Abstract
Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) have been associated with potential cardiovascular benefits, partly attributed to their bioactive metabolites. However, the underlying mechanisms responsible for these advantages are not fully understood. We previously reported that metabolites of the cytochrome P450 pathway derived from eicosapentaenoic acid (EPA) mediated the atheroprotective effect of ω-3 PUFAs. Here, we show that 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and its receptor, sphingosine-1-phosphate receptor 1 (S1PR1), in endothelial cells (ECs) can inhibit oscillatory shear stress- or tumor necrosis factor-α-induced endothelial activation in cultured human ECs. Notably, the atheroprotective effect of 17,18-EEQ and purified EPA is circumvented in male mice with endothelial S1PR1 deficiency. Mechanistically, the anti-inflammatory effect of 17,18-EEQ relies on calcium release-mediated endothelial nitric oxide synthase (eNOS) activation, which is abolished upon inhibition of S1PR1 or Gq signaling. Furthermore, 17,18-EEQ allosterically regulates the conformation of S1PR1 through a polar interaction with Lys34Nter. Finally, we show that Vascepa, a prescription drug containing highly purified and stable EPA ethyl ester, exerts its cardiovascular protective effect through the 17,18-EEQ–S1PR1 pathway in male and female mice. Collectively, our findings indicate that the anti-inflammatory effect of 17,18-EEQ involves the activation of the S1PR1–Gq–Ca2+–eNOS axis in ECs, offering a potential therapeutic target against atherosclerosis.
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Data availability
Raw data and source data in figures, extended data and tables are provided in the source data file. RNA sequencing data has been deposited in the Gene Expression Omnibus under accession number GSE259394. The details of commercial reagents, antibodies and primer sequences are provided with the supplementary data. Source data are provided with this paper.
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Acknowledgements
This work was supported by grants from the National Key R&D Program of China (2019YFA0904200 to J.-P.S. and 2019YFA0802003 to Y.Z.); the National Science Fund for Distinguished Young Scholars Grant (81925003 to D.A.); the National Natural Science Foundation of China (T2321004 to J.-P.S., 82330118 to J.-P.S., 81773704 to J.-P.S., 82300890 to J.C., 82330012 to Y.Z., 82130014 to D.A.); and the Key Research Project of the Beijing Natural Science Foundation, China (Z200019 to J.-P.S.); China National Postdoctoral Program for Innovative Talents (BX20230201 to J.C.); China Postdoctoral Science Foundation (2023M742066 to J.C.). J.-P.S. is also supported by the Tencent New Cornerstone Investigator Program.
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T.Z., J.C., S.H., C.Z., M.-X.G. and L.-J.Z. performed the experiments and data analysis. D.A., Y.Z. and J.-P.S. designed and supervised the study; D.A., Y.Z. and J.-P.S. wrote the manuscript. All the authors reviewed and approved the final version of the manuscript.
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Nature Metabolism thanks Aldons Lusis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.
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Extended data
Extended Data Fig. 1 Identification of the receptor of 17,18-EEQ and validation of siRNA knockdown efficiency.
a,b, HUVECs pretreated with 1 mM 17,18-EEQ for the indicated time point (6, 12, 24 h) were exposed to OSS (a) for 12 h or TNF-α(b) for 6 h. Representative Western blots and quantitative analysis of vascular cell adhesion molecule 1(VCAM-1) in whole cell lysates (n = 6). c, HUVECs transfected with scramble siRNA and GPR120 siRNA for 48 h were pretreated with 17,18-EEQ (1 mM) for 24 h, then with TNF-α (0.1 ng/ml) for 6 h. Western blot analysis of VCAM-1 and GPR120. Quantification of protein level of VCAM-1 (n = 3). d, HUVECs transfected with scramble siRNA and GPR132 siRNA for 48 h were pretreated with 17,18-EEQ (1 mM) for 24 h, then with TNF-α (0.1 ng/ml) for 6 h. Western blot analysis of VCAM-1 and GPR132. Quantification of protein level of VCAM-1 (n = 3). e, Quantitative PCR analysis was performed to detect 10 GPCRs mRNA levels in HUVECs transfected with scramble siRNA or specific siRNAs for 48 h (n = 3). f, Western blot analysis in candidate GPCRs (ADORA2A, CALCRL, GPR3, ADGRE5, ADRB2, GPR180, GPRC5A, GPR89A, P2RY1, S1PR1)-knockdown or -overexpressing HUVECs to validate antibodies for these 10 GPCRs. g, Whole-cell ELISA measuring the cell surface expression of VCAM-1 in HUVECs transfected with scramble siRNA or 10 GPCR-specific siRNAs for 48 h, then were pretreated with 17,18-EEQ (1 mM) for 24 h and TNF-α (0.1 ng/ml) for 6 h (n = 3). All data are mean±s.e.m. of biologically independent smaples, a-d, one-way ANOVA with Bonferroni’s multiple comparisons post hoc test. e, g, two-tailed Student’s t-tests.
Extended Data Fig. 2 Saturation binding assay of [3H]-S1P to interact with S1PR1 and incorporation of BRET sensors had no effect on S1PR1 expression.
a, Saturation binding of [3H]-S1P to the membrane fraction from S1PR1 overexpressing-HEK293 cell. The nonspecific binding in the presence of 1 μM unlabeled S1P was subtracted from the total binding to yield the specific binding curve. Representative curves from three biologically independent experiments were shown (n = 3). b, Saturation binding of [3H]-S1P to the membrane fraction from S1PR1 overexpressing-HEK293 cell. The nonspecific binding in the presence of 1 μM unlabeled 17,18-EEQ was subtracted from the total binding to yield the specific binding curve. Representative curves from three independent experiments were shown (n = 3). c, Elisa experiments to determine the expression levels of the wild-type S1PR1 and five FlAsH motif incorporated FLAsH-BRET sensors. Data from three biologically independent experiment (n = 3). All data are mean±s.e.m. of biologically independent smaples, c, one-way ANOVA with Bonferroni’s multiple comparisons post hoc test.
Extended Data Fig. 3 17,18-EEQ biasedly activated S1PR1-Gq-Ca2+ signaling.
a, HUVECs were treated with 1 μM 17,18-EEQ after S1PR1 knockdown by siNRA. ELISA were performed to measure cAMP levels (n = 3). b, Saturation binding of BODIPY-FL-GTPγS to the membrane fraction from HEK293 cell overexpressing S1PR1 combined with Gq, Gs, Gi, G12, G13 or control vehicle stimulated by 17,18-EEQ (n=3). c-h, Dose response curves of Gα (including Gs, Gi, Gq, G11, G12 and G13)-Gβγ dissociation in S1PR1-overexpressing HEK293 cells in response to stimulation with different agonists of S1PR1 (S1P,17,18-EEQ, AUY954 and SEW2871) (n = 3). i-m, Competitive binding curves of S1P or 17,18-EEQ to S1PR1-5 (n = 3). n,o, The potency of Gα (including Gs, Gi, Gq, G11 and G13)-Gβγ dissociation in response to stimulation with S1P (n) or 17,18-EEQ (o) in S1PR1 overexpressing HEK293 cells. The heatmap is colored according to the value of pEC50 (n = 3). p, The relative fpkm values of Gq and G11 in untreated HUVECs from RNA-seq (n = 3). q,r, HUVECs transfected with scramble siRNA and G11 siRNA were pretreated with 17,18-EEQ, then exposed to OSS (q) or TNF-α(r). The relative mRNA levels of VCAM-1 (vascular cell adhesion molecule 1) (n = 3). s,t, After G11 knockdown, HUVECs treated with 17,18-EEQ were stimulated by OSS (s) or TNF-α (t). Representative western blot and quantification of VCAM-1, G11 and GAPDH (n = 3). u, Effects of G11 siRNA on the 17,18-EEQ-induced IP3 production in HUVECs (n = 3). v, The changes of fluorescence intensity represents Ca2+ concentration in HUVECs treated with 1 μM 17,18-EEQ after S1PR1 silencing (left) (n = 24; 3 independent experiments). Quantification (right) of Ca2+ transient was represented by the relative AUC (n = 3). All data are mean±s.e.m. of biologically independent smaples, ns, no significant difference; ND, signal not detectable. a,q-v two-way ANOVA with Bonferrni’s post hoc test.
Extended Data Fig. 4 17,18-EEQ biasedly activated S1PR1-Gq-Ca2+ signaling to inhibit endothelial activation.
a, HUVECs transfected with scramble siRNA and S1PR1 siRNA for 48 h, were pretreated with 17,18-EEQ (1 mM) for 24 h, then with TNF-α (0.1 ng/ml) for another 6 h. Representative western blots and quantification of the protein levels of VCAM-1 (n = 6). b,c, Representative blots (b) and quantification (c) of the phosphorylation and protein levels of p-IKKα, p-p65, IκBα in HUVECs transfected by scramble siRNA or S1PR1 siRNA, and then treated with 1 mM 17,18-EEQ, then with 0.1 ng/ml TNF-α (n=6). d,e, Representative images of monocyte-endothelial adhesion (d). HUVECs transfected with scramble siRNA and S1PR1 siRNA for 48 h, were treated with 1 mM 17,18-EEQ for 24 h, then exposed to 0.1 ng/ml TNF-α for 6 h. The number of adhesive cells were quantified from 5 random fields with 10× objective in each experiment and was normalized to static HUVECs (e) (n = 3). f, Effects of YM254890 Gq inhibitor and Ionomycin calcium ionophore on TNF-α-induced VCAM-1 expression in HUVECs treated with 1 μM 17,18-EEQ or PBS. Representative blots of the protein levels of VCAM-1 (n = 6). g,h, After pretreatment with YM254890, Ionomycin, or their combination for 1 h, HUVECs were subjected to 1 μM 17,18-EEQ for 4 h, then exposed to TNF-α for 30 min. Representative blots (g) and quantification (h) of protein levels of p-IKKα, p-p65, IκBα (n = 6). All data are mean±s.e.m. of biologically independent smaples, ns, no significant difference. a,c,e, two-way ANOVA with Bonferroni’s multiple comparisons post hoc test. f,h, one-way ANOVA with Bonferroni’s multiple comparisons post hoc test.
Extended Data Fig. 5 17,18-EEQ dose and time dependently induced eNOS ser1177 phosphorylation, which was mediated by calcium release.
a,b, HUVECs were seeded on plates pretreated with various concentrations (a) of 17,18-EEQ (1, 10, 100, 1000 nM) for the indicated time points (b) (15, 30, 45, 60 min). Representative blots and quantification of the protein levels of p-eNOS (n = 3). c, HUVECs pretreated with EGTA or DMSO for 30 min were subjected to 1 μM 17,18-EEQ for another 45 min. Representative blot (left panel) of p-eNOS (Ser1177) and total eNOS. Quantification of protein levels of p-eNOS in the right panel (n = 6). d, Strategy for the generation of S1pr1fl/fl mice. e, S1pr1fl/fl mice were injected intraperitoneally with or without 75 mg/kg tamoxifen for five days. Representative immunofluorescence staining images of S1PR1, VE-cadherin and DAPI in mouse carotid arteries, with quantitative analysis in the right panel (n = 3). Scale bar: 20 μm. f, Plasma levels of 17,18-EEQ in S1pr1fl/fl mice followed by 17,18-EEQ (5 μg/kg) intraperitoneal injection (n = 6). All data are mean±s.e.m. of biologically independent smaples, a,b, Kruskal-Wallis test. c, two-way ANOVA with Bonferroni’s multiple comparisons post hoc test. e,f, two-tailed Student’s t-tests.
Extended Data Fig. 6 The potential binding sites at S1PR1 for 17,18-EEQ by docking.
a, The average Root-mean-square deviation (RMSD) value of 17, 18-EEQ during triplicate 500 ns of MD simulation. b, Dose response curves of key residue mutations for the toggle switch W2696.48 and E1413.49-R1423.50-Y1433.51 of S1PR1 in response to stimulation with 17,18-EEQ (n = 3). c, Effects of key residue mutations of binding pockets of S1PR1 on radioligand binding induced by S1P or 17,18-EEQ. Bars represent differences in calculated potency for each mutant compared with the wild type (n = 3). d, Competitive binding curves of S1P (left) or 17,18-EEQ (right) for the mutants in the binding pockets of S1PR1 (n = 3). e, Barcode representation of stronger interaction patterns in the ligand pocket of S1P-S1PR1 and 17,18-EEQ-S1PR1. Residues of S1PR1 formed both interactions with ligands were indicated in orange. Residues that only show interactions with S1P or 17,18-EEQ were indicated in light blue or light green, respectively. f, Effects of key residue mutations (V194A, L195A, F210A, W269A, L272A, L276A and L297A) of binding pockets of S1PR1 on FlAsH-BRET ratio changes induced by S1P or 17,18-EEQ. Bars represent differences in calculated potency for each mutant compared with the wild type (n = 3). g,h, Representative dose-response curves of the S1P or 17,18-EEQ-induced FlAsH-BRET ratio for the mutants (V194A, L195A, F210A, W269A, L272A, L276A and L297A) in the binding pockets of S1P-S1PR1 (g) or 17,18-EEQ-S1PR1 (h) (n = 3). All data are mean±s.e.m. of biologically independent smaples. c,f, two-sided one-way ANOVA with Tukey’s test (compared with wild type). All data are mean±s.e.m. of biologically independent smaples. c,f, two-sided one-way ANOVA with Tukey’s test (compared with wild type).
Extended Data Fig. 7 17,18-EEQ has no effect on serum lipid and other regioisomeric EEQs plasma levels in vivo.
a, Hepatic expression of low-density lipoprotein receptor from mice administrated by AAV-ctrl or AAV-PCSK9DY for 28 days (n = 3). b,c, Serum levels of triglyceride (b), total cholesterol (c) in S1pr1fl/fl and S1pr1ECKO mice followed by AAV-PCSK9DY (1 x 1011 VG) administration and western diet (WD). These mice were implanted with an osmotic minipump infusing with 17,18-EEQ (5 μg/kg) for 7 days and subjected to PCAL surgery for another 21 days (n = 8). (d-g) Eight-week-old S1pr1fl/fl and S1pr1ECKO mice administrated by AAV-PCSK9DY (1 x 1011 VG) and fed with WD, were implanted with an osmotic minipump infusing 17,18-EEQ (5 μg/kg) for 7 days and subjected to PCAL surgery for another 21 days. Plasma levels of CYP products 5,6-EEQ (d), 8,9-EEQ (e), 11,12-EEQ (f), 14,15-EEQ (g) were determined by LC-MS/MS (n = 8). h,i, Eight-week-old S1pr1fl/fl and S1pr1ECKO mice administrated by AAV-PCSK9DY (1 x 1011 VG) and fed with WD, were implanted with an osmotic minipump infusing 17,18-EEQ (5 μg/kg) for 7 days and subjected to PCAL surgery for another 7 days. Representative immunofluorescence staining images (h) of t-eNOS, VE-cadherin and DAPI in male mouse carotids. Quantification data (i) of t-eNOS expression (n = 6). Scale bar: 20 μm. The statistical analysis was performed on LCA groups. All data are mean±s.e.m. of biologically independent smaples, ns, no significant difference. a, two-tailed Student’s t-tests. b-g,i, two-way ANOVA with Bonferroni’s multiple comparisons post hoc test.
Extended Data Fig. 8 Vascepa has no effect on other regioisomeric EEQs plasma levels and t-eNOS expression in vivo.
a-f, Eight-week-old S1PR1fl/fl and S1PR1ECKO male mice followed by AAV-PCSK9DY (1 x 1011 VG) administration and western diet (WD). These mice were orally gavaged with Vascepa (666.7 mg/kg) or olive oil for 7 days and subjected to PCAL surgery for another 21 days. The levels of triglyceride (a), total cholesterol (b), 5,6-EEQ (c), 8,9-EEQ (d), 11,12-EEQ (e), 14,15-EEQ (f) in these mice (n = 8). g, Eight-week-old S1pr1fl/fl and S1pr1ECKO male mice administrated by AAV-PCSK9DY (1 x 1011 VG) and fed with WD, were treated with Vascepa (666.7 mg/kg) or olive oil for 7 days and subjected to PCAL surgery for another 7 days. Representative images (g) and of quantification of t-eNOS (h), VE-cadherin and DAPI in male mouse carotids. (n = 6). Scale bar: 20 μm. The statistical analysis was performed on LCA groups. h, Schematic diagram of the PCAL-induced atherosclerosis model. i-q, The levels of triglycerides (i), total cholesterol (j), EPA (k), 17,18-EEQ (l), 17,18-DHETE (m), 5,6-EEQ (n), 8,9-EEQ (o), 11,12-EEQ (p), 14,15-EEQ (q) in PCSK9DY injected-S1pr1fl/fl and -S1pr1ECKO mice performed with PCAL, were treated with Vascepa (666.7 kg/mg) or Olive oil, and fed with WD for 4 weeks (n = 5). r, Isolated carotid arteries in PCSK9DY injected-S1pr1fl/fl and -S1pr1ECKO mice treated with Vascepa (666.7 kg/mg) or Olive oil (n = 5). Scale bars: 1.5 mm. s-u, Representative images (s) and quantification (t-u) of plaques in ligated carotid arteries of mice were analyzed with H&E staining. Scale bars: 100 μm (n = 5). All data are mean±s.e.m. of biologically independent smaples, ns, no significant difference. a-g,i-q,t,u, two-way ANOVA with Bonferroni’s multiple comparisons post hoc test.
Extended Data Fig. 9 The beneficial effect of Vascepa on reducing aortic atherosclerosis is mediated by endothelial S1PR1.
a, Schematic diagram of the experimental strategy for the adeno-associated virus (AAV) administration, oral gavage, western diet (WD) feeding, and subsequent analyses for female mice. b-h, Plasma levels of EPA (b), 17,18-EEQ (c), 17,18-DHETE (d), 5,6-EEQ (e), 8,9-EEQ (f), 11,12-EEQ (g), 14,15-EEQ (h) from S1pr1fl/fl and S1pr1ECKO mice followed by PCSK9DY injection with WD feeding for 12 weeks. Vascepa (666.7 kg/mg) or olive oil were orally administered once a day (n = 5). i, Oil Red staining of aortas in PCSK9DY injected-S1pr1fl/fl and -S1pr1ECKO mice treated with Vascepa (666.7 kg/mg) or Olive oil (n = 5). Scale bars: 5 mm. j, Quantification of Oil Red staining of whole aortas, aortic arch, thoracic aorta from PCSK9DY injected-S1pr1fl/fl and -S1pr1ECKO mice (n = 5). k, Oil Red (upper panel) and H&E (lower panel) staining of atherosclerotic lesion areas in cross-sections at the aortic root (n = 5). Scale bars: 200 μm. l, Quantification of aortic root atherosclerotic plaques (n = 5). m-n, The levels of triglycerides (m) and total cholesterol (n) in serum collected from PCSK9DY injected-S1pr1fl/fl and -S1pr1ECKO mice treated with Vascepa (666.7 kg/mg) or Olive oil, and fed with WD for 12 weeks (n = 5). All data are mean±s.e.m. of biologically independent smaples, ns, no significant difference. b-h,j,l-n, two-way ANOVA with Bonferroni’s multiple comparisons post hoc test.
Extended Data Fig. 10 The mechanism of the anti-inflammatory effect mediated by 17,18-EEQ.
S1PR1 functions as a receptor for 17,18-EEQ, selectively activating Gq-Ca2+-eNOS-NO signaling to inhibit vascular endothelial inflammation and hinder the progression of atherosclerosis.
Supplementary information
Supplementary Tables 1 and 2
1. The expression profile of GPCRs in HUVECs from RNA-seq (left) and public dataset (right). 2. Statistical details in all figure panels.
Supplementary Data 1–4
1. Antibodies used for Western blots and Immunofluorescence staining in this study. 2. Chemical reagents used for this study. 3. Primer sequence. 4. siRNA sequences.
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Uncropped western blots
Uncropped western blots for both main figures and extended data figures.
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Zhou, T., Cheng, J., He, S. et al. The sphingosine-1-phosphate receptor 1 mediates the atheroprotective effect of eicosapentaenoic acid. Nat Metab 6, 1566–1583 (2024). https://doi.org/10.1038/s42255-024-01070-3
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DOI: https://doi.org/10.1038/s42255-024-01070-3
