Highlights
Blood-based DNA methylation patterns were associated with carotid, coronary, and peripheral atherosclerosis in two prospective cohorts involving 3,688 participants.
Most atherosclerosis-associated CpG sites overlapped with sites linked to smoking, inflammation, or metabolic risk traits, suggesting that the epigenetic signal largely reflects cumulative exposure.
Epigenetic risk scores modestly predicted major adverse cerebrovascular and cardiovascular events, but did not clearly indicate strong vascular-bed-specific biology.
Clinical Background and Unmet Need
Atherosclerotic cardiovascular disease remains the leading cause of death worldwide, but the disease does not manifest uniformly across vascular beds. Carotid plaque, coronary atherosclerosis, and peripheral artery disease are related yet biologically distinct phenotypes. Clinicians routinely assess traditional cardiovascular risk factors such as smoking, hypertension, dyslipidemia, diabetes, obesity, and systemic inflammation. However, these measures do not fully capture long-term biological exposure, nor do they explain why some individuals develop disease in one arterial territory more prominently than another.
Epigenetics, especially DNA methylation, has emerged as a potential bridge between environmental exposures and vascular disease. DNA methylation is a chemical modification of DNA that can alter gene expression without changing the DNA sequence. Because blood is accessible and methylation patterns can reflect lifetime exposures, investigators have hoped that blood-based epigenetic profiling could improve risk prediction, identify mechanistic pathways, and potentially uncover therapeutic targets.
This study addresses an important question: do blood DNA methylation signatures meaningfully distinguish between atherosclerosis in different vascular beds, or do they mainly capture cumulative exposure to conventional cardiovascular risk factors?
Study Design and Methods
In this analysis published in the Journal of the American College of Cardiology, Ingold and colleagues examined blood DNA methylation at 767,735 CpG sites in 3,688 individuals from two prospective cohort studies. The investigators performed epigenome-wide association studies (EWAS) to identify methylation sites associated with three atherosclerosis phenotypes: carotid, coronary, and peripheral disease.
Atherosclerosis was objectively assessed using medical diagnoses, ultrasonography-defined carotid plaque presence, and ankle-brachial index for peripheral disease. Significance was defined using 5% false discovery rate correction, which helps limit the number of false-positive findings when testing hundreds of thousands of sites.
To evaluate whether methylation signals had clinical utility beyond cross-sectional association, the team trained methylation scores using ridge regression in one cohort and tested their discriminatory and prognostic performance in a separate cohort. They also examined how much of the methylation signal overlapped with or attenuated after adjustment for cardiovascular risk factors, including smoking, inflammatory traits, and metabolic markers.
Key Findings
Extensive methylation associations were detected across all vascular territories
The investigators identified 1,687 CpG sites significantly associated with carotid atherosclerosis, 3,131 with coronary atherosclerosis, and 5,852 with peripheral atherosclerosis. In total, 2,155 CpG sites were significant in two or more settings, showing that there is both shared and phenotype-specific epigenetic architecture across vascular beds.
The most prominent loci were located in an intergenic region on chromosome 2 near ALPP/ALPG, and within or near AHRR, PRSS23, and F2RL3. Additional strong signals for coronary disease mapped to ABCG1 and DHCR24. Several of these loci, particularly AHRR and F2RL3, are well-established markers of smoking exposure, which immediately raises the possibility that the methylation findings may reflect accumulated tobacco exposure rather than direct atherosclerotic biology.
Epigenetic scores showed prognostic value, but the effect sizes were modest
Methylation-based scores derived from the EWAS were associated with 3-point major adverse cerebrovascular and cardiovascular events. Reported hazard ratios ranged from 1.23 to 1.39, all with P values below 0.001. This indicates statistically robust associations, but the magnitude of risk separation appears moderate rather than transformative.
In practical terms, these scores may carry some prognostic information, yet their incremental value over established clinical predictors still needs to be clarified. The study demonstrates biological signal and risk association, but not yet a validated clinical tool.
Most atherosclerosis-associated CpG sites overlapped with cardiovascular risk factor signals
A key result was the extensive overlap between atherosclerosis-associated CpG sites and CpG sites associated with cardiovascular risk factors. More than 90% of atherosclerosis-associated sites intersected with risk factor-associated sites. Smoking showed the strongest overlap, reaching up to 90%, followed by inflammatory traits at 60% and metabolic traits at 44%.
When the authors adjusted the atherosclerosis EWAS for smoking pack-years alone, the median absolute estimate of significant CpG sites decreased by 19.6% to 29.0%. Joint adjustment for all cardiovascular risk markers reduced the estimates further, by 25.5% to 32.8%. This attenuation suggests that much of the observed methylation signal is mediated by or at least closely linked to conventional exposures and risk states.
Signals were not strongly vessel-specific in blood
Although the study found numerous associations across carotid, coronary, and peripheral disease, the overall pattern did not support a sharply vessel-specific blood methylation signature. Instead, the findings suggest that blood methylation largely integrates systemic, long-term exposure history, particularly smoking and cardiometabolic dysregulation, rather than directly encoding localized vascular-bed biology.
This does not mean methylation is unimportant. Rather, it may be better understood as a biomarker of exposure burden and downstream pathobiology than as a direct map of where plaques are forming.
Expert Commentary and Interpretation
This is a carefully executed comparative EWAS with several strengths. It included a large number of methylation sites, used two prospective cohorts, corrected for multiple testing, and attempted both discovery and validation. The focus on three atherosclerosis phenotypes is clinically meaningful because carotid, coronary, and peripheral disease often coexist but have different screening strategies and prognostic implications.
The most important interpretive message is that blood methylation appears to be more of a systemic exposure record than a vessel-specific disease code. That conclusion is especially plausible given the dominance of smoking-related CpG loci such as AHRR and F2RL3. These sites have repeatedly been shown in prior studies to be strong markers of cigarette smoke exposure, often with persistent methylation changes years after cessation.
From a mechanistic perspective, this is not surprising. Smoking, inflammation, insulin resistance, dyslipidemia, and obesity all exert broad systemic effects on immune cells and circulating blood cells, which are the source of the DNA analyzed in this study. Therefore, blood methylation may capture the inflammatory and metabolic consequences of vascular risk more than the plaque biology itself.
That said, epigenetic profiling could still have value. If a methylation score reliably summarizes lifetime exposure burden, it may complement traditional risk factors, especially when exposure history is incomplete or underreported. It might also help identify patients whose disease progression is driven by cumulative toxic or inflammatory burden despite seemingly acceptable single time-point clinical measurements.
Clinical Implications
For clinicians, the study reinforces three practical points. First, smoking remains one of the most epigenetically visible and biologically important drivers of atherosclerosis. Second, peripheral blood methylation is promising as a research biomarker, but it is not yet ready to replace established risk assessment tools. Third, the path from epigenetic association to clinical utility requires demonstration of incremental predictive value, reproducibility across ancestries and populations, and evidence that methylation-guided intervention improves outcomes.
For researchers, these data argue for more refined tissue- and cell-type-specific approaches. Blood may be ideal for exposure surveillance, but arterial tissue, plaque samples, single-cell epigenomics, and longitudinal within-person designs may be needed to uncover true vessel-specific mechanisms. Integrating methylation with transcriptomics, proteomics, and imaging could also help separate causal biology from biomarkers of exposure.
Limitations
Several limitations should temper interpretation. First, the analysis is observational, so causality cannot be inferred. Second, blood methylation is a proxy tissue; it may not accurately reflect changes occurring within the arterial wall. Third, even though the investigators adjusted for many cardiovascular risk factors, residual confounding is likely, particularly for lifetime smoking exposure and socioeconomic or environmental determinants.
Fourth, epigenetic scores showed only modest prognostic performance, and their added value beyond standard risk calculators was not established in the abstract. Fifth, EWAS findings can be influenced by blood cell composition, batch effects, and cohort-specific characteristics, although large modern studies typically try to mitigate these issues. Finally, generalizability to more diverse ancestries and health systems remains uncertain unless externally validated in broader populations.
Conclusion
This comparative EWAS across carotid, coronary, and peripheral atherosclerosis provides a detailed blood methylation atlas and confirms that epigenetic signals are detectable across vascular territories. However, the central message is that most of these signals overlap with smoking and cardiometabolic risk factors. In blood, the methylation signature of atherosclerosis appears to be more a reflection of cumulative exposure than a sharp marker of vessel-specific disease biology.
That is an important negative as well as a positive finding. It suggests that blood methylation may be best developed as a biomarker of biological risk burden, not as a standalone signature of where atherosclerosis occurs. Future studies should determine whether integrating methylation with clinical risk models can improve prevention, and whether more tissue-specific epigenetic profiling can uncover actionable pathways.
Funding and ClinicalTrials.gov
The abstract provided here does not specify funding details or a ClinicalTrials.gov registration number. Those should be verified in the full article.
References
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