Now a team from the Université de Montréal, the Azrieli CHU Sainte-Justine Research Centre, and the Montreal Heart Institute has put that genetic support to its most rigorous test to date. Writing in Frontiers in Genetics, Tristan St-Laurent, Julien St-Pierre, Julien Quang Le Van, and Marc-André Legault revisited the association between ALOX5 and ALOX5AP variation and atherosclerotic cardiovascular disease using data from 378,962 participants in the UK Biobank, organized into a trans-ancestral meta-analysis spanning four genetically predicted ancestry groups. Their conclusion is blunt: when ancestry is adequately accounted for, the previously reported genetic associations simply do not replicate, and neither hypothesis-driven replication analyses nor agnostic association scans support a role for genetic variation in these two genes in cardiovascular risk.

The technical heart of the study lies in how the researchers handled a polymorphism that has confounded the field for twenty years. In the promoter region of ALOX5, a tandem repeat of the sequence 5′-GCGGGG-3′, known as the Sp1 motif, occurs in variable copy numbers ranging from one to eleven. A landmark 2004 study in the New England Journal of Medicine reported that people carrying non-5-repeat alleles had significantly greater carotid intima-media thickness, a measure of subclinical atherosclerosis, in a cohort of 470 participants from the Los Angeles Atherosclerosis Study. That finding became a cornerstone of the 5-LO hypothesis. But the distribution of Sp1 repeat counts varies dramatically across populations. The UK Biobank analysis confirmed that the most common total repeat count is eight in individuals of African ancestry versus ten in other groups, with the 5-repeat allele predominating in Europeans and deletion alleles being far more frequent in Africans. Because cardiovascular disease prevalence and mean carotid thickness also differ across ancestries, the authors argue that residual population stratification, the confounding of genetic variants with ancestry-linked disease risk, could have generated false positives in earlier studies that used the 5-repeat count as a universal reference point.

To genotype the tandem repeat at scale, the team relied on whole-genome sequencing calls from the UK Biobank’s DRAGEN pipeline, which achieves SNV and INDEL precision exceeding 99.8 percent against Genome in a Bottle standards. They then applied two association models: a direct replication of the original homozygous non-5-repeats coding, and an ancestry-specific dosage model in which copy number is coded relative to the most common allele within each ancestry group. The power calculations are striking. In the European ancestry group alone, the study had 80 percent power to detect a carotid thickness effect of roughly eight micrometers, while the original study reported mean differences exceeding 62 micrometers, meaning the new analysis had approximately 99.9 percent power to detect an effect fourfold smaller. For myocardial infarction, the model could detect an odds ratio of 1.30. Despite this power, the Sp1 repeat variant showed no statistically significant association with C-reactive protein, carotid thickness, LDL cholesterol, myocardial infarction, ischemic stroke, or revascularization procedures. The one nominally significant finding, an association with C-reactive protein in the African ancestry subgroup, did not survive correction for multiple testing and was absent from the trans-ancestral analysis.

There was, however, one biologically coherent signal that survived. Using the ancestry-specific dosage model, the researchers found a significant negative association between the number of Sp1 repeats and plasma omega-6 fatty acid levels. Arachidonic acid, an omega-6 fatty acid, is the primary substrate of 5-LO, so a reduction in enzyme activity would be expected to cause substrate accumulation. This association was directionally concordant across ancestries and consistent with prior nutrigenetic studies linking ALOX5 variants to eicosanoid production. In other words, the promoter polymorphism does appear to influence 5-LO activity, yet that influence does not translate into measurable differences in atherosclerosis or clinical cardiovascular events. The drug target may be real, but the genetic case that its variation drives disease is not.

The ALOX5AP story followed a similar arc. In 2004, an Icelandic team reported that a four-SNP haplotype called HapA in the gene encoding 5-LO activating protein was associated with myocardial infarction and stroke, a finding that was subsequently replicated in Scottish cohorts but contradicted by later meta-analyses of thirty studies finding no association with ischemic stroke. The Montreal team characterized substantial ancestral variability in HapA frequency, noting that one of its constituent SNPs is monomorphic in East Asian populations. Carriers of HapA under a dominant model showed no significantly increased risk of any cardiovascular outcome. A recessive model restricted to homozygous carriers, leveraging the full cohort of 378,962 participants, revealed a modest association with myocardial infarction in the European ancestry subgroup only, with an odds ratio of 1.12, but this did not remain significant after Bonferroni correction across the fourteen statistical models and outcomes assessed. HapA showed no association with omega-6 levels, unlike the ALOX5 promoter variant, further weakening the biological plausibility of the marginal signal.

The researchers then went beyond replication. They tested whether the variants behave differently in individuals at high genetic risk of coronary artery disease, using interaction terms with a CAD polygenic risk score, and found no effect heterogeneity. They examined 24,188 individuals of European ancestry with established atherosclerotic disease at baseline, of whom 5,862 experienced an incident event during follow-up, and found no association between either gene and incident events in this high-risk population. Regional association scans across the ALOX5 and ALOX5AP gene regions, padded by 100 kilobases and analyzed with the REGENIE mixed-effects model, identified five ALOX5 variants reaching the significance threshold for myocardial infarction in the discovery analysis, but three failed to replicate in the independent CARDIoGRAMplusC4D cohort and the remaining two were not present in that dataset, making them likely false positives.

The team also deployed a transcriptome-wide association study, or TWAS, to test whether genetically predicted expression of eighteen leukotriene pathway genes is associated with coronary artery disease. Using S-PrediXcan with expression prediction models derived from GTEx in whole blood, aorta, and coronary artery, and summary statistics from 113,937 individuals with CAD, none of the genes showed a significant association. A gene set enrichment analysis of the leukotriene pathway ranked by TWAS z-scores revealed no significant enrichment either, with a normalized enrichment score of 0.76 and a p-value of 0.77. Finally, rare variant association testing using the Genebass resource from 394,841 UK Biobank exomes and the All by All platform from 392,030 All of Us whole genomes, employing burden tests and SKAT, found no significant associations with any ASCVD-related phenotype.

The implications for drug development are considerable. Genetic validation has become an increasingly important predictor of clinical success for drug targets, and this study systematically dismantles the genetic support for 5-LO and its activating protein as targets in atherosclerotic cardiovascular disease. The authors are careful to note what their analysis does not rule out. It cannot exclude gene-gene interactions within the leukotriene pathway, nutrient-gene interactions involving arachidonic acid availability, or the possibility that inhibiting 5-LO redirects arachidonic acid toward clinically relevant alternate fates. Indeed, a clinical study of asthma patients treated with zileuton, a 5-LO inhibitor, observed increased spontaneous platelet aggregation, consistent with the hypothesis that blocking the enzyme pushes substrate into the cyclooxygenase pathway, generating thromboxane and prostaglandins instead of leukotrienes. The authors suggest that cysteinyl leukotriene receptors or enzymes downstream of 5-LO may be more viable targets, partly because they would spare 5-LO-mediated lipoxin production, which appears beneficial in the plaque environment.

There are also genuine limitations to what genetics can resolve here. The UK Biobank lacks circulating leukotriene measurements, so the team could not use direct molecular markers of pathway activity, relying instead on omega-6 as a proxy. The analysis excluded some ancestry groups not available through self-reporting, such as Middle Eastern populations. And 5-LO activity may be highly localized within the plaque itself, a context that current TWAS approaches, which rely on bulk gene expression atlases from cohorts without necessarily having ASCVD, lack the resolution to detect. The authors propose that future studies incorporating precise molecular measurements of leukotriene pathway products may provide more direct evidence on the impact of genetic variation in these genes. For now, the message is clear: the inflammatory biology of atherosclerosis remains a promising therapeutic frontier, as demonstrated by the success of IL-1β blockers and colchicine in randomized trials, but the specific genetic case for targeting 5-LO and its activating protein in cardiovascular disease has collapsed under the weight of a properly powered, ancestry-aware analysis.

Subject of Research: Revisiting the genetic support of leukotriene pathway drug targets in atherosclerotic cardiovascular disease

Article Title: Revisiting the genetic support of leukotriene pathway drug targets in atherosclerotic cardiovascular disease

Article References: St-Laurent, T., St-Pierre, J., Le Van, J. Q., & Legault, M.-A. (2026). Revisiting the genetic support of leukotriene pathway drug targets in atherosclerotic cardiovascular disease. Frontiers in Genetics, 17, Article 1812905. https://doi.org/10.3389/fgene.2026.1812905

Image Credits: AI Generated

DOI: 10.3389/fgene.2026.1812905

Keywords: Revisiting, genetic, support, leukotriene, pathway, drug, targets, atherosclerotic, cardiovascular, disease, scientific research

PFOS is a typical persistent emerging environmental contaminant, closely correlated with increased cardiovascular disease risks. Atherosclerosis serves as the core pathological basis of cardiovascular disorders, yet the explicit molecular mechanism underlying PFOS-induced pro-atherosclerotic toxicity has remained unclear, limiting the assessment of its environmental cardiovascular hazards. The new study aimed to close that gap by identifying the key signaling axis and pathogenic mechanism of PFOS-aggravated atherosclerosis through a combination of computational prediction and experimental validation.

The research team screened overlapping target genes of PFOS toxicity and atherosclerosis, identifying sixty overlapping target genes. From these, they pinpointed ten hub genes, predominantly CCL2 and CCR2, that were significantly enriched in cytokine-receptor interaction and chemokine signaling pathways. These hub genes represent the critical molecular nodes through which PFOS exposure may exert its pro-atherosclerotic effects, offering a focused target for further investigation into the contaminant’s cardiovascular toxicity.

To validate the computational predictions, the researchers performed molecular docking analyses to assess the binding affinity between PFOS and the core hub proteins. All core hub proteins exhibited high binding affinity with PFOS, with binding energies below −6 kcal/mol. This strong binding suggests a plausible physical interaction between the contaminant and the proteins encoded by the hub genes, lending mechanistic weight to the network toxicology predictions and supporting the hypothesis that PFOS directly engages the CCL2/CCR2 inflammatory axis.

The in vivo phase of the study relied on zebrafish exposure assays, a widely used model for cardiovascular research due to the transparency of embryos and the genetic conservation of key inflammatory pathways. The researchers exposed zebrafish to PFOS and observed dose-dependent developmental toxicity, lipid metabolism disorder, and excessive inflammatory infiltration. These phenotypic alterations mirror the early stages of atherosclerosis, providing a living model of how the contaminant may initiate vascular damage long before overt disease appears.

Crucially, the zebrafish experiments confirmed significant upregulation of CCL2 and CCR2 following PFOS exposure, aligning the in vivo results with the computational predictions. The researchers employed multiple transgenic zebrafish lines to visualize specific cell populations, including Tg(mpeg1:EGFP) with green fluorescent protein labeling macrophages, Tg(lyzc:dsRed) with red fluorescent protein labeling neutrophils, Tg(gata1a:mCherry) with red fluorescent protein labeling hematopoietic cells, and Tg(flk1:EGFP) with green fluorescent protein labeling vascular endothelial cells. They also used apoeb knockout zebrafish, a model of hypercholesterolemia, to examine atherosclerotic-like phenotypes. The convergence of gene expression changes and cellular infiltration patterns strengthens the case that the CCL2/CCR2 inflammatory cascade is a key pathogenic event in PFOS cardiovascular toxicity.

The study’s conclusion is that PFOS exposure was associated with exacerbated early atherosclerotic lesions, possibly through disrupted lipid homeostasis and activation of the CCL2/CCR2 inflammatory cascade, which may serve as an upstream driver of vascular inflammation and macrophage infiltration. This integrated computational and in vivo approach characterizes key pathogenic events associated with PFOS cardiovascular toxicity, provides correlative molecular insights into the pro-atherosclerotic effects of PFOS, and identifies potential biomarker candidates for the toxicological safety evaluation of emerging per- and polyfluoroalkyl substance (PFAS) alternatives.

The implications of the research extend beyond PFOS itself. As industries phase out older PFAS compounds and introduce alternatives, the toxicological safety of these emerging substitutes remains an open question. The biomarker candidates identified in this study, particularly the CCL2/CCR2 axis, could serve as a template for evaluating whether newer PFAS alternatives carry similar pro-atherosclerotic risks. The zebrafish model, with its suite of transgenic reporter lines and knockout strains, offers a scalable platform for such assessments, allowing researchers to visualize inflammatory cell recruitment and vascular alterations in living animals.

The work was supported by the National Natural Science Foundation of China, the Shandong Traditional Chinese Medicine Science and Technology Project, and the Natural Science Foundation of Shandong Province. The research team, led by corresponding authors Wenqing Yang and Dan Zhang at Shandong University of Traditional Chinese Medicine, acknowledges the zebrafish platform of the Animal Experiment Center for providing the multiple zebrafish strains used in the study. As PFOS and related PFAS compounds continue to be detected in water systems and human blood samples worldwide, studies like this one provide the mechanistic foundation needed to understand and ultimately mitigate their cardiovascular hazards.

Beyond the headline findings, the study’s methodological architecture deserves attention because it illustrates how modern toxicology increasingly blends computational screening with whole-organism validation. Network toxicology begins by mining databases for genes associated with a chemical’s known toxicity and with a disease of interest, then intersecting those gene sets to find targets where the compound and the pathology converge. In this case, the intersection yielded sixty overlapping genes, which were then narrowed to ten hub genes through topological analysis of protein-protein interaction networks. Hub genes occupy central positions in these networks, meaning they connect many other genes and are therefore more likely to represent biologically consequential control points rather than peripheral noise. The prominence of CCL2 and CCR2 among these hubs is consistent with decades of vascular biology research showing that chemokine signaling is a master regulator of leukocyte recruitment to the arterial wall.

The CCL2/CCR2 axis itself has a well-established role in atherogenesis. CCL2, also known as monocyte chemoattractant protein-1, is secreted by endothelial cells and smooth muscle cells in response to inflammatory stimuli, while CCR2 is the receptor expressed on the surface of circulating monocytes. When CCL2 binds CCR2, it guides these monocytes out of the bloodstream and into the vessel wall, where they differentiate into macrophages, ingest lipids, and transform into the foam cells that characterize early atherosclerotic plaques. Disrupting this axis in animal models has repeatedly been shown to reduce plaque formation, which is why the finding that PFOS upregulates both the ligand and its receptor is mechanistically significant. It suggests the contaminant may amplify the very signaling pathway that draws inflammatory cells into the arterial intima.

The molecular docking results add a complementary layer of evidence. Docking simulations predict how tightly a small molecule fits into the binding pockets of target proteins, and binding energies below −6 kcal/mol are generally interpreted as favorable interactions in this kind of screening context. While docking cannot replace experimental confirmation of binding, it provides a rational filter for prioritizing which protein targets merit in vivo follow-up. The consistency between the docking predictions and the observed gene expression changes in exposed zebrafish strengthens the overall inferential chain, even though the authors appropriately frame their conclusions in correlational rather than strictly causal terms.

The choice of zebrafish as the experimental platform also carries scientific weight. Zebrafish embryos develop externally and remain optically transparent during early life, allowing researchers to observe immune cell trafficking and vascular morphology in real time under fluorescence microscopy. The transgenic lines used in the study each label a distinct cell population: macrophages, neutrophils, hematopoietic cells, and vascular endothelial cells. By combining these reporter lines with the apoeb knockout background, which impairs lipid clearance and predisposes fish to hypercholesterolemia, the researchers could examine whether PFOS accelerates atherosclerotic-like changes in a susceptible model. This design reflects a broader trend in toxicology toward using vertebrate models that bridge the gap between cell culture studies and mammalian experiments, offering physiological complexity at a scale and cost that permits dose-response characterization.

The dose-dependent nature of the observed toxicity is another important dimension. Developmental toxicity, lipid metabolism disorder, and inflammatory infiltration all intensified with increasing PFOS concentration, which is the kind of monotonic relationship that supports a toxicological interpretation rather than random biological variation. Dose-response data also provide a foundation for future risk assessment work, since regulatory frameworks for environmental chemicals typically rely on identifying the levels at which adverse effects begin to emerge. It is worth noting, however, that the exposure concentrations used in laboratory zebrafish studies often exceed the levels measured in human blood, so translating these findings into human risk estimates requires careful consideration of exposure magnitude, duration, and life stage.

PFOS belongs to the broader family of per- and polyfluoroalkyl substances, synthetic chemicals valued for their resistance to heat, water, and grease, which made them ubiquitous in products ranging from firefighting foams to food packaging. That same chemical stability makes them extraordinarily persistent in the environment and resistant to degradation in the human body, where some PFAS compounds have elimination half-lives measured in years. Epidemiological studies have repeatedly associated PFAS exposure with elevated cholesterol, hypertension, and other cardiovascular risk factors, but the cellular mechanisms connecting exposure to disease have been harder to pin down. This study contributes a candidate mechanism, linking a specific chemokine pathway to the early vascular changes that precede clinically apparent atherosclerosis.

For the field of PFAS toxicology, the identification of biomarker candidates may prove to be one of the most durable contributions. As regulatory pressure phases out legacy compounds and manufacturers shift to shorter-chain alternatives, researchers face a growing backlog of replacement chemicals whose cardiovascular safety profiles remain largely uncharacterized. A validated set of molecular markers, such as CCL2 and CCR2 expression changes detectable in a rapid vertebrate screening model, could streamline comparative testing across the PFAS family. Whether the same inflammatory signature holds true in mammalian systems and in humans exposed at environmental levels remains an open question, and the authors’ framing of their results as correlative molecular insights reflects that appropriate scientific caution. Follow-up studies in mammalian models and human cohort data will be needed to determine how readily these zebrafish findings generalize to human cardiovascular risk.

Subject of Research: Early atherosclerotic phenotypic alterations induced by PFOS exposure: a network toxicology and zebrafish model study

Article Title: Early atherosclerotic phenotypic alterations induced by PFOS exposure: a network toxicology and zebrafish model study

Article References: Wang, H., Yin, J., Zhao, F., Li, M., Zhang, X., Peng, S., Jiang, T., Liang, D., Yang, W., & Zhang, D. (2026). Early atherosclerotic phenotypic alterations induced by PFOS exposure: a network toxicology and zebrafish model study. BMC Pharmacology and Toxicology. https://doi.org/10.1186/s40360-026-01262-5

Image Credits: AI Generated

DOI: 10.1186/s40360-026-01262-5

Keywords: PFOS, atherosclerosis, zebrafish, cardiovascular disease, environmental toxicology, CCL2, CCR2, PFAS, network toxicology, molecular docking, Early, atherosclerotic

The severity grading framework used in the radiology literature helps to contextualize why the blistering described in this case deserves attention. In the national registry analysis of 1,085 extravasation events, the overwhelming majority—94.6%—were classified as mild injuries, typically limited to swelling and discomfort that resolve without specific intervention. Moderate injuries accounted for 4.7% of events, and severe injuries for only 0.8%. Yet the outcome data attached to these categories reveal a striking pattern: in the observational cohort with detailed follow-up, 29 of 32 patients with moderate extravasation developed skin blisters, with a mean recovery time of five days and a range spanning two to twelve days. All five patients with severe extravasation developed blisters as well, and their recovery was markedly prolonged, averaging 23 days with a range of 17 to 30 days. Blister formation, in other words, functions as a clinical marker that the injury has crossed from trivial into a more consequential category, and it is precisely this transition that the present case illustrates.

Several patient-specific and procedural factors converge to explain why a relatively modest estimated extravasated volume of approximately 5 mL could produce tense, fluid-filled blisters within five hours. The osmolality and viscosity of the contrast agent, the injection rate, the volume delivered before the injection was halted, and the anatomical characteristics of the injection site all influence the degree of tissue injury. Iobitridol at 350 mgI/mL is a non-ionic low-osmolar agent, a class considerably safer than the older ionic high-osmolar compounds, but hyperosmolarity remains an inherent property of iodinated contrast. When such fluid enters the interstitial space, it creates an osmotic gradient that draws additional water into the tissue, amplifying swelling and pressure. The power injector flow rate of 3.5 to 4.0 mL/s used for lower-extremity CT angiography, while standard for vascular imaging, delivers contrast under sufficient pressure to dissect along tissue planes once the vessel wall is breached. The forearm, with its relatively tight fascial compartments and thin overlying skin, offers less accommodating space for leaked fluid than looser subcutaneous regions, which may partly explain the rapid emergence of blistering at this site.

The temporal course of the injury is also instructive. The patient reported distending pain at the puncture site during injection—an early warning sign that prompted immediate termination of the injection at 80 mL delivered—and bedside ultrasound subsequently quantified the residual extravasated volume. Yet the visible skin changes did not appear immediately. Only at routine nursing rounds five hours later did the blisters become evident, on a background of erythema and mild swelling. This delayed presentation reflects the physiology of contrast-induced tissue injury: osmotic fluid shifts, inflammatory mediator release, and endothelial changes evolve over hours rather than seconds. The practical implication is that a patient who appears unharmed immediately after an extravasation event still requires structured follow-up observation, as the source case demonstrates through hourly nursing reassessment of the affected limb.

Equally important is what the clinical course did not show. At no point did the patient develop evidence of compartment syndrome—the most feared complication of large-volume extravasation, in which rising interstitial pressure within a confined fascial space compromises capillary perfusion and threatens nerve and muscle viability. The assessment of capillary refill, distal sensation, and passive finger movement, repeated throughout the observation period, represents the bedside evaluation that nursing and medical staff should perform whenever significant extravasation occurs. Pain that intensifies with passive stretch, progressive swelling with tenseness of the compartment, paresthesia, and diminished pulse are the classic warning features, and their absence in this case allowed management to remain entirely conservative. The distinction matters because compartment syndrome from contrast extravasation, while rare, may necessitate urgent surgical fasciotomy, whereas blistering injuries without compartment involvement can generally be managed with topical and supportive measures.

The management regimen applied in this case draws on two distinct therapeutic traditions. Hirudoid cream, whose active constituent is mucopolysaccharide polysulfate, has long held a place in European and Asian practice for superficial phlebitis, bruising, and extravasation injuries. Its proposed mechanisms include anti-inflammatory effects on local tissue and promotion of connective tissue repair and resorption of edema. Guidelines from both the American College of Radiology and the European Society of Urogenital Radiology emphasize conservative first-line measures—cessation of injection, limb elevation, appropriate compresses, and close monitoring—with topical agents such as mucopolysaccharide polysulfate commonly incorporated into that framework. The intermittent ice compresses used here, applied for 15 to 20 minutes every 2 to 4 hours with a cloth barrier protecting the skin, serve the dual purpose of vasoconstriction, which limits further spread of the osmotically active contrast within the tissue, and mild analgesia. Cold application must be timed and barriered carefully, since prolonged direct icing risks adding a cold injury to the existing chemical one.

The third component, Jinhuang San—Golden Yellow Powder, also known by its fuller name Ru-Yi-Jin-Huang-San—is a classical traditional Chinese medicine external formula with a documented history of use for traumatic swelling, carbuncles, and drug extravasation injuries. The supporting evidence cited in the source literature is twofold. A clinical study found that early wet compresses with the formula shortened mean healing time of extravasation-induced skin injury to 6.44 days. Separately, an experimental model of iohexol extravasation demonstrated significant reductions in swelling area and inflammatory infiltration at 24 and 48 hours respectively. The preparation method used in this case—mixing the powder with green-tea water to form a wet poultice—follows customary practice, in which the tea may contribute additional astringent and anti-inflammatory properties while providing the moisture needed for transcutaneous delivery of the herbal constituents.

The rationale for combining these three modalities is plausible at the level of mechanism. Mucopolysaccharide polysulfate addresses inflammation and tissue repair; cold compresses limit the osmotic spread of the extravasated agent and relieve pain; and the herbal poultice, according to the experimental data, reduces both edema and the cellular inflammatory response. Each component targets a different phase or aspect of the injury process, and their combination is conceptually complementary rather than redundant. Nevertheless, the authors are appropriately careful to frame the outcome as hypothesis-generating. A single uncontrolled case cannot establish efficacy, and the natural history of even moderate extravasation injuries includes spontaneous improvement in many patients, making attribution of the rapid 48-hour resolution to the specific regimen inherently uncertain. Prospective, controlled studies would be required to determine whether this integrated approach genuinely accelerates healing compared with standard conservative care alone.

The patient’s underlying vascular disease adds a further dimension of clinical interest. Lower-extremity atherosclerotic occlusive disease, with the severe aortoiliac and iliac stenoses and occlusions documented on the completed CT angiography, indicates widespread arterial compromise. Although the extravasation occurred in the forearm rather than the diseased lower limbs, the systemic atherosclerotic burden raises questions about whether impaired microvascular reserve might influence tissue tolerance to interstitial contrast exposure. The patient’s preserved renal function, with estimated glomerular filtration rates above 125 mL/min/1.73 m² on both assessments, and her regimen of clopidogrel, atorvastatin, nifedipine, and telmisartan reflect the standard pharmacological management of high-risk atherosclerotic disease. Whether antiplatelet therapy, statin-associated effects on endothelial function, or antihypertensive agents modify the local inflammatory response to extravasated contrast remains unexplored, but such interactions represent a legitimate direction for future investigation.

From a prevention standpoint, the case reinforces several established principles of contrast administration. Power injectors deliver contrast at flow rates and pressures that exceed what manual injection can achieve, and they demand correspondingly careful venous access assessment before use. A 20-G peripheral cannula in a forearm vein is an appropriate choice for CT angiography, but cannula position, vein caliber, and patient factors such as fragile veins in older adults all influence extravasation risk. The patient’s complaint of distending pain at the puncture site, rated 6 on a 10-point numeric scale, functioned exactly as an early warning should: the injection was stopped immediately, the cannula removed, and the examination completed through a new puncture site. This responsiveness likely limited the extravasated volume and, by extension, the severity of the injury. Patient-reported symptoms during power injection should never be dismissed, because the interval between the first symptom and tissue damage can be short.

The reporting of this case according to the CARE guidelines also reflects a broader movement toward methodological rigor in case-based evidence. Single-case reports occupy the lowest tier of the evidence hierarchy, but they serve indispensable functions: they document rare presentations, describe novel management combinations, and generate testable hypotheses for controlled research. The observation that blistering from iobitridol extravasation resolved within 48 hours under a three-component topical regimen, without skin breakdown, provides a concrete clinical trajectory that future studies can measure against. Until such studies exist, the case stands as a well-documented example of early recognition, structured multidisciplinary management, and a favorable outcome in an uncommon manifestation of a common complication.

For clinicians and nursing teams, the practical takeaways are straightforward. Every contrast-enhanced examination carries a small but real extravasation risk; patients should be monitored both during and after injection, with particular vigilance in the hours following an event; blistering signals a moderate-to-severe injury requiring escalation of care; and conservative management—elevation, appropriate compresses, topical agents, and serial assessment for compartment syndrome—remains the cornerstone of treatment for injuries without surgical indications. The integration of traditional Chinese medicine external therapies with conventional topical treatment, while promising in this instance, should be viewed as an avenue for further research rather than a settled standard, and the authors’ call for prospective controlled studies is a fitting conclusion to a carefully documented clinical observation.

Subject of Research: Contrast media extravasation following CT angiography in a patient with lower-extremity atherosclerotic occlusive disease: a case report

Article Title: Contrast media extravasation following CT angiography in a patient with lower-extremity atherosclerotic occlusive disease: a case report

Article References: Yan, C., Zhang, T., Xu, Q., & Bi, S. (2026). Contrast media extravasation following CT angiography in a patient with lower-extremity atherosclerotic occlusive disease: a case report. Frontiers in Medicine, 13, Article 1888394. https://doi.org/10.3389/fmed.2026.1888394

Image Credits: AI Generated

DOI: 10.3389/fmed.2026.1888394

Keywords: Contrast, media, extravasation, following, angiography, patient, lower-extremity, atherosclerotic, occlusive, disease, case, report

DNA methylation has long served as one of molecular epidemiology’s most informative bridges between environmental exposures and disease. The addition of a methyl group to cytosine bases, typically at CpG dinucleotides, can silence genes, alter enhancer activity, and reshape chromatin architecture, and these marks are known to respond dynamically to external stimuli such as diet, stress, pollutants, and tobacco smoke. Tobacco smoking is widely regarded as one of the most powerful exogenous modifiers of the human epigenome, producing methylation changes that persist for years, and in some cases decades, after cessation. Because of this durability and sensitivity, methylation signatures of smoking have been proposed as objective biomarkers of exposure that could complement self-reported smoking history, which is known to be imperfect and prone to recall bias.

Until now, however, the field’s largest meta-analyses of smoking-related methylation have been constrained by limited ancestral diversity among participants and by sample sizes that, while impressive, could not support well-powered comparisons between populations. Most prior epigenome-wide association studies of smoking drew predominantly from cohorts of European ancestry, leaving open the question of whether the methylation marks discovered in those populations generalize to people of African, Admixed American, Asian, or other ancestries. Genetic ancestry influences patterns of genetic variation that in turn shape methylation, through mechanisms such as meQTLs, or methylation quantitative trait loci, where nearby genetic variants control methylation levels at specific sites. Differences in allele frequencies and linkage disequilibrium across populations therefore have the potential to alter both the detection and the magnitude of exposure-related methylation associations.

The new analysis, led by Dennis Khodasevich of VA Palo Alto Health Care System and Stanford University School of Medicine together with Andres Cardenas and colleagues, exploited the extraordinary scale and diversity of the Million Veteran Program to address these gaps directly. The team performed epigenome-wide association analyses, or EWAS, comparing methylation measured across the genome between current smokers, former smokers, and never smokers. In the overall study population, the researchers identified 40,529 methylation sites significantly associated with current smoking and 4,240 sites associated with former smoking after applying Bonferroni correction, the stringent multiple-testing threshold that guards against false discoveries when testing hundreds of thousands of positions simultaneously. These numbers represent by far the most comprehensive catalog of smoking-associated methylation ever assembled, dwarfing previous efforts and revealing an enormous breadth of low-magnitude signals that smaller studies could not reliably detect.

Crucially, the team then repeated the analyses within ancestry-specific strata, examining European, African, and Admixed American subgroups separately. The results revealed a striking degree of commonality: between 95 and 100 percent of the associations identified in each ancestry-specific analysis were also detected in the overall population, indicating that the fundamental epigenetic response to smoking is broadly shared across human populations. Yet the study also uncovered evidence of ancestry-specific associations and differences in the magnitude of methylation changes between groups, suggesting that while the core smoking response is conserved, genetic background and environmental context modulate its precise epigenomic expression. Across all analyses, a set of 31 CpG sites emerged as differentially methylated regardless of ancestry, forming a robust, universally responsive signature of smoking exposure.

The identification of these 31 common sites enabled one of the study’s most clinically oriented analyses: the construction of a methylation risk score for predicting atherosclerotic cardiovascular disease, or ASCVD. Smoking is a major driver of atherosclerosis, the pathological buildup of cholesterol-laden plaque in arterial walls that underlies coronary artery disease, peripheral arterial disease, and acute ischemic stroke. The researchers tested whether aggregating methylation values across the 31 shared CpG sites into a single quantitative score could improve prediction of time to ASCVD events beyond conventional three-category smoking status, which distinguishes current, former, and never smokers. The methylation risk score achieved a concordance statistic of 0.673, compared with 0.668 for smoking status alone, a modest but meaningful improvement that illustrates how epigenetic information can capture gradations of biological exposure and residual physiological effect that coarse behavioral categories miss.

The concordance difference, though small in absolute terms, carries conceptual significance for precision medicine. A person’s smoking history exists on a continuum of intensity, duration, and time since cessation, and the methylation mark integrates this exposure over biological time in ways that self-report cannot. By encoding the cumulative epigenetic consequence of smoking into a risk score, clinicians may eventually gain a more nuanced tool for cardiovascular risk stratification, particularly for patients whose reported smoking history is incomplete or inconsistent. The score’s performance also underscores that methylation is not merely a passive record of exposure; it may reflect downstream molecular pathways, such as inflammatory and endothelial processes, that mediate the link between smoking and arterial disease.

The study’s findings on former smoking are particularly noteworthy. The 4,240 significant sites associated with former smoking, together with what the authors describe as extensive low-magnitude associations, demonstrate that the epigenome retains detectable traces of past tobacco exposure long after smoking ceases. Some methylation changes at well-characterized loci, such as sites in the AHRR and F2RL3 genes that previous literature has repeatedly linked to smoking, are known to partially but incompletely revert after cessation. The persistence of these marks raises the possibility that they contribute to the elevated cardiovascular risk that former smokers continue to carry for years after quitting, although the current study establishes association rather than causation, and disentangling whether methylation changes drive disease or merely track it remains a central challenge for the field.

Methodologically, the work exemplifies the power of biobank-scale epigenomics. The Million Veteran Program, a nationwide cohort of US Veterans who consented to genomic research linked to comprehensive electronic health records, provided the sample size needed to detect effects of small magnitude and to stratify analyses by ancestry without sacrificing statistical power. The researchers acknowledged the Veterans whose participation made the study possible, and the analysis was conducted under VA research infrastructure with appropriate ethical oversight and informed consent from all participants. The consortium-based authorship, credited to the VA Million Veteran Program alongside individual investigators from Stanford, the University of Utah, the University of Pennsylvania, and VA medical centers, reflects the collaborative scale that modern genomic epidemiology increasingly demands.

The implications of the study extend across several domains. For basic epigenomics, the massive catalog of smoking-responsive sites provides a rich resource for investigating the biological pathways that tobacco combustion products perturb, including xenobiotic metabolism, oxidative stress, and immune regulation. For genetic epidemiology, the confirmation that ancestry-specific methylation signals exist alongside broadly shared ones reinforces the importance of diverse cohorts and ancestry-aware analytical designs in biomarker discovery, helping to prevent the inequities that arise when tools calibrated in one population are deployed uncritically in another. For clinical medicine, the methylation risk score, while not yet ready for routine deployment, offers a proof of concept that epigenetic biomarkers can add incremental predictive value to established risk factors for the leading cause of death worldwide.

Future work will need to validate the 31-site score in independent, ancestrally diverse cohorts, refine its calibration, and test whether it improves clinical decision-making in prospective settings. Researchers will also want to probe the ancestry-specific associations more deeply, examining whether genetic variation at methylation quantitative trait loci or differing exposure patterns explain the between-group differences in association magnitude. As epigenome-wide association studies continue to scale up, the smoking methylation signature documented in the Million Veteran Program stands as both a landmark catalog and a template for how large, diverse, deeply phenotyped cohorts can illuminate the molecular conversations between environment, genome, and disease.

Subject of Research: Ancestry-specific DNA methylation signatures of smoking and associations with atherosclerotic cardiovascular disease risk: findings from the Million Veteran Program

Article Title: Ancestry-specific DNA methylation signatures of smoking and associations with atherosclerotic cardiovascular disease risk: findings from the Million Veteran Program

Article References: Khodasevich, D., Hilliard, A. T., Barad, A., Zhou, J., Guarischi-Sousa, R., Clarke, S. L., Pridgen, K. M., Lynch, J. A., Chang, K.-M., Tsao, P. S., Assimes, T. L., Cardenas, A., & VA Million Veteran Program (2026). Ancestry-specific DNA methylation signatures of smoking and associations with atherosclerotic cardiovascular disease risk: findings from the Million Veteran Program. Genome Medicine. https://doi.org/10.1186/s13073-026-01761-4

Image Credits: AI Generated

DOI: 10.1186/s13073-026-01761-4

Keywords: Ancestry-specific, methylation, signatures, smoking, associations, atherosclerotic, cardiovascular, disease, risk, findings, Million, Veteran