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Canadian Journal of Cardiology

Do Oxidized Lipoproteins Cause Atherosclerotic Cardiovascular Diseases?

  • Benoit J. Arsenault
    Correspondence
    Corresponding author: Dr Benoit J. Arsenault, Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Y-2110, Pavillon Marguerite D'Youville, 2725 chemin Ste-Foy, Québec, Quebec G1V 4G5, Canada. Tel.: +1-418-656-8711 ×3498.
    Affiliations
    Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada

    Department of Medicine, Faculty of Medicine, Université Laval, Québec City, Québec, Canada
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  • Raphaëlle Bourgeois
    Affiliations
    Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada

    Department of Medicine, Faculty of Medicine, Université Laval, Québec City, Québec, Canada
    Search for articles by this author
  • Patrick Mathieu
    Affiliations
    Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada

    Department of Surgery, Faculty of Medicine, Université Laval, Québec City, Québec, Canada
    Search for articles by this author
Published:October 06, 2017DOI:https://doi.org/10.1016/j.cjca.2017.09.016
      Decades of research in cardiovascular disease (CVD) risk factors have yielded substantial evidence for a causal role for circulating apolipoprotein B-containing lipoproteins in the pathophysiology of a broad range of CVDs such as coronary artery disease, calcific aortic valve stenosis (CAVS), and ischemic stroke. Such evidence, which emerges from experimental preclinical models, follow-up of patients with inherited lipid disorders causing lifelong exposure to elevated low-density lipoprotein (LDL) cholesterol levels, and more importantly randomized clinical trials of LDL cholesterol-lowering therapy, now support the notion that interventions that reduce the number of circulating apolipoprotein B-containing particles translate into cardiovascular benefits.
      • Pedersen T.R.
      The success story of LDL cholesterol lowering.
      • Baigent C.
      • Keech A.
      • Kearney P.M.
      • et al.
      Cholesterol Treatment Trialists’ (CTT) Collaborators
      Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins.
      • Cannon C.P.
      • Blazing M.A.
      • Giugliano R.P.
      • et al.
      Ezetimibe added to statin therapy after acute coronary syndromes.
      • Sabatine M.S.
      • Giugliano R.P.
      • Keech A.C.
      • et al.
      Evolocumab and clinical outcomes in patients with cardiovascular disease.
      • Anderson T.J.
      • Grégoire J.
      • Pearson G.J.
      • et al.
      2016 Canadian Cardiovascular Society guidelines for the management of dyslipidemia for the prevention of cardiovascular disease in the adult.
      Most apolipoprotein B-containing lipoproteins in the circulation are LDL particles. Other apolipoprotein B-containing lipoproteins include very low-density lipoproteins, intermediate-density lipoproteins, and lipoprotein(a) [Lp(a)], which is a highly atherogenic lipoprotein particle that consists of an LDL particle bound to liver-derived apolipoprotein(a). Although LDL cholesterol levels predict cardiovascular events in the general population, the “penetrance” of high LDL cholesterol is far from perfect, with events occurring in individuals with untreated LDL cholesterol in the normal range and others with high LDL cholesterol free from CVD.
      • Arsenault B.J.
      • Boekholdt S.M.
      • Kastelein J.J.
      Lipid parameters for measuring risk of cardiovascular disease.
      Obviously, this observation could be attributable to other risk determinants (smoking, elevated blood pressure, type 2 diabetes, poor diet, physical inactivity, obesity, Lp[a]), but many studies have shown that such a discrepancy could also be attributable to interindividual variability in LDL particle properties. LDL particles are heterogeneous with respect to size, density, number, protein, and lipid cargo.
      • Scheffer P.G.
      • Bos G.
      • Volwater H.G.
      • et al.
      Associations of LDL size with in vitro oxidizability and plasma levels of in vivo oxidized LDL in type 2 diabetic patients.
      For example, it has been shown that smaller and denser LDL particles predict the risk of CVD in the general population.
      • Arsenault B.J.
      • Lemieux I.
      • Després J.P.
      • et al.
      Cholesterol levels in small LDL particles predict the risk of coronary heart disease in the EPIC-Norfolk prospective population study.
      Oxidized LDL (OxLDL) particles have also been suggested to play an important role in various steps of the pathological process that leads to atherosclerosis and CAVS
      • Nsaibia M.J.
      • Boulanger M.C.
      • Bouchareb R.
      • et al.
      OxLDL-derived lysophosphatidic acid promotes the progression of aortic valve stenosis through a LPAR1-RhoA-NF-κB pathway.
      (as shown in Fig. 1).
      • Mathieu P.
      • Boulanger M.C.
      Basic mechanisms of calcific aortic valve disease.
      LDL particles can be oxidatively modified by enzymatic and nonenzymatic mechanisms.
      • Christoph J.B.
      • Nikolina P.M.
      • Joseph L.W.
      Innate sensing of oxidation-specific epitopes in health and disease.
      Enzymes involved in OxLDL generation (mostly from polyunsaturated-containing phospholipids at the surface of LDL particles) include lipoxygenases, cyclo-oxygenase and some phospholipases, whereas free radicals might generate OxLDLs in a nonenzymatic manner.
      Figure thumbnail gr1
      Figure 1Pathobiological mechanisms through which oxidized (Ox) low-density lipoproteins (LDLs) and lipoprotein(a) promote atherosclerosis and calcific aortic valve stenosis (CAVS). (A) LDL, OxLDL, and oxidized phospholipids (OxPLs) enter into the subendothelial space. They are involved in the recruitment, activation, and proliferation of monocytes and macrophages. This process is observed in the early stages of atherosclerotic diseases. Activated macrophages or foam cells secrete growth factors, which leads to the migration and proliferation of smooth muscle cells (SMCs). OxLDL and OxPL also stimulate platelet adhesion and aggregation, which could explain to a certain extent the thrombotic complications observed in advanced atherosclerosis. (B) OxLDL and lipoprotein(a) are also involved in the early stages of CAVS. This pathology is also initiated by the infiltration of OxLDL into the aortic valve, which ultimately induces the transition of valve interstitial cells into osteoblast-like cells. At the molecular level, the calcification process is driven by the oxidized phospholipid content of OxLDLs, which leads to the enzymatic generation of lysophosphatidic acid (LysoPA) via autotaxin (ATX). In turn, LysoPA promotes osteogenic transition though the production of bone morphogenetic protein 2 (BMP2). Also, activation of macrophages in aortic valve by OxLDL promotes the production of tumour necrosis factor (TNF)α and interleukin (IL)1β with pro-osteogenic properties. LPAR, lysophosphatidic acid receptors; LP-PLA2, lipoprotein-associated phospholipase A2; LysoPC, lysophosphatidylcholine; sPLA2, secretory phospholipase A2.
      Circulating OxLDL are typically assessed using enzyme-linked immunosorbent assays using detecting antibodies for either oxidized phospholipids (OxPL) or malondialdehyde-modified amino groups on apolipoprotein B. Because OxLDL can be assessed in stored plasma samples with relatively high throughput, several investigations aimed at linking circulating levels of OxLDL with CVD severity or cardiovascular events have been conducted over the past 10-15 years. Although many of these studies have reported that OxLDLs are strongly associated with cardiovascular events and CAVS progression, an equal number reported modest and in many cases null associations between OxLDL and cardiovascular events, thereby bringing into question the OxLDL-hypothesis of CVD.
      In this issue of the Canadian Journal of Cardiology, Gao and colleagues
      • Gao S.
      • Zhao D.
      • Wang M.
      • et al.
      Association between circulating oxidized LDL and atherosclerotic cardiovascular disease: a meta-analysis of observational studies.
      report the results of a meta-analysis of observational studies that aimed at documenting the association between OxLDL and the risk of cardiovascular events from nested case-control or case-cohort studies as well as hospital-based or community-based cohort studies. The results of this study, which are on the basis of the analysis of 12 studies, show that circulating OxLDL is positively associated with the risk of CVD, the summary effect size of increased circulating OxLDL being 1.79 (95% confidence interval, 1.56-2.05). Sensitivity analyses revealed that the results are comparable upon investigating primary and secondary prevention separately. As expected, this study shares the limitations of most studies seeking to link a potential biomarker of interest with the risk of cardiovascular events (publication bias, reverse causality, lack of control for potential mediated risk factors), but the most important limitation of this analysis is that there are currently 3 different assays to measure OxLDL. Different epitopes, which are recognized by 4E6, DLH3, and E06 antibodies, are routinely used to measure circulating levels of OxLDL. Nine of the 12 studies used the 4E6 antibody, which is directed against oxidized apolipoprotein B, 1 study used the DLH3 antibody, which is directed against oxidized phosphatidylcholine, and 2 studies used the E06 antibody, which binds the phosphocholine headgroup of OxPLs transported by apolipoprotein B-containing lipoproteins, mostly Lp(a).
      • Leibundgut G.
      • Scipione C.
      • Yin H.
      • et al.
      Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a).
      However, sensitivity analyses performed according to the different assays revealed that the association between OxLDL and cardiovascular events was positive irrespective of how OxLDL was assessed, although the number of person-years and cardiovascular events was substantially reduced in these subanalyses.
      As described previously, there is strong evidence from experimental studies performed in vitro and in preclinical animal models of atherosclerosis and CAVS to suggest that oxidized lipoproteins likely cause atherosclerotic CVD.
      • Bouchareb R.
      • Mahmut A.
      • Nsaibia M.J.
      • et al.
      Autotaxin derived from lipoprotein(a) and valve interstitial cells promotes inflammation and mineralization of the aortic valve.
      However, it is unknown if the measurements of circulating OxLDL fully capture the atherogenicity of OxLDL, because a very significant proportion of LDL particles is most likely oxidized within the subintimal layer of arteries, exerting effects within the vascular wall rather than plasma. Additionally, to further support the oxidized lipoprotein hypothesis in atherosclerotic CVD, additional studies are needed to determine whether: (1) oxidized lipoproteins predict CVD risk independently from established risk factors; (2) the measurement of oxidized lipoproteins is clinically useful (does the assessment of oxidized lipoprotein allow reclassification of enough individuals at intermediate CVD risk in low categories if they have low levels and conversely in higher risk categories if they have high levels of oxidized lipoproteins); and (3) targeting oxidized lipoproteins specifically will ultimately translate into cardiovascular benefits.
      Most studies included in the meta-analysis of Gao et al.
      • Gao S.
      • Zhao D.
      • Wang M.
      • et al.
      Association between circulating oxidized LDL and atherosclerotic cardiovascular disease: a meta-analysis of observational studies.
      have shown that the association between oxidized lipoproteins and CVD events was independent of risk factors. Another observation supporting these findings stems from the recent study of Rao et al.,
      • Rao F.
      • Schork A.J.
      • Maihofer A.X.
      • et al.
      Heritability of biomarkers of oxidized lipoproteins: twin pair study.
      who showed that circulating levels of oxidized lipoproteins could be moderately heritable in the case of OxLDL and highly heritable in the case of OxPL, thereby suggesting that, at least in part, oxidized lipoproteins are less susceptible to environmental risk factors than previously anticipated.
      Only a few studies aimed at determining whether the assessment of oxidized lipoproteins could improve risk reclassification (both included in the meta-analysis of Gao and colleagues
      • Gao S.
      • Zhao D.
      • Wang M.
      • et al.
      Association between circulating oxidized LDL and atherosclerotic cardiovascular disease: a meta-analysis of observational studies.
      ). Gómez et al.
      • Gómez M.
      • Vila J.
      • Elosua R.
      • et al.
      Relationship of lipid oxidation with subclinical atherosclerosis and 10-year coronary events in general population.
      recently reported in the REGICOR study that the assessment of OxLDL (with the 4E6 antibody) could improve the net reclassification index in their sample of 2793 men and women from the general population followed for an average of 9 years. In another study, the Bruneck investigators showed that the assessment of OxPL on apolipoprotein B-containing lipoproteins could improve risk reclassification with a 15-year follow-up.
      • Tsimikas S.
      • Willeit P.
      • Willeit J.
      • et al.
      Oxidation-specific biomarkers, prospective 15-year cardiovascular and stroke outcomes, and net reclassification of cardiovascular events.
      It is, however, worth mentioning that these studies only included a small number of cardiovascular events (103 in the REGICOR study and 138 in the Bruneck study). Therefore, larger studies will ultimately be required to determine whether the assessment of oxidized lipoprotein helps in risk reclassification.
      Finally, and perhaps most importantly, the ultimate demonstration of a potential causal role of oxidized lipoproteins in the pathophysiology of atherosclerotic CVD will require a randomized clinical trial showing that targeting oxidized lipoproteins decreases the risk of cardiovascular events. Lipid-lowering therapy with statins has been shown to slightly decrease circulating OxLDL levels but to significantly increase OxPL and Lp(a) levels.
      • Ky B.
      • Burke A.
      • Tsimikas S.
      • et al.
      The influence of pravastatin and atorvastatin on markers of oxidative stress in hypercholesterolemic humans.
      • Capoulade R.
      • Chan K.L.
      • Yeang C.
      • et al.
      Oxidized phospholipids, lipoprotein(a), and progression of calcific aortic valve stenosis.
      Therefore, it is likely that statin-mediated cardiovascular protection is not mediated by effects on OxLDL, but rather on its ability to reduce apolipoprotein B-containing lipoproteins. Antioxidant trials (eg, with vitamin E, vitamin C, or β-carotene) did not provide any convincing evidence for cardiovascular prevention.
      • Sesso H.D.
      • Buring J.E.
      • Christen W.G.
      • et al.
      Vitamins E and C in the prevention of cardiovascular disease in men: the Physicians' Health Study II randomized controlled trial.
      • Cook N.R.
      • Albert C.M.
      • Gaziano J.M.
      • et al.
      A randomized factorial trial of vitamins C and E and beta carotene in the secondary prevention of cardiovascular events in women: results from the Women's Antioxidant Cardiovascular Study.
      • Hercberg S.
      • Galan P.
      • Preziosi P.
      • et al.
      The SU.VI.MAX study: a randomized, placebo-controlled trial of the health effects of antioxidant vitamins and minerals.
      • Guallar E.
      • Stranges S.
      • Mulrow C.
      • Appel L.J.
      • Miller 3rd, E.R.
      Enough is enough: stop wasting money on vitamin and mineral supplements [erratum in: 2014;160:143].
      However, it should be highlighted that these studies were not specifically designed to test the role of oxidized lipoproteins. Mendelian randomization studies have recently suggested that Lp(a) (which carries the larger part of OxPL in the bloodstream) might be a causal risk factor and a potential therapeutic target for coronary artery disease, stroke, heart failure, and CAVS.
      • Clarke R.
      • Peden J.F.
      • Hopewell J.C.
      • et al.
      Genetic variants associated with Lp(a) lipoprotein level and coronary disease.
      • Arsenault B.J.
      • Boekholdt S.M.
      • Dubé M.P.
      • et al.
      Lipoprotein(a) levels, genotype, and incident aortic valve stenosis: a prospective mendelian randomization study and replication in a case-control cohort.
      • Emdin C.A.
      • Khera A.V.
      • Natarajan P.
      • et al.
      Phenotypic characterization of genetically lowered human lipoprotein(a) levels.
      IONIS-APO(a)Rx is an antisense oligonucleotide targeting hepatic apolipoprotein(a) mRNA that is currently being developed to lower Lp(a) levels. Results from 2 recently reported phase II trials showed that in addition to reducing Lp(a) by 60%-70%, this compound decreases OxPL on apolipoprotein B-containing lipoproteins by approximately 50%-60%.
      • Viney N.J.
      • van Capelleveen J.C.
      • Geary R.S.
      • et al.
      Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.
      Although an important proportion of the atherogenic properties of Lp(a) is likely to be mediated by OxPL, the apolipoprotein(a) moiety of Lp(a) is also likely proatherogenic and prothrombotic. Therefore, even if this compound is successful at decreasing the risk of cardiovascular events in individuals at high risk or at slowing the progression of CAVS, we might never know if the beneficial effects of Lp(a)-targeting compounds such IONIS-APO(a)Rx will be attributable to the reduction in OxPL concentrations. One additional factor to consider is that OxPL, Lp(a), or OxLDL might have more biological relevance in the early stages of atherosclerosis and valvular calcification than in end-stage disease where other mechanisms are operative. In this regard, we have recently reported that OxPLs were positively associated with CAVS progression in young, but not in older patients with CAVS.
      • Capoulade R.
      • Chan K.L.
      • Yeang C.
      • et al.
      Oxidized phospholipids, lipoprotein(a), and progression of calcific aortic valve stenosis.
      Therefore, although a significant amount of recent research has established that oxidized lipoproteins are associated with the long-term risk of CVD, additional efforts are needed to determine whether oxidized lipoproteins are actually causal in atherosclerotic CVD.

      Funding Sources

      B.J.A. holds a junior scholarship from Fonds de Recherche du Québec-Santé. P.M. holds the Fonds de Recherche du Québec-Santé Research Chair on the Pathobiology of Calcific Aortic Valve Disease. The work of the authors is supported by the Canadian Institutes of Health Research grants to B.J.A. (FRN149068) and P.M. (FRN114893, FRN142244, FRN148778, FRN130254), and the Heart and Stroke Foundation of Canada (G-14-0005913 to P.M.).

      Disclosures

      B.J.A. is a consultant for Pfizer and has received research contracts from Ionis Pharmaceuticals and Pfizer. P.M. is a consultant for Ionis Pharmaceuticals. R.B. has no conflicts of interest to disclose.

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