The DNA methylome of human vascular endothelium and its use in liquid biopsies

Ayelet Peretz, Netanel Loyfer, Sheina Piyanzin, Bracha Lea Ochana, Daniel Neiman, Judith Magenheim, Agnes Klochendler, Zeina Drawshy, Ilana Fox-Fisher, Ori Fridlich, Joshua Moss, Daniel Cohen, Hai Zemmour, Gordon Cann, Joerg Bredno, Oliver Venn, Batia Avni, Tural Alekberli, Yaacov Samet, Amit KorachOri Wald, Vladimir Yutkin, Uzi Izhar, Nir Pillar, Markus Grompe, Zvi Fridlender, Ariel Rokach, David Planer, Giora Landesberg, Benjamin Glaser, Ruth Shemer, Tommy Kaplan, Yuval Dor*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Vascular endothelial cells (VECs) are an essential component of each tissue, contribute to multiple pathologies, and are targeted by important drugs. Yet, there is a shortage of biomarkers to assess VEC turnover. Methods: To develop DNA methylation-based liquid biopsies for VECs, we determined the methylome of VECs isolated from freshly dissociated human tissues. Findings: A comparison with a human cell-type methylome atlas yielded thousands of loci that are uniquely unmethylated in VECs. These sites are typically gene enhancers, often residing adjacent to VEC-specific genes. We also identified hundreds of genomic loci that are differentially methylated in organotypic VECs, indicating that VECs feeding specific organs are distinct cell types with a stable epigenetic identity. We established universal and lung-specific VEC markers and evaluated their presence in circulating cell-free DNA (cfDNA). Nearly 2.5% of cfDNA in the plasma of healthy individuals originates from VECs. Sepsis, graft versus host disease, and cardiac catheterization are associated with elevated levels of VEC-derived cfDNA, indicative of vascular damage. Lung-specific VEC cfDNA is selectively elevated in patients with chronic obstructive pulmonary disease (COPD) or lung cancer, revealing tissue-specific vascular turnover. Conclusions: VEC cfDNA biomarkers inform vascular dynamics in health and disease, potentially contributing to early diagnosis and monitoring of pathologies, and assessment of drug activity. Funding: This work was supported by the Beutler Research Program, Helmsley Charitable Trust, JDRF, Grail and the DON Foundation (to Y.D.). Y.D holds the Walter & Greta Stiel Chair in heart studies. B.G., R.S., J.M., D.N., T.K., and Y.D. filed patents on cfDNA analysis.

Original languageAmerican English
Pages (from-to)263-281.e4
JournalMed
Volume4
Issue number4
DOIs
StatePublished - 14 Apr 2023

Bibliographical note

Funding Information:
This paper is dedicated to the memory of Prof. Giora Landesberg. We thank Lilach Gavish, Noa Makhervax, and Dvora Rotnemer for help with sample acquisition; Dr. Eleonora Medvedev for help with flow cytometry; Drs Abed Nasereddin and Idit Shiff from the Genomics lab of the Core Research Facility (CRF) at the Faculty of Medicine, The Hebrew University of Jerusalem, for their support in DNA and sequencing analysis; and Dr. Miriam Grunewald for critical reading of the manuscript. This work was supported by grants from the Human Islet Research Network (HIRN UC4DK116274 and UC4DK104216 to R.S. and Y.D.); the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine; The Alex U Soyka Pancreatic Cancer Fund; The Israel Science Foundation; the Waldholtz/Pakula family; the Robert M. and Marilyn Sternberg Family Charitable Foundation; the Helmsley Charitable Trust; and Grail and the DON Foundation. Y.D. holds the Walter and Greta Stiel Chair and Research grant in heart studies. Graphical abstract created with BioRender.com. Conceptualization, A.P. N.L. B.G. R.S. T.K. and Y.D.; investigation, A.P. N.L. S.P. B.L.O. D.N. J. Magenheim, A. Klochendler, Z.D. I.F.-F. O.F. J. Moss, D.C. H.Z. G.C. J.B. O.V. B.A. T.A. Y.S. A. Korach, O.W. V.Y. U.I. N.P. M.G. Z.V. A.R. D.P. and G.L.; project administration, B.G. R.S. T.K. and Y.D.; supervision, R.S. B.G. T.K. and Y.D.; writing – original draft, A.P. and Y.D.; writing – review & editing, A.P. N.L. G.C. J.B. B.G. R.S. T.K. and Y.D.; statistical analyses, A.P. N.L. R.S. and T.K. A.P. R.S. and Y.D. had unrestricted access to all data. Y.D. and A.P. prepared the first draft of the manuscript, which was reviewed and edited by all other authors. All authors agreed to submit the manuscript, read and approved the final draft, and take full responsibility for its content including the accuracy of the data and statistical analysis. T.K. B.G. R.S. and Y.D. have filed patents related to DNA methylation markers. G.C. J.B. and O.V. are employees of GRAIL, LLC, a subsidiary of Illumina, LLC.

Funding Information:
This paper is dedicated to the memory of Prof. Giora Landesberg. We thank Lilach Gavish, Noa Makhervax, and Dvora Rotnemer for help with sample acquisition; Dr. Eleonora Medvedev for help with flow cytometry; Drs Abed Nasereddin and Idit Shiff from the Genomics lab of the Core Research Facility (CRF) at the Faculty of Medicine, The Hebrew University of Jerusalem, for their support in DNA and sequencing analysis; and Dr. Miriam Grunewald for critical reading of the manuscript. This work was supported by grants from the Human Islet Research Network (HIRN UC4DK116274 and UC4DK104216 to R.S. and Y.D.); the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine; The Alex U Soyka Pancreatic Cancer Fund ; The Israel Science Foundation ; the Waldholtz/Pakula family; the Robert M. and Marilyn Sternberg Family Charitable Foundation ; the Helmsley Charitable Trust ; and Grail and the DON Foundation . Y.D. holds the Walter and Greta Stiel Chair and Research grant in heart studies.

Publisher Copyright:
© 2023 Elsevier Inc.

Keywords

  • DNA methylation
  • Translation to patients
  • biomarkers
  • cfDNA
  • liquid biopsy
  • tissue dynamics
  • vascular endothelial cells

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