Within the spectrum of lipid abnormalities, elevated LDL-cholesterol (LDL-C) is the most firmly established causal risk factor for cardiovascular disease (CVD) [1,2]. Discovery of therapeutic targets and ensuing development of potent LDL-C lowering drugs (i.e. statins, PCSK9 inhibitors) can be counted among the success stories of modern medicine. Although cardiovascular risk reduction through LDL-C lowering has been in the limelight in recent decades, it should not be forgotten that multiple large epidemiological as well as genetic studies have firmly established that triglyceride levels are also independently associated with CVD [3,4▪▪].
Following the potential to eradicate LDL-C by applying combination therapies, efforts to reduce the residual cardiovascular-burden have shifted toward additional triglyceride lowering strategies. The search for novel therapeutic targets has revealed two promising candidates: apolipoprotein C-III (apoC-III) and angiopoietin like protein 3 (angptl3).
In the current article, we review the roles of apoC-III and angptl3 in triglyceride metabolism and their relation to cardiovascular disease. Next, we combine early clinical evidence from novel therapeutic strategies aimed at these targets to speculate whether apoC-III or angptl3 inhibition will indeed reduce CVD.
THE ROLE OF APOLIPOPROTEIN C-III AND ANGIOPOIETIN LIKE PROTEIN 3 IN TRIGLYCERIDE METABOLISM
Plasma triglycerides routinely measured in clinical laboratories entail triglycerides that are present in all lipoprotein classes, but predominantly in chylomicrons and VLDL. Chylomicrons are assembled in the intestine and deliver exogenous (dietary) triglycerides to peripheral tissues. VLDL is excreted into the circulation by the liver and contains endogenous triglycerides derived from lysosomal breakdown of other triglyceride-rich lipoproteins (TRL) or by de novo lipogenesis in the liver. Triglycerides in both chylomicrons and VLDLs undergo intravascular lipolysis by lipoprotein lipase (LPL), resulting in the release of free fatty acids to be used as fuel for peripheral tissues (e.g. muscles and heart) or stored as fat (i.e. adipose tissue). The left-over particles, so called remnant lipoproteins (i.e. chylomicron remnants and IDLs), can be cleared from the circulation by the liver. Alternatively, IDLs can be further hydrolyzed resulting in LDL particles. These processes are outlined in Fig. 1.
All processes involved in triglyceride metabolism are highly controlled by multiple proteins. Two pivotal proteins regulating triglyceride metabolism are apoC-III and angptl3 [5▪,6]. ApoC-III is a glycoprotein that is primarily synthesized in the liver and to a lesser extent in the intestine. ApoC-III binds to the surface of almost all types of lipoproteins, predominantly to HDL, but also to LDL and TRLs such as chylomicrons and VLDL [5▪]. Two mechanisms by which apoC-III increases triglyceride levels have been proposed: inhibition of LPL-mediated lipolysis of TRLs and/or attenuation of hepatic TRL uptake (Fig. 1). Which mechanism predominates in vivo in humans is a matter of continued debate . Hepatic TRL uptake appears important as patients with Familial Chylomicronemia Syndrome (i.e. in full absence of residual LPL activity) still displayed a 70% reduction of triglyceride levels following administration of an APOC3 antisense therapy . More recently, a stable isotope study compared lipoprotein metabolism in five heterozygous carriers of an APOC3 null allele with unaffected siblings matched for age and sex. It showed that partial loss of apoC-III synthesis had no significant effect on the uptake of VLDL remnants by the liver, whereas the conversion rate of VLDL to LDL particles was higher . Angptl3 is solely produced by the liver in humans and also functions as a potent inhibitor of LPL, especially in the presence of angptl8 . Its triglyceride raising effects may also originate from increased secretion of VLDL triglycerides from the liver  or increased apolipoprotein B (apoB) secretion from the liver . As angptl3 is not associated with increased hepatic apoB production, the exact mechanism leading to increased VLDL excretion remains to be elucidated [6,10].
CAUSES OF HYPERTRIGLYCERIDEMIA
VLDL particles harbor the largest part of plasma triglycerides, which is therefore used to reflect plasma VLDL levels. For clinical and epidemiological purposes, hypertriglyceridemia is classified as mild-to-moderate [triglyceride levels ranging from 2.0 to 10 mmol/l (177–886 mg/dl)] or severe hypertriglyceridemia [>10 mmol/l (886 mg/dl)] [12▪]. In the latter group, pancreatitis is the most predominant complication of hypertriglyceridemia . The rarest and most severe forms of hypertriglyceridemia are caused predominantly by homozygous defects in genes related to triglyceride metabolism (i.e. LPL, APOC2, APOA5, LMF1, GPIHBP1), termed Familial Hyperchylomicronemia Syndrome. With decreasing levels of triglycerides, the likelihood of finding a genetic cause for hypertriglyceridemia decreases from heterozygous large-effect variants in the above mentioned genes, via accumulated common small-effect variants in multiple genes (polygenic hypertriglyceridemia)  to multifactorial causes such as lifestyle related factors (high-fat or high-glycemic diet, alcohol consumption, metabolic syndrome, diabetes mellitus type 2) [12▪]. The pathophysiology and diagnosis of hypertriglyceridemia is extensively described elsewhere [12▪].
TRIGLYCERIDE-RICH LIPOPROTEINS AND CARDIOVASCULAR DISEASE RISK
Several lines of research link triglyceride levels to cardiovascular disease. Multiple large cohort studies have shown that both fasting and nonfasting triglyceride levels are independently associated with CVD. For example in the Copenhagen General Population and Copenhagen City Heart studies nonfasting triglycerides of 6.6 mmol/l were associated with a 5.1 [95% confidence interval (CI): 3.5–7.2] hazard ratio for myocardial infarction (MI) . Similarly, the Emerging Risk Factors Collaboration showed that 1-SD increase in fasting triglycerides was associated with a hazard ratio of 1.37 (95% CI: 1.31–1.42) for coronary heart disease (CHD) . However, this association was no longer significant after adjustment for HDL-cholesterol (HDL-C) and non-HDL-C. In fact, as non-HDL-C plasma concentration reflects the plasma cholesterol present in all apoB-containing particles, the latter finding highlights the importance of apoB as a measure for CVD risk in hypertriglyceridemia [17,18▪▪]. Recent genetic data lend further support to this observation. Thus, in the UK Biobank, Ference et al.[4▪▪] elegantly showed that the achieved CVD risk reduction over a lifetime is proportional to genetically reduced apoB levels using mendelian randomization methods to investigate the effect of genetically lowering triglycerides or cholesterol. Both triglyceride lowering genetic variants in the LPL gene (reflecting triglyceride clearance pathways) as well as genetic variants in the LDL receptor gene (reflecting cholesterol clearance pathway) were associated with an identically lower risk for CHD per 10 mg/dl (0.1 g/l) lower levels of apoB [odds ratios (OR) of 0.771 and 0.773, respectively].
To date, randomized controlled trials (RCTs) investigating potent triglyceride-lowering drugs (fibrates) have shown inconsistent results concerning VLDL–LDL and apoB-reduction. The vast majority of fibrate RCTs failed to demonstrate a benefit of (marked) triglyceride-lowering on CVD outcome . However, subsequent post-hoc analyses demonstrated a significant CVD-benefit in patients characterized by hypertriglyceridemia (>200 mg/dl) and/or low HDL-C (<40 mg/dl) . As pointed out by Sniderman et al. the fibrate trial results may support the notion that lowering of atherogenic apoB-particles but not the triglyceride reduction itself drives CVD benefit from fibrate therapy. More recently, the impact of fish oils on CVD outcome were also reported. The REDUCE-IT study revealed a modest triglyceride lowering of approximately 19% with a concomitant marked CVD reduction of −25% major adverse cardiovascular events [20▪], leading to the hypothesis that the benefits of icosapent ethyl (a highly purified eicosapentaenoic acid (EPA) ethyl ester) are likely due to pleiotropic effects of the fish oil rather than its triglyceride-lowering properties . In contrast, the STRENGTH study (using omega-3 carboxylic acid; a mixture of EPA and docosahexaenoic acid (DHA)) was recently discontinued due to futility and its results await presentation and publication .
Collectively, these RCTs imply that the benefit on CVD risk of novel triglyceride lowering drugs may not be proportional to the achieved triglyceride reduction and more likely relate to the total reduction of all atherogenic apoB containing lipoprotein particles [23▪].
TARGETING APOLIPOPROTEIN C-III
Ample genetic evidence links apoC-III levels with CVD risk. In 2008, Pollin et al. were the first to show how a rare APOC3 null-mutation found in the Lancaster county Amish population (n = 1033) correlated with reduced triglycerides levels and CVD risk as measured by lower coronary calcium score in mutation-carriers. This finding was later replicated in a multiethnic cohort in the US . In 2014, two landmark studies firmly linked APOC3 loss of function (LOF) mutations with around 40% lower triglycerides and 40% reduction in CHD [26,27]. In the Framingham heart study, every 1 mg/dl decrease in plasma apoC-III levels was associated with 4% decrease in CVD risk, adjusted for age and sex .
In 2018, a meta-analysis of 137 895 individuals showed that in the 776 APOC3 LOF carriers, remnant cholesterol was 43% lower whereas the mean LDL-C was only nonstatistically significantly decreased by 4% . 37 of the observed 41% lower risk of ischemic vascular disease could be explained by reduction in remnant cholesterol. Specifically, apolipoprotein B was 13% lower in heterozygous APOC3 LOF carriers compared with noncarriers. Significantly, homozygous loss of APOC3 function is not lethal. In the Pakistani kindred in which the homozygous mutations were discovered, individuals were healthy and postprandial triglycerides elevation was markedly blunted .
Van Capelleveen et al. measured plasma lipoproteins (including apoC-III and apoB) and assessed their predictive capacity for coronary artery disease (CAD) in the European Prospective Investigation of Cancer (EPIC)-Norfolk cohort comprising 1879 controls and 832 cases with CAD. The top apoC-III quintile predicted CAD after adjustment for traditional risk factors and lipid-lowering therapy (OR 1.50, 1.13–1.98), but lost statistical significance after adjusting for other lipoprotein variables. This may strengthen the notion that CVD risk is mediated by apoB containing TRL particles rather than by apoC-III or triglycerides per se.
Results from the first in-human apoC-III inhibiting therapy were published in 2014 . This antisense oligonucleotide therapy targets apoC-III expression at mRNA level and showed spectacular decrease in triglyceride levels in patients with familial chylomicronemia syndrome [31,32▪]. The therapy (volanesorsen) gained market access in several European countries in 2019 but was not approved in the United States, primarily due to concerns of drug-induced side effects comprising platelet count reduction, injection site reactions and flu-like symptoms following antisense administration [32▪].
To overcome these side effects, the antisense oligonucleotide sequence was coupled to a GalNAc moiety to improve specific uptake in the liver and increasing potency by at least 15× [33▪▪]. This AKCEA-APOCIII-LRx has recently completed a phase 1/2a dose escalating trial without safety concerns and was able to markedly reduce triglycerides and yielded a more favorable lipid profile (Table 1) [33▪▪]. ApoB reduction was significant (up to ∼30% in the highest dosed cohort). To date, no (plans for a) CVD endpoint trial have been announced.
TARGETING ANGIOPOIETIN-LIKE 3
Multiple studies have shown that rare LOF variants in the angptl3 coding gene ANGPTL3 are associated with decreased plasma levels of triglycerides, LDL cholesterol, HDL cholesterol and with decreased risk for CHD [35,40]. These findings were preceded by the discovery of combined hypolipidaemia phenotypes (i.e. low LDL-C, low HDL-C and low triglyceride levels) in ANGPTL3 knock-out mice  and later in a family with autosomal dominantly inherited hypolipoproteinemia with loss-of-function variants in ANGPTL3. In addition, angptl3 plasma levels have been shown to be associated with MI in the PROMIS cohort (a matched case–control study of Pakistani patients with MI comprising 1493 cases and 3231 matched controls) . Patients in the lowest two tertiles of angptl3 plasma levels had a reduced risk of MI compared with the highest tertile with OR of 0.75 (95% CI: 0.64–0.88) and 0.65 (95% CI: 0.55–0.77), respectively.
Unsurprisingly, these results have led to the development of two pharmacological compounds that target angptl3. The first one, IONIS-ANGPTL3-LRx, a GalNAc-conjugated antisense oligonucleotide directed against ANGPTL3 mRNA, showed dose-dependent and potent reductions in all lipid fractions in a phase 1 dose-finding study in 44 healthy volunteers . Especially triglycerides were lowered up to 63% and LDL-C up to 33%. This coincided with a maximum decrease in apoB of 26% (Table 1). No serious adverse events were observed in this trial. The IONIS-ANGPTL3-LRx, is currently being investigated in patients with hypertriglyceridemia, type 2 diabetes mellitus and nonalcoholic fatty liver disease (NCT03371355).
In parallel, a fully human mAb directed against angptl3, evinacumab, was developed and investigated for its efficacy in multiple patient categories. A phase 1, double-blind, single ascending dose study in 83 healthy volunteers with triglyceride levels between 1.7 and 5.1 mmol/l or LDL-C levels above 2.6 mmol/l received different doses of Evinacumab subcutaneously or intravenously, which resulted in 50 and 28% reduction in triglyceride and LDL-C in the highest dosed intravenous treatment arms, respectively . The full results of this single ascending dose study were later combined with a multiple ascending dose study in 56 healthy volunteers [37▪]. These results showed an increase in triglyceride- and LDL-C-lowering efficacy at day 15 when participants received multiple subcutaneously doses (Table 1). Evinacumab was subsequently administered to nine homozygous familial hypercholesterolemia patients, which resulted in a mean LDL-C reduction of almost 50% . Evinacumab was generally well tolerated and safe. In the three reported trials no serious adverse events were observed, although there were numerically more liver enzyme abnormalities and headaches reported in the groups receiving active treatment [34–36,37▪]. Evinacumab is now further investigated in patients with homozygous and heterozygous familial hypercholesterolemia in multiple trials (NCT03399786 and NCT03175367), as well as in patients with severe hypertriglyceridemia (NCT03452228).
For both the antisense oligonucleotide and mAb therapy, the observed apoB reductions ranged from −14 to −46%. Depending on the baseline apoB levels, these reductions can be expected to result in a proportional risk reduction for CVD, as predicted by mendelian randomization studies [4▪▪].
CONCLUSION AND FUTURE PERSPECTIVES
Triglyceride-rich particles causally and independently contribute to CVD risk. RCTs evaluating the effect of lowering TRLs have not (yet) provided sufficient evidence to demonstrate a consistent CVD risk reduction. Indirect genetic evidence implies a predominant effect of a reduction in TRL particle number, best reflected by apoB or non-HDL-C. ApoC-III and angptl3 are two novel targets in lipoprotein metabolism that are currently being investigated in clinical trials for reducing triglyceride levels and TRL particle number. The limited data available show marked triglyceride reduction with moderate apoB reduction following apoC-III inhibition, whereas both triglycerides and apoB are reduced following angptl3 inhibition. Combining these RCT data with recent mendelian randomization studies, it can be postulated that the angptl3 inhibition offers the best profile for reducing CVD risk with a marked apoB reduction ranging from −14 to −46% in RCTs. Conversely, both apoC-III and angptl3 genetic data revealed CVD benefit. Further studies using these compounds are eagerly awaited to evaluate whether and to what extent reduction of TRLs will be able to further reduce the residual cardiovascular burden in patients.
Financial support and sponsorship
Conflicts of interest
L.F.R. and T.R.T. declare no conflicts of interest. E.S.G.S. reports personal fees from Novartis, personal fees from Amgen, personal fees from Sanofi-Regeneron, personal fees from Mylan, personal fees from Esperion, unrelated to the submitted work. All fees were paid to the institution.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
1. Ference BA, Ginsberg HN, Graham I, 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.
2. Borén J, Williams KJ. The central role of arterial retention of cholesterol-rich apolipoprotein-b-containing lipoproteins in the pathogenesis of atherosclerosis: a triumph of simplicity. Curr Opin Lipidol 2016; 27:473–483.
3. Vallejo-Vaz AJ, Kondapally Seshasai SR, Cole D, et al. Familial hypercholesterolaemia: a global call to arms. Atherosclerosis 2015; 243:257–259.
4▪▪. Ference BA, Kastelein JJP, Ray KK, et al. Association of triglyceride-lowering LPL variants and LDL-C-lowering LDLR variants with risk of coronary heart disease. JAMA 2019; 321:364–373.
5▪. Taskinen MR, Packard CJ, Borén J. Emerging evidence that apoC-III inhibitors provide novel options to reduce the residual CVD. Curr Atheroscler Rep 2019; 21:27.
6. Kersten S. Angiopoietin-like 3 in lipoprotein metabolism. Nat Rev Endocrinol 2017; 13:731–739.
7. Ramms B, Gordts PLSM. Apolipoprotein C-III
in triglyceride-rich lipoprotein metabolism. Curr Opin Lipidol 2018; 29:171–179.
8. Gaudet D, Brisson D, Tremblay K, et al. Targeting APOC3 in the familial chylomicronemia syndrome. N Engl J Med 2014; 371:2200–2206.
9. Reyes-Soffer G, Sztalryd C, Horenstein RB, et al. Effects of APOC3 heterozygous deficiency on plasma lipid and lipoprotein metabolism. Arterioscler Thromb Vasc Biol 2019; 39:63–72.
10. Wang Y, Gusarova V, Banfi S, et al. Inactivation of ANGPTL3 reduces hepatic VLDL-triglyceride Secretion. J Lipid Res 2015; 56:1296–1307.
11. Xu Y-X, Redon V, Yu H, et al. Role of angiopoietin-like 3 (ANGPTL3) in regulating plasma level of low-density lipoprotein cholesterol. Atherosclerosis 2018; 268:196–206.
12▪. Laufs U, Parhofer KG, Ginsberg HN, Hegele RA. Clinical review on triglycerides
. Eur Heart J 2020; 41:99c–109c.
13. Zafrir B, Saliba W, Jubran A, et al. Severe hypertriglyceridemia-related pancreatitis: characteristics and predictors of recurrence. Pancreas 2019; 48:182–186.
14. Brown WV, Gaudet D, Goldberg I, Hegele R. Roundtable on etiology of familial chylomicronemia syndrome. J Clin Lipidol 2018; 12:5–11.
15. Nordestgaard BG, Varbo A. Triglycerides
and cardiovascular disease. Lancet 2014; 384:626–635.
16. Di Angelantonio E, Sarwar N, Perry P, et al. The Emerging Risk Factors CollaborationMajor lipids, apolipoproteins, and risk of vascular disease. JAMA 2009; 302:1993–2000.
17. Sniderman AD, Couture P, Martin SS, et al. Hypertriglyceridemia and cardiovascular risk: a cautionary note about metabolic confounding. J Lipid Res 2018; 59:1266–1275.
18▪▪. Sniderman AD, Thanassoulis G, Glavinovic T, et al. Apolipoprotein B particles and cardiovascular disease: a narrative review. JAMA Cardiol 2019; 4:1287–1295.
19. Jun M, Foote C, Lv J, et al. Effects of fibrates on cardiovascular outcomes: a systematic review and meta-analysis. Lancet 2010; 375:1875–1884.
20▪. Bhatt DL, Steg PG, Miller M, et al. Cardiovascular risk reduction with icosapent ethyl for hypertriglyceridemia. N Engl J Med 2019; 380:11–22.
21. Kastelein JJP, Stroes ESG. FISHing for the miracle of eicosapentaenoic acid. N Engl J Med 2019; 380:89–90.
22. Astrazeneca. Update on Phase III STRENGTH trial for Epanova in mixed dyslipidaemia. [Online]. Available from: https://www.astrazeneca.com/media-centre/press-releases/2020/update-on-phase-iii-strength-trial-for-epanova-in-mixed-dyslipidaemia-13012020.html
[Last accessed 14 April 2020].
23▪. Marston NA, Giugliano RP, Im KA, et al. Association between triglyceride lowering and reduction of cardiovascular risk across multiple lipid-lowering therapeutic classes: a systematic review and meta-regression analysis of randomized controlled trials. Circulation 2019; 140:1308–1317.
24. Pollin TI, Damcott CM, Shen H, et al. A null mutation in human apoc3 confers a favorable plasma lipid profile and apparent cardioprotection. Science 2008; 322:1702–1705.
25. Natarajan P, Kohli P, Baber U, et al. Association of APOC3 loss-of-function mutations with plasma lipids and subclinical atherosclerosis: the multiethnic bioimage study. J Am Coll Cardiol 2015; 66:2053–2055.
26. Crosby J, Peloso GM, Auer PL, et al. Loss-of-function mutations in APOC3, triglycerides
, and coronary disease. N Engl J Med 2014; 371:22–31.
27. Jørgensen AB, Frikke-Schmidt R, Nordestgaard BG, Tybjræg-Hansen A. Loss-of-function mutations in APOC3 and risk of ischemic vascular disease. N Engl J Med 2014; 371:32–41.
28. Wulff AB, Nordestgaard BG, Tybjaærg-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.
29. Saleheen D, Natarajan P, Armean IM, et al. Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity. Nature 2017; 544:235–239.
30. van Capelleveen JC, Lee SR, Verbeek R, et al. Relationship of lipoprotein-associated apolipoprotein C-III
with lipid variables and coronary artery disease risk: the EPIC-norfolk prospective population study. J Clin Lipidol 2018; 12:1493–1501.e11.
31. Gaudet D, Alexander VJ, Baker BF, et al. Antisense inhibition of apolipoprotein C-III
in patients with hypertriglyceridemia. N Engl J Med 2015; 373:438–447.
32▪. Witztum JL, Gaudet D, Freedman SD, et al. Volanesorsen and triglyceride levels in familial chylomicronemia syndrome. N Engl J Med 2019; 381:531–542.
33▪▪. Alexander VJ, Xia S, Hurh E, et al. N
-Acetyl galactosamine-conjugated antisense drug to APOC3 mRNA, triglycerides
and atherogenic lipoprotein levels. Eur Heart J 2019; 40:2785–2796.
34. Graham MJ, Lee RG, Brandt TA, et al. Cardiovascular and metabolic effects of ANGPTL3 antisense oligonucleotides. N Engl J Med 2017; 377:222–232.
35. Dewey FE, Gusarova V, Dunbar RL, et al. Genetic and pharmacologic inactivation of ANGPTL3 and ccardiovascular disease. N Engl J Med 2017; 377:211–221.
36. Gaudet D, Gipe DA, Pordy R, et al. ANGPTL3 inhibition in homozygous familial hypercholesterolemia. N Engl J Med 2017; 377:296–297.
37▪. Ahmad Z, Banerjee P, Hamon S, et al. Inhibition of angiopoietin-like protein 3 with a monoclonal antibody reduces triglycerides
in hypertriglyceridemia. Circulation 2019; 140:470–486.
38. Graham MJ, Lee RG, Bell TA, et al. Antisense oligonucleotide inhibition of apolipoprotein C-III
reduces plasma triglycerides
in rodents, nonhuman rrimates, and humans. Circ Res 2013; 112:1479–1490.
39. Gouni-Berthold I, Alexander V, Digenio A, et al. Apolipoprotein C-III
inhibition with volanesorsen in patients with hypertriglyceridemia (COMPASS): a randomized, double-blind, placebo-controlled trial. J Clin Lipidol 2017; 11:794–795.
40. Stitziel NO, Khera AV, Wang X, et al. ANGPTL3 deficiency and protection against coronary artery disease. J Am Coll Cardiol 2017; 69:2054–2063.
41. Koishi R, Ando Y, Ono M, et al. Angptl3 regulates lipid metabolism in mice. Nat Genet 2002; 30:151–157.
42. Musunuru K, Pirruccello JP, Do R, et al. Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia. N Engl J Med 2010; 363:2220–2227.