Sphingosine-1-Phosphate In the Plasma Compartment Regulates Basal and Inflammation-Induced Vascular Leak In Mice (original) (raw)
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Engagement of S1P1-degradative mechanisms leads to vascular leak in mice
Journal of Clinical Investigation, 2011
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-1phosphate (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 (S1P 1 ) 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.
Journal of Pharmacology and Experimental Therapeutics, 2011
The sphingosine kinase (SPK)/sphingosine-1-phosphate (S1P) pathway recently has been associated with a variety of inflammatory-based diseases. The majority of these studies have been performed in vitro. Here, we have addressed the relevance of the SPK/S1P pathway in the acute inflammatory response in vivo by using different well known preclinical animal models. The study has been performed by operating a pharmacological modulation using 1) L-cycloserine and DL-threo-dihydrosphingosine (DTD), S1P synthesis inhibitors or 2) 2-undecyl-thiazolidine-4-carboxylic acid (BML-241) and N-(2,6-dichloro-4-pyridinyl)-2-[1,3-dimethyl-4-(1-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-hydrazinecarboxamide (JTE-013), specific S1P 2 and S1P 3 receptor antagonists. After local injection of carrageenan in mouse paw S1P release significantly increases locally and decreases during the resolution phase. Expression of SPKs and S1P 2 and S1P 3 receptors is increased in inflamed tissues. Administration of L-cycloserine or DTD caused a significant anti-inflammatory effect. By using different animal models we have also demonstrated that the SPK/S1P pathway contributes to changes in vascular permeability and promotes cell recruitment. The S1P effect on cell recruitment results is receptor-mediated because both JTE-013 and BML-241 inhibited zymosan-induced cell chemotaxis without effect on vascular leakage. Conversely, changes in vascular permeability involve mainly SPK activity, because compound 48/80-induced vascular leakage was significantly inhibited by DTD. In conclusion, the SPK/S1P pathway is involved in acute inflammation and could represent a valuable therapeutic target for developing a new class of antiinflammatory drugs. This work was supported by the Ministero della Università e della Ricerca PRIN 2006 Italy.
PLoS ONE, 2014
Sphingosine-1-phosphate (S1P) is an agonist for five distinct G-protein coupled receptors, that is released by platelets, mast cells, erythrocytes and endothelial cells. S1P promotes endothelial cell barrier function and induces release of endothelial cell-specific storage-organelles designated Weibel-Palade bodies (WPBs). S1P-mediated enhancement of endothelial cell barrier function is dependent on S1P receptor 1 (S1PR1) mediated signaling events that result in the activation of the small GTPase Rac1. Recently, we have reported that Rac1 regulates epinephrine-induced WPB exocytosis following its activation by phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1). S1P has also been described to induce WPB exocytosis. Here, we confirm that S1P induces release of WPBs using von Willebrand factor (VWF) as a marker. Using siRNA mediated knockdown of gene expression we show that S1PR1 is not involved in S1P-mediated release of WPBs. In contrast depletion of the S1PR3 greatly reduced S1P-induced release of VWF. S1P-mediated enhancement of endothelial barrier function was not affected by S1PR3-depletion whereas it was greatly impaired in cells lacking S1PR1. The Rho kinase inhibitor Y27632 completely abrogated S1P-mediated release of VWF. Also, the calcium chelator BAPTA-AM significantly reduced S1P-induced release of VWF. Our findings indicate that S1P-induced release of haemostatic, inflammatory and angiogenic components stored within WPBs depends on the S1PR3.
Journal of Pharmacology and Experimental Therapeutics, 2007
Ca 2ϩ signaling plays an important role in endothelial cell (EC) functions including the regulation of barrier integrity. Recently, the endogenous lipid derivative, sphingosine-1-phosphate (S1P), has emerged as an important modulator of EC barrier function. We investigated the role of endogenously generated S1P in Ca 2ϩ metabolism and barrier function in human umbilical endothelial cells (HUVECs) stimulated by thrombin, histamine, or other agonists. Barrier function was assessed by dextran diffusion through HUVEC monolayers, and Ca 2ϩ transients were measured using a fluoroprobe. Thrombin or histamine increased Ca 2ϩ release from the endoplasmic reticulum (ER) and Ca 2ϩ entry through store-operated channels (SOCs) that was accompanied by increased EC permeability. Inhibition of S1P synthesis by a specific sphingosine kinase inhibitor (SKI) decreased thrombin or histamine-induced increased permeability and decreased Ca 2ϩ entry via SOC in a concentration-This work was supported by National Institutes of Health-National Institute of General Medical Sciences Grants GM59841 and 69790 (to E.A.D.), GM69861 (to Z.S.), and GM059179 (to C.J.H.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
Circulation Research, 2008
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. (Circ Res. 2008;103:1164-1172.) Running title: Sphingosine kinase-1 activation prevents vascular leak *, equally contributed to the work Subject Codes: [95] Endothelium, [176] Thrombin, [138] Cell signaling/signal transduction, [18] Pulmonary circulation and disease
PloS one, 2016
Homeostasis of vascular barriers depends upon sphingosine 1-phosphate (S1P) signaling via the S1P1 receptor. Accordingly, S1P1 competitive antagonism is known to reduce vascular barrier integrity with still unclear pathophysiological consequences. This was explored in the present study using NIBR-0213, a potent and selective S1P1 competitive antagonist. NIBR-0213 was tolerated at the efficacious oral dose of 30 mg/kg BID in the rat adjuvant-induced arthritis (AiA) model, with no sign of labored breathing. However, it induced dose-dependent acute vascular pulmonary leakage and pleural effusion that fully resolved within 3-4 days, as evidenced by MRI monitoring. At the supra-maximal oral dose of 300 mg/kg QD, NIBR-0213 impaired lung function (with increased breathing rate and reduced tidal volume) within the first 24 hrs. Two weeks of NIBR-0213 oral dosing at 30, 100 and 300 mg/kg QD induced moderate pulmonary changes, characterized by alveolar wall thickening, macrophage accumulation...
Journal of Biological Chemistry, 2008
and eventually enter an irreversible growth arrest state called "cellular senescence." It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P 1-3 receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P 1 and S1P 2 was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P 2 receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P 2 to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P 2 -mediated activation of the lipid phosphatase PTEN. . 2 The abbreviations used are: S1P, sphingosine 1-phosphate; GPCR, G-protein-coupled receptor; S1P 1 and S1P 2 (old nomenclature EDG-1, endothelial differentiating gene-1, and EDG-5, respectively), two high affinity GPCRs for S1P; PTEN, phosphatase and tensin homolog deleted on chromosome 10; WT-and DN-PTEN, wild-type and dominant-negative PTEN constructs (PTEN-C/S, the catalytic Cys-124 is mutated to Ser); FAK, focal adhesion kinase; TEER, transendothelial electrical resistance; ECIS, electrical cell-substrate impedance-sensing technique; HUVEC, human umbilical vein endothelial cell; EC, cultured endothelial cell; PDs, population doublings of cultured cells; CPDL, cumulative population doubling level; SA, senescent-associated; PECAM, platelet/endothelial cell adhesion molecule; m.o.i., multiplicity of infection; BSA, bovine serum albumin; ERK, extracellular signal-regulated kinase; siRNA, small interference RNA; BrdUrd, bromodeoxyuridine; RT, reverse transcription. Downloaded from