One-carbon metabolism and cardiovascular disease: Molecular mechanisms, genetic influences, and epigenetic regulation

Abstract

Hyperhomocysteinemia, characterized by elevated levels of circulating homocysteine, has emerged as a significant risk factor for cardiovascular disease (CVD). This literature review explores the multifactorial relationship between one-carbon metabolism (OCM) and cardiovascular health, focusing on the biological mechanisms that link OCM disruptions to vascular dysfunction and disease development. Emphasis is placed on the biochemical interplay between genetic polymorphisms, nutritional deficiencies (particularly B vitamins and folate), and epigenetic processes including DNA methylation, histone modifications, and non-coding RNAs. These alterations can affect gene expression, endothelial integrity, lipid metabolism, and inflammatory pathways, thereby increasing the risk of atherosclerosis, hypertension, and other CVDs. Promising therapeutic interventions—such as targeted supplementation and epigenetic-modulating strategies—are discussed as future tools for prevention and personalized treatment of cardiovascular disorders.

Highlights

• •One-carbon metabolism links homocysteine to cardiovascular risk.
• •Genetic variants in OCM pathways modulate CVD susceptibility.
• •Epigenetic changes connect OCM imbalance to vascular dysfunction.
• •Nutritional B-vitamin status shapes OCM and CVD outcomes.
• •Personalized OCM-targeted strategies may improve CVD prevention.

1. Introduction

Cardiovascular diseases (CVDs), which encompass a range of conditions affecting the heart and blood vessels, constitute the leading cause of mortality worldwide. They result in a significant burden of deaths and disabilities. In 2021, CVDs were responsible for 20.5 million fatalities, representing approximately one-third of all global deaths [1]. Once considered primarily diseases of affluent nations, cardiovascular diseases (CVDs) now predominantly affect low- and middle-income countries (LMICs), accounting for over 75% of CVD-related deaths [2]. These diseases are the leading cause of premature mortality from non-communicable diseases (NCDs). Ischemic heart disease, in particular, is the foremost cause of premature death in 146 countries for men and 98 countries for women [3]. The global burden is exacerbated by disparities, with LMICs experiencing higher rates of premature CVD mortality compared to high-income countries (HICs), and a slower reduction in age-standardized mortality rates.

In May 2012, the World Health Assembly set a target to reduce premature mortality from non-communicable diseases (NCDs) by 25% by 2025 [4]. To achieve this objective, the NCD Global Monitoring Framework was introduced during the World Health Assembly in May 2013. This framework is designed to enhance NCD prevention and control, serve as a basis for advocacy, increase awareness, bolster political commitment, and encourage global action against NCDs. It includes nine voluntary global targets addressing behavioral and metabolic risk factors, as well as responses from national healthcare systems [4].

Despite over a decade of efforts by international organizations and governments to meet these global targets and reduce premature mortality, the current pace of decline remains inadequate. Trends observed between 2010 and 2016 indicate that fewer than 20% of countries will achieve the UN Sustainable Development Goal Target 3.4 (SDG 3.4), which aims for a one-third reduction in premature mortality from NCDs by 2030 [3]. Most countries, especially those classified as low- or lower-middle-income, need to accelerate their progress to meet these targets.

Despite breakthroughs in treatment, including medications like lipid-lowering agents, anticoagulants, anti-platelet drugs, beta-receptor antagonists, and angiotensin-converting enzyme inhibitors, there remains a considerable gap in therapies capable of preventing disease progression and development [5,6]. In this context, emerging biomaterial-based approaches have gained attention as complementary strategies for cardiovascular repair and regeneration. Recent advances include the development of multifunctional cardiovascular biomaterials and heparin-loaded silk fibroin/bacterial nanocellulose conduits for small-caliber vascular grafts, highlighting the potential for improved hemocompatibility and vascular integration. Epidemiological research indicates that lifestyle and dietary changes can reduce cardiovascular disease (CVD) risk [7]. Other CVD risk factors include gene-environment interactions and gene imprinting changes, mostly mediated by epigenetics [8]. Understanding epigenetics and its involvement in CVD development may lead to new insights and improved disease management strategies [9,10].

Notably, homocysteine-lowering with folic acid and other B vitamins reliably reduces circulating homocysteine, yet large secondary-prevention randomized trials have generally shown limited or no reduction in composite major cardiovascular events, fueling ongoing debate about whether homocysteine is a modifiable causal driver or primarily a biomarker. Recent evidence syntheses suggest that any clinical benefit may be endpoint- and context-specific (for example, a more consistent signal for stroke prevention in settings without or with partial folic-acid fortification), emphasizing the need to interpret homocysteine-lowering interventions within population, baseline-status, and background-fortification contexts [11].

Recent reviews have advanced the field by re-evaluating hyperhomocysteinemia and the mixed outcomes of homocysteine-lowering strategies in cardiovascular disease, as well as synthesizing evidence on genetic susceptibility related to one-carbon metabolism variants. However, these domains are often addressed in parallel rather than within an integrated framework linking one-carbon flux, epigenetic regulation, and clinically relevant heterogeneity in response to nutritional interventions. To address this gap, the present review systematically integrates molecular and epigenetic mechanisms connecting one-carbon metabolism to vascular dysfunction with genetic influences shaping pathway activity and disease susceptibility, and emerging evidence that nutritional interventions interact with genetic background to produce variable biological responses, including differential homocysteine reduction and DNA methylation changes following high-dose folic acid supplementation in hyperhomocysteinemic patients [12].

One-carbon metabolism (OCM) is a critical biological process where one-carbon units, particularly methyl groups, are transferred from donor molecules to acceptor molecules. This process supports essential biological functions such as cell growth, differentiation, and development, maintaining the body's balance. OCM consists of a complex network of reactions that involve interconnected folate and activated methionine cycles. These cycles are vital for cellular activities, underscoring the importance of OCM in preserving health and facilitating tissue repair [13]. By providing substrates, this process aids in lipid and protein synthesis, oxidation and reduction reactions, methylation activities, and nucleotide anabolism [14,15]. Amino acids like glycine, serine, and threonine are sources of the methyl groups central to one-carbon metabolism (OCM). The serine synthesis pathway (SSP) facilitates the entry of these amino acids into cells and their production from glucose. These methyl groups are known as S-adenosylmethionine (SAM), the universal methyl donor in methylation reactions. SAM is synthesized in vivo by l-methionine adenosyltransferase (MATs) from adenosine triphosphate (ATP) and l-methionine (l-Met) during the methionine cycle [16]. When the methyl group from SAM is transferred to an acceptor substrate, S-adenosylhomocysteine (SAH) is formed [17]. One-carbon metabolism (OCM) generates cofactors necessary for various biochemical and metabolic pathways, including redox electron carriers such as NADH and NADPH, and it also produces ATP [18]. Deviations from OCM can increase the risk of aging and age-related disorders [19]. The production of serine/glycine and OCM are crucial for the survival and rapid proliferation of cancer cells, making them significant in therapeutic contexts. Excessive stimulation of serine/glycine synthesis promotes cancer by supplying a one-carbon unit for OCM [20]. Thus, different dietary inputs act as precursors for one-carbon units, which serve as building blocks for biosynthesis, methylation, and redox activities [21]. Nutritional or genetic imbalances in OCM can affect development, leading to decreased embryo growth and poor pregnancy outcomes [22]. In eukaryotes, one-carbon metabolism (OCM) processes are compartmentalized, with each compartment following a distinct metabolic pathway, unlike in prokaryotes. Despite the different routes, folate cofactors are always involved in these pathways. Unlike most bacteria, yeast, and plants, mammals, including humans, cannot synthesize folate and thus depend on dietary folate intake to maintain adequate levels [23]. Folic acid (vitamin B9), a synthetic food additive, is typically the main source of folates for humans [24]. Insufficient folate intake can lead to adult anemia and increase the risk of neural tube defects in newborns [23]. Folic acid must first be converted to dihydrofolate (DHF) and then, via the enzyme dihydrofolate reductase (DHFR), to tetrahydrofolate (THF). THF is the active form of folate that serves as a methyl donor in one of the carbon metabolism routes. It carries chemically reactive one-carbon units, primarily derived from amines like serine and glycine, and choline breakdown products such as dimethylglycine and methylglycine [25,26]. In most human tissues, the primary form of one-carbon-loaded folate is 5-methyl tetrahydrofolate [22]. This form can be modified by enzymes in the cytosol and mitochondria to convert between different oxidation states, with each oxidation state donating a one-carbon unit to drive various biochemical processes [27].

In the early 1950s, the role of mitochondria in the oxidation of one-carbon donors was uncovered, revealing the importance of folate-dependent enzymes in mitochondrial functions. This discovery linked cytoplasmic and mitochondrial one-carbon metabolism, showing that mitochondria house about 40% of total cellular folate [28]. One-carbon-loaded folates struggle to cross intracellular membranes, a characteristic feature of one-carbon metabolism, necessitating their production both within mitochondria and in the cytoplasm [25]. Serine, glycine, and formate are key molecules that facilitate reactions between these two compartments [29,30]. Mitochondrial one-carbon flow primarily involves oxidative processes, producing formate from single-carbon units derived from these connecting molecules [31]. Consequently, mitochondria are considered the primary site for generating glycine and single-carbon units, which are crucial for cell division [32,33].

Cell division and numerous other biological processes rely on B vitamins acting as cofactors, precursors, and substrates. The eight water-soluble B vitamins include B1, B2, B3, B5, B6, B7, B9, and B12. These vitamins are categorized based on their solubility in water and their interdependent roles in cellular coenzyme functions, rather than their chemical structure similarities. Adequate dietary intake of B vitamins is essential for maintaining human health [34]. Deficiencies in these vitamins can lead to serious health issues if there is a disruption in their metabolic pathways [35]. Most B vitamins participate in one-carbon metabolism, either directly or indirectly [34].

2. Biochemical pathways of one-carbon metabolism

One-carbon (1C) metabolism is a fundamental biochemical network that supports essential cellular functions by facilitating the transfer of single-carbon units required for nucleotide biosynthesis, DNA methylation, and redox homeostasis [23,36]. This system is primarily governed by two interconnected cycles: the folate cycle and the methionine cycle, which operate within the cytosol, mitochondria, and nucleus to sustain critical biological processes [25] (cf. Fig. 1).

Fig. 1.

Overview of One-Carbon Metabolism pathways and their role in cardiovascular health.

The folate cycle plays a vital role in 1C metabolism by enabling the activation and transfer of carbon units through different oxidation states. Tetrahydrofolate (THF), the active form of folate, serves as a carrier of these one-carbon units, which are derived predominantly from serine and glycine metabolism [23]. The enzyme serine hydroxymethyltransferase (SHMT) catalyzes the conversion of serine to glycine while transferring a methyl group to THF, generating 5,10-methylene-THF (CH₂-THF), a crucial metabolic intermediate [37]. CH₂-THF serves as a branching point in the folate cycle, undergoing oxidation to 10-formyl-THF (CHO-THF) for purine biosynthesis, or reduction to 5-methyl-THF (5-mTHF), which is required for homocysteine re-methylation in the methionine cycle [38].

The methionine cycle is closely linked to the folate cycle, ensuring the availability of S-adenosylmethionine (SAM), the principal methyl donor for DNA, RNA, and histone methylation. The conversion of homocysteine to methionine is mediated by methionine synthase (MS), which requires 5-mTHF as a methyl donor and vitamin B12 as a cofactor [25]. Methionine is then activated to SAM by methionine adenosyltransferase (MAT), enabling its role in methylation reactions [23]. Once SAM donates its methyl group, it is converted to S-adenosylhomocysteine (SAH), which is hydrolyzed back into homocysteine, completing the cycle.

Apart from its roles in nucleotide synthesis and methylation, 1C metabolism is pivotal for maintaining redox balance through the production of NADPH, a key reducing agent that protects against oxidative stress [39]. The mitochondrial branch of 1C metabolism, driven by methylenetetrahydrofolate dehydrogenase (MTHFD2), generates 10-formyl-THF and NADPH, supporting antioxidant defense and biosynthetic pathways [40]. Dysfunction in these pathways has been implicated in various metabolic and age-related disorders, emphasizing their significance in cellular homeostasis.

Recent studies have highlighted the role of one-carbon metabolism in cardiovascular diseases (CVDs), particularly through its regulation of homocysteine levels and epigenetic modifications [41,42]. Hyperhomocysteinemia, resulting from impaired folate metabolism or genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR), has been identified as a risk factor for atherosclerosis, hypertension, and stroke [39]. Elevated homocysteine contributes to endothelial dysfunction, oxidative stress, and inflammation, all of which promote vascular damage and increase the likelihood of cardiovascular events [41]. Additionally, alterations in DNA methylation patterns regulated by one-carbon metabolism may influence the expression of genes involved in lipid metabolism, vascular remodeling, and immune responses, further linking this pathway to cardiovascular health [42].

Given its essential role in cellular physiology and disease pathology, one-carbon metabolism remains a key area of interest in understanding the molecular basis of cardiovascular diseases and potential therapeutic interventions aimed at modulating these metabolic pathways.

3. Review of epidemiological evidence linking aberrations in one-carbon metabolism

Cardiovascular diseases (CVD) are to be the leading cause of mortality worldwide, accounting for more than one-third of all deaths [43]. CVD encompasses alterations in the cardiovascular system, which consists of the heart and the blood vessels. This term is usually associated with fatty acid (FA) deposition (atherosclerosis) in the blood vessels of the heart (coronary artery disease -CAD) and/or the periphery (peripheral artery disease-PAD), blood clots formation, stroke, arrythmias, and valve disease. Because one carbon metabolism (OCM) interferes in so many cellular mechanisms, OCM dysregulations were associated with cardiovascular and other age-related diseases [44]. Yuan et al. mentioned that HTN is one of the most important risk factors for CVD [45]. American Heart Associations (AHA) defines HTN when a patient SBP is equal or above 140 mmHg and/or diastolic blood pressure equal or above 90 mmHg [46]. Rooney et al. elaborated that a 10 mmHg increase in Blood pressure (BP) is associated with 9% increased risk for CVD [47]. Contrarily, a decrease of 6-13 mmHg Bp is anticipated to lower CVD risk by 30% [18]. We will discuss the role of OCM intermediates in CVD and in HTN in the upcoming paragraphs.

3.1. Homocysteine (Hcy)'s effect on both CVD and HTN

Homocysteine, a non-proteinogenic sulfhydryl-containing amino acid, is a homolog of cysteine. The serum concentration is regulated by two different pathways: remethylation back to methionine or trans-sulfuration to cysteine [48]. A lot of studies were conducted to determine if there is a correlation between OCM and CVD. The most relevant studies focused mainly on homocysteine (Hcy) as a good CVD marker. Ganguly et al. and Kang et al. approved by a meta-analysis that there is a correlation between high levels of Hcy and an increased risk of hemorrhagic stroke and CVD [49,50]. Pawlak et al. and Xiong et al. discussed two pathways for Hcy breakdown, both require adequate amount of vitamin B6, B9 and B12 [43,51]. In the case of vitamin B deficiency, hyperhomocysteinemia (HHcy) results. The latter statement was supported by Yang et al. who elaborated the incidence of decreased Hcy serum concentration by vitamin B6 supplementation [52]. Liu et al. supported that this increase in Hcy levels has been associated with increased risk of CVD [53]. Furthermore, vitamin B9 deficiency had little significance on CVD when Hcy levels were adjusted by supplementation [53]. This suggests that the association is significantly mediated by circulating Hcy. Moreover, elevated homocysteine in the blood increased atherosclerotic plaque formation and arterial thrombosis formation. Not only that, but Yuan et al. mentioned the association between HHcy and hypertriglyceridemia [45]. Plasma Hcy showed a positive and negative association with LDL-C and HDL-C respectively [45]. Hypercoagulability was also shown to correlate with HHcy [54]. Patients with HHcy are present with increased platelet adhesions, procoagulant factors (V, XII) and decreased anticoagulants (reduced protein C and tissue plasminogen activator activity). These studies conclude a correlation between HHcy and plaques formation, hypercoagulability, increased LDL-C, and arterial thrombosis, all of which increased risk of MI and mortality.

HHcy was not only associated with CVD but also with HTN. Yuan et al. showed that the majority of hypertensive patients in the study were associated with HHcy [45]. HHcy may influence the development of HTN via four different mechanisms: first by affecting vasorelaxation due to HHcy cytotoxic effect on smooth muscle cells, thus, affecting blood vessels’ contractility. Second, HHcy impaired nitric oxide (NO) production by endothelial cells, thus affecting vasorelaxation. Third, HHcy altered elastin/collagen ration, thus affecting elasticity. Fourth, HHcy induction of inflammatory cytokine and/or chemokine in monocytes and endothelial cultures inducing T cell proliferation, leading to T cell mediated HTN [55]. Alkaissi et al. added that HHcy affect HTN by reducing the expression of nitric oxide synthase (NOS), decrease NO and increase reactive oxygen species (ROS) production [54]. This results in accumulation of peroxynitrite (ONOO-), a ROS, causing an increase in vasoconstrictive eicosanoid (thromboxane A2-TxA2) production. Wu et al. also noted amplified oxidative stress and induced vascular inflammation in HHcy [56]. The overall effect of HHcy on blood vessels is an increase in vasoconstriction and a decrease in vasodilation. This results in an increase in peripheral vascular resistance contributing to elevated high blood pressure.

3.2. B-vitamins’ effect on both CVD and HTN

3.2.1. B-vitamin status, deficiency, and metabolite levels in relation to CVD and HTN

B vitamins are a group of eight water-soluble micronutrients that play essential roles in cellular metabolism and homeostasis, several of which are integral to one-carbon metabolism (OCM) [57]. Deficiencies in vitamins B6, B9 (folate), and B12 have been implicated in cardiovascular disease (CVD) pathogenesis, largely through disruption of homocysteine metabolism and methylation processes [43,53,58].

Low circulating levels of vitamin B6 have been associated with increased cardiovascular morbidity and mortality [52]. Proposed mechanisms include heightened inflammation, oxidative stress, impaired lipid and glucose metabolism, and increased reactive oxygen species generation [56,59]. Similarly, folate and vitamin B12 deficiencies impair homocysteine remethylation, resulting in hyperhomocysteinemia (HHcy), which has been consistently associated with endothelial dysfunction, atherosclerosis, thrombosis, and hypertension [43,45,53].

Observational studies further suggest that both low and excessively high circulating concentrations of folate and vitamin B12 are associated with increased CVD mortality, particularly among hypertensive or metabolically compromised individuals [53]. Vitamin B12 deficiency is also associated with hematologic abnormalities, including elevated mean corpuscular volume (MCV) and red cell distribution width (RDW), which have been linked to venous thrombosis and coronary artery disease risk [43,60]. Together, these findings support an association between altered B-vitamin status, disturbed one-carbon metabolism, and adverse cardiovascular outcomes.

3.2.2. Dietary intake of B vitamins and associations with CVD and HTN

Epidemiological studies evaluating habitual dietary intake of B vitamins have generally reported inverse associations between folate, vitamin B6, and vitamin B12 intake and cardiovascular risk. Higher dietary intake of these vitamins has been associated with lower prevalence of hypertension, improved lipid profiles, and reduced risk of CVD events in population-based studies [51,59,61].

However, these associations are not uniform across studies and appear to be influenced by baseline nutritional status, genetic background, and circulating homocysteine levels. Several analyses suggest that the apparent cardiovascular benefit of higher B-vitamin intake is attenuated after adjustment for homocysteine, indicating that homocysteine may act as an important mediator rather than an independent causal factor [53]. Thus, while adequate dietary intake of B vitamins is associated with favorable cardiovascular profiles, the strength and independence of these associations vary across populations.

3.2.3. B-vitamin supplementation and primary or secondary prevention of CVD and HTN

B-vitamin supplementation, particularly with folic acid, vitamin B6, and vitamin B12, has been extensively studied for the primary and secondary prevention of cardiovascular disease. Supplementation consistently lowers circulating homocysteine levels, and several studies and meta-analyses have reported reductions in stroke risk, along with improvements in endothelial function and vascular reactivity [59,62,63].

Folic acid supplementation has been shown to enhance nitric oxide (NO) bioavailability, prevent nitric oxide synthase (NOS) uncoupling, and reduce oxidative stress, contributing to improved endothelial function and modest blood pressure reductions in selected populations, including pregnant women and individuals with low baseline folate status [62,64].

Nevertheless, large randomized controlled trials and comprehensive reviews have demonstrated that homocysteine lowering through B-vitamin supplementation does not consistently translate into reductions in major cardiovascular outcomes. Several secondary prevention trials reported null effects on myocardial infarction, composite cardiovascular endpoints, and all-cause mortality despite significant reductions in homocysteine levels [61,65]. A detailed review of clinical trial evidence concluded that, although vitamin supplementation reliably lowers homocysteine, it generally fails to reduce cardiovascular risk, supporting the interpretation that homocysteine may function primarily as a biomarker rather than a causal factor in atherothrombotic disease [66].

Discrepancies across studies likely reflect heterogeneity in baseline homocysteine levels, background folic acid fortification, vitamin doses and combinations, genetic variation in one-carbon metabolism enzymes, duration of follow-up, and differences between surrogate and clinical endpoints [53,63,65]. Importantly, evidence suggests that B-vitamin supplementation provides limited cardiovascular benefit when baseline homocysteine levels are not elevated or when baseline vitamin status is adequate [53,66].

Overall, the totality of evidence indicates that while B-vitamin supplementation effectively modifies one-carbon metabolism and lowers homocysteine, its impact on cardiovascular disease prevention is context-dependent and most evident in populations with deficiencies or hyperhomocysteinemia rather than as a universal preventive strategy.

3.3. Limitations and null effects of B vitamin supplementation

Although there is compelling mechanistic evidence that OCM perturbation could lead to vascular dysfunction, a remarkable clinical paradox has emerged from randomized controlled trials of B-vitamin supplementation. Several largeRCTs have consistently observed no appreciable cardiovascular benefit of B-vitamin therapy for major cardiovascular outcomes [58,67]. While vitamin B6, B9, and B12 supplementation consistently decreases circulating Hcy concentrations, this biochemical correction has not consistently translated into a significant lowering of CVD events or mortality [66]. These findings question previous assumptions that Hcy lowering by B-vitamin supplementation directlycauses cardiovascular protection.

Seminal trials, including Heart Outcomes Prevention Evaluation-2 (HOPE-2) and Vitamin Intervention for Stroke Prevention (VISP), detected no decrease in CVD events among high risk individuals with Hcy-lowering therapy [[68], [69], [70]]. Several reasons have been suggested for such null findings, including sufficient baseline folate status of populations following folic acid fortification, late onset supplementation in CVD failure (F), and interindividual genetic variation modulating the efficiency of OCM with gene polymorphisms particularly in MTHFR enzyme [58,[71], [72], [73]]. In line with this interpretation, a meta-analysis observed little or no effect of secondary prevention B-vitamin supplementation, and particularly among folate-replete populations [74].

There is also accumulating evidence that cardioprotective influence of B-vitamins is epigenetically mediated and appears to be related more to long-term or early exposures rather than short-term intervention after the development of advanced CVD [75]. Altogether, these observations highlight the intricate regulation of OCM and suggest that its relevance to cardiovascular risk is highly context-specific and depends on nutritional status, genetic background, disease stage [41,76,77]. The heterogeneity of clinical findings emphasizes the shortcoming for focusing on OCM as a therapeutic approach in the context of end-stage vascular pathology.

As public focus on hyperhomocysteinemia (HHcy) as a modifiable CVD risk factor has increased, several reports have tried to generalize findings in small studies. But meta-analyses of B-vitamin trials did not show consistent cardiovascular benefit, even with successful homocysteine lowering [78]. However, recent data suggest that prophylactic B-vitamin supplementation may reduce CVD risk and a measure of sub-clinical atherosclerosis (carotid intima-media thickness) in healthy individuals [76]. Our data imply that interventions to lower homocysteine may be most beneficial prior to the development of manifest cerebro- and cardiovascular disease.

In these high-risk CVD populations, any homocysteine-lowering strategy may need to be complemented with other pharmacological approaches in order to confer vascular benefit. For instance, classes of antihypertensive drugs like ACE inhibitors were associated with reduction of homocysteine induced endothelial dysfunction [79]. Likewise, additional to its lipid-lowering effect, statins have been reported to enhance endothelial function via a HDAC-inhibiting activity mediated via Akt-activation [80,81]. However, clinical studies that have controlled for statin use failed to find further benefit of combined with compared with statins alone [82]. These results support the principle that B-vitamin treatment for advanced atherosclerotic disease is less promising for preserving endothelial integrity than it is to achieve itsregression [83].

Finally, while a comprehensive discussion of metabolic syndrome is beyond the scope of this review, increasing evidence point to an involvement of one-carbon metabolites in the chronic inflammatory background associated with metabolic disorders and their cardiovascular implications [84]. In summary, we suggest that the deviation between mechanistic understanding and clinical impression of hyperhomocysteinemia is due to incomplete knowledge of OCM and previously over simplifying paradigms based on single-pathway modifications that do not reflect the many levels of metabolic and pathologic interaction driving OCM toward chronic consequences.

• •MTHFR C667T ’s effect on both CVD and HTN

Methylene tetrahydrofolate reductase (MTHFR) is an enzyme in the OCM catalyzing 5,10 methylene THF into 5-methyl THF, this enzyme proved to be vital for cellular homeostasis especially due to its role in the metabolism of both methionine and folate and in the synthesis of protein, RNA, and DNA as well [85]. In addition, this enzyme maintains methionine and Homocysteine (Hcy) balance and prevent any cellular dysfunction [85]. MTHFR C677T polymorphism will cause an amino acid change from Alanine 222 to Valine. This mutation decreases the optimal functioning of the enzyme, especially after 37 °C, make it thermolabile [85,86]. This polymorphism results in loss of function of the enzyme, therefore, a loss of balance between methionine and Hcy leading to hyperhomocysteinemia (HHcy), which in turn, has been associated with increased risk of CVD [45,85,87]. The increase in homocysteine (Hcy) levels, a sulfur-containing amino acid, reflect a decrease in DNA methylation levels [88,89]. The reduction in genome-wide methylation level was closely related to atherosclerosis formation. Therefore, MTHFR C667T polymorphism is associated with a higher risk of atherosclerosis and MI [88]. Yuan et al. stated how this polymorphism was linked with hypercholesterolemia and abnormal LDL-C levels which increased the risk for atherosclerosis and MI [45]. In addition, Raghubeer et al. mentioned that this mutation was also associated with cancer, inflammatory conditions and vascular disorders [85].

MTHFR polymorphism is not only associated with CVD but also associated with hypertension (HTN) [47,90]. Rooney et al. showed that brachial BP in individuals 18-65 years is increased in MTHFR C667T polymorphism compared to normal genotype [47]. The risk for HTN is even doubled in vitamin B, riboflavin, and MTHFR cofactor deficiency. Mcnulty et al. identified eight genetic loci associated with blood pressure, one of which is near the MTHFR gene [91,92]. Therefore, associating C677T polymorphism with an increased risk of hypertension.

In addition, riboflavin, MTHFR co-factor/vitamin B2, seems to play a crucial role in blood pressure modulation, improving BP control, especially in hypertensive patients [47,91,92]. Low or deficient riboflavin biomarker was associated with a three-fold risk HTN in MTHFR polymorphism [47]. Vitamin B2 was proven to be an effective anti-hypertensive drug, especially for patients with the MTHFR polymorphism C677T [93]. Therefore, riboflavin supplementation demonstrated a good response in lowering BP, especially in MTHFR C667T genotype independently [47,92]. This supplementation did not decrease in BP alone, studies reported a decrease in homocysteine levels as well, suggesting a stabilizing effect of riboflavin on the variant enzyme, thus, restoring its activity [92].

• •Endothelial damage

One Carbon Metabolism (OCM) is a complex network of metabolic cycles and pathways; namely the methionine cycle, the folate cycle and the transulfuration pathway [23].

Aberrations of the one carbon metabolism leads to depletion/accumulation of its various constituent metabolites (such as homocysteine) due to enzymatic insufficiency, impaired excretion, or dietary habits. Homocysteine is a non-proteinogenic amino acid known for its significant toxicity at elevated levels. Hyperhomocysteinemia, a complex state with complex etiologies [94], has become a representative and a marker of metabolic defects within the one carbon cycle.

Impairments in one carbon metabolism have become a well-established contributor to cardiovascular disease. Indeed, not only have the past two decades revealed a wealth of academic literature [[95], [96], [97], [98], [99]] that corroborates this most significant role in the development and progression of various cardiovascular pathologies, but recent works have shifted towards OCM metabolites as diagnostic and therapeutic targets [[100], [101], [102], [103]]. As will be discussed in the following section OCM disruption is an independent risk factor for endothelial dysfunction, a known progenitor of atherogenesis, and by extension, cardiovascular disease.

Vascular endothelial cells form cellular membranes that serve as a tightly regulated selectively permeable barriers. Far from being passive walls, endothelial membranes actively participate in the regulation and wellbeing of the vascular system by modulating constriction, inflammation and thrombosis with important implications in atherosclerosis and its sequelae [104].

Homocysteine is a well-recognized disruptor of endothelia [105,106]. Utilizing an ex vivo model of endothelial dysfunction, hyperhomocysteinemia impaired vasodilation in sectioned porcine [107,108], and murine arteries [109]. Stuhlinger et al., Zheng et al., and Haloul et al. [[90], [91], [92]] revealed homocysteine as a culprit in endothelial dysfunction none-invasively by measuring peripheral vessel dilation via ultrasonography in human subjects [[110], [111], [112]]. Meanwhile, the authoritative meta-analysis by Bokayeva et al. assayed randomized controlled trials finding a significant effect of folate supplementation, possibly, by homocysteine lowering, on flow-mediated vasodilation, a key indicator of endothelial function [113].

Impaired endothelia kick off a complex signaling cascade ultimately leading to a pro-inflammatory/pro-oxidative shift. Monocytes are thus activated, adhering to and infiltrating into vascular intimae. Chronic local upregulation of inflammatory chemokines dysregulates lipid metabolism within these monocytes, leading to the formation of so-called “foam cells” (cf. Fig. 2). Foam cell accumulation exerts significant oxidative and inflammatory damage on neighboring tissue leading to deleterious tissue remodeling. This remodeling is characterized by progressive endothelial damage and apoptosis accompanied by smooth muscle proliferation, elastin degradation and collagen accumulation forming a fibromuscular lesion nidus. This latter matures into an atherosclerotic plaque with a fibrinous layer encasing a lipid-rich core of necrotic foam cells, accumulated lipids, as well as reactive oxygen species (ROS) and their free radical progeny [114,115] (see Fig. 3).

Fig. 2.

Mechanistic pathways linking hyperhomocysteinemia to endothelial and atherosclerosis.

Fig. 3.

Role of P66shc in atherosclerotic cardiovascular disease. Hcy: Homocysteine, SAH: Adenosylhomocysteine, ROS: Reactive Oxygen Species, eNOS: Endothelial Nitric Oxide Pathway, CVD: Cardio-Vascular Disease.

As seen throughout this work, one of the primary markers of endothelial function is arterial dilatation, often measured in response to flow-rate variation. Indeed, this ability of vessels to dilate is a central feature of healthy endothelia. At a molecular level, vasodilation is mediated by a specialized nitric oxide-mediated pathway referred to as “endothelial nitric oxide synthase pathway” (eNOS). NO is a remarkable secondary metabolite with extensive roles in various signaling pathways. Pertinent to our study are two forms of nitric oxide synthase: the eNOS and the inducible NOS (iNOS). Whereas the former is a constitutive, calcium dependent pathway in which NO performed paracrine signaling within the endothelium. The iNOS is an important inflammatory pathway produced by leukocytes in response to pro-inflammatory cytokines and with far reaching consequences on chronic inflammation and pathological states. As such, discussion of the NOS pathways goes beyond simple up- and down-regulation and more into the grey-scale of mechanistic dysregulation and disruption. Within the pathophysiology of cardiovascular disease, iNOS is rarely discussed in isolation. Instead, it often rears its head within the greater context of the inflammatory processes of atherosclerosis. As compared with eNOS, iNOS leads to a significantly larger, albeit transient, NO release, which exerts massive ROS (ONOO⁻) stress and primes surrounding leukocytes leading to monocyte recruitment and ultimately the formation of foamy macrophages [116].

More important to our analysis of the one-carbon metabolic pathways is the eNOS axis. Normal mechanical (laminar shear stress), endocrine (VEGF, insulin, estrogen, bradykinin …) and physiological stimuli nominally induce protein kinase B (akt) via the action of phosphoinositide 3-Kinase (PI3K) on phosphatidylinositol-4,5-bisphosphate (PIP2). Akt kinase activation, primed by anchoring at the phosphatidylinositol (3,4,5)-trisphosphate (PIP3) moiety, further proceeds with akt phosphorylation by PIP₃-dependent kinase 1 (PDK1) and mechanistic target of rapamycin complex 2 (mTORC2). The activated akt kinase then mediates the phosphorylation of NO synthase, actuating NO release [117].

1C disruptions fundamentally disturb the tight balance that keeps endothelial NO in check. This disbalance, which has been termed “eNOS uncoupling”, refers to disruptions of the synthetic cascade nominally producing NO from l-arginine leading to the production of reactive oxidative species, particularly superoxides (O₂⁻), well known for their central role in atherogenesis. Depletion of tetrahydrobiopterin (BH4), an essential co-factor of NO synthase, is particularly involved in eNOS uncoupling especially in the context of heritable 1C metabolism disorders [118]. Likewise, enzymatic arginine wasting via Hcy-activated arginase is an important mediator of disease. Lastly, increasing oxidative and inflammatory burden lead to further endothelial dysfunction and further oxidative damage in the now all-too-familiar deleterious cycle [119]. A wealth of literature, described in detail in Table 1, has progressed this link between 1C metabolism and endothelial dysfunction far beyond barebones mechanistic interplay and into in vivo studies that bring these pathways into the midst of clinical relevance.

Table 1.

Mechanisms and effects of homocysteine (Hcy) on endothelial dysfunction and atherogenesis.

Mechanism	Effect	References
Inflammatory Cytokine regulation
Hcy ↑ TNF-α, NF-κB, MCP-1, IL-1β, IL-6, IL-8, IL-10, IL-12	Promotes inflammation and atherogenesis	[[120], [121], [122], [123], [124], [125]]
Hcy ↑↑ Receptor for Advanced Glycation End-products (RAGE), VCAM-1, MMP-9, and tissue factor	Activates NF-κB	[126,127]
Hcy ↑ Angiotensin II Type I receptor, ↑ PKC & ERK1/2, ↑ IL-6, MCP-1	Macrophage infiltration and adventitial fibroblast migration; promotes atherogenesis	[120]
Hcy ↑ TNF-α, IL-6, MCP-1, and superoxide ROS	Induces pro-inflammatory bone marrow and monocyte differentiation via rIFN-γ and Ly6C, leading to macrophage accumulation	[128]
Hcy ↑ ROS, α-SMA, N-cadherin, p-p65 protein; ↓ VE-cadherin	Facilitates endothelial-mesenchymal transition (EndMT), contributing to atherogenesis progression. Catalpol may reduce these effects.	[129,130]
Nitric Oxide Suppression
Hcy ↑ Protein Kinase C; ↓ eNOS, NO production	Leads to endothelial dysfunction	[131]
Methionine diet ↑ Hcy, ↓ Akt & eNOS, ↑ VEGF	Promotes endothelial dysfunction	[109,132]
Obesity-related Hcy ↑; Folate & B12 ↓	Decreases NO and endothelial function (in vivo)	[110]
Hcy ↓ DDAH, ↑ ADMA; ↓ eNOS, NO	Possible amelioration by 5-aza-2′-deoxycytidine (Aza) via ↑ DDAH and ↓ DNMT & ADMA	[[133], [134], [135], [136]]
Endoplasmic Reticulum Stress
Hcy ↓ Cystathionine-β-synthase (CBS) & cystathionine-γ-lyase (CSE)	Reduces H2S, leading to inflammation, ER stress, and endothelial damage. Estradiol-17β offers protection.	[137,138]
Hcy dysregulates protein folding via PDI overexpression	Causes ER stress and apoptosis. H2S restored PDI activity, mitigating effects.	[139]
Autophagy Suppression
Hcy ↑ ROS, suppressed autophagy markers	Induces endothelial senescence	[140]
Hcy & metabolic derivatives (Hcy-thiolactone, N-Hcy-protein)	Downregulate PHF8/H4K20me1/mTOR/autophagy pathway	[141,142]
Endothelial Apoptosis
Hcy ↑ Death-Associated Protein Kinase (DAPK); ↓ Bcl2, ↑ Bax, and Caspase-2	Induces endothelial apoptosis. Selenium supplementation shows protective effects.	[143,144]
Hcy Gasdermin cleavage	Induces pyroptosis, exacerbating local inflammatory response	[122]
Oxidation
Hcy ↑ ROS	Drives oxidative stress and endothelial cell death via redox interaction with copper	[[145], [146], [147], [148], [149]]
Hcy ↑ TLR4/NF-κB/DNMT1, ↑ ROS	Accumulates oxidized LDL, promoting atherogenesis	[148]
Hcy & Homocysteine Thiolactone (HTL)	Reduces plasminogen and plasmin activity, leading to fibrinolysis dysfunction and thrombotic risk.	[[150], [151], [152]]
Other Mechanisms
CR6 interacting factor 1 (CRIF1)	Disrupts DHFR expression, increasing Hcy levels and promoting atherogenesis	[153]
Hcy dysregulates macrophage lipid metabolism	Inhibits cholesterol efflux via PCSK9 synergism, contributing to atherogenesis	[154,155]
Hcy contributes to lipid metabolism disruption	Involves paraoxonase 1 (PON1) and possibly the mevalonate pathway	[156,157]

Abbreviations: TNF-α: Tumor Necrosis Factor-α; NF-κB: Nuclear Factor-κB; ROS: Reactive Oxygen Species; NO: Nitric Oxide; eNOS: Endothelial Nitric Oxide Synthase; VEGF: Vascular Endothelial Growth Factor; ER: Endoplasmic Reticulum.

An obstacle commonly faced when studying atherogenesis is the sheer diversity and complexity of the molecular pathways involved. Indeed, the concise pathophysiology provided above grows into gargantuan dimensions when seen through a mechanistic lens. As such, a sufficiently detailed coverage of the latter and its interplay with one-carbon metabolites makes for a tedious and verbose reading. To remedy this, a thorough coverage of recent pertinent works has been compiled into Table 1. While we attempt to break down the mechanisms into discrete entities, the reality of molecular signaling often treads over semantic borders. The reader will thus notice a tightly woven interplay of pathways, in this sense, all roads, unfortunately, lead to atherogenesis.

4. Genetics factors influencing OCM and their potential effects on CVD

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that catalyzes the conversion of 5′,10′-methylenetetrahydrofolate to 5′-methyltetrahydrofolate and it is also involved in the remethylation of homocysteine to methionine. These are two major reactions that are involved in folate metabolism and methylation pathways [158]. Homocysteine-Methionine (HM) cycle yields S-adenosylmethionine (SAM) which is a universal methyl group donor. It also produces methyltransferase inhibitor S-adenosylhomocysteine (SAH) and homocysteine (Hcy) [159]. MTHFR is the enzyme that is responsible for the metabolism of methionine into homocysteine which is a sulfur amino acid [160].

There are several mutations in the MTHFR gene that alter its activity. One of these polymorphisms is the variant of the MTHFR gene that involves a replacement of alanine by valine (C to T) at position 677 [161]. MTHFR C677T is a single nucleotide polymorphism (SNP) that has been rotated to increase levels of homocysteine which is an important risk factor for coronary artery disease (CAD) [158]. This polymorphism is widely considered as the main cause of hyperhomocysteinemia [85,161], since MTHFR is a gene involved in the catabolism of Hcy [162]. It is established that hyperhomocysteinemia (HHcy) is an independent and important risk factor for cardiovascular diseases (CVD) [85,159], including CAD [163], atherosclerosis [164], and stroke [165].

Variants of C677T MTHFR has been associated with HHcy as the catabolic activity of this enzyme has been decreased to 65% in CT carriers and 30% in TT carriers as compared to CC (wild type) [162]. This SNP prevents the optimal functioning of the enzyme at temperatures above 37 °C [85]. A study that was done to test the association between C677T MTHFR, levels of circulating Hcy, and the coronary lesions’ severity in patients with acute coronary syndrome (ACS), showed that TT patients have higher levels of Hcy and more severe coronary lesions as compared to CT and CC groups suggesting its important role as coronary artery disease (CAD) marker [166]. It has been suggested that C677T MTHFR polymorphism, also known as rs1801133 polymorphism, could affect the DNA methylation state, therefore altering lipid metabolism leading to CAD [167]. The association between MTHFR gene and cardiovascular diseases played a role as well in autoimmune disorders, especially systemic lupus erythematosus, where it was shown that the MTHFR 677 TT genotype is an independent predictor for arterial wall thickening as well as plaque formation [168]. Moreover, two studies have investigated the relationship between Rheumatoid Arthritis patients with 677 TT genotype and plasma homocysteine level as compared to those with 677CC genotype, and their findings showed that those with the 677 TT genotype have higher plasma homocysteine level [169,170].

There is an association between an elevated plasma Hcy and an elevation in hemorrhagic stroke risk [50], with a higher risk of developing heart disease in patients with C677T polymorphism [171]. Moreover, Frosst et al. proposed that this mutation might have a role in vascular diseases [172]. Furthermore, the homozygous C677T MTHFR gene polymorphism correlates with triple the likelihood of premature development of CVD [173]. HHcy coincides with a higher incidence of venous thrombosis but research has offered contradictory findings as to whether the C677T MTHFR polymorphism is responsible for the thrombosis [174]. Some studies stated that there was no link between thrombosis and the C677T polymorphism [174,175]. Conversely, other studies documented a significant correlation between these two or that the C677T polymorphism acts as one of the risk factors for the development of thrombosis [[176], [177], [178], [179]]. Furthermore, a study conducted by Xuan et al. concluded that there is a correlation between myocardial infarction (MI) risk and the C677T polymorphism but another contradicting study by Alizadeh et al. documented no overall link in the CT as compared to TT models [86,87]. MI occurs when coronary arteries get occluded by clots that are formed when atherosclerotic plaques rupture and it is considered as one of the most prevalent CVDs [180]. MI is a complex process that involves environmental and genetic factors and it is also believed to be affected by the levels of Hcy [87].

Prothrombin is a precursor of thrombin that has an important role in the formation of fibrin and coagulation. And multiple studies have documented there is a correlation between increased plasma prothrombin levels and G20210A which is the single base-pair change in the prothrombin gene [33,[181], [182], [183]]. G20210A variant involves a single base change from guanine (G) to adenine (A) at position 20210 [184]. Carriers of a single copy of this polymorphism have approximately 5 times greater risk of developing a blood clot. And this risk is even elevated to 50 times greater in patients with 2 copies of the G20210A allele making them more susceptible to venous and arterial thrombosis [182,185,186]. Another study was conducted to assess the potential correlation of C677T MTHFR and G20210A factor II prothrombin polymorphisms with the likelihood of developing MI. The results of this study documented that there was an association of G20210T FII polymorphism and MI risk, but no correlation was obtained between C677T MTHFR and MI risk [187].

Cystathionine-β-synthase (CBS) is the first enzyme in the transsulfuration pathway of Hcy. It catalyzes the reaction that converts serine and Hcy into cystathionine and water [188]. Certain studies isolated hearts from wild type mice and from HHcy mice which were heterozygous CBS deficient mice. And the results reported that the hearts of HHcy mice have impaired contractile function, defective cardiac relaxation and elevated death of the cells following reperfusion after ischemia [189]. A study documented that certain CBS alleles act as risk factors for vascular disease development [190]. A study determined that there was no association between T833C mutation in the CBS gene and Myocardial Infarction (MI) in African Americans (Dilley et al., 2001). A meta-analysis was conducted to study the correlation of G919A, T833C and 844ins68 polymorphisms in the 8th exon region of the CBS gene with coronary artery disease (CAD) [191].

Therefore, there are multiple genetic factors that influence the One-Carbon Metabolism and have a potential effect on CVDs.

5. The association between MTHFR C677T and CVD across different populations

The association between the variants of the MTHFR gene and cardiovascular diseases has been widely studied in the general population and it is believed that those with the MTHFR 677 TT genotype have significantly higher CVD risk [167,171,173,192].

As for the ethnic and geographical distribution of the MTHFR C667T polymorphism reported to be frequent in US and Europe, intermediate in Asia, and less prevalent in Africa [193]. Alizadeh et al. found a statistical significance relating MTHFR polymorphism to myocardial infarction (MI) in African populations but not in European, Asian and American populations [87]. Furthermore, Alizadeh et al. reported a decreased risk of MI in North American population, possibly due to different genetic background [87]. In contrast, this polymorphism did not increase risk of CVD in the elderly but had a protective effect against MI [87].

A meta-analysis that included 47 studies concluded that there was no statistically significant correlation between MI risk and MTHFR polymorphisms [87]. On the contrary, a sub-group analysis by ethnicity concluded that there is an increased MI risk in carriers of the T allele by 63% in African populations as compared to the C allele. However, the CT genotypes showed a decrease in the risk of MI in North American (Caucasian) populations as compared to the CC genotype [87]. Another study reported that the 677T MTHFR polymorphism is considered as a genetic risk factor that leads to the development of congenital heart disease (CHD) in the Chinese pediatric population [194]. A further meta-analysis showed a remarkable association between MTHFR C677T and increasing the risk for coronary artery disease in the Chinese population [195].

Furthermore, another study population that included 13,748 adults taken from a pooling of 4 population-based studies that took place in Denmark showed that there was no association between TT genotype and CVD (hypertension, dyslipidemia, stroke and all-cause mortality). However, this study showed that there is an increased risk for ischemic heart disease in TT genotype [196].

Moreover, a meta-analysis was conducted on Turkish population in order to estimate the risk of CVDs associated with MTHFR C677T and it showed a strong association between the T allele and increased risk for CVD suggesting that this polymorphism acts as a significant risk factor for cardiovascular diseases in the Turkish population [197].

Therefore, a remarkable association has been shown between MTHFR C677T polymorphism and cardiovascular diseases across various populations and ethnicities.

6. Epigenetics factors influencing OCM and their potential effects on CVD

Epigenetic regulation of gene expression has an important role in vascular disease development [198]. Atherosclerosis is influenced by multiple factors including genetics and non-genetic factors which renders it as a complex disorder [199]. Atherosclerotic vascular disease and vascular endothelial dysfunction are a result of the independent risk factor hyperhomocysteinemia and cellular methylation reactions are modulated by homocysteine [200].

DNA methylation is a post-synthetic modification of DNA that plays a crucial role in the epigenetic modulation of gene expression. DNA methylation, the covalent addition of a methyl group at the 5 carbon of a cytosine ring resulting in 5-methylcytosine, is considered as a leading epigenetic modification that is involved in the pathological consequences of S-adenosylhomocysteine (SAH) accumulation [201]. It is considered as the most important epigenetic mechanism in the pathogenesis of atherosclerosis and it involves gene modification without the actual change in the sequence of the gene [199]. There is a positive association between elevated S-adenosylhomocysteine (SAH) levels, which is the precursor of homocysteine, and the development and progression of atherosclerosis as well as with the increased risk of CVD [202]. Inhibition of the SAH hydrolase (SAHH) leads to an elevation in plasma SAH. The ensuing SAH-associated endothelial injury plays a role in the development of atherosclerosis through the epigenetic regulation of the p66shc-mediated oxidative stress pathway [202]. Elevations in the plasma level of Hcy is considered to be an independent risk factor for atherosclerosis and venous thrombosis.

DNA methylation process is catalyzed by a minimum of 5 independent DNA methyltransferases which includes DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L. De novo methylation is done by DNMT3A and DNMT3B while the maintenance of DNA is performed by DNMT1 [203]. In vivo, as well as in vitro, DNA methylation can be regulated at the SAH level, since SAH is a potent inhibitor of DNA methyltransferases. SAH reduces the expression as well as the activity of these DNA methyltransferases. An example of this was portrayed in the association of high SAH levels and decreased mRNA expressions of DNMT3A and DNMT3B [204,205].

Nutrients such as B vitamins and folic acid are involved in low-carbon metabolism (OCM) and they interact to regulate DNA methylation [199]. Folic acid is known to re-methylate Hcy to methionine by methylene tetrahydrofolate reductase-dependent pathways [206]. Moreover, folic acid produces high-energy phosphates, acts as an antioxidant and improves nitric oxide (NO) production by the enzyme endothelial NO synthase [206]. Deficiencies in folic acid, B6 and B12 can cause an increase in homocysteine levels which in turn induce endothelial dysfunction, thus accelerating the pathological process of atherosclerosis [199]. During epigenetic modifications, the methylation of DNA/RNA clearly produces homocysteine. Additionally, an increased production of Hcy is due to the enhanced overexpression of methyl transferase. The high level of Hcy and its accumulation induces vascular dysfunction [206]. Supplementation with these nutrients can make the DNA methylation status better and it can reduce the levels of inflammatory factors as well, and delay the atherosclerotic process [199].

There are multiple mechanisms in which HHcy leads to endothelial dysfunction and these include oxidative inactivation of nitric oxide (NO) [207,208]. Another mechanism is the generation of reactive oxygen species (ROS) when it is auto-oxidized to Hcy in plasma [209]. Multiple pieces of evidence suggest that Hcy stimulates a change in the methylation process of DNA and proteins which may mediate the effects of Hcy on vascular endothelial cells. The transfer of methyl groups from S-adenosylmethionine (SAM) to substrates is catalyzed by cellular methyltransferases. These substrates include dC bases in CpG dinucleotides. This leads to the formation of S-adenosylhomocysteine (SAH). SAH, in turn, inhibits the trans-methylation reaction [210]. The cellular pool of SAH is increased by HHcy and that decreases the SAM/SAH ratio. This mechanism has been involved in Hcy-induced p21ras-mediated inhibition of the growth of endothelial cells [211]. Moreover, hypomethylation of cyclin A locus and its subsequent decrease in the expression of cyclin A, has also played a role in Hcy-induced endothelial cell growth inhibition [212]. In endothelial cells, increased levels of SAH are correlated with DNA hypomethylation [213].

P66shc is a protein that is a part of the shcA family of adaptor proteins, that increases intracellular oxidative stress by dysregulating mitochondrial function. Upon entering the mitochondria, p66shc oxidizes cytochrome c leading to generation of reactive oxygen species from oxygen. P66shc knockout cells, including endothelial and cardiac cells have demonstrable lower levels of ROS even under pro-oxidant exposure. Additionally, p66shc-mediated pro-oxidant profile was associated with mitochondrial swelling, dysfunction and rupture with important consequences on cell apoptosis and tissue function [214]. It was shown that mice that are deficient for p66shc have increased lifespan [215]. These mice are protected against the age-associated endothelial dysfunction [216] and the high-fat drug-induced atheroma [217]. Furthermore, p66shc has interesting interactions with the endothelial nitric oxide pathway essential for endothelial tissue function. P66shc knockout cells exhibited increased eNOS expression and subsequent improved vaso-relaxation [216] and endothelial function [218].

The expression of p66shc displays significant variability in different tissues, and methylation of CpG dinucleotides in the promoter region of the p66shc long being recognized as responsible for regulating the transcription of this gene [219,220]. The partial failure of B vitamin supplementation in the prevention and treatment of cardiovascular disease despite appropriate homocysteine levels control now points towards larger 1-C metabolic dysfunction, and not isolated HHcy, as a main culprit in the pathophysiology of cardiovascular disease. Indeed, within various murine models expressing elevated S-adenosylhomocysteine (SAH), a Hcy precursor metabolized by S-adenosylhomocysteine hydrolase (SAHH), p66shc upregulation was noted within the larger context of oxidative stress, impaired eNOS and subsequent vascular dysfunction. This effect was greatly ameliorated by DNA methyltransferase 1, p66shc siRNAs and antioxidant supplementation, confirming the role of both p66shc within the endothelial dysfunction cascade and that of 1-C metabolism dysregulation in the up-regulation of p66shc via epigenetic disruption [202].

Within human populations, patients with coronary artery disease had increased SAH, p66shc expression which was associated with increased oxidative markers and impaired flow-wave dilation when compared to healthy controls. Both populations demonstrated an inverse association between SAH accumulation and flow-wave dysfunction, and a positive association between SAH levels and p66shc hypomethylation [202].

Similarly, p66shc mRNA levels were associated with Hcy levels and both were strongly correlated with greater endothelial disease burden in CAD patients using various markers of endothelial dysfunction and atherosclerosis [221].

HHcy acts as a risk factor for CVD particularly affecting the vasculature as in atherosclerosis. However, the exact mechanism of this process is still not well understood. Epigenetic modifications like DNA methylation and histone acetylation have key roles in modulating gene expression during atherosclerosis development. Moreover, noncoding RNAs, such as microRNAs (miRNAs) have been implicated in gene expression regulation [222]. The small, single-stranded miRNAs are endogenous, noncoding RNA with 18-22 nucleotides. These play crucial roles in various biological processes [223,224]. Abnormal expression of miRNAs has been involved in multiple pathophysiological processes that lead to atherosclerosis development and CVD. These processes include various changes in endothelial function, macrophage function, foam cell formation, and vascular smooth muscle cell formation and proliferation [225,226]. Several studies have shown that epigenetic modifications of miRNAs trigger the pathogenicity of HHcy in vascular diseases. A seminal study done by Zhang et al. has shown that the upregulation of DNMT3a (DNA Methyltransferase 3a) results in the direct hypermethylation of miR-143 genes in homocysteine-induced vascular smooth muscle cells (VSMCs). It also showed that downregulation of its function leads to the proliferation of VSMCs. Both of these processes influence the pathogenesis and the progression of atherosclerosis [227]. Once the lysine residues on histones are modified by methylation, the chromatin structure is altered regulating gene expression. For example, there is an elevation in H3K27me3 modification in aortas that are affected by Hcy-induced atherosclerotic plaques. This process was accompanied by histone methyltransferase EZH2 alterations which were regulated by miR-92a [228].

One possible molecular basis of the relation between Hcy and atherosclerosis involves its effect on the cellular transmethylation reactions [[229], [230], [231]]. Several metabolic pathways tightly regulate the intracellular concentration of Hcy. This intracellular concentration of Hcy affects the cell methylating capacity. The cell methylating capacity is defined as the ratio of SAM to SAH in methionine metabolism [106,230]. SAM is the methyl donor to multiple methyltransferases that affect countless biomolecules such as proteins and DNA. Elevated levels of SAH inhibit the actions of these SAM-dependent methyltransferases. This would therefore decrease the intracellular methylating capacity [105,232]. SAH can be further converted into Hcy in a reversible reaction that strongly favors the synthesis of SAH rather than its hydrolysis. Therefore, once Hcy accumulates, there will also be an accumulation in SAH and this will eventually lead to a decrease in the SAM:SAH ratio, thus causing hypomethylation stress [213,231,233]. A cohort study found that there is a positive association between higher plasma SAH concentrations and CVD risk in patients undergoing coronary angiography [234]. Another study showed that the plasma concentration of SAH (but not Hcy) has a strong association with CVD risk factors and subclinical atherosclerosis in those with low CVD risk [235]. Furthermore, plasma levels of SAH, in a small sample of patients of coronary artery disease (CAD), were found to be inversely associated with endothelial dysfunction [202]. Multiple studies have shown that decreased SAM:SAH ratios have a hypomethylating effect on epigenetically relevant targets which might lead to the vast vascular effects of the accumulation of Hcy [105,231,232]. Histone methylation is one of the epigenetic modulators of gene expression [229]. A study conveyed that hypomethylating stress favor the formation of a proatherogenic environment and that is by causing the down-regulation of the activity of an enhancer of zeste homolog 2 (EZH2) which is a SAM-dependent histone lysine methyltransferase [236]. EZH2 catalyzes the tri-methylation of histone H3 at lysine 27 (H3K27me3) which leads to silencing of gene expression. A hypomethylating environment also affects EZH2 which leads to a lower endothelial content of H3K27me3 [236], thus promoting a phenotype that is pro-atherogenic [106,236]. Another study showed that a significant reduction of the methylation of H3K9 and H3K27 was seen in atherosclerotic plaques in inflammatory cells and in smooth muscle cells (SMCs). The severity of atherosclerosis has been associated with H3K4 methylation. This study also revealed that in advanced atherosclerotic lesions there was an increased histone acetylation on H3K9, and H3K27 in SMCs compared to healthy vessels. Moreover, H3K9 acetylation in macrophages and MSCs was associated with the severity of the plaques in atherosclerosis [237].

Interestingly it was noted that, in vivo, mild HHcy was not enough to cause vascular hypomethylating stress or to cause the development of atherosclerosis. Therefore, only markedly elevated levels of total plasma homocysteine induce vascular toxicity and epigenetic dysregulation [238]. Atherosclerosis is accelerated by elevated plasma SAH levels associated with endoplasmic reticulum stress through histone methylation modifications [239]. Histone modification is another epigenetic modification that might be a part of the pathological mechanism of SAH. Methylation of histones occurs on arginine (R) or lysine (K) residues. In vivo, lysine residues can either be monomethylated, dimethylated, or trimethylated [240]. Therefore, there are multiple epigenetic factors that influence the One-Carbon Metabolism and have a potential effect on CVD.

7. Sex and age-related modulation of OCM and epigenetic responses in cardiovascular disease

Biological sex and aging represent two key determinants of cardiovascular disease risk, acting partially through regulation of the molecular pathways of the OCM. Biological sex, defined by gonosomal composition (XX versus XY) and sex hormones exposure (particularly estrogen, testosterone) shapes metabolic and epigenetic regulation across human's lifespan [241,242]. Aging, by contrast, is a time-dependent process characterized by progressive physiological function deterioration, cumulative molecular damage, and metabolic efficiency drop [243,244]. Importantly, aging is found to have divergent impact on CVD across sexes due to estrogen's role and effect in gene regulation and metabolic changes [245,246].

Sex-specific differences in DNA methylation patterns and in homocysteine (Hcy) metabolism were reported, especially with premenopausal women expressing lower Hcy concentration and more favorable gene-specific DNA methylation profiles compared to men of the same age group [[247], [248], [249]]. These disparities are largely related to estrogen-mediated regulations within the one-carbon-metabolism (OCM). The latter regulations exert marked cardioprotective influence by regulating both OCM pathways and endothelial function [250,251]. This influence becomes possible by enhancing remethylation and upregulating transsulfuration through cystathionine β-synthase, an estrogen receptor-mediated enzyme, thereby estrogen lowers circulating Hcy levels and promotes cysteine and glutathione synthesis, which will reflect in decreasing oxidative stress, improving endothelial function, and supporting vascular integrity [137,252,253]. Concomitantly, estrogen increases nitric oxide (NO) bioavailability by activating endothelial nitric oxide synthase (NOS), therefore, improving vasodilation, reducing vascular inflammation and inhibiting smooth muscle proliferation [254,255]. In addition, estrogen regulates DNA methyltransferase expression and activity, maintains an optimal S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) ratio, and preserves a homeostatic DNA methylation pattern across genes found to be critical for vascular reactivity, redox balance, and inflammatory signaling [28]. Through this regulation of metabolic, redox, and inflammatory signaling, estrogen sustains endothelial integrity and delays atherogenesis.

In contrast, men with chronically low circulating estrogen levels, lack sustained activation of estrogen-mediated protective mechanisms, resulting in a higher basal Hcy circulating concentrations, a reduced antioxidant capacity, and a less favorable DNA methylation profile [6]. Similarly, a sharp decline in estrogen during the menopausal transition disrupts the homeostasis in the OCM pathways, impairing DNA methylation and methyl group availability, leading to Hcy and S-adenosylhomocysteine accumulation together with reduced NO bioavailability, thereby accelerating endothelial dysfunction, oxidative stress, and vascular inflammation [[256], [257], [258]]. Consequently, postmenopausal women's risk for CVD increases progressively and converges toward that observed in men [259]. Therefore, highlighting the essential role of estrogen in OCM and its epigenetic stability in sex-specific cardioprotection. Beyond biological sex, aging represents an independent, non-modifiable, and potent modifier of OCM and epigenetic regulation, predisposing individuals to cardiovascular disease including cerebral ischemia [19,260]. Multiple progressive impairments in OCM efficiency were associated with normal physiological changes observed in elderly people. Most of them are rooted in the anorexia of elderlies, in parallel with age related decline in gastric secretions, intestinal absorption, renal impairment, and enzyme function [261,262]. These age-related physiological changes will reflect in a reduced dietary intake, decreased intestinal folate and vitamin B12 absorption, and decreased key remethylation enzymes such as methionine synthase and methylenetetrahydrofolate reductase (MTHFR) [[261], [262], [263], [264]]. Consequently, all these changes compromise methionine recycling and favor accumulation of Homocysteine (Hcy) and S-adenosylhomocysteine levels, causing a sustained decline in S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) ratio resulting in a widespread epigenetic modification and consequence [19,265–267]. These modifications are translated by a decline in methylation potential leading to global DNA hypomethylation and epigenetic alterations of genes involved in endothelial function, inflammatory signaling, lipid metabolism, and oxidative stress response [262,[265], [266], [267]]. This phenomenon is referred to as the epigenetic drift reflecting the status of cumulative metabolic stress, excessive demethylation, and progressive decline in methyl donor availability [268]. In vascular cells, these age-related epigenetic drifts have been linked to endothelial nitric oxide synthase (NOS) dysregulation, reduced nitric oxide (NO) bioavailability, increased pro-inflammatory mediators' synthesis, impaired antioxidant defenses, all reflets in vascular aging and cardiovascular disease [[269], [270], [271], [272], [273]]. Concomitantly, reduced transsulfuration flux limits glutathione synthesis, which will further exacerbate oxidative stress and endothelial dysfunction [274]. Altogether, these age-driven metabolic dysregulation and disturbances create a pro-inflammatory environment promoting arterial stiffness, vascular inflammation, endothelial dysfunction, nitric oxide (NO) dysregulation, and an accelerated atherosclerotic progression [269,270,272,275].

Finally, it is important to mention that aging and sex-specific hormonal changes act synergistically and concomitantly in amplifying OCM dysregulation, increasing CVD susceptibility, and precipitating the sharp increase in CVD incidence and death.

8. The role of epigenetic modifications in mediating one-carbon metabolism effects on cardiovascular health

OCM integrates folate-mediated nucleotide synthesis with methionine cycling to produce S-adenosylmethionine (SAM), the universal methyl donor for epigenetic reactions. DNA methylation, catalyzed by DNA methyltransferases (DNMTs), silences genes by adding methyl groups to CpG islands, while histone modifications, mediated by histone methyltransferases (HMTs), dynamically regulate chromatin accessibility and transcription. These processes are crucial for maintaining cardiovascular health, controlling genes involved in endothelial function, lipid metabolism, inflammation, and cardiac remodeling. Disruptions in OCM, such as folate or B12 deficiency, hyperhomocysteinemia, or genetic variants like MTHFR C677T, impair SAM production, leading to epigenetic variations linked to cardiovascular disease (CVD). For instance, disruptions in one-carbon metabolism due to hyperhomocysteinemia (>15 μmol/L), folate deficiency (<7 nmol/L), or vitamin B12 insufficiency (<150 pmol/L) have been linked to aberrant DNA methylation and histone modifications that contribute to vascular dysfunction, atherosclerosis, hypertension, and stroke [199,232,276].

• •One-Carbon Metabolism and Epigenetic Regulation

A study by Jakubowski demonstrated that hyperhomocysteinemia is associated with a 15–20% reduction in global DNA methylation levels, leading to endothelial dysfunction and vascular inflammation. The hypermethylation of the endothelial nitric oxide synthase (eNOS) promoter by 30% results in decreased nitric oxide bioavailability, leading to endothelial dysfunction and increased oxidative stress [232]. Similarly, Zhao et al. reported that folate deficiency correlates with a 25–40% increase in the hypermethylation of inflammatory genes, IL6, TNF-α, and CRP, which enhances vascular inflammation and atherosclerosis progression [199]. While patients with adequate folate intake have exhibited a 35% lower risk of atherosclerosis, likely due to the maintenance of normal DNA methylation patterns.

• •DNA Methylation and Cardiovascular Disease

Aberrant DNA methylation patterns have been identified as key contributors to the pathogenesis of various cardiovascular conditions. Keene et al. conducted an epigenome-wide association study (EWAS) in the Vitamin Intervention for Stroke Prevention (VISP) trial, identified a 45% reduction in GSTP1 (Glutathione S-Transferase Pi 1) gene methylation among recurrent stroke patients. This reduction led to increased oxidative stress, suggesting that altered DNA methylation may serve as a biomarker for stroke risk prediction. Moreover, hypermethylation of NRF2, by approximately 20%, in stroke survivors was associated with impaired antioxidant defense mechanisms, further exacerbating vascular injury [277].

In CAD, Muskiet et al. found that affected individuals showed an 18% reduction in global DNA methylation compared to healthy age-matched controls. The ABCA1 gene, which plays a crucial role in cholesterol efflux and lipid homeostasis, was found to be hypomethylated by 22%, resulting in increased lipid accumulation and accelerated atherosclerosis progression [278]. Such findings suggest that epigenetic dysregulation in lipid metabolism pathways contributes significantly to CAD pathophysiology. Mentch et al. used cancer cell lines to show that methionine restriction lowered SAM, reducing H3K4me3 genome-wide by 30–70%, with parallel effects on metabolic gene expression, an effect that might be translatable to cardiovascular cells [279].

• •Histone Modifications and Cardiovascular Disease

Histone modifications, including methylation, acetylation, and phosphorylation, play crucial roles in regulating chromatin remodeling and gene expression, influencing numerous physiological and pathological processes. Among these modifications, histone methylation notably modulates vascular biology by altering gene transcription in endothelial and smooth muscle cells. Specifically, increased trimethylation of histone H3 at lysine 4 (H3K4me3), typically linked with transcriptional activation, has been extensively documented in vascular diseases such as atherosclerosis [280]. Wei et al. showed that histone methylation levels, including elevated H3K4me3, significantly enhanced the sustained activation of pro-inflammatory genes in atherosclerotic plaques. Conversely, they noted that decreased levels of repressive histone marks, particularly methylation at histone H3 lysine 9 (H3K9me), led to increased and prolonged expression of inflammatory cytokines, exacerbating vascular inflammation and promoting disease progression [280].

Histone deacetylases (HDACs), particularly HDAC1, HDAC2, and HDAC3, critically regulate endothelial cell proliferation, migration, and survival, contributing to endothelial dysfunction and the pathology of vascular diseases including atherosclerosis [281]. Luan et al. found that HDAC activity is significantly elevated in atherosclerotic patients, contributing directly to endothelial dysfunction by repressing key endothelial-protective genes, including Kruppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (NOS3) [219]. This repression resulted in disrupted vascular homeostasis, characterized by increased endothelial permeability, leukocyte adhesion, and decreased nitric oxide production. Moreover, increased HDAC activity notably exacerbated smooth muscle cell proliferation, migration, and apoptosis; critical steps in plaque formation and progression of atherosclerosis [281].

Notably, HDAC3 has been shown to mediate inflammation by modulating the acetylation status of key transcription factors like NF-κB and STAT1. Watson et al. demonstrated that increased HDAC3 expression was consistently associated with enhanced inflammatory gene expression across multiple disease contexts, including vascular inflammation and neuroinflammatory conditions [220]. Selective inhibition of HDAC3 led to significant anti-inflammatory effects by reducing pro-inflammatory cytokine production, particularly interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), by approximately 30–40% [282]. Moreover, Jasim et al. emphasized that histone acetylation and deacetylation mediated by histone acetyltransferases (HATs) and HDACs dynamically modulate chromatin structure, thereby critically influencing inflammatory responses through transcriptional regulation. Treatment strategies involving HDAC inhibitors, trichostatins [283], as well as such as valproic acid, have demonstrated notable clinical potential by reducing inflammation through modulation of acetylation patterns on histones and non-histone substrates, ultimately influencing cellular differentiation, survival, and inflammatory gene expression [282,284].

Within healthy endothelium, uniform laminar shear stress caused by normal blood flow continuously inhibits histone deacetylation by inhibiting HDACs. Thus, the acetylated regions are made more accessible for transcription leading to up-regulation of key genes involved in endothelial maintenance and function. Notably, laminar flow-mediated HDAC inhibition increased the expression of so-called Krüppel-like Factors 2 and 4 (KLF2/4) under the effect of myocyte enhancer factor-2 (MEF2). KLF, most pertinent of which is KLF 2, serve as central transcriptional hubs for arrays of genes involved in vascular health and homeostasis. KLF 2 is a well-known activator of eNOS promotors. It likewise exhibits significant synergy with nuclear factor erythroid 2-related factor 2 (NRF2), a central promotor of ROS scavenging via the activation of antioxidant response elements (AREs). Thus, the healthy endothelium exists within the folds of a constant anti-oxidative and cytoprotective cascade in which eNOS is a main effector of vascular function [285]. This has pathophysiological sequela as regions of disrupted or turbulent flow such as bifurcation points, tortuous vessel regions, and fatty streak sites serve as important loci of early dysregulation of the HDAC-KLF2/eNOS pathway. Atherosclerosis can thus be seen as a vicious cycle of deviation from healthy cellular signaling. Indeed, as discussed above, disinhibition of HDAC 3 and 5 led to down-regulation of the KLF2/eNOS pathway leading to impaired endothelial response to inflammation and oxidation [283].

As discussed elsewhere in this work, hyperhomocysteinemia has major cyto-toxic sequela on endothelial tissue. The fact that homocysteine exerts much of its effects through similar pathways to those discussed above means that 1C metabolic disruptions fit insidiously into the greater atherosclerosis inflammatory cycle. disruptions of methionine metabolism were shown to potentiate the deleterious effects of high fat diets in a process that impaired hydrogen disulfide production [286]. The interplay between homocysteine and H₂S have been extensively delineated in Table 1. Regardless, a clear epigenetic link between HHcy and deacetylation factors remains a lucrative target of future investigation.

9. Therapeutic and nutritional implications

Given the integral role of OCM in CV health, dietary interventions and pharmacological epigenetic therapies have emerged as promising strategies for preventing and managing cardiovascular diseases. Folic acid and vitamin B12 are critical cofactors involved in OCM pathways, influencing DNA methylation patterns that regulate gene expression. Kok et al. reported significant changes in genome-wide DNA methylation following long-term supplementation with folic acid combined with vitamin B12, observing differential methylation at 162 genomic sites in elderly individuals compared to only 14 sites in the placebo group [287]. This supplementation resulted in a significant median increase in serum folate levels from 16.2 nmol/L to 52.3 nmol/L, serum vitamin B12 from 279 pmol/L to 595 pmol/L, and a substantial reduction in plasma homocysteine levels from 14.7 μmol/L to 9.6 μmol/L, indicating a meaningful improvement in methylation-related metabolic homeostasis [287]. Furthermore, Kaye et al. showed that folic acid supplementation effectively reduced plasma homocysteine concentrations by approximately 25%, highlighting that this metabolic change could potentially lower cardiovascular disease risk given the strong association between elevated homocysteine and CAD [288].

Variant DNA methylation patterns have also been associated with hypertension, specifically in the context of angiotensin-converting enzyme 2 (ACE2), a key regulator of blood pressure through the renin-angiotensin system. Fan et al. demonstrated significant hypermethylation at specific CpG sites within the ACE2 promoter in patients with essential hypertension, with methylation frequencies at CpG4 and CpG5 significantly increased to 97.56% and 12.75%, respectively, compared to 95.73% and 11.47% in healthy controls [289]. Notably, the methylation at these sites significantly predicted hypertension risk, underlining the therapeutic potential of epigenetic modulation for hypertension [289]. Additionally, aberrant methylation of ACE2 has also been correlated with altered gene expression and cardiovascular pathology, suggesting the potential for DNA methylation modulation as a novel treatment strategy for hypertension-related cardiovascular risk [290].

10. Conclusion

The interplay between one-carbon metabolism (OCM) and cardiovascular disease (CVD) illustrates how genetic predispositions, nutritional status, and epigenetic mechanisms converge to influence cardiovascular health. OCM, through its role in folate-mediated nucleotide synthesis and the methionine cycle, governs DNA and histone methylation, thereby regulating gene expression related to vascular integrity, lipid metabolism, and inflammation. Disruptions in this network—such as hyperhomocysteinemia, deficiencies in folate and vitamin B12, or genetic variants like the MTHFR C677T polymorphism—can impair methylation capacity and contribute to endothelial dysfunction, oxidative stress, and atherosclerosis [128,287].

Epigenetic dysregulation is now recognized as a central mechanism linking OCM imbalance to cardiovascular pathology. DNA methylation changes have been observed in critical genes such as eNOS, IL-6, and TNF-α, which are key regulators of vascular tone and inflammation [287]. These modifications reflect and drive disease progression, offering potential biomarkers for early risk detection. Additionally, evidence suggests that methylation of the ACE2 promoter is associated with increased risk of hypertension, pointing to epigenetic regulation as a therapeutic target beyond atherosclerotic disease [289].

Histone modifications, particularly through the activity of histone deacetylases (HDACs), have also been implicated in vascular remodeling, smooth muscle cell proliferation, and chronic inflammation, further linking epigenetic regulation to CVD pathogenesis [281]. These findings underscore the broader role of chromatin remodeling in cardiovascular dysfunction and highlight novel epigenetic targets for intervention.

Nutritional interventions remain a promising avenue for modifying these epigenetic trajectories. Folic acid and vitamin B12 supplementation have been shown to significantly reduce homocysteine levels and restore DNA methylation homeostasis, potentially mitigating CVD risk [288]. Long-term supplementation has been associated with differential methylation at numerous genomic sites, demonstrating the plasticity of the epigenome in response to modifiable dietary inputs [287].

In conclusion, OCM represents a crucial biological interface between metabolism, gene regulation, and cardiovascular disease. Advances in our understanding of its molecular and epigenetic pathways offer promising strategies for personalized prevention and therapy. Continued investigation into the modulation of OCM—through diet, supplementation, or epigenetic pharmacology—may yield novel approaches to reducing the global burden of cardiovascular disease [287,288].

Declaration of non-use of AI tools

I hereby declare that no generative artificial intelligence (AI) software, large language models, or automated writing tools were used in the conception, drafting, writing, or editing of this manuscript. All content has been prepared exclusively by the authors.

Funding

The authors declare that this work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

Mohamad Al Qassab: Data curation, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing. Ayman Bou Ghanem: Data curation, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing. Yaman Hussayni: Data curation, Investigation, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing. Raghid Kadbey: Data curation, Investigation, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing. Yara Ratel: Data curation, Investigation, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing. Shereen Yehya: Data curation, Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing. Maysaa Zahr: Data curation, Investigation, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing. Hilda E. Ghadieh: Investigation, Methodology, Writing – review & editing. Ziad Abi Khattar: Investigation, Methodology, Writing – review & editing. Sami Azar: Investigation, Project administration, Supervision, Writing – review & editing. Amjad Kanaan: Investigation, Methodology, Project administration, Supervision, Writing – review & editing. Frederic Harb: Conceptualization, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.