ALA can be gradually converted to stearidonic acid (SDA), EPA, docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) in vivo by the action of the desaturase system and the elongation enzyme. The downstream metabolites of EPA and DHA are physiologically crucial for the organism. Specifically, EPA and DHA are catalyzed by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 oxidase (CYP450) to produce a series of specialized pro-resolving mediators (SPMs). SPMs include separate families of molecules: resolvins, protectins, and maresins. These act as stimulatory cell agonists, arresting neutrophil infiltration and enhancing macrophage uptake of apoptotic cells [22]. Resolvins can inhibit neutrophil infiltration, inhibit platelet aggregation, and reduce the production of proinflammatory factors [23]. Protectins can promote the expression and activity of antiapoptotic proteins and inhibit the expression and activity of proapoptotic proteins [24]. In addition, EPA is catalyzed by COX to produce thromboxane A3, which inhibits platelet aggregation, prostacyclin I3, which promotes vasodilatation, as well as prostaglandin E3 and leukotriene LT5, which has anti-inflammatory properties. Thus, both EPA and DHA metabolites can exert anti-inflammatory effects (Fig. 1).

Fig. 1.

Classification and metabolism of PUFAs. ALA, $\alpha$-linolenic acid; SDA, stearidonic acid; ETA, eicosatetraenoic acid; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid; LA, linoleic acid; GLA, gamma-linoleic acid; DGLA, dihomogamma linoleic acid; AA, arachidonic acid; DTA, docosatetraenoic acid; DPA, docosapentaenoic acid; ELOVL, elongation of very long fatty acids; FADS, fatty acid desaturase; COX, cyclooxygenase; LOX, lipoxygenase; CYP450, cytochrome p450; LT, leukotriene; PGE3, prostaglandin E3; PGI3, prostacyclin I3; TX, thromboxane synthase; PUFAs, polyunsaturated fatty acids; FADS2, fatty acid desaturase 2.

LA can be converted to gamma-linolenic acid (GLA), double-high gamma-linolenic acid (dihomo-$\gamma$-linolenic acid, DGLA), arachidonic acid (AA), and DPA in vivo, under the action of the desaturase system and elongation enzyme. AA has essential biological functions and is catalyzed by COX to produce prostaglandin E2 and thromboxane A2, which promotes the inflammatory response, platelet aggregation, and vasoconstriction. AA can also produce proinflammatory factors from the leukotriene four-family, which, in the presence of LOX, play an essential role in the development and maintenance of the inflammatory response. In addition, AA is also catalyzed by LOX to produce lipoxin A4 (lipoxin) and lipoxin B4, which have a pro-resolving role in the abrogation of inflammatory responses (Fig. 1). However, in cardiovascular diseases, metabolites of AA exert deleterious effects that are proinflammatory, prothrombotic, and proplatelet aggregation to promote the development of atherosclerosis [25].

The controversy over the effect of $\omega$-3 PUFAs on ASCVD lies in the fact that different clinical studies have yielded different results. REDUCE-IT, a multicenter RCT that included and followed more than 8000 patients with cardiovascular disease for almost five years, showed that compared to the placebo, patients who ingested EPA (4 g/d) underwent a significant reduction in the risk of cardiovascular death and nonfatal risk of myocardial infarction [31]. However, two similar RCTs (OMEMI (Omega-3 Fatty acids in Elderly with Myocardial Infarction), STRENGTH study (the Long-Term Outcomes Study to Assess Statin Residual Risk with Epanova in High Cardiovascular Risk Patients with Hypertriglyceridemia)) concluded that the intake of omega-3 PUFAs (EPA+DHA) did not reduce the risk of nonfatal myocardial infarction, stroke, or cardiovascular death [32, 33]. A systematic evaluation of the effects of $\omega$-3 PUFAs on cardiovascular health showed that although the increased intake of EPA and DHA lowered plasma triglycerides and increased high-density lipoprotein (HDL) levels, they did not reduce the incidence of coronary artery disease (CAD), stroke, or other ASCVD events, nor the risk of death. In addition, increased intake of ALA reduces the risk of death from CAD, and increased intake of ALA has a preventive effect on ASCVD [34, 35, 36]. An RCT of the CVD population in the United States showed that increased omega-3 PUFAs (EPA+DHA) did not significantly reduce the risk of major cardiovascular events (myocardial infarction, stroke, and cardiovascular death) [8]. Circulating DHA, total omega-3, LA, and total omega-6 concentrations had no protective effect on the risk of cardiovascular disease in a Mendelian randomization study from the UK Biobank [37].

Although the above studies yielded different results, an analysis of these results from the different studies found that the composition of $\omega$-3 PUFAs being consumed by the patients was different in each study, which may be one of the reasons for the controversy over the effects of $\omega$-3 PUFAs on ASCVD. It has been suggested that EPA intake alone may be more effective in reducing the risk of cardiovascular disease than EPA+DHA [38], that serum EPA levels may need to reach a certain threshold to exert a preventive effect on ASCVD, and that a high dose ($>$1 g/d) significantly reduces the risk of cardiovascular events compared to low-dose EPA levels [39, 40]. In addition, when EPA was combined with DHA, higher DHA levels attenuated the preventive effect of EPA on ASCVD [30]. Therefore, the intake of EPA in $\omega$-3 PUFAs and EPA blood levels in the study population may be important reasons for the observed variability in the effects of $\omega$-3 PUFAs on ASCVD [41]. At present, the effect of omega-3 PUFAs on atherosclerosis remains a focus of clinical research. An ongoing RCT (NCT05365438) from Korea will assess the effects of combination therapy using atorvastatin and omega-3 PUFAs (EPA+DHA) compared with atorvastatin and ezetimibe combination therapy in diabetes mellitus type 2 (T2DM) patients with asymptomatic carotid atherosclerosis. The progression of carotid intima-media thickness and carotid artery plaques will be evaluated by three-dimensional (3D) carotid ultrasound. Another ongoing RCT (NCT05725486) from Croatia will investigate the influence of n-3 PUFAs enriched chicken on vascular and endothelial functions in a population of healthy young subjects and active athletes. Specifically, whether the intake of omega-3 PUFAs affects lipid profiles, oxidative stress, and inflammation. Further elucidating the mechanisms of vascular protection for omega-3 PUFAs may lead to new interventions for atherosclerosis in clinical practice.

The effect of the intake of $\omega$-6 PUFAs on ASCVD is equally controversial. A meta-analysis involving 44 prospective cohort studies showed that higher LA intake was associated with a reduced risk of death from CAD. It supported the potential long-term benefits of $\omega$-6 PUFAs in reducing the risk of cardiovascular disease [9]. In addition, replacing a saturated fatty acid diet with an $\omega$-6 PUFA (LA) may reduce the risk associated with CAD [42]. However, a systematic evaluation of the effects of $\omega$-6 PUFAs on cardiovascular health suggests that increased intake of $\omega$-6 PUFAs (LA+GLA) may reduce total cholesterol levels in the blood but does not significantly affect the risk of cardiovascular disease or mortality; therefore, the potential benefits in terms of reduced myocardial infarction remain to be demonstrated [43]. Another meta-analysis of randomized controlled trials showed that increasing the intake of omega-6 PUFAs (LA, GLA, DGLA, and AA) did not affect the incidence of myocardial infarction, stroke, CAD, and mortality [44]. An RCT studying the effects following the intake of different types of PUFAs from vegetable oils on cardiovascular disease in a population of hypercholesterolemic adults in China showed that after one year of measuring the intake of oleic acid (saturated fatty acid)-rich peanut oil, LA-rich corn oil, and ALA-rich blended oils, on fasting lipids, glucose, insulin concentration, and high sensitivity C-reactive protein levels of the different populations, the intake of different fatty acids did not affect cardiovascular risk factors [45].

Although some studies supported beneficial outcomes for cardiovascular disease following an increased intake of $\omega$-6 PUFAs, several studies still produced conflicting results. The intake of $\omega$-6 PUFAs may negatively impact ASCVD by causing an increase in downstream metabolites, such as proinflammatory 2-series prostaglandins and 4-series leukotriene. However, it is difficult to show a direct effect of $\omega$-6 PUFAs on ASCVD when the impact of $\omega$-6 PUFA metabolites on ASCVD is studied [42]. Therefore, more rigorous RCTs are needed to elucidate whether $\omega$-6 PUFAs play a preventive or promotional role in ASCVD.

The competitive inhibition of enzymes between $\omega$-3 and $\omega$-6 PUFAs makes balancing the intake ratio between both $\omega$-3 and $\omega$-6 PUFAs complex. In addition, PUFA metabolism is also affected by genetic factors, which lead to alterations in the activity of fatty acid desaturase and the subsequent conversion of its products. Therefore, focusing only on the intake of a particular PUFA without considering the proportion of $\omega$-3/$\omega$-6 PUFAs in the diet and the influence of genetic factors on PUFA metabolism makes it difficult to explain the variability in the results from these studies.

The primary function of fatty acid desaturases is to dehydrogenate and introduce a double bond between the carbon atoms of the fatty acyl chain. In humans, membrane-bound fatty acid desaturases are known as “front-end” desaturases, and introduce a nascent double bond between an existing double bond, usually between the carboxyl group and the ninth carbon atom of the terminal methyl group, with front-end desaturation occurring at the $\mathrm{\Delta }$4, $\mathrm{\Delta }$5, $\mathrm{\Delta }$6, and $\mathrm{\Delta }$8 positions, while they are responsible for the endogenous biosynthesis of PUFAs. Therefore, fatty acid desaturases are categorized into four different types based on the location where desaturation occurs, namely, $\mathrm{\Delta }$4 fatty acid desaturases, $\mathrm{\Delta }$5 fatty acid desaturases, $\mathrm{\Delta }$6 fatty acid desaturases, and $\mathrm{\Delta }$8 fatty acid desaturases [46].

The fatty acid desaturase (FADS) gene cluster that encodes fatty acid desaturase is located on human chromosome 11 (11q12-13.1) [47]. Data from the National Center of Biotechnology Information (NCBI) database demonstrates that FADS1, FADS2, and FADS3 are composed of 13 exons and 11 introns, and the total lengths of FADS1, FADS2, and FADS3 are 17.2, 39.1, and 18.7 kb, respectively (Fig. 2). FADS1 encodes $\mathrm{\Delta }$5 fatty acid desaturase; FADS2 encodes $\mathrm{\Delta }$4, $\mathrm{\Delta }$6, and $\mathrm{\Delta }$8 fatty acid desaturase; FADS3 encodes $\mathrm{\Delta }$9 and $\mathrm{\Delta }$13 fatty acid desaturase. Since the gene clusters have the same location and similar structures, it is hypothesized that they have evolved based on gene duplication, meaning they have acquired substrate specificity [46, 48, 49].

Fig. 2.

Function of FADS cluster and location of FADS cluster in chromosome 11. FEN1, flap endonuclease 1; FADS1, fatty acid desaturase 1; FADS2, fatty acid desaturase 2; FADS3, fatty acid desaturase 3; PUFA, polyunsaturated fatty acid; hs-CRP, high-sensitivity C-reactive protein.

Fatty acid desaturase is a critical enzyme in PUFA metabolism, and gene polymorphism in FADS affects the activity and function of fatty acid desaturase [50, 51], which in turn affects metabolic activities in the body, such as lipid concentrations, cardiovascular disease risk, pregnancy, cognitive function, Alzheimer’s disease, overweight, and type 2 diabetes mellitus [20, 52, 53].

The first exploratory study of variants in the FADS gene cluster found that single nucleotide polymorphisms (SNPs) in FADS1 and FADS2affect the composition of serum phospholipid fatty acids in healthy adults, as evidenced by changes in the levels of LA, DGLA, AA, and DPA [54]. Other studies have obtained consistent results. Mid-FADS gene cluster variants in Caucasian populations are associated with higher levels of precursor PUFAs and lower levels of AA and EPA, less inflammation, and lower risks of cardiovascular disease [55, 56]. A recent cohort study on FADS SNPs and fetal growth and development in pregnant women in China’s Han population showed that pregnant women with the FADS1/rs174448G, FADS3/rs174455T, and FADS3/rs174464A allele should be supplemented with DHA-rich $\omega$-3 PUFAs exogenously during pregnancy due to the blockage of the endogenous synthesis of DHA [57].

These findings suggest that FADS gene cluster variants have a direct impact on PUFA metabolism, which in turn affects the risk of inflammation and cardiovascular disease, and that FADS gene cluster variants in pregnant women have a direct effect on PUFA levels in breast milk, which in turn affects infant growth and development. Several studies on the association between variants in the FADS gene cluster and child development have shown that the FADS SNPs are strongly associated with the synthesis of DHA and AA in vivo and can influence intelligence quotient (IQ) as well as cognitive ability in infants and young children, as shown by the fact that genetic restriction of endogenous PUFA synthesis results in poorer cognitive development in infants fed formulas that do not provide DHA and AA. This developmental deficit can be eliminated when infants are fed AA. European legislation mandating the addition of AA and DHA to infant formula may address developmental deficiencies due to insufficient synthesis of endogenous PUFAs in infants or mutations in the FADS gene cluster [20]. A case–control study reported that the FADS1/rs174556 genotype significantly increased the susceptibility to Alzheimer’s disease by regulating the efficiency of AA synthesis in $\omega$-6 PUFAs [58]. However, this study had a small sample size and did not analyze AA derivatives in detail. Another case–control study reported an association between FADS1/rs174556, FADS2/rs174617, and obesity, by demonstrating that plasma levels of omega-6 PUFAs and AA were higher in overweight and obese patients; however, the difference in $\omega$-3 PUFA levels was not significant. The study concluded that mutations in the FADS1/FADS2 locus could cause metabolic disorders and increase the risk of cardiovascular disease [59]. Genome-wide association studies (GWAS) in European and Asian populations have shown that FADS1/rs174546 is associated with reduced $\mathrm{\Delta }$5 fatty acid desaturase activity, obesity, and the risk of insulin resistance [60]. A study of the genetic etiology of type 2 diabetes mellitus showed that people with the FADS1/rs174546G allele had higher fasting insulin levels and higher HOMA-IR (an indicator to assess the level of insulin resistance) and that the increase in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was more pronounced with elevated plasma DGLA and AA levels [61]. FADS1/rs174547 effects on dyslipidemia have been reported in many races, and a study of the Chinese adult population showed that FADS1/rs174547 was significantly associated with high triglyceride levels in men and negatively associated with low density lipoprotein (LDL) cholesterol levels in women, thereby suggesting that it may be a sex-specific SNP locus [62].

The FADS gene cluster affects a variety of diseases by regulating the metabolism of PUFAs. Genetic variants in the FADS gene cluster are associated with an increased risk of dyslipidemia, obesity, and insulin resistance. These outcomes are strongly associated with atherosclerotic disease and produce a state of hypersecretion of proinflammatory cytokines, which increases the body’s susceptibility to atherosclerotic disease.

Previous studies on the association between differences in dietary PUFAs and the metabolism of PUFAs in humans and ASCVD suggest that both $\omega$-3 and $\omega$-6 PUFAs and their downstream metabolites can affect ASCVD. However, there are relatively few studies on the effect of the FADS gene cluster on ASCVD.

Genetic studies have shown that variants in FADS1, encoding the $\mathrm{\Delta }$5 fatty acid desaturase, and FADS2, encoding the $\mathrm{\Delta }$6 fatty acid desaturase, are most directly genetically linked to plasma PUFA levels and that the FADS gene cluster is the most important locus for influencing PUFA metabolism [56, 63, 64, 65]. Therefore, the FADS SNP can alter the accumulation of PUFAs and directly affect ASCVD. Genetic studies have provided a theoretical foundation for exploring the impact of the FADS gene cluster on the pathogenesis of ASCVD. Several studies have found a strong association between FADS gene cluster variants and ASCVD (Table 1, Ref. [10, 51, 66, 67, 68, 69, 70, 71, 72, 73]). For example, a Mendelian randomization study of European and American populations showed that plasma ALA and LA levels were higher in the FADS1/rs174547 sub-allele population and that this population was less likely to have CAD, stroke, and aortic stenosis, thereby suggesting that the FADS1 SNP drives the association between plasma levels of PUFAs and ASCVD [66]. An RCT on the effect of the FADS2 SNP on left ventricular remodeling after acute myocardial infarction (AMI) demonstrated that six months of high doses of $\omega$-3 PUFAs after an AMI resulted in a significant attenuation of adverse left ventricular remodeling, noninfective myocardial fibrosis, and amelioration of the FADS2 rs1535GG-imposed hyperinflammatory response [51]. A case–control study in the Han Chinese population in northern China showed a strong association between the FADS3 SNP and CAD. The recessive G allele of FADS3 rs1000778 was associated with a higher risk of CAD, whereas the minor AA allele was associated with a lower risk of CAD; however, the plasma cholesterol and triglyceride levels remained similar between the two genotypes [67]. Aortic stenosis contributes to cardiovascular mortality and morbidity, and recent studies found that FADS1 SNP is associated with the risk of aortic stenosis. A GWAS of 44703 participants in the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort, one of the largest collections of aortic stenosis in the world, demonstrated that the FADS1/FADS2 locus variants (rs174547) are associated with aortic stenosis, while higher levels of AA and a higher ratio of AA/LA were associated with increased odds of calcification of the aortic valve leaflets [68]. A study of the relationship between localized PUFA in aortic valves and FADS genotypes by expression quantitative loci (eQTL) found that the minor C allele of rs174547, which corresponds to the protective genotype for aortic stenosis, was associated with higher FADS2 mRNA levels in calcified valve tissues, whereas FADS1 mRNA and other transcripts in proximity of the SNP were unaltered. In contrast, the $\mathrm{\Delta }$5 desaturase activity and the $\mathrm{\Delta }$6 desaturase activity were decreased. The authors concluded that the association between the FADS1 genotype and lower risk for aortic stenosis may implicate DHA and DHA-derived specialized pro-resolving mediators that contribute to a protective effect [74]. In addition, the diet–gene interaction for ASCVD is crucial. A recent cross-sectional population-based cohort study demonstrated that differential associations between the FADS1 locus variant and carotid–femoral pulse wave velocity (for assessing atherosclerosis) were observed depending on the intake of omega-3 PUFAs, with a high intake of omega-3 PUFAs attenuating the FADS1 locus variant-dependent associations. This suggests that the high intake of omega-3 PUFAs (EPA/DHA) may compensate for an unfavorable FADS1 locus genotype [75].

The effect of FADS gene cluster variants on ASCVD is mainly due to regulating the metabolism of PUFAs in the body, by altering fatty acid desaturase activity. In addition, exogenous supplementation of PUFAs can reverse the adverse effects of certain FADS gene cluster variants, suggesting that certain FADS gene-deficient disorders can be treated by increasing the intake of PUFAs that can prevent or alter the course of cardiovascular diseases.

Various clinical data indicate that the FADS gene cluster has an essential effect on PUFAs metabolism, ASCVD, and glucose metabolism. Since FADS1 encodes $\mathrm{\Delta }$5 fatty acid desaturase and its metabolites, EPA and AA play essential roles in the inflammatory response and atherosclerosis. In a study investigating the effect of FADS1 on atherosclerosis and its mechanism of action, Powell et al. [14] showed that under high-fat dietary conditions, FADS1 knockout mice had weight loss, improved blood glucose, and reduced atherosclerotic plaques compared with wild-type mice. This study hypothesized that the low expression of FADS1 is associated with a reduced inflammatory response in the arterial wall, which is mainly manifested as a reduced AA/LA ratio in plasma and adipose tissue, and plays a positive role in preventing AS. Shuichi et al. [76] found that oral administration of a $\mathrm{\Delta }$5 fatty acid desaturase inhibitor to apolipoprotein E (ApoE) KO mice on a high-fat dietary background resulted in a reduction in atherosclerotic plaques, a decrease in the levels of AA and DHA, and an increase in the levels of DGLA. This finding is in general agreement with Powell’s conclusions. Gromovsky et al. [15] used antisense oligonucleotides (ASOs) to specifically knockdown FADS1 in the liver, adipose, and reticuloendothelial systems of low density lipoprotein receptor (LDLR) KO mice. However, they produced a different result, whereby the specific knockdown of the FADS1 in LDLR KO mice aggravated atherosclerosis and the hepatic inflammatory response, which differed from the previous two studies. The authors highlight several possibilities for their differing conclusions. The study by Powell et al. [14] did not validate the hepatic levels of FADS1 expression. Second, the Powell et al. [14] mouse model produced sub-alleles; therefore, the functionality of FADS1 was not completely lost. In addition, the different genetic backgrounds of the two mice may be another reason for the inconsistent results. The study also noted that a diet of $\omega$-3 PUFAs (ALA+SDA) reduced the area of the atherosclerotic plaque in the aortic root of the mice compared with a saturated fatty acid diet.

Studies have shown that mice of different genetic backgrounds present different outcomes after FADS1 knockdown and that the percentage of PUFAs in the diet affects the survival and progression of atherosclerosis in mice. The downstream metabolites and signal transduction pathways regulated by FADS1 have yet to be thoroughly studied. Whether FADS1 can directly affect AS by affecting the production of inflammatory factors, such as PGs, LTs, and TXs is worthy of further exploration.