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The pleiotropic benefits of statins include the ability to reduce CD47 and amplify the effect of pro-efferocytic therapies in atherosclerosis

Nature Cardiovascular Research volume 1, pages 253–262 (2022) Cite this article

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Abstract

The pleiotropic benefits of statins may result from their impact on vascular inflammation. The molecular process underlying this phenomenon is not fully elucidated. In the present study, RNA-sequencing designed to investigate gene expression patterns after CD47–SIRPα inhibition identifies a link of statins, efferocytosis and vascular inflammation. In vivo and in vitro studies provide evidence that statins augment programmed cell removal by inhibiting the nuclear translocation of NF-κB1 p50 and suppressing the expression of the critical ‘don’t-eat-me’ molecule, CD47. Statins amplify the phagocytic capacity of macrophages, and thus the anti-atherosclerotic effects of CD47–SIRPα blockade, in an additive manner. Analyses of clinical biobank specimens suggest a similar link between statins and CD47 expression in humans, highlighting the potential translational implications. Taken together, our findings identify efferocytosis and CD47 as pivotal mediators of statin pleiotropy. In turn, statins amplify the anti-atherosclerotic effects of prophagocytic therapies independently of any lipid-lowering effect.

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Fig. 1: RNA-seq revealed HMG-CoA reductase inhibitor as one of the top upstream regulators of SHP-1 inhibition in macrophages.
The alternative text for this image may have been generated using AI.
Fig. 2: Combined treatment of CD47–SIRPα blockade and atorvastatin showed additive effects on atherosclerotic plaque activity in vivo.
The alternative text for this image may have been generated using AI.
Fig. 3: Combined treatment of CD47–SIRPα blockade and atorvastatin showed additive effects on efferocytosis rate in vitro and in vivo.
The alternative text for this image may have been generated using AI.
Fig. 4: Atorvastatin inhibited NF-κB1 p50 nuclear translocation under atherogenic conditions and thus directly regulated gene expression of Cd47.
The alternative text for this image may have been generated using AI.
Fig. 5: The pleiotropic benefits of statins include the activation of efferocytosis in atherosclerosis.
The alternative text for this image may have been generated using AI.

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Data availability

The authors declare that all data supporting the findings of the present study are available within the paper, its supplementary information files or publicly available. Raw RNA-seq data are available from the National Center for Biotechnology Information under accession no. PRJNA7337400. Source data are provided with this paper.

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Acknowledgements

We thank U. Hedin and L. Matic for the input on the clinical data. We thank M. Käller for assistance with the RNA-seq. We thank the Stanford Cell Science Imaging Facility and Servier Medical Art (www.smart.servier.com) for providing components of Fig. 5. This work was supported by the Deutsche Forschungsgemeinschaft (nos. JA 2869/1-1:1 to K.-U.J. and SFB1123 to L.M.), the Deutsche Herzstiftung e.V. (no. S/09/19 to K.-U.J.), the NIH (no. R35 HL144475 to N.J.L.), the American Heart Association (no. EIA34770065 to N.J.L.) and the German Centre for Cardiovascular Research (DZHK, Junior Research group to L.M.).

Author information

Authors and Affiliations

  1. Department of Surgery, Division of Vascular Surgery, Stanford University School of Medicine, Stanford, CA, USA

    Kai-Uwe Jarr, Jianqin Ye, Yoko Kojima, Zhongde Ye, Hua Gao, Lingfeng Luo, Richard A. Baylis, Mozhgan Lotfi, Nicolas Lopez, Anne V. Eberhard & Nicholas J. Leeper

  2. Department for Vascular and Endovascular Surgery, Klinikum rechts der Isar, Technical University Munich, Munich, Germany

    Sofie Schmid & Lars Maegdefessel

  3. Department of Biomedical Engineering, Michigan State University, East Lansing, MI, USA

    Bryan Ronain Smith

  4. Institute for Quantitative Health Science and Engineering, East Lansing, MI, USA

    Bryan Ronain Smith

  5. Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA

    Irving L. Weissman

  6. German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Berlin, Germany

    Lars Maegdefessel

  7. Department of Medicine, Karolinska Institute, Stockholm, Sweden

    Lars Maegdefessel

  8. Stanford Cardiovascular Institute, Stanford University, Stanford, CA, USA

    Nicholas J. Leeper

  9. Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Nicholas J. Leeper

Authors
  1. Kai-Uwe Jarr
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  2. Jianqin Ye
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Contributions

K.-U.J., J.Y., Y.K., Z.Y., H.G., L.L., S.S., M.L., N.L. and A.V.E. conducted experiments and collected and analyzed data. K.-U.J., J.Y., Y.K., Z.Y., H.G., L.L., R.A.B., B.R.S., I.L.W., L.M. and N.J.L. conceptualized and designed experiments, discussed results and interpreted data. K.-U.J. and N.J.L. designed figures and wrote the manuscript. K.-U.J. directed the study. N.J.L. supervised the study.

Corresponding author

Correspondence to Nicholas J. Leeper.

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Competing interests

I.L.W. and N.J.L. are cofounders and directors of Bitterroot Bio Incorporated, a cardiovascular company studying macrophage checkpoint inhibition. K.-U.J., Y.K., I.L.W. and N.J.L. have filed a provisional patent (US Application serial no. 63/106,794): ‘CD47 Blockade and Combination Therapies Thereof For Reduction Of Vascular Inflammation’. The remaining authors declare no competing interests.

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Nature Cardiovascular Research thanks Chiara Giannarelli, Peter Libby, Tobias Deuse and Chris Jones for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 RNA sequencing revealed HMG-CoA reductase inhibitor as one of the top upstream regulators of SHP-1 inhibition in macrophages.

a, Flow cytometry gating strategy for cell sorting to isolate Cy5.5-positive bone marrow-derived macrophages in each group (SHP1i versus SWNT), which were then subjected to RNA sequencing. b, Rbl1, Xiap, ApoE, Rhob, and Gpx3 expression by quantitative polymerase chain reaction in bone marrow-derived macrophages upon atorvastatin treatment (n = 6 biologically independent samples per group). c, Functional pathways enriched among all differential expressed genes (false-discovery rate < 0.10) as determined by pathway analysis (p-value of overlap). d–e, Samples from vascular smooth muscle cells, bone marrow-derived macrophages, and RAW 264.7 macrophages were collected to rule out mycoplasma contamination by polymerase chain reaction and/or biochemical detection (n = 3 biologically independent samples per group). Each datapoint represents a biologically independent sample. Data and error bars present mean ± 95 % CI for parametric results. Data of b were analyzed using a two-tailed, unpaired Student’s t-test. Data of c were analyzed using a Fisher’s Exact Test, calculated by IPA.

Source data

Extended Data Fig. 2 Combined treatment of CD47-SIRPα blockade and atorvastatin showed additive effects on atherosclerotic plaque activity in vivo.

a, Quantification of atherosclerotic lesion area (n = 9 for PBS; n = 10 for statin). TVA, total vessel area. b, Quantification of necrotic core size (n = 9 for PBS; n = 10 for statin). c, Quantification of total cholesterol, HDL, LDL, and glucose in the blood (n = 9 for PBS; n = 7 for statin). d, Quantification of atherosclerotic lesion area (n = 13 for IgG; n = 13 for anti-CD47; n = 13 for anti-CD47 + statin). e, Quantification of necrotic core size (n = 13 for IgG; n = 13 for anti-CD47; n = 13 for anti-CD47 + statin). f, Quantification of total cholesterol, HDL, LDL, and glucose in the blood (n = 10 for IgG; n = 11 for anti-CD47; n = 10 for anti-CD47 + statin). g, Quantification of atherosclerotic lesion area (n = 12 for SWNT; n = 11 for SHP1i; n = 15 for SHP1i + statin). h, Quantification of necrotic core size (n = 12 for SWNT; n = 11 for SHP1i; n = 15 for SHP1i + statin). i, Quantification of total cholesterol, HDL, LDL, and glucose in the blood (n = 11 for SWNT; n = 10 for SHP1i; n = 12 for SHP1i + statin). j–l, Quantification of atherosclerotic lesion area and necrotic core size (n = 10 for Statin; n = 13 for anti-CD47; n = 13 for anti-CD47 + statin; n = 11 for SHP1i; n = 15 for SHP1i + tatin). Each datapoint represents a biologically independent animal. Data and error bars present mean ± 95% CI for parametric and median ± IQR for nonparametric results. Data of a were analyzed using a two-tailed, unpaired Student’s t-test. Data of b–c were analyzed using a two-tailed Mann–Whitney U test. Data of d,g, and j–l were analyzed using one-way ANOVA with Sidak’s multiple comparisons test. Data of e–f and h–l were analyzed using Kruskal-Wallis with Dunn’s multiple comparisons test.

Source data

Extended Data Fig. 3 Combined treatment of CD47-SIRPα blockade and atorvastatin showed additive effects on efferocytosis rate in vitro and in vivo.

a, Flow cytometry plots depicting the staining controls for the conditions. b, Apoptosis assay to quantify the rate of programmed cell death in vitro in the presence or absence of atorvastatin, SHP1i, and dual treatment after staurosporine (STS) stimulation (n = 5 biologically independent samples per group). c, Immunofluorescence images depicting cleaved caspase-3 activity (n = 9 for PBS; n = 10 for Statin; n = 11 for SHP1i; n = 15 for SHP1i + statin). White line depicts intima. Scale bar, 50 µm; scale bar inset, 10 µm. d, Immunofluorescence images depicting the ratio of free to macrophage associated cleaved caspase-3 activity (n = 9 for PBS; n = 10 for Statin; n = 11 for SHP1i; n = 15 for SHP1i + statin). White line depicts intima. *free cleaved caspase-3. #macrophage-associated cleaved caspase-3. Scale bar, 50 µm; scale bar inset, 10 µm. Each datapoint represents a biologically independent sample. Data and error bars present mean ± 95 % CI for parametric results. Data of b were analyzed using one-way ANOVA test.

Source data

Extended Data Fig. 4 Atorvastatin inhibited NF-κB1 p50 nuclear translocation under atherogenic conditions and thus directly regulated gene expression of Cd47.

a, Cd47 expression by quantitative polymerase chain reaction in bone marrow-derived macrophages (n = 6 biologically independent samples per group). TNF-α, tumor necrosis factor-α. b, Cd47 expression by flow cytometry in bone marrow-derived macrophages (n = 4 biologically independent samples per group). RFI, ratio of median fluorescence intensity. c, Cd47 expression by immunofluorescence in smooth muscle cells (n = 10 cells for vehicle and n = 15 cells for TNF-α or TNF-α + Statin examined over three biologically independent samples per group). SMC, smooth muscle cells. Scale bar, 10 µm. d, NF-κB1 p50 nuclear translocation by immunofluorescence in smooth muscle cells (n = 3 biologically independent samples per group). M, mevalonate. Scale bar and scale bar inset, 10 µm. Each datapoint represents a biologically independent sample. Data and error bars present mean ± 95 % CI for parametric and median +/− IQR for non-parametric results. Data of a were analyzed using one-way ANOVA test. Data of b were analyzed using Kruskal-Wallis test.

Source data

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Differentially expressed genes that regulate the response to SHP-1i in bone marrow-derived macrophages.

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Jarr, KU., Ye, J., Kojima, Y. et al. The pleiotropic benefits of statins include the ability to reduce CD47 and amplify the effect of pro-efferocytic therapies in atherosclerosis. Nat Cardiovasc Res 1, 253–262 (2022). https://doi.org/10.1038/s44161-022-00023-x

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