP) was used to construct the sepsis model. The lung tissue damage was observed by hematoxylin-eosin (H&E) staining, and the wet-to-dry (W/D) ratio was used to evaluate the degree of pulmonary edema. Inflammatory cell counting and protein concentration in bronchoalveolar lavage fluid (BALF) were, respectively, detected by hemocytometer and bicinchoninic acid (BCA) method. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and IL-18 in BALF were detected by their commercial enzyme-linked immunosorbent assay (ELISA) kits. The myeloperoxidase (MPO) activity and expression of apoptosis-related proteins and SphK1/S1P/S1PR1 pathway-related proteins were, respectively, analyzed by MPO ELISA kit and Western blot analysis. The cell apoptosis in lung tissues was observed by TUNEL assay. Acute ethanol intoxication (EtOH) decreased the survival rate of mice and exacerbated the lung injury caused by sepsis through increasing pulmonary vascular permeability, neutrophil infiltration, release of inflammatory factors, and cell apoptosis. In addition, EtOH could activate the SphK1/S1P/S1PR1 pathway in CLP mice. However, PF-543, as a specific inhibitor of SphK1, could partially reverse the deleterious effects on lung injury of CLP mice. PF-543 alleviated lung injury caused by sepsis in acute ethanol intoxication rats by suppressing the SphK1/S1P/S1PR1 signaling pathway.

Topics: Alcoholic Intoxication; Animals; Apoptosis; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Inflammation Mediators; Lung; Lung Injury; Lysophospholipids; Male; Methanol; Mice, Inbred C57BL; Neutrophil Infiltration; Oxidative Stress; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Pulmonary Edema; Pyrrolidines; Sepsis; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Sulfones

Sepsis and acute lung injury (ALI) have devastatingly high mortality rates. Both are associated with increased vascular leak, a process regulated by complex molecular mechanisms.. We hypothesized that integrin αvβ3 could be an important determinant of vascular leak and endothelial permeability in sepsis and ALI.. β3 subunit knockout mice were tested for lung vascular leak after endotracheal LPS, and systemic vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. Possible contributory effects of β3 deficiency in platelets and other hematopoietic cells were excluded by bone marrow reconstitution experiments. Endothelial cells treated with αvβ3 antibodies were evaluated for sphingosine-1 phosphate (S1P)–mediated alterations in barrier function, cytoskeletal arrangement, and integrin localization.. β3 knockout mice had increased vascular leak and pulmonary edema formation after endotracheal LPS, and increased vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. In endothelial cells, αvβ3 antibodies inhibited barrier-enhancing and cortical actin responses to S1P. Furthermore, S1P induced translocation of αvβ3 from discrete focal adhesions to cortically distributed sites through Gi- and Rac1-mediated pathways. Cortical αvβ3 localization after S1P was decreased by αvβ3 antibodies, suggesting that ligation of the αvβ3 with its extracellular matrix ligands is required to stabilize cortical αvβ3 focal adhesions.. Our studies identify a novel mechanism by which αvβ3 mitigates increased vascular leak, a pathophysiologic function central to sepsis and ALI. These studies suggest that drugs designed to block αvβ3 may have the unexpected side effect of intensifying sepsis- and ALI-associated vascular endothelial leak.

Topics: Actins; Acute Lung Injury; Animals; Disease Models, Animal; Endothelium, Vascular; Female; Integrin alphaVbeta3; Lysophospholipids; Mice; Mice, Knockout; Pulmonary Edema; Sepsis; Signal Transduction; Sphingosine; Vascular Diseases

GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the sphingosine-1-phosphate (S1P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S1P receptor 1 (S1P₁) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S1P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics.

Topics: Animals; Capillary Leak Syndrome; Dose-Response Relationship, Drug; Endocytosis; Fingolimod Hydrochloride; Gene Knock-In Techniques; Lymphopenia; Lysophospholipids; Mice; Organophosphates; Peptide Hydrolases; Phosphorylation; Propylene Glycols; Protein Processing, Post-Translational; Protein Structure, Tertiary; Pulmonary Edema; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; Recombinant Fusion Proteins; Sphingosine; Sphingosine-1-Phosphate Receptors; Ubiquitin-Protein Ligases; Ubiquitination

Two mammalian sphingosine kinase (SphK) isoforms, SphK1 and SphK2, possess identical kinase domains but have distinct kinetic properties and subcellular localizations, suggesting each has one or more specific roles in sphingosine-1-phosphate (S1P) generation. Although both kinases use sphingosine as a substrate to generate S1P, the mechanisms controlling SphK activation and subsequent S1P generation during lung injury are not fully understood. In this study, we established a murine lung injury model to investigate LPS-induced lung injury in SphK1 knockout (SphK1(-/-)) and wild-type (WT) mice. We found that SphK1(-/-) mice were much more susceptible to LPS-induced lung injury compared with their WT counterparts, quantified by multiple parameters including cytokine induction. Intriguingly, overexpression of WT SphK1 delivered by adenoviral vector to the lungs protected SphK1(-/-) mice from lung injury and attenuated the severity of the response to LPS. However, adenoviral overexpression of a SphK1 kinase-dead mutant (SphKKD) in SphK1(-/-) mouse lungs further exacerbated the response to LPS as well as the extent of lung injury. WT SphK2 adenoviral overexpression also failed to provide protection and, in fact, augmented the degree of LPS-induced lung injury. This suggested that, in vascular injury, S1P generated by SphK2 activation plays a distinctly separate role compared with SphK1-dependent S1P generation and survival signaling. Microarray and real-time RT-PCR analysis of SphK1 and SphK2 expression levels during lung injury revealed that, in WT mice, LPS treatment caused significantly enhanced SphK1 expression ( approximately 5x) levels within 6 h, which declined back to baseline levels by 24 h posttreatment. In contrast, expression of SphK2 was gradually induced following LPS treatment and was elevated within 24 h. Collectively, our results for the first time demonstrate distinct functional roles of the two SphK isoforms in the regulation of LPS-induced lung injury.

Topics: Adenoviridae; Animals; Gene Deletion; Gene Expression Regulation, Enzymologic; Gene Transfer Techniques; Lipopolysaccharides; Lung; Lung Injury; Lysophospholipids; Membrane Proteins; Mice; Mice, Inbred C57BL; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Pulmonary Edema; Sphingosine; Time Factors; Tumor Necrosis Factor-alpha

Little is known about the contribution of bone marrow-derived progenitor cells (BMPCs) in the regulation endothelial barrier function as defined by microvascular permeability alterations at the level of adherens junctions (AJs).. We investigated the role of BMPCs in annealing AJs and thereby in preventing lung edema formation induced by endotoxin (LPS).. We observed that BMPCs enhanced basal endothelial barrier function and prevented the increase in pulmonary microvascular permeability and edema formation in mice after LPS challenge. Coculture of BMPCs with endothelial cells induced Rac1 and Cdc42 activation and AJ assembly in endothelial cells. However, transplantation of BMPCs isolated from sphingosine kinase-1-null mice (SPHK1(-/-)), having impaired S1P production, failed to activate Rac1 and Cdc42 or protect the endothelial barrier.. These results demonstrate that BMPCs have the ability to reanneal endothelial AJs by paracrine S1P release in the inflammatory milieu and the consequent activation of Rac-1 and Cdc42 in endothelial cells.

Topics: Adherens Junctions; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Capillary Permeability; cdc42 GTP-Binding Protein; Cell Movement; Cell Separation; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Flow Cytometry; Humans; Lipopolysaccharides; Lung; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuropeptides; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Edema; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; Signal Transduction; Sphingosine; Stem Cells; Time Factors

Topics: Adherens Junctions; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Capillary Permeability; cdc42 GTP-Binding Protein; Cell Movement; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Humans; Lung; Lysophospholipids; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Edema; rac GTP-Binding Proteins; Signal Transduction; Sphingosine; Stem Cells; Time Factors

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(-/-) 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(-/-) lungs or SPHK1-deficient endothelial cells restored endothelial barrier function. Our results suggest the crucial role of activation of the SPHK1-->S1P-->S1P1 signaling pathway in response to inflammatory mediators in endothelial cells in regulating endothelial barrier homeostasis.

Topics: Animals; Capillary Permeability; Cells, Cultured; Enzyme Activation; Hemostatics; Humans; Inflammation Mediators; Intercellular Junctions; Lipopolysaccharides; Lung; Lysophospholipids; Mice; Mice, Knockout; Neuropeptides; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Edema; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; Receptor, PAR-1; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Thrombin

To investigate the effects of sphingosine-1-phosphate (S1P) and its analogue FTY720 on the lung injury induced by acute necrotizing pancreatitis in rats.. Acute necrotizing pancreatitis was induced by retrogradely injection of 5% sodium taurocholate of biliopancreatic duct in rats. Sphingosine-1-phosphate (100 microg/kg) or FTY720 (1 mg/kg) was administered immediately after the model induction by peritoneal injection. Six hours after the model induction, bronchoalveolar lavage protein concentration, total cell count, polymorphonuclear neutrophil percentage, proinflammatory cytokines (interleukin 1beta, interleukin 6, and tumor necrosis factor alpha), nuclear factor kappaB activation of alveolar macrophages, and lung myeloperoxidase (MPO) activity were examined. Serum amylase and lipase were tested. In addition, histopathological changes of the pancreas and lung were observed.. Bronchoalveolar lavage protein concentration, total cell count, PMN percentage, proinflammatory cytokines, nuclear factor kappaB activation, lung capillary leakage, and lung myeloperoxidase were all reduced significantly in both S1P and FTY720 groups. The pulmonary pathological injury in both S1P and FTY720 groups was ameliorated obviously. Nevertheless, the serum amylase, lipase, and the pancreatic pathological damages were not decreased.. Sphingosine-1-phosphate and its analogue FTY720 significantly decreased pulmonary inflammation and injury in a rat model of acute lung injury caused by acute necrotizing pancreatitis and may represent a novel therapeutic strategy for the acute necrotizing pancreatitis-associated lung injury.

Topics: Amylases; Animals; Bronchoalveolar Lavage Fluid; Capillary Permeability; Cytokines; Fingolimod Hydrochloride; Lipase; Lung; Lung Injury; Lysophospholipids; Macrophages, Alveolar; Male; NF-kappa B; Pancreatitis, Acute Necrotizing; Peroxidase; Propylene Glycols; Pulmonary Edema; Rats; Rats, Wistar; Sphingosine

S1P acts via the S1PR family of G protein-coupled receptors to regulate a variety of physiological responses. Whereas S1P1R activates G(i)- and PI-3-kinase-dependent signals to inhibit vascular permeability, the related S1P2R inhibits the PI-3-kinase pathway by coupling to the Rho-dependent activation of the PTEN phosphatase. However, cellular consequences of S1P2R signaling in the vascular cells are not well understood.. Selective signaling of the S1P2R was achieved by adenoviral-mediated expression in endothelial cells. Secondly, endogenously expressed S1P2R was blocked by the specific pharmacological antagonist JTE013. Activation of S1P2R in endothelial cells resulted in Rho-ROCK- and PTEN-dependent disruption of adherens junctions, stimulation of stress fibers, and increased paracellular permeability. JTE013 treatment of naive endothelial cells potentiated the S1P1R-dependent effects such as formation of cortical actin, blockade of stress fibers, stimulation of adherens junction assembly, and improved barrier integrity. This observation was extended to the in vivo model of vascular permeability in the rat lung: the S1P2R antagonist JTE013 significantly inhibited H2O2-induced permeability in the rat lung perfused model.. S1P2R activation in endothelial cells increases vascular permeability. The balance of S1P1 and S1P2 receptors in the endothelium may determine the regulation of vascular permeability by S1P.

Topics: Adherens Junctions; Animals; Antigens, CD; Cadherins; Capillary Permeability; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Humans; Hydrogen Peroxide; Intracellular Signaling Peptides and Proteins; Lysophospholipids; Phosphorylation; Protein Serine-Threonine Kinases; PTEN Phosphohydrolase; Pulmonary Edema; Pyrazoles; Pyridines; rac GTP-Binding Proteins; Rats; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Stress Fibers; Time Factors; Transfection

Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo-active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S1P1 tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S1P-S1P1 'set points' among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention.

Topics: Anilides; Animals; Capillary Permeability; Cells, Cultured; CHO Cells; Cricetinae; Disease Models, Animal; Evans Blue; Humans; Lymph Nodes; Lymphocytes; Lysophospholipids; Mice; Mice, Inbred C57BL; Models, Biological; Organophosphonates; Phenotype; Pulmonary Edema; Receptors, Lysosphingolipid; Sphingosine; Stereoisomerism

Excessive mechanical stress is a key component of ventilator-associated lung injury, resulting in profound vascular leak and an intense inflammatory response. To extend our in vitro observations concerning the barrier-protective effects of the lipid growth factor sphingosine 1-phosphate (Sph 1-P), we assessed the ability of Sph 1-P to prevent regional pulmonary edema accumulation in clinically relevant rodent and canine models of acute lung injury induced by combined intrabronchial endotoxin administration and high tidal volume mechanical ventilation. Intravenously delivered Sph 1-P significantly attenuated both alveolar and vascular barrier dysfunction while significantly reducing shunt formation associated with lung injury. Whole lung computed tomographic image analysis demonstrated the capability of Sph 1-P to abrogate significantly the accumulation of extravascular lung water evoked by 6-hour exposure to endotoxin. Axial density profiles and vertical density gradients localized the Sph 1-P response to transitional zones between aerated and consolidated lung regions. Together, these results indicate that Sph 1-P represents a novel therapeutic intervention for the prevention of pulmonary edema related to inflammatory injury and increased vascular permeability.

Topics: Acute Disease; Animals; Bronchoalveolar Lavage Fluid; Capillary Permeability; Disease Models, Animal; Dogs; Extravascular Lung Water; Female; Linear Models; Lung Injury; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Probability; Pulmonary Circulation; Pulmonary Edema; Respiration, Artificial; Respiratory Function Tests; Severity of Illness Index; Sphingosine; Tomography, X-Ray Computed