Research ArticleVASCULAR BIOLOGY

HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation

See allHide authors and affiliations

Science Signaling  11 Aug 2015:
Vol. 8, Issue 389, pp. ra79
DOI: 10.1126/scisignal.aaa2581
  • Fig. 1 S1P1 activation in the aortic endothelium exposed to disturbed shear forces.

    Aortae from S1P1-GFP signaling mice (27) were dissected, and nuclei were counterstained in en face preparations. Nuclear GFP abundance 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 thoracic aorta branch point (blue lining). Images are representative of eight mice obtained in five independent experiments.

  • Fig. 2 Genetic modulation of endothelial S1P1 expression and inflammatory marker expression.

    Aortae from wild-type (WT) (S1pr1f/f) and S1pr1ECKO (S1pr1f/f Cdh5-CreERT2+/−) mice were dissected, and intima was stained in en face preparations. (A) Immunostaining of S1P1 and VE-cadherin. S1P1 immunofluorescence was undetectable in S1pr1ECKO aortae. (B and C) Immunostaining for VCAM-1 (B) and ICAM-1 (C) in the presence or absence of endothelial S1P1. Images are representative of 10 mice per genotype obtained in 8 to 10 independent experiments. Aortae from WT (S1pr1f/stop/f) and S1pr1ECGOF (S1pr1f/stop/f Cdh5-CreERT2+/−) mice were dissected, and intima was stained in en face preparations. (D) Immunostaining of S1P1 and VE-cadherin. S1P1 immunofluorescence was increased in S1pr1ECGOF aortae. (E) Immunostaining for ICAM-1 with and without endothelial S1P1 overexpression. Images are representative of 10 mice per genotype obtained in five to seven independent experiments.

  • Fig. 3 S1P chaperone–dependent inhibition of TNFα-induced increase in ICAM-1 abundance.

    (A) Serum-starved HUVECs were stimulated with human recombinant TNFα for the indicated times, in the absence or presence of HDL isolated from WT (ApoM+HDL) or Apom−/− (ApoMHDL) mice or albumin (Alb)–S1P. ICAM-1 abundance was assessed by immunoblot of total cell lysates. (B) ICAM-1 abundance was quantified from three independent 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 time point). A.U., arbitrary unit. (C) Serum-starved HUVECs were stimulated with human recombinant TNFα and with increasing doses of ApoMHDL or ApoM+HDL as indicated. ICAM-1 abundance was determined by immunoblot analysis. (D) Data are shown as means ICAM abundance ± SD from three independent experiments. *P < 0.05, compared to TNFα alone by Kruskal Wallis test followed by Dunnett’s multiple comparison.

  • Fig. 4 HDL-S1P suppresses cytokine-induced NF-κB activation.

    (A and C) Serum-starved HUVECs were stimulated with TNFα with or without increasing doses of human HDL (A) or WT mouse HDL (ApoM+HDL) and mouse HDL from Apom−/− animals (ApoMHDL) (C). Phosphorylation (p) of the NF-κB subunit p65 was analyzed by immunoblot of total (tot) cell lysates. (B and D) Data in (B) and (D) represent combined analysis from three independent experiments and are shown as means ± SD. *P < 0.05, **P < 0.01, compared to TNFα alone by one-way ANOVA followed by Dunnett’s multiple comparison.

  • Fig. 5 HDL-S1P acts as a biased agonist on S1P1.

    (A) HUVECs were transduced with lentivirus encoding GFP-tagged S1P1 and after serum starvation, were stimulated with TNFα or with human HDL (huHDL), albumin-S1P, or P-FTY720 for the indicated times. Confocal microscopy was performed to assess the localization of GFP-tagged S1P1. Images are representative of three independent experiments. (B) Serum-starved HUVECs were stimulated with albumin-S1P or HDL-S1P for the indicated times. MAPK phosphorylation was assessed by immunoblot of total lysates. (C) Serum-starved HUVECs were preincubated for 30 min with 3-isobutyl-1-methylxanthine (IBMX), and then stimulated with increasing doses of albumin-S1P, HDL-S1P, or ApoMHDL in the presence of forskolin for 30 min. Results are expressed as picomole of cAMP per microgram of protein. Data represent combined analysis of five separate experiments and are expressed as means ± SD. **P < 0.01 by Friedman test followed by Dunnett’s multiple comparisons.

  • Fig. 6 The role of β-arrestin 2 in the biased signaling of HDL-S1P.

    (A) Serum-starved HUVECs were treated with albumin-S1P or human HDL-S1P for the indicated times. Cell lysates were subjected to immunoprecipitation for S1P1 and immunoblotted (IB) with β-arrestin 2 antibody. Immunoblots are representative of three independent experiments. (B) HUVECs stably overexpressing β-arrestin 2 complementary DNA or an shRNA directed against β-arrestin 2 were serum-starved and stimulated with TNFα, with or without human HDL at the indicated doses. p65 phosphorylation was analyzed by immunoblot assay. (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 TNFα in the absence or presence of albumin-S1P or ApoMHDL or human HDL as indicated. In the P-FTY720–treated experiments, serum-starved HUVECs were pretreated with P-FTY720 in the absence or presence of albumin-S1P, ApoMHDL, or human HDL before TNFα stimulation in the presence of P-FTY720. ICAM-1 abundance was assessed by immunoblotting. (E) Data represent the combined analysis of ICAM-1 abundance from three independent experiments. **P < 0.01 by unpaired Student’s t test.

  • Fig. 7 Endothelial S1P1 suppresses atherosclerotic lesion development.

    Male Apoe−/− S1pr1 WT and Apoe−/− S1pr1ECKO littermates were placed on high-fat diet for 16 weeks. (A) Aortae were dissected; en face preparation followed by Oil Red O staining was done. Shown here is a representative image of aorta from each group. (B) Quantification of plaque areas in each section of aorta (aortic arch, descending aorta, and total aorta) is shown (six Apoe−/− WT mice and eight Apoe−/− S1pr1ECKO mice). ns, not significant. (C) Aortic root sections from Apoe−/− S1pr1 WT and Apoe−/− S1pr1ECKO mice were immunostained with MOMA-2 to visualize the infiltration of macrophages and foam cells. (D) Quantification of MOMA-2 infiltration in the aortic valve was assessed (n = 5 mice for each genotype). (E) Plaques from descending aorta of Apoe−/− S1pr1 WT and Apoe−/− S1pr1ECKO mice were isolated, and macrophage was visualized by MOMA-2 staining followed by confocal microscopy. (F) Quantification of MOMA-2 staining in the descending aorta lesion was assessed (n = 2 animals, two separate plaques analyzed for each aorta). Data are shown as means ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/389/ra79/DC1

    Fig. 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 TNFα-induced increases in ICAM-1 and VCAM-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 β-arrestin 2 depends on the S1P carrier.

    Fig. S8. Genetic modulation of S1P1 expression in endothelial cells and modification of ICAM-1 abundance.

    Fig. S9. Analysis of weight, plasma cholesterol, and cell populations in Apoe−/− S1pr1 wild-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.

  • Supplementary Materials for:

    HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation

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

    *Corresponding author. E-mail: tih2002{at}med.cornell.edu

    This PDF file includes:

    • Fig. 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 TNFα-induced increases in ICAM-1 and VCAM-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 β-arrestin 2 depends on the S1P carrier.
    • Fig. S8. Genetic modulation of S1P1 expression in endothelial cells and modification of ICAM-1 abundance.
    • Fig. S9. Analysis of weight, plasma cholesterol, and cell populations in Apoe−/− S1pr1 wild-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.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.12 MB


    Citation: S. Galvani, M. Sanson, V. A. Blaho, S. L. Swendeman, H. Conger, B. Dahlbäck, M. Kono, R. L. Proia, J. D. Smith, T. Hla, HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation. Sci. Signal. 8, ra79 (2015).

    © 2015 American Association for the Advancement of Science

Navigate This Article