Sage Journals: Discover world-class research

Abstract

An elevated low-density lipoprotein cholesterol concentration is a classical risk factor for cardiovascular disease. This has led to pharmacotherapy in patients with atherosclerotic heart disease or high heart disease risk with statins to reduce serum low-density lipoprotein cholesterol. Even in patients in whom the target levels of low-density lipoprotein cholesterol are reached, there remains a significant residual cardiovascular risk; this is due, in part, to a focus on low-density lipoprotein cholesterol alone and neglect of other important aspects of lipoprotein metabolism. A more refined lipoprotein analysis will provide additional information on the accumulation of very low-density lipoproteins, intermediate density lipoproteins, chylomicrons, chylomicron-remnants and Lp(a) concentrations. Instead of measuring the cholesterol and triglyceride content of the lipoproteins, measurement of their apolipoproteins (apos) is more informative. Apos are either specific for a particular lipoprotein or for a group of lipoproteins. In particular measurement of apos in atherogenic particles is more biologically meaningful than the measurement of the cholesterol concentration contained in these particles. Applying apo profiling will not only improve characterization of the lipoprotein abnormality, but will also improve definition of therapeutic targets. Apo profiling aligns with the concept of precision medicine by which an individual patient is not treated as ‘average’ patient by the average (dose of) therapy. This concept of precision medicine fits the unmet clinical need for stratified cardiovascular medicine. The requirements for clinical application of proteomics, including apo profiling, can now be met using robust mass spectrometry technology which offers desirable analytical performance and standardization.

Keywords

Mass spectrometry laboratory methods cardiology clinical studies proteins analytes

Introduction

Individuals with risk factors for cardiovascular disease (CVD) (which include smoking, hypertension, diabetes mellitus (DM), central adiposity and dyslipidaemia) and patients suffering from CVD may be treated with a combination of life style modification and pharmacotherapy. Currently, cardiovascular risk stratification is, in part, based on classical lipid parameters cholesterol (C), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C). Although guidelines outline an impressive list of preventive and therapeutic measures,¹ risk stratification and treatment are not always successful in the long term. There are several indicators of sub-optimal patient management, including, but not limited to: (1) residual risk despite successful therapy, e.g. with statins,² (2) underdiagnosis of CVD in women,³ and (3) the presence of atherogenic remnant disease that is hard to diagnose with current diagnostic procedures.⁴

Currently, the traditional evidence based medicine approach is used for risk stratification and therapy in patients with CVD. However, it is time for a paradigm shift from ‘reactive medicine’ that generally treats one particular item, e.g. LDL-C, to ‘proactive P4 medicine’ (predictive, preventive, personalized, and participatory), that is person-centric, based on disease mechanism and systems biology.^5,6 In this holistic approach medical decisions are based on individual patient characteristics (including biomarkers) rather than on averages over a whole population. In this review, we summarize evidence for and propose the use of a multiplexed panel of apolipoproteins (apos) as an adjunct for cardiovascular risk stratification, to facilitate implementation of precision medicine (Figure 1).

Figure 1.

Conceptual view of the proposed paradigm shift from traditional, population-based medicine with prescription roulette due to statin intolerance/poor patient medicine concordance rather than lack of statin efficacy in attaining an LDL-C target and inadequate targeting of the individual lipoprotein risk factors, to precision medicine with personalized therapies, based on biology-driven test results.

A checklist to identify clinical management decisions linking biomarker testing to health outcomes was recently developed by the Test Evaluation Working Group of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM).⁷ The present review has been structured in line with this checklist and is organized into four domains: (a) identification of the unmet clinical need (deficiencies in current clinical care and described in the present section) (b) verification of the unmet clinical need i.e. are there existing biomarkers that might fill this unmet need (in particular the roles of apos in lipid metabolism as described in the section Biochemistry and pathobiology of apolipoproteins: Insight into dyslipidaemia and atherosclerosis), (c) validation of the intended use of the proposed biomarker marker (e.g. in sections on the current evidence for the role of apos in a clinical setting based on outcome studies at both the protein level and the genetic level) and (d) assessment of the feasibility of the new biomarker to influence clinical practice and health outcomes (recommendations for medical laboratories on the role of apo quantitation in the clinical care pathway, to be addressed in the section Embedding apolipoprotein profiling in cardiovascular precision clinical care pathways).⁷

Residual CV risk beyond conventional CVD therapies

In the management of CVD the use of the most appropriate medication requires a clear definition of the underlying metabolic problem in a particular patient. Although the diagnosis may look simple, say diabetes mellitus type 2 (DM2) or ischemic heart disease, the most appropriate therapeutic measures may be difficult to select for each individual patient. Currently, clinical chemistry testing for dyslipidaemia in plasma/serum is, besides clinical history, echocardiography, electrocardiography and angiography, a cornerstone in the investigation of patients with CVD. The concentrations of cholesterol, TG, HDL-C and LDL-C are risk factors used to identify, stratify and characterize dyslipidaemia. This enables prescription of specific therapy directed towards these risk factors.

However, even if all risk factors present are treated, there remains residual CVD risk. In trials that studied statin vs. placebo therapy residual CVD risk was shown to be 25–40% in the statin-treated groups.^8–13 Interestingly though, intensive statin therapy resulted only in a slight decrease in incidence of major CVD events.^14–16 In 2005, Libby stipulated that the post-statin era suffers from a residual burden of cardiovascular mortality in about two-thirds of the patients with CAD, indicating the need for better understanding of factors contributing to this unexplained residual risk.² Superko similarly concluded that a therapy that focusses on LDL-C reduction alone neglects other important aspects of lipoprotein metabolism.¹⁷ In the Multi-Ethnic Study of Atherosclerosis, the overall residual risk in statin-treated individuals was >17% in a 10 year period. Even in a subset of patients with a baseline LDL-C < 2.58 mmol/L, DM, LDL-particle size, hs-CRP and coronary artery calcification (CAC) were the most important predictors of residual risk.¹⁸ This raises the question of how to diminish residual risk.

CVD in women

The treatment of CVD in women remains insufficient: compared to men, the prevalence and mortality of CVD in women is increasing. CVD generally manifests itself 10 years later in women than in men. Furthermore in addition to atherosclerotic coronary artery disease, non-atherosclerotic coronary artery disease may be an important cause of myocardial infarction, particularly in younger women. Risk factors in women include DM, endothelial dysfunction, leukocyte activation, platelet activation, renal dysfunction, and hypertension. The presenting symptoms are often non-specific in women, resulting in delayed diagnoses. In pre-menopausal women, severe manifestations of CVD, such as acute myocardial infarction (MI) and sudden death, are relatively rare. Women tend to develop asymptomatic heart failure secondary to CVD more frequently than men.¹⁹ Specific differences in CVD risk factors related to blood lipids have been described in women: (1) low HDL-C is more predictive for cardiovascular events in women than a high LDL-C; (2) high Lp(a) is a risk determinant for CVD in pre-menopausal and post-menopausal women <66 year; and (3) the concentration of total cholesterol seems to be associated with CVD only in pre-menopausal women and the concentration of TG only in older women.²⁰

Inadequate cholesterol-based tests do not identify atherogenic remnant disease

Remnant-cholesterol (remnant-C) includes primarily VLDL-C, IDL-C and Lp(a). Calculated remnant-C shows a skewed distribution with a tail towards higher concentrations in a general population.²¹ Hypertriglyceridemia (HTG) is associated with elevated concentrations of TG-rich lipoproteins (TRLs), apoB100, apoCIII and small dense LDL (sdLDL-C), in the setting of a normal or low concentration of LDL-C. Elevations of these (apo)lipoproteins, as are often observed in patients with DM or metabolic syndrome, are associated with increased CVD risk. In a model that included established risk factors, sdLDL-C was associated with incident CHD with a hazard ratio (HR) of 1.51 (95% confidence interval (CI), 1.21–1.88) for the highest vs. the lowest quartile. Even in individuals considered to be at low CVD risk based on their LDL-C concentrations, sdLDL-C predicted risk for incident CVD (HR 1.61; 95% CI, 1.04–2.49).²² LDL-C and remnant-C in ≈90,000 individuals from the Danish general population who were followed for up to 22 years were associated equally with risk of CHD and MI. Non-fasting remnant-C concentrations were associated stepwise with all-cause mortality ranging from HR of 1.0 (95% CI, 0.9–1.1) to HR of 1.6 (95% CI, 1.4–1.9) (P = 0.001), whereas LDL-C concentrations were associated with decreased all-cause mortality risk in a U-shaped pattern, with HRs ranging from 0.8 (95% CI, 0.7–0.8) to 0.9 (95% CI, 0.8–1.0) (P = 0.002). Only non-fasting remnant-C concentrations were associated with increased all-cause mortality risk.²¹

Ineffective therapy due to average conclusions and average dosing not considering the individual patient

Statin group drugs to reduce LDL-C are widely used in the management of CVD. Clinical practice guidelines for the most part stipulate the titration of statin doses to achieve pre-specified ‘group’ (i.e. rather than individualized patient) LDL-C targets.¹ A key question however is what is the personalized target for the patient? Treatment of comorbidities, such as other dyslipidaemias, DM, metabolic syndrome, hypertension or renal insufficiency, deserves attention in the individual patient. In other words, guidelines mention average target levels for the average patient, but an individual patient is likely not to be the average patient.

In recent years, several novel lipid lowering pharmacological therapies other than statins have been developed and results from clinical trials are now emerging.²³ These include ezetimibe, a cholesterol absorption inhibitor, and the very potent PCSK9 inhibitors, that have already been licenced in Europe and the US.²⁴ More recently, initial studies with anti-sense oligonucleotides (ASOs) blocking apoCIII and apo(a) have shown promising results.²⁵ With these new therapies emerging, there is a need for specific guidance in selection of the most appropriate therapy and selection of the patients for this therapy.

We believe the time has come to change from generic therapy tailored towards the average patient to guidelines that address the specific goals to be met in the individual patient. Molecular markers which reveal the underlying biology and pathology can give better clues for definition and treatment of disease than average target levels of LDL-C. To that purpose, the use of a multiplexed apo panel is advocated over the traditional lipoprotein profile that is based on cholesterol and TG, and evidence for the use of such an apo panel is presented.

Apolipoprotein quantitation by mass spectrometry

To accommodate the proposed paradigm shift to personalized healthcare, laboratory professionals explored alternative analytical strategies to define metabolic risk factors in individual patients. Mass spectrometry (MS) is a strategic (antibody-independent) technology for highly multiplexed protein quantification, that has recently entered the medical laboratory.^26,27 Multiple clinically relevant (apolipo)proteins can simultaneously be enzymatically digested,²⁸ upon which protein-specific signature peptides can be quantified using MS.

Newly developed (multiplex) biomarker methods should meet basic clinical chemistry principles.²⁹ Criteria for imprecision and bias should be pre-defined, e.g., based on biological variation,³⁰ and the methods should be robust and high-throughput to facilitate measurement of larger volumes of samples. The use of commutable and value-assigned serum-based calibrators should facilitate harmonization of assay methods and may contribute to a more rapid transition of biomarker discovery to clinical utility with benefit for the patient’s treatment and improvement of general health care.

We and others have now reported the development of liquid chromatography-mass spectrometry (LC-MS) based methods for the multiplexed quantitation of serum apos.^31–35 We were able to show that our method demonstrates stable performance over a longer period of time.³⁶ Moreover we showed that LC-MS based apoE phenotyping (qualitative analysis of apo ε2, ε3 and ε4 alleles) using unique signature peptides performs equally well compared to genotyping.³⁷ These results show the strength of MS in metrology, as the technique allows for unequivocal identification and quantitation of well-defined measurands at the proteoform level through the use of specific and unique peptides. This has now also been recognized by the clinical chemistry community, as an IFCC working group has been initiated to establish metrological traceability and standardization of apo quantitation and phenotyping using MS (http://www.ifcc.org/ifcc-scientific-division/sd-working-groups/wg-apo-ms/).

Inadequate standardization of Lp(a) measurement

Apo(a) is a heterogeneous glycoprotein with large size heterogeneity ranging from 200 to 800 kDa, due to a size polymorphism. The measurement of Lp(a) is indicated in intermediate CVD risk patients, patients with inherited dyslipidaemias and patients with premature vascular disease.^1,38 However, the size polymorphism of apo(a) hampers the accurate quantitation op Lp(a) in g/L,^39,40 as well as standardization. While Lp(a) has been known to be a risk factor for CVD for a long time, the poor quality of quantitative tests has hampered adoption of Lp(a) in clinical care pathways so far. The National Heart Blood and Lung Institute Working Group on Future Research Directions on Lipoprotein(a) and Cardiovascular Disease therefore recently recommended the development of a standardized measurement of Lp(a), in which apo(a) values should be quantified in nmol/L.⁴¹ Here too, MS seems to be an excellent technology to achieve this, as selection of a non-kringle IV-2 peptide would allow for quantitation of apo(a) at the molar level.

Biochemistry and pathobiology of apolipoproteins: Insight into dyslipidaemia and atherosclerosis

ApoB100

Human apoB100 is a 4536-amino acid secretory glycoprotein, and a single molecule is present in each VLDL particle when secreted by the liver. Lipoprotein lipase (LPL) and hepatic lipase (HL) are responsible for lipolysis of TG of the VLDL particle in the circulation, leading to formation of VLDL-remnants. Although VLDL-remnants (also called IDL) are predominantly cleared by the liver (through apoE-mediated pathways, see below) a large part of VLDL-remnants is converted – via IDL – into LDL by further lipolysis of TG. ApoB100 is responsible for uptake of VLDL, IDL and LDL by the hepatic LDL-receptor (LDLR).⁴² Generally, the concentration of LDL in the circulation is relatively high, compared to that of VLDL and IDL. LDL is relatively poor in TG and rich in cholesterol, whereas VLDL and IDL are particles that are relatively rich in TG.

Each particle of VLDL, IDL and LDL contains – in theory – one copy of apoB100. In the small intestine, a truncated form of apoB is formed corresponding to its N-terminal 48% (apoB48), which directs the formation of CMs. These TG-rich and cholesterol-poor particles undergo lipolysis of TG in the circulation, leading to uptake of CM-remnants by the liver and – in certain circumstances – accumulation of CM-remnants in the blood.⁴³

A high level of total cholesterol and particularly of LDL-C is a strong risk factor for atherosclerosis underlying several diseases such as CHD. In the initiation of atherosclerosis crucial events are the subendothelial retention and modification of apoB100-containing lipoproteins. However, not only abundancy of LDL but also abundancy of remnant particles trigger the atherosclerotic process, although the latter by mechanisms that are currently understood poorly.⁴⁴ LDL-lowering drugs (e.g., statins, ezetimibe and PCSK9 inhibitors) are currently the most effective lipid lowering therapies in routine clinical use and are shown to reduce coronary events and improve survival, which is attributed to the reduction in LDL-C.

Apart from VLDL, IDL and LDL, a fourth lipoprotein type, Lp(a), carries apoB100 (Figure 2). Lp(a) is composed of an apoB100-containing LDL-like particle, covalently linked to the plasminogen-like glycoprotein apo(a). Lp(a) is a highly prevalent genetic risk factor for CVD and calcific aortic valve disease.⁴⁵

Figure 2.

Density and particle size of the different lipoproteins. Their major apolipoprotein constituents are indicated.

In patients with high concentrations of sdLDL, the risk of CHD is best assessed by apoB concentrations (thus including apoB100 and apoB48), as the presence of sdLDL is not reflected by elevated LDL-C concentrations. Thus, an apoB measurement includes, besides LDL, also Lp(a), IDL, VLDL and CM-remnants,⁴⁶ and is recommended as (1) assessment of risk of CHD and (2) target of lipid-lowering therapy.^47,48

ApoCI

ApoCI is a single-chain polypeptide of 57 amino acids. ApoCI is produced in the liver and is a constituent of VLDL and HDL. ApoCI is an inhibitor of LPL activity, and inhibits the binding of VLDL to LDL-receptor related protein (LRP),^49,50 as well as the apoE-mediated binding of VLDL to the LDLR and the LRP.^51,52 ApoCI is a potent activator of lecithin:cholesterol acyltransferase (LCAT),^53,54 leading to elevated HDL-cholesterol ester concentrations.^55–57 In apoCI-deficient mice, the primary metabolic defect is an impaired VLDL uptake by the liver in vivo.⁵⁸ Interestingly, whereas overexpression of human APOC1 in transgenic mice predominantly inhibits the uptake of VLDL by the liver, the absence of endogenous apoCI in mice appears to have the same effect, although to a lesser extent. ApoCI has been reported to inhibit cholesteryl ester transfer protein (CETP) activity.

ApoCII

ApoCII is a single chain polypeptide of 79 amino acids. ApoCII is an essential cofactor of LPL, the rate-limiting enzyme for the hydrolysis and removal of TG in CMs and VLDL.⁵⁹ Like apoCI, but less effectively, apoCII inhibits the apoE-mediated binding of VLDL to the LRP.⁴⁹ The plasma TG concentration is independently associated with the apoCII concentration in a model including waist, circumference, apoCIII, apoE, glucose, insulin and mean LDL size in hyperlipidaemic patients.⁶⁰

Statin treatment resulted in significant apoCII reduction by ≈20%.^61–63 It was found that the apoCII-lowering effect of rosuvastatin occurred only in the subgroup with TG concentration ≥1200 mg/L.⁶³

In a study of 27 patients with primary HTG (TG >3500 mg/L) who received atorvastatin (20 mg/day or 80 mg/day) for four weeks, dose-dependent reductions of total cholesterol and TG were observed. In addition significant reductions in apoCII (by 28% and 42%), apoCIII (by 18% and 30%), and apoE (by 37% and 49%) were observed.⁶²

Individuals who are completely deficient in apoCII have chylomicronaemia and grossly elevated plasma TG concentrations, although their parents, who have half normal plasma concentrations of apoCII, have normal TG concentrations.⁶⁴ However, transgenic mice overexpressing human APOC2 had HTG, due to an accumulation of TG-rich VLDL in their circulation, caused by an impaired clearance of VLDL-TG, consistent with a defective LPL-mediated hydrolysis of VLDL-TG.⁶⁵ These results are in contrast with the LPL-activating action of apoCII: it may be that apoCII is an activator of LPL at low apoCII concentration, whereas a high apoCII concentration directly inhibits VLDL lipolysis.⁴⁹ Thus, both an excess and a deficiency of apoCII are associated with reduced LPL activity and HTG.⁶⁶

ApoCIII

ApoCIII is a 79-amino acid polypeptide that is produced mainly in the liver and to a lesser extent in the intestines. ApoCIII is a significant component of TRLs and a minor component of HDL.⁶⁷ ApoCIII exerts its impact on TG metabolism by four distinct mechanisms: (1) apoCIII functions as an inhibitor of HL and LPL, key enzymes that catalyse the hydrolysis of TG in VLDL and CMs;^68–70 (2) apoCIII acts to retard apoE-mediated hepatic uptake of TRLs;^71,72 (3) apoCIII serves to facilitate VLDL-TG assembly and secretion from the liver;^73–76 and (4) apoCIII completely abolishes the apoB100-mediated binding of lipoproteins to the LDLR, probably due to a masking of the receptor domain of apoB100 by apoCIII.^77,78

In the fasting state, apoCs are mainly associated with HDL, whereas in the fed state, as well as in patients with HTG, the apoCs preferentially redistribute to the surface of CMs and VLDL particles.⁷⁹ Elevated plasma apoCIII concentrations are associated with augmented production and retarded clearance of TRLs, characteristic of HTG. Plasma apoCIII concentrations were positively correlated with (1) plasma TG and (2) VLDL-TG.⁸⁰

Fibrates, a class of drugs that reduce plasma TG concentrations, effectively decrease apoCIII synthesis rate in humans⁸¹ as well as APOC3 mRNA concentrations in isolated human hepatocytes and rat livers via a peroxisome proliferator activated receptor (PPAR)-dependent pathway.^82–84 The development of ASOs targeted to the hepatic mRNA of apoCIII holds considerable promise, as dose-dependent reductions in TG levels of up to 80% are attainable.⁸⁵

ApoE

ApoE, a 299-amino acid polypeptide, is a ligand for the LDLR and LRP, binds to (hepatic and endothelial) cell surface heparin sulphate proteoglycans, and plays a key role in the receptor-mediated uptake of apoB100-containing lipoproteins including remnant lipoproteins.⁷⁹ In addition to its well-established function, apoE is proposed to modulate TG lipolysis.⁸⁶ ApoE can specifically inhibit the LPL-mediated hydrolysis of emulsion TG both in vitro and in vivo and inhibits the LPL-mediated processing of TRLs, including VLDL-TG.^87–89 Plasma apoE is synthesized primarily by liver hepatocytes, which account for ≈75% of the body’s apoE production. Various cell types throughout the body, including macrophages, also synthesize apoE.⁹⁰ The hepatic uptake of VLDL-remnants and CM-remnants is facilitated by apoE, but is inhibited by apoCI, apoCII and apoCIII.⁹¹

Epidemiologic studies have shown that apoE polymorphism is associated with interindividual variations in plasma lipid concentrations as well as susceptibility to atherosclerosis.^86,91 ApoE4 is associated with higher plasma total cholesterol, LDL-C, and apoB100 concentrations than apoE3. In addition, apoE4 is proposed to be an independent risk factor for CHD regardless of its effect on plasma cholesterol concentrations.^92–94 On the other hand, homozygotes of apoE2, insofar as they do not develop type III hyperlipoproteinaemia (also called dysbetalipoproteinaemia), show lower plasma total cholesterol and LDL-C concentrations, lower apoB100 concentrations, and higher plasma TG concentrations than homozygotes of apoE3. Homozygotes of apoE2 demonstrate higher postprandial plasma TG concentrations than those of apoE3 and apoE4.^95,96 Furthermore, apoE2 is proposed to reduce the risk of CVD.⁹⁷ However, homozygotes for apoE2 who develop type III hyperlipoproteinaemia due to secondary precipitating factors are susceptible to CVD.^98,99

Apo(a)

Lp(a) is composed of an apoB100-containing LDL-like particle, covalently linked to the plasminogen-like glycoprotein apo(a). Lp(a) is highly polymorphic in size due to the number of kringle IV type 2 (KIV2)-encoding sequences, giving origin to >40 apo(a) isoforms varying in number among individuals and populations. Although its exact role in lipid metabolism is unknown, Lp(a) is a highly prevalent genetic risk factor for CVD and calcific aortic valve disease (CAVD). Lp(a) concentrations in the atherothrombotic range are generally accepted as >300 to 500 mg/L (>75 to 125 nmol/L). Such concentrations affect 20% to 30% of the global population, with possibly higher incidence in patients with established CVD and CAVD.⁴¹

The atherogenicity of Lp(a) is caused by interference with the fibrinolytic system, the affinity to secretory phospholipase A2, the interaction with extracellular matrix glycoproteins and the binding to scavenger receptors on macrophages. An association between high Lp(a) and incidence of venous thromboembolism (VTE) has been described. Apo(a) KIV-2 repeat number was significantly lower in VTE patients (excl. hereditary and acquired thrombophilia) than in controls (11 vs. 15, respectively). KIV-2 repeat number was independently associated with VTE.¹⁰⁰ Elevated Lp(a) concentration has a strong association with angiographically documented CAD^45,101 and with risk of cardiovascular death, non-fatal MI and ischemic stroke.¹⁰² Nordestgaard et al. advocate screening for elevated Lp(a) in those with intermediate or high CVD/CHD risk, and use of pharmacotherapy for Lp(a) and CVD/CHD risk reduction, accepting a desirable concentration <500 mg/L.¹⁰³

Apo A-I

ApoA-I is the major apo in HDL, accounting for 70% of all HDL-associated proteins, and mediates many of the anti-atherogenic functions of HDL. ApoA-I is produced both in the liver and intestines, and is responsible for initiating reverse cholesterol transport, whereby excess cholesterol in peripheral tissues is carried back to the liver for excretion. Patients with low concentrations of apoA-I (<1.2 g/L) are more likely to have CVD than those with high apoA-I concentrations (≥1.6 g/L).¹⁰⁴

As with LDL, the cholesterol content of HDL particles varies among patient types and is influenced by plasma TG concentrations; patients with high TG tend to have low apoA-I concentrations. Nonetheless, it is unclear whether apoA-I alone is a predictor of CVD risk independently of its association with HDL.¹⁰⁵

Genetic evidence for the role of apolipoproteins in CVD

Three types of studies are typically used to determine whether a certain parameter is a risk factor for CHD. These studies include primary evidence through observational studies, randomized controlled trials (RCT), and lately Mendelian randomization trials (MRTs).¹⁰⁶ Because genetic evidence from MRTs is most compelling, in this review we specifically focus on evidence for the role of apos in CHD obtained from such study designs. Mendelian randomization was first proposed in 1986 to evaluate whether the association between cholesterol concentrations and cancer was really causative using apoE genotypes (ε2, ε3, ε4),¹⁰⁷ and has been reviewed extensively.¹⁰⁸ It is a method in which measured variation in genes of interest is used to evaluate the effects of a measure of exposure (e.g., concentrations of cholesterol or TG) on disease states (e.g., incidence of CHD).¹⁰⁹ The design is highly resistant to reverse causation and confounding, which often impede or mislead epidemiological studies. Most striking are occasions where observational epidemiological studies have highlighted a seemingly substantial causal association that later failed to be confirmed in large-scale RCTs. An example is the hypothesis that β-carotene protects against cancer, which was derived from several observational epidemiologic studies.¹¹⁰ This hypothesis was tested in the ATBC and CARET studies, both RCTs in which a 16%¹¹¹ and 28%¹¹² increased incidence of lung cancer was reported in individuals treated with β-carotene, respectively. Genotypes are randomly inherited from parents to offspring, known as Mendelian randomization, and as such, the genotype distribution used in RCTs should be unrelated to confounders that may affect conventional observational studies.¹⁰⁸ Moreover, a genetic polymorphism can, by definition, only be causative to a change in the measure of exposure, thus avoiding reverse causation. However, genetic pleiotropy may confound results from MRTs, and a number of methods to identify pleiotropy in MRTs have been described.¹¹³

ApoB

There is a large body of evidence for a direct implication of LDL and other apoB-containing particles (VLDL, IDL and Lp(a)) on the development of CVD in a concentration dependent manner.¹¹⁴ Specifically, a number of meta-analyses, such as the Prospective Studies Collaboration and the Emerging Risk Factors Collaboration, have provided evidence that there is an association between exposure to LDL and risk of CVD.^115,116 Even though mutations in the APOB gene might be prone to pleiotropy due to association with other lipoproteins or risk factors for CVD, some MRT studies have used single nucleotide polymorphisms (SNPs) in APOB to evaluate CVD risk. Kathiresan et al. evaluated the role of LDL-C and HDL-C in CVD through common SNPs in nine genes.¹¹⁷ APOB SNP rs693 was shown to be individually associated with LDL-C concentrations in 5287 subjects of the cardiovascular cohort of the Malmo¨ Diet and Cancer Study. Subsequently, a panel of nine SNPs from nine genes, including APOB rs693 could be associated with incident CVD, providing evidence for the causal association of LDL-C concentrations with CVD. In a similar approach, APOB rs562338 was included in a multi-SNP panel to assess the relation between LDL-C and CVD risk.¹¹⁸

Besides the MRTs, there is evidence for the role of apoB from observational studies towards inherited forms of CVD. Individuals with a loss-of-function mutation in the apoB gene typically have familial hypercholesterolemia (FH), which is characterized by substantially elevated concentrations of LDL-C and early-onset CVD, specifically CAD (e.g. Pang et al.¹¹⁹), further validating the causative role for apoB in CVD.

ApoCII

The second most frequently reported cause of chylomicronaemia, often resulting in severe pancreatitis, is the presence of homogeneous APOC2 loss-of-function mutations.¹²⁰ Indeed, several case reports indicate chylomicronaemia and decreased concentrations of apoCII in individuals with APOC2 loss-of-function mutations.^121–125 However, so far, these numbers are too small for full observational studies, and MRTs have not yet been conducted. In 38 patients with very premature STEMI, 4 patients with low apoCII concentrations (≤5.0 mg/L) had worse reinfarction-free or revascularization-free survival than those with apoCII >5.0 mg/L during follow-up of 10 years (Figure 5(a) and (b)).¹²⁶ These findings point towards a role for apoCII in CVD, and might warrant further evaluation of the role of apoCII in an MRT.

ApoCIII

There is a long-lasting debate on whether or not TG concentrations are a risk factor for CHD, and several studies indicated that TG concentrations are associated with CHD. However, adjusting for confounding variables (e.g., DM) weakened these associations. Because apoCIII is one of the major constituents of TRLs, MRT studies were performed to further evaluate the relationship between TG concentrations and CHD risk.

In one study, four rare apoCIII variants were identified to be associated with lower TG concentrations: nonsense mutation R19X, two splice-site mutations (IVS2 + 1G→A and IVS3 + 1G→T) and a missense mutation A43T. Carriers of these mutations had 39% decreased TG concentrations and circulating concentrations of apoCIII were 46% lower. When these mutations were subsequently tested in 110,097 individuals from several studies, carriers had a 40% reduction in CHD.¹²⁷ In a similar study, three apoCIII variants could be associated with a 44% reduction in TG concentrations. In a Danish cohort of 75,725 individuals, it was subsequently observed that carriers of these variants had a 41% reduction in CHD.¹²⁸ These studies clearly indicate that reduction of apoCIII reduces CHD risk. However, whether this reduction in CHD is TG mediated is unsure, as reduced concentrations of apoCIII have also been associated with lower concentrations of LDL-C.¹²⁹

Further evidence for the role of apoCIII as a risk factor for CHD can be found in a study which found that about 5% of the Lancaster Amish are heterozygous carriers of an APOC3 null mutation (R19X). Lower TG and LDL-C concentrations were observed in mutation carriers and the incidence of subclinical atherosclerosis was decreased, indicating a cardioprotective effect of apoCIII deficiency.¹³⁰ The same mutation has subsequently been found to be enriched in an isolated Greek population, in which it could be associated with reduced TG concentrations.^131,132

Apo(a)

Apo(a) is the characteristic polymorphic protein carried by Lp(a) particles, and evidence for the role of Lp(a) as a risk factor for CVD has recently been reviewed.⁴⁵ A number of observational studies have clearly identified Lp(a) concentration to be associated with CVD incidence,^102,133,134 indicating a potential causal role for Lp(a). In a meta-analysis in which 36 studies with 7385 cases and 8514 controls were included, small apo(a) isoforms (approximately 22 or fewer KIV2 repeats) were shown to be associated with a two-fold increased risk for CVD when compared to larger apo(a) isoforms.¹³⁵

To further assess the role of Lp(a) particles in CVD risk, MRTs have been performed (e.g. ^{133,136–138}), and results were recently reviewed.⁴⁵ Here the most important, and novel findings are summarized. In 2009, LPA SNPs rs10455872 and rs3798220 were shown to be associated with Lp(a) concentration. These same SNPs were subsequently shown to also be associated with CVD risk,¹³⁶ thus directly indicating a causal effect for apo(a) concentration on CVD. These results were corroborated by Kamstrup et al.¹³³ Recently, the Mendelian randomization strategy was also used to estimate the required change in plasma Lp(a) concentration that should therapeutically be reached to produce a clinically meaningful reduction in CVD risk.¹³⁸ A panel of 43 genetic variants, which explained 51–63% of the variance in Lp(a) concentration, was used to predict Lp(a) concentration, and it was modelled that each 100 mg/L lower Lp(a) was associated with a 5.8% lower CVD risk. By comparing the risk reduction with the same parameter for LDL-C risk reduction, it could be inferred that an 1 g/L reduction in apo(a) concentrations would provide a similar reduction in CVD risk as a 1 mmol/L reduction in LDL-C concentrations, which is generally regarded clinically meaningful.¹³⁸

There is an inverse relation between apo(a) concentration and the number of apo(a) KIV2 repeats. However, there is large interindividual variation in this correlation,⁴⁵ which is indicated by a study that showed that the size variation of apo(a) could explain 17% of the apo(a) concentration in Sudanese, but 77% in Mexicans.¹³⁹ Because of this correlation, it has long been unclear whether just Lp(a) concentration, or also apo(a) KIV2 number influences CVD risk. To address this question, Salaheen et al. performed an MRT by identifying genetic variants associated independently with either apo(a) KIV2 number (rs2457564) or apo(a) concentration (rs3777392) in the Pakistan Risk of Myocardial Infarction Study (PROMIS).¹⁴⁰ Genetic data from 60,801 CHD cases and 123,504 controls from the CARDIoGRAMplusC4D consortium revealed that the OR for MI was 0.96 per 1SD increment of apo(a) KIV2 number, while an OR of 1.27 was observed per 1SD increment of apo(a) concentration. Because the SNPs were chosen to be specific for their apo(a) characteristic, it could be concluded that both apo(a) KIV2 numbers as well as apo(a) concentration are independent causal risk factors for CVD.¹⁴⁰

ApoE

In a study evaluating the effect of long-term exposure to lower LDL-C concentrations on the risk of CVD, nine SNPs in six genes, among which APOE rs4420638, were studied.¹⁴¹ The APOE SNP was shown to be associated with reduced LDL-C concentrations in a meta-analysis involving 126,788 samples. In a subsequent meta-analysis, these nine SNPs, including APOE rs4420638 were associated with a decreased risk of CHD. The correlation of proportional CHD risk reduction and lower LDL-C revealed 18% CHD risk reduction per 0.25 mmol/L lower LDL-C (≈72% lower risk per mmol/L lower LDL-C).¹⁴¹ In a meta-analysis of 26 statin trials, treatment with a statin was associated with a 24% reduction of CHD risk per mmol/L reduction of LDL-C. Thus, compared with treatment with a statin later in life, prolonged exposure to lower LDL-C beginning early in life was associated with a two- to three-fold greater reduction in the risk of CHD for each unit lower LDL-C (Figure 3).^129,141 This was further corroborated by Kathiresan et al., who monitored the same APOE rs4420638 SNP to study the relation between LDL-C and CVD risk in the Malmo¨ Diet and Cancer Study.¹¹⁷

Figure 3.

Overview of the relation between reduction in LDL-C and reduction in CVD in genetics and intervention studies. A lifetime reduction of LDL-C by genetic traits (studies indicated by the green dots, mutations indicated) reduces the risk of CVD two to three times more than pharmacological reduction of LDL-C by statin therapy during an average of five years (studies indicated by the blue dots).Source: reproduced with permission from Elsevier.¹²⁹

ApoE has three common alleles encoded by the APOE gene. These alleles occur at different frequencies in humans (ε2, 5–10%; ε3, 65–70%; and ε4, 15–20%) and give rise to three homozygous (apoE2/2, apoE3/3, and apoE4/4) and three heterozygous (apoE3/2, apoE4/2, and apoE4/3) phenotypes.¹⁴² The structural basis for the three isoforms occurs through amino acid interchanges (single base changes in the apoE gene) at residues 112 and 158: apoE2 has cysteine at both sites, apoE4 has arginine at both sites, and apoE3 has cysteine (Cys)-112 and arginine (Arg)-158.¹⁴³ The hypothesis that the APO ε2/3/4 genotype affects plasma Lp(a) concentration was tested by Kritharides et al.¹⁴⁴ They found that the APOE ε2 genotype is a strong determinant of low Lp(a) concentrations, but the APOE ε2 genotype does not modify the causal association of Lp(a) with MI or CAVD.¹⁴⁴

ApoA1

Haase et al. sequenced the regulatory and coding regions of APOA1 in 190 individuals from the Copenhagen City Heart Study with the lowest 1% (n = 95) and highest 1% (n = 95) apoA-I concentrations.¹⁴⁵ Genotype combinations of two SNPs were associated with increases in apoA-I and HDL-C concentrations of up to 6.6 and 8.5%, respectively. Although these genotypes would theoretically predict reductions in 9 and 8% for CHD risk and 14 and 12% for MI risk, these genetic variations of APOA1 did not associate with decreased risk of CHD or MI in daily practice. The authors suggest that the atheroprotective effects of high concentrations of apoA-1 and HDL-C in epidemiological studies may not be due to the apoA-I and HDL-C elevations itself, but may be caused by the concomitant reduction in TG concentrations and thus by the concomitant reduction in atherogenic remnant lipoproteins.¹⁴⁵

ApoA-IV

ApoA-IV has been observed to be associated with lipid concentrations, renal function, and adiposity- and diabetes-related parameters. Mack et al. conducted bidirectional Mendelian randomization analysis to assess the causal relationship of apoA-IV with these phenotypes.¹⁴⁶ These causal relationships were found with estimated glomerular filtration rate, and serum TG concentration, independently from HDL-C and LDL-C concentrations. Evaluating the inverse direction of causality revealed a possible causal association of apoA-IV on HDL-C.⁴⁶

ApoA-V

The Triglyceride Coronary Disease Genetics Consortium and Emerging Risk Factor Collaboration assessed the −1131T>C (rs662799) promoter polymorphism of the apoA-V (APOA5) gene in relation to TG concentration, several other risk factors, and risk of CHD.¹⁴⁷ −1131T>C was modestly associated with lower HDL-C, lower apoA-I, and higher apoB, and strongly associated with elevated TG. The OR for CHD was 1.18 (95% CI 1.11–1.26; p = 2.6 × 10⁻⁷) per C allele. −1131T>C was significantly associated with higher VLDL particle number and smaller HDL particle size, factors believed to mediate the effects of TG.¹⁴⁷

In an evaluation of the contributions of rare mutations to MI risk in the population, Do et al. sequenced the protein-coded regions of 9793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls.¹⁴⁸ Carriers of rare APOA5 mutations (1.4 of cases vs. 0.6% of controls) were at 2.2-fold increased risk of MI. APOA5 mutation carriers had higher plasma TG, which suggested that elevated concentrations of TRLs contribute to MI risk.¹⁴⁸

Apolipoprotein profiles and cardiovascular risk: Evidence from outcome based studies

Besides evidence for the role of apos in CVD from genetics studies, there is now also ample literature describing associations between protein concentrations and CVD outcome. Initially, these studies were performed using immunoassays, but recently studies using MS have emerged.¹⁴⁹ Specifically, several studies have attempted to improve current LDL-C measurements by targeting apoB100, the major apo of LDL. In a large cohort study of 175,553 individuals Walldius et al. reported that the relative risk (RR) of MI per 1SD increment of LDL-C was substantially reduced after additional adjustment for apoB100; in contrast, apoB100 was still associated with substantial risk when adjusted for LDL-C, indicating that the risk of atherosclerotic disease is best predicted by apoB100, instead of LDL-C and non-HDL-C.¹⁵⁰ Pischon et al. found that apoB was more strongly related to CHD risk than was non-HDL-C. This is in line with previous studies showing that apoB100 is superior to non-HDL-C in predicting subclinical atherosclerosis. Based on the now disputed assumption that each LDL particle contains one apoB100 molecule, these findings led Pischon et al. to state that ‘direct measurement of the concentration of atherogenic particles is more biologically meaningful than the measurement of the cholesterol concentration contained in these particles’.¹⁵¹ In a meta-analysis based on 12 published epidemiological studies that contained estimates of the relative risks of non-HDL-C and apoB for CVD, it was concluded that over a 10-year period, a non-HDL-C strategy would prevent 300,000 more events than an LDL-C strategy, whereas an apoB100 strategy would prevent 500,000 more events than a non-HDL-C strategy.¹⁵² These findings indicate that apoB100 is superior to LDL-C and non-HDL-C as a predictor of CVD risk. These results are consistent with CHD risk being more closely related to the number of atherogenic apoB100 particles than to the mass of cholesterol within them, as previously stated (Figure 4).⁴⁷

Figure 4.

Relative LDL particle size and plasma lipids/lipoprotein concentrations in three patients with identical low LDL-C (1.8 mmol/L) but with discordant non-HDL-C and apoB with regard to desirable treatment targets for very high-risk patients. Patient 1 has all three targets at goal and normal numbers of (predominantly larger sized) LDL particles. Patient 2 with moderate HTG has discordant non-HDL-C above target (2.6 mmol/L) because of increased remnant-cholesterol. Patient 3 also has moderate HTG and increased remnant-cholesterol but concurrently higher apoB concentration than patient 2 because of a high number of sdLDL particles not detected by standard LDL-C measurement.Source: reproduced with permission from American Association for Clinical Chemistry.⁴⁶

The risk of CVD is inversely proportional to HDL-C and apoA-I concentrations, but this seems to be mostly relative to LDL-C.¹⁵³ Therefore, quantitation of apoA-I may be most useful in conjunction with apoB100 to assess the balance between atherogenic and atheroprotective cholesterol transport. A higher apoB100/apoA-I ratio likely indicates that more atherogenic particles are circulating in plasma leading to more plaque build-up in arteries, atherosclerosis and higher risk of CVD events.¹⁵⁴ Compared to other lipid ratios, the apoB100/apoA-1 ratio may be more accurate in risk prediction, particularly among high-risk individuals, than high LDL-C and low HDL-C concentrations.¹⁵⁵ Concordantly, we reported recently that the apoB/apoA-1 ratio was strongly associated with risk of ST-segment elevation myocardial infarction (STEMI) (Figure 5(c)).¹⁵⁶

Figure 5.

(a) ApoCII concentration in 38 patients with very premature STEMI. (b) In this group of patients with very premature CAD, 4 patients with apoCII ≤5 mg/L had a high risk of reinfarction or revascularization over ten years compared to those with apoCII >5 mg/L. (c) In the MISSION Intervention Study the serum concentrations of apoA1, apoB and the apoB/apoA1 ratio were strongly associated with risk of STEMI.Source: reproduced with permission from Elsevier¹²⁶ and SpringerNature.¹⁵⁶

While apoB100 is mostly associated with LDL particles, and apoA-I partially reflects HDL-C, the situation for apos associated with TRLs is more complicated. For instance, in normolipidaemic subjects, apoCII is mainly distributed in HDL, whereas in subjects with HTG, apoCII is predominantly found in the VLDL and LDL. TG concentrations are generally inversely related to HDL-C, and for a long time, it was assumed that the measurement of HDL-C enabled calculation of CVD risk. However, there is now ample evidence from genetic studies (Genetic evidence for the role of apolipoproteins in CVD Section) that not low HDL-C, but high concentrations of TRLs are a causal factor for CVD.¹⁰⁶ Therefore, interest in TRLs and TRL-associated apos, such as CI, CII, CIII and E,⁴⁹ as markers for CVD risk has surged.

In 2002, Gerber et al. reported that increased apoCII concentrations are associated with CHD after adjustment for cardiovascular risk factors in a case–control study including 352 CHD patients and 395 controls.¹⁵⁷ This evidence supports an association between apoCII and CVD risk, but causality between apoCII concentrations and CVD was not established.⁶⁶ Interestingly, we recently identified a phenotype of relatively young women with a combination of very low apoCII (≤5 mg/L), normal TG concentrations, and low concentrations of LDL-C, apoB100, and apoE who presented with STEMI. Despite their low a priori risk for CAD, these women presented with MI and had a high relative risk of 10-year reinfarction or revascularization.¹²⁶ These finding further point towards a role for apoCII in CVD.

In a nested case–control study in the prospective EPIC-Norfolk study comprising 2711 apparently healthy study participants, of whom 832 subsequently developed CAD, Van Capelleveen et al. demonstrated that increased apoCIII concentrations were significantly associated with incidence of CAD.¹⁵⁸ The significance was retained after adjustment for traditional CAD risk factors, but it was proposed that elevated concentrations of remnant lipoproteins, sdLDL and low-grade inflammation could explain the association with CAD.¹⁵⁸

Recently, Pechlaner et al.¹⁴⁹ used a targeted MS-based method to evaluate the association between apoA-I, apoA-II, apoA-IV, apoB100, apoCI, apoCII, apoCIII, apoD, apoE, apoH, apoL-I, apoM and apoJ with incident CVD in 688 participants of the Bruneck Study. During 10 years follow-up, the apos that were most significantly associated with incident CVD were apoCII (hazard ratio (HR) per 1 SD: 1.40; 95% CI, 1.17–1.67), apoCIII (HR per 1 SD: 1.38; 95% CI, 1.17–1.63), and apoE (HR per 1 SD: 1.31; 95% CI, 1.13–1.52). These associations remained significant even after adjustment for traditional risk factors, and demonstrate the advantage of using apos to identify dyslipidaemic states, particularly related to TRLs.¹⁴⁹

Further evidence for the direct role of apoCIII in the development of CVD comes from the novel anti-APOCIII ASO therapy with volanesorsen. ApoCIII inhibition by volanesorsen reduced plasma concentrations of apoCII, apoCIII, and TG, without affecting concentrations of apoB100.^149,159,160 Whether targeting of apoCIII in patients with elevated concentrations of TRLs will reduce risk of CVD is subject of ongoing studies, but a recent report on the use of volanesorsen in individuals with familial chylomicronaemia syndrome, a rare genetic disorder characterized by marked chylomicronaemia leading to pancreatitis, indicated a significant reduction in disease burden.¹⁶¹

There is very limited evidence for a direct causal role of apo(a) in CVD, mostly because direct measurement of apo(a) is not performed routinely. However, a 1:1 stoichiometry of apo(a) on Lp(a) particles is assumed, and a large body of evidence exists for the association between Lp(a) concentrations and the risk for CAD, regardless of other risk factors. For example Kostner et al.¹⁶² have estimated that CAD risk is 2.3 times higher in patients with Lp(a) concentrations >500 mg/L, while Riches and Porter have calculated that risk was twice greater for Lp(a) concentrations >200 mg/L.¹⁶³ In a meta-analysis of 40 prospective studies with 58,000 participants, a two-fold increase in the risk for developing CAD and cerebral vascular accident was found in individuals with smaller apo(a) isoforms, regardless of the Lp(a) concentration and the classical risk factors.¹³⁵

Recently, an anti-APOA ASO (IONIS-APO(a)-LRx) therapy has been developed. On treatment, Lp(a) concentrations fell by an average of 68%.¹⁶⁴ Studies further evaluating the efficacy for this therapy in lowering CVD incidence are currently ongoing. Chronic lipoprotein apheresis is an alternative treatment that was previously developed, and 154 subjects with isolated Lp(a)-hyperlipoproteinaemia with progressive CVD who underwent apheresis showed reduced Lp(a) concentrations by 68%, associated with a decline of major adverse cardiac events by 78% after five years.¹⁶⁵

Embedding apolipoprotein profiling in cardiovascular precision clinical care pathways

Conventional clinical care pathway

Currently, the standard procedure for patients that present with symptoms of CVD is to monitor the patient’s lipid profile by determining the concentrations of total cholesterol, TG, and HDL-C, followed by calculation of LDL-C using Friedewald’s formula.¹⁶⁶ Several risk assessment systems have been developed, among which the widely applied Systemic Coronary Risk Estimation (SCORE),¹⁶⁷ which is recommended by the European Guidelines on CVD prevention in clinical practice,¹⁶⁸ because it is based on large European cohort datasets.

Quantities of total cholesterol, TG, HDL-C and LDL-C allow us to calculate (1) non-HDL-C (=total cholesterol – HDL-C) which quantity includes the cholesterol of all atherogenic lipoproteins (VLDL, IDL, LDL, CM, CM-remnants and Lp(a)) and (2) remnant-C (=total cholesterol – HDL-C – LDL-C) which quantity includes the cholesterol of all lipoproteins except LDL and HDL. Non-HDL-C is a global measure of all atherogenic lipoproteins and has been suggested as a predictor of CHD risk. LDL-C, a major component of non-HDL-C, is the current target to be treated and this is achieved with statins, ezetimibe and PCSK9 inhibitors. Remnant-C is included in non-HDL-C but has merit in being measured separately: these remnants, mostly TG-rich remnant lipoproteins (in the non-fasting state CM-remnants and IDL) contribute also to risk of CHD.⁴⁶

Several studies confirm that non-HDL-C is superior to LDL-C in predicting CHD.^151,169 In a prospective study participants with high non-HDL-C concentrations were at increased CHD risk, independent of LDL-C concentrations.¹⁷⁰ An analysis of the treating to new targets (TNT) and Incremental Decrease in End Points Through Aggressive Lipid Lowering (IDEAL) trials found that on-treatment, concentrations of non-HDL-C and apoB100 were better predictors of CVD risk than LDL-C.¹⁷¹ Puri et al. reported that the achieved concentrations of on-treatment non-HDL-C and TG concentrations were closely associated with coronary atheroma progression and regression, irrespective of achieved concentrations of LDL-C, CRP and DM status.¹⁷²

While the current clinical care pathway does indeed improve cardiovascular risk, considerable residual risk remains, particularly in women and individuals with remnant lipoprotein particles.

Proposed cardiovascular precision care pathway

The use of novel markers to improve cardiovascular risk stratification as well as newly developed therapeutics targeting apoCIII and apo(a), pose a challenge for laboratory professionals. The use of novel markers based on improved understanding of pathophysiology would also help target therapy. Based on our current understanding of lipoprotein pathophysiology and the knowledge acquired from MRT-studies and protein-based outcome studies, we suggest that a multiplexed panel including apoA-I, apoB, apoCI, apoCII, apoCIII, apoE and apo(a) might allow improved risk stratification (see Figure 6 for a proposed clinical care pathway). Currently, a Consensus Conference Report from the American Diabetes Association and the American College of Cardiology suggested that measurement of apoB100 be added to measures of LDL-C and non-HDL-C in patients at high cardiometabolic risk, with target apoB100 concentrations set at <900 mg/L in high risk patients and <800 mg/L in highest risk patients.¹⁵⁴ Moreover, two studies have reported that quantitation of Lp(a) in intermediate risk patients could potentially reclassify 15 to 40% of individuals into a lower or higher risk groups.^134,173 It is highly likely that similar results will be obtained using apo(a). Guidelines from the European Atherosclerosis Society¹ and the Canadian Cardiovascular Society³⁸ recommend screening for apo(a) in patients with a family history of CVD in addition to quantitation of apoB100. While these guidelines originating from 2016 do not mention apoCs, we believe the very recent compelling evidence for the role of apoCs in CVD both at the genetic and protein concentration^106,174 (sections on Genetic and Proteomic evidence for the role of apolipoproteins in CVD) warrants their inclusion in risk stratification.

Figure 6.

Clinical test-treatment pathways for CVD reduction according to current and new practices.Source: reproduced with permission from Elsevier.⁷

Another group of patients that might benefit from a multiplexed apo panel in conjunction with conventional lipid measurements are individuals with a family history of CVD. For instance, individuals with FH typically suffer their first CVD event prior to the age of 50.¹⁷⁵ Given that apo concentrations are mainly genetically defined (Figure 6) there may be a role for apo measurement in screening family members of young CVD patients as a first line strategy prior to genotyping. Therefore, it is likely that measurement of an apo panel in a single serum sample might be sufficient to characterize a particular familial dyslipidaemia. Indeed, recently the intra-individual variability of serum apo(a) concentrations was shown to be low.¹⁷⁶

While there is substantial evidence that apos may be better predictors for CVD, there is as yet insufficient evidence from prospective outcome studies on the use of multiplexed apo panel as compared with a conventional lipoprotein panel. Therefore, we would currently propose the use of an apo multiplex panel as an add-on test, even though this will carry an associated additional health care cost. Future studies should be directed towards a comparison of the traditional lipoprotein panel and a multiplexed apo panel, so that ultimately, the apo panel could potentially replace the traditional lipoprotein panel. Based on the evidence outlined in the previous three sections, we believe a biology-driven multiplexed apo panel including apoA-I, apoB100, apoCI, apoCII, apoCIII, apoE and apo(a) will provide better CVD risk stratification and allow for targeted and more effective treatment (e.g., high apoCIII could be treated directly with anti-apoCIII ASO valonesorsen) than current cholesterol, TG, HDL-C and LDL-C measurement (Figure 6).

Inclusion of such a multiplexed apo panel in the clinical care pathway would be feasible from a technological perspective, as we previously developed an MS-based test that allows for the simultaneous quantitation of apos,³¹ and were recently able to show that this test can be delivered with stable analytical performance over a long period of time.³⁶ The volume of serum required for the test is only 25 µL. Although the current turnaround time of 48 hours to perform the multiplexed apo panel is considerably slower than that of the traditional lipid panel, this must be offset against the possibility of more accurate risk assessment.

Conclusions and future outlook

The delivery of healthcare to the highest standard without a further increase in cost, requires that clinical practice should become more efficient. The current clinical care pathway for cardiovascular risk assessment is unsatisfactory, in that there remains considerable unexplained residual risk, even if LDL-C targets are met. It is time for a paradigm shift from traditional reactive medicine, often based on statin therapy, to proactive ‘P4 medicine’ in which disease mechanisms and systems biology allow for a targeted and patient-centric approach.^5,6 We believe that the use of a multiplexed apo panel will allow better characterization of a patient’s dyslipidaemic status than that given by the traditional lipid profile (total cholesterol, TG, HDL-C, and calculated LDL-C). Apo profiling can be performed using an MS-based strategy, since the requirements for applying clinical proteomics, including apo profiling, in medical laboratories can be met using low resolution MS with applications that fulfil desirable performance criteria and that are standardized according to the calibration hierarchies outlined in ISO 17511. Because the apo profiles provide insight in the pathophysiology of the dyslipidaemia, it will facilitate the refining of therapeutic targets and will allow the use of more targeted therapies such as ASOs which may help reduce residual cardiovascular risk. In the near future it should be possible to demonstrate that apo profiling should be clinically effective and cost effective, and can be run with adequate throughput.

Further developments in risk stratification and tailoring of therapy may emerge in the near future. It is now clear that one gene can present itself in multiple proteoforms due to genetic modifications, alternative splicing and post-translational modifications.¹⁷⁷ Because different proteoforms are likely to have different functionality, quantitation of proteoforms might be of importance, specifically in light of precision medicine and targeted therapeutics.¹⁷⁸ Proteoforms have been found for several apos. Specifically, differential O-glycosylation of apoCIII was initially observed using isoelectric focusing,¹⁷⁹ and, more recently, using intact protein MS analysis.¹⁸⁰ Concentrations of sialylation on apoCIII are associated with lipid profiles in DM2,¹⁸¹ and lower concentrations of non- and mono-sialylated apoCIII were associated with larger LDL particles after dietary interventions.¹⁸² Proteoforms of apos CI and CII have also been reported,¹⁸³ and an in-depth analysis of apoA-1 recently revealed large proteoform heterogeneity.¹⁸⁴ Currently the clinical relevance of the proteoforms of apos for CVD are unknown, and more research is warranted. MS is an excellent technique for the in-depth characterization of an individual’s proteoform-profile,^178,185 as mass-based differences either at the intact protein or at the peptide concentration can easily be identified. Specifically the introduction of high resolution MS-instruments, which can provide accurate mass information at resolutions >60,000, would be beneficial for the analysis of proteoforms.¹⁸⁶ We believe that in the future, proteoform specific analysis in an extended apo-form panel might provide additional benefits, but such analyses are far from clinical implementation and require further technical development before clinical evaluation could be considered.

Overall, we believe that implementation of a multiplex apo panel for refined diagnosis of CVD would fit the unmet clinical need for enhancing stratified cardiovascular medicine. There is compelling evidence as outlined in this review, that the use of a biology-driven apo panel fits the concept of precision medicine, with potential benefit for patients and society. A carefully selected and biology-derived apo panel will likely enable tailored treatment of individual patients rather than standard treatment of average patients.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

Review and Ethics Committee: not applicable (invited review)

Guarantor

CC.

Contributorship

CC researched literature and made a content table encompassing the structure, relevant topics and chapters to cover in this apo review. The content was shared and mutually agreed upon among the co-authors. Specific chapters were assigned to specific authors. In that way CC, LR and AvdL all were lead authors for specific chapters. In regular follow-up meetings, the progression of all chapters was evaluated and discussed in the entire group. Consequently, all authors contributed equally.

References

Catapano

Graham

De Backer

et al . 2016 ESC/EAS guidelines for the management of dyslipidaemias. Eur Heart J 2016; 37: 2999–3058.

Libby

The forgotten majority: unfinished business in cardiovascular risk reduction. J Am Coll Cardiol 2005; 46: 1225–1228.

Mehta

Beckie

DeVon

et al . Acute myocardial infarction in women: a scientific statement from the American Heart Association. Circulation 2016; 133: 916–947.

Varbo

Benn

Nordestgaard

BG.

Remnant cholesterol as a cause of ischemic heart disease: evidence, definition, measurement, atherogenicity, high risk patients, and present and future treatment. Pharmacol Ther 2014; 141: 358–367.

Hood

Systems biology and P4 medicine: past, present, and future. Rambam Maimonides Med J 2013; 4: e0012.

Mathur

Sutton

Personalized medicine could transform healthcare. Biomed Rep 2017; 7: 3–5.

Monaghan

Lord

St John

et al . Biomarker development targeting unmet clinical needs. Clinica Chimica Acta 2016; 460: 211–219.

Scandinavian Simvastatin Survival Study Group. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994; 344: 1383–1389.

Long-Term Intervention with Pravastatin in Ischaemic Disease Study Group. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. N Engl J Med 1998; 339: 1349–1357.

10.

Sacks

Pfeffer

Moye

et al . The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events Trial investigators. N Engl J Med 1996; 335: 1001–1009.

11.

Heart Protection Study Collaborative Group. MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20 536 high-risk individuals: a randomised placebocontrolled trial. Lancet 2002; 360: 7–22.

12.

Shepherd

Cobbe

Ford

et al . Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. West of Scotland Coronary Prevention Study Group. N Engl J Med 1995; 333: 1301–1307.

13.

Downs

Clearfield

Weis

et al . Primary prevention of acute coronary events with lovastatin in men and women with average cholesterol levels. JAMA 1998; 279: 1615–1622.

14.

Cannon

Braunwald

McCabe

et al . Intensive versus moderate lipid lowering with statins after acute coronary syndromes. N Engl J Med 2004; 350: 1495–1504.

15.

Pedersen

Faergeman

Kastelein

et al . High-dose atorvastatin vs usual-dose simvastatin for secondary prevention after myocardial infarction: the IDEAL study: a randomized controlled trial. JAMA 2005; 294: 2437–2445.

16.

LaRosa

Grundy

Waters

et al . Intensive lipid lowering with atorvastatin in patients with stable coronary disease. N Engl J Med 2005; 352: 1425–1435.

17.

Superko

HR.

The failure of LDL cholesterol reduction and the importance of reverse cholesterol transport. The role of nicotinic acid. Br J Cardiol 2006; 13: 131–136.

18.

Wong

Zhao

Quek

RGW

et al . Residual atherosclerotic cardiovascular disease risk in statin-treated adults: the multi-ethnic study of atherosclerosis. J Clin Lipidol 2017; 11: 1223–1233.

19.

Rich-Edwards

Manson

Hennekens

et al . The primary prevention of coronary heart disease in women. N Engl J Med 1995; 332: 1758–1766.

20.

Orth-Gomer

Mittleman

Schenck-Gustafsson

et al . Lipoprotein(a) as a determinant of coronary heart disease in young women. Circulation 1997; 95: 329–334.

21.

Varbo

Freiberg

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.

22.

Hoogeveen

Gaubatz

Sun

et al . Small dense low-density lipoprotein-cholesterol concentrations predict risk for coronary heart disease: the Atherosclerosis Risk In Communities (ARIC) study. Arterioscler Thromb Vasc Biol 2014; 34: 1069–1077.

23.

Lloyd-Jones

Morris

Ballantyne

et al . 2017 Focused update of the 2016 ACC expert consensus decision pathway on the role of non-statin therapies for LDL-cholesterol lowering in the management of atherosclerotic cardiovascular disease risk: a report of the American College of Cardiology Task Force on Expert Consensus Decision Pathways. J Am Coll Cardiol 2017; 70: 1785–1822.

24.

Rosenson

Hegele

Fazio

et al . The evolving future of PCSK9 inhibitors. J Am Coll Cardiol 2018; 72: 314–329.

25.

Tsimikas S. RNA-targeted therapeutics for lipid disorders. Curr Opin Lipidol 2018; 29(6): 459–466.

26.

Hoofnagle

Wener

MH.

The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry. J Immunol Methods 2009; 347: 3–11.

27.

Lehmann

Hoofnagle

Hochstrasser

et al . Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application. Clin Chem Lab Med 2013; 51: 919–935.

28.

van den Broek

Smit

Romijn

et al . Evaluation of interspecimen trypsin digestion efficiency prior to multiple reaction monitoring-based absolute protein quantification with native protein calibrators. J Proteome Res 2013; 12: 5760–5774.

29.

Horvath

Lord

StJohn

et al . From biomarkers to medical tests: the changing landscape of test evaluation. Clin Chim Acta 2014; 427: 49–57.

30.

Sandberg

Fraser

Horvath

et al . Defining analytical performance specifications: consensus statement from the 1st Strategic Conference of the European Federation of Clinical Chemistry and Laboratory Medicine. Clin Chem Lab Med 2015; 53: 833–835.

31.

van den Broek

Romijn

Nouta

et al . Automated multiplex LC-MS/MS assay for quantifying serum apolipoproteins A-I, B, C-I, C-II, C-III, and E with qualitative apolipoprotein E phenotyping. Clin Chem 2016; 62: 188–197.

32.

Smit

Romijn

van den Broek

et al . Metrological traceability in mass spectrometry-based targeted protein quantitation: a proof-of-principle study for serum apolipoproteins A-I and B100. J Proteomics 2014; 109: 143–161.

33.

Agger

Marney

Hoofnagle

AN.

Simultaneous quantification of apolipoprotein A-I and apolipoprotein B by liquid-chromatography-multiple-reaction-monitoring mass spectrometry. Clin Chem 2010; 56: 1804–1813.

34.

Dittrich J, Adam M, Maas H, Hecht M, Reinicke M, Ruhaak LR, et al. Targeted On-line SPE-LC-MS/MS Assay for the Quantitation of 12 Apolipoproteins from Human Blood. Proteomics 2018; 18(3-4).

35.

Toth

Kuklenyik

Jones

et al . On-column trypsin digestion coupled with LC-MS/MS for quantification of apolipoproteins. J Proteomics 2017; 150: 258–267.

36.

Ruhaak

Smit

NPM

Romijn

et al . Robust and accurate 2-year performance of a quantitative mass spectrometry-based apolipoprotein test in a clinical chemistry laboratory. Clin Chem 2018; 64: 747–749.

37.

Ruhaak

Smit

NPM

Suchiman

HED

et al . MS-based proteomics: a metrological sound and robust alternative for apolipoprotein E phenotyping in a multiplexed test. Clin Chem Lab Med. Epub ahead of print 21 September 2018. DOI: https://doi.org/10.1515/cclm-2018-0782

38.

Anderson

Gregoire

Pearson

et al . 2016 Canadian Cardiovascular Society Guidelines for the management of dyslipidemia for the prevention of cardiovascular disease in the adult. Can J Cardiol 2016; 32: 1263–1282.

39.

Tsimikas

Fazio

Viney

et al . Relationship of lipoprotein(a) molar concentrations and mass according to lipoprotein(a) thresholds and apolipoprotein(a) isoform size. J Clin Lipidol 2018; 12: 1313–1323.

40.

Marcovina

Albers

JJ.

Lipoprotein (a) measurements for clinical application. J Lipid Res 2016; 57: 526–537.

41.

Tsimikas

Fazio

Ferdinand

et al . NHLBI working group recommendations to reduce lipoprotein(a)-mediated risk of cardiovascular disease and aortic stenosis. J Am Coll Cardiol 2018; 71: 177–192.

42.

Diffenderfer

Schaefer

EJ.

The composition and metabolism of large and small LDL. Curr Opin Lipidol 2014; 25: 221–226.

43.

Cooper

AD.

Hepatic uptake of chylomicron remnants. J Lipid Res 1997; 38: 2173–2192.

44.

Varbo

Benn

Tybjaerg-Hansen

et al . Remnant cholesterol as a causal risk factor for ischemic heart disease. J Am Coll Cardiol 2013; 61: 427–436.

45.

Nordestgaard

Langsted

Lipoprotein (a) as a cause of cardiovascular disease: insights from epidemiology, genetics, and biology. J Lipid Res 2016; 57: 1953–1975.

46.

Langlois

Chapman

Cobbaert

et al . Quantifying atherogenic lipoproteins: current and future challenges in the era of personalized medicine and very low concentrations of LDL cholesterol. A consensus statement from EAS and EFLM. Clin Chem 2018; 64: 1006–1033.

47.

Pencina

D'Agostino

Zdrojewski

et al . Apolipoprotein B improves risk assessment of future coronary heart disease in the Framingham Heart Study beyond LDL-C and non-HDL-C. Eur J Prev Cardiol 2015; 22: 1321–1327.

48.

Brown

Myers

Sniderman

et al . Should we use apoB for risk assessment and as a target for treatment? J Clin Lipidol 2010; 4: 144–151.

49.

Jong

Hofker

Havekes

LM.

Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2. Arterioscler Thromb Vasc Biol 1999; 19: 472–484.

50.

Weisgraber

Mahley

Kowal

et al . Apolipoprotein C-I modulates the interaction of apolipoprotein E with beta-migrating very low density lipoproteins (beta-VLDL) and inhibits binding of beta-VLDL to low density lipoprotein receptor-related protein. J Biol Chem 1990; 265: 22453–22459.

51.

Sehayek

Eisenberg

Mechanisms of inhibition by apolipoprotein C of apolipoprotein E-dependent cellular metabolism of human triglyceride-rich lipoproteins through the low density lipoprotein receptor pathway. J Biol Chem 1991; 266: 18259–18267.

52.

Kowal

Herz

Weisgraber

et al . Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein. J Biol Chem 1990; 265: 10771–10779.

53.

Shachter

Ebara

Ramakrishnan

et al . Combined hyperlipidemia in transgenic mice overexpressing human apolipoprotein Cl. J Clin Invest 1996; 98: 846–855.

54.

Jong

Dahlmans

van Gorp

et al . In the absence of the low density lipoprotein receptor, human apolipoprotein C1 overexpression in transgenic mice inhibits the hepatic uptake of very low density lipoproteins via a receptor-associated protein-sensitive pathway. J Clin Invest 1996; 98: 2259–2267.

55.

Vaisman

Klein

H-G

Rouis

et al . Overexpression of human lecithin cholesterol acyltransferase leads to hyperalphalipoproteinemia in transgenic mice. J Biol Chem 1995; 270: 12269–12275.

56.

Mehlum

Staels

Duverger

et al . Tissue-specific expression of the human gene for lecithin: cholesterol acyltransferase in transgenic mice alters blood lipids, lipoproteins and lipases towards a less atherogenic profile. Eur J Biochem 1995; 230: 567–575.

57.

Francone

Gong

et al . Expression of human lecithin-cholesterol acyltransferase in transgenic mice. Effect of human apolipoprotein AI and human apolipoprotein all on plasma lipoprotein cholesterol metabolism. J Clin Invest 1995; 96: 1440–1448.

58.

Jong

Ree JHv Dahlmans

VEH

et al . Reduced very-low-density lipoprotein fractional catabolic rate in apolipoprotein C1-deficient mice. Biochem J 1997; 321: 445–450.

59.

Breckenridge

Little

Steiner

et al . Hypertriglyceridemia associated with deficiency of apolipoprotein C-II. N Engl J Med 1978; 298: 1265–1273.

60.

Kostapanos

Milionis

Lagos

et al . Baseline triglyceride levels and insulin sensitivity are major determinants of the increase of LDL particle size and buoyancy induced by rosuvastatin treatment in patients with primary hyperlipidemia. Eur J Pharmacol 2008; 590: 327–332.

61.

Mabuchi

Kamon

Fujita

et al . Effects of Cs-514 on serum-lipoprotein lipid and apolipoprotein levels in patients with familial hypercholesterolemia. Metabolism 1987; 36: 475–479.

62.

N-A

Innis-Whitehouse

et al . Lipid and apolipoprotein levels and distribution in patients with hypertriglyceridemia: Effect of triglyceride reductions with atorvastatin. Metabolism 2000; 49: 167–177.

63.

Kostapanos

Milionis

Filippatos

et al . A 12-week, prospective, open-label analysis of the effect of rosuvastatin on triglyceride-rich lipoprotein metabolism in patients with primary dyslipidemia. Clin Ther 2007; 29: 1403–1414.

64.

Shen

Lookene

Nilsson

et al . Functional analyses of human apolipoprotein CII by site-directed mutagenesis: identification of residues important for activation of lipoprotein lipase. J Biol Chem 2002; 277: 4334–4342.

65.

Shachter

Hayek

Leff

et al . Overexpression of apolipoprotein CII causes hypertriglyceridemia in transgenic mice. J Clin Invest 1994; 93: 1683–1690.

66.

Kei

Filippatos

Tsimihodimos

et al . A review of the role of apolipoprotein C-II in lipoprotein metabolism and cardiovascular disease. Metab Clin Exp 2012; 61: 906–921.

67.

Peter

Kantartzis

Machicao

et al . Visceral obesity modulates the impact of apolipoprotein C3 gene variants on liver fat content. Int J Obes (Lond) 2012; 36: 774–782.

68.

Wang

McConathy

Kloer

et al . Modulation of lipoprotein lipase activity by apolipoproteins. Effect of apolipoprotein C-III. J Clin Invest 1985; 75: 384–390.

69.

McConathy

Gesquiere

Bass

et al . Inhibition of lipoprotein lipase activity by synthetic peptides of apolipoprotein C-III. J Lipid Res 1992; 33: 995–1003.

70.

Kinnunen

PKJ

Ehnholm

Effect of serum and C-apoproteins from very low-density lipoproteins on human postheparin plasma hepatic lipase. FEBS Lett 1976; 65: 354–357.

71.

Quarfordt

Michalopoulos

Schirmer

The effect of human C apolipoproteins on the in vitro hepatic metabolism of triglyceride emulsions in the rat. J Biol Chem 1982; 257: 14642–14647.

72.

Mann

Troussard

Yen

et al . Inhibitory effects of specific apolipoprotein C-III isoforms on the binding of triglyceride-rich lipoproteins to the lipolysis-stimulated receptor. J Biol Chem 1997; 272: 31348–31354.

73.

Qin

Sundaram

Wang

et al . Missense mutation in APOC3 within the C-terminal lipid binding domain of human ApoC-III results in impaired assembly and secretion of triacylglycerol-rich very low density lipoproteins: evidence that ApoC-III plays a major role in the formation of lipid precursors within the microsomal lumen. J Biol Chem 2011; 286: 27769–27780.

74.

Chan

Watts

Nguyen

et al . Apolipoproteins C-III and A-V as predictors of very-low-density lipoprotein triglyceride and apolipoprotein B-100 kinetics. Arterioscler Thromb Vasc Biol 2006; 26: 590–596.

75.

Taskinen

Adiels

Westerbacka

et al . Dual metabolic defects are required to produce hypertriglyceridemia in obese subjects. Arterioscler Thromb Vasc Biol 2011; 31: 2144–2150.

76.

Cohn

Patterson

Uffelman

et al . Rate of production of plasma and very-low-density lipoprotein (VLDL) apolipoprotein C-III is strongly related to the concentration and level of production of VLDL triglyceride in male subjects with different body weights and levels of insulin sensitivity. J Clin Endocrinol Metab 2004; 89: 3949–3855.

77.

Agnani

Bard

Candelier

et al . Interaction of LpB, LpB:E, LpB:C-III, and LpB:C-III:E lipoproteins with the low density lipoprotein receptor of HeLa cells. Arterioscler Thromb 1991; 11: 1021–1029.

78.

Clavey

Lestavel-Delattre

Copin

et al . Modulation of lipoprotein B binding to the LDL receptor by exogenous lipids and apolipoproteins CI, CII, CIII, and E. Arterioscler Thromb Vasc Biol 1995; 15: 963–971.

79.

Mahley

Innerarity

Rall

Jr et al . Plasma lipoproteins: apolipoprotein structure and function. J Lipid Res 1984; 25: 1277–1294.

80.

Le NA

GJC

Ginsberg

HN.

Independent regulation of plasma apolipoprotein-C-II and apolipoprotein-C-III concentrations in very low-density and high-density lipoproteins – implications for the regulation of the catabolism of these lipoproteins. J Lipid Res 1988; 29: 669–677.

81.

Malmendier

Lontie

Delcroix

et al . Apolipoproteins C-II and C-III metabolism in hypertriglyceridemic patients. Effect of a drastic triglyceride reduction by combined diet restriction and fenofibrate administration. Atherosclerosis 1989; 77: 139–149.

82.

Staels

Vu-Dac

Kosykh

et al . Fibrates downregulate apolipoprotein C-III expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the hypolipidemic action of fibrates. J Clin Invest 1995; 95: 705–712.

83.

Hertz

Bisharashieban

Bartana

Mode of action of peroxisome proliferators as hypolipidemic drugs – suppression of apolipoprotein C-III. J Biol Chem 1995; 270: 13470–13475.

84.

Auwerx

Schoonjans

Fruchart

et al . Transcriptional control of triglyceride metabolism: fibrates and fatty acids change the expression of the LPL and apo C-III genes by activating the nuclear receptor PPAR. Atherosclerosis 1996; 124 Suppl: S29–S37.

85.

Graham

Lee

Bell

3rd et al . Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans. Circ Res 2013; 112: 1479–1490.

86.

Mahley

Rall

Jr.

Apolipoprotein E: far more than a lipid transport protein. Annu Rev Genomics Hum Genet 2000; 1: 507–537.

87.

Rensen

van Berkel

TJ.

Apolipoprotein E effectively inhibits lipoprotein lipase-mediated lipolysis of chylomicron-like triglyceride-rich lipid emulsions in vitro and in vivo. J Biol Chem 1996; 271: 14791–14799.

88.

Jong

Dahlmans

VEH

Hofker

et al . Nascent very-low-density lipoprotein triacylglycerol hydrolysis by lipoprotein lipase is inhibited by apolipoprotein E in a dose-dependent manner. Biochem J 1997; 328: 745–750.

89.

van Dijk

van Vlijmen

BJM

van't Hof

et al . In LDL receptor-deficient mice, catabolism of remnant lipoproteins requires a high level of apoE but is inhibited by excess apoE. J Lipid Res 1999; 40: 336–344.

90.

Mahley

RW.

Apolipoprotein E: from cardiovascular disease to neurodegenerative disorders. J Mol Med 2016; 94: 739–746.

91.

Horejsi

Ceska

Apolipoproteins and atherosclerosis. Apolipoprotein E and apolipoprotein(a) as candidate genes of premature development of atherosclerosis. Physiol Res 2000; 49: S63–S69.

92.

Yamakawa-Kobayashi

Arinami

et al . Methylenetetrahydrofolate reductase and apolipoprotein E polymorphisms are independent risk factors for coronary heart disease in Japanese: a case-control study. Atherosclerosis 1998; 137: 23–28.

93.

Scuteri

Bos

AJG

Zonderman

et al . Is the apoE4 allele an independent predictor of coronary events? Am J Med 2001; 110: 28–32.

94.

Chen

Reis

Kammerer

et al . APOE polymorphism and angiographic coronary artery disease severity in the Women's Ischemia Syndrome Evaluation (WISE) study. Atherosclerosis 2003; 169: 159–167.

95.

Weintraub

Eisenberg

Breslow

JL.

Dietary-fat clearance in normal subjects is regulated by genetic-variation in apolipoprotein-E. J Clin Invest 1987; 80: 1571–1577.

96.

Orth

Wahl

Hanisch

et al . Clearance of postprandial lipoproteins in normolipemics: role of the apolipoprotein E phenotype. Biochim Biophys Acta 1996; 1303: 22–30.

97.

Zhao

Zhang

et al . Apolipoprotein E gene variants and risk of coronary heart disease: a meta-analysis. Biomed Res Int 2016; 2016: 3912175.

98.

Kuo

Wilson

Kostis

et al . Treatment of type III hyperlipoproteinemia with gemfibrozil to retard progression of coronary artery disease. Am Heart J 1988; 116: 85–90.

99.

Feussner

Wagner

Ziegler

Relation of cardiovascular risk factors to atherosclerosis in type III hyperlipoproteinemia. Hum Genet 1993; 92: 122–126.

100.

Sticchi

Magi

Kamstrup

et al . Apolipoprotein(a) Kringle-IV type 2 copy number variation is associated with venous thromboembolism. PLoS One 2016; 11: e0149427.

101.

Tsimikas

Brilakis

Miller

et al . Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease. N Engl J Med 2005; 353: 46–57.

102.

Emerging Risk Factors

Collaboration

Erqou

Kaptoge

et al . Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality. JAMA 2009; 302: 412–423.

103.

Nordestgaard

Chapman

Ray

et al . Lipoprotein(a) as a cardiovascular risk factor: current status. Eur Heart J 2010; 31: 2844–2853.

104.

Tuteja

Rader

DJ.

High-density lipoproteins in the prevention of cardiovascular disease: changing the paradigm. Clin Pharmacol Ther 2014; 96: 48–56.

105.

Emerging Risk Factors Collaboration, Di Angelantonio

Sarwar

et al . Major lipids, apolipoproteins, and risk of vascular disease. JAMA 2009; 302: 1993–2000.

106.

Nordestgaard

BG.

Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology. Circ Res 2016; 118: 547–563.

107.

Katan

MB.

Apolipoprotein-E isoforms, serum-cholesterol, and cancer. Lancet 1986; 1: 507–508.

108.

Davey Smith

Ebrahim

Mendelian randomization’: can genetic epidemiology contribute to understanding environmental determinants of disease?

Int J Epidemiol 2003; 32: 1–22.

109.

Lawlor

Harbord

Sterne

et al . Mendelian randomization: using genes as instruments for making causal inferences in epidemiology. Statist Med 2008; 27: 1133–1163.

110.

Peto

Doll

Buckley

et al . Can dietary beta-carotene materially reduce human cancer rates? Nature 1981; 290: 201–208.

111.

Alpha-Tocopherol BCCPSG. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med 1994; 330: 1029–1035.

112.

Omenn

Goodman

Thornquist

et al . Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. N Engl J Med 1996; 334: 1150–1155.

113.

Bowden

Hemani

Davey Smith

Detecting individual and global horizontal pleiotropy in Mendelian randomization: a job for the humble heterogeneity statistic?

Am J Epidemiol 2018; 187: 2681–2685.

114.

Ference

Ginsberg

Graham

et al . Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel. Eur Heart J 2017; 38: 2459–2472.

115.

Emerging Risk Factors Collaboration, Di Angelantonio

Gao

et al . Lipid-related markers and cardiovascular disease prediction. JAMA 2012; 307: 2499–2506.

116.

Prospective Studies Collaboration. Blood cholesterol and vascular mortality by age, sex, and blood pressure: a meta-analysis of individual data from 61 prospective studies with 55 000 vascular deaths. Lancet 2007; 370: 1829–1839.

117.

Kathiresan

Melander

Anevski

et al . Polymorphisms associated with cholesterol and risk of cardiovascular events. N Engl J Med 2008; 358: 1240–1249.

118.

Voight

Peloso

Orho-Melander

et al . Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomisation study. Lancet 2012; 380: 572–580.

119.

Pang

Poulter

Bell

et al . Frequency of familial hypercholesterolemia in patients with early-onset coronary artery disease admitted to a coronary care unit. J Clin Lipidol 2015; 9: 703–708.

120.

Brahm

Hegele

RA.

Chylomicronaemia-current diagnosis and future therapies. Nat Rev Endocrinol 2015; 11: 352–362.

121.

Lam

Yuen

Cheng

et al . Missense mutation Leu72Pro located on the carboxyl terminal amphipathic helix of apolipoprotein C-II causes familial chylomicronemia syndrome. Clinica Chimica Acta 2006; 364: 256–259.

122.

Okubo

Toromanovic

Ebara

et al . Apolipoprotein C-II Tuzla: a novel large deletion in APOC2 caused by Alu-Alu homologous recombination in an infant with apolipoprotein C-II deficiency. Clinica Chimica Acta 2015; 438: 148–153.

123.

Ueda

Dunbar

Wolska

et al . A novel APOC2 missense mutation causing apolipoprotein C-II deficiency with severe triglyceridemia and pancreatitis. J Clin Endocrinol Metab 2017; 102: 1454–1457.

124.

Streicher

Geisel

Weisshaar

et al . A single nucleotide substitution in the promoter region of the apolipoprotein C-II gene identified in individuals with chylomicronemia. J Lipid Res 1996; 37: 2599–2607.

125.

Baggio

Manzato

Gabelli

et al . Apolipoprotein C-II deficiency syndrome. Clinical features, lipoprotein characterization, lipase activity, and correction of hypertriglyceridemia after apolipoprotein C-II administration in two affected patients. J Clin Invest 1986; 77: 520–527.

126.

Hermans

MPJ

Bodde

Jukema

et al . Low levels of apolipoprotein-CII in normotriglyceridemic patients with very premature coronary artery disease: observations from the MISSION! Intervention study. J Clin Lipidol 2017; 11: 1407–1414.

127.

Tg, HDL Working Group of the Exome Sequencing Project NHL, Blood I,et al. Loss-of-function mutations in APOC3, triglycerides, and coronary disease. N Engl J Med 2014; 371: 22–31.

128.

Jorgensen

Frikke-Schmidt

Nordestgaard

et al . Loss-of-function mutations in APOC3 and risk of ischemic vascular disease. N Engl J Med 2014; 371: 32–41.

129.

Cohen

Stender

Hobbs

HH.

APOC3, coronary disease, and complexities of Mendelian randomization. Cell Metab 2014; 20: 387–389.

130.

Pollin

Damcott

Shen

et al . A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection. Science 2008; 322: 1702–1705.

131.

Tachmazidou

Dedoussis

Southam

et al . A rare functional cardioprotective APOC3 variant has risen in frequency in distinct population isolates. Nat Commun 2013; 4: 2872.

132.

Wulff

Nordestgaard

Tybjaerg-Hansen

A. APOC3 loss-of-function mutations, remnant cholesterol, low-density lipoprotein cholesterol, and cardiovascular risk: mediation- and meta-analyses of 137 895 individuals. Arterioscler Thromb Vasc Biol 2018; 38: 660–668.

133.

Kamstrup

Tybjaerg-Hansen

Steffensen

et al . Genetically elevated lipoprotein(a) and increased risk of myocardial infarction. JAMA 2009; 301: 2331–2339.

134.

Willeit

Kiechl

Kronenberg

et al . Discrimination and net reclassification of cardiovascular risk with lipoprotein(a): prospective 15-year outcomes in the Bruneck Study. J Am Coll Cardiol 2014; 64: 851–860.

135.

Erqou

Thompson

Di Angelantonio

et al . Apolipoprotein(a) isoforms and the risk of vascular disease: systematic review of 40 studies involving 58,000 participants. J Am Coll Cardiol 2010; 55: 2160–2167.

136.

Clarke

Peden

Hopewell

et al . Genetic variants associated with Lp(a) lipoprotein level and coronary disease. N Engl J Med 2009; 361: 2518–2528.

137.

Arsenault

Boekholdt

Dube

et al . Lipoprotein(a) levels, genotype, and incident aortic valve stenosis: a prospective Mendelian randomization study and replication in a case-control cohort. Circ Cardiovasc Genet 2014; 7: 304–310.

138.

Burgess

Ference

Staley

et al . Association of LPA variants with risk of coronary disease and the implications for lipoprotein(a)-lowering therapies: a Mendelian randomization analysis. JAMA Cardiol 2018; 3: 619–627.

139.

Sandholzer

Hallman

Saha

et al . Effects of the apolipoprotein(a) size polymorphism on the lipoprotein(a) concentration in 7 ethnic groups. Hum Genet 1991; 86: 607–614.

140.

Saleheen

Haycock

Zhao

et al . Apolipoprotein(a) isoform size, lipoprotein(a) concentration, and coronary artery disease: a Mendelian randomisation analysis. Lancet Diabetes Endocrinol 2017; 5: 524–533.

141.

Ference

Yoo

Alesh

et al . Effect of long-term exposure to lower low-density lipoprotein cholesterol beginning early in life on the risk of coronary heart disease: a Mendelian randomization analysis. J Am Coll Cardiol 2012; 60: 2631–2639.

142.

Mahley

RW.

Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 1988; 240: 622–630.

143.

Mahley

Huang

Apolipoprotein E: from atherosclerosis to Alzheimer's disease and beyond. Curr Opin Lipidol 1999; 10: 207–217.

144.

Kritharides

Nordestgaard

Tybjaerg-Hansen

et al . Effect of APOE epsilon genotype on lipoprotein(a) and the associated risk of myocardial infarction and aortic valve stenosis. J Clin Endocrinol Metab 2017; 102: 3390–3399.

145.

Haase

Tybjaerg-Hansen

Grande

et al . Genetically elevated apolipoprotein A-I, high-density lipoprotein cholesterol levels, and risk of ischemic heart disease. J Clin Endocrinol Metab 2010; 95: E500–E510.

146.

Mack

Coassin

Vaucher

et al . Evaluating the causal relation of apoA-IV with disease-related traits – a bidirectional two-sample Mendelian randomization study. Sci Rep 2017; 7: 8734.

147.

Triglyceride Coronary Disease Genetics Consortium and Emerging Risk Factors Collaboration, Sarwar

et al . Triglyceride-mediated pathways and coronary disease: collaborative analysis of 101 studies. Lancet 2010; 375: 1634–1639.

148.

Stitziel

Won

et al . Exome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction. Nature 2015; 518: 102–106.

149.

Pechlaner

Tsimikas

Yin

et al . Very-low-density lipoprotein-associated apolipoproteins predict cardiovascular events and are lowered by inhibition of APOC-III. J Am Coll Cardiol 2017; 69: 789–800.

150.

Walldius

Jungner

Holme

et al . High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet 2001; 358: 2026–2033.

151.

Pischon

Girman

Sacks

et al . Non-high-density lipoprotein cholesterol and apolipoprotein B in the prediction of coronary heart disease in men. Circulation 2005; 112: 3375–3383.

152.

Sniderman

Williams

Contois

et al . A meta-analysis of low-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol, and apolipoprotein B as markers of cardiovascular risk. Circ Cardiovasc Qual Outcomes 2011; 4: 337–345.

153.

Vaisar

Pennathur

Green

et al . Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL. J Clin Invest 2007; 117: 746–756.

154.

Davidson

MH.

Apolipoprotein measurements: is more widespread use clinically indicated?

Clin Cardiol 2009; 32: 482–486.

155.

Sandhu

Musaad

Remaley

et al . Lipoprotein biomarkers and risk of cardiovascular disease: a laboratory medicine best practices (LMBP) systematic review. Jrnl App Lab Med 2016; 1: 214–229.

156.

Bodde

Hermans

MPJ

Jukema

et al . Apolipoproteins A1, B, and apoB/apoA1 ratio are associated with first ST-segment elevation myocardial infarction but not with recurrent events during long-term follow-up. Clin Res Cardiol. Epub ahead of print 8 October 2018. doi: 10.1007/s00392-018-1381-5.

157.

Gerber

Goldbourt

Cohen

et al . Association between serum apolipoprotein CII concentration and coronary heart disease. Prev Med 2002; 35: 42–47.

158.

van Capelleveen

Bernelot Moens

Yang

et al . Apolipoprotein C-III levels and incident coronary artery disease risk: the EPIC-Norfolk prospective population study. Arterioscler Thromb Vasc Biol 2017; 37: 1206–1212.

159.

Yang

Lee

Choi

et al . Reduction in lipoprotein-associated apoC-III levels following volanesorsen therapy: phase 2 randomized trial results. J Lipid Res 2016; 57: 706–713.

160.

Digenio

Dunbar

Alexander

et al . Antisense-mediated lowering of plasma apolipoprotein C-III by volanesorsen improves dyslipidemia and insulin sensitivity in type 2 diabetes. Dia Care 2016; 39: 1408–1415.

161.

Arca

Hsieh

Soran

et al . The effect of volanesorsen treatment on the burden associated with familial chylomicronemia syndrome: the results of the ReFOCUS study. Expert Rev Cardiovasc Ther 2018; 16: 537–546.

162.

Kostner

Avogaro

Cazzolato

et al . Lipoprotein Lp(a) and the risk for myocardial infarction. Atherosclerosis 1981; 38: 51–61.

163.

Riches

Porter

KE.

Lipoprotein(a): cellular effects and molecular mechanisms. Cholesterol 2012; 2012: 923289.

164.

Viney

van Capelleveen

Geary

et al . Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials. Lancet 2016; 388: 2239–2253.

165.

Roeseler

Julius

Heigl

et al . Lipoprotein apheresis for lipoprotein(a)-associated cardiovascular disease: prospective 5 years of follow-up and apolipoprotein(a) characterization. Arterioscler Thromb Vasc Biol 2016; 36: 2019–2027.

166.

Friedewald

Levy

Fredrickson

DS.

Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18: 499–502.

167.

Conroy

Pyorala

Fitzgerald

et al . Estimation of ten-year risk of fatal cardiovascular disease in Europe: the SCORE project. Eur Heart J 2003; 24: 987–1003.

168.

Perk

De Backer

Gohlke

et al . European Guidelines on cardiovascular disease prevention in clinical practice (version 2012). The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts). Eur Heart J 2012; 33: 1635–1701.

169.

Ridker

Rifai

Cook

et al . Non-HDL cholesterol, apolipoproteins A-I and B100, standard lipid measures, lipid ratios, and CRP as risk factors for cardiovascular disease in women. JAMA 2005; 294: 326–333.

170.

Arsenault

Rana

Stroes

et al . Beyond low-density lipoprotein cholesterol: respective contributions of non-high-density lipoprotein cholesterol levels, triglycerides, and the total cholesterol/high-density lipoprotein cholesterol ratio to coronary heart disease risk in apparently healthy men and women. J Am Coll Cardiol 2009; 55: 35–41.

171.

Kastelein

van der Steeg

Holme

et al . Lipids, apolipoproteins, and their ratios in relation to cardiovascular events with statin treatment. Circulation 2008; 117: 3002–3009.

172.

Puri

Nissen

Shao

et al . Non-HDL cholesterol and triglycerides: implications for coronary atheroma progression and clinical events. Arterioscler Thromb Vasc Biol 2016; 36: 2220–2228.

173.

Verbeek

Sandhu

Hovingh

et al . Lipoprotein(a) improves cardiovascular risk prediction based on established risk algorithms. J Am Coll Cardiol 2017; 69: 1513–1515.

174.

Varbo

Nordestgaard

BG.

Remnant cholesterol and triglyceride-rich lipoproteins in atherosclerosis progression and cardiovascular disease. Arterioscler Thromb Vasc Biol 2016; 36: 2133–2135.

175.

Goldstein

Hobbs

Brown

Familial hypercholesterolemia. In: Scriver

Beaudet

Sly

et al . (eds) The metabolic and molecular basis of inherited disease. New York: McGraw-Hill, 2001, pp. 2863–2913.

176.

Marcovina

Viney

Hughes

et al . Temporal variability in lipoprotein(a) levels in patients enrolled in the placebo arms of IONIS-APO(a)Rx and IONIS-APO(a)-LRx antisense oligonucleotide clinical trials. J Clin Lipidol 2018; 12: 122–129 e2.

177.

Smith

Kelleher

; Consortium for Top Down Preoteomics. Proteoform: a single term describing protein complexity. Nat Methods 2013; 10: 186–187.

178.

van der Burgt

YEM

Cobbaert

CM.

Proteoform analysis to fulfill unmet clinical needs and reach global standardization of protein measurands in clinical chemistry proteomics. Clin Lab Med 2018; 38: 487–497.

179.

Wopereis

Grunewald

Morava

et al . Apolipoprotein C-III isofocusing in the diagnosis of genetic defects in O-glycan biosynthesis. Clin Chem 2003; 49: 1839–1845.

180.

Nicolardi

van der Burgt

Wuhrer

et al . Mapping O-glycosylation of apolipoprotein C-III in MALDI-FT-ICR protein profiles. Proteomics 2013; 13: 992–1001.

181.

Koska

Yassine

Trenchevska

et al . Disialylated apolipoprotein C-III proteoform is associated with improved lipids in prediabetes and type 2 diabetes. J Lipid Res 2016; 57: 894–905.

182.

Mendoza

Trenchevska

King

et al . Changes in low-density lipoprotein size phenotypes associate with changes in apolipoprotein C-III glycoforms after dietary interventions. J Clin Lipidol 2017; 11: 224–233 e2.

183.

Trenchevska

Schaab

Nelson

et al . Development of multiplex mass spectrometric immunoassay for detection and quantification of apolipoproteins C-I, C-II, C-III and their proteoforms. Methods 2015; 81: 86–92.

184.

Seckler

HDS

Fornelli

Mutharasan

et al . A targeted, differential top-down proteomic methodology for comparison of apoA-I proteoforms in individuals with high and low HDL efflux capacity. J Proteome Res 2018; 17: 2156–2164.

185.

Nedelkov

Human proteoforms as new targets for clinical mass spectrometry protein tests. Expert Rev Proteomics 2017; 14: 691–699.

186.

Ruhaak

van der Burgt

Cobbaert

CM.

Prospective applications of ultrahigh resolution proteomics in clinical mass spectrometry. Expert Rev Proteomics 2016; 13: 1063–1071.

Apolipoprotein profiling as a personalized approach to the diagnosis and treatment of dyslipidaemia

Abstract

Keywords

Introduction

Residual CV risk beyond conventional CVD therapies

CVD in women

Inadequate cholesterol-based tests do not identify atherogenic remnant disease

Ineffective therapy due to average conclusions and average dosing not considering the individual patient

Apolipoprotein quantitation by mass spectrometry

Inadequate standardization of Lp(a) measurement

Biochemistry and pathobiology of apolipoproteins: Insight into dyslipidaemia and atherosclerosis

ApoB100

ApoCI

ApoCII

ApoCIII

ApoE

Apo(a)

Apo A-I

Genetic evidence for the role of apolipoproteins in CVD

ApoB

ApoCII

ApoCIII

Apo(a)

ApoE

ApoA1

ApoA-IV

ApoA-V

Apolipoprotein profiles and cardiovascular risk: Evidence from outcome based studies

Embedding apolipoprotein profiling in cardiovascular precision clinical care pathways

Conventional clinical care pathway

Proposed cardiovascular precision care pathway

Conclusions and future outlook

Footnotes

Declaration of conflicting interests

Funding

Ethical approval

Guarantor

Contributorship

References