Research ArticlePharmacology

An engineered S1P chaperone attenuates hypertension and ischemic injury

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Science Signaling  15 Aug 2017:
Vol. 10, Issue 492, eaal2722
DOI: 10.1126/scisignal.aal2722
  • Fig. 1 Production, purification, and characterization of S1P binding by ApoM-Fc and ApoM-Fc–TM fusion proteins.

    (A) Top: Cocrystal structure of S1P bound to ApoM. Three residues (Arg98, Trp100, and Arg116) that contact the phosphate head group region of S1P (red and green) are labeled in yellow. Bottom: Space-filling model of the head group region of S1P in the ApoM molecule. (B) ApoM-Fc and ApoM-Fc–TM fusion proteins in the conditioned medium of HEK293 or Sf9 cells were separated by nonreducing or reducing 10% SDS–polyacrylamide gel electrophoresis (PAGE) and detected by anti-ApoM antibody. (C) Sf9-derived purified proteins (4 μg) were analyzed by reducing 10% SDS-PAGE and stained with Coomassie Brilliant Blue. WT, wild type. (D) Purified IgG1-Fc (Fc), ApoM-Fc, or ApoM-Fc–TM (TM) was analyzed for S1P binding by fluorescence spectrofluorimetry as described (n = 4 independent experiments; mean ± SD). ****P < 0.001, Student’s t test and two-way analysis of variance (ANOVA) followed by Dunnett’s posttest comparing ApoM-Fc or ApoM-Fc–TM to Fc alone (ApoM-Fc and ApoM-Fc–TM). (E) Purified ApoM-Fc and ApoM-Fc–TM (5 μM) were incubated or not with S1P as described for 24 to 48 hours, purified by gel filtration chromatography, and analyzed for sphingolipids by electrospray ionization–MS/MS. The resulting data are expressed as means ± SD; n = 4 independent experiments.

  • Fig. 2 ApoM-Fc activates S1P receptors.

    (A) Increasing doses of albumin (Alb)–S1P, ApoM-Fc, or ApoM-Fc–TM were incubated with mouse embryonic fibroblasts (MEFs) isolated from S1P1–green fluorescent protein (GFP) signaling mice for 24 hours and analyzed by flow cytometry. S1P and protein concentrations are indicated. (B) Quantitative analysis of results from (A). n = 3 independent experiments; mean ± SD. ***P < 0.01, ****P < 0.001, two-way ANOVA followed by Tukey’s posttest comparing ApoM-Fc or Alb-S1P to ApoM-Fc–TM. (C) CHO cells or CHO cells stably transduced with S1P1, S1P2, or S1P3 were treated for 5 to 30 min using albumin-S1P, ApoM-Fc–S1P (both 100 nM S1P), or ApoM-Fc–TM (12 μg/ml) (top) or by a dose response with albumin-S1P or ApoM-Fc–S1P diluted to 0 to 200 nM S1P or ApoM-Fc–TM (12 μg/ml) (bottom). Samples were analyzed for phospho-p44/42 ERK (pERK) and total p42/44 ERK (tERK) by immunoblotting. (D) HUVECs were treated with albumin-S1P (333 nM S1P), ApoM-Fc–S1P (20 μg/ml; 240 nM S1P) (ApoM), or ApoM-Fc–TM (20 μg/ml) for indicated times and analyzed by immunoblotting for activation of p44/42 ERK, Akt, and eNOS. (E) CRISPR/Cas9-derived S1P1, S1P3, or S1P1/3 knockout (KO) HUVECs were treated with ApoM-Fc–S1P (12 μg/ml; 100 nM S1P) and analyzed by immunoblotting for activation of p44/42 ERK. n = 3 independent experiments with a representative blot shown.

  • Fig. 3 Effect of ApoM-Fc on endothelial cell barrier function and S1P receptor endocytosis.

    (A) HUVECs were analyzed for barrier function by real-time measurement of TEER. At time 0, either albumin-S1P (200 nM), ApoM-Fc (20 μg/ml; 200 nM S1P), or ApoM-Fc–TM (20 μg/ml) was added. All data were compared to baseline ApoM-Fc–TM. n = 3 independent experiments; expressed as mean ± SEM. ****P < 0.0001, two-way ANOVA followed by t test. (B) HUVECs or S1P1 KO HUVECs (S1P1-CRISPR) were treated with ApoM-Fc (10 μg/ml; 100 nM S1P) and analyzed for barrier function by real-time measurement of TEER. ****P < 0.0001, one-way ANOVA followed by t test. n = 3 independent experiments. (C) U2OS cells expressing S1P1-GFP were treated with indicated concentrations of FTY720-P, Alb-S1P, ApoM-Fc, or ApoM-Fc–TM for 30 min at 37°C. Receptor internalization was quantified. All data were compared to baseline ApoM-Fc–TM. n = 2 independent experiments, n = 8 wells analyzed in total; expressed as mean ± SEM. **P < 0.01, *P < 0.05, t test; P < 0.01, one-way ANOVA.

  • Fig. 4 Effect of ApoM-Fc administration on plasma S1P concentrations and circulating hematopoietic cells.

    (A) WT mice were treated with purified ApoM-Fc (4 mg/kg) or ApoM-Fc–TM (4 mg/kg) (n = 4 mice per treatment) by intraperitoneal injection, and plasma ApoM concentrations were determined by immunoblot analysis. (B) Apom−/− mice (n = 4 mice; expressed as mean ± SD) were administered ApoM-Fc–S1P (4 mg/kg), and plasma sphingolipids at 24 hours after administration were quantified. Sph, sphingosine; dhSph, dihydrosphingosine. (C) WT mice (n = 4 mice per treatment; expressed as mean ± SD) were administered purified ApoM-Fc–S1P (4 mg/kg) or ApoM-Fc–TM (4 mg/kg) by intraperitoneal injection, and plasma sphingolipids at 24 hours after administration were quantified. (B and C) *P = 0.05; **P < 0.01; ***P < 0.005, two-tailed Student’s t test. (D) WT mice were administered either phosphate-buffered saline (PBS) (n = 5 mice), purified ApoM-Fc–S1P (4 mg/kg) (n = 5 mice), or ApoM-Fc–TM (4 mg/kg) (n = 5 mice) by intraperitoneal injection, and blood was collected at 6 and 24 hours after injection. Blood cells were isolated by centrifugation, and lymphocytes, white blood cells (WBCs), red blood cells (RBCs), and platelets were quantified as described. The observed variations in relative blood cells counts were statistically insignificant as judged by two-way ANOVA and Tukey’s posttest. For ApoM-Fc, N.S. was P > 0.55 (lymphocytes), P > 0.33 (WBCs), P > 0.07 (platelets), and P > 0.15 (RBCs).

  • Fig. 5 ApoM-Fc administration leads to a sustained antihypertensive effect in mice.

    (A) Systolic blood pressure (SBP) was measured at the indicated times in AngII-treated WT mice administered with either vehicle (PBS) (n = 5 mice), ApoM-Fc (4 mg/kg) (n = 7 mice), or ApoM-Fc–TM (4 mg/kg) (n = 4 mice) by intraperitoneal injection. (B) AngII-treated mice were injected with either ApoM-Fc or ApoM-Fc–TM and the S1P1 antagonist W146 (10 mg/kg) every 24 hours (arrows) followed by measurement of SBP (n = 5 mice per treatment). (C) ApoM-Fc or ApoM-Fc–TM was administered to AngII-treated mice (N = 6 mice per treatment) for 72 hours, and plasma nitrite concentrations were measured as described. (D) SBP was measured in normotensive mice administered with either ApoM-Fc (4 mg/kg) (n = 6 mice) or ApoM-Fc–TM (4 mg/kg) (n = 4 mice) by intraperitoneal injection. (E) SBP was measured in WT (n = 12 mice) or Apom−/− (n = 11 mice) mice as described. All data are expressed as means ± SEM. **P < 0.01; ***P < 0.005; ****P < 0.001 compared to WT (A to D). Statistical significance was determined by two-way ANOVA followed by Bonferroni’s post hoc test or one-way ANOVA.

  • Fig. 6 ApoM-Fc administration attenuates I/R injury in the heart.

    WT mice were administered PBS (n = 7 mice) or either ApoM-Fc (4 mg/kg) (n = 9 mice) or ApoM-Fc–TM (4 mg/kg) (n = 9 mice) after I/R injury. (A) Representative images of left ventricular (LV) slices with Alcian blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining (red), which indicates viable myocardium. (B) Quantitative measurement of area at risk (AAR)/LV area and infarct/AAR area was performed in a blinded manner. Median values (one-way nonparametric ANOVA followed by Tukey’s test); **P < 0.01; N.S. was P > 0.7 (left graph) and P > 0.27 (ApoM-Fc) and P > 0.97 (ApoM-Fc–TM) (right graph). (C) Heart sections were stained for Ly6G and IB4, and neutrophils and capillary density were quantified for pixel density. n = 9 mice per treatment; mean ± SEM. P < 0.03 (left graph) and P > 0.85 (right graph), unpaired two-tailed parametric t test. DAPI, 4′,6-diamidino-2-phenylindole. (D) Representative images of two-dimensional (2D) guided M-mode echocardiography of the LV at baseline and 7 days after MI/R injury. n = 6 mice per treatment. (E) LV end-diastolic (LVDd) diameter, LV end-systolic (LVDs) diameter, and fractional shortening (FS) of (D) were measured at the indicated time points after MI/R injury. n = 6 mice per treatment; means ± SEM. Two-way ANOVA and Sidak’s multiple-comparisons test; *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

  • Fig. 7 ApoM-Fc administration attenuates I/R injury in the brain.

    (A) WT mice were subjected to 60 min of focal cerebral ischemia by MCAO. Right after reperfusion, mice received ApoM-Fc (4 mg/kg) (n = 11 mice), ApoM-Fc–TM (4 mg/kg) (n = 11 mice), or PBS (n = 10 mice) by intraperitoneal injection. Representative images of TTC staining of seven, 1-mm-thick brain coronal slices 23 hours after reperfusion. Two mice per group are shown. (B and C) Infarct (B) and edema (C) ratios were calculated by image analysis and reported as a ratio of the non-ischemic hemisphere. Infarct ratios were corrected for edema. (D) Total infarct volume in cubic millimeters, corrected for edema. (E) Neurological deficit scores were assessed 23 hours after reperfusion. (F) Relative cerebral blood flow (rCBF) in the MCA territory was measured by laser speckle flowmetry during MCAO surgery. rCBF during occlusion (I) and after reperfusion (R) is shown. CL, contralateral. The individual values and the median are shown. *P < 0.05 (one-way nonparametric ANOVA followed by Dunn’s test). (G) Evans blue dye (EBD) extravasation into the brain after MCAO stroke was quantified as described. IL, ipsilateral. Individual values and mean ± SEM are shown from n = 8 to 10 mice per treatment. P < 0.05, one-way ANOVA nonparametric test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/492/eaal2722/DC1

    Fig. S1. Sphingolipid base content of purified ApoM-Fc and ApoM-Fc–TM.

    Fig. S2. Plasma lipid concentrations in mice after ApoM-Fc administration.

    Fig. S3. ApoM-Fc is found in the soluble protein fraction of plasma in vivo.

    Fig. S4. ApoM-Fc does not induce lymphopenia.

    Fig. S5. ApoM-Fc does not induce lymphocyte accumulation in lymphoid tissues.

    Table S1. Physiological variables in mice treated with PBS, ApoM-Fc, or ApoM-Fc–TM.

  • Supplementary Materials for:

    An engineered S1P chaperone attenuates hypertension and ischemic injury

    Steven L. Swendeman, Yuquan Xiong, Anna Cantalupo, Hui Yuan, Nathalie Burg, Yu Hisano, Andreane Cartier, Catherine H. Liu, Eric Engelbrecht, Victoria Blaho, Yi Zhang, Keisuke Yanagida, Sylvain Galvani, Hideru Obinata, Jane E. Salmon, Teresa Sanchez, Annarita Di Lorenzo, Timothy Hla*

    *Corresponding author. Email: timothy.hla{at}childrens.harvard.edu

    This PDF file includes:

    • Fig. S1. Sphingolipid base content of purified ApoM-Fc and ApoM-Fc–TM.
    • Fig. S2. Plasma lipid concentrations in mice after ApoM-Fc administration.
    • Fig. S3. ApoM-Fc is found in the soluble protein fraction of plasma in vivo.
    • Fig. S4. ApoM-Fc does not induce lymphopenia.
    • Fig. S5. ApoM-Fc does not induce lymphocyte accumulation in lymphoid tissues.
    • Table S1. Physiological variables in mice treated with PBS, ApoM-Fc, or ApoMFc–TM.

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    Citation: S. L. Swendeman, Y. Xiong, A. Cantalupo, H. Yuan, N. Burg, Y. Hisano, A. Cartier, C. H. Liu, E. Engelbrecht, V. Blaho, Y. Zhang, K. Yanagida, S. Galvani, H. Obinata, J. E. Salmon, T. Sanchez, A. Di Lorenzo, T. Hla, An engineered S1P chaperone attenuates hypertension and ischemic injury. Sci. Signal. 10, eaal2722 (2017).

    © 2017 American Association for the Advancement of Science

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