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Sphingosine kinase 1 is integral for elastin deficiency-induced arterial hypermuscularization

Nature Cardiovascular Research volume 5, pages 34–50 (2026) Cite this article

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Abstract

Deficiency of elastin (ELN), the major component of elastic fibers, leads to excess smooth muscle cells (SMCs), which characterizes arterial diseases (for example, supravalvular aortic stenosis (SVAS)) as well as physiological ductus arteriosus (DA) closure. Here we demonstrate that sphingosine kinase 1 (SPHK1) is a key node in these contexts. Sphk1 is the most upregulated transcript in Eln(−/−) aortic SMCs at embryonic day 15.5 when these cells are initially hyperproliferative. The aorta of humans with SVAS also upregulates SPHK1. Reduced ELN increases levels of transcription factor early growth response 1, resulting in increased SPHK1 levels. SMC-specific Sphk1 deletion or pharmacological inhibition of SPHK1 attenuates SMC proliferation and mitigates aortic disease. Furthermore, treatment with a SPHK1 inhibitor reduces DA SMC accumulation, leading to DA patency in wild-type mice. These findings indicate that inhibiting SPHK1 may be a therapeutic strategy for SVAS and select congenital heart diseases in which patent DA maintains circulation.

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Fig. 1: Sphk1 is upregulated before morphological differences develop in elastin-deficient aorta.
The alternative text for this image may have been generated using AI.
Fig. 2: Elastin insufficiency upregulates SPHK1 in mouse and human aortic SMCs.
The alternative text for this image may have been generated using AI.
Fig. 3: Sphk1 deletion with Acta2-CreERT2 attenuates elastin aortopathy.
The alternative text for this image may have been generated using AI.
Fig. 4: Pharmacological inhibition of SPHK1 mitigates aortopathy in elastin mutants.
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Fig. 5: Sphk1 is upregulated by elastin deficiency-induced active transcription.
The alternative text for this image may have been generated using AI.
Fig. 6: SPHK1 and EGR1 are upregulated in murine ductus arteriosus.
The alternative text for this image may have been generated using AI.
Fig. 7: SPHK1 inhibition attenuates hypermuscularization, leading to DA patency.
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Fig. 8: Schematic representation of working model.
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Data availability

The bulk RNA-seq data are publicly available on the Gene Expression Omnibus under accession numbers GSE270682 and GSE270681. All other data associated with this study are present in the paper or available in the Source data.

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Acknowledgements

We thank D. Montefusco at Virginia Commonwealth University for providing Sphk1(flox/flox) mice and M. Schwartz and R. Garcia-Milian at Yale University for providing fibronectin and supporting bulk RNA-seq analyses, respectively. We acknowledge the Stony Brook Cancer Center Biological Mass Spectrometry Shared Resource for expert assistance with S1P measurements. We extend our thanks to Greif laboratory members for their input. J.S. was supported by the Overseas Research Fellowship from the Japan Society for the Promotion of Science (number 202260284), the Congenital Heart Defect Research Award from the American Heart Association and the Children’s Heart Foundation (23POSTCHF1022933) and the Pathway to Independence Award from the NIH (K99HL171838, R00HL171838). Funding was also provided by the NIH (R35HL150766, R01HL125815 and R01HD110059 to D.M.G. and R01AG078602 to T.H.) and American Heart Association (Established Investigator Award, 19EIA34660321, Collaborative Sciences Award, 23CSA1051139 to D.M.G.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Department of Internal Medicine, Section of Cardiovascular Medicine, Yale Cardiovascular Research Center, New Haven, CT, USA

    Junichi Saito, Jui M. Dave, Eunate Gallardo-Vara, Nandhini Sadagopan, Inamul Kabir & Daniel M. Greif

  2. Department of Genetics, Yale University, New Haven, CT, USA

    Junichi Saito, Jui M. Dave, Eunate Gallardo-Vara, Nandhini Sadagopan, Inamul Kabir & Daniel M. Greif

  3. Stem Cell Center, Yale University, New Haven, CT, USA

    Junichi Saito, Jui M. Dave, Eunate Gallardo-Vara, Nandhini Sadagopan, Inamul Kabir & Daniel M. Greif

  4. Department of Surgery, Yale University, New Haven, CT, USA

    George Tellides

  5. Congenital Division, Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA, USA

    Robert K. Riemer

  6. Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA

    Zsolt Urban

  7. Department of Cellular, Molecular and Genetic Medicine, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA

    Sarah Spiegel

  8. Department of Surgery, Harvard Medical School, Boston, MA, USA

    Timothy Hla

  9. Vascular Biology Program, Boston Children’s Hospital, Boston, MA, USA

    Timothy Hla

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Contributions

J.S., J.M.D. and D.M.G. conceived of and designed the experiments. J.S., J.M.D., E.G-V. and N.S. performed the experiments. G.T., R.K.R. and Z.U. provided human aortic samples. J.M.D., I.K. and T.H. provided input for study direction. T.H. provided S1pr1(knock-in/knock-in), H2B-GFP mice, and S.S. provided Sphk1 plasmids. J.S. and D.M.G. analyzed the results, prepared the figures and wrote the paper. All authors reviewed and provided input on the paper.

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Correspondence to Junichi Saito or Daniel M. Greif.

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Nature Cardiovascular Research thanks Bert Callewaert, Besim Ogretmen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 In aortic SMCs, Sphk1 levels are regulated by elastin gene dosage, and Sphk1 deletion does not induce apoptosis.

(a) Aortic lysates from Eln(+/+), Eln(+/−) or Eln(−/−) embryos at E13.5 were subjected to qRT–PCR for Sphk1. n = 6 mice per group. (b) Aortic SMCs were isolated from Eln(+/+), Eln(+/−) or Eln(−/−) pups at P0.5 and subjected to immunostaining for markers of SMCs (SMA) and nuclei (DAPI). Scale bar, 100 μm. (c) Lysates from isolated aortic SMCs of indicated genotypes were subjected to qRT–PCR for Eln or Sphk1. For Eln mRNA, n = 7 per group, biological replicates. For Sphk1 mRNA, n = 6 Eln(+/+) SMCs, n = 4 Eln(+/−) SMCs, n = 6 Eln(−/−) SMCs, biological replicates. (d, e) Aortic lysates from Eln(+/+), Eln(+/−) or Eln(−/−) mice at P0.5 (d) or from human aortic SMCs treated with scrambled (Scr) RNA or siRNA targeting ELN (e) were subjected to qRT–PCR for SPHK2. In d, n = 4 Eln(+/+) mice, n = 5 Eln(+/−) mice, n = 5 Eln(−/−) mice. In e, n = 4 per group, biological replicates. (f-j) Dams pregnant with Sphk1(flox/flox) embryos also carrying Acta2-CreERT2 or no Cre were injected daily with tamoxifen from E13.5 to E17.5. In (f-h), aortas were harvested from pups at P0.5 and subjected to lysis after adventitia and EC removal. In (f), extracted genomic DNA was subjected to PCR with primers flanking Sphk1. The 120, 250, and 600 PCR products represent the wild-type, floxed, and deleted Sphk1 alleles, respectively. In (g), isolated RNA was reverse transcribed and subjected to qRT–PCR. Histogram represents Sphk1 transcript levels relative to Gapdh. n = 4 non-Cre mice, n = 6 Acta2-CreERT2 mice. In (h), Western blot was performed on aortic lysates with densitometry of protein bands relative to β-actin shown on right. n = 4 mice per group. (i) Transverse aortic sections from mice of indicated genotypes at P0.5 were stained for markers of apoptosis (cleaved caspase-3), SMCs (SMA), and nuclei (DAPI). Section of the thymus from Eln(+/+), non-Cre pup at P0.5 is a positive control for cleaved caspase-3 staining. Lu, lumen. Scale bar, 50 μm. (j) Percent of SMCs that are cleaved caspase-3+ from sections represented in i. n = 5 Eln(+/+) mice per group, n = 6 Eln(+/−) mice, n = 6 Eln(−/−) mice per group. Bar graphs are shown as mean ± SD. P values were determined by two-tailed unpaired Student’s t test (e, g, h) or one-way ANOVA with Tukey’s post hoc test (a, c, d, j).

Source data

Extended Data Fig. 2 Sphk1 is essential for elastin deficiency-induced SMC hyperproliferation.

Aortic SMCs were isolated from Eln(+/+), Eln(+/−) or Eln(−/−) pups at P0.5. (a-c) SMCs were cultured for 8 h either with EdU (a, b) and stained for EdU and nuclei (Hoechst), n = 4–6 (n = 5 Eln(+/+), n = 4 Eln(+/−), n = 6 Eln(−/−)) or with BrdU (c) and subjected to BrdU ELISA, n = 40 per group, biological replicates. (d) SMCs were treated with scrambled (Scr) RNA or siSphk1 and then subjected to qRT–PCR. n = 4 per group, biological replicates. (e, f) SMCs treated with Scr RNA or siSphk1 were cultured with EdU for 8 h and then stained for EdU and nuclei (Hoechst). n = 5 per group, biological replicates. (g, h) SMCs were treated with siRNA and then subjected to scratch assay. Percent of wound area relative to time 0 was quantified. n = 5 per group, biological replicates. (i-k) SMCs were treated with siRNA and then transfected with vector control or vector containing wild-type Sphk1 (Sphk1-WT) or catalytically inactive Sphk1 (Sphk1-CI). These SMCs were subjected to qRT–PCR for Sphk1 (i) and EdU cell proliferation assay (j, k). In i, n = 10 SMCs with Scr RNA and vector; n = 5 SMCs with Scr RNA and Sphk1-WT; n = 8 SMCs with Scr RNA and Sphk1-CI; n = 8 SMCs with siSphk1 and vector; n = 5 SMCs with siSphk1and Sphk1-WT; n = 7 SMCs with siSphk1and Sphk1-CI, biological replicates. In j, n = 9 per group, biological replicates. Bar graphs are shown as mean ± SD. P values were determined by one-way ANOVA with Tukey’s post hoc test (b-d, f, i, k) or two-tailed unpaired Student’s t test (h). Scale bars, 100 μm.

Source data

Extended Data Fig. 3 Sphk1 deletion in SMCs does not improve emphysema in Eln(−/−) mice.

(a, c) Transverse lung sections from mice of indicated genotypes at P0.5 were subjected to H&E staining. Of note in c, pregnant dams were injected with tamoxifen daily from E13.5 to E17.5. (b, d) Histograms represent mean linear intercept from sections as represented by a and c. In b, n = 10 Eln(+/+)mice, n = 12 Eln(+/−)mice, n = 12 Eln(−/−)mice. In d, n = 9 Eln(+/+), non-Cre mice; n = 6 Eln(+/+), Acta2-CreERT2 mice; n = 9 Eln(−/−), non-Cre mice; n = 8 Eln(−/−), Acta2-CreERT2 mice. Bar graphs are shown as mean ± SD. P values were determined by one-way ANOVA with Tukey’s post hoc test. Scale bars, 200 μm (a, c).

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Extended Data Fig. 4 Sphk1 deletion with Cdh5-CreERT2 modestly attenuates elastin aortopathy.

(a) Experimental strategy for (b-h). Pregnant dams were injected with tamoxifen daily from E13.5 to E17.5, and pups were collected at P0.5. Graphical objects were created with BioRender (https://biorender.com/). (b) Extracted genomic DNA was subjected to PCR with primers flanking Sphk1. The 120, 250, and 600 bp PCR products represent the wild-type, floxed, and deleted Sphk1 alleles, respectively. (c) Isolated RNA was reverse transcribed and subjected to qRT–PCR. Histograms represent Sphk1 transcript levels relative to Gapdh. n = 5 mice per group. (d) Western blot was performed on lysates of ECs isolated from the aorta. Densitometry of protein bands relative to β-actin is shown on right. n = 4 mice per group. (e) Transverse aortic sections of indicated genotypes were stained for SMA, CD31, and nuclei (DAPI). (f) Histograms represent aortic medial thickness and lumen area from sections as shown in e. n = 10 Eln(+/+) mice per group; n = 12 Eln(+/−), non-Cre mice; n = 6 Eln(+/−), Cdh5-CreERT2 mice; n = 11 Eln(−/−), non-Cre mice; n = 9 Eln(−/−), Cdh5-CreERT2 mice. (g) Transverse aortic sections were stained for proliferation marker Ki67, SMC marker SMA, and nuclei (DAPI). (h) Histograms represent percent of SMCs that are Ki67+ as in g. n = 10 Eln(+/+), non-Cre mice; n = 7 Eln(+/+), Cdh5-CreERT2 mice; n = 15 Eln(+/−), non-Cre mice; n = 12 Eln(+/−), Cdh5-CreERT2 mice; n = 11 Eln(−/−), non-Cre mice; n = 15 Eln(+/−), Cdh5-CreERT2 mice. Bar graphs are shown as mean ± SD. P values were determined by two-tailed unpaired Student’s t test (c, d) or one-way ANOVA with Tukey’s post hoc test (f, h). Scale bars, 200 μm (e), 50 μm (g).

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Extended Data Fig. 5 SPHK1 selective inhibition neither induces apoptosis in aortic SMCs nor improves emphysema in Eln(−/−) mice.

Pregnant dams were injected with vehicle or SPHK1 inhibitor (PF-543) daily from E13.5 to E19.5, and pups were collected at P0.5. (a) Transverse aortic sections of Eln(+/+) and Eln(−/−) pups were stained for cleaved caspase-3 (apoptosis marker), SMA, and nuclei (DAPI). Section of thymus from Eln(+/+) pup injected with vehicle at P0.5 was a positive control for cleaved caspase-3 staining. (b) Percent of SMCs that are cleaved caspase-3+ from sections represented in a. n = 5 Eln(+/+) mice per group; n = 5 Eln(+/−) mice per group; n = 6 Eln(−/−) mice per group. (c) Lung transverse sections from Eln(+/+) and Eln(−/−) pups were subjected to H&E staining. (d) Histograms represent mean linear intercept from sections as shown in c. Bar graphs are shown as mean ± SD. n = 10 Eln(+/+) mice with vehicle; n = 12 Eln(+/+) mice with PF-543; n = 12 Eln(−/−) mice with vehicle; n = 10 Eln(−/−) mice with PF-543. P values were determined by one-way ANOVA with Tukey’s post hoc test (b, d). Scale bars, 50 μm (a), 200 μm (c).

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Extended Data Fig. 6 SPHK1 selective inhibitor mitigates established aortic muscularization in Eln(+/−) mice.

(a) Transverse aortic sections from Eln(+/+) or Eln(+/−) pups at P0.5 were stained for markers of proliferation (Ki67), SMCs (SMA), and nuclei (DAPI). (b) Histograms represent aortic medial wall thickness from sections as shown in a. n = 10 mice per group. (c) Percent of SMCs that are Ki67+ from sections represented in a. n = 5 Eln(+/+) mice, n = 7 Eln(+/−) mice. (d) Experimental strategy for (e-i). Eln(+/+) or Eln(+/−) pups were injected daily with PF-543 (20 mg/kg body weight) or vehicle from P2.5 to P6.5 and analyzed at P8.5. Graphical objects were created with BioRender (https://biorender.com/). (e, h) Transverse aortic sections were stained for SMA, nuclei (DAPI) and either Ki67 in e or cleaved caspase-3 (apoptosis marker) in h. (f) Histograms represent aortic medial wall thickness from sections at P8.5 as shown in e. n = 9 Eln(+/+) mice with vehicle; n = 6 Eln(+/+) mice with PF-543; n = 8 Eln(+/−) mice with vehicle; n = 7 Eln(+/−) mice with PF-543. (g) Percent of SMCs that are Ki67+ from sections represented in e. n = 5 mice per group. (i) Percent of SMCs that are cleaved caspase-3+ from sections represented in h. n = 6 Eln(+/+) mice with vehicle; n = 5 Eln(+/+) mice with PF-543; n = 5 Eln(+/−) mice with vehicle; n = 5 Eln(+/−) mice with PF-543. Bar graphs are shown as mean ± SD. P values were determined by two-tailed unpaired Student’s t test (b, c) or one-way ANOVA with Tukey’s post hoc test (f, g, i). Scale bars, 50 μm (a, e, h).

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Extended Data Fig. 7 Elastin insufficiency increases S1P levels and activates S1PR1, leading to SMC proliferation.

(a) Aortic SMCs were isolated from Eln(+/+) or Eln(−/−) mice, cultured for 72 h and subjected to mass spectrometry to S1P measure levels. Pi, inorganic phosphate. n = 7 Eln(+/+) SMCs, n = 10 Eln(−/−) SMCs, biological replicates. (b, c) SMCs were transfected with Scr RNA or siSphk1 and then treated with vehicle (BSA) or S1P (10−2 µM). Cells were subjected to EdU cell proliferation assay. n = 6 Eln(+/+) SMCs per group; n = 6 Eln(+/−) SMCs per group; n = 9 Eln(−/−) SMCs with Scr RNA per group; n = 6 Eln(−/−) SMCs with siSphk1 and vehicle; n = 9 Eln(−/−) SMCs with siSphk1 and S1P, biological replicates. (d, e) Lysates from aortas of Eln(+/+), Eln(+/−) or Eln(−/−) mice at E15.5 and P0.5 were subjected to qRT–PCR for S1pr1, S1pr2 and S1pr3 (d) or Western blotting for S1PR1 (e). Densitometry of protein bands relative to β-actin is shown in e, right. For mRNA at E15.5 (d, left), n = 6 Eln(+/+) mice, n = 4 Eln(+/−) mice, n = 6 Eln(−/−) mice. For mRNA at P0.5 (d, right), n = 6 Eln(+/+) mice, n = 7 Eln(+/−) mice, n = 6 Eln(−/−) mice. For S1PR1 protein at P0.5 (e), n = 4 mice per group. (f) Ascending aortic sections of Eln(+/+) or Eln(−/−) mice at P0.5 stained for S1PR1, SMA, and nuclei (DAPI). Lu, lumen. (g-i) Aortic SMCs isolated from Eln(+/+), S1pr1(knock-in/knock-in), H2B-GFP pups at P0.5 were treated with Scr RNA or siEln. In (g), lysates from SMCs treated with siRNA as indicated were subjected to qRT–PCR for Eln and Sphk1. n = 8 per group, biological replicates. In (h), SMCs with indicated genotypes and treatments were stained for GFP (marker for S1PR1 activity), SMA, and nuclei (DAPI) with histograms (i) representing percent of SMCs that are GFP+. n = 8 SMCs with Scr RNA, n = 10 SMCs with siEln, biological replicates. (j, k) SMCs were treated with vehicle (DMSO), PF-543 (SPHK1-selective inhibitor, 1 µM), Ex26 (potent and selective S1PR1 antagonist, 1 µM) or fingolimod (functional antagonist of S1PR1, 1 µM) and then subjected to EdU cell proliferation assay. n = 10 per group, biological replicates. Bar graphs are shown as mean ± SD. P values were determined by two-tailed unpaired Student’s t test (a, e, g, i) or one-way ANOVA with Tukey’s post hoc test (c, d, k). Scale bars, 100 μm (b, h, j) and 50 μm (f).

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Extended Data Fig. 8 Eln(+/+) SMC-derived extracellular matrices attenuate Sphk1 expression in elastin mutant SMCs.

(a, b) Isolated aortic SMCs from Eln(+/+), Eln(+/−), or Eln(−/−) pups at P0.5 were cultured for 5 or 10 days and subjected to staining for ELN, fibronectin (FN), SMA, and nuclei (DAPI). (c, d) After 10 days of incubation, cells were decellularized and immunostained for ELN, FN, and SMA. (e, f) After decellularization, aortic SMCs isolated from Eln(+/+), Eln(+/−), or Eln(−/−) pups at P0.5 were re-seeded on the resultant matrices. After incubation for 48 h, lysates from indicated SMCs on each substrate were subjected to qRT–PCR for Sphk1. n = 4 per group, biological replicates. (g, h) Medium was cultured with aortic SMCs of each genotype for 10 days and then collected. Aortic SMCs of the indicated genotypes were cultured with this conditioned medium for 48 h and subjected to qRT–PCR for Sphk1. n = 4 per group, biological replicates. Bar graphs are shown as mean ± SD. P values were determined by one-way ANOVA with Tukey’s post hoc test. Scale bars, 100 μm. Graphical objects were created with BioRender (https://biorender.com/).

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Extended Data Fig. 9 Soft substrates attenuate Sphk1 and Egr1 expression in elastin mutant SMCs.

(a) PDMS substrate A and B are liquid oligomeric base and crosslinker reagents (Sylgard184) with estimated stiffness. (b-d) Aortic SMCs isolated from Eln(−/−) pups at P0.5 were cultured on PDMS substrates for 72 h and then subjected to qRT–PCR for Sphk1 (b) and Egr1 (c) and Western blotting for SPHK1 (d). Densitometry of bands relative to β-actin and normalized to this ratio on plastic is shown in d, right. In b, from left to right, n = 6, 5, 3, 3, 4, 5, biological replicates. In c, n = 4 per group, biological replicates. In d, n = 6 per group, biological replicates. Bar graphs are shown as mean ± SD. P values were determined by one-way ANOVA with Tukey’s post hoc test.

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Extended Data Fig. 10 Postnatal persistent PDA is induced by inhibition of SPHK1 signaling, which is independent of prostaglandin E pathway.

(a) Experimental strategy for (b, c). Pregnant dams bearing wild-type embryos were injected daily from E13.5-17.5 with SPHK1 inhibitor (PF-543, 20 mg/kg) or vehicle and subjected to cesarean section at E18.5 to standardize the time of delivery. Neonates were kept alive and warm on a heating pad and harvested at postnatal ages up to 6 h as indicated to evaluate the kinetics of postnatal DA closure. Graphical objects were created with BioRender (https://biorender.com/). (b) Representative images of transverse sections of DA at postnatal 6 h stained for markers of SMCs (SMA), ECs (CD31), and nuclei (DAPI). Scale bar, 200 μm. (c) Medial wall and lumen area of DA at indicated time points after cesarean section. For the medial wall area, at sequential timepoints from 0 min to 6 h, n = 10, 8, 8, 7 mice with vehicle; n = 10, 10, 10, 10 mice with PF-543. For the lumen area, at sequential timepoints from 0 min to 6 h, n = 10, 8, 8, 6 mice with vehicle; n = 10, 10, 10, 10 mice with PF-543. (d, e) Descending aortic and DA SMCs were isolated from wild-type mice at P0.5. In d, SMCs were treated with either vehicle (DMSO), PGE2 (10−6 M), or indomethacin (10−5 M), and lysates were subjected to qRT–PCR for fibulin 1 (Fbln1), Egr1, and Sphk1. For Fbln1 expression, n = 7 aortic SMCs per group, n = 6 DA SMCs per group, biological replicates. For Egr1 expression, n = 7 aortic SMCs per group, n = 7 DA SMCs per group, biological replicates. For Sphk1 expression, n = 7 aortic SMCs with vehicle; n = 6 aortic SMCs with PGE2; n = 6 aortic SMCs with indomethacin; n = 7 DA SMCs with vehicle; n = 7 DA SMCs with PGE2; n = 6 DA SMCs with indomethacin, biological replicates. In e, SMCs were treated with Scr RNA or siSphk1, and lysates were subjected to qRT–PCR for Sphk1, Fbln1, and cyclooxygenase 2 (Cox2). For Sphk1 expression, n = 5 aortic SMCs with Scr RNA; n = 6 aortic SMCs with siSphk1; n = 5 DA SMCs with Scr RNA; n = 6 DA SMCs with siSphk1, biological replicates. Bar graphs are shown as mean ± SD. P values were determined by two-tailed unpaired Student’s t test (c) or one-way ANOVA with Tukey’s post hoc test (d, e).

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Supplementary Table 1. Demographic information for patients with WBS and controls. Supplementary Table 2. Primer pair sequences used for quantitative PCR with reverse transcription. Supplementary Table 3. Primer sequences used for genotyping.

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Saito, J., Dave, J.M., Gallardo-Vara, E. et al. Sphingosine kinase 1 is integral for elastin deficiency-induced arterial hypermuscularization. Nat Cardiovasc Res 5, 34–50 (2026). https://doi.org/10.1038/s44161-025-00762-7

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