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. 2025 Aug;120(4):799-814.
doi: 10.1007/s00395-025-01105-0. Epub 2025 Jul 1.

A macrophage gene-regulatory network linked to clinical severity of coronary artery disease : The STARNET and NGS-PREDICT primary blood macrophage studies

Lijiang Ma #  1   2 , Jacqueline E Tamis-Holland #  3 , Giuseppe Mocci  4 , Kathryn Wolhuter  5   6 , Nicole S Bryce  5   6 , Swathy Sajja  2   7 , Letizia Amadori  2   7 , Payal Pradhan  2 , Peik Sean Chong  2 , Katyayani Sukhavasi  8   9 , Haoxiang Cheng  2 , Ling Li  10 , Shichao Pang  10 , Eric E Schadt  2 , Heribert Schunkert  10   11 , Moritz von Scheidt  10   11 , Arno Ruusalepp  8   9   12 , Pedro R Moreno  13 , Ke Hao  2 , Chiara Giannarelli  1   2   7 , Clint L Miller  14   15   16 , Jason C Kovacic #  17   18   19   20 , Johan L M Björkegren #  2   4   12   13
Affiliations

A macrophage gene-regulatory network linked to clinical severity of coronary artery disease : The STARNET and NGS-PREDICT primary blood macrophage studies

Lijiang Ma et al. Basic Res Cardiol. 2025 Aug.

Abstract

Coronary artery disease (CAD) is a major cause of global morbidity and mortality. Macrophages play a central role in orchestrating this disease process. In 2016, we initiated the STARNET primary blood macrophage study, followed by the multi-ethnic NGS-PREDICT primary blood macrophage study in 2018. We applied integrative systems genetics analysis to explore and validate the role of macrophage gene regulatory co-expression networks (GRNs) in clinically significant CAD. This study included 318 CAD cases and 134 CAD-free controls in STARNET, and 95 CAD cases and 35 CAD-free controls in NGS-PREDICT. Primary leukocytes were isolated from blood and differentiated into macrophages in vitro, followed by RNA extraction and deep sequencing (RNAseq). In STARNET, we analyzed differentially expressed genes, inferred macrophage GRNs, assessed the phenotypic associations and functions of these GRNs, and determined their key driver genes. Integrative analysis of STARNET expression quantitative traits (eQTLs) with genotype data from genome-wide association studies was performed to determine the content of CAD candidate genes in these GRNs, and their contributions to CAD heritability. Five independent RNAseq datasets were used to retrospectively validate CAD-associated macrophage GRNs, followed by prospective validation in the NGS-PREDICT study. Using the STARNET datasets, we identified 23 macrophage GRNs. Of these, GRNGREEN stood out as being causally associated with CAD severity (SYNTAX score) and comprised 729 genes and 90 key drivers, with the top key driver being NEIL1. GRNGREEN accounted for 3.73% of CAD heritability and contained 34 candidate genes previously identified by GWAS of CAD. Functional analysis of GRNGREEN revealed a large portion of genes involved in the biological process of SRP-dependent co-translational protein targeting to the membrane. GRNGREEN replicated retrospectively in five independent human arterial wall RNAseq datasets, and prospectively in the NGS-PREDICT study. To prevent clinically significant CAD, GRNGREEN and its top key driver NEIL1 may be suitable therapeutic targets to modify SRP-dependent co-translational targeting of proteins to the endoplasmic reticulum in macrophages.

Keywords: Atherosclerosis; Coronary artery disease; Macrophage; Mitochondria.

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Conflict of interest statement

Declarations. Conflict of interest: JCK is the recipient of an Agilent Thought Leader Award (January 2022), which includes funding for research that is unrelated to the current manuscript. JLMB (the Principal Investigator in the STARNET study) is the founder and former CEO of Clinical Gene Networks (CGN), a privately-held company which seeks to commercialize clinical gene networks underlying coronary artery disease, myocardial infarction, and stroke. JLMB serves as Chairman of the Board, and owns equity in the company. JLMB also receives financial compensation as a consultant for CGN. The overall aim of the research collaboration is to investigate the activity of genes and their respective proteins in the cells of organs associated with ischemic heart disease/atherosclerosis and in vascular walls and metabolic organs such as skeletal muscles, fat tissue, and the liver. The ultimate aim is to use new knowledge to identify more accurate diagnostic methods and more effective therapies. The creation of biobanks is performed at Tartu University Hospital. The resources to study DNA, RNA, biopsy material and blood proteins are provided mainly by the Icahn School of Medicine at Mount Sinai (ISMMS) in New York and third parties. AR is also on the board of directors and a shareholder in CGN. Neither ISMMS nor CGN have made claims to results presented in this study. CLM received grant support from AstraZeneca for work unrelated to the current study.

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