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

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.

Graphical abstract

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

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

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

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

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

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

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.