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

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.

Cross-sectional analyses of high-dimensional protein data deemed associations significant according to a false discovery rate q value below 0.05. Other results were not adjusted for multiplicity (6).
To estimate effects of apoC-III synthesis inhibition, for each subject, measurements at day 1 (baseline) and

Apolipoproteins and Cardiovascular Events
When investigating whether apoC-III, apoC-II, and apoE could improve on traditional risk factors in 10-year cardiovascular risk prediction (Online Table 2), no significant change in the c-index was found; however, a significantly positive net reclassification index indicated that 12.3% of subjects could be more appropriately classified into the clinically relevant risk categories of 0.0% to 5.0%, 5.0% to 7.5%, or more than 7.5% when including apolipoproteins.    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 ¼ apolipoprotein; CI ¼ confidence interval; CVD ¼ cardiovascular disease; Pechlaner et al.    Figures 6A and 6B, bottom row) and, to a lesser extent, upon adjustment for apoE (Online Figure 6C). This is consistent with the notion that apoC-II and apoC-III were more strongly correlated with total cholesterol, non-HDL-C, and TGs than apoE (Online Figure 4).   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.
This is thought to be due to inhibiting hepatic clearance of TRL lipoproteins mediated by LDLR or LRP-1 (18,21,23). Thus, it is now apparent that apoC-III regulates TRL metabolism by both an LPL-dependent and LPL-independent pathway, and is thus a central regulator of plasma TG levels (2,7,18,22).
ApoC-II, apoC-III, and apoE were associated with obesity, hypertension, impaired glucose metabolism, and most strongly with lipid parameters (Online  suggest that loss-of-function mutations of apoC-III confer cardiovascular protection (12,13,24,25). The Framingham Study has linked apoC-III, as measured by immunoassays, to incident myocardial infarction or angina pectoris (12). We now provide the first data that apoC-III, along with apoC-II and apoE, associates The reduction in apoC-III levels was profound, leading to >75% decreases at the dose of antisense used. This was associated not only with marked reductions in plasma TGs of w70%, but there were nearly 50% decreases in both apoC-II and apoE. These changes are consistent with lowering of VLDL and remnant lipoproteins (7,21), and are in line with observations after apoC-III inhibition in mice, nonhuman primates, and humans (2,3,29). However, the disparity between the extent of reduction in  on other lipoproteins, such as HDL (30). Indeed, the observed increase in apoA-I, apoA-II, and apoM is consistent with the reported rise of HDL-C following apoC-III inhibition (3,29) and lower HDL-C levels in apoC-III transgenic mice (31). A potential mechanistic explanation is reduced exchange of HDL-C with VLDL-TG mediated via cholesterol ester transfer protein (CETP) (7,29). Notably, apoM has been reported to mark an HDL subpopulation that stimulates particularly efficient cholesterol efflux (32).
Although apoC-III delays clearance of VLDL remnants that contain apoB-100, apoC-III inhibition by volanesorsen did not reduce total apoB-100 levels. This would explain why only 16% lower apoB-100 levels were reported in carriers of apoC-III loss-offunction mutations with normal TG levels (13), although a recent report of such subjects did not find lower LDL levels (25). Following apoC-III inhibition, TAGs and DAGs were decreased ( Figure 5B).
Although the former is expected, the latter is notable because DAGs are precursors of TAGs in the last step of TG synthesis (33

CONCLUSIONS
Our data provide strong epidemiological support to the concept that TRLs contribute to atherosclerosis. Pechlaner et al.