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A biological rationale for the disparate effects of omega-3 fatty acids on cardiovascular disease outcomes

  • Samuel C.R. Sherratt
    Affiliations
    Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH 03823, USA

    Elucida Research LLC, Beverly, MA 01915-0091, USA
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  • Peter Libby
    Affiliations
    Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115-6110, USA
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  • Deepak L. Bhatt
    Affiliations
    Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115-6110, USA
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  • R. Preston Mason
    Correspondence
    Corresponding author at: Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115-6110, USA.
    Affiliations
    Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115-6110, USA

    Elucida Research LLC, Beverly, MA 01915-0091, USA
    Search for articles by this author
Open AccessPublished:May 20, 2022DOI:https://doi.org/10.1016/j.plefa.2022.102450

      Highlights

      • Identified differences in cardiovascular outcome effects of IPE in clinical trials and how this differed from EPA/DHA mixed formulations;
      • Discussed how different n3-FAs, specifically EPA and DHA, interact with the membrane at the molecular level;
      • Compared the effects of n3-FAs on membrane oxidative stress and cholesterol crystalline domain formation during hyperglycemia;
      • Reviewed effects of n3-FA on endothelial function and nitric oxide bioavailability, and the role of n3-FA-generated metabolites in inflammation resolution;
      • Discussed ongoing and future clinical investigations exploring treatment targets for n3-FAs, including COVID-19.

      Abstract

      The omega-3 fatty acids (n3-FAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) rapidly incorporate into cell membranes where they modulate signal transduction pathways, lipid raft formation, and cholesterol distribution. Membrane n3-FAs also form specialized pro-resolving mediators and other intracellular oxylipins that modulate inflammatory pathways, including T-cell differentiation and gene expression. Cardiovascular (CV) trials have shown that EPA, administered as icosapent ethyl (IPE), reduces composite CV events, along with plaque volume, in statin-treated, high-risk patients. Mixed EPA/DHA regimens have not shown these benefits, perhaps as the result of differences in formulation, dosage, or potential counter-regulatory actions of DHA. Indeed, EPA and DHA have distinct, tissue-specific effects on membrane structural organization and cell function. This review summarizes: (1) results of clinical outcome and imaging trials using n3-FA formulations; (2) membrane interactions of n3-FAs; (3) effects of n3-FAs on membrane oxidative stress and cholesterol crystalline domain formation during hyperglycemia; (4) n3-FA effects on endothelial function; (5) role of n3-FA-generated metabolites in inflammation; and (6) ongoing and future clinical investigations exploring treatment targets for n3-FAs, including COVID-19.

      Keywords

      Abbreviations

      n3-FA
      omega-3 fatty acids
      EPA
      eicosapentaenoic acid
      DHA
      docosahexaenoic acid
      AA
      arachidonic acid
      IPE
      icosapent ethyl
      CV
      cardiovascular
      PL
      phospholipid
      PL-EPA
      1-palmitoyl-2-eicosapentaenoyl-sn-glycero-3-phosphocholine
      PL-DHA
      1-palmitoyl-2-docosahexaenyol-sn-glycero-3-phosphocholine
      PL-AA
      1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine

      1. Clinical outcome and imaging trials using different omega-3 fatty acid formulations

      Omega-3 fatty acids (n3-FAs), along with their bioactive lipid metabolites, have tissue-selective effects that influence inflammation, cell signaling, oxidative stress, and mechanisms of chronic disease, including atherosclerosis [
      • Borow KM
      • Nelson JR
      • Mason RP.
      Biologic plausibility, cellular effects, and molecular mechanisms of eicosapentaenoic acid (EPA) in atherosclerosis.
      ,
      • Mason RP
      • Libby P
      • Bhatt D L
      Emerging mechanisms of cardiovascular protection for the omega-3 fatty acid eicosapentaenoic acid.
      ]. Two n3-FAs evaluated for treatment of cardiovascular (CV) disease are eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6). These essential fatty acids differ only by one double bond and two carbon atoms and derive from dietary sources such as oily marine fish [
      • Bhatt DL
      • Budoff MJ
      • Mason RP.
      A Revolution in Omega-3 Fatty Acid Research∗.
      ]. EPA and DHA have been recently tested for their ability to reduce residual CV risk among statin-treated subjects with well-controlled LDL but elevated triglyceride (TG) levels [
      • Libby P.
      Triglycerides on the rise: should we swap seats on the seesaw?.
      ,
      • Ganda OP
      • Bhatt DL
      • Mason RP
      • Miller M
      • Boden WE.
      Unmet need for adjunctive dyslipidemia therapy in hypertriglyceridemia management.
      ]. CV outcome trials (CVOTs) have demonstrated that benefits with n3-FAs have been highly inconsistent potentially due to differences in dosage, formulation, and composition [
      • Mason RP
      • Sherratt SCR
      • Eckel RH.
      Rationale for different formulations of omega-3 fatty acids leading to differences in residual cardiovascular risk reduction.
      ]. In particular, CVOTs using IPE, a highly purified ethyl ester of EPA, have shown consistent reductions in CV events and progression of atherosclerosis compared with mixed EPA/DHA treatments despite similar TG-lowering actions as we describe below [
      • Bhatt DL
      • Budoff MJ
      • Mason RP.
      A Revolution in Omega-3 Fatty Acid Research∗.
      ,
      • Mason RP
      • Sherratt SCR
      • Eckel RH.
      Rationale for different formulations of omega-3 fatty acids leading to differences in residual cardiovascular risk reduction.
      ,
      • Budoff MJ
      • Bhatt DL
      • Kinninger A
      • Lakshmanan S
      • Muhlestein JB
      • Le VT
      • et al.
      Effect of icosapent ethyl on progression of coronary atherosclerosis in patients with elevated triglycerides on statin therapy: final results of the EVAPORATE trial.
      ,

      Bhatt D, L., Steg PG, Miller M, Brinton EA, Jacobson TA, Ketchum SB, et al. Cardiovascular risk reduction with Icosapent Ethyl for hypertriglyceridemia. New England Journal of Medicine. 2019;380:11-22.

      ,
      • Yokoyama M
      • Origasa H
      • Matsuzaki M
      • Matsuzawa Y
      • Saito Y
      • Ishikawa Y
      • et al.
      Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis.
      ,
      • Watanabe T
      • Ando K
      • Daidoji H
      • Otaki Y
      • Sugawara S
      • Matsui M
      • et al.
      A randomized controlled trial of eicosapentaenoic acid in patients with coronary heart disease on statins.
      ,
      • Nishio R
      • Shinke T
      • Otake H
      • Nakagawa M
      • Nagoshi R
      • Inoue T
      • et al.
      Stabilizing effect of combined eicosapentaenoic acid and statin therapy on coronary thin-cap fibroatheroma.
      ,
      • Alfaddagh A
      • Elajami TK
      • Ashfaque H
      • Saleh M
      • Bistrian BR
      • Welty FK.
      Effect of eicosapentaenoic and docosahexaenoic acids added to statin therapy on coronary artery plaque in patients with coronary artery disease: a randomized clinical trial.
      ,
      • Group ASC
      • Bowman L
      • Mafham M
      • Wallendszus K
      • Stevens W
      • Buck G
      • et al.
      Effects of n-3 fatty acid supplements in diabetes mellitus.
      ,
      • Manson JE
      • Cook NR
      • Lee IM
      • Christen W
      • Bassuk SS
      • Mora S
      • et al.
      Marine n-3 fatty acids and prevention of cardiovascular disease and cancer.
      ,
      • Kalstad AA
      • Myhre PL
      • Laake K
      • Tveit SH
      • Schmidt EB
      • Smith P
      • et al.
      Effects of n-3 fatty acid supplements in elderly patients after myocardial infarction: a randomized, controlled trial.
      ,
      • Nicholls SJ
      • Lincoff AM
      • Garcia M
      • Bash D
      • Ballantyne CM
      • Barter PJ
      • et al.
      Effect of High-Dose Omega-3 Fatty Acids vs Corn Oil on Major Adverse Cardiovascular Events in Patients at High Cardiovascular Risk: The STRENGTH Randomized Clinical Trial.
      ].
      A review of recent CVOTs provides insight into this debate and, in particular, the relationship between risk reduction and TG lowering (Table 1Table 1) [
      • Mason RP
      • Sherratt SCR
      • Eckel RH.
      Rationale for different formulations of omega-3 fatty acids leading to differences in residual cardiovascular risk reduction.
      ]. The Reduction of Cardiovascular Events with Icosapent Ethyl–Intervention Trial (REDUCE-IT) demonstrated that IPE (4 g/d) significantly reduced CV events in at-risk patients with elevated TG levels (>150 mg/dL) [

      Bhatt D, L., Steg PG, Miller M, Brinton EA, Jacobson TA, Ketchum SB, et al. Cardiovascular risk reduction with Icosapent Ethyl for hypertriglyceridemia. New England Journal of Medicine. 2019;380:11-22.

      ]. First ischemic events fell by 25% (p<0.0001) and total (first and subsequent) ischemic events by 32% (p<0.0001). The large relative and absolute risk reductions observed in the study exceeded that attributable to the degree of TG lowering (19.7% decrease) and, instead, suggest pleiotropic benefits for EPA beyond lipid modification [
      • Bhatt DL
      • Steg PG
      • Miller M.
      Cardiovascular risk reduction with icosapent ethyl. Reply.
      ,
      • Mason RP.
      New insights into mechanisms of action for omega-3 fatty acids in atherothrombotic cardiovascular disease.
      ,
      • Bhatt DL
      • Steg PG
      • Miller M
      • Brinton EA
      • Jacobson TA
      • Jiao L
      • et al.
      Reduction in first and total ischemic events with icosapent ethyl across baseline triglyceride tertiles.
      ]. In fact, in REDUCE-IT, on-treatment serum EPA levels strongly correlated with CV outcomes compared with other traditional biomarkers such as levels of ApoB particles, including LDL [
      • Pisaniello AD
      • Nicholls SJ
      • Ballantyne CM
      • Bhatt DL
      • Wong ND.
      Eicosapentaenoic acid: atheroprotective properties and the reduction of atherosclerotic cardiovascular disease events.
      ]. Other CVOTs and clinical imaging studies using IPE formulations have yielded data consistent with REDUCE-IT while also providing mechanistic insights [
      • Budoff MJ
      • Bhatt DL
      • Kinninger A
      • Lakshmanan S
      • Muhlestein JB
      • Le VT
      • et al.
      Effect of icosapent ethyl on progression of coronary atherosclerosis in patients with elevated triglycerides on statin therapy: final results of the EVAPORATE trial.
      ,
      • Yokoyama M
      • Origasa H
      • Matsuzaki M
      • Matsuzawa Y
      • Saito Y
      • Ishikawa Y
      • et al.
      Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis.
      ,
      • Watanabe T
      • Ando K
      • Daidoji H
      • Otaki Y
      • Sugawara S
      • Matsui M
      • et al.
      A randomized controlled trial of eicosapentaenoic acid in patients with coronary heart disease on statins.
      ,
      • Nishio R
      • Shinke T
      • Otake H
      • Nakagawa M
      • Nagoshi R
      • Inoue T
      • et al.
      Stabilizing effect of combined eicosapentaenoic acid and statin therapy on coronary thin-cap fibroatheroma.
      ].
      By contrast, CVOTs using EPA/DHA combination therapy with prescription or dietary supplement products have not demonstrated consistently favorable CV effects especially in more recent trials where baseline statin usage was ≥96% (STRENGTH, OMEMI) [
      • Alfaddagh A
      • Elajami TK
      • Ashfaque H
      • Saleh M
      • Bistrian BR
      • Welty FK.
      Effect of eicosapentaenoic and docosahexaenoic acids added to statin therapy on coronary artery plaque in patients with coronary artery disease: a randomized clinical trial.
      ,
      • Group ASC
      • Bowman L
      • Mafham M
      • Wallendszus K
      • Stevens W
      • Buck G
      • et al.
      Effects of n-3 fatty acid supplements in diabetes mellitus.
      ,
      • Manson JE
      • Cook NR
      • Lee IM
      • Christen W
      • Bassuk SS
      • Mora S
      • et al.
      Marine n-3 fatty acids and prevention of cardiovascular disease and cancer.
      ,
      • Kalstad AA
      • Myhre PL
      • Laake K
      • Tveit SH
      • Schmidt EB
      • Smith P
      • et al.
      Effects of n-3 fatty acid supplements in elderly patients after myocardial infarction: a randomized, controlled trial.
      ,
      • Aung T
      • Halsey J
      • Kromhout D
      • Gerstein HC
      • Marchioli R
      • Tavazzi L
      • et al.
      Associations of omega-3 fatty acid supplement use with cardiovascular disease risks: meta-analysis of 10 trials involving 77 917 individuals.
      ,
      • Mason RP
      • Eckel RH.
      Is there a role for omega-3 fatty acids in cardiovascular disease risk reduction?.
      ]. In the Long-Term Outcomes Study to Assess Statin Residual Risk with Epanova in High Cardiovascular Risk Patients with Hypertriglyceridemia (STRENGTH) trial, mixed EPA/DHA carboxylic acids (4 g/d) treatment did not reduce primary CV outcomes (5-point major adverse cardiac events) compared with placebo despite effective TG lowering (19%) [
      • Nicholls SJ
      • Lincoff AM
      • Garcia M
      • Bash D
      • Ballantyne CM
      • Barter PJ
      • et al.
      Effect of High-Dose Omega-3 Fatty Acids vs Corn Oil on Major Adverse Cardiovascular Events in Patients at High Cardiovascular Risk: The STRENGTH Randomized Clinical Trial.
      ]. This result is consistent with those obtained with other TG-lowering agents, such as fibrates and niacin, in statin-treated patients [
      • Elam MB
      • Ginsberg HN
      • Lovato LC
      • Corson M
      • Largay J
      • Leiter LA
      • et al.
      Association of Fenofibrate Therapy With Long-term Cardiovascular Risk in Statin-Treated Patients With Type 2 Diabetes.
      ,
      Effects of combination lipid therapy in type 2 diabetes mellitus.
      ,
      Effects of extended-release niacin with laropiprant in high-risk patients.
      ,
      Niacin in patients with low HDL cholesterol levels receiving intensive statin therapy.
      ]. While some have raised concerns over the possible negative effects of the placebo (pharmaceutical grade mineral oil) used in REDUCE-IT compared with STRENGTH (corn oil), subsequent analyses showed no significant difference in plaque progression between mineral oil and typical cellulose-based placebos [
      • Lakshmanan S
      • Shekar C
      • Kinninger A
      • Dahal S
      • Onuegbu A
      • Cai AN
      • et al.
      Comparison of mineral oil and non-mineral oil placebo on coronary plaque progression by coronary computed tomography angiography.
      ] while any rise in LDL-C or hs-CRP in the placebo group could not explain the reduction in CV events in the IPE arm [
      • Olshansky B
      • Chung MK
      • Budoff MJ
      • Philip S
      • Jiao L
      • Doyle J
      • Ralph T
      • et al.
      Mineral oil: safety and use as placebo in REDUCE-IT and other clinical studies.
      ]. Additionally, the randomized JELIS trial, which showed a 19% relative risk reduction in the primary endpoint of any major coronary event with 1.8 grams of IPE daily versus no IPE, did not use any placebo treatment [
      • Yokoyama M
      • Origasa H
      • Matsuzaki M
      • Matsuzawa Y
      • Saito Y
      • Ishikawa Y
      • et al.
      Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis.
      ]. The consistent results between JELIS and REDUCE-IT makes any confounding role that placebo treatment may have played in the results of REDUCE-IT highly unlikely.
      The Pemafibrate to Reduce Cardiovascular Outcomes by Reducing Triglycerides in Patients with Diabetes (PROMINENT) trial investigated the effects of the selective peroxisome proliferator-activated receptor alpha modulator (SPPARM-α) pemafibrate on major CV events in statin-treated patients with type 2 diabetes and mild to moderate hypertriglyceridemia (200-499 mg/dL) and low HDL-C (≤40 mg/dL) [
      • Pradhan AD
      • Paynter NP
      • Everett BM
      • Glynn RJ
      • Amarenco P
      • Elam M
      • et al.
      Rationale and design of the Pemafibrate to Reduce Cardiovascular Outcomes by Reducing Triglycerides in Patients with Diabetes (PROMINENT) study.
      ]. Pemafibrate differs in its affinity and specificity for PPAR-α, with a more than 2,500-fold increase in potency compared with fenofibrate. With phase II trial data showing median reductions of TG-levels between 40-50% compared with placebo, PROMINENT was designed to convey this robust effect to a patient population in which fibrate derivatives had previously shown clinical benefits within prespecified subgroups defined with elevated TGs and low HDL-C (see: ACCORD and FIRST) [
      Effects of combination lipid therapy in type 2 diabetes mellitus.
      ,
      • Davidson MH
      • Rosenson RS
      • Maki KC
      • Nicholls SJ
      • Ballantyne CM
      • Mazzone T
      • et al.
      Effects of fenofibric acid on carotid intima-media thickness in patients with mixed dyslipidemia on atorvastatin therapy: randomized, placebo-controlled study (FIRST).
      ]. However, this trial was recently halted prematurely citing the unlikelihood of meeting its primary endpoint [
      Kowa Research Institute I. Kowa to discontine K-877 (pemafibrate) "PROMINENT" cardiovascular outcomes study.
      ]. This result, along with the lack of TG-dependent benefit in other failed trials targeting TG-lowering in statin treated patients, casts doubt on the causative role TGs play in CV disease. Finally, dietary supplements of n3-FAs have not produced clinical benefits in patients with CV risk. While these products are widely consumed due to perceived CV benefits, they often contain oxidized fatty acids and other unfavorable components, including saturated fat, due to lack of regulatory standards and manufacturing oversight [
      • Sherratt SCR
      • Lero M
      • Mason RP.
      Are dietary fish oil supplements appropriate for dyslipidemia management? A review of the evidence.
      ,
      • Albert BB
      • Derraik JG
      • Cameron-Smith D
      • Hofman PL
      • Tumanov S
      • Villas-Boas SG
      • et al.
      Fish oil supplements in New Zealand are highly oxidised and do not meet label content of n-3.
      ,
      • Mason RP
      • Sherratt SCR.
      Omega-3 fatty acid fish oil dietary supplements contain saturated fats and oxidized lipids that may interfere with their intended biological benefits.
      ]. Taken together, a review of CVOTs indicate that n3-FA-mediated clinical event reduction depends critically on the formulation and dose of EPA rather than TG lowering efficacy [
      • Bhatt DL
      • Budoff MJ
      • Mason RP.
      A Revolution in Omega-3 Fatty Acid Research∗.
      ].
      Imaging studies performed in high-risk patients with coronary atherosclerosis provide mechanistic insights into the clinical benefits of IPE treatment [
      • Budoff MJ
      • Bhatt DL
      • Kinninger A
      • Lakshmanan S
      • Muhlestein JB
      • Le VT
      • et al.
      Effect of icosapent ethyl on progression of coronary atherosclerosis in patients with elevated triglycerides on statin therapy: final results of the EVAPORATE trial.
      ,
      • Yokoyama M
      • Origasa H
      • Matsuzaki M
      • Matsuzawa Y
      • Saito Y
      • Ishikawa Y
      • et al.
      Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis.
      ,
      • Watanabe T
      • Ando K
      • Daidoji H
      • Otaki Y
      • Sugawara S
      • Matsui M
      • et al.
      A randomized controlled trial of eicosapentaenoic acid in patients with coronary heart disease on statins.
      ,
      • Nishio R
      • Shinke T
      • Otake H
      • Nakagawa M
      • Nagoshi R
      • Inoue T
      • et al.
      Stabilizing effect of combined eicosapentaenoic acid and statin therapy on coronary thin-cap fibroatheroma.
      ,
      • Kita Y
      • Watanabe M
      • Kamon D
      • Ueda T
      • Soeda T
      • Okayama S
      • et al.
      Effects of Fatty Acid Therapy in Addition to Strong Statin on Coronary Plaques in Acute Coronary Syndrome: An Optical Coherence Tomography Study.
      ,
      • Motoyama S
      • Nagahara Y
      • Sarai M
      • Kawai H
      • Miyajima K
      • Sato Y
      • et al.
      Effect of Omega-3 Fatty Acids on Coronary Plaque Morphology - A Serial Computed Tomography Angiography Study.
      ]. In the Combination Therapy of Eicosapentaenoic Acid and Pitavastatin for Coronary Plaque Regression Evaluated by Integrated Backscatter Intravascular Ultrasonography (CHERRY) trial, changes in coronary thin-cap fibroatheroma were evaluated using integrated backscatter intravascular ultrasound (IB-IVUS) in patients treated with both EPA ethyl esters (IPE) and pitavastatin versus pitavastatin alone [
      • Watanabe T
      • Ando K
      • Daidoji H
      • Otaki Y
      • Sugawara S
      • Matsui M
      • et al.
      A randomized controlled trial of eicosapentaenoic acid in patients with coronary heart disease on statins.
      ]. The EPA/statin treatment group demonstrated a significant decrease in total atheroma volume. This change in lipid volume correlated with an increase in the ratio of EPA to arachidonic acid (AA), suggesting that EPA facilitates coronary plaque stabilization through anti-inflammatory pathways that counter the specifically pro-inflammatory effects of AA metabolism [
      • Nishio R
      • Shinke T
      • Otake H
      • Nakagawa M
      • Nagoshi R
      • Inoue T
      • et al.
      Stabilizing effect of combined eicosapentaenoic acid and statin therapy on coronary thin-cap fibroatheroma.
      }. Animal studies suggest that the benefits of EPA may arise in part from its preferential plaque incorporation and anti-inflammatory effects on dendritic cells and circulating T-lymphocytes [
      • Nakajima K
      • Yamashita T
      • Kita T
      • Takeda M
      • Sasaki N
      • Kasahara K
      • et al.
      Orally Administered Eicosapentaenoic Acid Induces Rapid Regression of Atherosclerosis Via Modulating the Phenotype of Dendritic Cells in LDL Receptor-Deficient Mice.
      ,
      • Sato T
      • Horikawa M
      • Takei S
      • Yamazaki F
      • Ito TK
      • Kondo T
      • et al.
      Preferential incorporation of administered eicosapentaenoic acid into thin-cap atherosclerotic plaques.
      ].
      Computed tomographic angiography also assessed the effects of IPE intervention on plaque regression in statin-treated patients with coronary disease and dyslipidemia in the randomized, double-blind, placebo-controlled Effect of Vascepa on Improving Coronary Atherosclerosis in People With High Triglycerides Taking Statin Therapy (EVAPORATE) trial [
      • Budoff MJ
      • Bhatt DL
      • Kinninger A
      • Lakshmanan S
      • Muhlestein JB
      • Le VT
      • et al.
      Effect of icosapent ethyl on progression of coronary atherosclerosis in patients with elevated triglycerides on statin therapy: final results of the EVAPORATE trial.
      ]. Treatment with IPE (4 g/day) for 18 months reduced low attenuation plaque (LAP) volume by 17% compared with baseline while the placebo (statin-only) group demonstrated an increase in LAP over the same study period (p = 0.006). Changes in lipid-rich, LAP volume is a predictor of fatal versus nonfatal myocardial infarction (MI); patients with a LAP burden of more than 4% are five-fold more likely to suffer MI [
      • Williams MC
      • Kwiecinski J
      • Doris M
      • McElhinney P
      • D'Souza MS
      • Cadet S
      • et al.
      Low-Attenuation Noncalcified Plaque on Coronary Computed Tomography Angiography Predicts Myocardial Infarction.
      ]. Reduced plaque progression with intervention in EVAPORATE was consistent in several measurements of plaque volume including total plaque, which regressed compared with the placebo group (−9% vs. 11%, respectively; p = 0.002). These favorable changes with IPE treatment in vulnerable plaque could not be accounted for by TG or other lipid changes and were maintained after multivariable adjustment for CV risk factors [
      • Budoff MJ
      • Bhatt DL
      • Kinninger A
      • Lakshmanan S
      • Muhlestein JB
      • Le VT
      • et al.
      Effect of icosapent ethyl on progression of coronary atherosclerosis in patients with elevated triglycerides on statin therapy: final results of the EVAPORATE trial.
      ].
      The mechanism for these effects of EPA on plaque progression and stability may relate to its ability to incorporate into the arterial wall where it may modulate inflammation. In atherosclerosis-prone animals, EPA and DHA, along with their various metabolites, associate with lesions with vulnerable characteristics in different ways. In apolipoprotein E-deficient mice fed a Western diet supplemented with either EPA or DHA, mass spectrometry and histological approaches evaluated arterial plaque distribution of these n3-FAs and their metabolites [
      • Sato T
      • Horikawa M
      • Takei S
      • Yamazaki F
      • Ito TK
      • Kondo T
      • et al.
      Preferential incorporation of administered eicosapentaenoic acid into thin-cap atherosclerotic plaques.
      ]. EPA and a common metabolite, 12-hydroxy-eicosapentaenoic acid (12-HEPE), associated preferentially with thin-cap plaques with an increase in M2 macrophages that exhibit an anti-inflammatory slant. By contrast, DHA distributed more evenly, appearing in thin-cap as well as thick-cap plaques. In the aortic arch, EPA followed a concentration gradient from the endothelium to the media. Additional insights into the mechanism(s) of action for EPA come from a short-term (median 21 days) intervention study using an EPA/DHA combination regimen in patients scheduled to undergo carotid endarterectomy [
      • Cawood AL
      • Ding R
      • Napper FL
      • Young RH
      • Williams JA
      • Ward MJ
      • et al.
      Eicosapentaenoic acid (EPA) from highly concentrated n-3 fatty acid ethyl esters is incorporated into advanced atherosclerotic plaques and higher plaque EPA is associated with decreased plaque inflammation and increased stability.
      ]. Post-surgical analysis of plaque tissue demonstrated elevated and preferential incorporation of EPA. Indeed, the EPA content of plaque phospholipids correlated inversely and significantly with characteristics of plaque instability, plaque inflammation, and T-cell number. Thus, EPA appears to incorporate preferentially into atherosclerotic lesions where it increases plaque stability while reducing foam cell and T-cell content and associated tissue inflammation.

      2. Model membrane interactions of EPA and DHA

      Following dietary intake, n3-FAs are repackaged and transported in chylomicrons from the gut to the blood in the lymphatic system through specialized vessels (Fig. 1). n3-FAs are then processed in the liver where they are packaged as constituents of TG-rich lipoproteins in esterified form or, to lesser extent, bound to albumin as free fatty acids [
      • Sawazaki S
      • Hamazaki T
      • Yamazaki K
      • Taki H
      • Kaneda M
      • Yano S
      • et al.
      Comparison of the increment in plasma eicosapentaenoate concentrations by fish oil intake between young and middle-aged volunteers.
      ]. EPA and DHA are both incorporated into TGs, cholesteryl esters and plasma phospholipids, including phosphatidylcholine and phosphatidylethanolamine [
      • Subbaiah PV
      • Kaufman D
      • Bagdade JD.
      Incorporation of dietary n-3 fatty acids into molecular species of phosphatidyl choline and cholesteryl ester in normal human plasma.
      ]. Released from these lipoproteins by lipases at the cell surface in various tissues, the n3-FAs enter the smooth endoplasmic reticulum where they are esterified at the sn-2 position of phospholipids. When the n3-FA-modified phospholipids enter the plasma membrane, they influence lipid structure and fluid dynamics (Fig. 2) while also serving as a reservoir for intracellular lipid metabolites following release by phospholipase A2 (PLA2). These membrane interactions have been characterized using various biophysical methodologies, including x-ray diffraction, micropipette aspiration, nuclear magnetic resonance (NMR) spectroscopy, and fluorescence polarization analyses.
      Fig 1
      Fig. 1Icosapent ethyl (IPE) distributes broadly following oral intake. (1) Following ingestion, the ethyl ester of IPE is cleaved by lipases in the intestinal lumen, thus forming free fatty acid EPA. This is absorbed by enterocytes lining the intestinal wall, wherein EPA is esterified into triglycerides and repackaged into chylomicrons. The chylomicrons then enter the lymphatic vessels before entering the circulation. (2) In the liver, the esterified EPA is packaged into very low dense lipoproteins (VLDL) and transported in the circulation. (3) In the aterial wall, glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1 (GPIHBP1) expressed on the endothelial cell surface recruits lipoprotein lipase (LPL). The LPL faciliates the release of EPA free fatty acid which is absorbed by the target cell and used in phospholipid biosynthesis.
      Fig 2
      Fig. 2EPA and DHA have distinct effects of membrane phospholipid dynamics and cholesterol distribution. EPA and DHA are esterified to membrane phospholipids and are known to often concentrate in different tissues with DHA enriched in the cell membranes of the retina and brain compared to EPA. Due to their distinct biophysical properties, EPA and DHA differentially effect membrane structure and dynamics. EPA has a stable, extended conformation within the membrane while DHA disorders surrounding lipid due to rapid isomerizations, resulting in increased fluidity and exclusion of cholesterol into distinct cholesterol domains.
      The interactions of n3-FAs with membranes containing physiologic levels of cholesterol and phospholipids, including phosphatidylcholine esterified to EPA (PL-EPA), DHA (PL-DHA) or arachidonic acid (PL-AA), were recently examined using small angle x-ray diffraction approaches [
      • Sherratt SCR
      • Juliano RA
      • Copland C
      • Bhatt DL
      • Libby P
      • Mason RP
      Eicosapentaenoic acid and docosahexaenoic acid containing phospholipids have contrasting effects on membrane structure.
      ]. The results showed that PL-EPA increased membrane hydrocarbon core electron density over a broad area ± 0-10 Å from the center, consistent with an extended orientation and stabilizing effects on surrounding phospholipid acyl chains. By contrast, PL-DHA interacts with the phospholipid head group region while producing molecular disorder in the hydrocarbon core by disrupting van der Waals interactions. PL-EPA and PL-DHA were also tested together at equimolar levels and observed to have little effect on membrane structure compared with their separate effects. This finding suggested that opposite effects of PL-EPA and PL-DHA were diminished or “neutralized” when combined. The contrasting effects of PL-EPA, PL-DHA, and their combination on membrane structure may contribute to a better explanation of their disparate effects in recent CVOTs and experimental models [
      • Mason RP
      • Sherratt SCR
      • Eckel RH.
      Rationale for different formulations of omega-3 fatty acids leading to differences in residual cardiovascular risk reduction.
      ].
      These differential n3-FA membrane interactions were independently confirmed in a series of micropipette aspiration studies [
      • Jacobs ML
      • Faizi HA
      • Peruzzi JA
      • Vlahovska PM
      • Kamat NP.
      EPA and DHA differentially modulate membrane elasticity in the presence of cholesterol.
      ]. Using this technique, Jacobs et al. determined the effects of EPA and DHA on the apparent membrane area expansion modulus (Kapp), which measures the ability of a membrane to stretch in response to applied force. In vesicles composed of cholesterol at levels approximating those seen in plasma membranes, DHA decreased the Kapp and promoted cholesterol segregation into discrete regions. DHA-induced cholesterol separation reduced the bulk lipid stabilizing effects of its rigid sterol ring structure. The loss of membrane stability due to uneven distribution of cholesterol resulted in increased lateral movement in the phospholipid acyl chains. By contrast, EPA maintained the normal distribution of cholesterol. These contrasting membrane interactions of EPA and DHA in the presence of cholesterol agree with the results of x-ray diffraction studies [
      • Sherratt SCR
      • Juliano RA
      • Copland C
      • Bhatt DL
      • Libby P
      • Mason RP
      Eicosapentaenoic acid and docosahexaenoic acid containing phospholipids have contrasting effects on membrane structure.
      ,
      • Sherratt SCR
      • Mason RP.
      Eicosapentaenoic acid and docosahexaenoic acid have distinct membrane locations and lipid interactions as determined by X-ray diffraction.
      ,
      • Mason RP
      • Jacob RF
      • Shrivastava S
      • Sherratt SC
      • Chattopadhyay A.
      Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes.
      ].
      Solid-state 2H NMR spectroscopy has shown that PL-DHA concentrates in disordered lipid rafts that are rich in sphingolipids and lower cholesterol content compared with more ordered, cholesterol-enriched microdomains [
      • Soni SP
      • LoCascio DS
      • Liu Y
      • Williams JA
      • Bittman R
      • Stillwell W
      • et al.
      Docosahexaenoic acid enhances segregation of lipids between: 2H-NMR study.
      ]. This distinct effect of DHA is attributed to its ability to “repel” cholesterol due to its disordering effects caused by rapid trans-gauche isomerizations. Such physicochemical actions of DHA may be essential for normal raft formation in the plasma membrane of various tissues where it is concentrated. In particular, DHA makes up 50-60% of total membrane fatty acids in retinal rod photoreceptors where its “fluidizing” actions facilitate rapid conformational changes of rhodopsin and overall folding of the plasma membrane [
      • Querques G
      • Forte R
      • Souied EH.
      Retina and omega-3.
      ]. DHA is also the most abundant PUFA in neuronal membranes where it modifies lipid rafts and fluidity in domains rich in sphingolipids and cholesterol [
      • Dyall SC.
      Long-chain omega-3 fatty acids and the brain: a review of the independent and shared effects of EPA, DPA and DHA.
      ,
      • Innis SM.
      Dietary (n-3) fatty acids and brain development.
      ,
      • Uauy R
      • Dangour AD.
      Nutrition in brain development and aging: role of essential fatty acids.
      ]. In the myocardium, n3-FAs influence ion channel activity in a manner dependent on lipid composition and fluid dynamics [
      • Chang HM
      • Reitstetter R
      • Mason RP
      • Gruener R.
      Attenuation of channel kinetics and conductance by cholesterol: an interpretation using structural stress as a unifying concept.
      ,
      • Heijman J
      • Voigt N
      • Nattel S
      • Dobrev D.
      Cellular and molecular electrophysiology of atrial fibrillation initiation, maintenance, and progression.
      ,
      • Maixent JM
      • Duran MJ
      • Pierre S
      • Sennoune S
      • Robert K
      • Bernard M
      • et al.
      Remodeling of Na,K-ATPase, and membrane fluidity after atrial fibrillation in sheep.
      ]. These effects on ion transport function may contribute to the various effects of n3-FAs on atrial and ventricular fibrillation as reported in clinical studies [

      Bhatt D, L., Steg PG, Miller M, Brinton EA, Jacobson TA, Ketchum SB, et al. Cardiovascular risk reduction with Icosapent Ethyl for hypertriglyceridemia. New England Journal of Medicine. 2019;380:11-22.

      ,
      • Nicholls SJ
      • Lincoff AM
      • Garcia M
      • Bash D
      • Ballantyne CM
      • Barter PJ
      • et al.
      Effect of High-Dose Omega-3 Fatty Acids vs Corn Oil on Major Adverse Cardiovascular Events in Patients at High Cardiovascular Risk: The STRENGTH Randomized Clinical Trial.
      ,
      • Doi M
      • Nosaka K
      • Miyoshi T
      • Iwamoto M
      • Kajiya M
      • Okawa K
      • et al.
      Early eicosapentaenoic acid treatment after percutaneous coronary intervention reduces acute inflammatory responses and ventricular arrhythmias in patients with acute myocardial infarction: a randomized, controlled study.
      ,
      • Myhre P
      • Smith EB
      • Kalstad AA
      • Tveit S
      • Laake K
      • Smith P
      • et al.
      Changes in EPA and DHA during supplementation with omega-3 fatty acids and incident cardiovascular events: secondary analysis from the OMEMI trial.
      ].
      Fluorescence anisotropy techniques have also been used to measure differences between EPA and DHA on membrane fluidity [
      • Mason RP
      • Jacob RF
      • Shrivastava S
      • Sherratt SC
      • Chattopadhyay A.
      Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes.
      ]. Changes in membrane fluidity were ascertained by monitoring the apparent rotational correlation time (ARCT) of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), which intercalates into the membrane where its rotational properties are measured as a function of treatment. In membrane prepared at 50 mol% cholesterol, EPA was found to have no significant effect on ARCT over a broad range of concentrations (1–10 mol%); however, DHA significantly reduced ARCT from 19.35 ns (control) to 15.56 ns (10 mol%) in a dose-dependent manner, consistent with a significant increase in membrane fluidity [
      • Sato T
      • Horikawa M
      • Takei S
      • Yamazaki F
      • Ito TK
      • Kondo T
      • et al.
      Preferential incorporation of administered eicosapentaenoic acid into thin-cap atherosclerotic plaques.
      ]. Collectively, these various biophysical approaches indicate that EPA and DHA have differential effects on cholesterol organization, lipid dynamics, and phospholipid structure that may contribute to tissue specific effects on raft formation, protein function, and signal transduction pathways.

      3. Effects of n3-FAs on membrane oxidative stress and cholesterol crystalline domain formation under hyperglycemic conditions

      Oxidative modification of membrane lipids and lipoproteins contributes to vascular injury and inflammation during atherosclerosis [
      • Witztum JL.
      The oxidation hypothesis of atherosclerosis.
      ,
      • Chisolm GM
      • Steinberg D.
      The oxidative modification hypothesis of atherogenesis: an overview.
      ]. Circulating levels of oxidized LDL correlate with acute coronary syndromes and increased risk for ischemic events and metabolic disease [
      • Ehara S
      • Ueda M
      • Naruko T
      • Haze K
      • Itoh A
      • Otsuka M
      • et al.
      Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes.
      ,
      • Walter MF
      • Jacob RF
      • Bjork RE
      • Jeffers B
      • Buch J
      • Mizuno Y
      • et al.
      Circulating lipid hydroperoxides predict cardiovascular events in patients with stable coronary artery disease: the PREVENT study.
      ,
      • Walter MF
      • Jacob RF
      • Jeffers B
      • Ghadanfar MM
      • Preston GM
      • Buch J
      • et al.
      Serum levels of TBARS predict cardiovascular events in patients with stable coronary artery disease: A longitudinal analysis of the PREVENT study.
      ,
      • Holvoet P
      • Kritchevsky SB
      • Tracy RP
      • Mertens A
      • Rubin SM
      • Butler J
      • et al.
      The metabolic syndrome, circulating oxidized LDL, and risk of myocardial infarction in well-functioning elderly people in the health, aging, and body composition cohort.
      ]. Oxidative damage of unsaturated phospholipid acyl segments promotes the disruption of membrane structural integrity, including changes in bilayer width and the formation of cholesterol crystalline domains [
      • Mason RP
      • Walter MF
      • Mason PE.
      Effect of oxidative stress on membrane structure: Small angle x-ray diffraction analysis.
      ,
      • Mason RP
      • Walter MF
      • Day CA
      • Jacob RF.
      Active metabolite of atorvastatin inhibits membrane cholesterol domain formation by an antioxidant mechanism.
      ,
      • Self-Medlin Y
      • Byun J
      • Jacob RF
      • Mizuno Y
      • Mason RP.
      Glucose promotes membrane cholesterol crystalline domain formation by lipid peroxidation.
      ,
      • Wratten ML
      • van-Ginkel G
      • van't Veld AA
      • Bekker A
      • van Faassen EE
      • Sevanian A.
      Structural and dynamic effects of oxidatively modified phospholipids in unsaturated lipid membranes.
      ]. n3-FAs scavenge reactive oxygen species (ROS) by “trapping” and stabilizing unpaired, free radicals via the conjugated double-bonds along the acyl chain—a process known as resonance stabilization. These double bonds are optimally situated in the membrane bilayer to absorb or trap lipid-centered free radicals and interrupt their propagation. EPA in vitro shows potent, dose-dependent antioxidant properties in lipid membrane bilayers and various apolipoprotein B-containing particles (LDL, VLDL, small dense LDL). These effects were not reproduced by other pharmacologic TG-lowering agents or long chain fatty acids, such as DHA, under identical conditions [
      • Mason RP
      • Jacob RF
      • Shrivastava S
      • Sherratt SC
      • Chattopadhyay A.
      Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes.
      ,
      • Sherratt SCR
      • Juliano RA
      • Mason RP.
      Eicosapentaenoic acid (EPA) has optimal chain length and degree of unsaturation to inhibit oxidation of small dense LDL and membrane cholesterol domains as compared to related fatty acids in vitro.
      ,
      • Mason RP
      • Sherratt SC
      • Jacob RF.
      Eicosapentaenoic acid inhibits oxidation of ApoB-containing lipoprotein particles of different size in vitro when administered alone or in combination with atorvastatin active metabolite compared with other triglyceride-lowering agents.
      ,
      • Mason RP
      • Jacob RF.
      Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism.
      ] and are facilitated by the stable and extended orientation of EPA across the membrane bilayer as previously described in various biophysical studies. We hypothesize that the attenuated scavenging capacity of DHA over time results from rapid changes in orientation and disruption in the phospholipid acyl chain region of the membrane bilayer [
      • Sherratt SCR
      • Juliano RA
      • Copland C
      • Bhatt DL
      • Libby P
      • Mason RP
      Eicosapentaenoic acid and docosahexaenoic acid containing phospholipids have contrasting effects on membrane structure.
      ,
      • Sherratt SCR
      • Juliano RA
      • Mason RP.
      Eicosapentaenoic acid (EPA) has optimal chain length and degree of unsaturation to inhibit oxidation of small dense LDL and membrane cholesterol domains as compared to related fatty acids in vitro.
      ,
      • Shaikh SR.
      Biophysical and biochemical mechanisms by which dietary N-3 polyunsaturated fatty acids from fish oil disrupt membrane lipid rafts.
      ,
      • Williams JA
      • Batten SE
      • Harris M
      • Rockett BD
      • Shaikh SR
      • Stillwell W
      • et al.
      Docosahexaenoic and eicosapentaenoic acids segregate differently between raft and nonraft domains.
      ,
      • Shaikh SR
      • Kinnun JJ
      • Leng X
      • Williams JA
      • Wassall SR.
      How polyunsaturated fatty acids modify molecular organization in membranes: insight from NMR studies of model systems.
      ]. Interestingly, the antioxidant actions of EPA are enhanced when combined with the lipophilic, active metabolite form of atorvastatin [
      • Mason RP
      • Sherratt SC
      • Jacob RF.
      Eicosapentaenoic acid inhibits oxidation of ApoB-containing lipoprotein particles of different size in vitro when administered alone or in combination with atorvastatin active metabolite compared with other triglyceride-lowering agents.
      ]. These agents share a preferential membrane location that facilitates additional resonance stabilization activity in a potentially synergistic manner [
      • Mason RP
      • Walter MF
      • Day CA
      • Jacob RF.
      Active metabolite of atorvastatin inhibits membrane cholesterol domain formation by an antioxidant mechanism.
      ,
      • Jacob RF
      • Walter MF
      • Self-Medlin Y
      • Mason RP.
      Atorvastatin active metabolite inhibits oxidative modification of small dense low-density lipoprotein.
      ,
      • Aviram M
      • Rosenblat M
      • Bisgaier CL
      • Newton RS.
      Atorvastatin and gemfibrozil metabolites, but not the parent drugs, are potent antioxidants against lipoprotein oxidation.
      ].
      The membrane antioxidant benefits of EPA are also observed under conditions of hyperglycemia, which is associated with the increased generation of ROS and carbonyl species [
      • Self-Medlin Y
      • Byun J
      • Jacob RF
      • Mizuno Y
      • Mason RP.
      Glucose promotes membrane cholesterol crystalline domain formation by lipid peroxidation.
      ,
      • Mason RP
      • Jacob RF.
      Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism.
      ]. These actions of glucose are associated with an increase in cell permeability as well as a loss in membrane integrity [
      • Borow KM
      • Nelson JR
      • Mason RP.
      Biologic plausibility, cellular effects, and molecular mechanisms of eicosapentaenoic acid (EPA) in atherosclerosis.
      ,
      • Mason RP
      • Jacob RF.
      Membrane microdomains and vascular biology: emerging role in atherogenesis.
      ]. High glucose levels cause membrane cholesterol to aggregate into discrete domains that precipitate extracellular cholesterol monohydrate crystals, which are crucial components of advanced atherosclerotic lesions [
      • Self-Medlin Y
      • Byun J
      • Jacob RF
      • Mizuno Y
      • Mason RP.
      Glucose promotes membrane cholesterol crystalline domain formation by lipid peroxidation.
      ,
      • Mason RP
      • Jacob RF.
      Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism.
      ]. Formation of cholesterol crystals may also be induced experimentally through enzymatic inhibition of cholesterol esterification [
      • Kellner-Weibel G
      • Yancey PG
      • Jerome WG
      • Walser T
      • Mason RP
      • Phillips MC
      • et al.
      Crystallization of free cholesterol in model macrophage foam cells.
      ]. Microscopic cholesterol crystals have been observed in early diet-induced atherosclerotic lesions and coincide with the emergence of inflammatory cells [
      • Duewell P
      • Kono H
      • Rayner KJ
      • Sirois CM
      • Vladimer G
      • Bauernfeind FG
      • et al.
      NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.
      ]. Cholesterol crystals activate the intracellular NLR family pyrin domain containing 3 (NLRP3) inflammasome, resulting in caspase-1 mediated activation of the proinflammatory cytokines interleukin-1 beta and IL-18 [
      • Duewell P
      • Kono H
      • Rayner KJ
      • Sirois CM
      • Vladimer G
      • Bauernfeind FG
      • et al.
      NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.
      ,
      • Rajamäki K
      • Lappalainen J
      • Oörni K
      • Välimäki E
      • Matikainen S
      • Kovanen PT
      • et al.
      Cholesterol crystals activate the NLRP3 inflammasome in human macrophages: a novel link between cholesterol metabolism and inflammation.
      ]. In mice deficient in the expression of the NLRP3 inflammasome or cathepsin molecules, the inflammatory effects of these crystals are inhibited [
      • Duewell P
      • Kono H
      • Rayner KJ
      • Sirois CM
      • Vladimer G
      • Bauernfeind FG
      • et al.
      NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.
      ]. Membrane cholesterol domains are also stimulated by angiotensin II, leading to loss of eNOS function and increased oxidative stress [
      • Amiya E
      • Watanabe M
      • Takeda N
      • Saito T
      • Shiga T
      • Hosoya Y
      • et al.
      Angiotensin II impairs endothelial nitric-oxide synthase bioavailability under free cholesterol-enriched conditions via intracellular free cholesterol-rich membrane microdomains.
      ]. Over time, these microscopic shards grow into various shapes that can destabilize and puncture the protective fibrous cap of an atherosclerotic lesion [
      • Kellner-Weibel G
      • Yancey PG
      • Jerome WG
      • Walser T
      • Mason RP
      • Phillips MC
      • et al.
      Crystallization of free cholesterol in model macrophage foam cells.
      ,
      • Abela GS
      • Aziz K.
      Cholesterol crystals rupture biological membranes and human plaques during acute cardiovascular events—a novel insight into plaque rupture by scanning electron microscopy.
      ,
      • Phillips JE
      • Geng YJ
      • Mason RP.
      7-Ketocholesterol forms crystalline domains in model membranes and murine aortic smooth muscle cells.
      ,
      • Abela GS
      • Aziz K.
      Cholesterol crystals cause mechanical damage to biological membranes: a proposed mechanism of plaque rupture and erosion leading to arterial thrombosis.
      ]. Histopathological analysis shows cholesterol crystals to be present in areas of necrosis and in regions with high concentrations of immune cells [

      Small DM. George Lyman Duff memorial lecture. Progression and regression of atherosclerotic lesions. Insights from lipid physical biochemistry. Arteriosclerosis: An Official Journal of the American Heart Association, Inc. 1988;8:103-29.

      ].
      High glucose levels promote the development of cholesterol crystalline domains through increased membrane ROS generation. These effects are not observed with other monosaccharides (e.g., mannose) at comparable concentrations [
      • Mason RP
      • Walter MF
      • Day CA
      • Jacob RF.
      Active metabolite of atorvastatin inhibits membrane cholesterol domain formation by an antioxidant mechanism.
      ,
      • Self-Medlin Y
      • Byun J
      • Jacob RF
      • Mizuno Y
      • Mason RP.
      Glucose promotes membrane cholesterol crystalline domain formation by lipid peroxidation.
      ]. Glucose has unique physicochemical properties that make it particularly reactive with singlet oxygen or other ROS to form glucose radicals [
      • Jakus V
      • Rietbrock N.
      Advanced glycation end-products and the progress of diabetic vascular complications.
      ,
      • Pennathur S
      • Heinecke JW.
      Mechanisms for oxidative stress in diabetic cardiovascular disease.
      ]. Due to its radical scavenging activity, EPA inhibits cholesterol crystal formation during hyperglycemia in a manner that directly correlates with reduced lipid peroxidation [
      • Mason RP
      • Jacob RF.
      Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism.
      ]. The antioxidant actions of EPA and associated inhibition of cholesterol domains are superior to less potent lipophilic antioxidants or other long chain fatty acids, including DHA and alpha-linolenic acid (ALA), while docosapentaenoic acid (DPA, 22:5 n-3) had similar cholesterol domain inhibition [
      • Sherratt SCR
      • Juliano RA
      • Mason RP.
      Eicosapentaenoic acid (EPA) has optimal chain length and degree of unsaturation to inhibit oxidation of small dense LDL and membrane cholesterol domains as compared to related fatty acids in vitro.
      ,
      • Mason RP
      • Jacob RF.
      Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism.
      ].
      As glucose metabolism is central to various metabolic syndromes, including diabetes and obesity, therapeutic interventions directed toward improved insulin sensitivity are needed in conjunction with lifestyle modifications to reduce CV risk. Preclinical animal studies have shown that when normal and obese mice were fed a high fat diet enriched with EPA, glucose tolerance and insulin sensitivity were improved in a manner not replicated in mice fed the same diet enriched with DHA in place of EPA [86]. Additionally, mice fed a high fat diet experienced significantly improved glucose tolerance, reduced weight, and reduced fasting blood glucose following treatment with IPE but not an EPA/DHA mixed formulation [
      • Al Rijjal D
      • Liu Y
      • Lai M
      • Song Y
      • Danaei Z
      • Wu A
      • et al.
      Vascepa protects against high-fat diet-induced glucose intolerance.
      ]. This may be due, in part, to the actions of resolvin E1 and its receptor ERV1/ChemR23, as another study showed that treatment with resolvin E1 improved fasting glucose levels compared with control treatment in obese mice, and knockdown of this receptor ablated this benefit [
      • Pal A
      • Al-Shaer AE
      • Guesdon W
      • Torres MJ
      • Armstrong M
      • Quinn K
      • et al.
      Resolvin E1 derived from eicosapentaenoic acid prevents hyperinsulinemia and hyperglycemia in a host genetic manner.
      ]. How these mechanisms translate to clinical benefits remains unclear but there are emerging data from REDUCE-IT related to this question. In REDUCE-IT, approximately 59% of patients who underwent randomization had diabetes and 57% had a body mass index ≥30 [

      Bhatt D, L., Steg PG, Miller M, Brinton EA, Jacobson TA, Ketchum SB, et al. Cardiovascular risk reduction with Icosapent Ethyl for hypertriglyceridemia. New England Journal of Medicine. 2019;380:11-22.

      ]. Recently, analysis of primary outcome prevention across waist circumference tertiles from REDUCE-IT found significant reductions in events with IPE that were independent of abdominal obesity [
      • Bhatt DL
      • Brinton EA
      • Miller M
      • Steg PG
      • Jacobson TA
      • Ketchum S
      • et al.
      Icosapent ethyl reduces cardiovascular risk substantially and consistently regardless of waist circumference.
      ]. These findings support metabolic benefits with IPE treatment in patients having diabetes or obesity.
      Cholesterol is an essential component of mammalian cell membranes where it influences membrane structure, phospholipid mobility, and intrinsic protein function. Due to its rigid sterol nucleus, cholesterol reduces phospholipid acyl chain trans-gauche isomerization and increased membrane width in a concentration-dependent manner [
      • Tulenko TN
      • Chen M
      • Mason PE
      • Mason RP.
      Physical effects of cholesterol on arterial smooth muscle membranes: Evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis.
      ]. These structural changes reversibly influence the kinetics of transmembrane ion channels and signal transduction activity as observed in experimental hypercholesterolemia [
      • Paragh G
      • Kovács É
      • Seres I
      • Keresztes T
      • Balogh Z
      • Szabó J
      • et al.
      Altered signal pathway in granulocytes from patients with hypercholesterolemia.
      ,
      • Fang Y
      • Mohler ER
      • Hsieh E
      • Osman H
      • Hashemi SM
      • Davies PF
      • et al.
      Hypercholesterolemia Suppresses Inwardly Rectifying K+Channels in Aortic Endothelium In Vitro and In Vivo.
      ]. In membrane-isolated ion channels, the addition of cholesterol favors closed channel confirmation by increasing stress energy in the bilayer and reducing channel activation energy by up to 50% [
      • Chang HM
      • Reitstetter R
      • Mason RP
      • Gruener R.
      Attenuation of channel kinetics and conductance by cholesterol: an interpretation using structural stress as a unifying concept.
      ]. Cholesterol-induced changes in membrane structure and function are also influenced by polyunsaturated fatty acid (PUFA) content [
      • Sherratt SCR
      • Juliano RA
      • Copland C
      • Bhatt DL
      • Libby P
      • Mason RP
      Eicosapentaenoic acid and docosahexaenoic acid containing phospholipids have contrasting effects on membrane structure.
      ,
      • Brzustowicz MR
      • Cherezov V
      • Caffrey M
      • Stillwell W
      • Wassall SR.
      Molecular organization of cholesterol in polyunsaturated membranes: microdomain formation.
      ,
      • Kucerka N
      • Marquardt D
      • Harroun TA
      • Nieh MP
      • Wassall SR
      • de Jong DH
      • et al.
      Cholesterol in bilayers with PUFA chains: doping with DMPC or POPC results in sterol reorientation and membrane-domain formation.
      ,
      • Kucerka N
      • Marquardt D
      • Harroun TA
      • Nieh MP
      • Wassall SR
      • Katsaras J.
      The functional significance of lipid diversity: orientation of cholesterol in bilayers is determined by lipid species.
      ]. Specific changes in membrane width varies based on phospholipid acyl chain length and degree of saturation [
      • Sherratt SCR
      • Juliano RA
      • Copland C
      • Bhatt DL
      • Libby P
      • Mason RP
      Eicosapentaenoic acid and docosahexaenoic acid containing phospholipids have contrasting effects on membrane structure.
      ,
      • Tulenko TN
      • Chen M
      • Mason PE
      • Mason RP.
      Physical effects of cholesterol on arterial smooth muscle membranes: Evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis.
      ,
      • Ruocco MJ
      • Shipley GG.
      Interaction of cholesterol with galactocerebroside and galactocerebroside-phosphatidylcholine bilayer membranes.
      ,
      • Mason RP
      • Moisey DM
      • Shajenko L.
      Cholesterol alters the binding of Ca2+ channel blockers to the membrane lipid bilayer.
      ]. Under hypercholesterolemic conditions, the accumulation of excess free cholesterol in membranes of vascular smooth muscle cells and macrophages leads to the formation of cholesterol crystalline domains as also shown in experimental atherosclerosis [
      • Mason RP
      • Walter MF
      • Day CA
      • Jacob RF.
      Active metabolite of atorvastatin inhibits membrane cholesterol domain formation by an antioxidant mechanism.
      ,
      • Mason RP
      • Jacob RF.
      Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism.
      ,
      • Tulenko TN
      • Chen M
      • Mason PE
      • Mason RP.
      Physical effects of cholesterol on arterial smooth muscle membranes: Evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis.
      ,
      • Ruocco MJ
      • Shipley GG.
      Interaction of cholesterol with galactocerebroside and galactocerebroside-phosphatidylcholine bilayer membranes.
      ]. Multiple lines of evidence support the abundance of such crystals in many atherosclerotic plaques [
      • Stary HC
      • Chandler AB
      • Dinsmore RE
      • Fuster V
      • Glagov S
      • Insull W
      • et al.
      A Definition of Advanced Types of Atherosclerotic Lesions and a Histological Classification of Atherosclerosis.
      ,
      • Falk E.
      Pathogenesis of Atherosclerosis.
      ,
      • Virmani R
      • Kolodgie FD
      • Burke AP
      • Farb A
      • Schwartz SM.
      Lessons From Sudden Coronary Death.
      ].

      4. n3-FA effects on endothelial function

      Increased CV risk and adverse outcomes correlate with loss of endothelial-derived nitric oxide (NO) and NO-mediated vasodilation, independent of traditional risk factors [
      • Vlachopoulos C
      • Aznaouridis K
      • Stefanadis C.
      Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis.
      ]. As a signaling molecule, NO activates soluble guanylyl cyclase, a heterodimeric protein found in arterial smooth muscle cells that produces cyclic guanosine monophosphate (cGMP), which activates downstream signaling molecules leading to vasodilation and reduced inflammatory changes (Fig. 3). Mutations in genes that facilitate vasodilation and inhibit platelet activation during NO stimulation are associated with greater risk for CV disease [
      • Erdmann J
      • Stark K
      • Esslinger UB
      • Rumpf PM
      • Koesling D
      • de Wit C
      • et al.
      Dysfunctional nitric oxide signalling increases risk of myocardial infarction.
      ]. These findings demonstrate an essential link between impaired NO signaling and MI risk. By contrast, genetic predisposition to enhanced NO signaling is associated with reduced risk of coronary disease, stroke, and peripheral artery disease [
      • Emdin CA
      • Khera AV
      • Klarin D
      • Natarajan P
      • Zekavat SM
      • Nomura A
      • et al.
      Phenotypic Consequences of a Genetic Predisposition to Enhanced Nitric Oxide Signaling.
      ]. Conversely, animals genetically predisposed to NO deficiency present with increased insulin resistance, hyperlipidemia and associated vascular changes [
      • Duplain H
      • Burcelin R
      • Sartori C
      • Cook S
      • Egli M
      • Lepori M
      • et al.
      Insulin resistance, hyperlipidemia, and hypertension in mice lacking endothelial nitric oxide synthase.
      ].
      Fig 3
      Fig. 3Endothelial nitric oxide bioavailability depends on eNOS coupling efficiency. In the healthy endothelium, nitric oxide (NO) is produced by the eNOS dimer which couples oxidation of L-arginine with the reduction of molecular oxygen. The essential cofactor tetrahydrobiopterin (BH4) facilitates this redox reaction to generate NO. NO then binds to guanylate cyclase in vascular smooth muscle cells to convert guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) which relaxes smooth muscle cells. Under disease-like conditions that increase oxidative stress (i.e. high glucose, smoking, hypertension, etc.), there is eNOS “uncoupling” that favors production of superoxide (O2−) which reacts with NO to form peroxynitrite (ONOO−), a cytotoxic radical. The ratio of [NO]/[ONOO−] is a key indicator of eNOS coupling efficiency and loss of NO bioavailability is associated with atherothrombosis.
      Treatment with n3-FAs improves endothelial function by enhancing endothelial NO synthase (eNOS) activity and reduced oxidative stress. In human endothelial cells (ECs), DHA can suppress the expression of cytokine-induced proatherogenic and proinflammatory proteins [
      • De Caterina R
      • Cybulsky MI
      • Clinton SK
      • Gimbrone MA
      • Libby P
      The omega-3 fatty acid docosahexaenoate reduces cytokine-induced expression of proatherogenic and proinflammatory proteins in human endothelial cells.
      ,
      • Weber C
      • Erl W
      • Pietsch A
      • Danesch U
      • Weber PC.
      Docosahexaenoic acid selectively attenuates induction of vascular cell adhesion molecule-1 and subsequent monocytic cell adhesion to human endothelial cells stimulated by tumor necrosis factor-alpha.
      ]. Similar effects have been observed with EPA as it also inhibited lipopolysaccharide (LPS)-induced monocyte adhesion and expression of adhesion molecules both in vitro and in vivo [
      • Yamada H
      • Yoshida M
      • Nakano Y
      • Suganami T
      • Satoh N
      • Mita T
      • et al.
      In vivo and in vitro inhibition of monocyte adhesion to endothelial cells and endothelial adhesion molecules by eicosapentaenoic acid.
      ]. Treatment with EPA improves vasomotor control and arterial compliance in patients with CV risk, including those receiving a statin or with diabetes, in a manner that also correlated with plasma EPA/AA ratios [
      • Haiden M
      • Miyasaka Y
      • Kimura Y
      • Tsujimoto S
      • Maeba H
      • Suwa Y
      • et al.
      Effect of eicosapentaenoic acid on regional arterial stiffness: Assessment by tissue Doppler imaging.
      ,
      • Ito R
      • Satoh-Asahara N
      • Yamakage H
      • Sasaki Y
      • Odori S
      • Kono S
      • et al.
      An increase in the EPA/AA ratio is associated with improved arterial stiffness in obese patients with dyslipidemia.
      ,
      • Takaki A
      • Umemoto S
      • Ono K
      • Seki K
      • Ryoke T
      • Fujii A
      • et al.
      Add-on therapy of EPA reduces oxidative stress and inhibits the progression of aortic stiffness in patients with coronary artery disease and statin therapy: a randomized controlled study.
      ,
      • Mita T
      • Watada H
      • Ogihara T
      • Nomiyama T
      • Ogawa O
      • Kinoshita J
      • et al.
      Eicosapentaenoic acid reduces the progression of carotid intima-media thickness in patients with type 2 diabetes.
      ]. The ability of EPA to reduce arterial stiffness did not depend on changes in blood pressure or LDL levels but instead associated with reduced biomarkers of inflammation and oxidative stress. Thus, the vascular benefits of EPA may relate specifically to improvement in endothelial-dependent NO release.
      The membrane and subcellular distribution of proteins in various organelles, including mitochondria, is influenced by exposure to n3-FAs and other long chain fatty acids. Treatment with n3-FAs specifically modifies the acyl chain composition of phospholipids in caveolae and other lipid rafts in ECs [
      • Li Q
      • Zhang Q
      • Wang M
      • Zhao S
      • Ma J
      • Luo N
      • et al.
      Eicosapentaenoic acid modifies lipid composition in caveolae and induces translocation of endothelial nitric oxide synthase.
      ]. Additionally, EPA can displace caveolin-1, a known inhibitor of eNOS, from membrane caveolae and eNOS, thus enhancing eNOS activity [
      • Li Q
      • Zhang Q
      • Wang M
      • Zhao S
      • Ma J
      • Luo N
      • et al.
      Eicosapentaenoic acid modifies lipid composition in caveolae and induces translocation of endothelial nitric oxide synthase.
      ,
      • Mason RP
      • Dawoud H
      • Jacob RF
      • Sherratt SC
      • Malinski T.
      Eicosapentaenoic acid improves endothelial function and nitric oxide bioavailability in a manner that is enhanced in combination with a statin.
      ]. By modulating membrane lipid dynamics, n3-FAs also influence the organization of lipid rafts enriched in cholesterol and sphingolipids. These highly-ordered lipid assemblies, in turn, control the clustering of proteins required for cell signaling during CD4+ T lymphocyte activation and differentiation [
      • Hou TY
      • McMurray DN
      • Chapkin RS.
      Omega-3 fatty acids, lipid rafts, and T cell signaling.
      ]. Another potential mechanism of endothelial protection and increased NO bioavailability with EPA is mediated through heme oxygenase-1 (HO-1). In cultured ECs, EPA increased HO-1 expression via p38 MAPK signaling and translocation of transcription factor nuclear factor-erythroid factor 2-related factor 2 (Nrf2) to the nucleus [
      • Lee SE
      • Kim G-D
      • Yang H
      • Son GW
      • Park HR
      • Cho J-J
      • et al.
      Effects of eicosapentaenoic acid on the cytoprotection through Nrf2-mediated heme oxygenase-1 in human endothelial cells.
      ,
      • Zhang L
      • Xiao K
      • Zhao X
      • Sun X
      • Zhang J
      • Wang X
      • et al.
      Quantitative proteomics reveals key proteins regulated by eicosapentaenoic acid in endothelial activation.
      ]. HO-1 is an important cytoprotective enzyme that is rate limiting for catabolism of heme to the signaling products biliverdin, free iron, and carbon monoxide (CO), and critical for cellular response to oxidative stress (Fig. 4) [
      • Fredenburgh LE
      • Merz AA
      • Cheng S.
      Haeme oxygenase signalling pathway: implications for cardiovascular disease.
      ,
      • Duckers HJ
      • Boehm M
      • True AL
      • Shaw-Fang Y
      • Hong S
      • Park JL
      • et al.
      Heme oxygenase-1 protects against vascular constriction and proliferation.
      ,
      • Otterbein LE
      • Soares MP
      • Yamashita K
      • Bach FH.
      Heme oxygenase-1: unleashing the protective properties of heme.
      ]. Products of HO-1-mediated heme breakdown provide antioxidant activity (specifically CO and biliverdin which is further converted to bilirubin), improve eNOS function and activated by NO donors, thereby decreasing expression of pro-inflammatory adhesion molecules [
      • Luo W
      • Wang Y
      • Yang H
      • Dai C
      • Hong H
      • Li J
      • et al.
      Heme oxygenase-1 ameliorates oxidative stress-induced endothelial senescence via regulating endothelial nitric oxide synthase activation and coupling.
      ,
      • Polte T
      • Abate A
      • Dennery PA
      • Schröder H.
      Heme Oxygenase-1 Is a cGMP-Inducible Endothelial Protein and Mediates the Cytoprotective Action of Nitric Oxide.
      ,
      • Choi BM
      • Pae HO
      • Chung HT.
      Nitric oxide priming protects nitric oxide-mediated apoptosis via heme oxygenase-1 induction.
      ,
      • Soares MP
      • Seldon MP
      • Gregoire IP
      • Vassilevskaia T
      • Berberat PO
      • Yu J
      • et al.
      Heme Oxygenase-1 Modulates the Expression of Adhesion Molecules Associated with Endothelial Cell Activation.
      ]. Experimentally, exogenous induction of HO-1 decreased atherosclerotic lesion size and lipid hydroperoxide formation in mice on Western and high cholesterol diets [
      • Ishikawa K
      • Sugawara D
      • Wang X-p
      • Suzuki K
      • Itabe H
      • Maruyama Y
      • et al.
      Heme Oxygenase-1 Inhibits Atherosclerotic Lesion Formation in LDL-Receptor Knockout Mice.
      ]. In ECs, expression and activity of HO-1 was linked to eNOS function, where exogenous induction of HO-1 and overexpression of HO-1 increased Ser1177 phosphorylation on eNOS by the kinase Akt and improved eNOS coupling efficiency [
      • Luo W
      • Wang Y
      • Yang H
      • Dai C
      • Hong H
      • Li J
      • et al.
      Heme oxygenase-1 ameliorates oxidative stress-induced endothelial senescence via regulating endothelial nitric oxide synthase activation and coupling.
      ]. Thus, increased HO-1 expression and activity associated with EPA treatment may further improve endothelial function.
      Fig 4
      Fig. 4EPA induces heme oxygenase-1 expression in endothelial cells, a mechanism of cytoprotection. Heme oxygenase-1 (HMOX1) converts primary heme into carbon monoxide, biliverdin, and free iron. These signaling molecules provide antioxidant, anti-apoptotic and anti-inflammatory actions either directly (carbon monoxide) or indirectly (biliverdin and free iron).
      Endothelial cells modulate vasodilation in response to changes in blood flow, endogenous dilators like NO, interaction with HDL and other lipoproteins, and stimulation with various pharmacologic agents [
      • Ignarro LJ
      • Buga GM
      • Wood KS
      • Byrnes RE
      • Chaudhuri G.
      Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.
      ,
      • Ignarro LJ
      • Napoli C
      • Loscalzo J.
      Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide.
      ,
      • Anderson TJ
      • Meredith IT
      • Yeung AC
      • Frei B
      • Selwyn AP
      • Ganz P.
      The effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion.
      ,
      • Mason RP
      • Kalinowski L
      • Jacob RF
      • Jacoby AM
      • Malinski T.
      Nebivolol reduces nitroxidative stress and restores nitric oxide bioavailability in endothelium of black Americans.
      ,
      • Paniagua OA
      • Bryant MB
      • Panza JA.
      Role of endothelial nitric oxide in shear stress-induced vasodilation of human microvasculature: diminished activity in hypertensive and hypercholesterolemic patients.
      ]. In addition to vasodilation, NO inhibits platelet activation and interferes with leukocyte adhesion, hemostasis, thrombosis, and fibrinolysis [
      • Nishizaki Y
      • Shimada K
      • Tani S
      • Ogawa T
      • Ando J
      • Takahashi M
      • et al.
      Significance of imbalance in the ratio of serum n-3 to n-6 polyunsaturated fatty acids in patients with acute coronary syndrome.
      ,
      • Szmitko PE
      • Wang CH
      • Weisel RD
      • de Almeida JR
      • Anderson TJ
      • Verma S.
      New markers of inflammation and endothelial cell activation: Part I.
      ]. Conversely, ECs release potent vasoconstrictors such as angiotensin II, thromboxane A2, prostaglandin H2, and endothelin 1. Loss of NO bioavailability promotes inflammation and thrombosis while interfering with normal vasodilation [
      • Forstermann U
      • Munzel T.
      Endothelial nitric oxide synthase in vascular disease: from marvel to menace.
      ,
      • Panza JA
      • Quyyumi AA
      • Brush JE
      • Epstein SE.
      Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension.
      ]. Without adequate levels of substrate or co-factors (e.g., tetrahydrobiopterin), eNOS in its uncoupled state will donate electrons to molecular oxygen to produce superoxide (O2) [
      • Landmesser U
      • Dikalov S
      • Price SR
      • McCann L
      • Fukai T
      • Holland SM
      • et al.
      Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension.
      ,
      • Huk I
      • Nanobashvili J
      • Neumayer C
      • Punz A
      • Mueller M
      • Afkhampour K
      • et al.
      L-Arginine treatment alters the kinetics of nitric oxide and superoxide release and reduces ischemia/reperfusion injury in skeletal muscle.
      ]. The uncoupling of eNOS can also promote oxidative stress, including increased peroxynitrite (ONOO) production, in the endothelium. The reaction of NO with O2 is diffusion limited to produce the highly reactive ONOO [
      • Dawoud H
      • Malinski T.
      A nanomedical approach to understanding the mechanism of endothelial function and dysfunction – clinical implications.
      ,
      • Malinski T.
      Understanding nitric oxide physiology in the heart: a nanomedical approach.
      ]. n3-FAs and n6-FAs have differential effects on endothelial NO release that correlate with changes in cellular fatty acid composition [
      • Sherratt SCR
      • Dawoud H
      • Bhatt DL
      • Malinski T
      • Mason RP.
      Omega-3 and omega-6 fatty acids have distinct effects on endothelial fatty acid content and nitric oxide bioavailability.
      ]. EPA treatment can increase endothelial NO release and reduce ONOO levels. DHA, however, increased NO release with no effect on ONOO; and AA had no effect on either NO or ONOO levels. Consistent with these results, EPA-treated cells showed a pronounced improvement in the NO/ONOO release ratio, an indicator of eNOS coupling efficiency, as compared to other fatty acids or vehicle alone [
      • Sherratt SCR
      • Dawoud H
      • Bhatt DL
      • Malinski T
      • Mason RP.
      Omega-3 and omega-6 fatty acids have distinct effects on endothelial fatty acid content and nitric oxide bioavailability.
      ]. These different effects on eNOS function correlated with overall changes in cellular fatty acid content. EPA treatment increased EC levels of EPA and its immediate metabolic product, DPA by 10-fold and 2-fold, respectively, with no effects on DHA or AA levels. DHA treatment, by contrast, had no effect on DPA levels while AA actually reduced DPA synthesis due to competition for common biosynthetic enzymes. The conversion of EPA to DPA occurs via elongase enzymes which add two carbons to the acyl chain [
      • Bhatt DL
      • Budoff MJ
      • Mason RP.
      A Revolution in Omega-3 Fatty Acid Research∗.
      ,
      • Drouin G
      • Rioux V
      • Legrand P.
      The n-3 docosapentaenoic acid (DPA): A new player in the n-3 long chain polyunsaturated fatty acid family.
      ]. EPA is eventually converted to DHA through DPA via one more elongase, a Δ6-desaturase, and a final β-oxidation step which occurs in peroxisomes. Similar FA changes with EPA were observed in vitro in human THP-1 macrophages compared with DHA or AA treatments and in vivo in mice on high fat diets enriched with EPA but not diets enriched in DHA [
      • Pinel A
      • Pitois E
      • Rigaudiere J-P
      • Jouve C
      • De Saint-Vincent S
      • Laillet B
      • et al.
      EPA prevents fat mass expansion and metabolic disturbances in mice fed with a Western diet [S].
      ,
      • Dakroub H
      • Nowak M
      • Benoist JF
      • Noel B
      • Vedie B
      • Paul JL
      • et al.
      Eicosapentaenoic acid membrane incorporation stimulates ABCA1-mediated cholesterol efflux from human THP-1 macrophages.
      ]. EPA, but not DHA or AA, was also found to increase ABCA1-mediated cholesterol efflux to nascent HDL particles without altering aqueous diffusion or the macrophage phenotype. As the primary mechanism for transporting cholesterol back to the liver, this effect of EPA on ABCA1 macrophage function has important implications for reducing plaque volume.
      In recent CVOTs, n3-FA treatment in high-risk patients with dyslipidemia was conducted on top of contemporary therapy including statins [
      • Singh N
      • Bhatt DL
      • Miller M
      • Steg PG
      • Brinton EA
      • Jacobson TA
      • et al.
      Consistency of Benefit of Icosapent Ethyl by Background Statin Type in REDUCE-IT.
      ]. Laboratory experiments have investigated the combined effects of n3-FAs and statins on NO metabolism in human ECs as well as isolated vessels. When combined with a high-intensity statin (atorvastatin) intervention, EPA reduced endothelial dysfunction caused by exposure to either oxLDL or high glucose levels [
      • Mason RP
      • Dawoud H
      • Jacob RF
      • Sherratt SC
      • Malinski T.
      Eicosapentaenoic acid improves endothelial function and nitric oxide bioavailability in a manner that is enhanced in combination with a statin.
      ]. The benefits of EPA were superior to those of DHA and were not associated with changes in eNOS expression; instead, EPA was found to increase eNOS coupling efficiency and the NO/ONOO release ratio. The favorable interactions between EPA and hydroxylated active metabolite of atorvastatin may also contribute to complementary benefits on eNOS activity [
      • Mason RP
      • Jacob RF.
      Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism.
      ]. The complementary effects of EPA and a statin on EC cell function have also been observed in a rodents [
      • Mason RP
      • Dawoud H
      • Jacob RF
      • Sherratt SC
      • Malinski T.
      Eicosapentaenoic acid improves endothelial function and nitric oxide bioavailability in a manner that is enhanced in combination with a statin.
      ]. While both EPA and atorvastatin showed separate benefits, their combination augmented NO bioavailability under disease conditions, including hyperglycemia.

      5. Role of n3-FA-generated lipid metabolites in inflammation

      Membrane n3-FAs are available to intracellular cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP) to form bioactive lipids that modulate and resolve inflammation in various tissues (Fig. 5) [
      • O'Connell TD
      • Mason RP
      • Budoff MJ
      • Navar AM
      • Shearer GC.
      Mechanistic insights into cardiovascular protection for omega-3 fatty acids and their bioactive lipid metabolites.
      ]. Also referred to as oxylipins, these metabolites are produced by the enzymatic conversion of PUFAs and have multifactorial roles during endothelial inflammation and atherosclerosis [
      • Shearer GC
      • Newman JW.
      Impact of circulating esterified eicosanoids and other oxylipins on endothelial function.
      ]. Specifically, COX1, COX2, and LOXs produce pro-inflammatory eicosanoids, such as prostaglandin E2 and leukotriene B4, respectively, using AA as a substrate in the absence of aspirin, while CYP enzymes produce epoxyeicosatetraenoic acids and hydroxyeicosatetraenoic acids for various signaling pathways [
      • Bonafini S
      • Fava C.
      Omega-3 fatty acids and cytochrome P450-derived eicosanoids in cardiovascular diseases: Which actions and interactions modulate hemodynamics?.
      ]. The pro-inflammatory effects of AA can be countered by aspirin-triggered lipoxins (e.g., lipoxin A4 and B4) due to acetylation of COX2 and modified conversion of AA [
      • Hansen TV
      • Vik A
      • Serhan CN.
      The protectin family of specialized pro-resolving mediators: potent immunoresolvents enabling innovative approaches to target obesity and diabetes.
      ,
      • Serhan CN.
      Novel pro-resolving lipid mediators in inflammation are leads for resolution physiology.
      ]. Although COX enzymes are more selective for AA than EPA, especially in the absence of aspirin, the LOX and CYP enzymes convert many PUFAs, including AA, DHA, EPA, in a complex network of overlapping signaling pathways.
      Fig 5
      Fig. 5Specialized pro-resolving mediators (SPMs) derived from omega-3 fatty acids promote resolution of inflammation. The acute inflammatory response to injury or infection is initiated by certain eicosanoids (leukotriene B4, prostaglandin E2, etc.) derived from the omega-6 fatty acid arachidonic acid (AA) via cyclooxygenase (COX) and lipoxygenase (LOX) enzymes. To attenuate the inflammatory response, a switch in lipid mediator class occurs to favor the pro-resolving mediators of EPA, DHA and DPA, including resolvins, protectins, and maresins.
      The n3-FAs in membrane phospholipids are made available to enzymes by the activity of PLA2, which releases the long-chain fatty acids into the cytoplasm where COXs, LOXs, and CYPs convert them into a broad array of metabolites. The anti-thrombotic metabolites, such as thromboxane A3 and prostaglandin I3 (PGI3), are generated from both EPA and DHA while n6-FAs give rise to thromboxane A2, a platelet activator and key contributor to atherothrombosis [
      • Mitchell JA
      • Kirkby NS.
      Eicosanoids, prostacyclin and cyclooxygenase in the cardiovascular system.
      ]. The n3-FAs reduce n6-FA-generated vasoconstrictor and pro-aggregatory mediators by competing for COX enzymes that synthesize these thromboxanes [
      • Arterburn LM
      • Hall EB
      • Oken H.
      Distribution, interconversion, and dose response of n-3 fatty acids in humans.
      ,
      • Braeckman RA
      • Manku MS
      • Bays HE
      • Stirtan WG
      • Soni PN.
      Icosapent ethyl, a pure EPA omega-3 fatty acid: effects on plasma and red blood cell fatty acids in patients with very high triglyceride levels (results from the MARINE study).
      ]. Of the various eicosanoids produced by COX, prostacyclin uses a paracrine signaling pathway on nearby platelets and ECs. Prostacyclin counteracts the prothrombotic effects of thromboxane by receptor binding and subsequent inhibition of platelet activation. Smooth muscle relaxation and endothelial-dependent vasodilation are also promoted by prostacyclin generated from n3-FAs.
      Reducing and resolving inflammation is essential to restoring homeostatic balance and counteracting chronic disease. Maresins, protectins, and resolvins are specific families of bioactive lipids derived from n3-FAs and generated by specific lipoxygenases in macrophages and neutrophils [
      • Larsson SC
      • Kumlin M
      • Ingelman-Sundberg M
      • Wolk A.
      Dietary long-chain n-3 fatty acids for the prevention of cancer: a review of potential mechanisms.
      ]. These specialized pro-resolving mediators (SPMs) participate in a coordinated process that promotes resolution of inflammation and homeostasis after tissue injury [
      • Serhan CN
      • Savill J.
      Resolution of inflammation: the beginning programs the end.
      ]. EPA is metabolized by CYP and then 5- or 15-LOX to the E-series resolvins E1-E3 (among other bioactive metabolites), while DHA is converted to maresins, D-series resolvins, and protectins by LOX and acetylated COX2 enzymes [
      • Gabbs M
      • Leng S
      • Devassy JG
      • Monirujjaman M
      • Aukema HM.
      Advances in Our Understanding of Oxylipins Derived from Dietary PUFAs.
      ,
      • Djuricic I
      • Calder PC.
      Beneficial Outcomes of Omega-6 and Omega-3 Polyunsaturated Fatty Acids on Human Health: An Update for 2021.
      ]. Protectins, resolvins, and maresins are also derived from DPA [
      • Drouin G
      • Rioux V
      • Legrand P.
      The n-3 docosapentaenoic acid (DPA): A new player in the n-3 long chain polyunsaturated fatty acid family.
      ]. As a class, SPMs share many overlapping anti-inflammatory mechanisms, including reductions in cytokine production following acute inflammation and inhibition of granulocyte trafficking, all of which promotes tissue repair. For example, resolvin E1 from EPA, protectin D1 from DPA, and 14,20-DiHDoHE from DHA all inhibit neutrophil infiltration in murine models of peritonitis [
      • Drouin G
      • Rioux V
      • Legrand P.
      The n-3 docosapentaenoic acid (DPA): A new player in the n-3 long chain polyunsaturated fatty acid family.
      ,
      • Gabbs M
      • Leng S
      • Devassy JG
      • Monirujjaman M
      • Aukema HM.
      Advances in Our Understanding of Oxylipins Derived from Dietary PUFAs.
      ]. Macrophage-mediated clearance of cellular debris is also promoted by these lipid mediators during the resolution of inflammation [
      • Fredman G
      • Tabas I.
      Boosting Inflammation Resolution in Atherosclerosis: The Next Frontier for Therapy.
      ]. Finally, multiple PUFAs including EPA and DHA can bind the G-protein-coupled receptor 120 (GPR120) found on macrophages. Binding these receptors by n3-FAs inhibits the activation of nuclear factor kappa (NF-κB), a key regulator of inflammatory gene transcription [
      • Oh DY
      • Talukdar S
      • Bae EJ
      • Imamura T
      • Morinaga H
      • Fan W
      • et al.
      GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects.
      ,
      • Yamada H
      • Umemoto T
      • Kakei M
      • Momomura SI
      • Kawakami M
      • Ishikawa SE
      • et al.
      Eicosapentaenoic acid shows anti-inflammatory effect via GPR120 in 3T3-L1 adipocytes and attenuates adipose tissue inflammation in diet-induced obese mice.
      ]. Supporting the role of inflammation resolution in CV disease, inadequate levels of resolvins and impaired T-cell responses have been measured in patients with chronic inflammation in heart failure [
      • Chiurchiù V
      • Leuti A
      • Saracini S
      • Fontana D
      • Finamore P
      • Giua R
      • et al.
      Resolution of inflammation is altered in chronic heart failure and entails a dysfunctional responsiveness of T lymphocytes.
      ].
      The n3-FAs oxylipins may also limit chronic inflammation and activation of the adaptive immune system in a coordinated fashion. This results from beneficial changes in the T helper type 1 (Th1) cell to T helper type 2 (Th2) cell balance through polarization of CD4+ T-cells toward a Th2 bias [
      • Zhang P
      • Smith R
      • Chapkin RS
      • McMurray DN.
      Dietary (n-3) polyunsaturated fatty acids modulate murine Th1/Th2 balance toward the Th2 pole by suppression of Th1 development.
      ]. This leads to regulation of T-cell function, including homeostatic levels of Th1 and T helper type 17 (Th17) cells [
      • Zhang P
      • Smith R
      • Chapkin RS
      • McMurray DN.
      Dietary (n-3) polyunsaturated fatty acids modulate murine Th1/Th2 balance toward the Th2 pole by suppression of Th1 development.
      ,
      • Monk JM
      • Hou TY
      • Turk HF
      • Weeks B
      • Wu C
      • McMurray DN
      • et al.
      Dietary n-3 polyunsaturated fatty acids (PUFA) decrease obesity-associated Th17 cell-mediated inflammation during colitis.
      ,
      • Monk JM
      • Hou TY
      • Turk HF
      • McMurray DN
      • Chapkin RS.
      n3 PUFAs reduce mouse CD4+ T-cell ex vivo polarization into Th17 cells.
      ]. While Th2 and regulatory T cells (Tregs) promote repair and resolution, Th1 or Th17 cells produce responses that favor inflammation. Resolvins reduce cytokine production by activated CD8+ T-cells and CD4+ Th1 cell and limit CD4+ T-cell differentiation into Th1 and Th17 cells in blood lymphocytes [
      • Chiurchiu V
      • Leuti A
      • Dalli J
      • Jacobsson A
      • Battistini L
      • Maccarrone M
      • et al.
      Proresolving lipid mediators resolvin D1, resolvin D2, and maresin 1 are critical in modulating T cell responses.
      ]. Resolvin E1 (RvE1) is a specialized member of the SPM family that is produced from EPA and serves a central role in restoring tissue homeostasis. RvE1 has been shown to block T-cell activation, Th17 cell stimulation and chemoattraction [
      • Oner F
      • Alvarez C
      • Yaghmoor W
      • Stephens D
      • Hasturk H
      • Firatli E
      • et al.
      Resolvin E1 Regulates Th17 Function and T Cell Activation.
      ]. RvE1 also attenuated the activation of dendritic cells by reducing their release of pro-inflammatory cytokines. Thus, RvE1 specifically targets Th17 activation to promote resolution of inflammation.

      6. Ongoing and future clinical investigations with n3-FAs

      Clinical investigations have also shown beneficial effects with EPA only treatment in acute CV disease. Doi et al. tested early administration (<24 h) of EPA ethyl esters (IPE) on top of pitavastatin in patients (N = 115) undergoing percutaneous coronary intervention (PCI) following acute MI [
      • Doi M
      • Nosaka K
      • Miyoshi T
      • Iwamoto M
      • Kajiya M
      • Okawa K
      • et al.
      Early eicosapentaenoic acid treatment after percutaneous coronary intervention reduces acute inflammatory responses and ventricular arrhythmias in patients with acute myocardial infarction: a randomized, controlled study.
      ]. Compared with statin alone, IPE (1.8 g/d) significantly reduced prespecified primary endpoints (p = 0.01), which included a composite of CV events, with the largest contribution due to decreased ventricular arrythmias at a 1-month follow-up. Peak CRP values following PCI were also significantly lower in the EPA plus statin group compared with statin alone (p = 0.001). Another open-label, randomized trial investigated early (<24 h) IPE (1.8 g/d) intervention on top of pitavastatin following percutaneous coronary intervention (PCI) in patients with acute coronary syndrome (ACE) and acute MI [
      • Nosaka K
      • Miyoshi T
      • Iwamoto M
      • Kajiya M
      • Okawa K
      • Tsukuda S
      • et al.
      Early initiation of eicosapentaenoic acid and statin treatment is associated with better clinical outcomes than statin alone in patients with acute coronary syndromes: 1-year outcomes of a randomized controlled study.
      ]. Once again, EPA plus statin treatment significantly reduced the primary endpoint, which was a composite of CV events requiring PCI or coronary artery bypass graft (CABG), at 12 month follow up compared to statin alone (p = 0.02). Interestingly, although REDUCE-IT investigated IPE in more stable patients, recent sub-analyses revealed that IPE treatment significantly reduced CV events in patients with a history of CABG, PCI, or MI (24%, 34%, 26% relative risk reduction, respectively, p = 0.004, p<0.001, p<0.001) [
      • Verma S
      • Bhatt DL
      • Steg PG
      • Miller M
      • Brinton EA
      • Jacobson TA
      • et al.
      Icosapent Ethyl Reduces Ischemic Events in Patients With a History of Previous Coronary Artery Bypass Grafting: REDUCE-IT CABG.
      ,
      • Peterson BE
      • Bhatt DL
      • Steg PG
      • Miller M
      • Brinton EA
      • Jacobson TA
      • et al.
      Treatment with icosapent ethyl to reduce ischemic events in patients with prior percutaneous coronary intervention- insights from REDUCE-IT PCI.
      ,
      • Gaba P
      • Bhatt DL
      • Steg PG
      • Miller M
      • Brinton EA
      • Jacobson TA
      • et al.
      Prevention of Cardiovascular Events and Mortality With Icosapent Ethyl in Patients With Prior Myocardial Infarction.
      ]. Importantly, the trials from Japan were single center and open-label. The study populations also did not include large cohorts, so additional multicentered, placebo-controlled trials with larger cohorts are necessary to confirm the benefits of EPA in the context of acute CV events such as post-MI/stroke and post-PCI/CABG.
      In addition to CV disease, expanded uses of EPA to combat other medical needs, including COVID-19. The COVID-19 pandemic, caused by the novel virus SARS-CoV-2, has caused a health crisis unprecedented in the last century, with more than 400 million cases and 5.8 million deaths worldwide, according to the World Health Organization as of this writing [
      WHO
      Coronavirus (COVID-19) Dashboard.
      ]. Even with development and administration of multiple vaccines against COVID-19, there is an ongoing need to find safe, effective therapies to combat SARS-CoV-2 infection, especially in high-risk patients and those with comorbidities. Specifically, patients with underlying respiratory, immunity or cardiovascular (CV) complications have greater risk for developing severe and life threatening symptoms from SARS-CoV-2 infection [
      • Varga Z
      • Flammer AJ
      • Steiger P
      • Haberecker M
      • Andermatt R
      • Zinkernagel AS
      • et al.
      Endothelial cell infection and endotheliitis in COVID-19.
      ]. Thus, existing therapies for CV disease merit consideration for mitigating severe COVID-19 disease sequelae, including IPE. To date, three trials have reported out results from using IPE in a COVID-19 setting, namely Vascepa COVID-19 CardioLink-9 (NCT04412018, hereafter referred to as “CardioLink-9”), PREPARE-IT 1 & 2 (NCT04460651). Each of these trials tested the effects of a loading dose (8 g/d for 3 days, followed by 4 g/day for the remainder of the treatment protocols) on COVID-19, including inflammatory biomarker levels and symptom progression (CardioLink-9), rates of SARS-CoV-2 infection in patients without prior positive test for SARS-CoV-2 but in high-risk settings (PREPARE-IT 1), and rates of COVID-19-related hospitalization and mortality in patients positive for SARS-CoV-2 (PREPARE-IT 2). The inclusion criteria for these trials did not include established CV disease. Despite significant reductions in hsCRP and D-dimer levels from baseline within the IPE arm, along with significant reductions in total symptoms (from FLU-PRO score) compared with standard of care, IPE treatment did not prevent SARS-CoV-2 infection or rates of COVID-19-related hospitalizations compared with placebo. This suggests that EPA lacks direct effects on SARS-CoV-2 uptake and replication.
      However, a trial currently underway with high CV risk patients is evaluating IPE treatment. This trial, MITIGATE (NCT04505098), is enrolling patients with established atherosclerotic CV disease (ASCVD) that will be randomized to IPE (4 g/d) or usual care and measure both rates of viral upper respiratory infection (URI) and worsening clinical status due to viral URI [
      • Ambrosy AP
      • Malik UI
      • Thomas RC
      • Parikh RV
      • Tan TC
      • Goh CH
      • et al.
      Rationale and design of the pragmatic randomized trial of icosapent ethyl for high cardiovascular risk adults (MITIGATE).
      ]. In contrast to the studies cited above, the inclusion criteria of MITIGATE resembles those of REDUCE-IT, and patients with established ASCVD have greater risk for severe complications from URIs including COVID-19, hence the rationale for this study IPE. Additionally, COVID-19 and CV disease share pathophysiology, including vascular inflammation and thrombus formation, with the arterial endothelium at the crossroads of mediating these responses. Preclinical studies, along with the MARINE and ANCHOR trials, show broad anti-inflammatory and antioxidant effects with EPA described above [
      • Mason RP
      • Libby P
      • Bhatt D L
      Emerging mechanisms of cardiovascular protection for the omega-3 fatty acid eicosapentaenoic acid.
      ,
      • Sherratt SCR
      • Juliano RA
      • Copland C
      • Bhatt DL
      • Libby P
      • Mason RP
      Eicosapentaenoic acid and docosahexaenoic acid containing phospholipids have contrasting effects on membrane structure.
      ,
      • Jacobs ML
      • Faizi HA
      • Peruzzi JA
      • Vlahovska PM
      • Kamat NP.
      EPA and DHA differentially modulate membrane elasticity in the presence of cholesterol.
      ,
      • Sherratt SCR
      • Mason RP.
      Eicosapentaenoic acid and docosahexaenoic acid have distinct membrane locations and lipid interactions as determined by X-ray diffraction.
      ,
      • Mason RP
      • Jacob RF
      • Shrivastava S
      • Sherratt SC
      • Chattopadhyay A.
      Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes.
      ,
      • Sherratt SCR
      • Juliano RA
      • Mason RP.
      Eicosapentaenoic acid (EPA) has optimal chain length and degree of unsaturation to inhibit oxidation of small dense LDL and membrane cholesterol domains as compared to related fatty acids in vitro.
      ,
      • Mason RP
      • Sherratt SC
      • Jacob RF.
      Eicosapentaenoic acid inhibits oxidation of ApoB-containing lipoprotein particles of different size in vitro when administered alone or in combination with atorvastatin active metabolite compared with other triglyceride-lowering agents.
      ,
      • Mason RP
      • Jacob RF.
      Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism.
      ,
      • Li Q
      • Zhang Q
      • Wang M
      • Zhao S
      • Ma J
      • Luo N
      • et al.
      Eicosapentaenoic acid modifies lipid composition in caveolae and induces translocation of endothelial nitric oxide synthase.
      ,
      • Mason RP
      • Dawoud H
      • Jacob RF
      • Sherratt SC
      • Malinski T.
      Eicosapentaenoic acid improves endothelial function and nitric oxide bioavailability in a manner that is enhanced in combination with a statin.
      ,
      • Lee SE
      • Kim G-D
      • Yang H
      • Son GW
      • Park HR
      • Cho J-J
      • et al.
      Effects of eicosapentaenoic acid on the cytoprotection through Nrf2-mediated heme oxygenase-1 in human endothelial cells.
      ,
      • Sherratt SCR
      • Dawoud H
      • Bhatt DL
      • Malinski T
      • Mason RP.
      Omega-3 and omega-6 fatty acids have distinct effects on endothelial fatty acid content and nitric oxide bioavailability.
      ,
      • Mason RP
      • Eckel RH.
      Mechanistic Insights from REDUCE-IT STRENGTHen the Case Against Triglyceride Lowering as a Strategy for Cardiovascular Disease Risk Reduction.
      ,
      • Sherratt SCR
      • Mason RP.
      Eicosapentaenoic acid inhibits oxidation of high density lipoprotein particles in a manner distinct from docosahexaenoic acid.
      ,
      • Pisaniello AD
      • Psaltis PJ
      • King PM
      • Liu G
      • Gibson RA
      • Tan JTM
      • et al.
      Omega-3 fatty acids ameliorate vascular inflammation: A rationale for their atheroprotective effects.
      ,
      • Bays HE
      • Ballantyne CM
      • Braeckman RA
      • Stirtan WG
      • Soni PN.
      Icosapent ethyl, a pure ethyl ester of eicosapentaenoic acid: effects on circulating markers of inflammation from the MARINE and ANCHOR studies.
      ]. Together, these results indicate that IPE treatment may offer anti-inflammatory activity in the context of viral infections such as influenza or COVID-19, which is characterized by systemic EC dysfunction, multisystem inflammation, thrombus formation, and the cytokine storm, thus providing incremental benefit for patients at elevated risk for CV disease [
      • Varga Z
      • Flammer AJ
      • Steiger P
      • Haberecker M
      • Andermatt R
      • Zinkernagel AS
      • et al.
      Endothelial cell infection and endotheliitis in COVID-19.
      ,
      • Libby P
      • Lüscher T.
      COVID-19 is, in the end, an endothelial disease.
      ,
      • Teuwen L-A
      • Geldhof V
      • Pasut A
      • Carmeliet P.
      COVD-19: the vasculature unleashed.
      ].
      Other pre-clinical and observational studies have shown potential benefit of EPA in the treatment of pulmonary diseases including pulmonary arterial hypertension (PAH). Kurahara et al. (2020) showed that treatment with EPA ethyl ester reversed cardiac remodeling, right ventricular hypertrophy, and dysfunction in a mouse model of pulmonary arterial hypertension (PAH) [
      • Kurahara LH
      • Hiraishi K
      • Yamamura A
      • Zhang Y
      • Abe K
      • Yahiro E
      • et al.
      Eicosapentaenoic acid ameliorates pulmonary hypertension via inhibition of tyrosine kinase Fyn.
      ]. The researchers probed mechanistic underpinnings for this effect using cultured pulmonary arterial ECs and found that EPA and its metabolite RvE1 both significantly blunted IL-6-induced activation of the Src-kinase pY416 and phosphorylation of STAT3, a known mechanism of PAH pathogenesis. Another study investigated EPA treatment effects on lung inflammation and oxidative stress induced by cigarette smoke and found that EPA decreased expression of pro-inflammatory chemokines, including monocyte chemoattractant protein-1 (CCL2) and macrophage inflammatory protein 2 (CXCL2), in lung tissue from mice exposed to cigarette smoke [
      • Liu M-H
      • Lin A-H
      • Lu S-H
      • Peng R-Y
      • Lee T-S
      • Kou YR.
      Eicosapentaenoic acid attenuates cigarette smoke-induced lung inflammation by inhibiting ROS-sensitive inflammatory signaling.
      ]. These results support findings from a cross-sectional observational trial of patients with COPD with varying levels of n3-FA and n6-FA intake as described by Lemoine et al. [
      • Lemoine C
      • Brigham E
      • Woo H
      • Koch A
      • Hanson C
      • Romero K
      • et al.
      Relationship between Omega-3 and Omega-6 Fatty Acid Intake and Chronic Obstructive Pulmonary Disease Morbidity.
      ]. They found a negative correlation between n3-FA intake and COPD morbidity, while n6-FA correlated positively. Taken together, EPA exerts beneficial anti-inflammatory actions that specifically target pulmonary endothelium and interrupts inflammatory and oxidative stress pathways, which may be favorable for patients diagnosed with COVID-19 and underlying CV co-morbidities. Confirmation of this pulmonary benefit for patients will require prospective randomized trials.
      Finally, several trials are investigating the effects of IPE in patients with cancer, including colorectal cancer and breast cancer. The EPA for Metastasis Trial 2 (EMT2, NCT03428477) is a phase III multicentered, randomized trial investigating the effects of 4 g/d IPE versus placebo on progression free survival (PFS) and overall survival (OS) in patients undergoing liver resection for colorectal cancer liver metastasis (CRCLM). Importantly, patients in this trial began IPE or placebo administration before resection and then continue treatment regimen for the duration of the study. Previous phase II trial data showed that treatment with EPA as a free fatty acid in this patient population showed reductions in vascularity score compared to placebo, indicating a possible anti-angiogenic effect of EPA [
      • Cockbain AJ
      • Volpato M
      • Race AD
      • Munarini A
      • Fazio C
      • Belluzzi A
      • et al.
      Anticolorectal cancer activity of the omega-3 polyunsaturated fatty acid eicosapentaenoic acid.
      ]. The larger patient cohort and longer duration of EMT2 should provide a clearer understanding of the effects of EPA in preventing CRCLM following resection. Another smaller phase II trial, the Prevention Using EPA Against Colorectal Cancer (PREPARE) trial (NCT04216251), is currently recruiting patients to investigate administration of IPE as a chemopreventive agent to reduce colorectal cancer risk in patients with at least one colorectal adenoma diagnosed in their lives. Recently, a new phase II clinical trial was announced to test the combination of IPE and dasatinib on triple negative metastatic inflammatory breast cancer (NCT05198843). The primary endpoint of this trial is to determine the maximum tolerated dose of IPE and dasatinib, with PFS and OS among the key secondary endpoints. This trial is not yet recruiting, but it shows another potential use for IPE intervention beyond CV disease.

      7. Conclusion

      The n3-FA, EPA, demonstrated broad CV benefit in large randomized CVOTs such as REDUCE-IT and JELIS. Mixed EPA/DHA formulations have yet to show incremental clinical benefit on top of statin therapy, possibly due to differences in formulation and the distinct effects of these n3-FAs on lipid oxidation, inflammation, membrane structure/organization, cholesterol domain formation, and EC function. The benefits of EPA ethyl ester monotherapy in CV disease suggest broad anti-inflammatory and anti-oxidative effects that may interrupt other disease processes, including URIs and cancer. Further investigation is needed to fully elucidate the multifaceted mechanisms of action of EPA in CV disease and expand its possible clinical use. Preclinical studies assessing protein and gene expression changes in models of CV disease and inflammation may yield novel mediators or pathways for EPA-induced benefit. The unique orientation of EPA within the membrane and consequent stabilizing effects compared with DHA may also influence integral membrane proteins in various tissues including cardiac myocytes. These investigations may be essential to our understanding of n3-FA effects on atrial and ventricular fibrillation. The role of DPA in CV disease also warrants further study given its metabolic relationship to changes in EPA levels. Finally, clinical investigation distinct other biomarkers that are uniquely associated with IPE treatment may lead to other potential mechanisms of action and further elucidate the basis for differences in CVOTs using IPE versus EPA/DHA mixed formulations.
      Table 1Summary of Recent n3-FA CVOTs. The trials included in the table were chosen based on the high background statin use. Abbreviations: LDL-C, low density lipoprotein cholesterol; TG, triglycerides; ASCVD, atherosclerotic cardiovascular disease; HDL-C, high density lipoprotein cholesterol; AMI, acute myocardial infarction; IPE, icosapent ethyl; FFA, free fatty acid; Revasc, revascularization; HR, hazard ratio; CI, confidence interval.
      Trial (year published)No. PatientsInclusion Criterian3-FA FormulationTherapeutic DosePer Capsule ContentStatin BackgroundPrimary EndpointPrimary Outcome HR (95% CI), P-value
      JELIS

      (2007)
      18,645Hypercholesterolemia, LDL-C ≥170 mg/dLIPE1.8 g/d≥294 mg IPE100%Any major coronary event0.81 (0.69-0.95)

      P = 0.011
      REDUCE-IT

      (2019)
      8,179TG Levels 150-499 mg/dL w/ ASCVD or diabetes w/≥1 other risk factorsIPE4 g/d≥960 mg IPE100%CV death, non-fatal MI, non-fatal stroke, coronary revasc, or unstable angina0.75 (0.68-0.83)

      P <0.001
      STRENGTH

      (2020)
      13,078TG Levels 180-499 mg/dL and HDL-C ≤40 mg/dL w/ ASCVD or diabetes w/≥1 other risk factorsEPA + DHA FFA4 g/d≥750 mg EPA + DHA FFA100%CV death, non-fatal MI, non-fatal stroke, coronary revasc, or unstable angina0.99 (0.90-1.09)

      P = 0.84
      OMEMI

      (2021)
      1,027Elderly patients (70-82 years) w/recent AMIEPA + DHA FFA1.8 g/d310 mg EPA +

      220 mg DHA
      96%AMI, revasc, stroke, all-cause death, or HF hospitalization1.08 (0.82-1.41)

      P = 0.60

      Funding

      No funding was obtained for this review.

      Declaration of Competing Interest

      Mr. Sherratt has no disclosures. Dr. Mason has received research funding or consulting from Amarin, Lexicon, Esperion, and HLS Therapeutics. PL is an unpaid consultant to, or involved in clinical trials for Amgen, AstraZeneca, Baim Institute, Beren Therapeutics, Esperion Therapeutics, Genentech, Kancera, Kowa Pharmaceuticals, Medimmune, Merck, Norvo Nordisk, Novartis, Pfizer, and Sanofi-Regeneron. Dr. Libby is a member of the scientific advisory board for Amgen, Caristo, Cartesian, CSL Behring, DalCor Pharmaceuticals, Dewpoint, Kowa Pharmaceuticals, Olatec Therapeutics, Medimmune, Novartis, PlaqueTec, TenSixteen Bio, and XBiotech, Inc. Dr. Libby's laboratory has received research funding in the last 2 years from Novartis. Dr. Libby is on the Board of Directors of XBiotech, Inc. Dr. Libby has a financial interest in Xbiotech, a company developing therapeutic human antibodies. Dr. Libby's interests were reviewed and are managed by Brigham and Women's Hospital and Partners HealthCare in accordance with their conflict-of-interest policies. Dr. Libby receives funding support from the National Heart, Lung, and Blood Institute (1R01HL134892), the American Heart Association (18CSA34080399), the RRM Charitable Fund, and the Simard Fund. DLB serves as the Chair and International Principal Investigator for REDUCE-IT, with research funding from Amarin to Brigham and Women's Hospital. Dr. Bhatt discloses the following relationships - Advisory Board: Bayer, Boehringer Ingelheim, Cardax, CellProthera, Cereno Scientific, Elsevier Practice Update Cardiology, Janssen, Level Ex, Medscape Cardiology, Merck, MyoKardia, NirvaMed, Novo Nordisk, PhaseBio, PLx Pharma, Regado Biosciences, Stasys; Board of Directors: Boston VA Research Institute, DRS.LINQ (stock options), Society of Cardiovascular Patient Care, TobeSoft; Chair: Inaugural Chair, American Heart Association Quality Oversight Committee; Data Monitoring Committees: Acesion Pharma, Assistance Publique-Hôpitaux de Paris, Baim Institute for Clinical Research (formerly Harvard Clinical Research Institute, for the PORTICO trial, funded by St. Jude Medical, now Abbott), Boston Scientific (Chair, PEITHO trial), Cleveland Clinic (including for the ExCEED trial, funded by Edwards), Contego Medical (Chair, PERFORMANCE 2), Duke Clinical Research Institute, Mayo Clinic, Mount Sinai School of Medicine (for the ENVISAGE trial, funded by Daiichi Sankyo; for the ABILITY-DM trial, funded by Concept Medical), Novartis, Population Health Research Institute; Rutgers University (for the NIH-funded MINT Trial); Honoraria: American College of Cardiology (Senior Associate Editor, Clinical Trials and News, ACC.org; Chair, ACC Accreditation Oversight Committee), Arnold and Porter law firm (work related to Sanofi/Bristol-Myers Squibb clopidogrel litigation), Baim Institute for Clinical Research (formerly Harvard Clinical Research Institute; RE-DUAL PCI clinical trial steering committee funded by Boehringer Ingelheim; AEGIS-II executive committee funded by CSL Behring), Belvoir Publications (Editor in Chief, Harvard Heart Letter), Canadian Medical and Surgical Knowledge Translation Research Group (clinical trial steering committees), Cowen and Company, Duke Clinical Research Institute (clinical trial steering committees, including for the PRONOUNCE trial, funded by Ferring Pharmaceuticals), HMP Global (Editor in Chief, Journal of Invasive Cardiology), Journal of the American College of Cardiology (Guest Editor; Associate Editor), K2P (Co-Chair, interdisciplinary curriculum), Level Ex, Medtelligence/ReachMD (CME steering committees), MJH Life Sciences, Piper Sandler, Population Health Research Institute (for the COMPASS operations committee, publications committee, steering committee, and USA national co-leader, funded by Bayer), Slack Publications (Chief Medical Editor, Cardiology Today's Intervention), Society of Cardiovascular Patient Care (Secretary/Treasurer), WebMD (CME steering committees); Other: Clinical Cardiology (Deputy Editor), NCDR-ACTION Registry Steering Committee (Chair), VA CART Research and Publications Committee (Chair); Research Funding: Abbott, Afimmune, Aker Biomarine, Amarin, Amgen, AstraZeneca, Bayer, Beren, Boehringer Ingelheim, Bristol-Myers Squibb, Cardax, CellProthera, Cereno Scientific, Chiesi, CSL Behring, Eisai, Ethicon, Faraday Pharmaceuticals, Ferring Pharmaceuticals, Forest Laboratories, Fractyl, Garmin, HLS Therapeutics, Idorsia, Ironwood, Ischemix, Janssen, Javelin, Lexicon, Lilly, Medtronic, Merck, Moderna, MyoKardia, NirvaMed, Novartis, Novo Nordisk, Owkin, Pfizer, PhaseBio, PLx Pharma, Recardio, Regeneron, Reid Hoffman Foundation, Roche, Sanofi, Stasys, Synaptic, The Medicines Company, 89Bio; Royalties: Elsevier (Editor, Cardiovascular Intervention: A Companion to Braunwald's Heart Disease); Site Co-Investigator: Abbott, Biotronik, Boston Scientific, CSI, St. Jude Medical (now Abbott), Philips, Svelte; Trustee: American College of Cardiology; Unfunded Research: FlowCo, Takeda.

      Acknowledgement

      The authors wish to thank Robert F. Jacob, Ph.D., Elucida Research LLC, for providing editorial assistance and Luke Groothoff, Elucida Research LLC, for preparing figure artwork.

      References

        • Borow KM
        • Nelson JR
        • Mason RP.
        Biologic plausibility, cellular effects, and molecular mechanisms of eicosapentaenoic acid (EPA) in atherosclerosis.
        Atherosclerosis. 2015; 242: 357-366
        • Mason RP
        • Libby P
        • Bhatt D L
        Emerging mechanisms of cardiovascular protection for the omega-3 fatty acid eicosapentaenoic acid.
        Arteriosclerosis, Thrombosis, and Vascular Biology. 2020; 40: 1135-1147
        • Bhatt DL
        • Budoff MJ
        • Mason RP.
        A Revolution in Omega-3 Fatty Acid Research∗.
        Journal of the American College of Cardiology. 2020; 76: 2098-2101
        • Libby P.
        Triglycerides on the rise: should we swap seats on the seesaw?.
        Eur Heart J. 2015; 36: 774-776
        • Ganda OP
        • Bhatt DL
        • Mason RP
        • Miller M
        • Boden WE.
        Unmet need for adjunctive dyslipidemia therapy in hypertriglyceridemia management.
        J Am Coll Cardiol. 2018; 72: 330-343
        • Mason RP
        • Sherratt SCR
        • Eckel RH.
        Rationale for different formulations of omega-3 fatty acids leading to differences in residual cardiovascular risk reduction.
        Metabolism - Clinical and Experimental. 2022; : 130
        • Budoff MJ
        • Bhatt DL
        • Kinninger A
        • Lakshmanan S
        • Muhlestein JB
        • Le VT
        • et al.
        Effect of icosapent ethyl on progression of coronary atherosclerosis in patients with elevated triglycerides on statin therapy: final results of the EVAPORATE trial.
        European Heart Journal. 2020; 41: 3925-3932
      1. Bhatt D, L., Steg PG, Miller M, Brinton EA, Jacobson TA, Ketchum SB, et al. Cardiovascular risk reduction with Icosapent Ethyl for hypertriglyceridemia. New England Journal of Medicine. 2019;380:11-22.

        • Yokoyama M
        • Origasa H
        • Matsuzaki M
        • Matsuzawa Y
        • Saito Y
        • Ishikawa Y
        • et al.
        Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis.
        The Lancet. 2007; 369: 1090-1098
        • Watanabe T
        • Ando K
        • Daidoji H
        • Otaki Y
        • Sugawara S
        • Matsui M
        • et al.
        A randomized controlled trial of eicosapentaenoic acid in patients with coronary heart disease on statins.
        Journal of Cardiology. 2017; 70: 537-544
        • Nishio R
        • Shinke T
        • Otake H
        • Nakagawa M
        • Nagoshi R
        • Inoue T
        • et al.
        Stabilizing effect of combined eicosapentaenoic acid and statin therapy on coronary thin-cap fibroatheroma.
        Atherosclerosis. 2014; 234: 114-119
        • Alfaddagh A
        • Elajami TK
        • Ashfaque H
        • Saleh M
        • Bistrian BR
        • Welty FK.
        Effect of eicosapentaenoic and docosahexaenoic acids added to statin therapy on coronary artery plaque in patients with coronary artery disease: a randomized clinical trial.
        J Am Heart Assoc. 2017; 6
        • Group ASC
        • Bowman L
        • Mafham M
        • Wallendszus K
        • Stevens W
        • Buck G
        • et al.
        Effects of n-3 fatty acid supplements in diabetes mellitus.
        New England Journal of Medicine. 2018; 379: 1540-1550
        • Manson JE
        • Cook NR
        • Lee IM
        • Christen W
        • Bassuk SS
        • Mora S
        • et al.
        Marine n-3 fatty acids and prevention of cardiovascular disease and cancer.
        N Engl J Med. 2018; 380: 23-32
        • Kalstad AA
        • Myhre PL
        • Laake K
        • Tveit SH
        • Schmidt EB
        • Smith P
        • et al.
        Effects of n-3 fatty acid supplements in elderly patients after myocardial infarction: a randomized, controlled trial.
        Circulation. 2021; 143: 528-539
        • Nicholls SJ
        • Lincoff AM
        • Garcia M
        • Bash D
        • Ballantyne CM
        • Barter PJ
        • et al.
        Effect of High-Dose Omega-3 Fatty Acids vs Corn Oil on Major Adverse Cardiovascular Events in Patients at High Cardiovascular Risk: The STRENGTH Randomized Clinical Trial.
        JAMA. 2020; 324: 2268-2280
        • Bhatt DL
        • Steg PG
        • Miller M.
        Cardiovascular risk reduction with icosapent ethyl. Reply.
        N Engl J Med. 2019; 380: 1678
        • Mason RP.
        New insights into mechanisms of action for omega-3 fatty acids in atherothrombotic cardiovascular disease.
        Curr Atheroscler Rep. 2019; 21: 2
        • Bhatt DL
        • Steg PG
        • Miller M
        • Brinton EA
        • Jacobson TA
        • Jiao L
        • et al.
        Reduction in first and total ischemic events with icosapent ethyl across baseline triglyceride tertiles.
        J Am Coll Cardiol. 2019; 74: 1159-1161
        • Pisaniello AD
        • Nicholls SJ
        • Ballantyne CM
        • Bhatt DL
        • Wong ND.
        Eicosapentaenoic acid: atheroprotective properties and the reduction of atherosclerotic cardiovascular disease events.
        European Medical Journal. 2020; 5: 29-36
        • Aung T
        • Halsey J
        • Kromhout D
        • Gerstein HC
        • Marchioli R
        • Tavazzi L
        • et al.
        Associations of omega-3 fatty acid supplement use with cardiovascular disease risks: meta-analysis of 10 trials involving 77 917 individuals.
        JAMA Cardiology. 2018; 3: 225-233
        • Mason RP
        • Eckel RH.
        Is there a role for omega-3 fatty acids in cardiovascular disease risk reduction?.
        eClinicalMedicine. 2021; : 39
        • Elam MB
        • Ginsberg HN
        • Lovato LC
        • Corson M
        • Largay J
        • Leiter LA
        • et al.
        Association of Fenofibrate Therapy With Long-term Cardiovascular Risk in Statin-Treated Patients With Type 2 Diabetes.
        JAMA Cardiology. 2017; 2: 370-380
      2. Effects of combination lipid therapy in type 2 diabetes mellitus.
        New England Journal of Medicine. 2010; 362: 1563-1574
      3. Effects of extended-release niacin with laropiprant in high-risk patients.
        New England Journal of Medicine. 2014; 371: 203-212
      4. Niacin in patients with low HDL cholesterol levels receiving intensive statin therapy.
        New England Journal of Medicine. 2011; 365: 2255-2267
        • Lakshmanan S
        • Shekar C
        • Kinninger A
        • Dahal S
        • Onuegbu A
        • Cai AN
        • et al.
        Comparison of mineral oil and non-mineral oil placebo on coronary plaque progression by coronary computed tomography angiography.
        Cardiovascular Research. 2019; 116: 479-482
        • Olshansky B
        • Chung MK
        • Budoff MJ
        • Philip S
        • Jiao L
        • Doyle J
        • Ralph T
        • et al.
        Mineral oil: safety and use as placebo in REDUCE-IT and other clinical studies.
        European Heart Journal Supplements. 2020; 22: J34-J48
        • Pradhan AD
        • Paynter NP
        • Everett BM
        • Glynn RJ
        • Amarenco P
        • Elam M
        • et al.
        Rationale and design of the Pemafibrate to Reduce Cardiovascular Outcomes by Reducing Triglycerides in Patients with Diabetes (PROMINENT) study.
        Am Heart J. 2018; 206: 80-93
        • Davidson MH
        • Rosenson RS
        • Maki KC
        • Nicholls SJ
        • Ballantyne CM
        • Mazzone T
        • et al.
        Effects of fenofibric acid on carotid intima-media thickness in patients with mixed dyslipidemia on atorvastatin therapy: randomized, placebo-controlled study (FIRST).
        Arteriosclerosis, Thrombosis, and Vascular Biology. 2014; 34: 1298-1306
      5. Kowa Research Institute I. Kowa to discontine K-877 (pemafibrate) "PROMINENT" cardiovascular outcomes study.
        Cision PR Newswire. 2022;
        • Sherratt SCR
        • Lero M
        • Mason RP.
        Are dietary fish oil supplements appropriate for dyslipidemia management? A review of the evidence.
        Curr Opin Lipidol. 2020; 31: 94-100
        • Albert BB
        • Derraik JG
        • Cameron-Smith D
        • Hofman PL
        • Tumanov S
        • Villas-Boas SG
        • et al.
        Fish oil supplements in New Zealand are highly oxidised and do not meet label content of n-3.
        PUFA. Sci Rep. 2015; 5: 7928
        • Mason RP
        • Sherratt SCR.
        Omega-3 fatty acid fish oil dietary supplements contain saturated fats and oxidized lipids that may interfere with their intended biological benefits.
        Biochem Biophys Res Commun. 2017; 483: 425-429
        • Kita Y
        • Watanabe M
        • Kamon D
        • Ueda T
        • Soeda T
        • Okayama S
        • et al.
        Effects of Fatty Acid Therapy in Addition to Strong Statin on Coronary Plaques in Acute Coronary Syndrome: An Optical Coherence Tomography Study.
        Journal of the American Heart Association. 2020; 9e015593
        • Motoyama S
        • Nagahara Y
        • Sarai M
        • Kawai H
        • Miyajima K
        • Sato Y
        • et al.
        Effect of Omega-3 Fatty Acids on Coronary Plaque Morphology - A Serial Computed Tomography Angiography Study.
        Circ J. 2021;
        • Nakajima K
        • Yamashita T
        • Kita T
        • Takeda M
        • Sasaki N
        • Kasahara K
        • et al.
        Orally Administered Eicosapentaenoic Acid Induces Rapid Regression of Atherosclerosis Via Modulating the Phenotype of Dendritic Cells in LDL Receptor-Deficient Mice.
        Arteriosclerosis, Thrombosis, and Vascular Biology. 2011; 31: 1963-1972
        • Sato T
        • Horikawa M
        • Takei S
        • Yamazaki F
        • Ito TK
        • Kondo T
        • et al.
        Preferential incorporation of administered eicosapentaenoic acid into thin-cap atherosclerotic plaques.
        Arterioscler Thromb Vasc Biol. 2019; 39: 1802-1816
        • Williams MC
        • Kwiecinski J
        • Doris M
        • McElhinney P
        • D'Souza MS
        • Cadet S
        • et al.
        Low-Attenuation Noncalcified Plaque on Coronary Computed Tomography Angiography Predicts Myocardial Infarction.
        Circulation. 2020; 141: 1452-1462
        • Cawood AL
        • Ding R
        • Napper FL
        • Young RH
        • Williams JA
        • Ward MJ
        • et al.
        Eicosapentaenoic acid (EPA) from highly concentrated n-3 fatty acid ethyl esters is incorporated into advanced atherosclerotic plaques and higher plaque EPA is associated with decreased plaque inflammation and increased stability.
        Atherosclerosis. 2010; 212: 252-259
        • Sawazaki S
        • Hamazaki T
        • Yamazaki K
        • Taki H
        • Kaneda M
        • Yano S
        • et al.
        Comparison of the increment in plasma eicosapentaenoate concentrations by fish oil intake between young and middle-aged volunteers.
        J Nutr Sci Vitaminol (Tokyo). 1989; 35: 349-359
        • Subbaiah PV
        • Kaufman D
        • Bagdade JD.
        Incorporation of dietary n-3 fatty acids into molecular species of phosphatidyl choline and cholesteryl ester in normal human plasma.
        American Journal of Clinical Nutrition. 1993; 58: 360-368
        • Sherratt SCR
        • Juliano RA
        • Copland C
        • Bhatt DL
        • Libby P
        • Mason RP
        Eicosapentaenoic acid and docosahexaenoic acid containing phospholipids have contrasting effects on membrane structure.
        J Lipid Res. 2021; 100106
        • Jacobs ML
        • Faizi HA
        • Peruzzi JA
        • Vlahovska PM
        • Kamat NP.
        EPA and DHA differentially modulate membrane elasticity in the presence of cholesterol.
        Biophysical Journal. 2021; 120: 2317-2329
        • Sherratt SCR
        • Mason RP.
        Eicosapentaenoic acid and docosahexaenoic acid have distinct membrane locations and lipid interactions as determined by X-ray diffraction.
        Chemistry and Physics of Lipids. 2018; 212: 73-79
        • Mason RP
        • Jacob RF
        • Shrivastava S
        • Sherratt SC
        • Chattopadhyay A.
        Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes.
        Biochimica et Biophysica Acta. 2016; 1858: 3131-3140
        • Soni SP
        • LoCascio DS
        • Liu Y
        • Williams JA
        • Bittman R
        • Stillwell W
        • et al.
        Docosahexaenoic acid enhances segregation of lipids between: 2H-NMR study.
        Biophys J. 2008; 95: 203-214
        • Querques G
        • Forte R
        • Souied EH.
        Retina and omega-3.
        J Nutr Metab. 2011; 2011748361
        • Dyall SC.
        Long-chain omega-3 fatty acids and the brain: a review of the independent and shared effects of EPA, DPA and DHA.
        Frontiers in Aging Neuroscience. 2015; 7
        • Innis SM.
        Dietary (n-3) fatty acids and brain development.
        J Nutr. 2007; 137: 855-859
        • Uauy R
        • Dangour AD.
        Nutrition in brain development and aging: role of essential fatty acids.
        Nutr Rev. 2006; 64 (S24-33; discussion S72-91)
        • Chang HM
        • Reitstetter R
        • Mason RP
        • Gruener R.
        Attenuation of channel kinetics and conductance by cholesterol: an interpretation using structural stress as a unifying concept.
        J Membr Biol. 1995; 143: 51-63
        • Heijman J
        • Voigt N
        • Nattel S
        • Dobrev D.
        Cellular and molecular electrophysiology of atrial fibrillation initiation, maintenance, and progression.
        Circulation Research. 2014; 114: 1483-1499
        • Maixent JM
        • Duran MJ
        • Pierre S
        • Sennoune S
        • Robert K
        • Bernard M
        • et al.
        Remodeling of Na,K-ATPase, and membrane fluidity after atrial fibrillation in sheep.
        J Recept Signal Transduct Res. 2002; 22: 201-211
        • Doi M
        • Nosaka K
        • Miyoshi T
        • Iwamoto M
        • Kajiya M
        • Okawa K
        • et al.
        Early eicosapentaenoic acid treatment after percutaneous coronary intervention reduces acute inflammatory responses and ventricular arrhythmias in patients with acute myocardial infarction: a randomized, controlled study.
        Int J Cardiol. 2014; 176: 577-582
        • Myhre P
        • Smith EB
        • Kalstad AA
        • Tveit S
        • Laake K
        • Smith P
        • et al.
        Changes in EPA and DHA during supplementation with omega-3 fatty acids and incident cardiovascular events: secondary analysis from the OMEMI trial.
        European Heart Journal. 2021; : 42
        • Witztum JL.
        The oxidation hypothesis of atherosclerosis.
        Lancet. 1994; 344: 793-795
        • Chisolm GM
        • Steinberg D.
        The oxidative modification hypothesis of atherogenesis: an overview.
        Free Radic Biol Med. 2000; 28: 1815-1826
        • Ehara S
        • Ueda M
        • Naruko T
        • Haze K
        • Itoh A
        • Otsuka M
        • et al.
        Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes.
        Circulation. 2001; 103: 1955-1960
        • Walter MF
        • Jacob RF
        • Bjork RE
        • Jeffers B
        • Buch J
        • Mizuno Y
        • et al.
        Circulating lipid hydroperoxides predict cardiovascular events in patients with stable coronary artery disease: the PREVENT study.
        J Am Coll Cardiol. 2008; 51: 1196-1202
        • Walter MF
        • Jacob RF
        • Jeffers B
        • Ghadanfar MM
        • Preston GM
        • Buch J
        • et al.
        Serum levels of TBARS predict cardiovascular events in patients with stable coronary artery disease: A longitudinal analysis of the PREVENT study.
        J Am Coll Cardiol. 2004; 44: 1996-2002
        • Holvoet P
        • Kritchevsky SB
        • Tracy RP
        • Mertens A
        • Rubin SM
        • Butler J
        • et al.
        The metabolic syndrome, circulating oxidized LDL, and risk of myocardial infarction in well-functioning elderly people in the health, aging, and body composition cohort.
        Diabetes. 2004; 53: 1068-1073
        • Mason RP
        • Walter MF
        • Mason PE.
        Effect of oxidative stress on membrane structure: Small angle x-ray diffraction analysis.
        Free Radic Biol Med. 1997; 23: 419-425
        • Mason RP
        • Walter MF
        • Day CA
        • Jacob RF.
        Active metabolite of atorvastatin inhibits membrane cholesterol domain formation by an antioxidant mechanism.
        J Biol Chem. 2006; 281: 9337-9345
        • Self-Medlin Y
        • Byun J
        • Jacob RF
        • Mizuno Y
        • Mason RP.
        Glucose promotes membrane cholesterol crystalline domain formation by lipid peroxidation.
        Biochim Biophys Acta. 2009; 1788: 1398-1403
        • Wratten ML
        • van-Ginkel G
        • van't Veld AA
        • Bekker A
        • van Faassen EE
        • Sevanian A.
        Structural and dynamic effects of oxidatively modified phospholipids in unsaturated lipid membranes.
        Biochemistry. 1992; 31: 10901-10907
        • Sherratt SCR
        • Juliano RA
        • Mason RP.
        Eicosapentaenoic acid (EPA) has optimal chain length and degree of unsaturation to inhibit oxidation of small dense LDL and membrane cholesterol domains as compared to related fatty acids in vitro.
        Biochimica et Biophysica Acta - Biomembranes. 2020; : 1862
        • Mason RP
        • Sherratt SC
        • Jacob RF.
        Eicosapentaenoic acid inhibits oxidation of ApoB-containing lipoprotein particles of different size in vitro when administered alone or in combination with atorvastatin active metabolite compared with other triglyceride-lowering agents.
        Journal of Cardiovascular Pharmacology. 2016; 68: 33-40
        • Mason RP
        • Jacob RF.
        Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism.
        Biochimica et Biophysica Acta. 2015; : 1848
        • Shaikh SR.
        Biophysical and biochemical mechanisms by which dietary N-3 polyunsaturated fatty acids from fish oil disrupt membrane lipid rafts.
        J Nutr Biochem. 2012; 23: 101-105
        • Williams JA
        • Batten SE
        • Harris M
        • Rockett BD
        • Shaikh SR
        • Stillwell W
        • et al.
        Docosahexaenoic and eicosapentaenoic acids segregate differently between raft and nonraft domains.
        Biophys J. 2012; 103: 228-237
        • Shaikh SR
        • Kinnun JJ
        • Leng X
        • Williams JA
        • Wassall SR.
        How polyunsaturated fatty acids modify molecular organization in membranes: insight from NMR studies of model systems.
        Biochim Biophys Acta. 2015; 1848: 211-219
        • Jacob RF
        • Walter MF
        • Self-Medlin Y
        • Mason RP.
        Atorvastatin active metabolite inhibits oxidative modification of small dense low-density lipoprotein.
        J Cardiovasc Pharmacol. 2013; 62: 160-166
        • Aviram M
        • Rosenblat M
        • Bisgaier CL
        • Newton RS.
        Atorvastatin and gemfibrozil metabolites, but not the parent drugs, are potent antioxidants against lipoprotein oxidation.
        Atherosclerosis. 1998; 138: 271-280
        • Mason RP
        • Jacob RF.
        Membrane microdomains and vascular biology: emerging role in atherogenesis.
        Circulation. 2003; 107: 2270-2273
        • Kellner-Weibel G
        • Yancey PG
        • Jerome WG
        • Walser T
        • Mason RP
        • Phillips MC
        • et al.
        Crystallization of free cholesterol in model macrophage foam cells.
        Arterioscler Thromb Vasc Biol. 1999; 19: 1891-1898
        • Duewell P
        • Kono H
        • Rayner KJ
        • Sirois CM
        • Vladimer G
        • Bauernfeind FG
        • et al.
        NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.
        Nature. 2010; 464: 1357-1361
        • Rajamäki K
        • Lappalainen J
        • Oörni K
        • Välimäki E
        • Matikainen S
        • Kovanen PT
        • et al.
        Cholesterol crystals activate the NLRP3 inflammasome in human macrophages: a novel link between cholesterol metabolism and inflammation.
        PLoS One. 2010; 5: e11765
        • Amiya E
        • Watanabe M
        • Takeda N
        • Saito T
        • Shiga T
        • Hosoya Y
        • et al.
        Angiotensin II impairs endothelial nitric-oxide synthase bioavailability under free cholesterol-enriched conditions via intracellular free cholesterol-rich membrane microdomains.
        J Biol Chem. 2013; 288: 14497-14509
        • Abela GS
        • Aziz K.
        Cholesterol crystals rupture biological membranes and human plaques during acute cardiovascular events—a novel insight into plaque rupture by scanning electron microscopy.
        Scanning. 2006; 28: 1-10
        • Phillips JE
        • Geng YJ
        • Mason RP.
        7-Ketocholesterol forms crystalline domains in model membranes and murine aortic smooth muscle cells.
        Atherosclerosis. 2001; 159: 125-135
        • Abela GS
        • Aziz K.
        Cholesterol crystals cause mechanical damage to biological membranes: a proposed mechanism of plaque rupture and erosion leading to arterial thrombosis.
        Clin Cardiol. 2005; 28: 413-420
      6. Small DM. George Lyman Duff memorial lecture. Progression and regression of atherosclerotic lesions. Insights from lipid physical biochemistry. Arteriosclerosis: An Official Journal of the American Heart Association, Inc. 1988;8:103-29.

        • Jakus V
        • Rietbrock N.
        Advanced glycation end-products and the progress of diabetic vascular complications.
        Physiol Res. 2004; 53: 131-142
        • Pennathur S
        • Heinecke JW.
        Mechanisms for oxidative stress in diabetic cardiovascular disease.
        Antioxid Redox Signal. 2007; 9: 955-969
        • Pinel A
        • Pitois E
        • Rigaudiere J-P
        • Jouve C
        • De Saint-Vincent S
        • Laillet B
        • et al.
        EPA prevents fat mass expansion and metabolic disturbances in mice fed with a Western diet [S].
        Journal of Lipid Research. 2016; 57: 1382-1397
        • Al Rijjal D
        • Liu Y
        • Lai M
        • Song Y
        • Danaei Z
        • Wu A
        • et al.
        Vascepa protects against high-fat diet-induced glucose intolerance.
        insulin resistance, and impaired β-cell function. iScience. 2021; : 24
        • Pal A
        • Al-Shaer AE
        • Guesdon W
        • Torres MJ
        • Armstrong M
        • Quinn K
        • et al.
        Resolvin E1 derived from eicosapentaenoic acid prevents hyperinsulinemia and hyperglycemia in a host genetic manner.
        The FASEB Journal. 2020; 34: 10640-10656
        • Bhatt DL
        • Brinton EA
        • Miller M
        • Steg PG
        • Jacobson TA
        • Ketchum S
        • et al.
        Icosapent ethyl reduces cardiovascular risk substantially and consistently regardless of waist circumference.
        Journal of the American College of Cardiology. 2022; 79: 1556
        • Tulenko TN
        • Chen M
        • Mason PE
        • Mason RP.
        Physical effects of cholesterol on arterial smooth muscle membranes: Evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis.
        J Lipid Res. 1998; 39: 947-956
        • Paragh G
        • Kovács É
        • Seres I
        • Keresztes T
        • Balogh Z
        • Szabó J
        • et al.
        Altered signal pathway in granulocytes from patients with hypercholesterolemia.
        Journal of Lipid Research. 1999; 40: 1728-1733
        • Fang Y
        • Mohler ER
        • Hsieh E
        • Osman H
        • Hashemi SM
        • Davies PF
        • et al.
        Hypercholesterolemia Suppresses Inwardly Rectifying K+Channels in Aortic Endothelium In Vitro and In Vivo.
        Circulation Research. 2006; 98: 1064-1071
        • Brzustowicz MR
        • Cherezov V
        • Caffrey M
        • Stillwell W
        • Wassall SR.
        Molecular organization of cholesterol in polyunsaturated membranes: microdomain formation.
        Biophys J. 2002; 82: 285-298
        • Kucerka N
        • Marquardt D
        • Harroun TA
        • Nieh MP
        • Wassall SR
        • de Jong DH
        • et al.
        Cholesterol in bilayers with PUFA chains: doping with DMPC or POPC results in sterol reorientation and membrane-domain formation.
        Biochemistry. 2010; 49: 7485-7493
        • Kucerka N
        • Marquardt D
        • Harroun TA
        • Nieh MP
        • Wassall SR
        • Katsaras J.
        The functional significance of lipid diversity: orientation of cholesterol in bilayers is determined by lipid species.
        J Am Chem Soc. 2009; 131: 16358-16359
        • Ruocco MJ
        • Shipley GG.
        Interaction of cholesterol with galactocerebroside and galactocerebroside-phosphatidylcholine bilayer membranes.
        Biophys J. 1984; 46: 695-707
        • Mason RP
        • Moisey DM
        • Shajenko L.
        Cholesterol alters the binding of Ca2+ channel blockers to the membrane lipid bilayer.
        Mol Pharmacol. 1992; 41: 315-321
        • Stary HC
        • Chandler AB
        • Dinsmore RE
        • Fuster V
        • Glagov S
        • Insull W
        • et al.
        A Definition of Advanced Types of Atherosclerotic Lesions and a Histological Classification of Atherosclerosis.
        Circulation. 1995; 92: 1355-1374
        • Falk E.
        Pathogenesis of Atherosclerosis.
        Journal of the American College of Cardiology. 2006; 47: C7-C12
        • Virmani R
        • Kolodgie FD
        • Burke AP
        • Farb A
        • Schwartz SM.
        Lessons From Sudden Coronary Death.
        Arteriosclerosis, Thrombosis, and Vascular Biology. 2000; 20: 1262-1275
        • Vlachopoulos C
        • Aznaouridis K
        • Stefanadis C.
        Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis.
        J Am Coll Cardiol. 2010; 55: 1318-1327
        • Erdmann J
        • Stark K
        • Esslinger UB
        • Rumpf PM
        • Koesling D
        • de Wit C
        • et al.
        Dysfunctional nitric oxide signalling increases risk of myocardial infarction.
        Nature. 2013; 504: 432-436