Sphingosine-1-Phosphate Prevents Tumor Necrosis Factor-α–Mediated Monocyte Adhesion to Aortic Endothelium in Mice

Abstract

Objective-Endothelial activation and monocyte adhesion to endothelium are key events in inflammation. Sphingosine-1-phosphate (S1P) is a sphingolipid that binds to G protein-coupled receptors on endothelial cells (ECs). We examined the role of S1P in modulating endothelial activation and monocyte-EC interactions in vivo. Methods and Results-We injected C57BL/6J mice intravenously with tumor necrosis factor (TNF)-␣ in the presence and absence of the S1P1 receptor agonist SEW2871 and examined monocyte adhesion. Aortas from TNF-␣-injected mice had a 4-fold increase in the number of monocytes bound, whereas aortas from TNF-␣ plus SEW2871-treated mice had few monocytes bound (PϽ0.0001). Using siRNA, we found that inhibiting the S1P1 receptor in vascular ECs blocked the ability of S1P to prevent monocyte-EC interactions in response to TNF-␣. We examined signaling pathways downstream of S1P1 and found that 100 nM S1P increased phosphorylation of Akt and decreased activation of c-jun. Conclusions-Thus, we provide the first evidence that S1P signaling through the endothelial S1P1 receptor protects the vasculature against TNF-␣-mediated monocyte-EC interactions in vivo. Key Words: endothelium Ⅲ sphingosine-1-phosphate Ⅲ inflammation Ⅲ Endothelium differentiation gene (Edg) receptors I nflammation is a hallmark of atherosclerosis and diabetes. 1 Monocyte-endothelial interactions are key initiating events of inflammation. 2 Activated monocytes release tumor necrosis factor (TNF)-␣ that mediates a variety of pathological vascular responses. 3 Monoclonal antibody therapies to reduce TNF-␣ have reduced inflammation in several chronic diseases, 4 but these therapies do not prevent the initiation of inflammation.Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid in circulation that evokes a variety of cellular responses, including cell migration, vascular maturation, and lymphocyte homing. 5 However, the role of S1P in vascular inflammation is unknown. S1P binds to 5 G protein-coupled receptors (Edg receptors) on endothelium and activates these receptors in the low nanomolar range. 6,7 In the vasculature, S1P receptors are found on both endothelial cells (ECs) and monocytes. 8,9 S1P1 is involved in endothelial migration and blood vessel formation. 10 S1P3 regulates heart rate in mice 11 and vascular tone through activation of endothelial nitric oxide synthase (eNOS). 12,13 In the current study, we report that S1P prevents TNF-␣-mediated monocyte adhesion to intact aorta and to cultured primary isolates of aortic endothelial cells. We found that the protective, antiinflammatory action of S1P is primarily mediated through binding to the endothelial S1P1 receptor. MethodsDetailed Methods for all experiments can be found online. Please see http://atvb.ahajournals.org. Results S1P and an S1P1 Receptor Agonist Prevent TNF-Mediated Monocyte-Endothelial InteractionsWe injected C57BL/6J (B6) mice with 0.5 g of recombinant murine TNF-␣ intravenously, and either 5 mg/kg of the selective S1P1 receptor agonist, SEW2871 (5...

FTY720 Promotes Local Microvascular Network Formation and Regeneration of Cranial Bone Defects

“…Studies have shown that S1P prevents monocyte adhesion to the endothelium via the S1P 1 receptor, thereby limiting its extravasation into the tissue space. 17 Thus, we hypothesized that treatment with S1P, FTY720, and VPC01091 would diminish the amount of the CD45 þ cells in the defect space compared with controls. …”

Induction of Scavenger Receptor Class B Type I Is Critical for Simvastatin Enhancement of High-Density Lipoprotein-Induced Anti-Inflammatory Actions in Endothelial Cells

“…The cells were washed by perfusion of RPMI 1640 medium containing 0.1% BSA at 37°C for 10 min. THP-1 monocytic cell suspension at the concentration of 10 6 cells/ml was then allowed to flow over HUVECs at the shear stress of 0.75 dymes/cm 2 as described (30). Five minutes later, the number of THP-1 monocytic cells attached to HUVECs was counted.…”

“…At 2 h postinjection, aortas were harvested from mice and immediately placed into 10% FBS containing DMEM. The ex vivo monocyte adhesion assay was performed as described by Bolick et al (30). In brief, the aortas were opened up longitudinally and pinned onto sterile agar.…”

Sphingosine-1-Phosphate Reduces CD4+ T-Cell Activation in Type 1 Diabetes Through Regulation of Hypoxia-Inducible Factor Short Isoform I.1 and CD69

“…Our laboratory routinely uses a monocyte:endothelial adhesion assay that uses primary mouse endothelial cells and monocytes (3,21,24). We modified this method to study T-cell:endothelial adhesion.…”