jte-013 and Inflammation

The endothelium, as the interface between blood and all tissues, plays a critical role in inflammation. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, highly abundant in plasma, that potently regulates endothelial responses through interaction with its receptors (S1PRs). Here, we studied the role of S1PR2 in the regulation of the proadhesion and proinflammatory phenotype of the endothelium. By using genetic approaches and a S1PR2-specific antagonist (JTE013), we found that S1PR2 plays a key role in the permeability and inflammatory responses of the vascular endothelium during endotoxemia. Experiments with bone marrow chimeras (S1pr2(+/+) → S1pr2(+/+), S1pr2(+/+) → S1pr2(-/-), and S1pr2(-/-) → S1pr2(+/+)) indicate the critical role of S1PR2 in the stromal compartment, in the regulation of vascular permeability and vascular inflammation. In vitro, JTE013 potently inhibited tumor necrosis factor α-induced endothelial inflammation. Finally, we provide detailed mechanisms on the downstream signaling of S1PR2 in vascular inflammation that include the activation of the stress-activated protein kinase pathway that, together with the Rho-kinase nuclear factor kappa B pathway (NF-kB), are required for S1PR2-mediated endothelial inflammatory responses. Taken together, our data indicate that S1PR2 is a key regulator of the proinflammatory phenotype of the endothelium and identify S1PR2 as a novel therapeutic target for vascular disorders.

Topics: Acute Disease; Animals; Biomarkers; Blood Coagulation; Blood Vessels; Capillary Permeability; Endothelium, Vascular; Endotoxemia; Enzyme Activation; Human Umbilical Vein Endothelial Cells; Humans; Immunohistochemistry; Inflammation; Inflammation Mediators; Kidney; Mice; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phenotype; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Signal Transduction; Stromal Cells

Sphingosine-1-phosphate (S1P) is a bioactive sophospholipid with various S1P receptor (S1PR) expression profiles in cells of different origin. S1PR1, R3 and - to a lesser extent - R2 were the main receptors expressed in most of endothelial cells (ECs). The balances in the expression and activation of S1PR1, R2 and R3 help to maintain the physiological functions of ECs. Reverse transcription-PCR and Western blotting were used to detect the mRNA transcript level and protein expression of S1PR. Endothelial barrier function was measured by transflux of tracer protein through endothelial monolayer. Human dermal microvascular ECs predominantly expressed S1PR1 and S1PR3. Lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α) significantly upregulated S1PR2 mRNA and protein levels. The application of S1PR2 antagonist JTE-013 decreased the endothelial monolayer hyper-permeability response induced by LPS and TNF-α. Inflammatory mediators LPS and TNF-α induce S1PR2 expression in endothelium, suggesting that S1PR2 up-regulation may be involved in LPS and TNF-α elicited endothelial barrier dysfunction.

Topics: Blotting, Western; Capillary Permeability; Cells, Cultured; Dermis; Dose-Response Relationship, Drug; Endothelial Cells; Humans; Inflammation; Inflammation Mediators; Lipopolysaccharides; Microvessels; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine-1-Phosphate Receptors; Time Factors; Tumor Necrosis Factor-alpha; Up-Regulation

Sphingomyelin deposition and metabolism occurs in the atherosclerotic plaque, leading to the formation of sphingosine-1-phosphate (S1P), which activates G protein-coupled receptors to regulate vascular and immune cells. The role of S1P receptors in atherosclerosis has not been examined.. We tested the hypothesis that S1P receptor-2 (S1PR2) regulates atherosclerosis. Apoe(-/-) S1pr2(-/-) mice showed greatly attenuated atherosclerosis compared with the Apoe(-/-) mice. Bone marrow transplant experiments indicate that S1PR2 function in the hematopoietic compartment is critical. S1PR2 is expressed in bone marrow-derived macrophages and in macrophage-like foam cells in atherosclerotic plaques. Reduced macrophage-like foam cells were found in the atherosclerotic plaques of Apoe(-/-)S1pr2(-/-) mice, suggesting that S1PR2 retains macrophages in atherosclerotic plaques. Lipoprotein profiles, plasma lipids, and oxidized low-density lipoprotein uptake by bone marrow-derived macrophages were not altered by the S1pr2 genotype. In contrast, endotoxin-induced inflammatory cytokine (interleukin [IL]-1β, IL-18) levels in the serum of S1PR2 knockout mice were significantly reduced. Furthermore, treatment of wild-type mice with S1PR2 antagonist JTE-013 suppressed IL-1β and IL-18 levels in plasma.. These data suggest that S1PR2 signaling in the plaque macrophage regulates macrophage retention and inflammatory cytokine secretion, thereby promoting atherosclerosis.

Topics: Animals; Aortic Diseases; Apolipoproteins E; Atherosclerosis; Bone Marrow Transplantation; Disease Models, Animal; Endotoxins; Inflammation; Inflammation Mediators; Interleukin-18; Interleukin-1beta; Lipids; Lipoproteins; Lipoproteins, LDL; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Sphingosine-1-Phosphate Receptors

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 anti-inflammatory drugs.

Topics: Animals; Capillary Permeability; Chemotaxis, Leukocyte; Cycloserine; Edema; Inflammation; Lysophospholipids; Male; Mice; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Thiazolidines