European Heart Journal

Crea, Filippo

doi: 10.1093/eurheartj/ehab844pmid: 34905602

Penson, Peter E; Banach, Maciej

doi: 10.1093/eurheartj/ehab358pmid: 34151941

In the field of cardiovascular disease prevention, much recent attention has naturally focused on the remarkable opportunities afforded by novel lipid-lowering drugs, including monoclonal antibody inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9),1 and inclisiran.2 Equally important are efforts to optimize the use of existing therapies. Statins are commonly available, cheap, safe and effective drugs, which reduce the risk of cardiovascular (CV) events by ∼25% per year, for each mmol/L reduction in LDL-C.3 Whilst acknowledging that statins might cause adverse effects (including muscle symptoms, new-onset diabetes, and elevation of liver enzymes) in small numbers of treated individuals, it is increasingly clear that statin therapy is strongly associated with the ‘nocebo effect’, whereby adverse effects result from the expectation that an inert substance will relieve or cause a particular symptom. In the case of statin therapy, the ‘expectation’ of harm is fuelled by often hostile and unfounded reports on the internet, social media, and in the lay press.4 The extent of adverse effects is overestimated owing to the misattribution of unrelated symptoms (such as musculoskeletal injury).5 The resultant poor rates of compliance with statin therapy inevitably result in unnecessary cardiovascular events.4 Whilst it has long been recognized that reported rates of adverse effects of statin therapy are greater in open label than randomized trials (a fact strongly suggestive of the nocebo effect), the absolute proportion of adverse effects caused by nocebo has been hard to quantify.5 Two recent trials have shed light on the issue (Table 1), and important forthcoming guidelines from the International Lipid Expert Panel (ILEP) for the first time aim to offer practical guidelines to help patients and prescribers overcome the nocebo problem. Table 1 A summary of major studies investigating the nocebo/drucebo effect with statin therapy . Design . Participants . Intervention . Comparator . Nocebo/drucebo contribution to SAMS . Resumption of therapy . ILEP and LBPMCG Meta-analysis 11 180 Open-label statin (various) Blinded statin (various) Drucebo contributed to between 38% and 78% of muscle pain. NR SAMSON Series of n-of-1 RCTs 60 patients reporting statin side-effects Atorvastatin 20 mg/day Placebo and no treatment No difference in symptom intensity between statin and placebo. 57% had, or intended to restart statin 6 months after trial StatinWISE Series of n-of-1 RCTs 200 with SAMS Atorvastatin 20 mg/day Placebo No difference in symptom intensity between statin and placebo. 66% had or intended to restart statin at trial end. . Design . Participants . Intervention . Comparator . Nocebo/drucebo contribution to SAMS . Resumption of therapy . ILEP and LBPMCG Meta-analysis 11 180 Open-label statin (various) Blinded statin (various) Drucebo contributed to between 38% and 78% of muscle pain. NR SAMSON Series of n-of-1 RCTs 60 patients reporting statin side-effects Atorvastatin 20 mg/day Placebo and no treatment No difference in symptom intensity between statin and placebo. 57% had, or intended to restart statin 6 months after trial StatinWISE Series of n-of-1 RCTs 200 with SAMS Atorvastatin 20 mg/day Placebo No difference in symptom intensity between statin and placebo. 66% had or intended to restart statin at trial end. ILEP, International Lipid Expert Panel; LBPMCG, Lipid and Blood Pressure Meta-analysis Collaboration Group; NR, not reported; RCT, randomized controlled trial; SAMS, statin-associated muscle symptoms; SAMSON, Statin Side-effects Or Nocebo Trial. Open in new tab Table 1 A summary of major studies investigating the nocebo/drucebo effect with statin therapy . Design . Participants . Intervention . Comparator . Nocebo/drucebo contribution to SAMS . Resumption of therapy . ILEP and LBPMCG Meta-analysis 11 180 Open-label statin (various) Blinded statin (various) Drucebo contributed to between 38% and 78% of muscle pain. NR SAMSON Series of n-of-1 RCTs 60 patients reporting statin side-effects Atorvastatin 20 mg/day Placebo and no treatment No difference in symptom intensity between statin and placebo. 57% had, or intended to restart statin 6 months after trial StatinWISE Series of n-of-1 RCTs 200 with SAMS Atorvastatin 20 mg/day Placebo No difference in symptom intensity between statin and placebo. 66% had or intended to restart statin at trial end. . Design . Participants . Intervention . Comparator . Nocebo/drucebo contribution to SAMS . Resumption of therapy . ILEP and LBPMCG Meta-analysis 11 180 Open-label statin (various) Blinded statin (various) Drucebo contributed to between 38% and 78% of muscle pain. NR SAMSON Series of n-of-1 RCTs 60 patients reporting statin side-effects Atorvastatin 20 mg/day Placebo and no treatment No difference in symptom intensity between statin and placebo. 57% had, or intended to restart statin 6 months after trial StatinWISE Series of n-of-1 RCTs 200 with SAMS Atorvastatin 20 mg/day Placebo No difference in symptom intensity between statin and placebo. 66% had or intended to restart statin at trial end. ILEP, International Lipid Expert Panel; LBPMCG, Lipid and Blood Pressure Meta-analysis Collaboration Group; NR, not reported; RCT, randomized controlled trial; SAMS, statin-associated muscle symptoms; SAMSON, Statin Side-effects Or Nocebo Trial. Open in new tab Both recent studies employed so-called ‘n-of-one trials’ in which each participant is exposed to interventions and comparators in a randomized fashion, effectively serving as their own control. The Self-Assessment Method for Statin Side-effects Or Nocebo (SAMSON) Trial recruited 60 patients who had recently discontinued statin therapy because of side-effects. Participants had their symptoms measured over a 12-month period during which they randomly alternated between receiving statins, placebo, or no treatment.6 The reported intensity of symptoms did not differ between the periods of statin use and placebo. However, when patients were taking statin or placebo, they reported a greater intensity of symptoms than during the periods of no treatment. Patients were shown their scores at the end of the trial period, and the results were used to inform patient-centred decision-making. Six months after the trial was completed, over half of the participants had restarted statin therapy or planned to do so. The inclusion of a period without treatment in SAMSON was very important. The term ‘nocebo’ properly refers to effects elicited by an inert substance (i.e. placebo), and can be problematic when applied to drugs. The magnitude of the nocebo effect can only be properly estimated when a ‘no-treatment’ group is included in a study—as it was in SAMSON, but this is rare. Therefore, in 2018, ILEP introduced the concept of ‘drucebo’ (DRUg + noCEBO) to overcome this difficulty and to allow existing clinical trial data to be used to calculate the proportion of adverse effects attributable to expectation, rather than pharmacological effects (Figure 1).5 In the case of muscle pain on statin therapy, we found that this proportion may be as high as 78%.5 A similar study, statinWISE enrolled 200 patients who had stopped or were considering stopping statin therapy, and randomized them to six 2-month periods of atorvastatin 20 mg daily, or placebo. Similarly to SAMSON, there was no difference between the severity of adverse effects on statin therapy or placebo. Two-thirds of participants were able to resume statin therapy.7 The dramatic results of statinWISE and SAMSON demonstrate the importance of identifying and managing the nocebo/drucebo effect to avoid exposing patients to cardiovascular risk by unnecessarily ceasing lipid-lowering therapy. With respect to LDL-C ‘lower is better for longer’8 and periods of non-treatment result in higher LDL-C and greater risk of cardiovascular events. The forthcoming ILEP guidelines are therefore important and urgently needed. Whilst the ‘n-of-1’ approach used in trials provides an extremely useful demonstration of the power of the nocebo/drucebo effect, it may be difficult to implement in the clinical practice. Placebo tablets may not be available, and randomization and blinding may not be practical in routine patient care. In any event, allocating patients to periods of placebo or no treatment is undesirable as it unnecessarily exposes them to LDL-C and cardiovascular risk. The forthcoming ILEP recommendations will focus on identifying patients with serious adverse effects, and the use of objective, step-by-step approaches to identify patients with symptoms likely to result from the nocebo/drucebo effect, in whom we will recommend a range of approaches, including MEDS and SLAP, which were previously briefly presented at European Society of Cardiology (ESC) Congress 2019 as the ILEP guidance on statin intolerance.9 Briefly, MEDS is a mnemonic encompassing essential considerations in all patients reporting adverse effects with statin therapy: Minimising disruption to lipid-lowering therapy—the cornerstone of management of cardiovascular risk. Providing high-quality, accessible, personalized, continuous Education relating to the benefits of statin therapy, and an objective assessment of risks. Patients should receive evidence-based advice about Diet, lifestyle changes, and nutraceuticals to reduce cardiovascular risk, and careful attention should be made to the intensity of Symptoms and biomarkers. SLAP provides a series of interventions, which can be used in patients with partial intolerance, who may be still able to tolerate statin therapy, but not at guideline-recommended doses. These include: Switch statins (a patient may have an adverse reaction to a particular drug, or even formulation). Lower dose (and add non-statin therapy, e.g. ezetimibe) or Alternate day dosing, which may be employed when adverse effects appear to be dose-dependent. However, care should be taken that such approaches do not reinforce the patients view that symptoms are caused by the statin—as they may be employed when symptoms are at their worst, and spontaneous resolution is likely. Finally, Polypharmacy (immediate combination lipid-lowering therapy or non-statin therapy), using ezetimibe, PCSK9 inhibitors, bempedoic acid, inclisiran, and other evidence-based therapies (including nutraceutical polypills) may be necessary to reach lipid-targets.10 The abovementioned recommendations of the ILEP experts will be published in the coming months, and we hope that they will benefit physicians and patients alike and improve access to life-saving lipid-lowering therapies. Conflict of interest: P.E.P. owns four shares in AstraZeneca PLC and has received honoraria and/or travel reimbursement for events sponsored by AKCEA, Amgen, AMRYT, Link Medical, Mylan, Napp, and Sanofi; M.B.—speakers bureau: Amgen, Esperion, Herbapol, Kogen, KRKA, Novartis, Polpharma, sanofi-aventis, Servier, Teva, Viatris, and Zentiva; consultant to Akcea, Amgen, Daichii Sankyo, Esperion, Freia Pharmaceuticals, Polfarmex, and sanofi-aventis; grants from Amgen, Viatris, Sanofi, and Valeant. Figure 1 Open in new tabDownload slide Nocebo, drucebo, and pharmacological effects explained. The nocebo effect refers to adverse effects experienced when taking an inert substance (i.e. the difference in symptom intensity between no treatment, and an inert tablet) and is analogous to the placebo effect (albeit with adverse rather than desired symptoms). The drucebo effect is defined as the difference in the frequency or intensity of symptoms between blinded and open-label use of a drug. The difference between symptoms experienced with an inert tablet and an apparently identically drug-containing tablet represents the true pharmacological effect of the drug. Image created using Biorender.com. Figure 1 Open in new tabDownload slide Nocebo, drucebo, and pharmacological effects explained. The nocebo effect refers to adverse effects experienced when taking an inert substance (i.e. the difference in symptom intensity between no treatment, and an inert tablet) and is analogous to the placebo effect (albeit with adverse rather than desired symptoms). The drucebo effect is defined as the difference in the frequency or intensity of symptoms between blinded and open-label use of a drug. The difference between symptoms experienced with an inert tablet and an apparently identically drug-containing tablet represents the true pharmacological effect of the drug. Image created using Biorender.com. References 1 Banach M , Penson PE. What have we learned about lipids and cardiovascular risk from PCSK9 inhibitor outcome trials: ODYSSEY and FOURIER? Cardiovasc Res 2019 ; 115 : e26 – e31 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Dyrbuś K , Gąsior M, Penson P, Ray KK, Banach M. Inclisiran-New hope in the management of lipid disorders? J Clin Lipidol 2020 ; 14 : 16 – 27 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Collins R , Reith C, Emberson J, Armitage J, Baigent C, Blackwell L, Blumenthal R, Danesh J, Smith GD, DeMets D, Evans S, Law M, MacMahon S, Martin S, Neal B, Poulter N, Preiss D, Ridker P, Roberts I, Rodgers A, Sandercock P, Schulz K, Sever P, Simes J, Smeeth L, Wald N, Yusuf S, Peto R. Interpretation of the evidence for the efficacy and safety of statin therapy . Lancet 2016 ; 388 : 2532 – 2561 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Nissen SE. Statin Denial: an internet-driven cult with deadly consequences . Ann Intern Med 2017 ; 167 : 281 – 282 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Penson PE , Mancini GBJ, Toth PP, Martin SS, Watts GF, Sahebkar A, Mikhailidis DP, Banach M; Lipid and Blood Pressure Meta-Analysis Collaboration (LBPMC) Group; International Lipid Expert Panel (ILEP) . Introducing the ‘Drucebo’ effect in statin therapy: a systematic review of studies comparing reported rates of statin-associated muscle symptoms, under blindedand open-label conditions . J Cachexia Sarcopenia Muscle 2018 ; 9 : 1023 – 1033 . Google Scholar Crossref Search ADS PubMed WorldCat 6 Wood FA , Howard JP, Finegold JA, Nowbar AN, Thompson DM, Arnold AD, Rajkumar CA, Connolly S, Cegla J, Stride C, Sever P, Norton C, Thom SAM, Shun-Shin MJ, Francis DP. N-of-1 trial of a statin, placebo, or no treatment to assess side effects . N Engl J Med 2020 ; 383 : 2182 – 2184 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Herrett E , Williamson E, Brack K, Beaumont D, Perkins A, Thayne A, Shakur-Still H, Roberts I, Prowse D, Goldacre B, van Staa T, MacDonald TM, Armitage J, Wimborne J, Melrose P, Singh J, Brooks L, Moore M, Hoffman M, Smeeth L; StatinWISE Trial Group . Statin treatment and muscle symptoms: series of randomised, placebo controlled n-of-1 trials . BMJ 2021 ; 372 : n135 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 8 Penson PE , Pirro M, Banach M. LDL-C: lower is better for longer-even at low risk . BMC Med 2020 ; 18 : 320 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Penson P , Toth P, Mikhailidis D, Ezhov M, Fras Z, Mitchenko O, Pella D, Sahebkar A, Rysz J, Reiner Z, Jozwiak J, Mazidi M, Banach M. P705Step by step diagnosis and management of statin intolerance: position paper from an International Lipid Expert Panel . Eur Heart J 2019 ; 40 (Suppl 1):ehz747.0310. Google Scholar OpenURL Placeholder Text WorldCat 10 Banach M , Penson PE, Vrablik M, Bunc M, Dyrbus K, Fedacko J, Gaita D, Gierlotka M, Jarai Z, Magda SL, Margetic E, Margoczy R, Durak-Nalbantic A, Ostadal P, Pella D, Trbusic M, Udroiu CA, Vlachopoulos C, Vulic D, Fras Z, Dudek D, Reiner Z; ACS EuroPath Central & South European Countries Project . Optimal use of lipid-lowering therapy after acute coronary syndromes: a Position Paper endorsed by the International Lipid Expert Panel (ILEP) . Pharmacol Res 2021 ; 166 : 105499 . Google Scholar Crossref Search ADS PubMed WorldCat Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Pirillo, Angela; Catapano, Alberico Luigi

doi: 10.1093/eurheartj/ehab511pmid: 34389860

This editorial refers to ‘Directly measured vs. calculated remnant cholesterol identifies additional overlooked individuals in the general population at higher risk of myocardial infarction’, by A. Varbo and B.G. Nordestgaard, doi: 10.1093/eurheartj/ehab293. Graphical Abstract Open in new tabDownload slide ApoB-containing lipoproteins are secreted either by the intestine (chylomicrons, apoB48) or the liver (VLDL, apoB100) and immediately undergo lipolysis to generate triglyceride (TG)-rich lipoprotein remnants. Lipoprotein classification is based on diameter (nm) and density (g/mL); the TG to cholesterol ratio decreases with the density and size of lipoproteins. What does remnant cholesterol estimation quantify? Graphical Abstract Open in new tabDownload slide ApoB-containing lipoproteins are secreted either by the intestine (chylomicrons, apoB48) or the liver (VLDL, apoB100) and immediately undergo lipolysis to generate triglyceride (TG)-rich lipoprotein remnants. Lipoprotein classification is based on diameter (nm) and density (g/mL); the TG to cholesterol ratio decreases with the density and size of lipoproteins. What does remnant cholesterol estimation quantify? Elevated LDL is a recognized causal factor for cardiovascular disease (CVD) and we have effective therapeutic approaches to its lowering, allowing a robust reduction of cardiovascular (CV) events. However, residual lipoprotein-dependent CV risk remains even in the presence of an optimal lipid-lowering therapy.1 In search for other ‘suspects’, plasma triglycerides (TGs) are back on the scene given that a number of experimental and clinical studies support their role in CVD. Furthermore, the notion that a subspecies of proatherogenic TG-rich lipoproteins (the so-called remnant lipoproteins), and more specifically their associated cholesterol (remnant cholesterol, RC), can accumulate in plasma under some circumstances has emerged from recent data. But how to estimate their levels? The biochemical, biological, and pathophysiological properties of remnants are ill defined, and measured levels often do not go beyond being merely the very-low-density lipoprotein (VLDL)- and intermediate-density lipoprotein (IDL)-associated cholesterol (plus some chylomicron cholesterol in non-fasting conditions). As newly secreted VLDLs and chylomicrons immediately undergo lipolysis, any residual VLDL and chylomicron present in the blood can be defined as a remnant (Graphical Abstract). Furthermore, the definition of a remnant is a matter of semantics, as LDLs are also remnants, at least in the great majority, of the catabolism of VLDL. Epidemiological studies have suggested that abnormal RC levels are defined as ≥0.9 mmol/L (≥35 mg/dL) in non-fasting conditions and ≥0.8 mmol/L (≥30 mg/dL) in fasting conditions.2 Elevated levels of RC have been associated with an increased risk of ischaemic heart disease (IHD),3 myocardial infarction (MI),4 ischaemic stroke,5 and all-cause mortality in the general population.6 The determination of RC levels as total cholesterol minus LDL-cholesterol (LDL-C) minus HDL-cholesterol (HDL-C) concentrations has, however, limitations related to the evaluation of LDL-C and TG levels.7 High plasma TG levels, or very low levels of LDL-C, make the Friedewald equation unreliable in estimating LDL-C, thus leading to a misclassification of patients, and an increase in treatment issues, calling for an additional evaluation in high-risk patients.7 Furthermore, Friedewald-estimated LDL-C includes IDL-associated cholesterol, thus not accounting for this fraction when RC is calculated. An additional equation for the measurement of LDL-C has been proposed, which, by means of an ‘adjustable factor’, takes into account a patient’s TG and non-HDL-C values, thus increasing the accuracy of LDL-C estimation in individuals with very low levels of LDL-C or high TGs (as well as in patients already on LDL-C-lowering therapy).8 To overcome these limitations, an assay for the direct measurement of cholesterol content of remnants would help. Such an assay is available, and is based on a two-step process that includes (i) the degradation of LDL and HDL, and (ii) the measurement of cholesterol in the remaining lipoproteins (remnants). In a recent study, published in this issue of the European HeartJournal,9 Varbo and Nordestgaard explored in 16 207 individuals from the Copenhagen General Population Study whether directly measured (with the above-described method) vs. calculated RC levels in non-fasting conditions may improve the identification of individuals at increased CV risk. Their analysis showed that ∼5% of the study population with a normal calculated RC level (<0.8 mmol/L) instead had an elevated directly measured RC level, that was significantly associated with a substantial increased risk of IHD and MI.9 Based on the levels of TGs and apolipoprotein B (apoB) observed in this group, the authors suggested that the increased risk might be attributed to a subspecies of remnant particles that, although present in lower number (as demonstrated by the lower apoB level), appear to be enriched in cholesterol, which would make these particles more atherogenic. The authors concluded that directly measured RC levels can re-stratify 5% of individuals in the general population who are at increased CV risk, but would be lost with the ‘simple’ calculation of RC levels. It must be noted that, independently of the method used to obtain RC levels (calculated vs. measured), subjects with RC above the 80th percentile have similar increased risks of IHD and MI.9 However, when considering the two groups each characterized by having only measured or calculated levels of RC above the 80th percentile, these two parameters showed a discordant association with the risk of IHD and MI. In fact, elevated measured RC levels were associated with an increased risk of both IHD and MI [hazard ratio (HR) 1.42 and 1.83, respectively], whereas elevated calculated RC levels were not (HR 1.14 and 1.14, respectively). This is at odds with previous studies where calculated levels similar to those observed in this study (1.0 mmol/L) were associated with a significantly increased risk of IHD and MI.3,6 Further, measured and calculated levels of RC in this study population are quite different, with directly measured levels being much higher than the calculated levels. Why is there a lack of correspondence between calculated and measured RC levels? Significant discrepancies were also observed by using different methodologies, such as nuclear magnetic resonance.10–12 Thus, it appears that the results are heavily dependent on the method used for RC determination. The authors state that this assay, at variance with calculated RC, allows the inclusion of cholesterol carried by IDL; however, beside the lack of any accurate characterization of the lipoproteins measured by the assay, it appears that the discrepancy is excessive and may not be explained solely by the inclusion of IDL. Furthermore, the correlation with CV risk appears to remain the same, meaning that IDL will have exactly the same atherogenic impact as ‘remnants’ in the chylomicron–VLDL range. To find another possible explanation for these discrepancies, the authors measured RC in samples frozen for 2 years and compared them with the values obtained in fresh samples from the same 101 individuals: although the association was linear, there was a marked divergence, with the results from frozen samples being much higher, albeit the obtained values correlated quite well. It would also have been of interest to compare, in the same population, calculated and freshly measured RC values: this information could help to answer the question of whether directly measured RC has an additional predictive value. The same authors have studied the association between calculated and directly measured RC (on fresh samples) and reported that measured RC increased 0.91 mmol/L per 1 mmol/L increase in calculated RC.13 The great difference reported here9 raises a further question: as this study used samples from individuals recruited between 2003 and 2010, can their measured RC levels be compared with each other? In other words, can a different storage duration influence the assay? We have no answer to these questions, as the definition of a percentile scale, and thus the categorization of individuals, is based on values determined during the study, and thus it might have been influenced by storage time. Discrepancies in the categorization of individuals were also reported: when subjects were divided by categories of calculated and measured RC, 91% of individuals in the lowest group [<0.5 mmol/L (<19 mg/dL)] of calculated RC were also in the lowest category of measured RC, but this did not apply to higher categories of RC concentrations.13 Of note, in the present study, the group with only high directly measured RC also showed elevated LDL-C levels, well above those recommended by current guidelines for a low-risk population (3.7 mmol/L vs. 3.0 mmol/L).9,14 How can these findings be translated at the individual level? The definition of the individual CV risk is based on the determination of several factors. People from the general population (considered to be at low CV risk) having LDL-C levels within the normal range may be at increased CV risk, due to the presence of concomitant risk factors other than LDL-C, and RC determination may help to identify subjects that may escape this definition. In clinical practice, calculated RC level is available at no extra cost, and several studies have unequivocally shown its causal association with IHD, MI, and ischaemic stroke.3–5 What does the directly measured RC level add? Identifying people at increased CV risk despite having a ‘normal’ LDL-C profile in clinical practice represents a plus in the prevention of CVD, and every factor that can improve the definition of the individual CV risk is a step towards the prevention. Altogether, these observations, on the one hand, support the predictive value of RC levels, and on the other hand they further highlight the complexity of this approach, and call for the need to establish a carefully standardized and reproducible definition of RC quantification, with a clear understanding of what the proposed assay determines. In fact, there is currently no consensus assay or established objective definition of RC for their quantification, and the difficulty of accurately quantifying the levels of these lipoproteins represents a significant challenge in studying remnants and their involvement in CVD. As it stands now, the definition of remnant lipoprotein is quite vague. No clear understanding of the origin of excess cholesterol (lipoprotein?) as compared with that present in VLDL is available, neither is the nature of the lipoproteins termed remnants unequivocally available. From the data that are available, it must be beyond the VLDL range. Is this the so-called IDL? Or a subfraction of LDL? And how does it relate to atherosclerosis? Again, the need for a clear definition of remnants has raised its head, as it is implicit in the concept of something that is remaining after delipidation, but at the same time is accumulating, as its removal is impaired. Further research is warranted to obtain a recognized clinical method for accurate measuring of RC levels, which should include an in-depth characterization of measured lipoproteins and the validation in other populations. This will allow the comparison between studies and the validation of RC as a potential target for therapy. Conflict of interest: none declared The opinions expressed in this article are not necessarily those of the Editors of the European Heart Journal or of the European Society of Cardiology. References 1 Shaya GE , Leucker TM, Jones SR, Martin SS, Toth PP. Coronary heart disease risk: low-density lipoprotein and beyond . Trends Cardiovasc Med 2021 ; doi: 10.1016/j.tcm.2021.04.002 . Google Scholar OpenURL Placeholder Text WorldCat Crossref 2 Nordestgaard BG , Langsted A, Mora S, Kolovou G, Baum H, Bruckert E, Watts GF, Sypniewska G, Wiklund O, Boren J, Chapman MJ, Cobbaert C, Descamps OS, von Eckardstein A, Kamstrup PR, Pulkki K, Kronenberg F, Remaley AT, Rifai N, Ros E, Langlois M; European Atherosclerosis Society (EAS) and the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) joint consensus initiative . Fasting is not routinely required for determination of a lipid profile: clinical and laboratory implications including flagging at desirable concentration cut-points—a joint consensus statement from the European Atherosclerosis Society and European Federation of Clinical Chemistry and Laboratory Medicine . Eur Heart J 2016 ; 37 : 1944 – 1958 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Varbo A , Benn M, Tybjaerg-Hansen A, Jorgensen AB, Frikke-Schmidt R, Nordestgaard BG. Remnant cholesterol as a causal risk factor for ischemic heart disease . J Am Coll Cardiol 2013 ; 61 : 427 – 436 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Jorgensen AB , Frikke-Schmidt R, West AS, Grande P, Nordestgaard BG, Tybjaerg-Hansen A. Genetically elevated non-fasting triglycerides and calculated remnant cholesterol as causal risk factors for myocardial infarction . Eur Heart J 2013 ; 34 : 1826 – 1833 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Varbo A , Nordestgaard BG. Remnant cholesterol and risk of ischemic stroke in 112,512 individuals from the general population . Ann Neurol 2019 ; 85 : 550 – 559 . Google Scholar Crossref Search ADS PubMed WorldCat 6 Varbo A , Freiberg JJ, Nordestgaard BG. Extreme nonfasting remnant cholesterol vs extreme LDL cholesterol as contributors to cardiovascular disease and all-cause mortality in 90000 individuals from the general population . Clin Chem 2015 ; 61 : 533 – 543 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Martin SS , Blaha MJ, Elshazly MB, Brinton EA, Toth PP, McEvoy JW, Joshi PH, Kulkarni KR, Mize PD, Kwiterovich PO, Defilippis AP, Blumenthal RS, Jones SR. Friedewald-estimated versus directly measured low-density lipoprotein cholesterol and treatment implications . J Am Coll Cardiol 2013 ; 62 : 732 – 739 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Martin SS , Blaha MJ, Elshazly MB, Toth PP, Kwiterovich PO, Blumenthal RS, Jones SR. Comparison of a novel method vs the Friedewald equation for estimating low-density lipoprotein cholesterol levels from the standard lipid profile . JAMA 2013 ; 310 : 2061 – 2068 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Varbo A , Nordestgaard BG. Directly measured vs. calculated remnant cholesterol identifies additional overlooked individuals in the general population at higher risk of myocardial infarction . Eur Heart J 2021 ; doi: 10.1093/eurheartj/ehab293 . Google Scholar OpenURL Placeholder Text WorldCat Crossref 10 Chen J , Kuang J, Tang X, Mao L, Guo X, Luo Q, Peng D, Yu B. Comparison of calculated remnant lipoprotein cholesterol levels with levels directly measured by nuclear magnetic resonance . Lipids Health Dis 2020 ; 19 : 132 . Google Scholar Crossref Search ADS PubMed WorldCat 11 Balling M , Langsted A, Afzal S, Varbo A, Davey Smith G, Nordestgaard BG. A third of nonfasting plasma cholesterol is in remnant lipoproteins: lipoprotein subclass profiling in 9293 individuals . Atherosclerosis 2019 ; 286 : 97 – 104 . Google Scholar Crossref Search ADS PubMed WorldCat 12 Remaley AT , Otvos JD. Methodological issues regarding: ‘A third of nonfasting plasma cholesterol is in remnant lipoproteins:lLipoprotein subclass profiling in 9293 individuals’ . Atherosclerosis 2020 ; 302 : 55 – 56 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Varbo A , Freiberg JJ, Nordestgaard BG. Remnant cholesterol and myocardial infarction in normal weight, overweight, and obese individuals from the Copenhagen General Population Study . Clin Chem 2018 ; 64 : 219 – 230 . Google Scholar Crossref Search ADS PubMed WorldCat 14 Mach F , Baigent C, Catapano AL, Koskinas KC, Casula M, Badimon L, Chapman MJ, De Backer GG, Delgado V, Ference BA, Graham IM, Halliday A, Landmesser U, Mihaylova B, Pedersen TR, Riccardi G, Richter DJ, Sabatine MS, Taskinen MR, Tokgozoglu L, Wiklund O; ESC Scientific Document Group . 2019 ESC/EAS Guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular risk . Eur Heart J 2020 ; 41 : 111 – 188 . Google Scholar Crossref Search ADS PubMed WorldCat Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

van Solingen, Coen; Moore, Kathryn J

doi: 10.1093/eurheartj/ehab640pmid: 34571536

This editorial refers to ‘Macrophage NFATc3 prevents foam cell formation and atherosclerosis: evidence and mechanisms’, by X. Liu et al., doi:10.1093/eurheartj/ehab660. Graphical Abstract Open in new tabDownload slide The transcription factor Nuclear Factor of Activated T-cell isoform c3 (NFATc3) acts as a key repressor of macrophage foam cell formation by inducing expression of microRNA-204 (miR-204). The two strands of the miR-204 duplex act through canonical and non-canonical functions to repress the expression scavenger receptors and thus, lipoprotein uptake. miR-204-5p binds the 3’-untranslated region (3’-UTR) of the SR-A mRNA to mediate translational repression and miR-204-3p is imported into the nucleus where it binds to the promoter region of Cd36 to repress its transcription. Graphical Abstract Open in new tabDownload slide The transcription factor Nuclear Factor of Activated T-cell isoform c3 (NFATc3) acts as a key repressor of macrophage foam cell formation by inducing expression of microRNA-204 (miR-204). The two strands of the miR-204 duplex act through canonical and non-canonical functions to repress the expression scavenger receptors and thus, lipoprotein uptake. miR-204-5p binds the 3’-untranslated region (3’-UTR) of the SR-A mRNA to mediate translational repression and miR-204-3p is imported into the nucleus where it binds to the promoter region of Cd36 to repress its transcription. Atherosclerosis is a chronic inflammatory disease occurring in the setting of dyslipidemia, in which lipid laden macrophages accumulate in the artery wall. Macrophage lipoprotein uptake by scavenger receptors contributes prominently to the formation of foam cells in atherosclerotic plaques, and strategies to block foam cell formation hold therapeutic promise. In this issue of the European Heart Journal, Liu et al.1 report that the transcription factor Nuclear Factor of Activated T-cell isoform c3 (NFATc3) represses macrophage foam cell formation by inducing expression of the microRNA miR-204, which acts via canonical and non-canonical functions to curb scavenger receptor expression. Atherosclerotic cardiovascular disease (CVD) remains a leading cause of death in developed nations.2 Despite marked advances in the fight against CVD in the late 20th century, CVD mortality is no longer declining, emphasizing the ongoing need to better understand the molecular mechanisms underlying atherosclerosis and to develop novel therapies for its treatment.3 Atherosclerosis results from an inflammatory response triggered by the accumulation of lipoproteins in the subendothelial space of arteries. Tissue resident and monocyte-derived macrophages attempt to clear these retained lipoproteins resulting in foam cell formation—a hallmark of atherosclerotic plaques.4 This transformation is driven by the internalization of modified lipoproteins through scavenger receptors, with the class A scavenger receptor (SR-A) and CD36 accounting for the majority of lipid uptake.5,6 In this issue of the European Heart Journal, Liu et al1 identify the transcription factor Nuclear Factor of Activated T-cell isoform c3 (NFATc3) as a key repressor of macrophage foam cell formation that curbs the expression of SR-A and CD36 via canonical and non-canonical actions of microRNA-204 (miR-204). NFAT family proteins have emerged as important transcriptional regulators of gene expression in CVD. NFATc3, an isoform highly expressed in macrophages,1 has previously been shown to inhibit foam cell formation in vitro,7 however the underlying mechanisms were unclear. Liu et al showed that NFATc3 mRNA levels are downregulated in peripheral blood mononuclear cells of patients with carotid atherosclerosis when compared to healthy controls, particularly in symptomatic patients compared to asymptomatic individuals.1 Furthermore, macrophages present in plaques from human carotid arteries or mouse models of atherosclerosis showed reduced levels of nuclear NFATc3 protein. Using myeloid-specific loss of function mouse models, the authors established that ablation of macrophage NFATc3 expression advances the development of atherosclerosis in mice on a high fat and high cholesterol western-style diet compared to control mice fed a similar diet. Conversely, macrophage-specific overexpression of NFATc3 diminished atherosclerotic plaque area and necrotic core formation, and increased collagen content indicating a more stable plaque phenotype. In vitro experiments performed in NFATc3-depleted and NFATc3-overexpressing macrophages revealed that higher NFATc3 levels correlated with reduced macrophage foam cell formation and inflammatory responses, a phenotype that would be expected to reduce atherosclerosis. Mechanistic studies revealed that NFATc3 regulates macrophage uptake of lipoproteins by modulating expression of the scavenger receptors SR-A and CD36 in vitro and in atherosclerotic plaques. Targeted deletion of NFATc3 in macrophages enhanced expression of these receptors, while other cholesterol transporters such as SR-B1 and ABCG1 remain unaffected. This effect was specific for NFATc3, as knock-down of other NFAT isoforms did not affect SR-A or CD36 levels. Notably, although four potential binding sites for NFATc3 were found in each of the promoter regions of SR-A and CD36, chromatin immunoprecipitation failed to show binding of NFATc3 at these sites, suggesting an alternative mechanism of NFATc3 regulation of scavenger receptor expression. The authors next explored whether NFATc3 could indirectly regulate SR-A and CD36 expression via induction of microRNAs (miRNA) that target these scavenger receptors. A microarray screen of NFATc3-knock-down and overexpressing macrophages identified miR-204 as a candidate NFATc3-regulated miRNA, and further studies confirmed that NFATc3 binds the miR-204 promoter and upregulates expression of the miR-204 precursor, resulting in increased levels of both strands of the mature miR-204 duplex: miR-204-5p and miR-204-3p. miRNAs have been widely studied as post-transcriptional regulators of gene expression, which can bind in a complex with Argonaute 2 (Ago2) to complementary sites in the 3’ untranslated regions (3’-UTR) of target mRNAs to repress translation or induce mRNA degradation.8,9 Liu et al. demonstrated that SR-A is repressed in this manner through direct binding of miR-204-5p to the SR-A 3’-UTR; however, neither miR-204-5p nor miR-204-3p were found to act on the CD36 3’-UTR. While these canonical actions of miRNAs occur in the cytoplasm, the authors found that miR-204-3p accumulated abundantly in the nucleus. The authors discovered that miR-204-3p binds in complex with Ago2 to the promoter region of Cd36, thereby restricting access of histone modifying complexes (e.g., H3K27ac and H3K4me3) and suppressing Cd36 transcription. Although this mode of miRNA regulation is atypical, non-canonical nuclear functions of the miRNA/Ago2 complex have previously been described.10,11 The discovery that the mature miR-204 duplex gives rise to two functional miRNAs that act in the cytoplasmic and nuclear compartments to coordinately restrict scavenger receptor expression underscores the versatility of miRNAs, and reinforces their roles as orchestrators of pathway regulation. The identification of the NFATc3-regulated nuclear miR-204-3p/CD36 and cytoplasmic miR-204-5p/SR-A axes (Graphical Abstract) adds new layers to our understanding of the regulation of foam cell formation and atherogenesis. The ability of NFATc3 to coordinately inhibit the expression of two important scavenger receptors for foam cell formation, and its down-regulation in monocytes and plaque macrophages of patients with CVD, identify NFATc3 as a potential therapeutic target for the treatment of atherosclerosis. However, altering the expression of transcription factors and/or miRNAs, molecules that are by definition able to influence expression of many different genes at the same time, can have unwanted consequences that present challenges for therapeutic development. Moving forward, it will also be important to further define the actions of NFATc3 in comparison to other NFAT isoforms which have been described to be drivers of inflammation and the progression of atherosclerosis.12,13 The opinions expressed in this article are not necessarily those of the Editors of the European Heart Journal or of the European Society of Cardiology. Acknowledgements K.J.M. and C.v.S. are supported by grants from the National Institutes of Health (R35HL135799 to K.J.M.) and the American Heart Association (19CDA346300066 to C.v.S). Conflicts of interest: None. References 1 Liu X , Guo JW, Lin XC, Tuo YH, Peng WL, He SY, Li ZQ, Ye YC, Yu J, Zhang FR, Ma MM, Shang JY, Lv XF, Zhou AD, Ouyang Y, Wang C, Pang RP, Sun JX, Ou JS, Zhou JG, Liang SJ. Macrophage NFATc3 prevents foam cell formation and atherosclerosis: evidence and mechanisms . European Heart Journal 2021 doi:10.1093/eurheartj/ehab660. Google Scholar OpenURL Placeholder Text WorldCat 2 Moore KJ , Tabas I. Macrophages in the pathogenesis of atherosclerosis . Cell 2011 ; 145 : 341 – 355 . 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