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V A S C U L A R B I O L O G Y

HDL-bound sphingosine 1-phosphate acts as abiased agonist for the endothelial cell receptorS1P1 to limit vascular inflammationSylvain Galvani,1 Marie Sanson,1 Victoria A. Blaho,1 Steven L. Swendeman,1

Hideru Obinata,1* Heather Conger,2 Björn Dahlbäck,3 Mari Kono,4 Richard L. Proia,4

Jonathan D. Smith,2 Timothy Hla1†

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The sphingosine 1-phosphate receptor 1 (S1P1) is abundant in endothelial cells, where it regulates vascu-lar development and microvascular barrier function. In investigating the role of endothelial cell S1P1 inadult mice, we found that the endothelial S1P1 signal was enhanced in regions of the arterial vasculatureexperiencing inflammation. The abundance of proinflammatory adhesion proteins, such as ICAM-1, wasenhanced in mice with endothelial cell–specific deletion of S1pr1 and suppressed in mice with endothelialcell–specific overexpression of S1pr1, suggesting a protective function of S1P1 in vascular disease. Thechaperones ApoM+HDL (HDL) or albumin bind to sphingosine 1-phosphate (S1P) in the circulation; there-fore, we tested the effects of S1P bound to each chaperone on S1P1 signaling in cultured human umbilicalvein endothelial cells (HUVECs). Exposure of HUVECs to ApoM+HDL-S1P, but not to albumin-S1P, promotedthe formation of a cell surface S1P1–b-arrestin 2 complex and attenuated the ability of the proinflammatorycytokine TNFa to activate NF-kB and increase ICAM-1 abundance. Although S1P bound to either chaperoneinduced MAPK activation, albumin-S1P triggered greater Gi activation and receptor endocytosis. Endothelialcell–specific deletion of S1pr1 in the hypercholesterolemic Apoe−/−mouse model of atherosclerosis enhancedatherosclerotic lesion formation in the descending aorta. We propose that the ability of ApoM+HDL to actas a biased agonist on S1P1 inhibits vascular inflammation, which may partially explain the cardiovascularprotective functions of HDL.

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INTRODUCTION

Vascular inflammation and endothelial dysfunction represent early eventsin cardiovascular disease (1). During vascular inflammation, endothelialfunction is altered, characterized by impaired nitric oxide release and anincrease in the abundance of leukocyte adhesion molecules such as inter-cellular adhesion molecule–1 (ICAM-1) and vascular cell adhesionmolecule–1 (VCAM-1), leading to the recruitment of proinflammatoryimmune cells into the vascular wall (2). Endothelial inflammation isaffected by hemodynamic shear forces. In the descending aorta, laminarshear forces promote endothelial cell alignment, which suppresses inflam-mation. In contrast, in the aortic arch, the lesser curvature, and the branchpoints, turbulent flow induces random alignment of cells and vascular in-flammation. Such regions are prone to atherosclerotic plaque formation(3). Plasma lipoproteins play a crucial role for either protection from orpromotion of atherosclerosis (4). Indeed, altered concentrations of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) represent awell-established risk factor for cardiovascular disease. High plasma concen-trations of LDL injures the endothelium and promotes vascular inflamma-tion (5, 6), whereas HDL is protective, partially by promoting endothelialfunction (7). Although the atheroprotective effect of HDL was initially

1Center for Vascular Biology, Department of Pathology and Laboratory Medicine,Weill Cornell Medical College, Cornell University, New York, NY 10065, USA. 2De-partment of Cellular and Molecular Medicine, Cleveland Clinic Lerner ResearchInstitute, Cleveland, OH 44195, USA. 3Department of Translational Medicine,Skåne University Hospital, Lund University, 214 28 Malmö, Sweden. 4Geneticsof Development and Disease Branch, National Institute of Diabetes and Di-gestive and Kidney Diseases, National Institutes of Health, Bethesda, MD20892, USA.*Present address: Gunma University, Gunma, 371-8510 Japan.†Corresponding author. E-mail: tih2002@med.cornell.edu

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attributed primarily to cholesterol removal from tissues, anti-inflammatoryand endothelial protective functions of HDL may also be important (8).HDL particles are composed of apolipoproteins (such as ApoA-I, ApoA-II,and ApoM), enzymes involved in antioxidative mechanisms such asparaoxonase-1, lecithin-cholesterol acyltransferase, and diverse lipid species,including cholesterol esters, triglycerides, phospholipids (9), and bioactivesphingolipids such as sphingosine 1-phosphate (S1P) (10).

S1P is a lipid mediator that regulates many physiologic functions, especiallythose in the vascular and immune systems (11–13). It is concentrated in plasma(~1 mM) and carried primarily by the ApoM+ subfraction of HDL (~65%;ApoM+HDL) (14), with the remainder bound to plasma albumin (~35%).S1P signals through specific G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) [sphingosine 1-phosphate recep-tors 1 to 5 (S1P1–5)] and regulates various cellular responses. In particular, S1P1inhibits vascular permeability and is required for vascular development (15) aswell as immune cell trafficking.Moreover, a functional antagonist of S1P1 (fingolimod/Gilenya) has been approved to treat relapsing-remitting multiple sclerosis (16).S1P1 is abundant in endothelial cells and is critical for laminar shear stresssignaling and stabilization of the nascent vascular network (17). The abilityof various chaperones to modify the functions of S1P is not well understood.

The role of the S1P1 receptor in vascular pathology is unclear. In non-obese diabetic (NOD) mice, S1P1 is an anti-inflammatory receptor, itsstimulation leads to reduced adhesion of monocytes to endothelial cells(18). In addition, S1P1 stimulation in macrophages promotes an anti-inflammatory M2-like phenotype in vitro (19). In vivo, pharmacologicalagents (KRP203, an S1P1-specific agonist, or FTY720, which targets allS1P receptors except S1P2) attenuate atherosclerotic plaque developmentin Ldlr−/− and Apoe−/−mouse models (20–22). In contrast, stimulation of en-dothelial cells with S1P increases the surface abundance of adhesion mole-cules (VCAM-1 and ICAM-1), although it inhibits tumor necrosis factor–a

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(TNFa)–induced increases in the abundance of thesemolecules (23,24). Thisinhibitory effect has been ascribed to S1P1 and/or S1P3 stimulation (24, 25).Additionally, S1P cooperateswith lipopolysaccharide and induced inflamma-tory molecules and leukocyte adhesion on endothelial cells in vitro (26).These somewhat disparate findings underscore the need to clarify the roleof this abundant lipid mediator in vascular inflammation and atherosclerosis.

Here, we report that mouse endothelial S1P1 acted as an anti-inflammatoryand anti-atherosclerotic GPCR in vivo. In addition, ApoM+HDL engagedhuman endothelial S1P1 to form an anti-inflammatory signaling complexwith b-arrestin 2 that suppressed nuclear factor kB (NF-kB)–dependent in-flammatory pathways. Collectively, these data suggest that S1P bound to thechaperone ApoM+HDL activates the endothelial S1P1 GPCR as a biasedagonist to suppress inflammatory responses and atherosclerosis.

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RESULTS

S1P1 is differentially activated along theaortic endotheliumWe have shown (17) that S1P1 is localized on the plasma membrane in thedescending aorta. In contrast, in the lesser curvature, S1P1 is intracellularand colocalized with endosomal markers (17) (fig. S1A). The abundanceof the proinflammatory cell adhesion molecules, ICAM-1 and VCAM-1 (fig.

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S1, B and C), was increased in the lesser curvature compared to the descend-ing aorta. To study the role of S1P1 signaling and its role in inflammation,we used the S1P1 reporter mice [S1P1–green fluorescent protein (GFP) re-porter mice] (27). In these mice, endogenous activation of S1P1 leads to therecruitment of a b-arrestin/TEV (tobacco etch virus) protease fusion pro-tein, resulting in the release of a transcription factor that stimulates theexpression of nuclear-localized GFP reporter. In the lesser curvature,subclavian-carotid bifurcation, thoracic branching point, and aortic valves(Fig. 1), substantial numbers of endothelial cells had nuclear GFP, suggest-ing an increase of S1P1 signaling. In contrast, nuclear GFP was not de-tected in the regions of the aortic tree exposed to laminar shear stress such asmedial aspect of the carotid branch point or descending aorta (Fig. 1). These

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Fig. 1. S1P1 activation in the aortic endothelium exposed to disturbed shearforces. Aortae from S1P -GFP signaling mice (27) were dissected, and

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nuclei were counterstained in en face preparations. Nuclear GFP abun-dance was analyzed by immunofluorescence in aortic valve (yellow lining),lesser curvature (red lining), subclavian-carotid bifurcation (purple lining),carotid artery (pink lining), descending aorta (green lining), and thoracicaorta branch point (blue lining). Images are representative of eight miceobtained in five independent experiments.

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(S1pr1f/f Cdh5-CreERT2+/−) mice were dissected, and intima was stained inen face preparations. (A) Immunostaining of S1P1 and VE-cadherin. S1P1immunofluorescence was undetectable in S1pr1ECKO aortae. (B and C)Immunostaining for VCAM-1 (B) and ICAM-1 (C) in the presence or ab-sence of endothelial S1P1. Images are representative of 10 mice per geno-type obtained in 8 to 10 independent experiments. Aortae fromWT (S1pr1f/stop/f)and S1pr1ECGOF (S1pr1f/stop/f Cdh5-CreERT2+/−) mice were dissected, andintima was stained in en face preparations. (D) Immunostaining of S1P1 andVE-cadherin. S1P1 immunofluorescence was increased in S1pr1ECGOFaortae. (E) Immunostaining for ICAM-1 with and without endothelial S1P1overexpression. Images are representative of 10 mice per genotype ob-tained in five to seven independent experiments.

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results suggest a correlation between disturbed shear forces, inflammation, andexaggerated S1P1 signaling/internalization.

Endothelial S1pr1 expression suppressesvascular inflammationTo study the role of endothelial S1P1 in vascular inflammation, we usedthe endothelial cell–specific inducible knockout mouse line (S1pr1f/f

Cdh5-CreERT2, referred to as S1pr1ECKO) (17) and deleted endothelialS1pr1 at 4 weeks of age. Efficiency of deletion has been previously as-sessed by quantitative reverse transcription polymerase chain reactionanalysis of endothelial cells isolated from lung tissue (17) as well as byimmunofluorescence of the aortic tissue (Fig. 2A), which displayed an al-most complete loss of S1P1 immunofluorescence in the aortic endothelium.We did not detect differences in vascular endothelial (VE)–cadherin immu-

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nofluorescence between wild-type and S1pr1ECKO aortic endothelium.However, S1pr1ECKO aorta showed an increase in VCAM-1 andICAM-1 immunofluorescence (Fig. 2, B and C), especially in the de-scending aorta.

To further validate the anti-inflammatory effect of S1P1, we immuno-blotted dissected aortic extracts for ICAM-1 and VCAM-1. The abundanceof ICAM-1 and to a lesser extent that of VCAM-1 was significantly increasedin S1pr1ECKO aortae compared to control, especially in the descending aorta(fig. S2, A and B). Cell surface biotinylation assays of excised aortae showedincreased ICAM-I abundance at the surface of S1pr1ECKO aorta (fig. S2C).Together, these results suggested that deletion of S1pr1 in endothelial cellspromoted a proinflammatory state in the aortic endothelium.

To further confirm the involvement of S1P1 in the control of vascularinflammation, we next used the inducible endothelial-specific S1P1 over-

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expression mouse line (S1pr1f /stop / f

Cdh5-CreERT2, referred to as S1P1 ECGOF)(17). Overexpression of endothelial S1pr1was induced at 4 weeks of age. Efficiencyof the overexpression was assessed byimmunofluorescence of the aortic tissue(Fig. 2D), which showed a strong, albeitmosaic increase in S1P1 staining. Overex-pression of S1P1 in the vascular endotheli-um led to increased membrane localization ofS1P1 in the lesser curvature of S1pr1ECGOFaorta. Similar to S1pr1ECKO aorta, we didnot detect substantial differences in VE-cadherin staining. In contrast, S1pr1ECGOFanimals showed a decrease in ICAM-1 stain-ing (Fig. 2E) in both lesser curvature and de-scending aorta, suggesting that S1P1 suppressedendothelial inflammatory gene expression.

HDL-bound S1P attenuatescytokine-induced increases inadhesion molecule abundance inendothelial cellsOn the basis of the anti-inflammatory prop-erties of endothelial S1P1 in vivo, we hy-pothesized that S1P1 induces an anti-inflammatory signal in endothelial cells. Inhuman umbilical vein endothelial cells(HUVECs) treated with the proinflamma-tory cytokine TNFa, ICAM-1 abundanceincreased in a time-dependent fashion, aresponse that was attenuated (~70%) byco-incubation with HDL from wild-typemice (ApoM+ HDL) (Fig. 3, A and B).HDLparticles contained ~4.79 ± 0.975 pmolS1P per microgram of protein, as deter-mined by liquid chromatography–tandemmass spectrometry.We estimated theApoMconcentration in HDL preparations to be~160 ng/mg protein. Thus, under our ex-perimental conditions, HUVECs were ex-posed to ~1.5 to 2 mM S1P and ~2.5 mMApoM. In contrast, HDL isolated fromApom−/− mice (ApoM−HDL) did not signif-icantly suppress the TNFa-induced increasein ICAM-1 expression. These HDL particles

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presence of HDL isolated fromWT (ApoM+HDL) orApom−/− (ApoM−HDL) mice or albumin (Alb)–S1P. ICAM-1 abun-dance was assessed by immunoblot of total cell lysates. (B) ICAM-1 abundance was quantified from threeindependent experiments. Data are shown as means ± SD. *P < 0.05 by two-way analysis of variance(ANOVA) followed by Dunnett’s multiple comparison (no treatment compared to each treatment for every timepoint). A.U., arbitrary unit. (C) Serum-starved HUVECs were stimulated with human recombinant TNFa and withincreasing doses of ApoM−HDL or ApoM+HDL as indicated. ICAM-1 abundance was determined by immu-noblot analysis. (D) Data are shown as means ICAM abundance ± SD from three independent experiments.*P < 0.05, compared to TNFa alone by Kruskal Wallis test followed by Dunnett’s multiple comparison.

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do not contain detectable ApoM and had very low S1P concentrations(0.038 ± 0.008 pmol S1P/mg protein). Albumin-S1P did not suppress theTNFa-induced increase in ICAM-1 abundance to the same extent as HDL-S1P (Fig. 3, A and B). These results suggest that the anti-inflammatoryeffect of S1P depended on its associationwith ApoM+HDL.Dose-responseanalysis of HDL isolated from wild-type and Apom−/− mice (Fig. 3, Cand D) and albumin-S1P (fig. S3, A and B) further illustrated that theTNFa-induced increase in ICAM-1 abundance was inhibited in a dose-dependent manner by ApoM+HDL but not by ApoM−HDL particlesor albumin-S1P. Flow cytometry revealed that VCAM-1 abundance wasincreased by TNFa treatment (fig. S3C), a response that was partially sup-pressed (~40%) by S1P bound to recombinant ApoM but not by recom-binant ApoM alone, and to a lesser extent by albumin-bound S1P (~20%)(fig. S3C). Together, these data suggest that S1P bound to ApoM in HDLparticles potently suppressed cytokine-induced increases in adhesionmolecule abundance in endothelial cells.

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Cytokine-induced NF-kBactivation is attenuatedby HDL-bound S1PThe NF-kB pathway is a key inflammatorysignaling pathway induced by TNFa inendothelial cells, and its activation requiresthe phosphorylation of the p65 NF-kB sub-unit (28). TNFa stimulation ofHUVECs in-creased the phosphorylation of p65 within10 min, a response that was suppressed byco-incubation with human HDL (fig. S4) ina dose-dependent manner (Fig. 4, A and B).Similarly, HDL isolated from wild-typemice (ApoM+HDL) inhibited the phospho-rylation of p65 in a dose-dependentmanner,whereas ApoM−HDL was less effective(Fig. 4, C and D). These observations sug-gest that S1P bound to HDL antagonizedthe cytokine-inducedNF-kBpathway in en-dothelial cells.

HDL-S1P induces the persistentpresence of S1P1at the cell membraneHDL-S1P enhances endothelial barrier func-tion by activating S1P1 at the cell membrane(29).We investigated the localization of S1P1in HUVECs expressing GFP-tagged S1P1(14) and stimulated with various chaperone-bound forms of S1P. GFP-tagged S1P1 waslocalized at the cell membrane under basalconditions, which was not affected by TNFastimulation or HDL treatment. In contrast,stimulation with albumin-S1P or exposureto P-FTY720 induced receptor endocytosis(Fig. 5A). Cell surface biotinylation assays(fig. S5, A and B) confirmed that albumin-S1P and P-FTY720, but not HDL treatment,induced the disappearance of membraneS1P1. These results suggested that HDL-S1P activation of endothelial cells promotedcell surface S1P1, whereas albumin-S1P pro-motes rapid endocytosis of S1P1.

Cellular responses to S1P are chaperone-dependentActivation of S1P1 by its ligand S1P leads to Gi-dependent signaling, whichactivates the mitogen-activated protein kinase (MAPK) extracellular signal–regulated kinase (ERK) and inhibits adenylate cyclase (30). Both albumin-S1P and HDL-S1P induced rapid phosphorylation of ERK [Fig. 5B and(14)], in an S1P1-dependent manner (fig. S6). In contrast, forskolin-activatedincreases in cyclic adenosine 3′,5′-monophosphate (cAMP) concentrationswere inhibited by albumin-S1P, but not by HDL-S1P (Fig. 5C), suggestingthat HDL-S1P acts as a biased agonist for S1P1.

b-Arrestin 2 plays a crucial role in HDL-induced S1P1anti-inflammatory signalingAfter ligand stimulation, b-arrestins associate with GPCRs to promotedesensitization and endocytosis (31, 32). b-Arrestins also provide anadditional GPCR signaling platform in addition to the heterotrimeric Gprotein–regulated mechanisms (32, 33), leading to the activation of intracellular

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and mouse HDL from Apom−/− animals (ApoM−HDL) (C). Phosphorylation (p) of the NF-kB subunit p65was analyzed by immunoblot of total (tot) cell lysates. (B and D) Data in (B) and (D) represent combinedanalysis from three independent experiments and are shown as means ± SD. *P < 0.05, **P < 0.01, com-pared to TNFa alone by one-way ANOVA followed by Dunnett’s multiple comparison.

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signaling responses including the inhibition of the NF-kB pathway (34, 35).We therefore asked whether b-arrestin was involved in mediating endo-thelial cell anti-inflammatory pathways.

HDL, but not albumin-S1P, treatment increased the association of S1P1with b-arrestin 2 (Fig. 6A). In HUVECs, cell membrane localization ofDsRed-tagged b-arrestin 2 was induced by HDL-S1P but not by albumin-S1P (fig. S7). The ability of HDL to suppress the phosphorylation of p65after TNFa treatment was blocked by short hairpin RNA (shRNA)–mediatedknockdown of b-arrestin 2 but was not affected by overexpression ofb-arrestin 2 (Fig. 6, B and C).

To confirm the involvement of S1P1 in this response, HUVECs weretreated with the S1P1 inhibitor P-FTY720, which attenuated the abilityof HDL-S1P to suppress TNFa-induced increases in ICAM-1 abundance(Fig. 6, D and E). These data suggest that a specific ApoM+HDL-induced

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signaling complex consisting of S1P1 and b-arrestin 2 was critical to in-hibit cytokine-induced inflammatory responses.

Endothelial S1pr1 abundance influencesatherosclerosis developmentExaggerated inflammation in the vascular wall promotes atherosclerosis inthe presence of hypercholesterolemia and hyperlipidemia (36). To deter-mine the role of endothelial S1P1 in atherosclerosis, we compared Apoe−/−

S1pr1ECKOmice with Apoe−/− S1pr1wild-type littermates. As for S1pr1E-CKOmice (Fig. 2C), ICAM-1 immunofluorescencewas increased inApoe−/−

S1pr1ECKO mice, especially in the descending aorta (fig. S8).When placed on a high-fat diet for 16 weeks, weight gain (fig. S9A)

and plasma cholesterol concentrations (fig. S9B) were similar betweenApoe−/− S1pr1ECKO andApoe−/− S1pr1wild-typemice. In addition,mono-cyte (CD11b+, CD115+, Ly6G−), neutrophil (CD11b+, Ly6Cint, Ly6Ghi),T lymphocyte (CD4+ and CD8+), and B lymphocyte (CD19+) numberswere similar in Apoe−/− S1pr1ECKO and Apoe−/− S1pr1 wild-type mice(fig. S9, C and D). Also, plasma concentrations of ceramide and sphingo-sine species did not differ (fig. S10, A and B).

We next examined the development of atherosclerotic plaques usingOil Red O staining of en face preparations of the aorta (Fig. 7A). Quan-tification of plaque areas in the aortic arch (Fig. 7B) did not show sig-nificant differences. However, plaque development was significantlygreater in the branch points of the descending aorta in Apoe−/− S1pr1ECKOmice than in Apoe−/− S1pr1 wild-type mice, likely as a consequence ofincreased ICAM-1 and VCAM-1 abundance in this specific area (Fig. 7Band fig. S8). Consequently, total plaque areawas significantly higher in theApoe−/− S1pr1ECKOmice than in the Apoe−/−mice (Fig. 7B). Histologicalanalysis of the lesions with hematoxylin-eosin, Masson’s trichrome, orelastin staining indicated that plaque composition was similar in the twogenotypes (fig. S11A). However, ICAM-1 staining was increased in plaquesin Apoe−/− S1pr1ECKO animals (fig. S11B). Plaque areas in aortic roots(fig. S12, A and B) were similar in Apoe−/− S1pr1ECKO and Apoe−/−

S1pr1 wild-type mice. Although macrophage immunofluorescent stainingin the aortic root did not appear to differ between the two genotypes (Fig.7, C andD), moremacrophage immunofluorescent signalswere detected inplaques of the descending aorta in Apoe−/− S1pr1ECKO mice (Fig. 7, Eand F). Together, these results suggest that endothelial S1P1 exerts aregion-specific antiatherosclerotic effect in the descending aorta.

DISCUSSION

Although sphingolipids, which function together with sterols in cellular mem-branes, are thought to be critical in cardiovascular disease, their specificfunctions in vascular pathophysiology remain unclear. S1P and its recep-tors are of particular interest, because the ligand is enriched in plasma, andthe receptors are present on vascular and immune cells. Indeed, S1P2 reg-ulates myeloid cell retention in tissues during sterile inflammatory responsesand in mouse models of atherosclerosis, and S1pr2 knockout mice exhibitreduced atherosclerotic plaques (37, 38). In contrast, S1P1 stimulation ofmonocytes-macrophages induces an anti-inflammatory phenotype (19),suggesting a potential function of S1P receptors in myeloid cells duringvascular disease. However, despite its high abundance in the endothelium,the role of S1P1 during vascular inflammation and atherosclerosis is stillunknown.

A major finding of this work is that S1P1 localization on the plasmamembrane of aortic endothelial cells was inversely correlated with inflam-matory adhesion molecule abundance. The surface amount of S1P1 wassubstantially lower in endothelial cells of the lesser curvature, which areexposed to disturbed flow, than in those in the descending aorta. Analysis

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starvation, were stimulated with TNFa or with human HDL (huHDL), albumin-S1P, or P-FTY720 for the indicated times. Confocal microscopy was performedto assess the localization of GFP-tagged S1P1. Images are representative ofthree independent experiments. (B) Serum-starved HUVECs were stimulatedwith albumin-S1P or HDL-S1P for the indicated times. MAPK phosphorylationwas assessed by immunoblot of total lysates. (C) Serum-starved HUVECswere preincubated for 30 min with 3-isobutyl-1-methylxanthine (IBMX), andthen stimulated with increasing doses of albumin-S1P, HDL-S1P, orApoM−HDL in the presence of forskolin for 30 min. Results are expressedas picomole of cAMP per microgram of protein. Data represent combinedanalysis of five separate experiments and are expressed as means ± SD.**P < 0.01 by Friedman test followed by Dunnett’s multiple comparisons.

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of the S1P1-GFP reporter mice revealed that areas exposed to laminar sheardid not show detectable S1P1 signaling, whereas areas exposed to disturbedshear forces showed high signaling, which correlated with enhanced inter-nalization of S1P1 in these areas. Supraphysiological activation of S1P1results in enhanced endocytosis followed by degradation of the receptor

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(39, 40). The reason for this gradient of signaling, despite a uniformdistribution of the ligand in the circulation, is not clear. A possible explana-tion for the enhanced signaling could be the increased permeability of dis-turbed shear areas of the endothelium (41), which would allow plasmaS1P to access S1P receptors on the basolateral surface of the endothelium.

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Alternatively, disturbed shear stress per se orlocal inflammatory response could induceexaggerated S1P1 signaling.

Endothelial S1P1 signaling was analyzedwith tissue-specific genetic loss of functionandoverexpression inmice.S1pr1ECKOmiceshowed increased VCAM-1 and ICAM-1abundance, whereas mice that overexpressedS1pr1 on endothelial cells had decreasedabundance of these inflammatory markersin the aortic endothelium, suggesting thatit suppressed inflammatory responses. InHUVECs, TNFa-induced NF-kB activa-tion and adhesion molecule expressionwere suppressed by HDL-S1P, specificallyApoM+HDL.HDL-S1P induced the associ-ation of b-arrestin 2 with S1P1 and pro-moted receptor localization on the plasmamembrane, whereas S1P bound to albumin,its other chaperone in the plasma, suppressedforskolin-induced increases in cAMP con-centrations. The ability of HDL-S1P to sup-press inflammation depended on S1P1,because P-FTY720 pretreatment, which trig-gers the degradation of S1P1, antagonizedit. However, both HDL-S1P and albumin-S1P activated the MAPK pathway in anS1P1-dependent manner. Together, thesedata suggest that HDL-S1P acts as a biasedagonist for S1P1. The ability of specificchaperones to impart unique biological ac-tions of S1Pmay be a general phenomenon,because we have described a unique func-tion ofHDL-S1P to suppress lymphopoiesisin the bone marrow (42). The mechanismunderlying the differences in receptor acti-vation by HDL-S1P and albumin-S1P is notknown but could depend on co-receptors forHDL such as SR-B1 (25, 43).

Using def ined HDL preparations(ApoM+HDL and ApoM−HDL), weshowed that the association betweenS1P1 and b-arrestin 2 correlated with theanti-inflammatory activity of HDL-S1P. Inaddition to the role of b-arrestins in thedesensitization and internalizationofGPCRs(33), they can act as G protein–independentsignal transducers (44).For instance,b-arrestinscan inhibit the proinflammatory cascade atdifferent levels, by interacting eitherwith in-hibitor of nuclear factor kB a (IkBa) to en-hance its stability (34, 35) or with the IkBkinase (IKK) complex directly (34). More-over, studies on the receptor GPR120,which is activated by anw-3 polyunsaturated

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immunoprecipitation for S1P1 and immunoblotted (IB) with b-arrestin 2 antibody. Immunoblots arerepresentative of three independent experiments. (B) HUVECs stably overexpressing b-arrestin 2complementary DNA or an shRNA directed against b-arrestin 2 were serum-starved and stimulated withTNFa, with or without human HDL at the indicated doses. p65 phosphorylation was analyzed by immunoblotassay. (C) Data represent combined analysis of p65 phosphorylation from four independent experiments.*P < 0.05, **P < 0.01, #P < 0.005 by two-way ANOVA followed by Dunnett’s multiple comparison. (D)Serum-starved HUVECs were stimulated with TNFa in the absence or presence of albumin-S1P orApoM−HDL or human HDL as indicated. In the P-FTY720–treated experiments, serum-starved HUVECswere pretreated with P-FTY720 in the absence or presence of albumin-S1P, ApoM−HDL, or human HDLbefore TNFa stimulation in the presence of P-FTY720. ICAM-1 abundance was assessed by immuno-blotting. (E) Data represent the combined analysis of ICAM-1 abundance from three independentexperiments. **P < 0.01 by unpaired Student’s t test.

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fatty acid, showed that recruitment of b-arrestins (45) prevented down-stream activation of the IKK complex and NF-kB activation. By usingshRNA down-regulation in HUVECs, our data showed that b-arrestin 2was essential for HDL-S1P to inhibit cytokine-induced inflammatoryresponses.

In the Apoe−/− background, S1pr1ECKO mice show enhanced plaquesize, especially at branch points that are subject to disturbed shear stress andshow exaggerated S1P1 signaling. These mice did not show alterations inplasma cholesterol or sphingolipids, suggesting that attenuated signaling ofthe S1P1 receptor in the endothelial compartment is responsible for theaccelerated atherosclerosis. This suggests that endothelial S1P1 protectsthe vessel wall from inflammation and atherosclerosis. These observationsare consistent with findings that show that P-selectin–dependent leukocyterolling is enhanced in postcapillary venules of S1P1 endothelial knockoutmice (46). In conclusion, our results highlight ApoM+HDL chaperone–dependent anti-inflammatory and antiatherosclerotic function of S1P inthe endothelium. This occurs through the activation of S1P1, which trans-duces an anti-inflammatory signal in response to ApoM+HDL-bound S1P.The ability of the S1P chaperone ApoM+HDL to impart specific signal trans-duction mechanisms and an anti-inflammatory function in the endotheliummay partially explain the cardiovascular protective functions of HDL.

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MATERIALS AND METHODS

MiceAnimal experiments were approved by theWeill Cornell Medical College InstitutionalAnimal Care and Use Committee. Allexperiments were done in male animals.S1pr1f/f mice were crossed to Cdh5-CreERT2

mice (a gift from R. Adams, Max Planck In-stitute) (47) to generate S1pr1f/f Cdh5-CreERT2

mice. Gene deletion was achieved by intrape-ritoneal injection of tamoxifen (150 mg/day)to mice at 4 weeks of age for five consec-utive days. For the atherosclerosis lesion de-velopment studies, S1pr1f/f Cdh5-CreERT2 micewere crossed with Apoe−/− mice (JacksonLaboratory) for >4 generations to obtainmice that were >95% C57Bl/6 background,as determined by single-nucleotide polymor-phism analysis. Apoe−/− S1pr1f/f littermates(Cdh5-CreERT2–negative) were used ascontrols. After S1P1 deletion, male mice re-ceived high-fat chow (Teklad, TD88137;adjusted calories diet, 42% from fat) for 16weeks.

S1pr1f/stop/f was generated as described(17) and crossed toCdh5-CreERT2mice to gen-erate S1pr1f/stop/f Cdh5-CreERT2 mice. Geneoverexpression was achieved by intraperi-toneal injection of tamoxifen (150 mg/day)at 4 weeks of age for five consecutive days.Apom−/− mice were a gift of L. B. Nielsenand C. Christoffersen of Rigshospitalet.S1P1-GFP reporter mice have been previ-ously described (27).

Cell cultureHUVECs (passages 3 to 8) (Clonetics)were grown in MEM1999 supplemented

with 10% heat-inactivated fetal bovine serum (FBS), 10 mM Hepes,heparin-stabilized, penicillin (50 mg/ml), streptomycin (50 mg/ml), and en-dothelial cell growth factor as previously described (48). GFP-tagged S1P1overexpression was done as previously described (49).

En face protocol and stainingAfter cervical dislocation, the superior vena cava was opened, and micewere flushed with cold phosphate-buffered saline (PBS) containing 5%heparin and a cocktail of phosphatase inhibitors (1 mM Na3VO4, 1 mMNaF, 10 mM b-glycerophosphate) followed by intracardiac injection ofcold 4% paraformaldehyde (PFA) in PBS. Aortae were dissected and thenfixed for 20 min in 4% PFA. After a brief wash in glycine solution (100mM),followed by a10-min incubation in 0.1% Triton X-100, arteries were care-fully opened, and descending aorta, lesser curvature, and greater curvaturewere isolated. Tissues were then incubated for 2 hours in blocking solution[75 mM NaCl, 18 mM Na3 citrate, 2% FBS, 1% bovine serum albumin(BSA), 0.05% Triton X-100] as described previously (50), followed byincubation with primary antibodies diluted in blocking solution at 4°C un-der constant agitation.

After a 24-hour incubation with primary antibodies, arteries were washedfor 2 hours in washing solution (75 mM NaCl, 18 mM Na3 citrate, and

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en face preparation followed by Oil Red O staining was done. Shown here is a representative image ofaorta from each group. (B) Quantification of plaque areas in each section of aorta (aortic arch, descendingaorta, and total aorta) is shown (six Apoe−/− WT mice and eight Apoe−/− S1pr1ECKO mice). ns, not sig-nificant. (C) Aortic root sections from Apoe−/− S1pr1 WT and Apoe−/− S1pr1ECKO mice were immuno-stained with MOMA-2 to visualize the infiltration of macrophages and foam cells. (D) Quantification ofMOMA-2 infiltration in the aortic valve was assessed (n = 5 mice for each genotype). (E) Plaques fromdescending aorta of Apoe−/− S1pr1 WT and Apoe−/− S1pr1ECKO mice were isolated, and macrophagewas visualized by MOMA-2 staining followed by confocal microscopy. (F) Quantification of MOMA-2 stain-ing in the descending aorta lesion was assessed (n = 2 animals, two separate plaques analyzed for eachaorta). Data are shown as means ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test.

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0.05% Triton X-100), incubated with fluorescent secondary antibodies(Alexa Fluor dyes, Molecular Probes) for 1 hour, counterstained withTO-PRO-3, mounted, and then imaged using an Olympus FluoView FV10iconfocal microscope.

In vivo aortic protein biotinylationMale age-matched mice were anesthetized with ketamine/xylene. Themice underwent thoracotomy, and an incision was made in the right ven-tricle. The left ventricle was perfused with cold PBS for 5 min and then bycell-impermeable Sulfo-NHS-Biotin (1mg/ml) for 10min using an infusionpump (VWR) at a constant rate of 1 ml/min. Excess biotin was rinsed outwith consecutive 10-ml infusions of cold 50mMNH4Cl in PBS followed bycold PBS alone. The thoracic aortawas harvested and immediately frozen inliquid nitrogen.

ImmunoprecipitationAfter the indicated treatments, HUVECs were washed in ice-cold PBScontaining phosphatase inhibitors and lysed using modified radio-immunoprecipitation assay (RIPA) buffer [50 mM tris (pH 7.5), 150 mMNaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,1% Triton X-100, 0.5% FOS-choline, containing also 1 mM Na3VO4,1 mM NaF, 10 mM b-glycerophosphate, and protease inhibitor cocktail].Lysates were then incubated for 20 min at 4°C using a tube rotator, andthen centrifuged for 20 min at 13,500 rpm. Lysates were then cleared byprotein A/G beads (Pierce Protein A Agarose, Thermo Scientific; ProteinG Sepharose, GE Healthcare) and subjected to overnight immuno-precipitation using a monoclonal anti-S1P1 immunoglobulin G antibody(H60, Santa Cruz Biotechnology). Bead-bound proteins were then separatedon NuPAGE Novex 4 to 12% bis-tris gel (Invitrogen).

Cell surface biotinylationAfter stimulation, cells were treated with a nonpermeable biotinylationagent (EZ-Link Sulfo-NHS-SS-Biotin) (Thermo Scientific) and lysed witha mild detergent following the supplier’s protocol. Labeled proteins were thenisolated using NeutrAvidin Agarose beads (Thermo Scientific) and subjectedto Western blotting analysis.

Oil Red O staining of atherosclerotic plaquesAortic lipid content was analyzed and quantified using Oil Red O stain-ing, as described previously (51). Briefly, after CO2 asphyxiation, aortaewere harvested from heart to iliac bifurcation, opened in situ to expose theintima, and fixed in 4% PFA overnight. Lipids in plaques were stainedusing 0.5% Oil Red O solution. Once stained, aortae were digitally photo-graphed, and plaque quantitation was performed using the Image-ProAnalyzer software. Aortic root lesion was determined as describedbefore (38).

Monocyte/macrophage staining ofatherosclerotic plaquesCryosections of aortic root were incubated for 1 hour in blocking solution(75 mM NaCl, 18 mM Na3 citrate, 2% FBS, 1% BSA, 0.05% Triton X-100)before immunostaining using rat anti-mouse MOMA-2 (Serotec). After incu-bation with fluorescent secondary antibody and nuclear counterstainingusing DAPI (4′,6-diamidino-2-phenylindole) solution, sections were im-aged using confocal microscope system (Olympus, FV10i).

Plasma cholesterol quantificationCholesterol was quantified using Cholesterol E CHOD-DAOS method kit(Wako) according to the manufacturer’s instructions. Results areexpressed as microgram of cholesterol per deciliter of plasma.

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Blood cell collection and flow cytometryMicewere euthanized with CO2 asphyxiation. Blood was recovered by hepaticportal vein puncture and collected in tubes containing 35 ml of 0.1 M EDTA.Whole blood (400 ml) was removed to a new tube, centrifuged at 2000g for15 min, and plasma was removed and stored at −20°C. Cells were subjected toammonium chloride erythrocyte lysis, washed with PBS, and stained witheFluor 718 fixable viability dye (eBioscience) according to the manufacturer’sinstructions. Cells were then briefly fixed for 30 min in 0.1% PFA, washed,and Fc receptors were blocked with TruStain FcX (BioLegend) in 5% FBSovernight at 4°C. Cells were then stained with the following antibody mix-tures for 30 min on ice: CD4–phycoerythrin (PE) (RM4-4; BioLegend), CD8-PerCP/eFluor 710 (H35-17.2; eBioscience), CD19–eFluor 450 (ID3;eBioscience), CD11b–APC–eFluor 780 (M1/70; eBioscience), Ly6C–FITC(fluorescein isothiocyanate) (HK1.4; Southern Biotechnology), Ly6G-PE-Cy7 (1A8; BioLegend), and CD115-APC (AFS98; BioLegend). Liquidcounting beads (BD) were added to each sample, and data were obtainedon a BD LSR II (Becton Dickson) and analyzed using FlowJo software (TreeStar Inc.).

HDL isolationHDL3 was isolated by fractionation of outdated pooled plasma (NewYork Blood Center) or freshly isolated mouse plasma from wild-type andApom−/−mice. Sequential ultracentrifugation, using increasing density solu-tions, was used to separate the different lipoprotein fractions according totheir flotation (52). Endotoxin contamination was assessed using theLimulus assay (Enzo Life Science). Albumin contamination was analyzedby Western blotting, and only endotoxin-free, albumin-free HDL3 isola-tions were used.

Immunoblot experimentsHUVECs were washed with cold PBS containing phosphate inhibitors(1 mMNa3VO4, 5 mMNaF, and 10 mM b-glycerophosphate) and lysedwith modified RIPA buffer [1 mM ETDA, 50 mM tris (pH 7.5), 150 mMNaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100,0.5% FOS-choline] containing phosphatase inhibitors (1 mM Na3VO4,1mMNaF, and 10mM b-glycerophosphate) and protease inhibitor cocktail(Calbiochem). After a freeze/thaw cycle, lysates were sonicated, proteinconcentrations were determined by bicinchoninic acid assay (Pierce), andsamples were briefly denatured at 95°C for 5 min after addition of 10%b-mercaptoethanol. Equal amounts of protein were separated into 10%polyacrylamide gel and transblotted on polyvinylidene difluoride mem-brane. Transferred proteinswere then probedwith the antibodies as indicated.Antibodies were obtained from Cell Signaling (pp65 NF-kB, b-arrestin2, phospho P44-42 MAPK, and total ERK) or Santa Cruz Biotechnology(S1P1, total p65 NF-kB, ICAM-1, and VCAM-1).

cAMP measurementCells were starved for 3 hours. Cells were then preincubated for 30 minwith 1 mM IBMX (Tocris Bioscience) followed by stimulation for 30 minwith albumin-S1P, ApoM− HDL, or ApoM+HDL, in the presence ofIBMX (1 mM) and forskolin (20 mM) (Tocris Bioscience). cAMP mea-surement was then assessed using the Direct cAMP ELISA (enzyme-linked immunosorbent assay) kit (Enzo Life Sciences) following thesupplier’s protocol.

Statistical analysisWestern blot quantification was done with ImageJ software (NationalInstitutes of Health). Data are expressed as means ± SD. Statistical anal-ysis was performed as mentioned using Prism software (Prism, GraphPad).P values <0.05 were considered significant.

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SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/8/389/ra79/DC1Fig. S1. S1P1 localization in en face preparations of the mouse aorta.Fig. S2. ICAM-1 and VCAM-1 abundance in aorta upon S1P1 deletion.Fig. S3. Effect of albumin and albumin-S1P on TNFa-induced increases in ICAM-1 andVCAM-1 abundance.Fig. S4. Characterization of the sphingolipid composition of human HDL.Fig. S5. Effect of S1P carrier on S1P1 retention at the cell surface.Fig. S6. Effect of S1P1 inhibition in S1P-induced activation of MAPK.Fig. S7. Subcellular localization of b-arrestin 2 depends on the S1P carrier.Fig. S8. Genetic modulation of S1P1 expression in endothelial cells and modification ofICAM-1 abundance.Fig. S9. Analysis of weight, plasma cholesterol, and cell populations in Apoe−/− S1pr1wild-type and ECKO animals after 16 weeks on a high-fat diet.Fig. S10. Plasma sphingolipid characterization.Fig. S11. Analysis of plaque composition.Fig. S12. Atherosclerotic root lesion quantification.

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www

Acknowledgments: We thank R. Adams, L. B. Nielsen, and C. Christoffersen for the giftof unique strains of mice. We also thank S. Narain and T. Prasad for their biostatisticaladvices and Kezhi Dai for help with the in vivo biotinylation assays. Funding: This workwas supported by NIH grants (PO1-HL70694 and RO1-HL89934 to T.H. and F32-CA142117 to V.A.B.). V.A.B. is a Leon Levy Research Fellow of the Weill Cornell MedicalCollege Brain and Mind Research Institute. Sphingolipid measurements are supported inpart by Lipidomics Shared Resource, Hollings Cancer Center, Medical University of SouthCarolina (P30 CA138313) and the Lipidomics Core in the South Carolina Lipidomics andPathobiology Centers of Biomedical Research Excellence (COBRE) (P20 RR017677).Author contribution: S.G. designed the studies, carried out most of the experiments,and analyzed the data. M.S. helped in the generation of Apoe−/− S1pr1 mutant mice. V.A.B.performed flow cytometry analysis on blood from Apoe−/− S1pr1 mutant animals. S.L.S.prepared and characterized mice HDL. H.O. constructed the lentiviral vectors used in this study.H.C. performed the aortic root Oil Red O staining and the root atherosclerotic plaque analysis.B.D., R.L.P., M.K., and J.D.S. provided conceptual input and critical reagents. T.H. providedconceptual input, supervised the study, and wrote the manuscript with S.G. Competing in-terests: The authors declare that they have no competing interests.

Submitted 7 November 2014Accepted 15 July 2015Final Publication 11 August 201510.1126/scisignal.aaa2581Citation: S. Galvani, M. Sanson, V. A. Blaho, S. L. Swendeman, H. Obinata, H. Conger,B. Dahlbäck, M. Kono, R. L. Proia, J. D. Smith, T. Hla, HDL-bound sphingosine 1-phosphateacts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation. Sci.Signal. 8, ra79 (2015).

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to limit vascular inflammation1S1PHDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor

Mari Kono, Richard L. Proia, Jonathan D. Smith and Timothy HlaSylvain Galvani, Marie Sanson, Victoria A. Blaho, Steven L. Swendeman, Hideru Obinata, Heather Conger, Björn Dahlbäck,

DOI: 10.1126/scisignal.aaa2581 (389), ra79.8Sci. Signal. 

cholesterol,'' in atherosclerosis.''biased'' signaling pathways, which may also contribute to the protective effect of HDL, commonly called ''good

HDL. Thus, S1P bound to different chaperones triggered distinct or+endothelial cells when bound to the lipoprotein ApoMmediator that is abundant in blood bound to different chaperone proteins. S1P suppressed inflammation in cultured

which is activated by S1P, a lipid1,atherosclerosis in mice were suppressed by the endothelial cell receptor S1P. found that vascular inflammation andet alinflammation, which contributes to atherosclerotic plaque formation. Galvani

Flow through blood vessels subjects endothelial cells to abnormal shear forces at specific locations that triggerMaintaining vascular health with HDL

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