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HDAC9 is implicated in atherosclerotic aortic calcification and affects vascular smooth muscle cell phenotype

Nature Genetics volume 51, pages 1580–1587 (2019)Cite this article

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Abstract

Aortic calcification is an important independent predictor of future cardiovascular events. We performed a genome-wide association meta-analysis to determine SNPs associated with the extent of abdominal aortic calcification (n = 9,417) or descending thoracic aortic calcification (n = 8,422). Two genetic loci, HDAC9 and RAP1GAP, were associated with abdominal aortic calcification at a genome-wide level (P < 5.0 × 10−8). No SNPs were associated with thoracic aortic calcification at the genome-wide threshold. Increased expression of HDAC9 in human aortic smooth muscle cells promoted calcification and reduced contractility, while inhibition of HDAC9 in human aortic smooth muscle cells inhibited calcification and enhanced cell contractility. In matrix Gla protein–deficient mice, a model of human vascular calcification, mice lacking HDAC9 had a 40% reduction in aortic calcification and improved survival. This translational genomic study identifies the first genetic risk locus associated with calcification of the abdominal aorta and describes a previously unknown role for HDAC9 in the development of vascular calcification.

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Fig. 1: Polymorphisms in the HDAC9 and RAP1GAP loci are associated with AAC.
Fig. 2: Inhibition of class IIa HDAC activity prevents osteogenic phenotype switch and calcification of cultured VSMCs.
Fig. 3: VSMC calcification, RUNX2 expression, proliferation and contractility are affected by changes in HDAC9 expression.
Fig. 4: HDAC9 deficiency protects against the development of vascular calcification and aortic elastin disruption and improves survival in Mgp−/− mice.

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Data availability

The summary statistics for the cohorts used in the meta-analysis and validation have been deposited with the database of Genotypes and Phenotypes (accession no. phs000930), according to the policy of the CHARGE consortium40. The remaining data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We acknowledge the essential role of the CHARGE Consortium in the development and support of this manuscript. The views expressed in this manuscript are those of the authors and do not necessarily represent the views of the National Heart, Lung, and Blood Institute (NHLBI), the NIH or the U. S. Department of Health and Human Services. The Framingham Heart Study was supported by the NHLBI (contract no. N01-HC-25195) and its contract with Affymetrix for genotyping services (contract No. N02-HL-6-4278). R.M. was supported by the NHLBI (grant nos. K08HL111210 and R01HL142809), the American Heart Association (grant no. 18TPA34230025), the Wild Family Foundation and the Hassenfeld Scholar Award. F.W. was supported by Deutsche Forschungsgemeinschaft (DFG; no. Wu 841/1-1). A.B. was supported by the Department of Defense Peer Reviewed Medical Research Program/Discovery Award (no. W81XWH-17-1-0058). S.U.N. was supported by the American Heart Association (grant no. 15FTF25980003). C.Song was supported by an international postdoctoral fellowship from the Swedish research council (no. 2016-00598). U.H. received research support unrelated to the current project: research grants on behalf of the institution from Kowa, MedImmune, HeartFlow, Duke University (Abbott), Oregon Health & Science University (American Hospital Association; no. 13FTF16450001), Columbia University (NIH; no. 5R01-HL109711) and NIH/NHLBI grant nos. 5K24HL113128, 5T32HL076136 and 5U01HL123339. M.E.L. was supported by the Fredman Fellowship, the Toomey Fund for Aortic Dissection Research and grant no. R01 HL130113. D.B.B. was supported by the Leducq Foundation and the National Institutes of Diabetes and Digestive and Kidney Diseases (no. 5R01DK082971). The Age, Gene/Environment Susceptibility-Reykjavik Study has been funded by National Institute on Aging (NIA) contract no. N01-AG-12100 with contributions from the National Eye Institute, National Institute on Deafness and Other Communication Disorders, NHLBI, NIA Intramural Research Program, Hjartavernd (the Icelandic Heart Association) and Althingi (Icelandic Parliament). MESA and the MESA SHARe project are conducted and supported by the NHLBI in collaboration with MESA investigators. MESA was supported by grant nos. R01 HL071739 and R01 HL72403, and by contract nos. HHSN268201500003I, N01-HC-95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169, UL1-TR-000040, UL1-TR-001079 and UL1-TR-001420. Funding for the SHARe genotyping was provided by NHLBI contract no. N02-HL-64278. Genotyping was performed at Affymetrix and the Broad Institute of Harvard and MIT using the Affymetrix Genome-Wide Human SNP Array 6.0. This work was also supported in part by the National Center for Advancing Translational Sciences, Clinical Translational Science Institute grant no. UL1TR001881 and the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) Diabetes Research Center (DRC) grant no. DK063491 to the Southern California Diabetes Endocrinology Research Center. M.B. was supported by the NIH grant no. R01HL071739. We thank the other investigators, staff and participants of the MESA study for their valuable contributions. A full list of participating MESA investigators and institutions can be found at http://www.mesa-nhlbi.org. The FamHS research was supported by NIH grant no. R01-HL-117078 from the NHLBI and grant no. R01-DK-089256 from the NIDDK. We are indebted to all the study participants and to the dedicated personnel of both the study center of the Heinz Nixdorf Recall study and the electron beam tomography scanner facilities (D. Grönemeyer and R. Seibel) as well as to the investigative group, in particular to U. Roggenbuck, U. Slomiany, E.M. Beck, A. Öffner, S. Münkel, M. Bauer, S. Schrader, R. Peter and H. Hirche. Scientific advisory board: We thank T. Meinertz (Chair), M. Blettner, C. Bode, P. J. de Feyter, B. Güntert, F. Gutzwiller, H. Heinen, O. Hess (deceased), B. Klein (deceased), H. Löwel, M. Reiser, G. Schmidt (deceased), M. Schwaiger, C. Steinmüller, T. Theorell and S.N. Willich. Criteria and end point committee: C. Bode (Chair), K. Berger, H.R. Figulla, C. Hamm, P. Hanrath, W. Köpcke, C. Weimar, A. Zeiher. Funding: We thank the Heinz Nixdorf Foundation (Chairman: M. Nixdorf; Past Chairman: G. Schmidt (deceased)), the German Ministry of Education and Science and the DFG (project nos. ER 155/6-1 and ER 155/6-2) for the generous support of this study. An additional research grant was received from Imatron, which provided the electron beam computerized tomography scanners, and GE-Imatron after the acquisition of Imatron. We acknowledge the support of the Sarstedt AG & Co. concerning laboratory equipment. We received generous support from the Ministry of Culture and Science of North Rhine-Westphalia and the Faculty of Medicine, University Duisburg-Essen for the genotyping of the Heinz Nixdorf Recall study participants. S.P. was supported by the DFG (project no. PE 2309/2-1). M.M.N. received support from the DFG (German Research Foundation) through the Transregional Collaborative Research Centre grant TRR259 (subproject B07 ”GUARD–Genes Underlying AoRtic valve Disease“; project number 426128588) and is a member of the Excellence Cluster ImmunoSensation2, which is funded by the DFG under Germany’s Excellence Strategy–EXC2151 (project number 390873048). Technical support for the imputation of the Heinz Nixdorf Recall Study data on the Supercomputer Cray XT6m was provided by the Center for Information and Media Services, University of Duisburg-Essen. The AA-DHS was supported by grant nos. R01-DK-071891 from the NIDDK, RO1-HL-67348 from the NHLBI and General Clinical Research Center of Wake Forest School of Medicine M01-RR-07122. N.F. is supported by NIH grant nos. MD012765, DK117445 and HL140385.

Author information

Author notes

  1. These authors contributed equally: Donald B. Bloch, Wendy S. Post, Christopher J. O’Donnell.

  2. These authors jointly supervised this work: Rajeev Malhotra, Christopher J. O’Donnell.

Authors and Affiliations

  1. Division of Cardiology, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA

    Rajeev Malhotra, Andreas C. Mauer, Christian L. Lino Cardenas, Christopher J. Nicholson, Georgia Shelton, Mary D. Buswell, Hanna J. Barnes, Haakon H. Sigurslid, Charles Slocum, Caitlin O’ Rourke, David K. Rhee, Mark E. Lindsay & Christopher J. O’Donnell

  2. Harvard Medical School, Boston, MA, USA

    Rajeev Malhotra, Christian L. Lino Cardenas, David K. Rhee, Aranya Bagchi, Sagar U. Nigwekar, Emmanuel S. Buys, Udo Hoffmann, Warren M. Zapol, Mark E. Lindsay, Donald B. Bloch & Christopher J. O’Donnell

  3. Institute for Translational Genomics and Population Sciences, Los Angeles Biomedical Research Institute and Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, CA, USA

    Xiuqing Guo, Jie Yao & Jerome I. Rotter

  4. National Heart, Lung, and Blood Institute Framingham Heart Study, Framingham, MA, USA

    Xiaoling Zhang, Shih-Jen Hwang, Andrew D. Johnson, Ci Song, George Thanassoulis & Christopher J. O’Donnell

  5. Department of Medicine (Biomedical Genetics Section), Boston University School of Medicine, Boston, MA, USA

    Xiaoling Zhang

  6. Department of Biostatistics, Boston University School of Public Health, Boston, MA, USA

    Xiaoling Zhang

  7. Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston, MA, USA

    Florian Wunderer, Aranya Bagchi, Emmanuel S. Buys, Warren M. Zapol & Donald B. Bloch

  8. Department of Anesthesiology, Intensive Care Medicine and Pain Therapy, University Hospital Frankfurt, Frankfurt am Main, Germany

    Florian Wunderer

  9. Icelandic Heart Association, Kópavogur, Iceland

    Albert V. Smith, Sigurdur Sigurdsson & Vilmundur Gudnason

  10. Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA

    Albert V. Smith

  11. Department of Biostatistics, University of Washington, Seattle, WA, USA

    Quenna Wong

  12. Institute for Medical Informatics, Biometry and Epidemiology, University Hospital Essen, Essen, Germany

    Sonali Pechlivanis, Karl-Heinz Jöckel, Susanne Moebus & Raimund Erbel

  13. National Heart, Lung and Blood Institute, Population Sciences Branch, Division of Intramural Research, Bethesda, MD, USA

    Shih-Jen Hwang, Andrew D. Johnson & Ci Song

  14. Division of Statistical Genomics, Washington University School of Medicine, St. Louis, MO, USA

    Judy Wang & Mary F. Feitosa

  15. Wake Forest School of Medicine, Winston-Salem, NC, USA

    Lingyi Lu, Barry I. Freedman & Donald W. Bowden

  16. Division of Nephrology, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA

    Sagar U. Nigwekar

  17. Mid-Atlantic Permanente Medical Group, Rockville, MD, USA

    Catherine Y. Campbell

  18. National Institute on Aging, Bethesda, MD, USA

    Tamara Harris

  19. Division of Cardiology, Department of Medicine and Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, CA, USA

    Matthew Budoff

  20. Department of Family Medicine and Public Health, University of California, San Diego, La Jolla, CA, USA

    Michael H. Criqui

  21. Department of Medical Sciences, Uppsala University, Uppsala, Sweden

    Ci Song

  22. Department of Epidemiology, University of North Carolina, Chapel Hill, NC, USA

    Nora Franceschini

  23. Inserm U1219, University of Bordeaux, Bordeaux, France

    Stephanie Debette

  24. Department of Neurology, University Hospital of Bordeaux, Bordeaux, France

    Stephanie Debette

  25. Department of Radiology, Massachusetts General Hospital, Boston, MA, USA

    Udo Hoffmann

  26. Department of Cardiology, Alfried Krupp Hospital, Essen, Germany

    Hagen Kälsch

  27. Witten/Herdecke University, Witten, Germany

    Hagen Kälsch

  28. Institute of Human Genetics, University of Bonn, Bonn, Germany

    Markus M. Nöthen

  29. Department of Genomics, Life & Brain GmbH, University of Bonn, Bonn, Germany

    Markus M. Nöthen

  30. Centre for Urban Epidemiology, University Hospital Essen, Essen, Germany

    Susanne Moebus

  31. Faculty of Medicine, University of Iceland, Reykjavik, Iceland

    Vilmundur Gudnason

  32. Department of Medicine, McGill University Health Centre, Montreal, Quebec, Canada

    George Thanassoulis

  33. Division of Rheumatology, Allergy, and Immunology; Department of Medicine, Massachusetts General Hospital, Boston, MA, USA

    Donald B. Bloch

  34. Division of Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA

    Wendy S. Post

  35. U.S. Department of Veterans Affairs, Boston, MA, USA

    Christopher J. O’Donnell

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  1. Rajeev Malhotra
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  2. Andreas C. Mauer
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  7. Florian Wunderer
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  18. Haakon H. Sigurslid
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Contributions

R.M., A.C.M., X.G., J.Y., C.Y.C., W.S.P. and C.J.O. conceived and designed the genome-wide association study. A.V.S., Q.W., S.P., S.-J.H., J.W., L.L., C.Y.C., T.H., M.B., M.H.C., J.I.R., A.D.J., C.Song, N.F., S.D., U.H., H.K., M.M.N., S.S., B.I.F., D.W.B., K.-H.J., S.M., R.E., M.F.F., V.G., G.T., W.S.P. and C.J.O. acquired the cohort-based phenotype and genotype data. R.M., A.C.M., X.G., J.Y., X.Z., W.S.P. and C.J.O. carried out the meta-analysis and sQTL analysis. R.M. conceived and designed the cell-based assays and in vivo studies. R.M., C.L.L.C., F.W., C.J.N., G.S., M.D.B., H.J.B., C.Slocum, H.H.S., C.O., D.K.R., A.B., S.U.N., E.S.B., W.M.Z., M.E.L. and D.B.B. acquired, analyzed or interpreted the data in the in cell-based assays and in vivo studies. R.M., A.C.M. and C.J.O. drafted the manuscript. All authors critically revised the manuscript for important intellectual content and approved the content of the manuscript.

Corresponding authors

Correspondence to Rajeev Malhotra or Christopher J. O’Donnell.

Ethics declarations

Competing interests

R.M. serves as a consultant for MyoKardia and Third Pole. A.B. is a consultant for Lungpacer Medical. U.H. is a consultant for Abbott, Duke University (NIH) and ReCor Medical.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Genome-wide association study does not identify polymorphisms associated with thoracic aortic calcification.

Manhattan (left) and Quantile-Quantile (right) plots for the association of thoracic aortic calcification with ~9 million SNPs in the GWAS meta-analysis of 8,422 participants. The hashed line indicates the genome-wide threshold for significance (P < 5 x 10-8).

Extended Data Fig. 2 MGP and HDAC9 deficiency do not result in aortic accumulation of HDAC4 or HDAC7.

Immunofluorescence staining of aortic sections from wild-type, Mgp-/- Hdac9+/+, Mgp-/- Hdac9+/-, and Mgp-/- Hdac9-/- mice. a, Genetic deletion of Mgp and Hdac9 had no influence on aortic HDAC4 expression (red), whereas the vascular smooth muscle contractile marker MYH11 (gold) was more abundant in sections from Mgp-/- Hdac9-/- mice when compared to Mgp-/- Hdac9+/+ mice. b, Immunofluorescence shows no accumulation of HDAC7 (red) in aortas from Mgp-/- Hdac9+/+, Mgp-/- Hdac9+/-, and Mgp-/- Hdac9-/- mice compared to wild-type mice. In contrast, as also shown in Fig. 4f, MMP9 expression was increased in Mgp-/- Hdac9+/+ mice compared to wild-type mice and HDAC9-deficient Mgp-/- mice. Three independent replicates in each group were assessed with representative images shown. Scale bars: 50 μm.

Extended Data Fig. 3 Primary aortic smooth muscle cells from Hdac9-/- mice are protected from calcification.

a, Primary aortic vascular SMCs isolated from Hdac9-/- mice displayed markedly lower mRNA expression of the osteogenic markers Runx2 (left) and tissue non-specific alkaline phosphatase (Tnap, middle) compared to aortic vascular SMCs isolated from wild-type mice. There was also a trend towards decreased expression of Col3a1 (right) in aortic vascular SMCs isolated from Hdac9-/- mice (n = 6 biologically independent samples in each group). b, Hdac9-/- primary aortic vascular SMCs failed to calcify when treated with calcifying media for 10 days compared with vascular SMCs isolated from wild-type mice as determined by Alizarin Red staining. Two independent experiments were performed with representative images shown. Scale bar is 1 cm. c, Hdac9-/- primary aortic vascular SMCs exhibited increased contractility compared with vascular SMCs isolated from wild-type mice (n = 6 biologically independent samples in each group). In a and c, statistical comparisons were made using a two-tailed Student t test. Mean ± s.e.m. is depicted in all plots.

Extended Data Fig. 4 Full scan of Western blots.

a,b, Full scans of the Western blots depicted in Fig. 3f (a) and Fig. 4c (b) are shown.

Supplementary information

Supplementary Information

Supplementary Tables 1–6 and Supplementary Note

Reporting Summary

Supplementary Table 7

SNPs associated with stroke, MI, or CAD were tested for association with AAC and TAC

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Malhotra, R., Mauer, A.C., Lino Cardenas, C.L. et al. HDAC9 is implicated in atherosclerotic aortic calcification and affects vascular smooth muscle cell phenotype. Nat Genet 51, 1580–1587 (2019). https://doi.org/10.1038/s41588-019-0514-8

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