Susceptible to oxidation yet resistant to atherosclerosis—reconciling the PUFA paradox via signalling

Another work in progress; any thoughts/feedback appreciated.

Atherosclerotic cardiovascular disease (ASCVD) is ubiquitous and a leading cause of death globally, while a cornerstone of dietary guidelines for prevention is replacing saturated fats (SFAs) with unsaturated fats (UFAs), especially plant-based PUFAs (e.g. from seed oils) ¹, and consuming more oily fish/n-3 PUFAs (FAO). Such public health recommendations are supported by the totality of evidence from observational and interventional studies (i.e. the mean of heterogeneous populations). Mechanistically however, this creates a potential paradox, since atherosclerosis is widely acknowledged to involve lipid oxidation, among which PUFAs are most susceptible, forming the basis of some concern ^2–6. At first glance, a simple dichotomous reconciliation is the benefits of PUFAs, such as lipid lowering or anti-inflammatory activity, may outweigh any putative negative effects. However, PUFA oxidation can take many paths and produce many molecules with diverse effects, including lipid lowering ^7,8 and anti-inflammatory activity ^9–14, suggesting context matters and opportunity for harmonisation.

At one extreme, free radical mediated autoxidation eventually degrades PUFAs into reactive aldehydes, such as n-6-derived 4-hydroxynonenal (4-HNE; C₉H₁₆O₂), n-3-derived 4-hydroxyhexenal (4-HHE; C₆H₁₀O₂), malondialdehyde (MDA; C₃H₄O₂) and acrolein (C₃H₄O), which can covalently bind proteins and exert toxicity. However, initial stages of peroxidation generate full-chain oxygenated metabolites (i.e. hydroperoxides), such as linoleic-derived HpODEs (C₁₈H₃₂O₄), arachidonic-derived HpETEs (C₂₀H₃₂O₄) and DHA-derived HpDHAs (C₂₂H₃₂O₄), before later cleavage and fragmentation. Further, these reactions are explicitly catalysed by enzymes, such as lipoxygenases (LOXs) and cyclooxygenases (COXs), with positional specificity and in the presence of antioxidants, thereby producing stable metabolites of physiological relevance. Indeed both early and late-stage PUFA peroxidation products exhibit signalling activity, as discussed herein. Hence it is important to consider the full scope and context of lipid peroxidation in vivo for pathological interpretation, and this article is particularly concerned with disentangling physiology.

The lab vs. plaque

Already in the 1950s lipid peroxidation was detected in human plaque, and much subsequent research supports the involvement of lipid and protein oxidation in atherosclerosis ¹⁵. Native and oxLDL were even found in fetal aortas with and without macrophages, suggesting an early event ¹⁶. OxLDL can also be detected in plasma where it normally represents a very small fraction of LDL ¹⁷ and associates with CVD ^18,19, although not always independently of apoB (e.g. CHD ²⁰ and MetS ²¹), likely due to 4E6 antibody cross-reactivity ²². On the other hand, oxidised phospholipids on apoB₁₀₀ (oxPL–apoB) are independently associated with CVD and mainly carried by lipoprotein(a), an LDL variant; indeed oxLDL donates its oxPL to lipoprotein(a) in vitro ²². Conversely, dietary and plasma antioxidant nutrients (esp. vitamins C, E and carotenoids—largely reflecting fruit/veg and seed oil intake) are inversely associated with CVD ^23,24, although the results of RCTs with high-dose (i.e. supra-physiological) supplements in general populations have mostly failed to show benefit ^15,25 (unlike general lipid-lowering). However, there is far less outcome data on more physiological antioxidant repletion/optimisation approaches and other phytochemicals (e.g. polyphenols) ²⁵.

Interestingly, plasma oxLDL and oxPL–apoB increase transiently with statins in humans, and preceding progression and regression of experimental atherosclerosis, suggesting exchange with plaque ^22,26. The high antioxidant capacity of plasma also suggests lipoprotein oxidation may occur elsewhere, such as within the arterial wall. Various catalysts are used in vitro, but most typically LDL is incubated with copper (Cu²⁺) sulfate, a transition metal mediating 1-electron oxidations ¹⁵. LDL oxidation induces many pro-atherogenic endothelial/inflammatory effects (e.g. eNOS ²⁷, CCL20 ²⁸, EPCs ²⁹ and HSPCs ³⁰), and most characteristically, macrophage uptake and cholesterol loading via scavenger receptors ¹⁹. Further, unlike native LDL, macrophage uptake of oxLDL results in lipid trapping within lysosomes ³¹, cholesterol crystallisation and NLRP3 activation ^32,33. Similar oxidation of HDL also induces macrophage uptake, reversing its protective activity ³⁴. In human tracer studies with autologous native 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 ³⁵. 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 most abundant lipophilic antioxidant) ³⁶.

However, despite much data apparently supporting a causal role of oxidative stress in atherogenesis, the general failure of high-dose vitamin E/antioxidant trials to improve hard outcomes suggests more complexity ¹⁵. Foremost, oxidation by free transition metals may have limited physiological analogy; although heme-iron (Fe²⁺) dysregulation may promote oxidation during advanced plaque haemorrhage and haemolysis ³⁷. On the other hand, human lesions express LOXs (non-heme iron-dependent dioxygenases), which oxidise PUFAs and can induce similar lipoprotein modification as copper oxidation ^38,39, but are not blocked by vitamin E ⁴⁰. Human plaques also express iNOS ^41,42 and MPO ⁴³ (both heme-dependent enzymes), while recovered LDL and apoA-I/HDL are highly enriched in their 2-electron protein oxidation products (i.e. nitrotyrosine and 3-chlorotyrosine, respectively) ^44,45, implicating immune-dependant redox modifications ¹⁵. These pathways are also not blocked by vitamin E ⁴⁶ (or serum ⁴⁷), and resulting NO₂–LDL stimulates macrophage uptake and loading via scavenger receptor CD36 ^47,48, while MPO-modified tryptophan residues within apoA-I/HDL inactivate its ABCA1-dependent acceptor activity ^45,49. However, other data present yet further challenges ^15,50. 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 ⁵¹. Indeed LDL aggregation greatly increases macrophage uptake by receptor-independent endocytosis ^51,52 and CE accumulation beyond native or oxLDL ^53,54. Accordingly, the ability of copper-oxidised LDL to induce lipid droplets may be somewhat limited by defective lysosomal processing (prior to cholesterol esterification) ³¹. More ‘minimally’ oxidised LDL still exhibits atherogenic effects, such as a tendency to aggregate ⁵⁵ and induce lysosomal crystals and NLRP3 activation ^32,33, so may contribute in these ways ⁵¹. On the other hand, a systematic study of LDL oxidised with copper for 0.5–24hr showed that 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 ⁵⁶. Furthermore, 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 ⁵⁷. Also, human ALOX15 variants if anything suggest increased enzyme activity is athero-protective ⁵⁸, consistent with ALOX15 overexpression increasing reverse cholesterol transport ⁵⁹.

Considering the specificity and spatiotemporal pattern of lipid oxidation in plaque may provide some reconciliation. 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 CEs ⁶⁰. Accordingly, 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 mono- and di-oxygenated derivatives of linoleate (i.e. HODEs and HpODEs, respectively) ⁶¹. The HODE-CE profile exhibited no regio- or stereo-specificity suggesting a dominance of non-enzymatic peroxidation (vs. 15-LOXs), although triglyceride PUFAs were not oxidised indicating some specificity ⁶¹. In several earlier studies 13-HODE stereoisomer ratios were consistent with ALOX15 activity, particularly in early lesions ⁶², while more recent studies on carotid plaques found increased expression of ALOX15B only, and in association with macrophages and HIF-1α ³⁹. Recent high-resolution imaging of advanced carotid plaques also found oxidised CEs co-localise with sphingomyelin in the necrotic core, while a metabolite resembling 7-ketocholesterol (representing 1-electron cholesterol oxidation) was uncorrelated ⁶³. Several earlier studies also found plaque lipid oxidation occurred despite normal levels of α-tocopherol ⁴⁶, and was similar in T2D ⁶⁴. In particular, an analysis of intimal lipoprotein-containing fractions of human 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-hydroxycholesterol 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 ⁶⁵. Another stage-dependent analysis of whole aortic lesions included tocopherol oxidation products and implicated 2-electron (enzymatic) oxidants ⁶⁶.

These observations contrast typical conditions in vitro ¹⁵, where under strong copper-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.) ⁶⁵. Conversely, under more mild conditions the α-tocopherol radical can initiate lipid peroxidation ⁶⁷, especially when there are insufficient regenerative co-antioxidants (e.g. CoQ₁₀ and carotenoids) ⁶⁸. Further, copper-oxidation of LDL generates substantial 7-ketocholesterol, which in macrophages inhibits lysosomal SMase causing accumulation of sphingomyelin–cholesterol particles ³¹, and dose-dependently induces cholesterol crystals ⁶⁹, thereby mediating key effects of oxLDL. However, in vivo 7-ketocholesterol is quantitatively and temporally overshadowed by 27-hydroxycholesterol, which is produced by sterol 27-hydroxylase, indicating a dominance of enzymatic oxidation ⁶⁵. In the liver this enzyme initiates the acidic pathway of bile acid synthesis, while elsewhere it may increase the polarity of cholesterol and facilitate efflux, before return to the liver for further conversion and excretion ⁷⁰. In this regard, in human lesion macrophages increased expression of sterol 27-hydroxylase is accompanied by the expression of genes functionally linked in vitro; specifically, RXR and PPARg ligands induce sterol 27-hydroxylase ⁷¹, which in turn induces 27-hydroxycholesterol and LXR—an oxysterol sensor mediating efflux ⁷². Further, the early linoleate peroxidation metabolite 13-HODE is a natural PPAR ligand ⁶² and also induces macrophage efflux to apoA-I via a PPARα/g–LXRα pathway ⁷. Upstream, 15-LOXs can directly oxidise CE-PUFAs, which are also preferred substrates for hydrolysis, and reincorporated into phospholipids ³⁹; while ALOX15 specifically is induced by Th2/M2 cytokines and apoptotic cells (via LXR ⁷³), consistent with a role in lipid/tissue homeostasis ^38,39. Therefore, considering all the above, mild enzymatic oxidation of trapped lipids may support cholesterol clearance ^62,65,74, whereas excessive oxidation may favour lipid trapping via 7-ketocholesterol ³¹ and inactivation of apoA-I/HDL ^45,49,57, perhaps in relation to advanced disease and inflammation ¹⁵.

Dietary fat and lipid oxidation

Dietary fat saturation has well recognised 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 ⁷⁵. Whereas fish oil/long-chain n-3 PUFA supplementation preferentially lowers triglycerides and non-HDL-cholesterol, particularly in those with hyperlipidemia or overweight/obesity ⁷⁶. In addition, recent trials find dietary SFAs can increase LDL sphingolipids and aggregation in vitro, whereas C18 UFAs lower LDL proteoglycan binding in vitro ^77,78. Clearly all these effects of UFAs may be beneficial by lowering arterial lipoprotein and lipid retention—the major prerequisite of atherogenesis ⁷⁹. On the other hand, the effect of dietary fats on lipoprotein oxidation is much more controversial. Tested since the early 90s in short-term trials, MUFA-rich diets (vs. n-6 PUFAs or oily fish/n-3) typically lower LDL and HDL oxidation, and susceptibility to copper oxidation (i.e. lag time and/or rate) and monocyte adhesion in vitro, which correlates lipoprotein phospholipid oleate/linoleate ratios ^80–82. Thus competition between C18:1/MUFAs and C18:2/PUFAs for membrane incorporation may modulate substrate for oxidation; similarly, fish oil/long-chain n-3 PUFAs may displace respective long-chain n-6 PUFAs (i.e. C20/22 species) with less double bonds ⁹. This has fuelled some concern, although doesn’t mirror hard outcome data or reflect the more nuanced redox biology in vivo, as above. Accordingly, in men ⁸³, monkeys ⁸⁴ and mice ⁸⁵ n-6 PUFA-rich diets increase linoleate/oleate ratios in plasma and plaques, and oxidation in vitro ⁸⁴ and in vivo ^85,86, yet are protective. The long-chain n-3 content of advanced carotid plaques is also increased by supplementation and correlates greater stability and lower inflammation, consistent with anti-inflammatory effects ^87,88.

The effect of PUFAs also depends on the food matrix. For instance, a 3-week diet of 31% sunflower oil/n-6 PUFAs (vs. olive oil/MUFAs) lowered LDL levels, oxidation susceptibility and proteoglycan binding, in relation to LDL antioxidant content and size ⁸⁹. Further, 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 (5-hour AUC) and oxLDL (at 2 hours) ⁹⁰, while longer trials show enrichment of PUFAs with preservation of oxidation status ^91,92, alongside many other cardio-protective effects (reviewed in ⁹³). Conversely, food storage and processing can oxidise lipids prior to ingestion. For instance, prolonged exposure of soybean oil to high cooking temperatures induces a gradual increase in peroxides before a decline (at 6hrs), while secondary aldehydes continue to increase ⁹⁴. When fed to humans oxidised linoleic acid could be detected in chylomicrons/remnants for 8 hours (esp. in diabetics with poor glycaemic control), whereas oxidised cholesterol appeared in all major lipoproteins and persisted for 72 hours; tested in vitro oxidised cholesterol was transferred to LDL and HDL, potentially via CETP ⁴. In animal models dietary oxidised linoleic acid can promote atherosclerosis ⁴, although has also been reported to lower blood lipids and atherosclerosis ⁷. More specifically, 13-HODE was shown to elevate blood lipids and atherosclerosis only in the presence of dietary cholesterol, possibly due to its increased solubilisation and absorption ⁹⁵. Also, in a unique RCT on healthy adults (with low TGs) comparing high quality to oxidised fish oil (vs. high-oleic sunflower oil/MUFAs), only the former lowered IDL/LDL particles and cholesterol content, which correlated CETP ⁹⁶.

In humans and mice red meat ingestion induced postprandial lipid peroxidation and plasma LDL–MDA modification, which was greatly inhibited by polyphenols ^97,98. 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 ⁹⁷. 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 ⁹⁷. The activity of many plant foods in this model has been indexed and correlates polyphenol content ⁹⁹; additionally, peroxidation was inhibited by oleate/MUFAs, opposite to fish oil/n-3 PUFAs ¹⁰⁰. Further, in APOE^–/– mice on a western diet 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 ⁶. Of note, iron deficiency is common and supplementation can also induce gastrointestinal lipid peroxidation ¹⁰¹.

Despite the oxidative stability of SFAs, and the responsiveness of serum stearate to diet ¹⁰², SFA-rich diets (vs. carbohydrates or MUFAs) may also increase LDL susceptibility to oxidation in relation to MUFA/PUFA ratios ⁸², vitamin E ⁸¹ and APOE promoter variants ¹⁰³. Further, excess dairy fat may 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 ¹⁰⁴. In mice a diet rich in dairy fat/SFAs also elevated oxidised HDL and LDL, while replacement with soybean oil/PUFAs (i.e. ~5:1 of n-6:n-3) enhanced HDL antioxidant activity ¹⁰⁵; and in another study replacement with olive oil and nuts lowered monocyte oxLDL uptake and CD36 expression, which was modulated correspondingly by TRLs from each diet ¹⁰⁶.

Dietary SFAs may affect lipid oxidation indirectly. For instance, 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 ¹⁰⁷. In a systematic review of RCTs dietary SFAs (vs. UFAs) induced postprandial LPS ¹⁰⁸, which at similar levels in vitro also induces LDL oxidation ¹⁰⁹. 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 ¹¹⁰. The glutathione system is also implicated in human atherosclerosis by associations with low plasma glutathione redox (i.e. GSH:GSSG) ^111,112 and arterial glutathione-related enzyme activity (i.e. GR, GPx and GSTs) ^113,114. Of interest here, in various human cohorts lower plasma glycine (a glutathione precursor) associates with metabolic and coronary disease, while in mice dietary glycine favourably modulated atherosclerosis, alongside lipids, glutathione and superoxide ¹¹⁵. Further, host glycine availability is itself dependent on the gut microbiome: 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 ¹¹⁶. Accordingly, 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 ¹¹⁷. 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 ¹¹⁸.

Oxylipin-induced homeostasis

Could the discordant effects of PUFAs on oxidation and atherosclerosis be further reconciled by oxylipin signalling? For instance, in human trials replacing SFAs with mostly n-6 PUFAs lowers plasma cholesterol, while inducing serum bile acids and PBMC LDLR/LXRα/ABCG1 gene expression ^118,119. Elsewhere, lowering dietary linoleic acid/n-6 PUFA (vs. mostly SFAs) also lowered plasma early peroxidation metabolites (i.e. HODEs and oxo-ODEs) ¹²⁰, while in vitro 13-HODE (but not linoleic acid) induced macrophage cholesterol transporter expression and efflux via PPARα/g–LXRα signalling ⁷. Extended to the liver LXRα could increase bile output and support systemic cholesterol lowering, underlying clinical effects of n-6 PUFAs ^118,121. In 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) ¹³; like mice on a high fat diet supplemented with DHA (alone/with EVOO) ¹²². Human trials with fish oil/n-3 PUFAs also increase plasma early peroxidation products (e.g. HDHAs) ^9,10,12,14 and downstream pro-resolving mediators (e.g. resolvins) ^11,13, at the expense of arachidonic-derived oxylipins ^9,11. Early cell studies found oxidation of EPA was required for inhibition of (cytokine-induced) NF-κB, which also required PPARα ¹²³; more recently, 7-HDHA (formed via ALOX5) was identified as a high-affinity PPARα ligand regulating brain morphology ¹²⁴. In the periphery n-3 PUFA induction of PPARα may also support triglyceride lowering ¹²¹ via suppression of SREBP-1 (which mediates hepatic lipogenesis) ¹²⁵ and apoC-III (which inhibits VLDL lipolysis) ¹²⁶.

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 ⁸⁶. Furthermore, 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) ¹²⁷. This might involve Nrf2 ^13,122, which induces 100s of genes supporting redox homeostasis (incl. HO-1, glutathione, catalase, etc.). As with NF-κB ¹²³, early cell studies found radical-mediated oxidation of EPA and DHA was required for induction of Nrf2–HO-1 (in contrast to sulforaphane), and potentially via formation of J₃-isoprostanes ¹²⁸. 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 ¹²⁹. 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 ¹³⁰. However, in the context of inflammation, prior injection of linoleic acid alleviated LPS-induced liver injury via Nrf2 ¹³¹. Moreover, low-level 4-HHE and 4-HNE (from n-6 PUFAs) similarly induce Nrf2 in endothelial cells ¹³², and in APOE^–/– mice on a high fat diet endothelial inflammation and 4-HNE precede Nrf2 activation, which then appears to exert negative feedback regulation and restrain atherosclerosis ¹³³. In addition, in LPS-treated mice 4-HNE inhibited inflammasome activation; tested in vitro this was independent of its effects on Nrf2/NF-κB signalling, but may involve direct binding to NLRP3 ¹³⁴—another major pathway mediating experimental atherosclerosis ³³.

Excess unsaturated aldehydes are ultimately toxic; indeed while low-level 4-HNE induces Nrf2 and supports homeostasis, high levels block Nrf2 and favour apoptosis ¹³⁵. Regardless, given the benefits of PUFAs even in advanced human disease (e.g. seed oils ¹ and fish oil ^87,88), an overall homeostatic effect seems likely. In healthy cells induction of Nrf2 supports antioxidant activity and aldehyde clearance in part via the glutathione system, which may be further reinforced by increased glycine availability, as above. In addition, in vitro studies with various phytochemicals show Nrf2 induces macrophage cholesterol efflux, via suppression of NF-κB signalling ¹³⁶ and induction of SR-B1 and ABCA1/G1 transporters ^137–139, and thus may also support lipid-lowering and prevent cholesterol sequelae. Conversely, in highly stressed cells Nrf2/glutathione exhaustion may favour aldehyde accumulation and apoptosis, which itself could indirectly exert homeostatic pressure via subsequent macrophage efferocytosis and induction of ALOX15 ^38,39. Further, 17-oxo-DHA (formed via COX-2) augmented efferocytosis via Nrf2/HO1-dependent expression of LOX/COX-2 and pro-resolving mediators ¹⁴⁰. Taken together, perhaps cellular PUFA status (n-3>n-6) could determine the threshold for Nrf2 induction under oxidative conditions, favouring earlier feedback and pleiotropic regulation of redox, immune and lipid homeostasis, which ultimately limits plaque growth and instability. 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 ¹⁴¹. Therefore, timely activation of Nrf2 may also limit systemic pathology.

Further hormetic insight may lie in other perspectives; for instance, the effects of PUFAs may somewhat overlap with exercise ⁸⁶. Firstly, exercise is well-documented to induce ROS/oxidative stress and adaptive/hormetic antioxidant responses ¹⁴². In mice exercise training also induces aortic catalase and sterol 27-hydroxylase ¹⁴³, as well as hepatic LXR and reverse cholesterol transport ¹⁴⁴. 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 ¹⁴⁵. 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) ¹⁴⁶. 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, which may be mediated in large part by ROS/Nrf2 ¹⁴². Intriguingly, exercise can also induce lipid peroxidation (incl. plasma HODEs ¹⁴⁷, isoprostanes and aldehydes ¹⁴⁸), 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 ^149,150. 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 ⁸.

In summary, PUFA oxidation is associated with atherosclerosis and induces toxic effects in vitro, supporting a pathogenic view; however, attention to specificity and signalling may unveil a precedent physiology. As such, the susceptibility of PUFAs to oxidation may not be dichotomous with their health benefits, rather biology couples enzymatic and non-enzymatic oxidation to adaptive responses via oxylipin signalling (incl. redox, immune and lipid homeostasis); even advanced peroxidation products may induce hormesis before toxicity. In this regard, the effects of PUFAs may have some analogy and synergy with exercise, which also generally benefits cardiovascular health. However, the PUFA balance and site of oxidation may be determinate; in particular, excessive peroxidation during food processing and/or digestion would presumably bypass the opportunity for physiologic signalling and instead favour accumulation of fragmented end products, with increasing potential for toxicity. This situation seems more analogous to the typical oxidative conditions used to create atherogenic lipoproteins in vitro and thus most harmonious with the traditional oxidative stress hypothesis. From a natural and practical perspective, wholefood PUFAs may be least susceptible to oxidation ex vivo, while still providing substrate for favourable oxidation in vivo, whereas the extent of any isolated oil peroxidation will be highly dependent on the degree of processing and dietary context. Controlling for these factors in human 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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