Susceptible to oxidation yet resistant to atherosclerosis—reconciling the PUFA paradox via PPARs and Nrf2

Another work in progress; any thoughts/feedback appreciated.

Atherosclerosis is a pervasive finding in humans and underlies most cardiovascular disease (CVD)—itself a leading cause of death globally. A long-standing cornerstone of many dietary guidelines for prevention of such diseases has been replacing saturated fats (SFAs) with unsaturated fats (UFAs), especially plant-based PUFAs (e.g. from seed oils), and consuming more oily fish/n-3 PUFAs (FAO). Such public health recommendations are based in the totality of evidence from observational and interventional studies, both of which are susceptible to confounding; with diet–heart trials being particularly old, heterogenous and debatable ^1–5. Mechanistic data can inform variables to aid interpretation and support biological plausibility, although here too a potential paradox arises: atherogenesis is generally thought to involve lipid peroxidation, yet PUFAs are most susceptible. This underlies the theoretical basis of some concern, particularly with the post-industrial increase in seed oil/n-6 intake ^2,5–11 (incl. the 2026 DGA report). Thus, a simple dichotomous discourse could weigh any putative benefits, such as lipid-lowering or anti-inflammatory activity, against susceptibility to oxidation. However, PUFA oxidation can take many paths and produce many molecules with diverse effects, including lipid-lowering ^12,13, anti-inflammatory ^14–18 and antioxidant activity ^19,20, suggesting context matters and opportunity for harmonisation.

PUFAs are defined chemically by their multiple double bonds, which confer fluidity and susceptibility to oxidation via adjacent bisallylic hydrogens with low dissociation energy. At one extreme, radical-mediated autoxidation eventually degrades and fragments PUFAs into reactive aldehydes, such as n-6-derived 4-hydroxynonenal (4-HNE), n-3-derived 4-hydroxyhexenal (4-HHE) and malondialdehyde (MDA), which can covalently bind proteins and exert toxicity. On the other hand, PUFA oxidation can initially generate various full-chain oxygenated metabolites (aka. oxylipins), and these reactions can be explicitly catalysed by enzymes, such as cyclooxygenases (COXs), lipoxygenases (LOXs) and cytochrome P450s (CYPs), in the presence of antioxidants and with positional specificity, thereby producing metabolites of physiological relevance. Indeed, both early and late-stage PUFA oxidation products exhibit signalling activity and modulate gene expression as ligands for transcription factors, such as PPARs ²¹ and Nrf2 ²²; hence it is important to consider the full scope and context of lipid oxidation in vivo for pathological interpretation. To this end, this article seeks to understand the role of PUFA oxidation in atherosclerosis by disentangling adaptive physiology, with a central focus on linoleic acid/n-6 as the major dietary/tissue PUFA, and with comparisons to n-3s for context.

Oxidation and atherogenesis

Atherosclerosis essentially involves the accumulation of lipids (esp. cholesterol) and leukocytes (esp. macrophages) in the arterial wall at susceptible sites due to imbalanced influx/efflux. At onset retention of plasma lipoproteins ²³ may precede macrophage infiltration ²⁴, while later cholesterol supersaturation ²⁵ and inefficient clearance of apoptotic cells (via efferocytosis) ²⁶ may underlie development of a lipid-rich necrotic core. Already in the 1950s lipid peroxidation was also detected in human plaque, with vast subsequent research supporting the involvement of both lipid and protein oxidation in atherosclerosis and inspiring causal hypotheses, as comprehensively reviewed elsewhere ^27,28. Both early and advanced PUFA peroxidation products are present in lesions; native and MDA/HNE-modified low-density lipoprotein (LDL) were even found in fetal aortas with/without macrophages, suggesting an early event ²⁹. Oxidised LDL (oxLDL) can also be detected in plasma and associates with CVD ^30,31, although not always independently of apoB (e.g. CHD ³² and MetS ³³), potentially due to 4E6 antibody cross-reactivity ³⁴. On the other hand, oxidised phospholipids on apoB₁₀₀ (oxPL–apoB), which normally represent a very small fraction of LDL ³⁵, are independently associated with CVD and mainly carried by lipoprotein(a), an LDL variant; indeed oxLDL donates its oxPL to lipoprotein(a) in vitro ³⁴.

Plasma oxLDL and oxPL–apoB increase transiently with statins in humans, and preceding progression and regression of experimental atherosclerosis, suggesting exchange with plaque ^34,36. The high antioxidant capacity of plasma also suggests LDL oxidation may occur elsewhere. Initial studies incubated LDL with arterial cells, beyond which many methods exist, but most typically LDL is incubated with copper (e.g. as CuSO₄), a transition metal mediating 1-electron oxidations ^27,28. In turn, incubation of oxLDL with various cells has many seemingly pro-atherogenic and pro-thrombotic effects. Most characteristically, LDL oxidation increasingly induces macrophage recognition and uptake via scavenger receptors, while decreasing arterial proteoglycan binding ³⁷, which favours cholesterol-loading (i.e. foam cell formation). Similar oxidation of HDL also induces macrophage uptake, reversing its protective activity ³⁸. Further, unlike native LDL, oxidation can favour lipid trapping within lysosomes ³⁹, cholesterol crystallisation and NLRP3 activation ^40,41. This may result in pyroptosis, while the presence of oxLDL (or peroxynitrite) also inhibited efferocytosis ²⁶. In human tracer studies with autologous and copper-oxidised LDL, the latter was cleared more quickly from plasma (T_1/2=85.8 vs. 124mins), but also detected more frequently (at 1hr) in areas of carotid lesions ⁴². In a subsequent study advanced carotid plaques (AHA type VI) were excised (at 24–72hrs) post-injection of labelled native LDL and revealed accumulation in foam cells specifically, which was suppressed in those on 4 weeks of high-dose α-tocopherol (aka. vitamin E)—the major endogenous lipophilic antioxidant ⁴³. Even in the pre-plaque stage, human native LDL injected into rodents appears as oxLDL in blood (after 30mins) ⁴⁴ and the arterial wall with endothelial activation (within 6hrs) ^45,46, which were suppressed by antioxidants; whereas direct oxLDL injection was rapidly cleared ⁴⁴ by the liver and did not appear in the arterial wall ^45,46. As such most endogenous oxLDL detected in plasma is likely mildly oxidised ²⁸.

Of lipids both UFAs and cholesterol are susceptible to oxidation at their double bonds, of which PUFAs have many and linoleic acid (C18:2n-6) is most abundant in plasma and plaque, particularly cholesteryl esters (CEs) ²⁵. The PUFA/linoleate content of LDL is largely responsible for its oxidative susceptibility; artificially ‘saturating’ human LDL with a reducing agent prior to injection into mice blocks its conversion to oxLDL in blood ⁴⁴. The characteristic atherogenic effects seen in vitro also involve lipid peroxidation ²⁸; MDA in particular reacts with lysine residues of apoB₁₀₀ (more than 4-HNE) resulting in recognition by scavenger receptors ⁴⁷ and dissociation from proteoglycans ³⁷, while also impairing apoA-I efflux (vs. other reactive carbonyls) ⁴⁸. CE aldehydes also have reduced macrophage hydrolysis ⁴⁹ and may be converted to 7-ketocholesterol ⁵⁰, which inhibits lysosomal SMase causing accumulation of sphingomyelin–cholesterol particles ³⁹, and dose-dependently induces cholesterol crystals ⁵¹. Supporting the relevance of these mechanisms, such aldehydes are detected in human arterial lesions (e.g. LDL ²⁹, HDL ⁴⁸ and CEs ^49,50). In comparing (LOX-derived) LDL hydroperoxides to direct LDL–MDA modification, only the latter induced macrophage uptake ⁴⁷. However, oxLDL may induce the CD36 scavenger receptor via the content of n-6 PUFA hydroxides (i.e. HODES and HETEs) ⁵² and 4-HNE ⁵³; CE hydroperoxides also induced TLR4-dependant macropinocytosis and bulk LDL uptake ⁵⁴, later attributed to oxidised arachidonate ⁵⁵. Further, VLDL is rich in triglycerides which may be released by lipolysis in the arterial wall ⁵⁶, and among fatty acids free linoleic acid can particularly induce endothelial activation ⁵⁶ and barrier disruption, which are inhibited by vitamin E ^57,58. This may involve linoleic peroxidation (via peroxisomes) ⁵⁹ and epoxidation (via CYP2C9) ⁶⁰, with further generation of superoxide and peroxynitrite (via eNOS) ¹¹. Thus, all such observations can support a more specific oxidised linoleic acid hypothesis of atherosclerosis ⁶.

Besides oxidation, in multiple prospective cohorts dietary and plasma antioxidant nutrients (esp. carotenoids and vitamins C/E—largely reflecting fruit/veg and seed oil intake) are inversely associated with CVD risk and mortality, from past to present ^61,62. However, the results of large RCTs with high-dose antioxidant supplements (i.e. vitamin E alone or combined) have mostly failed to show benefit ^27,63, despite improving oxidation markers ^64–67; but not precluding potential from more physiological and targeted approaches ^63,68,69. Regardless, this general failure to improve hard outcomes in humans (and animals) with ‘frontline’ antioxidants suggests more complexity ²⁷. Foremost, early studies on human plaque found lipid oxidation occurs despite no deficiency of antioxidants like α-tocopherol and ascorbate ^27,70—later extended to T2D ⁷¹; and short-term high-dose vitamin E alters markers in plasma but not plaque ^72,73. Moreover, oxidation by free transition metals in vitro may have limited analogy in plaque ²⁸, where particularly heme-iron (Fe²⁺) dysregulation may promote oxidation during advanced plaque haemorrhage and haemolysis ⁷⁴. On the other hand, early interest turned to 15-LOXs (i.e. non-heme iron-dependent dioxygenases) ⁷⁵, since they can initiate PUFA oxidation and lipoprotein modification ^76,77, which is not blocked by vitamin E ⁷⁸. Accordingly, human plaques express 15-LOXs (i.e. ALOX15 ^76,79 and ALOX15B ⁷⁷) and COXs ⁸⁰ within specific macrophage populations. Further, increased iNOS ^81,82 and MPO ⁸³, along with lipoprotein enrichment in their protein oxidation products (i.e. nitrotyrosine and 3-chlorotyrosine, respectively) ^84,85, also implicates immuno-oxidative activity ²⁷. These 2-electron pathways are also not blocked by vitamin E ⁷⁰ (or serum ⁸⁶) and resulting NO₂–LDL stimulates macrophage uptake and loading via scavenger receptors ^86,87, while MPO-modified tryptophan residues within apoA-I/HDL associate with lipid-poor particles in plaque and inactivate ABCA1-dependent acceptor activity ^85,88.

However, other data present more fundamental challenges ^27,28,89. For instance, in early studies LDL isolated from plaques was not always sufficiently oxidised for receptor-mediated uptake; rather LDL from human aortic fatty streaks and plaques exhibited increased macrophage uptake in a non-saturable manner attributable to aggregates ⁹⁰. Indeed LDL aggregation greatly increases macrophage uptake by receptor-independent endocytosis ^90,91 and CE accumulation beyond native or oxLDL ^92,93. Accordingly, the ability of copper-oxidised LDL to induce lipid droplets may be somewhat limited by defective lysosomal processing (prior to cholesterol esterification) ³⁹. More ‘minimally’ oxidised LDL still exhibits atherogenic effects, such as a tendency to aggregate ⁹⁴ and induce macropinocytosis ⁵⁴, lysosomal crystals and NLRP3 activation ^40,41, so may contribute in these ways ⁹⁰. On the other hand, studying LDL oxidised with copper for 0.5–24hr showed mild oxidation (>30min) initially inhibits macrophage selective CE uptake and native LDL-induced foam cell formation in relation to apoB fragmentation, before more extensive oxidation (>3hr) induces aggregation, CE oxidation and particle uptake ⁹⁵. Moreover, mild oxidation of HDL by copper, ALOX15 or HOCl (i.e. the product of MPO) actually increases efflux capacity by promoting formation of pre-β-migrating particles ⁹⁶. The early linoleic oxidation product 13-HODE also induces macrophage efflux ⁹⁷. And contrasting earlier studies ⁹⁸, copper-oxidation can induce HDL anti-platelet activity ^99,100 and suppress LDL and VLDL inflammatory signalling ^45,56. Of enzymatic pathways, human ALOX15 variants if anything suggest increased enzyme activity is athero-protective ¹⁰¹, consistent with ALOX15 overexpression increasing reverse cholesterol transport ¹⁰². Thus, the extent and type of oxidation may be important.

Lipid oxygenation and efflux

Considering the specificity and spatiotemporal pattern of lipid oxidation in plaque may provide some context. Of PUFA oxidation products, linoleic hydro(pero)xides dominate ²⁷. In particular, comprehensive analysis of CEs from human peripheral vascular plaques revealed a substantial proportion are oxidised (avg. 21%), with cholesteryl linoleate to the greatest extent (i.e. C18:2 > C20:4 > C22:6), and the most abundant species being full-chain mono- and dioxygenated derivatives (e.g. HODEs, KODEs, EpOMEs, etc.) ¹⁰³. The HODE-CE profile exhibited no regio- or stereo-specificity suggesting a dominance of non-enzymatic peroxidation, although triglyceride PUFAs were not oxidised indicating some specificity ¹⁰³. 15-LOX in particular can directly initiate CE oxidation ⁷⁵, while subsequent radical reactions may erode product specificity ^104,105; and at least in several earlier reports 13-HODE stereoisomer ratios were consistent with ALOX15 activity, particularly in early lesions ¹⁰⁶. Conversely, mouse models can lack oxidised CEs despite LOX activity ¹⁰⁷. Recent high-resolution imaging of advanced human carotid plaques also found oxidised CEs and sphingomyelin concentrate in the necrotic core, while a metabolite resembling 7-ketocholesterol was uncorrelated ¹⁰⁸. Further, from a temporal perspective, an analysis of intimal lipoprotein-containing fractions of aortic lesions from early to late-stage disease found accumulation of cholesterol (AHA types II–III) and CEs (types IV–V) preceded their major oxidised derivatives (i.e. 27-hydroxychoesterol and CE hydro(pero)xides, respectively), while 7-ketocholesterol only increased at late stages (types V–VI), and α-tocopherol and CoQ₁₀ levels remained relatively stable throughout ¹⁰⁹.

These observations in vivo notably contrast the typical situation in vitro ²⁷, where LDL oxidation generates hydroperoxides immediately followed by MDA ^37,47,87,110, before depletion of CEs with accumulation of 7-ketocholesterol ^50,111. Under such strong oxidising conditions α-tocopherol acts as a chain-breaking antioxidant and its depletion underlies the lipid oxidation lag phase and formation of secondary/advanced oxidation products (e.g. isoprostanes, aldehydes, etc.) ¹⁰⁹. Conversely, the profile in vivo implies more mildly oxygenating conditions where enzymes and the α-tocopherol radical can mediate lipid peroxidation ¹¹²; the latter being favoured by lower regenerative co-antioxidants ¹¹³ (e.g. CoQ₁₀ and carotenoids) ¹¹⁴. In particular, another stage-dependent analysis of whole aortic lesions also included tocopherol oxidation products, which exceeded lipid oxidation in early stages with a pattern implicating 2-electron oxidants and activated monocytes ¹¹³. Accordingly, MPO/iNOS-derived oxidants can directly induce protein modification and initiate lipid peroxidation, while in mice aortic lesions may lack MPO ⁸⁵.

Of oxysterols, human plaque is generally dominated by 27-hydroxychoesterol (27-HC), followed by 7-ketocholesterol ²⁷, although some reports suggested the opposite in human macrophages ^115,116 and absence of 27-HC in animals ¹¹⁷. As above, accumulation of cholesterol precedes 27-HC ¹⁰⁹ and at the fatty streak stage they are highly correlated ¹¹⁸. 27-HC is produced by mitochondrial sterol 27-hydroxylase (CYP27A1), which is also increased in plaque, particularly macrophages ^119–121. In the liver this enzyme initiates the ‘acidic pathway’ of bile acid synthesis, while elsewhere it can sequentially metabolise cholesterol to water-soluble cholestenoic acid ¹²². Moreover, sterol 27-hydroxylase has even greater activity on 7-ketocholesterol (i.e. macrophages ¹²³ and isolated enzyme ¹²⁴) and 27-hydroxylated 7-ketocholesterol was also detected at low levels in human plaque ¹²³. In extrahepatic tissues this pathway may facilitate efflux by increasing the polarity of cholesterol ¹²² and generating ligands for LXR—a nuclear oxysterol sensor. In particular, LDL/cholesterol loading of macrophages induces 27-HC and LXR, which may interact directly ¹²⁵ and within a feed-forward loop with autophagy ¹²⁶; whereas human CTX disorder ^123,125, 7-hydroperoxy-cholesterol ¹²⁷ (i.e. 7-ketocholesterol precursor) and isoLGE₂ (i.e. PGH₂ oxidation) ¹²⁸ can inhibit sterol 27-hydroxylase and efflux. Accumulation of cholesterol in plaque suggests this pathway is insufficient in vivo ¹²³. At least initially, incubation of human macrophages with human plaque (for 48hrs) induced LXR-dependent efflux transporters, whereas LXR inhibition increased intracellular free cholesterol, CE-SFAs/MUFAs and 27-HC, and endothelial inflammation via IL-6 ¹¹⁶. In the absence of LXR inhibition, 27-HC induced ABCA1/IL-1β and lowered IL-6/IL-18BP ¹¹⁶, and drives IL-10/M2 polarisation ¹²⁹. On the other hand, recent studies find 27-HC can also induce ROS ¹³⁰ and inflammation ¹³¹ in human pro-monocytes, and mediate plaque macrophage accumulation in APOE^–/– mice ¹¹⁹; although apoE is actually a target of LXR and required for efficient efflux (i.e. via secreted ¹³² and exogenous apoE ¹³³).

In human lesion macrophages expression of sterol 27-hydroxylase was accompanied by genes functionally linked in vitro, where RXR/PPARg ligands induced sterol 27-hydroxylase ¹²⁰, 27-HC and LXR ¹²¹. Plaque PPARg expression was also specifically associated with M2 macrophage markers distant from the lipid core ¹³⁴; although these macrophages actually had suppressed LXRα/ABCA1-dependent efflux, whereas PPARg supported phagocytosis ⁷⁹. From a temporal perspective, tracer studies in APOE^–/– mice revealed accumulation of M2 markers with PPARg and ABCA1 in early stages, whereas M1 markers with HIF-1α and NLRP3 in the advanced plaque microenvironment; suggesting an initial PPARg-based efflux attempt is followed by development of a hypoxic core favouring a shift to glycolysis, inflammation and pyroptosis ¹³⁵. Accordingly, in mouse models macrophage PPARg/LXR signaling is athero-protective ¹³⁶ and involved in athero-regression ^137,138. Regarding natural PPAR ligands, healthy arteries produce various oxylipins, of which COX-derived prostanoids are most abundant ²¹. Indeed, the first natural PPARg ligand discovered was 15d-PGJ₂, which was subsequently found in human plaque foam cells with COX-2, where it may mediate negative feedback ¹³⁹. Some years later, oxLDL was shown to induce a PPARg–CD36 uptake pathway via n-6 oxylipins (i.e. 13-KODE > 9/13-HODE > 15-HETE) ⁵², and subsequently a counterpoise PPARg–LXRα/ABCA1 efflux pathway ¹³⁶, similar to 13-HODE ⁹⁷. Such oxylipins may be largely in the CE fraction ⁵² and macrophages may preferentially hydrolyse oxygenated CEs ¹⁴⁰. Further, plasma LDL from patients with atherosclerosis was also shown to contain n-6 hydroxides (i.e. HODEs > HETEs), but mainly in the phospholipid fraction, and which activated PPARg at physiological levels in vitro ¹⁴¹. The potency of such PPARg ligands varies ^52,106,142 and 15-HETE may prefer PPARβ/δ ¹⁴³. In humans PPARg agonists induced an M2 marker in PBMCs, but not plaque macrophages, where it did still induce CD36 ¹³⁴.

Regarding enzymatic oxygenation, while ALOX15B is induced by hypoxia ⁷⁷ and mediates cholesterol biosynthesis ¹⁴⁴, ALOX15 is specifically induced by Th2/M2 cytokines and efferocytosis of apoptotic cells (via LXR ¹⁴⁵), consistent with a role in lipid/tissue homeostasis ⁷⁷. IL-4 induced ALOX15 may induce HODEs/HETEs, PPARg and CD36, while suppressing iNOS ¹⁴⁶; and may also suppress LXRα/ABCA1-dependent efflux (not PPARg-dependent efferocytosis) ⁷⁹. Conversely, in naive mouse macrophages ALOX15 overexpression increased CE hydrolysis and cholesterol efflux, but not via 15/13S-HETE (and 13S-HODE was undetectable) ¹⁰²; and also oxidised LDL via (LRP-dependent) selective uptake and efflux of CE linoleate ¹⁴⁷. 15-LOX-derived hydroxides also undergo reincorporation into specific phospholipids ¹⁴⁸, with specific functional implications ⁷⁷. In particular, macrophage oxidation of CEs from intra- and extracellular sources resulted in 13-HODE–oxPC ¹⁰⁵; this may involve LPCAT3 which mediates sn-2 incorporation of PUFAs ¹⁴⁸. LPCAT3 is also a target of LXR and hemopoietic knockout in LDLR^–/– mice impairs cholesterol efflux and exacerbates atherosclerosis ¹⁴⁹. Further, in human endothelial cells LXR induced both PUFA synthesis (i.e. 18:2–20:4 and 18:3–20:5/22:6) and phospholipid enrichment, while suppressing 9/13-HODE and 15-HETE but inducing 5-HETE (i.e. 5-LOX) ¹⁵⁰. Therefore, induction of PPARg–LXR may exert negative feedback on natural ligands and redirect linoleate toward elongation. In parallel, enrichment of PC in PUFAs may fine-tune substrate for CE synthesis by HDL-associated LCAT, which has specificity for sn-2 linoleate ¹⁵¹ (and possibly also 18:3 ¹⁵²) and may also act on hydro(pero)xides ¹⁵³. LDL oxidation also favoured net transfer of CEs to HDL (via CETP) ¹⁵⁴, while in rats HDL-associated CE hydro(pero)xides (i.e. [³H]Ch-18:2-O(O)H) were more rapidly removed by liver ¹⁵⁵ and excreted in bile (with the radioactivity in bile acids) ¹⁵⁶. As earlier, lipoprotein(a) is a major carrier of oxPLs ³⁴ and in hepatocytes induced PPARg/LXR/efflux by selective uptake of oxPLs via SR-B1, which may support HDL biosynthesis ¹⁵⁷, suggesting a novel physiological function at low levels ¹⁵⁸.

Endogenous antioxidant metabolism

Notwithstanding the potential for pro-oxidant effects ¹¹⁴, vitamin E supplementation dose-dependently increases the LDL lag time in vitro ^159,160 and decreases F₂-isoprostanes in vivo ¹⁶¹. However, this did not translate to clinical benefits long-term ⁶⁷; adding high-dose antioxidants (i.e. vitamins E and C, β-carotene and selenium) to statin–niacin lipid therapy even tended to blunt coronary regression and increases in HDL-C ⁶⁵. Notably, LDL lag time more strongly correlated α-tocopherol in plasma than LDL, suggesting indirect effects ¹⁵⁹, and oxidation by copper or cells in vitro requires pre-existing lipid peroxides ¹⁶², which might also be affected. Small-molecule antioxidants like α-tocopherol may particularly intercept and slow non-enzymatic peroxidation, but not affect decomposition of peroxides to aldehydes, contrasting α-keto acids (like pyruvate) which can reduce peroxides to alcohols ¹⁶³. This distinction may affect signalling, since all free linoleic hydro(pero)xides may induce PPARg, while 4-HNE does not ⁵², and reduction of HpODEs to HODEs could stabilise oxidation at the signal. In human peripheral plaque a preponderance of CE HODEs/KODEs over HpODEs was reported ¹⁰³, while in carotid plaque free HpODEs were increased in symptomatic (vs. asymptomatic) cases and correlated blood HbA1c, but inversely with HDL-C and paraoxonase-1 activity ¹⁶⁴, suggesting impaired reduction/clearance. Accordingly, lipid hydroperoxides can be transferred to HDL and reduced by apoA-I and associated enzymes, including paraoxonase-1 ¹⁶⁵, which is itself induced by hepatic PPARg ¹⁶⁶, thereby reinforcing reduction. In so doing HDL can remove seeding molecules for LDL oxidation ¹⁶². HDL, paraoxonase-1 and MPO may also exist within a ternary complex and reciprocally inhibit one another ¹⁶⁷, which could regulate acceptor/efflux activity, as above. Additionally, paraoxonase-1 may also protect LCAT from oxidative inactivation ¹⁶⁸.

In human atherosclerosis low arterial glutathione and related enzyme activity (i.e. GR, GPx and GST) was associated with 4-HNE-related markers and plaque severity ^169,170, while in plasma oxidation of glutathione redox (i.e. GSH/GSSG ratio) was associated with carotid intima–media thickening ¹⁷¹ independent of traditional markers ¹⁷². An RCT of n-acetyl cysteine (a glutathione precursor) for 1 week on 10 patients with CAD and hyperlipidemia selectively lowered oxLDL (not other lipids) ⁴⁴. Further, in various human cohorts lower plasma glycine (another glutathione precursor) associates with metabolic and coronary disease, while causality was shown in APOE^–/– mice on low and high glycine diets ⁶⁸. Here dietary glycine induced glutathione biosynthesis and effector enzymes (incl. GR, GPxs and GSTs), while lowering aortic/macrophage superoxide and atherosclerosis independent of plasma lipids ⁶⁸. Accordingly, depletion of glutathione and biosynthetic enzymes precedes plaque formation in APOE^–/– mice ¹⁷³, and in many studies augmenting glutathione via genes or supplements has a protective effect beyond lipid-lowering and involving macrophages ^44,174–176. Mechanistically, the glutathione system mediates reduction of ascorbate (and consequently tocopherol) and lipid hydroperoxides (e.g. Fig 1), as well as conjugation of KODEs ¹⁷⁷ and aldehydes ¹⁷⁸, and as such may regulate lipid oxidation, signalling and clearance. In macrophages glutathione deficiency increased ROS and CD36 expression independent of PPARg ¹⁷⁹, whereas glutathione supplementation induced efflux and PPARα ¹⁸⁰, and selenium supported IL-4 induced M2 polarisation via GPx1, PPARg and PGD₂ ¹⁸¹. Further, LDL and HDL also contain GPx activity ¹⁷⁵, while oxidised glutathione can inhibit HDL efflux activity via glutathionylation of paraoxonase-1 ¹⁸².

Importantly, antioxidant systems are highly regulated and may increase in early stages. For instance, in transgenic mice expressing human apoB and lipoprotein(a) a hepatic proteomic analysis prior to atherosclerosis revealed an increase in several antioxidant and efflux proteins ¹⁸³. In APOE^–/– mice the development of plaque is also preceded by an initial increase in many arterial antioxidant enzymes before a decline, suggesting a coordinated induction of antioxidant systems before a collapse with atherogenesis ¹⁸⁴. A subsequent proteomic study found oxidation of 1-Cys peroxiredoxin correlated lesion formation ¹⁸⁵. More recently, endothelial inflammation and 4-HNE were shown to precede Nrf2 activation, which then appeared to exert negative feedback regulation by restraining inflammation, peroxidation and atherosclerosis ¹⁸⁶. Accordingly, low-level 4-HNE induces Nrf2 ²², which in turn induces 100s of genes encoding antioxidants (incl. the glutathione system) and intermediary pathways to rewire redox metabolism for homeostasis ¹⁸⁷.

In human plaque Nrf2 expression was highest in macrophages of the lipid core and paralleled Myh9, while in APOE^–/– mice macrophage Nrf2 deficiency increased plaque severity, necrotic core size and accumulation of apoptotic cells, whereas Nrf2–Myh9 binding supported efferocytosis via the actin cytoskeleton ¹⁸⁸. Similarly, 15d-PGJ₂ and 17-oxo-DHA (both formed via COX-2) augmented efferocytosis via Nrf2/HO1-dependent expression of CD36 ¹⁸⁹, LOX/COX-2 and pro-resolving mediators ¹⁹⁰. Furthermore, oxLDL/4-HNE ⁵³ and 15d-PGJ₂ ¹⁸⁹ induce Nrf2-dependent CD36 expression independent of PPARg ^53,191 (similar to GSH depletion ¹⁷⁹), while phytochemicals induce Nrf2-dependent efflux via suppression of NF-κB signalling ¹⁹² and induction of SR-B1 and ABCA1/G1 transporters ^193–195. Thus, Nrf2 may support both apoptotic cell and cholesterol clearance in parallel to PPARg ^53,136. Some reciprocity is also suggested: Nrf2-dependent antioxidants/glutathione may support hydroperoxide reduction to affect PPAR signalling, while the intermediate 27-HC can induce autophagy via ROS/Nrf2 favouring cell survival ¹³⁰. Of note, macrophage Nrf2 status may have even broader impact: foam cell-derived exosomes were shown to propagate redox imbalance to brain microglia via Nrf2 exacerbating white matter injury and cognitive impairment ¹⁹⁶.

To summarise all the above, while advanced lipid oxidation and protein modifications are present in human plaque and may favour retention, the preponderance of cholesterol and PUFA oxygenation products, and even low-level advanced peroxidation products (e.g. 4-HNE), might initially support efflux and efferocytosis. However, clearly the very presence of plaque and lipid-poor apoA-I/HDL ⁸⁸ suggests this is insufficient, and such adaptive responses could depend on functional endogenous antioxidant systems, or else failed clearance may promote further pathology ^119,131. For instance, perhaps in response to arterial lipid accumulation, physiological oxidation-reduction responses could mediate efflux, but at the cost of reducing power, which may be overwhelmed. Thus, oxidation per se may not be the issue, but rather redox imbalance, due to excessive oxidation (e.g. via hyperlipidemia and inflammation/MPO) and/or insufficient reduction (e.g. glutathione/Nrf2 deficiency).

Dietary PUFAs on lipids and redox

Given the diversity of linoleate oxidation products in vivo, their differential effects in vitro and the potential involvement of whole-body physiology in CVD, it seems important to consult holistic human data on the impact of diet. As an essential fat, linoleic acid intake moderately correlates tissue levels and in recent meta-analyses of prospective cohorts higher dietary and blood/adipose linoleate is associated with reduced CVD incidence ¹⁹⁷ and all/CVD/cancer mortality ¹⁹⁸; and notably, some stronger associations were reported with blood CEs ¹⁹⁷—the major lipid in plaque ²⁵. Similarly, circulating long-chain n-3s are also inversely associated with all/CVD/cancer mortality ^199,200. Among diet–heart trials, in the Cochrane meta-analysis the most favourable effect was seen in replacing SFAs with PUFAs (which may include some n-3s ²), and meta-regression implicated reductions in serum cholesterol as a main source of heterogeneity ²⁰¹. Accordingly, dietary fat saturation has well-characterised quantitative effects on plasma lipids: replacing typical C12–16 SFAs with C18 MUFAs/PUFAs lowers apoB and total/LDL cholesterol, and to a smaller extent triglycerides, with PUFAs having the largest effect ²⁰², and without significantly affecting lipoprotein(a) ²⁰³. Whereas fish oil/long-chain n-3 PUFA supplementation preferentially lowers triglycerides and non-HDL-cholesterol, particularly in those with hyperlipidemia or overweight/obesity ²⁰⁴. In addition, recent trials find dietary SFAs can increase LDL sphingolipids and aggregation in vitro, whereas C18 UFAs lower LDL proteoglycan binding in vitro ^205,206. Clearly all these effects of UFAs may be beneficial by lowering arterial lipoprotein and lipid retention—the major prerequisite of atherogenesis ²³.

On the other hand, effects on lipoprotein oxidation-related markers seem more contentious. For instance, Lp-PLA₂ hydrolyses oxidised PUFAs, mostly travels with LDL and associates with CVD. In a subset of the healthy MESA cohort (n=2246) plasma phospholipid n-3 and n-6 fatty acids negatively and positivity correlated Lp-PLA₂ mass/activity, respectively ²⁰⁷. Consistent with this, in a meta-analysis of RCTs n-3s decreased Lp-PLA₂ mass ²⁰⁸, whereas a trial with soy oil capsules (replacing carbohydrates) increased Lp-PLA₂ activity in association with oxLDL (i.e. 4E6 antibody) and apoB ¹⁰; although an increase in the latter seems atypical ²⁰². Moreover, since the early 90s various short-term trials showed MUFA-rich diets (vs. n-6 PUFAs or oily fish/n-3) can lower LDL/HDL lipid oxidation markers, and susceptibility to copper oxidation (i.e. lag time and/or rate) and monocyte adhesion in vitro, which correlated lipoprotein phospholipid oleate/linoleate ratios ^209–211. Thus, competition between C18:1 and C18:2 may determine substrate for oxidation, while fish oil/long-chain n-3 PUFAs may displace respective long-chain n-6 PUFAs (i.e. C20/22 species) with less double bonds ¹⁴. Despite this, there are examples in men ²¹², monkeys ²¹³ and mice ²¹⁴ of n-6 PUFA-rich diets increasing linoleate/oleate ratios in plasma and plaques, and/or oxidation susceptibility in vitro ²¹³ and in vivo (i.e. MDA and isoprostanes ^214,215), yet being protective. In humans the long-chain n-3 content of advanced carotid plaques was also increased by supplementation (unlike vitamin E ^72,73) and correlated greater stability and lower inflammatory markers ^216,217. And a transgenic mouse model with deuterium-reinforced PUFAs (d-PUFAs), where deuterium replaced bisallylic hydrogens in C18:2 and C18:3, markedly lowered non-enzymatic isoprostane markers, yet only lowered the aortic lesion area (mean –26%) in proportion to plasma non-HDL-C (–28%) and cholesterol absorption ²¹⁸ (see below).

These PUFA trials are consistent with many other dissociations between general oxidation markers and atherosclerosis, as earlier ²⁷. Moreover, whether dietary PUFAs have an oxidative effect at all depends on the food matrix, and seeds are particularly rich in tocopherols, among other antioxidants. Accordingly, in the long-term LA Veterans trial the experimental seed oil group (vs. SFA-rich control) had much higher intake and blood levels of α-tocopherol, concomitant with a lower erythrocyte susceptibility to peroxide ²¹². Further, a 3-week diet of 31% sunflower oil/n-6 PUFAs (vs. olive oil/MUFAs) also lowered LDL levels, oxidation susceptibility and proteoglycan binding, in relation to LDL antioxidant content and size ²¹⁹. And in healthy adults an n-6/n-3 PUFA-rich walnut meal (i.e. 59g fat, 42g PUFAs) increased postprandial antioxidant capacity and lowered MDA (5hr AUC) and oxLDL (at 2hrs) ²²⁰, while longer trials show enrichment of PUFAs with preservation of oxidation status ^221,222, alongside many other cardio-protective effects (reviewed in ²²³).

Conversely, food storage and processing can oxidise lipids prior to ingestion, especially at the extreme end. For instance, compared to conventional corn oil, tocopherol depletion and 6–8wk air exposure resulted in a stepwise decrease in linoleic acid and increase in peroxides (i.e. conjugated dienes), which when fed to humans was reflected in postprandial chylomicrons/remnants for 8hrs ²²⁴. Subsequent studies revealed a greatly exaggerated response in poorly-controlled diabetes and differences with dietary oxidised cholesterol, which appeared in all major lipoproteins and persisted for 72hrs; in vitro oxidised cholesterol was transferred to LDL and HDL, potentially via CETP ⁷. Prolonged heating (i.e. 195°C for 9hrs) of refined tocopherol-depleted soybean oil induces a gradual increase in peroxides before a decline (at 6hrs), while secondary aldehydes continued to increase ²²⁵. In the context of deep-frying, thermo-oxidised (i.e. 20 cycles of 180°C for 5mins) sunflower oil/n-6 PUFAs fed to obese adults acutely increased protein carbonyls and lowered plasma glutathione redox (i.e. GSH/GSSG ratio) compared to oils rich in MUFAs and polyphenols ²²⁶. In animal models dietary oxidised linoleic acid can promote atherosclerosis ⁷, but also lower blood lipids ¹³ and atherosclerosis ⁹⁷, suggesting context is important. In this regard, 13-HODE was shown to elevate blood lipids and atherosclerosis only in the presence of dietary cholesterol, possibly due to increased solubilisation and absorption ²²⁷. Interestingly, in a unique RCT on healthy adults (with low TGs), comparing high quality to oxidised fish oil (both 1.6g/day of EPA+DHA) or control (high-oleic sunflower oil/MUFAs) for 7 weeks, only the former lowered IDL/LDL particles and cholesterol content, which correlated CETP ²²⁸. Thus, dietary PUFA oxidation may eventually affect blood lipid responses.

Regarding other dietary pro-oxidants, in humans and mice red meat ingestion also induced postprandial lipid peroxidation and plasma LDL–MDA modification, which was greatly inhibited by polyphenols ^229,230. Animal and in vitro models have localised this oxidative activity to the stomach, which has been conceptualised as a bioreactor that denatures foods and facilitates redox chemistry ²²⁹. Indeed under simulated conditions in vitro, incubation of red meat, metmyoglobin (contains heme-iron as Fe³⁺) or free iron exhibit pro-oxidant activity and can deplete antioxidant vitamins and induce advanced lipid peroxidation (like copper oxidation in vitro), whereas catalysis is inverted to antioxidant activity by polyphenols ²²⁹. The activity of many plant foods in this model has been indexed and correlates polyphenol content ²³¹; additionally, peroxidation was inhibited by oleate/MUFAs, opposite to fish oil/n-3 PUFAs ²³². Further, in APOE^–/– mice on a high fat diet (60% kcal) with red meat, addition of sunflower oil/n-6 PUFAs induced digestive and plasma 4-HNE and oxLDL, and worsened endothelial dysfunction and atherosclerosis (esp. necrotic core size), all of which was prevented by apple puree or polyphenol extract ⁹. Importantly, supplements containing free transition metals (i.e. iron and copper) can also induce gastrointestinal oxidation in humans ²³³ and animals ²³⁴, which might particularly confound rodent formula diets where antioxidants lower cholesterol.

Role of metabolism and microbiome

A simple PUFA-driven peroxidation paradigm is further challenged by other data. For instance, in a healthy Japanese cohort (n=130, median age=55) plasma oxPC–apoB was modestly and independently associated with LDL-C ³⁵. Further, in a young Finnish cohort (n=2196, age=24–39) serum PUFAs, particularly n-6 PUFAs, were negatively associated with LDL lipid oxidation and CRP, opposite to SFAs/MUFAs, which withstood adjustment for CVD risk factors and red meat intake ²³⁵. Moreover, despite long-chain n-3 PUFAs (e.g. DHA/C22:6) being most susceptible to oxidation, meta-analysis of 39 RCTs shows they can improve some peripheral redox markers (i.e. TAC, GPx and MDA) ¹⁹. Conversely, despite the oxidative stability of SFAs and the responsiveness of serum stearate to diet ²³⁶, in short-term trials SFA-rich diets (vs. carbohydrates or MUFAs) can also increase LDL susceptibility to oxidation in relation to MUFA/PUFA ratios ²¹¹, vitamin E ²¹⁰, apoB/LDL-C ^237,238 and APOE promoter variants ²³⁸. Excess dairy fat can also favour oxidation in vivo. For instance, in healthy adults a high fat milkshake (vs. low fat) induced pathological RBC remodelling and foamy monocytes, while elevating plasma and RBC-bound MPO in association with impaired flow-mediated dilation (FMD) and chlorination of HDL; tested in vitro major cow milk fatty acids (i.e. oleic or palmitic acid) induced MPO release by monocytes and uptake by porcine arteries ²³⁹. In mice a diet rich in dairy fat/SFAs (i.e. 21% weight) also elevated oxidised HDL and LDL, while replacement with soybean oil/PUFAs (i.e. ~5:1 of n-6:n-3) enhanced HDL antioxidant and Lp-PLA₂ activity, without affecting macrophage–faeces reverse transport or paraoxonase-1 ²⁴⁰. Elsewhere however, a diet rich in palm oil/SFAs (i.e. 45% kcal) vs. sunflower oil/MUFAs enriched the liver and HDL in acute-phase proteins and lowered paraoxonase-1 and faecal cholesterol excretion ²⁴¹.

Mechanistically, SFAs may affect lipid oxidation indirectly. For instance, LDL oxidation may occur secondary to arterial retention, which may be favoured by SFA-induced quantitative and qualitative changes, as earlier. Also, in an animal model lipoprotein susceptibility to oxidation increased with particle age (i.e. plasma residence) ²⁴², while in human tracer studies PUFAs (vs. SFAs) increase LDL catabolism (i.e. turnover) ²⁴³. Moreover, LDL susceptibility to oxidation is associated with small particle size (i.e. pattern B), which is associated with insulin resistance ²⁴⁴. In people with pattern B a SFA-rich diet (vs. MUFAs) increased small–medium LDL particles ²⁴⁵, while a meta-analysis of RCTs suggests exchanging SFAs for n-6 PUFAs may particularly improve glucose-insulin homeostasis ²⁴⁶. Further, a subsequent prospective study on the Finnish cohort above ²³⁵ found serum SFAs/MUFAs were positively, and PUFAs negatively, associated with cardiometabolic disorder outcomes (incl. obesity, fatty liver and HOMA-IR) over 10 years ²⁴⁷. Accordingly, such SFAs/MUFAs are also products of lipogenesis, and such metabolic disorders are associated with elevated hydroperoxides ^164,248, low glycine/glutathione ^68,170,248 and HDL antioxidant activity ¹⁶⁵.

Regarding microbes, human carotid plaque contains LPS from E.coli, which at similar levels in vitro induced TLR4/NOX2-dependent LDL oxidation; plaque LPS was also associated with plasma LPS and zonulin, implicating the gut microbiome as a source ²⁴⁹. In a systematic review of RCTs dietary SFAs (vs. UFAs) induced postprandial LPS ²⁵⁰, which at similar levels in vitro also induces LDL oxidation ²⁵¹. In mice a SFA-rich diet ²⁴¹ and low-dose LPS ²⁵² also similarly impair biliary excretion. Moreover, the bioactivity of LPS is actually mediated by acylated SFAs, which at high levels in vitro can directly induce TLR2/4 signalling, opposite to n-3 PUFAs ²⁵³. Regarding specific microbes, in twins an 8-week vegan diet (vs. omnivorous with a higher SFA/PUFA ratio) lowered faecal Bilophila wadsworthia in association with fasting insulin and increased serum glycine; tested in vitro B. wadsworthia consumed glycine (in Stickland fermentation) and its removal from mice increased serum glycine and hepatic Gstt2 expression, while decreasing body weight and LDL-C ²⁵⁴ (preprint). Accordingly, SFA intake is associated with Bilophila abundance ^255–257. In mice a milk fat/SFA-rich diet (vs. safflower oil/n-6 PUFAs) induced B. wadsworthia by favouring secretion of taurine-conjugated bile acids to fuel sulfite-based respiration, which was offset by supplementation of fish oil/n-3 PUFAs ²⁵⁸. This bile profile may be a result of the lower solubility of long-chain SFAs—another corollary of lipid saturation. Of note, in people with moderate hypercholesterolemia exchanging SFAs (6.5% kcals) for mostly n-6 PUFAs for 8 weeks did not significantly increase serum glycine, although in multivariate analysis it was a highly ranked variable of importance ²⁵⁹. In other trials the individual response to SFAs was related to baseline Bilophila ²⁵⁵ and diet ²⁵⁶, which may be sources of heterogeneity.

In summary, PUFA-rich diets increase the major substrate for peroxidation in general, although whether this occurs depends on the food matrix and dietary pattern. In particular, even prior to absorption non-enzymatic peroxidation is modulated by the balance of pro-oxidants (e.g. heat and heme-iron) vs. antioxidants (e.g. vitamins and polyphenols) during food processing and digestion. Moreover, dietary fats may also modulate systemic redox indirectly via metabolism (e.g. lipoprotein turnover and phenotype) and microbiome (e.g. LPS and glycine), where SFAs may particularly favour hyperlipidemia and immune-mediated oxidation, and lower antioxidant status.

Oxidation-dependant homeostasis?

While increased tissue linoleate is typically considered an oxidative liability ^2,6–10,218, could there be some more favourable effects? For instance, in healthy human trials replacing SFAs with mostly n-6 PUFAs lowers plasma cholesterol, while inducing serum bile acids and PBMC transcripts related to cholesterol uptake (e.g. LDLR/TLR4) and efflux (e.g. LXRα/ABCG1) ^259,260. Elsewhere, lowering dietary linoleic acid/n-6 PUFA (vs. mostly SFAs) also lowered respective plasma oxylipins (i.e. HODEs and KODEs) ²⁶¹. Linking these effects, linoleate oxygenation within LDL may favour macrophage CE uptake, hydrolysis and efflux via PPARg–27-HC–LXR signalling, as above. Similarly, oxygenated lipids within HDL may favour hepatic uptake and LXRα signalling to increase bile output and plasma cholesterol uptake (i.e. LDLR expression), underlying clinical effects of n-6 PUFAs ^259,262. Conversely, free linoleic acid itself does not induce efflux ⁹⁷ and can even suppress it ²⁶³, but may be lowered by LXR activation ¹⁵⁰. Regarding major sources of CE linoleate hydro(pero)xides in vivo, isomer analyses of both human plaque ¹⁰³ and healthy fasting plasma ²⁶⁴ suggest a dominance of radical-mediated oxidation, which could also be initiated by enzymes ¹⁰⁵, as earlier. From a biophysical perspective, the specificity of LPCAT3/LCAT for PUFAs may support CE fluidity ²⁵, while oxygenation (i.e. hydroxylation) of CEs/cholesterol may increase polarity/solubility to facilitate protein interactions and ‘fast-track’ transport to the liver ^122,123,156, before more extensive oxygenation to bile acids. In the pathological context, LDL is initially retained within the arterial wall via extracellular proteoglycans, with further aggregation and fusion making egress back to blood impossible ²³, at least without metabolism. Even in healthy rodents, native LDL appears in the arterial wall as oxLDL with endothelial activation ^45,46; perhaps this could be adaptive if it induces release from proteoglycans ^37,94, lipid transfer to HDL ¹⁵⁴ and monocyte recruitment with coupled uptake/efflux ¹³⁶. Further, plasma linoleate ²³⁵ and HODEs ²⁶⁵ have also been inversely associated with inflammatory markers in humans, while PPARg ¹³⁴ and 27-HC ¹²⁹ may favour M2 polarisation, supporting immune resolution.

Several studies suggest linoleic (18:2n-6) and α-linolenic acid (18:3n-3) have similar lipid-lowering effects, although in a recent trial comparing the 2 via seed oils the latter had stronger effects on cholesterol/apoB ²⁶⁶ and oxylipins (e.g. HOTrEs) ²⁶⁷, which also activate PPARs in vitro ²⁶⁸. By comparison, in women with obesity supplementation of n-3 PUFAs (~5:1 DHA:EPA) for 3 months lowered triglycerides (not cholesterol), insulin and inflammatory markers, while inducing PPARα and Nrf2-related antioxidant genes (incl. HO-1) ²⁰; similar to mice on a high fat diet supplemented with DHA (alone/with EVOO) ²⁶⁹. In human trials fish oil/n-3 PUFAs also increase plasma oxygenation products (e.g. HDHAs) ^14,15,17,18 and downstream pro-resolving mediators (e.g. resolvins) ^16,20, at the expense of arachidonic-derived oxylipins ^14,16. Early cell studies found (ambient) oxidation of EPA was required for inhibition of (cytokine-induced) NF-κB, which also required PPARα ²⁷⁰; more recently, 7-HDHA (formed via ALOX5) was identified as a high-affinity PPARα ligand regulating brain morphology ²⁷¹. In the periphery n-3 PUFA induction of PPARα may also support triglyceride lowering ²⁶² via suppression of SREBP-1 (which mediates hepatic lipogenesis) ²⁷² and apoC-III (which inhibits VLDL lipolysis) ²⁷³. Further, in animal models dietary oxidised linoleate can also lower hepatic and plasma triglycerides via PPARα ¹³. Presumably differences in n-6 and n-3-related lipid-lowering may relate to their differential lipid distribution, oxidative metabolism and signalling specificity. Also, in T2D ¹⁰⁰ and coronary syndrome ²⁷⁴ the anti-platelet activity of HDL was associated with content of several n-6 and n-3 hydroxides and causality was similarly shown in vitro (i.e. for HODEs, HEPEs and HDHAs).

Notably, the protective effects of linoleic acid/n-6 PUFAs (vs. MUFAs) on atherosclerosis in LDLR^–/– mice persisted even when switching to a cholesterol/SFA-rich diet, and lesions negatively correlated plasma isoprostanes (i.e. 8-iso-PGF_2α) and aortic catalase (decomposes H₂O₂), implicating oxidative stress-induced hormesis ²¹⁵. In endothelial cells free linoleic acid induced β-oxidation and catalase activity, which were sensitive to vitamin E ⁵⁹, and hydroperoxides with a decline in glutathione before an increase above baseline ⁵⁷. Further, 13-HpODE/HODE induced catalase expression in several arterial cells ²⁷⁵, which is regulated by PPARg ²⁷⁶ and blocks MPO-induced oxidation ⁸⁷. As above, the ability of n-3 PUFAs to improve redox markers ¹⁹ may involve Nrf2 ^20,269. Several n-3 oxylipins can activate Nrf2, such as 17-oxo-DHA, resolvins and maresins ²⁷⁷. In addition, early cell studies found radical-mediated oxidation of EPA and DHA was required for induction of Nrf2–HO-1 (in contrast to sulforaphane), and implicated formation of J₃-isoprostanes ²⁷⁸. Later, in mice fish oil/n-3 PUFAs increased aortic HO-1 expression and vasodilation, which were abolished by Nrf2 deletion; tested in vitro DHA-derived 4-HHE induced Nrf2–HO1 ²⁷⁹. Fish oil/n-3 PUFAs were further shown to induce 4-HHE and HO-1 in multiple organs, while safflower oil/n-6 PUFAs did not ²⁸⁰. However, in the context of inflammation, prior injection of linoleic acid alleviated LPS-induced liver injury via Nrf2 ²⁸¹. LPS induces various oxylipins ²¹ which are sensitive to n-3 status ¹⁷; and n-6 series Nrf2-inducers include EKODE ²⁸², 15d-PGJ₂ ²⁸³ and LXA₄ ²⁷⁷. Moreover, low-level 4-HHE and 4-HNE (from n-6 PUFAs) similarly induce Nrf2 in endothelial cells ²⁸⁴, and in APOE^–/– mice on a high fat diet 4-HNE precedes Nrf2 activation ¹⁸⁶; whereas the non-specific peroxidation product MDA may be less effective ⁵³.

Perhaps cellular PUFA status could lower the threshold for Nrf2 induction under oxidative conditions, favouring earlier feedback and pleiotropic regulation of redox, immune and lipid homeostasis, thereby limiting plaque growth and instability. On the other hand, excess unsaturated aldehydes are ultimately toxic; indeed while low-level 4-HNE induces Nrf2 and supports homeostasis, high levels block Nrf2 and favour apoptosis ²². In women supplemented with n-6 or n-3, LDL isolated and oxidised (via copper) from the latter group had less apoptotic activity on pro-monocytes, mirroring differential effects of HNE vs. HHE, respectively ²⁸⁵. However, in LPS-treated mice 4-HNE inhibited inflammasome activation and pyroptosis; tested in vitro this was independent of its effects on Nrf2/NF-κB signalling, but may involve direct binding to NLRP3 ²⁸⁶—another major pathway mediating experimental atherosclerosis ⁴¹. In mammalian cells 4-HNE is normally cleared extremely rapidly, especially by hepatocytes and enterocytes, and via several pathways including glutathione conjugation ²⁸⁷. Thus, induction of Nrf2/glutathione would provide feedback inhibition and moderate oxylipin/aldehyde levels, which could be reinforced by glycine availability, as earlier. Conversely, in metabolic disorders and highly stressed cells Nrf2/glutathione exhaustion may favour apoptosis, which itself could indirectly exert homeostatic pressure via oxPL-dependent efferocytosis and subsequent induction of ALOX15-dependent pro-resolving mediators ^76,77. In particular, enriching neutrophil-like cells and their phospholipids in linoleic acid displaced oleate and did not affect peroxide-induced apoptosis per se, but increased efferocytosis of intrinsic apoptosis via surface display of oxPS, which was enriched in di-oxygenated linoleate species, and subject to hydrolysis and abrogation by Lp-PLA₂ ²⁸⁸.

Context-dependant homeostasis?

Further homeostatic insight may lie in other perspectives; for instance, the effects of PUFAs may somewhat overlap with exercise ²¹⁵. Firstly, exercise is well-documented to induce ROS/oxidative stress and adaptive/hormetic antioxidant responses ²⁸⁹. In mice exercise training also induces aortic catalase and sterol 27-hydroxylase ¹¹⁷, as well as hepatic LXR and reverse cholesterol transport ²⁹⁰. Further, in LDLR^–/– mice on a high fat diet exercise training reversed endothelial (vasodilatory) dysfunction by increasing eNOS/nitric oxide and nNOS/hydrogen peroxide, while lowering NOX2/superoxide and inducing superoxide dismutase ²⁹¹. And in a similar mouse model exercise induced aortic catalase activity and eNOS, and lowered plasma cholesterol and atherosclerosis, all of which was thwarted by high-dose vitamin E (human equivalent 1000iu) ²⁹². This aligns with the more general finding that high-dose antioxidants (typically vitamins C and E) can block beneficial metabolic and functional adaptations to exercise in humans and mice ²⁸⁹, which may be mediated in large part by ROS/Nrf2 ²⁹³. Intriguingly, exercise can also induce lipid peroxidation (incl. plasma HODEs ²⁶⁵, isoprostanes and aldehydes ²⁹⁴), and preferentially in HDL ²⁹⁵, while some recent studies suggest a synergistic effect of exercise training and n-3 PUFA supplementation on antioxidant status, lipids and performance, among other factors ^296,297. Further, both cold exposure and moderate exercise induce release of linoleic-derived 12,13-diHOME (an epoxide synthesised via CYP) from brown adipose to stimulate fatty acid uptake and reduce serum triglycerides ¹².

In considering optimal PUFA intake for tissue homeostasis, post-industrial dietary and tissue linoleic acid/n-6 may seem high ^2,6, although still has favourable health associations ^197,198, but long-chain n-3s more so by mass ^199,200. Many seed oils are notoriously rich in linoleate, although have replaced animal source added fats (e.g. butter and lard) which would already amplify dietary SFAs/palmitate and MUFAs/oleate (Table S1) and potentially distort favourable ratios. Further, in the context of whole foods many nuts/seeds are naturally rich in fat and linoleate, where the PUFA/MUFA content is strongly inversely correlated, and olives represent the other extreme (Table S2). In the body C18:2/n-6 may also compete with C18:1/n-9 for esterification and C18:3/n-3 for desaturation. However, effects on tissue PUFAs, oxylipins ²⁶⁷ and inflammation ²⁹⁸ are also modified by FADS1 variants affecting Δ5-desaturase—perhaps here the dietary n-6/n-3 balance may become more important. In the context of a western diet already high in linoleate, there may be greater potential to shift lipid composition and physiology with n-3s. Indeed, n-3s appear to have substantial incorporation into plasma lipids ²⁶⁷, blood cells ¹⁴ (e.g. omega-3 index) and carotid plaque (i.e. vs. 18:1 ²¹⁶ or 18:2 ²¹⁷), and their increased susceptibly to non-enzymatic oxidation may support earlier Nrf2 activation ²⁸⁰ and moderate effects of 4-HNE ²⁸⁵. In contrast to PUFAs, serum oleate has unfavourable cardiometabolic associations ²⁴⁷, while in the diet plant sources (vs. animal) ²⁹⁹ and virgin olive oil (vs. common varieties) have been associated with lower all/CVD mortality ³⁰⁰, implicating non-lipid components. For instance, extra virgin olive oil may also support antioxidant/Nrf2 activity via its polyphenol content ²⁶⁹, which is actually much higher in whole olives ³⁰¹ (along with added sodium) and may involve generation of hydrogen peroxide ³⁰².

Importantly, the ability of PUFAs and ROS to support homeostasis may also rest upon the site of oxidation. In the body hydroperoxides are rapidly reduced to stable PPAR ligands and aldehydes induce Nrf2, both of which may support lipid flux and limit further oxidation. This could maintain spatiotemporal control over subcellular/organelle-specific ROS generation and transient/gradient-based redox signalling, resulting in a high signal-to-noise ratio. In contrast, during food processing and digestion the extent of PUFA oxidation essentially depends entirely on the chemistry of the food matrix and stomach before absorption by the body, resulting in a bolus of peroxidation products which can apparently overwhelm intestinal detoxification and incorporate into plasma lipoproteins for delivery to tissues ^7,229,230. Dietary thermo-oxidised oils can induce PPARs ¹³ and Nrf2 ^226,303,304, but alongside signs of inflammation, antioxidant depletion and DNA damage ³⁰⁵. Extensively oxidised LDL is rapidly cleared by hepatocytes ^45,46, but may also appear in arterial plaque ⁴², whereas more mildly seeded lipoproteins could linger increasing the probability of entering the arterial wall, wherein they may increase the oxidative burden and aggravate atherosclerosis ⁹. Thus, at least in theory, oxidation ‘ex vivo’ might eventually subvert homeostasis and promote pathogenesis in vivo.

In the natural context, wholefood plant-based PUFAs may be least susceptible to oxidation ex vivo, while still providing substrate for favourable oxidation in vivo, whereas in the post-industrial era the effect of any isolated oil peroxidation may be highly context dependent ⁵. Notably, significant dietary oil oxidation typically requires antioxidant depletion with prolonged air exposure ²²⁴ or intense heating ^225,226,303, whereas red meat may induce advanced peroxidation products within the normal digestive/postprandial phase ^229,230, which is exaggerated by addition of PUFA-rich oils ^9,232, making it a particularly relevant catalyst and potential confounder in PUFA studies. For instance, many old CVD trials had heterogeneous outcomes and reduced saturated fat via replacement with isolated seed oils to be used for cooking and incorporation into provided ‘filled’ foods ⁴, which included sausage products (i.e. Veterans study ²¹²) and beef (i.e. Minnesota study ²), and more recently liver pâté ²⁵⁹, suggesting direct contact with heme-iron. However, this could represent a small proportion of oil consumed and be offset by other dietary components.

Conclusion

PUFA peroxidation is associated with human atherosclerosis and induces atherogenic effects in vitro, supporting a pathogenic view and implicating the post-industrial increase in tissue linoleate. However, this clashes with much outcome and experimental data, hence this review sought to explore reconciliation through a more physiological systems-based perspective with attention to oxidative specificity and signalling in vivo. Indeed, human plaque expresses oxygenases and abundant oxygenated lipids which can induce efflux, secondary to cholesterol loading. Among PUFAs, linoleate seems particularly well positioned in this regard, although oxidation products of both n-6 and n-3 may induce differential and overlapping adaptive responses via PPARs and Nrf2, respectively; even advanced peroxidation products initially induce hormesis before toxicity. This could have some analogy and synergy with exercise and polyphenols, which may also generally benefit cardiovascular health. Dietary PUFAs can deliver both oxidative substrate and antioxidants via the plant food matrix. On the other hand, lipid peroxidation during food processing and/or digestion effectively bypasses the opportunity for physiologic signalling and feedback inhibition, thereby allowing accumulation of end products and increasing the potential for negative effects. Notably, this situation seems most analogous to the typical oxidative conditions used to create atherogenic lipoproteins in vitro, which inform classic oxidative stress/influx-based hypotheses. However, actual physiological effects may be heterogenous and depend on the extent of oxidation, dietary pattern and host health, those with metabolic/glutathione disorders perhaps being more susceptible, and even here PUFAs (vs. SFAs) may exert favourable effects via metabolism and microbiome.

As such, a context-dependant homeostatic hypothesis is suggested, wherein the inherent susceptibility of PUFAs to oxidation may not be dichotomous with their health benefits, but even underlie favourable modulation of redox, immune and lipid homeostasis—and opposite to typical SFAs—ultimately supporting efflux and efferocytosis to limit plaque growth and instability. However, incorporating the negative effects of excessive peroxidation, such adaptive responses may eventually be undermined by sufficient exogenous oxidation, or augmented by metabolic optimisation.

This review has notable limitations. Its mechanistic and theoretical nature make it susceptible to the reductionist trap, despite favouring a holistic framework via a human evidence-based hierarchy and biological systems-based reasoning (incl. dose-response, throughput/flux, homeostasis/feedback and compartmentalisation). The narrative synthesis is also inevitably limited by personal bias and incomplete research. Moreover, heterogeneity and inconsistencies within the current literature can challenge this and any coherent perspective at all; as such, some potential controversies, reconciliations and areas for further research are listed in the supplement. Regarding implications, this review provides mechanistic rationale to human outcome data on n-6/n-3 PUFAs, both in terms of complementary and overlapping health benefits, and modification by confounding factors. Controlling for all this in further studies may help refine and homogenise the evidence base; nonetheless, dietary guidelines already typically favour whole plant foods over processed foods and red meat, which may help safeguard PUFA quality.

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