Very-Low-Density Lipoprotein-Associated Apolipoproteins Predict Cardiovascular Events and Are Lowered by Inhibition of APOC-III

Raimund Pechlaner, Sotirios Tsimikas, Xiaoke Yin, Peter Willeit, Ferheen Baig, Peter Santer, Friedrich Oberhollenzer, Georg Egger, Joseph L Witztum, Veronica J Alexander, Johann Willeit, Stefan Kiechl, Manuel Mayr, Raimund Pechlaner, Sotirios Tsimikas, Xiaoke Yin, Peter Willeit, Ferheen Baig, Peter Santer, Friedrich Oberhollenzer, Georg Egger, Joseph L Witztum, Veronica J Alexander, Johann Willeit, Stefan Kiechl, Manuel Mayr

Abstract

Background: Routine apolipoprotein (apo) measurements for cardiovascular disease (CVD) are restricted to apoA-I and apoB. Here, the authors measured an unprecedented range of apolipoproteins in a prospective, population-based study and relate their plasma levels to risk of CVD.

Objectives: This study sought to measure apolipoproteins directly by mass spectrometry and compare their associations with incident CVD and to obtain a system-level understanding of the correlations of apolipoproteins with the plasma lipidome and proteome.

Methods: Associations of 13 apolipoproteins, 135 lipid species, and 211 other plasma proteins with incident CVD (91 events), defined as stroke, myocardial infarction, or sudden cardiac death, were assessed prospectively over a 10-year period in the Bruneck Study (N = 688) using multiple-reaction monitoring mass spectrometry. Changes in apolipoprotein and lipid levels following treatment with volanesorsen, a second-generation antisense drug targeting apoC-III, were determined in 2 human intervention trials, one of which was randomized.

Results: The apolipoproteins most significantly associated with incident CVD were apoC-II (hazard ratio per 1 SD [HR/SD]: 1.40; 95% confidence interval [CI]: 1.17 to 1.67), apoC-III (HR/SD: 1.38; 95% CI: 1.17 to 1.63), and apoE (HR/SD: 1.31; 95% CI: 1.13 to 1.52). Associations were independent of high-density lipoprotein (HDL) and non-HDL cholesterol, and extended to stroke and myocardial infarction. Lipidomic and proteomic profiles implicated these 3 very-low-density lipoprotein (VLDL)-associated apolipoproteins in de novo lipogenesis, glucose metabolism, complement activation, blood coagulation, and inflammation. Notably, apoC-II/apoC-III/apoE correlated with a pattern of lipid species previously linked to CVD risk. ApoC-III inhibition by volanesorsen reduced plasma levels of apoC-II, apoC-III, triacylglycerols, and diacylglycerols, and increased apoA-I, apoA-II, and apoM (all p < 0.05 vs. placebo) without affecting apoB-100 (p = 0.73).

Conclusions: The strong associations of VLDL-associated apolipoproteins with incident CVD in the general community support the concept of targeting triacylglycerol-rich lipoproteins to reduce risk of CVD.

Keywords: lipidomics; mass spectrometry; proteomics; triglycerides.

Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Associations of Apolipoproteins and Lipid Measures With Incident CVD Plasma levels of 13 apolipoproteins and of 4 conventional lipid measures were determined in 688 participants of the Bruneck Study. During 10 years of follow-up, 91 cardiovascular events occurred, comprising stroke, myocardial infarction, and sudden cardiac death. Model 1: Adjustment for age, sex, and statin therapy. Model 2: As in model 1, with additional adjustment for diabetes, systolic blood pressure, and smoking. Model 3: As in model 2, with additional adjustment for HDL-C and non–HDL-C. Quantitatively, for each variable, 1 SD corresponds to: ApoA-I, 607 mg/l; ApoA-II, 6.44 mg/l; ApoA-IV, 15.0 mg/l; ApoB-100, 363 mg/l; ApoC-I, 6.46 mg/l; ApoC-II, 6.30 mg/l; ApoC-III, 25.6 mg/l; ApoD, 7.98 mg/l; ApoE, 9.23 mg/l; ApoH, 38.2 mg/l; ApoL-I, 3.93 mg/l; ApoM, 2.42 mg/l; ApoJ, 23.1 mg/l; HDL-C, 15.2 mg/dl; LDL-C, 36.5 mg/dl; non-HDL-C, 41.4 mg/dl; triglycerides, 77.6 mg/dl. apo = apolipoprotein; CI = confidence interval; CVD = cardiovascular disease; HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol.
Figure 2
Figure 2
Correlations Among Apolipoproteins and Lipids Results are adjusted for age, sex, and statin therapy. Tile color codes for direction and magnitude of correlation, whereas tile text gives its sign and the first 2 decimal digits. Variables are arranged by similarity, as shown in the right-hand dendrogram. Only significant correlations are shown. Clustering gave rise to several groups of highly intercorrelated variables. High-level clusters were characterized by more extensive correlations with apoA-I (top right region) or with apoB-100 (bottom left region), with the latter containing subclusters likely representing VLDL (including apoC-III, apoC-II, apoE, TG, non–HDL-C, and HDL-C) and LDL (including apoB-100 and LDL-C). VLDL = very-low-density lipoprotein; other abbreviations as in Figure 1.
Figure 3
Figure 3
Associations of Apolipoproteins With Incident CVD Upon Additional Adjustment for ApoC-II, ApoC-III, or Apo-E Base adjustment consisted of adjustment for age, sex, and statin therapy and is shown for the significant apolipoproteins only in the first column (as in Figure 1). Additional adjustment for apoC-II, apoC-III, or apoE is shown in the other 3 columns, respectively. Note that apoB-100 loses its association with incident CVD upon adjustment for any of the 3 VLDL-associated apolipoproteins. Abbreviations as in Figures 1 and 2.
Figure 4
Figure 4
Associations of 135 Plasma Lipid Species With ApoC-II, ApoC-III, or ApoE Individual lipid species are depicted by filled circles and arranged by lipid class in 8 panels according to the number of total carbon atoms (x-axes) and number of double bonds (y-axes). Circle fill color represents the correlation of each lipid species with plasma concentrations of apoC-II, apoC-III, and apoE. Lipids with the same number of carbon atoms and double bonds are pulled apart vertically to increase their visibility. The distinguishing feature in this case is the presence of an alkyl ether linkage, signified in the formula as, for example, PC(O-38:3). Lipids possessing such a linkage are pulled upward, and their alkyl-ether-free counterparts are pulled downward. CE = cholesteryl ester; CI = confidence interval; LPC = lysophosphatidylcholine; LPE = lysophosphatidylethanolamine; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PS = phosphatidylserine; SM = sphingomyelin; TAG = triacylglycerol.
Figure 5
Figure 5
Effects of ApoC-III Synthesis Inhibition on Plasma Concentrations of Apolipoproteins and Lipid Classes In 2 independent intervention trials (IONIS1 and IONIS2), apoC-III synthesis was inhibited with the second-generation antisense oligonucleotide volanesorsen. In the randomized double-blind IONIS2 trial, 11 patients received treatment and 6 received placebo. *p values are for change from baseline in IONIS1, and for differential change from baseline in the treatment and placebo groups in IONIS2. (A) Effect on apolipoproteins. Among the apolipoproteins measured, apoC-III decreased most strongly, followed by apoC-II. An increase in plasma levels was observed for apoA-I, apoA-II, and apoM. (B) Effect on lipid classes. A substantial reduction in plasma levels was observed for TAG and DAG. CER = ceramide; DAG = diacylglycerol; FFA = free fatty acid; HCER = hexosylceramide; LCER = lactosylceramide; other abbreviations as in Figure 4.
Figure 5
Figure 5
Effects of ApoC-III Synthesis Inhibition on Plasma Concentrations of Apolipoproteins and Lipid Classes In 2 independent intervention trials (IONIS1 and IONIS2), apoC-III synthesis was inhibited with the second-generation antisense oligonucleotide volanesorsen. In the randomized double-blind IONIS2 trial, 11 patients received treatment and 6 received placebo. *p values are for change from baseline in IONIS1, and for differential change from baseline in the treatment and placebo groups in IONIS2. (A) Effect on apolipoproteins. Among the apolipoproteins measured, apoC-III decreased most strongly, followed by apoC-II. An increase in plasma levels was observed for apoA-I, apoA-II, and apoM. (B) Effect on lipid classes. A substantial reduction in plasma levels was observed for TAG and DAG. CER = ceramide; DAG = diacylglycerol; FFA = free fatty acid; HCER = hexosylceramide; LCER = lactosylceramide; other abbreviations as in Figure 4.
Central Illustration
Central Illustration
Associations of Apolipoproteins With Incident Cardiovascular Disease Plasma levels of 13 apolipoproteins were determined in 688 participants of the Bruneck Study. During 10 years of follow-up, 91 cardiovascular events occurred, comprising stroke, myocardial infarction, and sudden cardiac death. Results are adjusted for age, sex, and statin therapy. Apo = apolipoprotein; CI = confidence interval.

References

    1. Stegemann C., Pechlaner R., Willeit P. Lipidomics profiling and risk of cardiovascular disease in the prospective population-based Bruneck Study. Circulation. 2014;129:1821–1831.
    1. Gaudet D., Brisson D., Tremblay K. Targeting APOC3 in the familial chylomicronemia syndrome. N Engl J Med. 2014;371:2200–2206.
    1. Gaudet D., Alexander V.J., Baker B.F. Antisense inhibition of apolipoprotein C-III in patients with hypertriglyceridemia. N Engl J Med. 2015;373:438–447.
    1. IHD Register: Report of the Fifth Working Group. World Health Organization; Copenhagen, Denmark: 1971.
    1. Walker A.E., Robins M., Weinfeld F.D. The National Survey of Stroke: clinical findings. Stroke. 1981;12:I13–I44.
    1. Rothman K.J. No adjustments are needed for multiple comparisons. Epidemiology (Cambridge, Mass) 1990;1:43–46.
    1. Jong M.C., Hofker M.H., Havekes L.M. Role of ApoCs in lipoprotein metabolism functional differences between ApoC1, ApoC2, and ApoC3. Arterioscler Thromb Vasc Biol. 1999;19:472–484.
    1. Nordestgaard B.G., Varbo A. Triglycerides and cardiovascular disease. Lancet. 2014;384:626–635.
    1. Rapp J.H., Harris H.W., Hamilton R.L. Particle size distribution of lipoproteins from human atherosclerotic plaque: a preliminary report. J Vasc Surg. 1989;9:81–88.
    1. Do R., Willer C.J., Schmidt E.M. Common variants associated with plasma triglycerides and risk for coronary artery disease. Nat Genet. 2013;45:1345–1352.
    1. Jørgensen A.B., Frikke-Schmidt R., West A.S. Genetically elevated non-fasting triglycerides and calculated remnant cholesterol as causal risk factors for myocardial infarction. Eur Heart J. 2013;34:1826–1833.
    1. TG and HDL Working Group of the Exome Sequencing Project, National Heart, Lung, and Blood Institute Loss-of-function mutations in APOC3, triglycerides, and coronary disease. N Engl J Med. 2014;371:22–31.
    1. Jørgensen A.B., Frikke-Schmidt R., Nordestgaard B.G. Loss-of-function mutations in APOC3 and risk of ischemic vascular disease. N Engl J Med. 2014;371:32–41.
    1. Varbo A., Benn M., Tybjærg-Hansen A. Remnant cholesterol as a causal risk factor for ischemic heart disease. J Am Coll Cardiol. 2013;61:427–436.
    1. Varbo A., Benn M., Tybjærg-Hansen A. Elevated remnant cholesterol causes both low-grade inflammation and ischemic heart disease, whereas elevated low-density lipoprotein cholesterol causes ischemic heart disease without inflammation. Circulation. 2013;128:1298–1309.
    1. Sundaram M., Zhong S., Bou Khalil M. Expression of apolipoprotein C-III in McA-RH7777 cells enhances VLDL assembly and secretion under lipid-rich conditions. J Lipid Res. 2010;51:150–161.
    1. Mensenkamp A.R., Jong M.C., van Goor H. Apolipoprotein E participates in the regulation of very low density lipoprotein-triglyceride secretion by the liver. J Biol Chem. 1999;274:35711–35718.
    1. Sacks F.M. The crucial roles of apolipoproteins E and C-III in apoB lipoprotein metabolism in normolipidemia and hypertriglyceridemia. Curr Opin Lipidol. 2015;26:56–63.
    1. Gabelli C., Gregg R.E., Zech L.A. Abnormal low density lipoprotein metabolism in apolipoprotein E deficiency. J Lipid Res. 1986;27:326–333.
    1. Mahley R.W., Huang Y., Rall S.C., Jr. Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia): questions, quandaries, and paradoxes. J Lipid Res. 1999;40:1933–1949.
    1. Mendivil C.O., Zheng C., Furtado J. Metabolism of very-low-density lipoprotein and low-density lipoprotein containing apolipoprotein C-III and not other small apolipoproteins. Arterioscler Thromb Vasc Biol. 2010;30:239–245.
    1. Drenos F., Davey Smith G., Ala-Korpela M. Metabolic characterization of a rare genetic variation within APOC3 and its lipoprotein lipase-independent effects. Circ Cardiovasc Genet. 2016;9:231–239.
    1. Gordts P.L., Nock R., Son N.H. ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors. J Clin Invest. 2016;126:2855–2866.
    1. Pollin T.I., Damcott C.M., Shen H. A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection. Science. 2008;322:1702–1705.
    1. Natarajan P., Kohli P., Baber U. Association of APOC3 loss-of-function mutations with plasma lipids and subclinical atherosclerosis: the multi-ethnic BioImage study. J Am Coll Cardiol. 2015;66:2053–2055.
    1. Wyler von Ballmoos M.C., Haring B., Sacks F.M. The risk of cardiovascular events with increased apolipoprotein CIII: A systematic review and meta-analysis. J Clin Lipidol. 2015;9:498–510.
    1. Riwanto M., Rohrer L., Roschitzki B. Altered activation of endothelial anti- and proapoptotic pathways by high-density lipoprotein from patients with coronary artery disease role of high-density lipoprotein–proteome remodeling. Circulation. 2013;127:891–904.
    1. Hiukka A., Ståhlman M., Pettersson C. ApoCIII-enriched LDL in type 2 diabetes displays altered lipid composition, increased susceptibility for sphingomyelinase, and increased binding to biglycan. Diabetes. 2009;58:2018–2026.
    1. Graham M.J., Lee R.G., Bell T.A., III Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans. Circ Res. 2013;112:1479–1490.
    1. Yang X., Lee S.R., Choi Y.S. Reduction in lipoprotein-associated apoC-III levels following volanesorsen therapy: phase 2 randomized trial results. J Lipid Res. 2016;57:706–713.
    1. Han Y., Qi R., Liu G. Reduced high density lipoprotein cholesterol in severe hypertriglyceridemic ApoCIII transgenic mice via lowered hepatic ApoAI synthesis. Biochem Biophys Res Commun. 2015;462:420–425.
    1. Christoffersen C., Nielsen L.B., Axler O. Isolation and characterization of human apolipoprotein M-containing lipoproteins. J Lipid Res. 2006;47:1833–1843.
    1. Coleman R.A., Lee D.P. Enzymes of triacylglycerol synthesis and their regulation. Prog Lipid Res. 2004;43:134–176.

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