Genetics and Causality of Triglyceride-Rich …glycerides and the risk of coronary heart disease:...

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THE PRESENT AND FUTURE

STATE-OF-THE-ART REVIEW

Genetics and Causality ofTriglyceride-Rich Lipoproteins inAtherosclerotic Cardiovascular Disease

Robert S. Rosenson, MD,* Michael H. Davidson, MD,y Benjamin J. Hirsh, MD,z Sekar Kathiresan, MD,xDaniel Gaudet, MD, PHDk

ABSTRACT

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Triglycerides represent 1 component of a heterogeneous pool of triglyceride-rich lipoproteins (TGRLs). The reliance on

triglycerides or TGRLs as cardiovascular disease (CVD) risk biomarkers prompted investigations into therapies that lower

plasma triglycerides as a means to reduce CVD events. Genetic studies identified TGRL components and pathways

involved in their synthesis and metabolism. We advocate that only a subset of genetic mechanisms regulating TGRLs

contribute to the risk of CVD events. This “omic” approach recently resulted in new targets for reducing CVD

events. (J Am Coll Cardiol 2014;64:2525–40) © 2014 by the American College of Cardiology Foundation.

T riglyceride-rich lipoproteins (TGRLs) com-prise a vast array of intestinally derived andhepatically secreted particles with distinctcompositions and associations with risk for cardiovas-cular disease (CVD) or pancreatitis. Althoughthe contribution of plasma/serum triglycerides (tria-cylglycerols [TG]) to increased risk of coronary and ce-rebrovascular ischemic events was established inmultivariate models that adjust for major riskmarkers, including low-density lipoprotein choles-terol (LDL-C) and high-density lipoprotein cholesterol(HDL-C) (1,2), elevated TGRLs alter low-density lipo-protein (LDL) and high-density lipoprotein (HDL)

m the *Mount Sinai Heart, Cardiometabolic Disorders, Icahn School of Med

Cardiology, Pritzker School of Medicine, University of Chicago, Chicago, Illi

rk, New York; xMassachusetts General Hospital, Harvard Medical School,id Clinic, Department of Medicine, Université de Montreal, Chicoutim

eived research grants from Amgen, AstraZeneca, and Sanofi; he serv

traZeneca, Eli Lilly and Company, Regeneron, and Sanofi; serves as a c

norarium from Kowa; holds stock in and has received a travel award from

ToDate, Inc. Dr. Davidson has served as a consultant to Amgen, AstraZe

thera, a fully owned subsidiary of AstraZeneca. Dr. Kathiresan has served

d Company, Catabasis, and Regeneron; has served on the scientific ad

eived research grants from AstraZeneca, and Merck & Co. Dr. Gaudet s

tabasis, Isis, Regeneron, Sanofi, and Uniqure; and is a consultant to Chiesi,

has no relationships relevant to the contents of this paper to disclose. Mot

ten to this manuscript’s audio summary by JACC Editor-in-Chief Dr. Vale

u can also listen to this issue’s audio summary by JACC Editor-in-Chief D

nuscript received July 29, 2014; revised manuscript received September

composition and function, which may result in unac-counted risk in observational studies and clinicaltrials of lipid-modifying therapies. Despite the asso-ciation of circulating TGRL levels with atheroscle-rosis, whether abnormal TGRL metabolism and/orTGRL lipolytic products are causal remains uncertain.

The mechanisms underlying TGRLs and athero-sclerotic CVD risk are incompletely understood.Mendelian randomization studies provide evidencefor causal involvement of TG-mediated pathways incoronary heart disease (CHD); however, the contri-bution of TGRLs per se was not directly assessed (3).Furthermore, in clinical trials, TG-lowering therapies

icine at Mount Sinai, New York, New York; yDivisionnois; zMount Sinai Heart, Mount Sinai Hospital, NewBoston, Massachusetts; and the kECOGENE-21 andi, Quebec, Canada. Dr. Rosenson’s institution has

es on the advisory boards of Aegerion, Amgen,

onsultant to Novartis and Sanofi; has received an

LipoScience, Inc.; and has received royalties from

neca, Merck & Co., and Sanofi; and is employed by

as a consultant to Aegerion, Amgen, Novartis, Eli Lily

visory board of Catabasis and Regeneron; and has

erves on the advisory boards of Aegerion, Amgen,

Novartis, and Regeneron. Dr. Hirsh has reported that

i Kashyap, MD, served as Guest Editor for this paper.

ntin Fuster.

r. Valentin Fuster.

18, 2014, accepted September 21, 2014.

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CENT

(Top)

details

trated

DGAT

glycos

lipopr

monog

tilisin

lipopr

ABBR EV I A T I ON S

AND ACRONYMS

Apo = apolipoprotein

CHD = coronary heart disease

CVD = cardiovascular disease

DGAT = diacylglycerol

acyltransferase

HDL = high-density lipoprotein

LDL = low-density lipoprotein

PPAR = peroxisome

proliferator-activated receptor

TG = triacylglycerols

TGRLs = triglyceride-rich

lipoproteins

VLDL = very low-density

lipoprotein

Rosenson et al. J A C C V O L . 6 4 , N O . 2 3 , 2 0 1 4

Triglyceride-Rich Lipoproteins and Atherosclerosis D E C E M B E R 1 6 , 2 0 1 4 : 2 5 2 5 – 4 02526

not only alter TGRL concentration andcomposition, but also affect LDL, HDL, andinflammatory pathways.

This state-of-the-art review discusses thecomplexities of TGRL-associated atheroscle-rotic CVD risk and new directions in riskassessment and therapeutic responses toTG-lowering therapies from genetic studies.It is not intended to reiterate recent con-sensus statements on hypertriglyceridemiadefinitions, diagnosis, and management (4,5).

TGRL, HUMAN ATHEROSCLEROSIS,

AND ATHEROSCLEROTIC

CARDIOVASCULAR EVENTS

Chylomicron and very low-density lipopro-

tein (VLDL) remnants rapidly penetrate the arterialwall and contribute cholesterol to atherosclerotic le-sions (6–8). VLDL composition is a critical CVD riskdeterminant. In retrospective and prospective popu-lation studies, TG-associated CHD risk was limited to

RAL ILLUSTRATION Intestinal Synthesis and Hepat

Key pathways regulating intestinal synthesis and metabolism of t

of this pathway and its genetic regulation). (Bottom) Key pathw

(see the text for details about this pathway and its genetic regula

¼ diglyceride acyltransferase; ER ¼ endoplasmic reticulum; FA ¼ylphosphatidylinositol-anchored high-density lipoprotein-binding

otein receptor; LPL ¼ lipoprotein lipase; LRP1 ¼ low-density lipoplyceride acyl transferase; MTP ¼ microsomal transfer protein; NPkexin type 9; PCTV ¼ pre-chylomicron transport vesicle; TG ¼ triglotein transport vesicle.

apolipoprotein (Apo) C3-containing VLDL particlesand their metabolic remnants, small LDL particles (9–11). VLDL proteome analysis expanded the complex-ities of VLDL through identification of 33 functionalpathways, including 4 related to lipid transport andlipoprotein metabolism, and 8 associated with coagu-lation, hemostasis, and immunity (12).

METABOLISM OF INTESTINAL AND

HEPATIC-DERIVED TGRLS

Chylomicrons are intestinal-specific lipoproteins,formed mainly in the jejunum after a meal. Owing tothe large TG core (>90%), chylomicron density is

J A C C V O L . 6 4 , N O . 2 3 , 2 0 1 4 Rosenson et al.D E C E M B E R 1 6 , 2 0 1 4 : 2 5 2 5 – 4 0 Triglyceride-Rich Lipoproteins and Atherosclerosis

2527

diffusion or the action of specific transporters. At theendoplasmic reticulum (ER) membrane, mono-acylglycerol:acyl CoA transferase converts mono-acylglycerol and fatty acids into diacylglycerol acids,which diacylglycerol:acyl CoA transferase (DGAT)subsequently converts into TGs (14). Lipid droplets(LDs) are transiently formed in the cytosol, and thenfuse with apoB48 phospholipid-rich particles in theER lumen via the action of a chaperone, microsomaltransfer protein (MTP), which mediates primordialchylomicron particle formation. Proprotein con-vertase subtilisin kexin type 9 (PCSK9) enters andstimulates intestinal TGRL production by increasingboth apoB48 synthesis and TG production throughtranscriptional and post-transcriptional mechanismsinvolved in TRL assembly (MTP messenger ribonu-cleic acid [mRNA], protein, and lipid transfer activityand Niemann-Pick C1 like protein levels), and sterolresponse element binding protein 1 (SREBP1) targetgenes involved in fatty acid/triglyceride biosyntheticpathway (fatty acid synthase, stearoyl-CoA desa-turases, and diglyceride acyltransferase-2 [DGAT2])(15). Chylomicrons exit the ER to the Golgi via pre-chylomicron transport vesicles (16). After a meal,chylomicrons are exported into mesenteric lymphbefore entering the circulation, where they contributeto post-prandial TG concentrations (17,18). Lipopro-tein lipase (LPL) subsequently hydrolyzes chylomi-crons into fatty acids used as an energy supply by theheart and skeletal muscles or stored in adipose tissue.Chylomicron remnants are removed from the circu-lation after binding to the LDL receptor via the apoEligand, or by other routes including LDL receptor-related protein 1 and the heparan sulfate proteogly-can pathway (19). Although clearance is the primarymechanism for chylomicron remnants, ApoB48 over-production increases chylomicron production infructose-fed, insulin-resistant hamsters (20).

VLDL biogenesis occurs in hepatocytes by a 2-stepprocess involving synthesis of partially lipidatedapoB100 to form a primordial VLDL particle (through aprocess involving MTP), followed by TG addition to theprimordial particle in the ER lumen (Central Illustration,bottom). However, apoC3 inhibits VLDL assembly inde-pendent of MTP (21). After assembly, nascent VLDL par-ticles are transported to the Golgi by a rate-limiting stepmediatedby a specific ER-derivedVLDL transport vesicle(22). Cell death-inducingDFF45-like effector b (cideB) is aVTF-associated protein that mediates VLDL lipidationand maturation (23). CideB silencing results in hepaticsecretion of small VLDL particles, which is evidence thatVLDL heterogeneity is, in part, genetically regulated.

LPL is the principal enzyme that hydrolyzes TGin TGRLs. LPL is primarily synthesized in the

parenchyma of tissues that use fatty acids for energy(cardiac muscle, skeletal muscle) and TG storage (adiposetissue). LPL attaches to heparan sulfide side chains ofproteoglycans, and is transported to the capillary lumenby the endothelial protein glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein-1(GPIHBP1) (24). GPIHBP1 provides a platform for LPL toprocess TG from TGRL. TGRL hydrolysis at the capillarylumen releases free fatty acids and monoglycerides,resulting in partially catabolized chylomicrons (chylomi-cron remnants) and VLDL particles (VLDL remnants).

Due to its critical role in lipid homeostasis, LPL ac-tivity is highly regulated at the transcriptional, post-transcriptional, translational, and post-translationallevels. Various proteins regulate LPL including:apoC1 (25,26), apoC2 (27), apoC3 (26,28), apoA5 (29),angiopoietin-like protein 3 (ANGPTL3) (30), ANGPTL4(31,32), and ANGPTL8 (33). ANGPTL4 is a secretedprotein induced in adipose tissue by fasting (34). TGRLbinding stabilizes LPL; however, apoC1 and apoC3displace LPL from LDs, where ANGPTL4 inactivates itin the subendothelial space. This allows ANGPTL4 tobind LPL’s N-terminus, resulting in dissociation ofcatalytically active LPL homodimers to monomers(26). ANGPTL3 renders LPL more susceptible to pro-teolytic inactivation by proprotein convertases (35).

Low amounts of LPL are detected in human blood,where it is transported by both apoB48- and apoB100-containing lipoproteins. In humans, apoB-containinglipoproteins with LPL are cleared more rapidly thanapoB-containing particles without LPL (36). LPLwasmoreeffective in enhancing post-prandial clearance of apoB48-than apoB100-containing lipoproteins. Although apoE fa-cilitates receptor-mediated TGRL clearance pathways, itdid not affect TGRL clearance in this study.

The very low-density lipoprotein receptor (VLDLR), anLDL receptor family member (37), is expressed in heart,skeletal muscle, and adipose tissue (38,39). Under mostcircumstances, it is not detected in the liver; however,fenofibrate activation of the peroxisome proliferator-activated receptor (PPAR) alpha increases hepatic VLDLRtranslational activity through peroxisome proliferatorresponse element binding to the VLDL promoter (40).After VLDLs bind to VLDLR, VLDL-derived FA are deliv-ered to peripheral tissues, where they are used by heartand muscle for energy and in adipose tissue as an FAstorage reservoir. ApoC3 impairs VLDL binding to cellularreceptors, resulting in formation of small LDL particles(10,11), thereby counteracting the effects of apoE (41).

INFLAMMATION

TGRLs produced from both the exogenous (chy-lomicrons) or endogenous (VLDL) pathways are

TABLE 1

Endotheliu

Blood

Intestinalmicrofl

AP ¼ activproliferatorprotein; VCAVLDL ¼ ver



proinflammatory. Chylomicron accumulation favorspancreatic inflammation and is associated withincreased risk of acute pancreatitis. In contrast,VLDL, intermediate-density lipoprotein (IDL), VLDLremnants, and chylomicron remnants increaseendothelial inflammation and facilitate arterial wallinfiltration of monocytes. After a high-fat meal,hypertriglyceridemic subjects produce large, buoyantchylomicrons and VLDLs. VLDL particles producedpost-prandially have a distinct fatty acid compositionand increased apoC3 (42,43). TGRLs with high TGcontent up-regulate tumor necrosis factor-alpha,thereby inducing vascular cell adhesion molecule(VCAM)-1 expression in human aortic endothelial cellsand monocyte adhesion. By reducing VCAM-1 expres-sion and monocyte recruitment, TGRLs with lowtriglyceride content have an atheroprotective effect(42–44) (Table 1). The influence of TGRLs on VCAM-1transcription and expression depends on ER homeo-stasis. ER stress is the accumulation of unfolded ormisfolded proteins in the ER lumen, and is a unifyingmoniker for metabolic dysregulation linking obesity toinsulin resistance and inflammation in diabetes andatherosclerosis (45,46). ER stress is mitigated by theunfolded protein response, which involves dissocia-tion of binding immunoglobulin protein from ERtransmembrane sensor proteins and their downstreameffectors. In hypertriglyceridemic subjects, TGRLsregulate tumor necrosis factor alpha–induced VCAM-1expression kinetics through processes involving ERstress and the unfolded protein response (47).

TGRL lipolysis by LPL on the endothelial cell sur-face elaborates high concentrations of lipolysis prod-ucts along the blood endothelial interface, which maycontribute to atherosclerosis through mechanismsencompassing proinflammatory, procoagulant, andproapoptotic gene activation. TGRL lipolysis releasesneutral and oxidized free fatty acids that induceendothelial inflammation, vascular apoptosis (48),and reactive oxygen species production in endothe-lial cells, and altered lipid raft physiology (49).

TGRLs and Inflammation

m VCAM-1 transcription and expression

Monocyte infiltration

Increased permeability facilitates VLDL remnant uptake

Lipolytic products (oxidized fatty acids)

ROS-mediated AP-1 up-regulation of IL-1, IL-6, IL-8, VEGF,and PPAR-gamma

oraLPS transport into blood via dietary lipids

ator protein; IL ¼ interleukin; LPS ¼ lipopolysaccharide; PPAR ¼ peroxisome-activated receptor; ROS ¼ reactive oxygen species; TGRL ¼ triglyceride-rich lipo-M ¼ vascular cell adhesion molecule; VEGF ¼ vascular endothelial cell growth factor;y low-density lipoprotein.

The mechanism for vascular inflammation andapoptosis depends upon induction of transcriptionfactor ATF3-related genes in p38 and stress-activatedprotein kinase/c-Jun N-terminal kinase subset ofmitogen-activated protein kinase signaling, andcytokines associated with activator protein 1 sig-naling, including interleukin-1-alpha, interleukin-6,interleukin-8, vascular endothelial cell growth fac-tor, and PPAR-gamma (50). TGRL lipolysis productsincrease endothelial permeability and VLDL remnantuptake in the artery wall (51). The presence of VLDLRon macrophages, endothelium, and vascular smoothmuscle surfaces of the vessel wall may further facili-tate internalization of VLDL remnants.

Intestinal microflora were recently recognized asanother source of inflammatory mediators. Gram-negative bacteria in the intestinal microflora releaselipopolysaccharide (LPS) after lysis. Dietary lipidsfacilitate LPS entry into the circulation (52). Upon cellentry via Toll-like receptor 4, LPS activates redox-sensitive transcription factors that regulate proin-flammatory responses.

TGRLs increase atherosclerosis, inflammation, andthe lipid content of atherosclerotic lesions bothdirectly and indirectly. Causal pathways supportingdirect contributions of TGRLs to CVD events are dis-cussed in the following sections.

PHYSIOLOGY OF POST-PRANDIAL LIPEMIA:

CARDIOVASCULAR RISK OF FASTING

VERSUS NONFASTING TRIGLYCERIDE

MEASUREMENT

Current guidelines recommend screening and man-agement of patients with hypertriglyceridemia on thebasis of fasting TG measurement (53,54). However,prospective studies suggest that altered post-prandialTG metabolism contributes to atherosclerosis andmay predict CVD development. Furthermore, studiesdemonstrated variable post-prandial metabolic re-sponses in dyslipidemic patients compared withhealthy subjects, suggesting that common geneticpolymorphisms may underlie this difference (55,56).Nonfasting TG measurement was proposed as amarker for CVD risk stratification; however, furtherinvestigation into factors modulating the post-prandial response may prove valuable.

The recommendation for TG measurement in thefasting state arose due to early work demonstratingthe unreliability of fat tolerance testing, primarilydue to individual differences in gastric emptying,raising concerns regarding serum TG level variability(57–59). The lack of direct LDL-C assays promotedwidespread adoption of the Friedewald equation to


2529

estimate LDL-C, which depends on fasting-statemeasurements (60). Additionally, epidemiologicalevidence showed that fasting TGs are statisticallyindependent predictors of atherosclerosis and inci-dent CVD events (1,2). Studies also revealed a strongcorrelation between fasting and nonfasting TG,regardless of glucose status. Thus, fasting TG levelshave been used as a predictor of post-prandial TGlevels (61,62). However, the recommendation tomeasure fasting TG is not made on the basis of pro-spective studies demonstrating its superiority inpredicting CVD risk compared with nonfasting TG.Instead, following the implementation of screeningguidelines, epidemiologic investigations simplyadopted fasting TG level measurements (62).

In contrast to the observed variability with fattolerance tests, lipoprotein profiles change onlyminimally in response to normal food consumption inhealthy individuals, thereby calling into questionconcerns over post-prandial serum TG level variability(60,62). In addition, fasting TG measurement may notaccurately capture the effect of TGRLs and remnantparticles in the post-prandial period on multiple li-poprotein profile components (63). Furthermore,prospective studies suggested that nonfasting TGlevels are equal to or, in some populations, better thanfasting TG levels for predicting future CVD events

TABLE 2 Recent Studies Investigating the Use of Nonfasting TG and

Study/First Author (Ref. #)Primary Endpoint,

Sample SizePrimary Endpoin

Hazard Ratio (95%

The Women’s HealthStudy (64)

Incident CVD, nonfatalMI, nonfatal stroke,coronaryrevascularization, orcardiovascular death(n ¼ 26,509)

Fasting2.23 (1.82–2.74

Nonfasting2.53 (1.69–3.79

Nordestgaard et al. (65) Incident MI, CVD, and deathwith 1 mmol/l increase innonfasting TG(n ¼ 13,981)

CVD outcomeWomen

1.30 (1.22–1.40Men

1.14 (1.10–1.19

Varbo et al. (72) Incidence of CVD with 1mmol/l increase innonfasting RC(n ¼ 73,513)

1.4 (1.3–1.5)

Jørgensen et al. (73) Incidence of MI withgenetically elevateddoubling of nonfasting TGand RC (n ¼ 10,391)

1.87 (1.25–2.81

1 mmol/l ¼ 88.41 mg/dl.CCHS ¼ Copenhagen City Heart Study; CGPS ¼ Copenhagen General Population Study;

FTT ¼ fat tolerance test; HDL-C ¼ high-density lipoprotein cholesterol, LDL-C ¼ lowTG ¼ triglycerides.

(64,65). These studies controlled for variability inpost-prandial TG measures by stratifying by timesince last meal, in addition to other factors implicatedin affecting post-prandial measures including age,sex, body mass index, ethnicity, menopausal status,hormone use, and diabetes (64,65) (Table 2).

The post-prandial period is characterized bycirculation of potentially atherogenic lipoproteinparticles absorbed and processed through theintestine and liver, including chylomicrons, VLDL,and remnant particles (55,66). Their presence ismodulated by traditional epidemiologic and environ-mental factors including sex, age, body mass index,physical activity, and smoking, and by the amountand type of dietary fat in a meal (67,68). Because in-dividuals are in the nonfasting state most of the day(approximately 18 h) (68), the notion that alterationsin post-prandial TG concentration and lipoproteinclearance contribute to atherogenesis is plausible.Recent data showed that in normolipidemic controlsubjects, a post-prandial rise in TG and quantitativechanges in other lipoproteins and lipids are negligiblein response to dietary fat compared with baselinevalues (58). However, in patients with dyslipidemia,the rise in TGRL is 3-fold greater than normal, andpersists for up to 8 h (55,56). Studies demonstrated theatherogenic potential of remnant particles in these

RC Levels as Predictors of CVD Events

t,CI)

Subgroup,Sample Size

SubgroupPrimary Endpoint,

Hazard Ratio (95% CI)Fasting/Nonfasting Definition

(Limitations)

)

)

HDL-C $50 mg/dl Fasting1.32 (1.03–1.68)

Nonfasting1.94 (1.21–3.10)

Fasting: $8 h since last mealNonfasting:

FIGURE 1 Effect o

In patients with incre

(VLDL) is secreted w

triglyceride-rich lipop

dense low-density li

cholesterol (HDL-C).

HL ¼ hepatic lipase;high-density lipopro



patients, including chylomicron remnants, not previ-ously known to be proatherogenic (69–73); thislikely explains the strength of nonfasting TG mea-surements in identifying patients at greater CVD risk.

Post-prandial TG metabolism abnormalities arealso strongly associated with single nucleotide poly-morphisms (SNPs) at common genetic loci, suggestinggenetic mediation of interindividual variation inpost-prandial lipemia (5,70). These associationspersist after accounting for effects on other lipidtraits, including LDL-C, HDL-C, and lipoprotein con-centrations. Among the >150 deoxyribonucleic acid(DNA) sequence variants associated with serumlipids, candidate genes associated with post-prandiallipemia include apolipoprotein genes A1, A4, A5, C3,and E, as well as LPL, fatty acid binding protein 2,MTP, and scavenger receptor B1 (74). The ApoA5 locushas the strongest causal association between non-fasting TG and incident CVD (73,74).

ApoA5 is involved in multiple stages of TGRLmetabolism, including VLDL production, TGRLremnant clearance, and regulation of LPL activity (3).Early studies of apoA5 knockout mice reported a400% increase in serum TG levels (73,75,76). Genome-wide association studies (GWAS) reliably demon-strated that polymorphisms in or near the apoA5

f apoC3 on VLDL Metabolism

ased hepatic lipogenesis, enlarged very low-density lipoprotein

ith apolipoprotein (apo)C3. ApoC3 slows clearance of these

roteins (TGRLs), resulting in increased remnant cholesterol and small

poprotein cholesterol (LDL-C), and low high-density lipoprotein

CE ¼ cholesterol ester; CETP ¼ cholesterol ester transfer protein;LDL ¼ low-density lipoprotein; LPL ¼ lipoprotein lipase; sHDL ¼ smalltein; TG ¼ triglyceride.

locus are associated with nonfasting TG levels.Furthermore, associations between apoA5 geneticvariants and incident CHD in direct relation to theincrease in elevated nonfasting TG and remnantcholesterol were recently reported (76–78). In-vestigations into the additive effect of other SNPs,including glucokinase regulatory protein, apoE, andLPL, further enhanced the predictive ability of SNP-phenotype associations (67,74). Similarly, environ-mental factors, such as obesity, modify the effect ofthe apoA5 locus on post-prandial TGRL metabolismand atherosclerosis (77). Other studies demonstrateddifferential post-prandial responses to therapy on thebasis of genetic variants at this locus, suggesting thatSNP alterations may predict treatment efficacy (78).

Other gene loci thought to be important regulatorsof the TG response to dietary fat consumption werereported, including ANGPTL4 and PPAR alpha (67,74).With increasing appreciation of nonfasting TG levelsand CVD risk, further investigation into the roles ofgenetic variants and environmental factors in medi-ating the post-prandial response may provide prom-ising therapeutic approaches.

ATHEROGENICITY OF TGRL CHOLESTEROL

Non–HDL-C is an aggregate measure of the choles-terol content transported in atherogenic lipoproteins.The greater predictive value of non–HDL-C over LDL-C comes from the premise that VLDL-C (non–HDL-Cminus directly-measured LDL-C, also known asremnant cholesterol or TGRL cholesterol) is alsoatherogenic. Recent data supports the hypothesisthat VLDL-C or remnant cholesterol is even moreatherogenic than LDL-C. Remnants can cross theendothelial barrier and were identified in humanarteries (79). Because of their larger size, TGRL carries5 to 20 times more cholesterol/particle than LDL.Importantly, unlike native LDL, remnants can betaken up in an unregulated fashion by scavenger re-ceptors expressed by resident macrophages in thesubendothelial space, thus promoting foamcell formation. Chylomicron remnants and VLDLremnants rapidly penetrate the arterial wall andcontribute to atherogenesis (6,8). VLDL compositionis also a critical CVD risk determinant. In retrospectiveand prospective population studies, TG-associatedCHD risk was limited to apoC3-containing VLDL par-ticles and their metabolic remnants, small LDL parti-cles (9–11). ApoC3 impairs VLDL binding to cellularreceptors, resulting in small dense LDL particle for-mation (9,10), thereby counteracting the effects ofapoE (11) (Figure 1). In 2 prospective cohorts, theNurses’ Health Study and the Health Professionals


2531

Follow-up Study, CHD risk was higher in VLDL andLDL with apoC3 with a low apoE content than with ahigh apoE content (multivariable-adjusted relativerisk for apoE content in LDL with apoC3 for the top vs.lowest quintile: 0.45, 95% confidence interval [CI]:0.31 to 0.64; and for VLDL with apoC3: 0.50, 95% CI:0.35 to 0.72; p value for trend 73,000 Copenhagen participants enrolled in 1 of 2prospective studies (72), or a case-control study witha total of nearly 14,000 diagnosed with CHD (65).

NON-HDL-C, APOLIPOPROTEIN B, AND

LDL PARTICLE CONCENTRATION AS

TARGETS OF THERAPY FOR THE

PREVENTION OF ATHEROSCLEROSIS AND

ATHEROSCLEROTIC CVD

Hypertriglyceridemia is accompanied by elevatednon–HDL-C levels (due to increased VLDL-C), IDL,small and total LDL particles, and total apoC3, as wellas decreased HDL-C levels due to fewer smaller HDLparticles. All of these changes are associated withincreased risk, and which parameters are causal isdebated. Population studies consistently show thatnon–HDL-C more strongly correlates with CHD eventrisk than LDL-C in those with and without hyper-triglyceridemia (80). A large meta-analysis of statinoutcome trials found that the subgroup of patients onstatin therapy with non–HDL-C >130 mg/dl and withLDL-C >100 mg/dl had the highest relative risk (HR:1.32) compared with patients with both LDL-C andnon–HDL-C at the target levels of



of therapy (82,92–94). However, multiple studiesindicate that that VLDL is at least as (or more)atherogenic than LDL (73,79). Thus, according torecent professional society guidelines, combiningLDL-C and VLDL-C makes non–HDL-C a preferredtarget in patients with dyslipidemia (93,94). More-over, a recent analysis of contemporary statin trialsdemonstrated that on-treatment levels of non–HDL-Care more strongly associated with future risk of CVDevents than either apoB or LDL-C (80). In the sameanalysis, non–HDL-C explained a larger proportion ofthe atheroprotective effects of statin therapy thaneither apoB or LDL-C. These findings favor the use ofnon–HDL-C over LDL-C as a therapy target, particu-larly in hypertriglyceridemic individuals (93,94).Since apoB is the major apolipoprotein of both LDLand VLDL, some investigators propose total apoB asan alternative to non–HDL-C (95).

TGRLS: LESSONS FROM HUMAN GENETICS

Over the past 15 years, human genetic studies haveidentified new proteins involved in TGRL meta-bolism, revealed insights into the genetic architec-ture of plasma TG, and clarified the contribution ofTGRL to human CVD. At least 3 different humangenetic approaches—sequencing of biologic candidategenes, genetic analysis of Mendelian TG phenotypes,and GWAS of common DNA sequence variants—haveyielded new proteins and novel human DNA variantsinvolved in plasma TGRL regulation. Notable exam-ples include apoA5 (biologic candidate), ANGPTL3(biologic candidate and Mendelian low TG), LMF1(biologic candidate), apoC3 (GWAS), GCKR (GWAS),COL18A1 (GWAS), and TRIB1 (GWAS), among manyothers (96–106).

The genetic architecture for TG in the popula-tion appears to be a mosaic comprised of rare large-effect variants, common small-effect variants, and

TABLE 3 DNA Sequence Variants in LPL-Regulating Genes Associated

Gene rsID Protein

Effect ofVariant on

Protein Function

EffectMutatioLPL Act

LPL rs1801177rs328

D36NS474X

LossGain

DecreIncrea

APOA5 rs662799 Promoter-1131T/C

Loss Decre

ANGPTL4 rs116843064 E40K, T266M Loss Increa

APOC3 multiple R19X, IVS2þ1 G/A,IVS3þ1 G/T, A43T

Loss Increa

CHD ¼ coronary heart disease; LPL ¼ lipoprotein lipase; TG ¼ triglycerides.

environmental influences (107). Variants associatedwith common, complex traits like plasma TG rangefrom common (>1:20) frequency to low frequency(1:200 to 1:20) and to rare (150polymorphisms associated with plasma lipids (102).Across 185 polymorphisms, the strength of a variant’seffect on plasma TG highly correlated with themagnitude of its effect on CHD, even after accountingfor each variant’s potential effects on LDL-C and/orHDL-C (109). These results support the notion thatTGRL causally influences CHD risk.

Common, low frequency, and/or rare DNAsequence variants in 4 TGRL metabolism genes wereconvincingly associated with CHD risk, and all 4genes functionally relate to LPL (Table 3). Beyond

With TG and CHD

ofn onivity

LipidPhenotypeAssociation

CHDRisk

AlleleFrequencyin Whites

AlleleFrequencyin Blacks Ref. #

asese

[TGYTG

[

Y

2%11%

5%7%

(72)

ase [TG [ 8% Rare (3)

se YTG Y 1.6% 0.1% (96)

se YTG Y 0.7%(collective for all

4 mutations)

0.7%(collective for all

4 mutations)

(99,110,111)


2533

genes that alter LDL, this is the first set offunctionally related genes where naturally occurringgenetic variation alters the complex disease of CHD.We highlight 2 recent studies that establish a centralrole of apoC3 in TG and CHD risk (110,111). In the firststudy (110), the protein-coding regions (exome) of18,666 genes were sequenced in 3,734 individualsfrom the United States, and rare mutations in eachgene were tested for association with plasma TG. Thetop result was for APOC3. Approximately 1 in 150 in-dividuals carried any of 4 protein-altering or splice-site variants of APOC3. Heterozygous carriers of anyof these 4 APOC3 mutations had 39% lower plasma TG(p < 1 � 10�20), 46% lower circulating plasma APOC3(p ¼ 8 � 10�10), and 40% lower risk for CHD (oddsratio: 0.60, 95% CI: 0.47 to 0.75; p ¼ 4 � 10�6).

A second study (111), using data from 75,725 par-ticipants in 2 general population studies in Denmark,associated low levels of nonfasting TG with reducedrisks of ischemic vascular disease and ischemic heartdisease. Participants with nonfasting TG levels 1,000mg/dl). Although not TG-lowering agents, statinsreduce TG in patients with mild to severe hyper-triglyceridemia, and the more effective the statin in

decreasing LDL-C, the greater its effect on plasma TG(112). Pooled data from placebo-controlled trials alsosuggest that 2 to 7 years of statin treatmentmay reducethe risk of pancreatitis in patients with mild to mod-erate hypertriglyceridemia. However, because severehypertriglyceridemia is clinically, physiologically, andmetabolically distinct from mild to moderate hyper-triglyceridemia (5,113), statins have limited efficacy formanagement of severe hypertriglyceridemia and theclearance of large, buoyant, TG-rich lipoproteins suchas chylomicrons. When the fasting TG concentration is>1,000 mg/dl, chylomicrons are the predominantplasma lipoproteins. At TG levels between 500 and1,000 mg/dl, chylomicrons, VLDL, and remnant lipo-proteins cohabit in variable proportions, whereasminimal fasting chylomicronemia is observed whenTG values are



immediate-release niacin. In the Heart ProtectionStudy-2 and AIM-HIGH (Atherothrombosis Interven-tion in Metabolic Syndrome with Low HDL/High Tri-glycerides: Impact on Global Health Outcomes) trials,extended-release niacin was associated with in-creased infection rates (119,120).

OMEGA-3S FOR MANAGEMENT OF

HYPERTRIGLYCERIDEMIA

Since the initial observations by Bang et al. (121) in theearly 1970s that Greenland Eskimos with a diet rich inomega-3 fatty acids have a low incidence of CVD,clinical benefits of the major omega-3 fatty acids,eicosapentaenoic acid (EPA) and docosahexaenoicacid (DHA) have been investigated extensively. Thereis a better understanding that the complex mixture offatty acids found in fish oil contains multiple activecomponents, and that each of the major omega-3 fattyacids (EPA, DHA, and docosapentaenoic acid [DPA]),has overlapping, but distinct, biological activities(122). Health benefits of omega-3 fatty acids are linkedto fish intake, but EPA, DHA, and DPA plasma levelsare also determined by polymorphisms of the desa-turases (delta-5 and -6) that convert short-chainpolyunsaturated fatty acids (SC-PUFAs) to long-chain polyunsaturated fatty acids (LC-PUFAs). Notonly has the role of omega-3 fatty acids in humanhealth broadened since the initial observations inGreenland Eskimos, but there is also a better appre-ciation of the pharmacological and biological in-tricacies of the major fatty acid constituents of fishoil.

Recent advances in “omics” suggest new mecha-nisms of action for niacin and omega-3 fatty acids(123–129). Clinical trials conducted with GPR109A ag-onists, studies in mice lacking GPR109A, and analysesof niacin’s effect on gene expression profiles in skel-etal muscle of obese Zucker rats suggest that niacin’smechanism of action is not mainly driven by adipo-cyte triglyceride lipolysis, but involves genes andconsecutive metabolic effects in hepatocytes, skeletalmuscle cells, macrophages, neutrophils, and theendothelial wall (123–125).

Omega-3 fatty acids modify expression of acylgly-cerol synthesis pathway genes and their poly-morphisms (particularly GPAM, AGPAT3, andAGPAT4) modulate the effect of omega-3 fatty acidson plasma TG concentration (126). The EVOLVE(Epanova for Lowering Very High Triglycerides) clin-ical trial (127), demonstrated the safety and efficacy ofthe free fatty acid form of omega-3 fatty acidsin severe hypertriglyceridemia (plasma TG concen-tration from 500 to 2,000 mg/dl), confirming that

they are effective on post-prandial metabolism andthe exogenous (chylomicron-dependent) metabolicpathway. A recent study suggests that the biologiceffect of omega-3 fatty acids in enterocytes may beexerted through microribonucleic acids (miRNAs),particularly miR-192 and miR-30c. miRNAs are small,noncoding RNA molecules that play key roles intranscriptional and post-transcriptional regulation ofgene expression. Enterocyte targets of miR-192 andmiR-30c include genes encoding nuclear receptorcorepressor 2, isocitrate dehydrogenase 1, caveolin 1,ATP-binding cassette subfamily G member 4, andretinoic acid receptor b (128).

FADS POLYMORPHISMS AND PLASMA LEVELS

OF OMEGA-3 FATTY ACIDS

Until recently, dietary intake of long-chain marineomega-3 fatty acids was thought to be the majordeterminant of plasma concentrations. Short-chainomega-3 fatty acids, primarily alpha-linolenic acid,are abundant in the diet, but only small amounts areconverted to EPA and even less are elongated to DHAby desaturase enzymes (delta-5 and -6) (Figure 2A)(129). However, it is now widely recognized thatgenetic polymorphisms of the desaturase genes,FADS1 to 3, significantly affect LC-PUFA formation.FADS polymorphisms are relatively common andpotent, and may explain up to 30% of the variability inLC-PUFA levels (both omega-6 and -3). For example,about 80% of African Americans have a polymorphism(rs174537) associated with more effective conversionof SC-PUFA to LC-PUFA by desaturase-5, resulting inhigher levels of both arachidonic acid (AA) and EPA,even though their fish consumption is lower, onaverage, than that of whites. The “desaturase hy-pothesis” (130) is that, in populations following aWestern diet rich in omega-6 and relatively deficientin omega-3 PUFAs, FADS polymorphisms associatedwith high desaturase activity may lead to predominantproinflammatory, detrimental effects (131). It isintriguing to hypothesize that the higher levels ofdisorders such as hypertension, asthma, and aspirinresistance in African Americans are linked to a higherinflammatory state driven by overproduction of pros-taglandins and leukotrienes synthesized from AA. Incontrast, Hispanic individuals more commonly have adifferent FADS polymorphism (rs17454), resulting inless conversion of SC-PUFA to LC-PUFA, and theyconsequently have lower AA and EPA levels (130,132).This FADS polymorphism is associated with dyslipi-demia (high TG and HDL-C), modulated, in part, byPUFA intake. This polymorphismmay explain the highprevalence of hypertriglyceridemia in the Hispanic

FIGURE 2 FADS Polymorphisms and Fatty Acid Metabolism

(A) Long-chain polyunsaturated fatty acid (PUFA) plasma levels are determined by both

dietary intake and conversion of shorter-chain PUFAs by the delta-5 and -6 desaturases.

Polymorphisms of these fatty acid desaturases (FADS) result in either higher or lower long-

chain PUFA levels, possibly explaining differences in hypertriglyceridemia prevalence

between Hispanics and African Americans. (B) Long-chain omega-3 fatty acids in fish oils

decreasehepatic lipogenesis, thereby reducingVLDL-c levels, and enhance the conversionby

LPL of VLDL to LDL mediated in part by a decrease in apoC3. AA ¼ arachidonic acid;ALA ¼ alpha-linoleic acid; CHD ¼ coronary heart disease; DGLA ¼ dihomo-gamma-linoleicacid; DHA ¼ docosahexaenoic acid; DPA ¼ docosapentaenoic acid; EPA ¼ eicosapentaenoicacid; GLA ¼ gamma-linoleic acid; LA ¼ linoleic acid; other abbreviations as in Figure 1.


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population, whereas the polymorphism found pre-dominantly in African Americans and associated withhigher EPA levels may lead to their lower incidence ofelevated TG and higher HDL-C levels. Further studiesare needed to define the function of FADS1 and FADS2polymorphisms relative to mechanisms involved inthe development of atherosclerosis, dyslipidemia, andother diseases linked to either AA overproduction ormarine LC-PUFA deficiencies.

BIOLOGICAL DIFFERENCES IN EPA, DHA, ANDDPA

Omega-3 fatty acid treatment lowers the TG level byboth reducing hepatic TG secretion and enhancingthe rate of TG clearance from the circulation. ApoC3appears to play an important role in hyper-triglyceridemia pathogenesis, particularly with re-gard to inhibiting lipoprotein lipase and hepaticlipase, which slows TG hydrolysis (133). Apo C3 alsointerferes with the TGRL interactions with hepaticapoB/E receptors, slowing removal of these particlesfrom circulation (134). ApoC3 is regulated by the he-patic nuclear factor-4 alpha, forkhead box O tran-scription factor O1, and carbohydrate responseelement–binding protein in response to insulin(135). Increased apoC3 synthesis may represent acompensatory mechanism to reduce TGRL catabolismand uptake by hepatic receptors in an attempt tocope with a large influx of substrates for TG produc-tion. The effect of omega-3 fatty acids on apoC3, un-like that of fibrates, is independent of PPAR-alpha(136). Both EPA and DHA down-regulate SREBP-1c,the transcription factor that controls lipogenesis.EPA is a more potent agonist of PPAR-alpha thanDHA, whereas DHA appears to regulate hepatic nu-clear factor-4 alpha, forkhead box O transcriptionfactor O1, and carbohydrate response element–bindingprotein (137).

The severity of hypertriglyceridemia is typicallyassociated with the apoC3 level. Fish oil containinga complex mixture of omega-3 fatty acids loweredapoC3 in patients with hypertriglyceridemia(138). This is of potential clinical importance, becauseelevated apoC3 levels associated with VLDL þ LDLparticles were independent predictors of CVDevent risk, and loss-of-function apoC3 gene poly-morphisms were associated with reduced CVD eventrisk (110,111) (Figure 2B).

EMERGING THERAPIES FOR MANAGEMENT

OF HYPERTRIGLYCERIDEMIA

A number of new therapies to address the challengesand unmet needs of patients with high TG and mixed

dyslipidemia are being developed. Some are newformulations or improved delivery systems of pre-existing drugs, whereas others target new mecha-nisms for TGRL metabolism and hypertriglyceridemiamanagement. Cell-penetrating peptides or linkertechnologies combining niacin and omega-3, PCSK9inhibitors, apoC3-antisense, DGAT1 inhibitors, MTPinhibitors, peptide mimetics, and LPL gene replace-ment therapy represent the diversity of mechanismsand targets for management of TGRL and severe



hypertriglyceridemia. These emerging drugs decreasefasting TG concentration in different proportions:some are more effective on post-prandial lipidmetabolism and the exogenous (chylomicron) path-way, and others are more effective on the endoge-nous pathway, on liver fat management (includingnonalcoholic fatty liver disease and insulin resis-tance), or on fat metabolism in adipocytes andperipheral tissues (including lipodystrophies).

Cell-penetrating peptides represent a new mecha-nism of drug delivery. When conjugated to macro-molecules or when used at low concentrations,cell-penetrating peptides enter cells via the endo-cytic pathway. CAT-2003, a novel niacin and EPAconjugate, is currently in a phase II clinical trial fortreatment of severe hypertriglyceridemia of differentcauses (139). In pre-clinical studies, CAT-2003 syner-gistically reduced TG superior to niacin and EPA,individually or combined. CAT-2003 inhibits thetranscription factor SREBP in vitro and in vivo (140),resulting in reduced PCSK9 expression. SiRNA inhi-bition of PCSK9 gene expression reduces productionand assembly of multiple cholesterol biosyntheticenzymes in the cellular SREBP2 pathway, as well as ofseveral enzymes involved in intestinal TGRL synthe-sis and assembly (15).

ISIS-APOCIIIRx is a second-generation 20-O-(2-methoxyethyl) modified antisense inhibitor of apoC3synthesis. ISIS-APOCIIIRx selectively inhibits hepaticapoC3 protein synthesis by binding to a smallsequence of apoC3 mRNA to elicit its degradation byRNase H1, an endogenous ribonuclease expressedubiquitously in mammalian cells. In phase 2 studies,ISIS-APOCIIIRx was highly effective in loweringapoC3, fasting plasma TG, and non–HDL-C in patientswith elevated VLDL-TG or chylomicron-TG due to avariety of conditions, including familial chylomicro-nemia due to lipoprotein lipase deficiency (LPLD),suggesting that apoC3 might play a key role in a non-LPL–dependent TGRL metabolic pathway (141).Because hepatic apoC3 potentially promotes clear-ance of excess hepatic TG, ASO inhibition of apoC3could theoretically exacerbate hepatic lipid accumu-lation (142,143).

Lomitapide is an MTP inhibitor that interferes withapoB-containing lipoprotein assembly in the apoB100and apoB48 pathways, thus reducing both chylomi-cron and VLDL secretion. It is currently available forthe management of homozygous FH, and has beenused long-term (>13 years) to treat a single patientwith extremely severe hypertriglyceridemia due toLPLD (144). Lomitapide 40-mg daily eliminatedchronic abdominal pain and prevented pancreatitis;however, the fatty liver present before treatment

progressed to steatohepatitis and fibrosis after 12 to13 years.

DGAT1 catalyzes the final step of TG synthesis andis highly expressed in the gut wall, where it plays akey role in dietary fat absorption as chylomicron-TG(145,146). DGAT1 and DGAT2 are also required forLD formation in adipocytes, they are active in seba-ceous glands, and their dysregulation contributes tothe imbalance between lipid supply and demandand nonalcoholic fatty liver disease development(147,148). Pradigastat (formerly LCQ908), a selectiveDGAT1 inhibitor, is being evaluated for treatment offamilial chylomicronemia (149). In healthy humanvolunteers, pradigastat decreases post-prandialchylomicron particle numbers and prevents post-prandial hypertriglyceridemia (150). Gastrointestinalevents such as diarrhea, abdominal pain, and nauseaare the most commonly reported adverse events, andare related to dose and dietary fat content.

AAV1-LPLS447X gene replacement therapy (alipo-gene tiparvovec) was developed for treatment offamilial chylomicronemia due to LPLD (151–155).LPL gene therapy adds extra copies of the geneencoding a functionally potent enzyme to muscletissue of affected patients, specifically homozygotesor compound heterozygotes with documented null,LPLD-causing LPL gene mutations. In pivotal clinicaltrials, intramuscular administration of alipogenetiparvovec was generally well tolerated and associ-ated with signs of clinical improvement and reduc-tion in overall pancreatitis incidence up to 5 yearsafter administration (155).

CONCLUSIONS

Elevated fasting and nonfasting TG levels were asso-ciated with incident CVD events in multiple studies;however, they also accompany a multitude of bio-markers (lipoproteins, inflammatory mediators andproteins, and hemostatic measures) and CVD-associated conditions, including type 2 diabetes mel-litus. Recent clinical trials failed to demonstrate areduction in CVD events in statin-treated patients alsotreated with fibrates and niacin, challenging elevatedTG as an independent risk factor. However, clinicaltrials with fibrates demonstrated risk reduction insubgroups with fasting TG $200 mg/dl, particularlythose with low HDL-C, indicating a threshold effectthat should mandate future TG-lowering therapytrials.

Our understanding of TG-associated CVD riskevolved with the recognition that TG is just 1component of a vastly heterogeneous TGRL pool.Genetic studies demonstrate a causal relationship of


2537

certain TGRL components and TGRL synthetic andmetabolic pathways with atherosclerotic CVD events.Specific genetic mechanisms that increase plasmaTGRL and affect CVD include loss of LPL function,loss of APOA5 function, gain of ANGPTL4 function,and gain of APOC3 function.

Importantly, these genetic studies identified spe-cific targets in the causal pathway that may be can-didates for pharmacological intervention. Ongoingclinical trials targeting causal pathways in TGRL

metabolism should clarify the role of TGRLs andCVD. In the interim, we advocate the use of avai-lable TG-lowering therapy for prevention of acutepancreatitis.

REPRINT REQUESTS AND CORRESPONDENCE: Dr.Robert S. Rosenson, Mount Sinai Heart, CardiometabolicDisorders, Icahn School of Medicine at Mount Sinai,One Gustave L. Levy Place, Box 1030, New York, NewYork 10029. E-mail: [email protected].

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