REVIEW

CURRENT
OPINION The central role of arterial retention of cholesterol-
rich apolipoprotein-B-containing lipoproteins in the
pathogenesis of atherosclerosis: a triumph
of simplicity
Jan Borén a,b and Kevin Jon Williams a,b,c

Purpose of review
Today, it is no longer a hypothesis, but an established fact, that increased plasma concentrations of
cholesterol-rich apolipoprotein-B (apoB)-containing lipoproteins are causatively linked to atherosclerotic
cardiovascular disease (ASCVD) and that lowering plasma LDL concentrations reduces cardiovascular
events in humans. Here, we review evidence behind this assertion, with an emphasis on recent studies
supporting the ‘response-to-retention’ model – namely, that the key initiating event in atherogenesis is the
retention, or trapping, of cholesterol-rich apoB-containing lipoproteins within the arterial wall.
Recent findings
New clinical trials have shown that ezetimibe and anti-PCSK9 antibodies – both nonstatins – lower
ASCVD events, and they do so to the same extent as would be expected from comparable plasma LDL
lowering by a statin. These studies demonstrate beyond any doubt the causal role of apoB-containing
lipoproteins in atherogenesis. In addition, recent laboratory experimentation and human Mendelian
randomization studies have revealed novel information about the critical role of apoB-containing
lipoproteins in atherogenesis. New information has also emerged on mechanisms for the accumulation in
plasma of harmful cholesterol-rich and triglyceride-rich apoB-containing remnant lipoproteins in states of
overnutrition. Like LDL, these harmful cholesterol-rich and triglyceride-rich apoB-containing remnant
lipoprotein remnants become retained and modified within the arterial wall, causing atherosclerosis.
Summary
LDL and other cholesterol-rich, apoB-containing lipoproteins, once they become retained and modified
within the arterial wall, cause atherosclerosis. This simple, robust pathophysiologic understanding may
finally allow us to eradicate ASCVD, the leading killer in the world.
Keywords
apolipoprotein-B, atherosclerosis, Mendelian randomization studies, prospective randomized controlled
trials, proteoglycan

INTRODUCTION
Atherosclerotic cardiovascular disease (ASCVD) is
the leading cause of death globally. Widespread
in developed areas of the world and now becoming
more common in poorer countries, deaths from
ASCVD are projected to increase to more than
2.3  107 per year by 2030. It is tempting to think
of atherosclerosis as a disease of the modern
lifestyle. Yet atherosclerosis has plagued humans
for thousands of years and has been detected in
4000-year-old Egyptian mummies.
Today, it is no longer a hypothesis, but an
established fact, that increased plasma concen-
trations of cholesterol-rich apolipoprotein-B
a
Department of Molecular and Clinical Medicine, University of Gothen-
burg, bSahlgrenska University Hospital, Gothenburg, Sweden and
c
Section of Endocrinology, Diabetes, & Metabolism, Department of
Medicine, Lewis Katz School of Medicine at Temple University,
Philadelphia, Pennsylvania, USA
Correspondence to Jan Borén, MD, PhD, Wallenberg Laboratory,
Sahlgrenska University Hospital, Gothenburg, Sweden.
Tel: +46 733 764264; e-mail: jan.boren@wlab.gu.se
Curr Opin Lipidol 2016, 27:473–483
DOI:10.1097/MOL.0000000000000330
This is an open-access article distributed under the terms of the Creative
Commons Attribution-Non Commercial-No Derivatives License 4.0, where
it is permissible to download and share the work provided it is properly
cited. The work cannot be changed in any way or used commercially.

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Atherosclerosis: cell biology and lipoproteins
KEY POINTS
 Recent studies provide new support for the ‘response-
to-retention’ hypothesis – namely, that the key initiating
event in atherosclerotic cardiovascular disease
(ASCVD) is the retention, or trapping, of cholesterol-rich
apoB-containing lipoproteins within the arterial wall.
 Accordingly, the most effective therapies against
ASCVD – LDL-lowering drugs – are based upon the
principle that decreasing the concentration of apoB-
lipoproteins in the circulation decreases the probability
that they will enter and become retained in
the subendothelium.
 With the rise of overnutrition, obesity, and the
atherometabolic syndrome, cholesterol-rich and
triglyceride-rich apoB-containing remnant lipoproteins
(C-TRLs) have emerged as major drivers of human
ASCVD worldwide, also due to their retention within
the arterial wall.
 Using our extensive knowledge of the pathogenesis of
atherosclerosis, we can now reclassify nearly all
epidemiologic risk factors for ASCVD events into
causative agents, exacerbating factors, and mere
bystander phenomena, thereby focusing therapeutic
efforts on causative and exacerbating factors.
 At this point, we know enough about the
pathophysiology of atherosclerosis that eradication of
this major killer has become a genuine possibility.
(apoB)-containing lipoproteins are causatively
linked to ASCVD and that lowering LDL concen-
trations with statins and nonstatins reduces athero-
&&
sclerotic cardiovascular events in humans [1,2 –4 ,
& &
5 ,6 ]. Despite the clarity of the evidence today, the
role of LDL was questioned and even ridiculed for
many years [7]. During that period, numerous com-
peting hypotheses [8–11] were articulated to explain
the initiating events in atherogenesis. After decades
of research, however, there is now a large body of
evidence to support the ‘response-to-retention hy-
pothesis’ – namely, that the key initiating event in
atherogenesis is the retention, or trapping, of choles-
terol-rich apoB-containing lipoproteins within the
arterial wall. The retained lipoproteins and their
byproducts provoke a series of strikingly maladaptive
local responses that cause plaque initiation, growth,
and evolution (Fig. 1).
Modern research on atherosclerosis began
with the insightful and pioneering ‘cholesterol
hypothesis’ of Anichkov and Khalatov [12,13]. As a
point of clarification, however, we now know
that many things that carry cholesterol near
arteries – such as normal HDL and erythrocytes
(which transport a similar amount of cholesterol in
blood as LDL does) – do not cause atherosclerosis.
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Thus, cholesterol per se is not the cause of this disease.
In contrast, cholesterol-rich apoB-lipoproteins
possess special properties that render them uniquely
pathogenic in ASCVD.
The response-to-retention hypothesis of
atherogenesis
The response-to-retention hypothesis drew on work
from the 1940s to the 1980s showing that lipopro-
teins can interact with proteoglycans of the arterial
wall [14–17]. Lipids and apoB accumulate at lesion-
prone sites before gross morphological changes
occur [18–21], and retention of apoB-lipoproteins
is seen throughout the progression of atherosclero-
sis. The consequences of the retention of apoB-lip-
oproteins include, not only an accumulation of
lipid, but also prolonged exposure of these particles
to local enzymes and other factors within the vessel
wall. The retained and modified apoB-lipoproteins
trigger cellular responses within the artery wall that
accelerate further lipoprotein retention and lesion
development [9,11,22].
Influx of apolipoprotein-B-lipoproteins
into the artery wall
Lipoproteins normally flux into and out of the
arterial wall [20,23]. The molecular mediators of this
transendothelial movement of lipoproteins remain
incompletely characterized. Recent evidence has
suggested roles for caveolin-1 [24,25] and the scav-
enger receptor class B type I (SR-BI) [26], but
additional mediators are likely [27].
&&
ApoB-lipoproteins up to only approximately
70 nm in diameter [i.e., chylomicron remnants,
smaller VLDL, IDL, LDL, and lipoprotein(a)] can
efficiently cross an intact endothelium, and among
these, the smaller ones pass more readily than the
larger ones [23]. This size limitation explains why
patients with lipoprotein lipase deficiency do not
develop atherosclerosis despite very high plasma
levels of large apoB-containing chylomicrons.
Studies in-vivo of the influx and egress of LDL
particles into and out of the arterial intima
have shown that, although the endothelial layer
remains intact over most atherosclerotic lesions,
its permeability to LDL becomes abnormally high
[20,28,29]. The precise stimuli and molecular mech-
anism(s) responsible for this increased permeability
are still not elucidated, and it was not known until
recently if the increased endothelial permeability
for LDL could be reversed. To examine this issue,
Bartels et al. [30 ] fed LDL receptor null (Ldlr / )
&&
mice on a high-cholesterol diet for up to 5 months
to increase their plasma concentrations of apoB-
lipoproteins, thereby provoking the formation of
Volume 27  Number 5  October 2016
 The pathogenic simplicity of atherosclerosis Borén and Williams

LUMEN
LDL IDL Remnant

Monocyte
ap
sc
rou
Fib

Ps
MM
INTIMA

LPL Necrotic
debris
SMase T-cell
TF
UC-rich material
PGs IL-4,
IFN-γ Lipid-rich core

se s
Ea a s e
Ch rot e
SM
Ci

P
n-m Foam cell with LP-derived
igr
ati lipids, unable to emigrate
on
MEDIA

Retention Responses

FIGURE 1. The response-to-retention model of initiation and progression of atherosclerosis. Arrows are color-coded to indicate
crucial mechanisms in the retention of cholesterol-rich atherogenic apolipoprotein-B-lipoproteins within the arterial wall, which
is the key initiating step in atherogenesis (yellow) and then local responses to the retained and modified lipoproteins that lead
to plaque growth and evolution (red). The main text describes molecular mechanisms for early retention and aggregation of
apoB-lipoproteins within the arterial wall and then the acceleration of lipoprotein retention after plaque initiation and other
subsequent maladaptive responses. Two key enzymes implicated in the retention and aggregation of apoB-lipoproteins and
hence atherogenesis are the secretory sphingomyelinase (produced by endothelium, particularly when activated, and by
plaque macrophages) and lipoprotein lipase (produced by plaque macrophages and acting as a ‘bridge’ to facilitate further
adherence of apoB-lipoproteins to arterial matrix). ChEase, cholesteryl esterase; LP, lipoprotein; LPL, lipoprotein lipase; MMPs,
matrix metalloproteinases; PGs, proteoglycans; Remnant, cholesterol-rich and triglyceride-rich apoB-containing remnant
lipoprotein (C-TRL remnant); SMase, sphingomyelinase; SMC, smooth muscle cell, including immature SMCs; TF, tissue factor;
UC, unesterified cholesterol. Source: Adapted from [11].

atherosclerotic plaques. To investigate the effect of
an abrupt lowering of plasma apoB-lipoprotein con-
centrations on aortic endothelial permeability to
LDL and the fractional degradation rates of apoB-
lipoproteins within the atherosclerotic lesions,
these authors used an anti-apoB antisense oligonu-
cleotide (ASO) to reduce hepatic apoB synthesis.
Concentrations of apoB-containing lipoproteins in
plasma fell sharply to nearly undetectable levels
within 3 days after administration of the ASO.
The authors then analyzed the aortic plaques after
1 or 4 weeks of ASO treatments. Lowering of plasma
apoB-lipoprotein concentrations reduced LDL per-
meability into the arterial wall within 1 week of ASO
treatment, and this effect preceded any detectable
changes in the cellular composition or size of the
&&
atherosclerotic lesions [30 ]. The molecular mech-
anisms involved still remain unclear, and effects on
caveolin-1 and SR-BI were not examined. Bartels
&&
et al. [30 ] ruled out a nonspecific process by
showing that the anti-apoB ASO treatment had no
effect on endothelial permeability to Evans blue dye.
In addition, correction of the hypercholesterolemia
caused the fractional degradation rates of apoB
within the plaques to fall sharply. Most importantly,
the rapid decreases in permeability to LDL and in the
fractional degradation of LDL that enters the plaque
may turn out to be crucial for subsequent plaque
&
stabilization and atherosclerosis regression [6 ].
Retention of atherogenic apolipoprotein-
B-lipoproteins within the artery wall as the
key pathogenic event in atherosclerosis
Because the capacity of transendothelial transport
is high even in normal arterial segments, it is
unlikely that influx of LDL and other smaller

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Atherosclerosis: cell biology and lipoproteins
apoB-lipoproteins from the bloodstream is rate
limiting in determining their concentrations within
the artery wall. Instead, mechanisms for the selec-
tive retention of a tiny percentage of the apoB-lip-
oproteins that enter the artery wall drive their local
accumulation and hence atherogenesis. Support for
this model came from early work by Schwenke and
Carew showing that LDL entry did not differ
between normal arterial sites that are susceptible
versus resistant to subsequent plaque development.
Although the same amount of LDL entered, the
susceptible sites, though healthy, already showed
greater LDL accumulation [20]. More recent support
comes from molecular studies by Tran-Lundmark
et al. who crossed glycosaminoglycan (GAG)-
deficient perlecan (Hspg2D3/D3) mice with hypercho-
lesterolemic apolipoprotein-E (apoE)-null (Apoe / )
mice. Because perlecan participates both as a per-
meability barrier and as a proretentive molecule
within the murine arterial wall, the influx of LDL
and other apoB-lipoproteins into the aortic wall in
vivo was increased in Hspg2D3/D3/Apoe / mice com-
pared with Apoe / controls, whereas subendothe-
lial retention of LDL was paradoxically lower. In
other words, this ingenious genetic manipulation
of perlecan separated the two processes. Impor-
tantly, these authors demonstrated a significant
reduction of atherosclerotic lesions in Hspg2D3/D3/
Apoe / mice compared with Apoe / controls, indi-
cating the key role of decreased apoB-lipoprotein
retention regardless of the increase in endothelial
permeability to LDL entry. Thus, most apoB-
lipoprotein particles that entered the arterial wall
left without contributing to the growth of the
atherosclerotic lesion [31].
In earliest atherogenesis, negatively charged
proteoglycans in the extracellular matrix of the
arterial intima bind and trap apoB-lipoproteins via
electrostatic interactions with specific positively
charged aminoacyl residues in the full-length hep-
atic form of apoB, apoB100 (residues 3359–3369)
[32], and in the truncated intestinal form, apoB48
(residues 84–94) [33]. Moreover, apoB48-lipopro-
teins typically contain numerous molecules of apoE,
an apoprotein that has a proteoglycan-binding
domain almost identical to the proteoglycan-
binding sequence in apoB100. Proteoglycans are
negatively charged due to the sulfate and carboxylic
acid groups in their GAG side-chains. In experimen-
tal studies, several arterial-wall proteoglycans have
been shown to be important for apoB-lipoprotein
retention and atherosclerosis, most notably the
matrix-associated proteoglycans biglycan, perlecan,
and versican [9,34,35].
Consistent with the response-to-retention
model, numerous other interventions have been
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performed that do not change plasma concen-
trations of apoB-lipoproteins in hypercholesterole-
mic animals but specifically decrease or increase
the retention of these particles within the arterial
wall. These interventions correspondingly decrease
or increase atherogenesis. Such interventions
include mutagenesis of the proteoglycan/GAG-
binding domain of apoB100, thereby slowing LDL
retention and atherogenesis [36], manipulations
of arterial matrix to alter its affinity for apoB
&& &
[31,37–39,40 ,41 ], and knockouts of arterial-wall
enzymes specifically implicated in apoB-lipoprotein
retention and aggregation [42–45].
The first of these studies, which used mutagen-
esis of apoB100 to block its direct adherence
to arterial matrix, emphasizes the importance of
LDL quality, as opposed to simply focusing on its
concentrations in plasma. Other variations in LDL
quality may also affect its atherogenicity, such as
surface lipid composition [46] and core lipid com-
position (which influences the conformation of
apoB and hence its interactions with proteoglycans)
&&
[47,48 ,49]. In addition, the protein composition of
apoB-lipoproteins influences their interactions
with proteoglycans. As discussed above, apoE medi-
ates binding of lipoproteins to arterial GAGs,
although apoE also facilitates hepatic disposal of
atherogenic lipoproteins. In contrast, the small
exchangeable apolipoprotein, apolipoprotein-C
(apoC)-III, increases the affinity of LDL for arte-
rial-wall proteoglycans, whereas decreasing hepatic
uptake of apoB-lipoproteins [50], thereby increasing
the accumulation of atherogenic lipoproteins
within the vessel wall [51,52]. The mechanism has
been unclear because apoC-III by itself lacks posi-
tively charged domains to bind arterial-wall proteo-
glycans [51]. Hiukka et al. [53] demonstrated that an
increased apoCIII : apoB molar ratio in LDL isolated
from patients with type 2 diabetes correlated with
lower contents of unesterified cholesterol, sphingo-
myelin, ceramide, and GM1. Importantly, this com-
bination of compositional changes was associated
with increased proteoglycan binding [53]. The
mechanism remains unclear but one possibility is
that a more fluid monolayer on the LDL particle
surface may facilitate the insertion of more mol-
ecules of apoC-III and permit conformational
changes in apoB100 that increase its proteoglycan-
binding affinity. In contrast, however, the apoC-
III : apoB ratio in LDL from nondiabetic control
patients showed no correlations with the content
of these lipids, indicating less global changes in LDL
composition [53].
Regarding the subendothelial arterial matrix,
recent work has implicated proteases [54] and other
&
factors [55,56 ] in the regulation of the arterial-wall
Volume 27  Number 5  October 2016
 The pathogenic simplicity of atherosclerosis Borén and Williams
content of LDL-binding proteoglycans and hence
&& &
atherosclerosis. She et al. [40 ,41 ] recently focused
on a less well known proteoglycan in the vascula-
ture, the neural/glia antigen 2 (NG2) proteoglycan.
Although NG2 is undetectable in the intima
of normal murine arteries, early atheromata in
Apoe / mice contain immature synthetic smooth
&&
muscle cells (s-SMCs) that express NG2 [40 ]. Thus,
the atherogenic effects of NG2 seem to emerge with
accumulation of s-SMCs [21,57]. She et al. also dem-
onstrated a remarkable role for NG2-positive s-SMCs
in trapping LDL and then presenting it to macro-
phages. It is a novel mechanism for the presentation
of undesirable material to macrophages that, we
speculate, may have evolved to facilitate the
disposal of certain infectious agents or cellular
debris [58,59]. In the context of retained apoB-
lipoproteins, however, the process becomes malad-
aptive. Thus, NG2-positive s-SMCs dramatically
increase the uptake of LDL by macrophages within
the arterial wall, promoting foam-cell formation,
whereas NG2-deficient s-SMCs reduce LDL uptake.
Despite worse obesity and worse hyperlipidemia,
double knockout mice (Ng2 / /Apoe / ) developed
less atherosclerosis than did Apoe / controls.
Surprisingly, the NG2 GAG chains were not required
&&
for the binding of LDL to NG2 [40 ]. Furthermore,
NG2 also bound acetylated LDL, indicating that
NG2 has the capacity to bind LDL through hydro-
phobic interactions. These features make NG2
&&
unique amongst LDL-binding proteoglycans [40 ]
and may be related to its normal biologic functions.
Why does atherosclerosis preferentially develop
at specific sites in the arterial tree? These sites are
often associated with branches and bifurcations,
which expose the endothelium to disturbed laminar
blood flow and shear stress [60]. Most conventional
explanations for increased atherosclerosis suscepti-
bility at these sites have focused on biomechanical
alterations of endothelial cells, such as increased
permeability and elaboration of cytokines [61],
but none of these proposed mechanisms has been
definitely demonstrated. In a recent advance, Stef-
fensen et al. showed that induction of disturbed
laminar flow in the straight segment of the other-
wise atherosclerosis-resistant common carotid
artery of mice provokes matrix proliferation in the
nearby arterial wall, transforming it into a lipopro-
tein-retaining site – but without altering LDL influx.
This provides experimental evidence for a causal
chain linking disturbed laminar flow patterns
to increased arterial matrix deposition, apoB-
&&
lipoprotein retention, and atherosclerosis [62 ].
Like the prior work cited above [20,31], this study
contradicts the notion of a key role for disturbed
endothelial permeability to LDL in early phases of
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atherogenesis. Unraveling the molecular mechan-
isms from disturbed flow to matrix proliferation and
increased lipoprotein retention will be a fruitful area
for future research.
New work has focused on apoB-lipoprotein
retention as a potential therapeutic target. One
strategy is the creation of mAbs that block apoB-
binding sites on GAG chains within the arterial
&
matrix [63,64 ]. Interestingly, the antigen-binding
site (the CDR3 of the heavy chain) of one of
these antibodies contains an arginine motif
that resembles the major GAG-binding sequence
&
in apoB100 [33,36,64 ]. The antibodies decrease
intra-arterial lipoprotein retention and hence the
progression of atherosclerosis in hypercholesterole-
mic Apoe / mice [63,64 ].
&
Cholesterol-rich and triglyceride-rich
apolipoprotein-B-containing remnant
lipoproteins, which are metabolically
distinct from LDL, have emerged as a major
driver of human atherosclerosis
Lipid and lipoprotein concentrations are often
assessed in fasting plasma, but the idea that particles
originating in the postprandial state may contribute
to atherosclerosis appeared in the literature at least
as far back as the 1940s [65,66]. In that era, however,
the overwhelming driver of human atherosclerosis
was LDL, which is not primarily a postprandial
particle.
The situation has changed dramatically since
then. With the rise of overnutrition, obesity, and
the atherometabolic syndrome, harmful choles-
terol-rich and triglyceride-rich apoB-containing
remnant lipoproteins (C-TRLs) have emerged as a
&
major driver of human ASCVD worldwide [67 ]. The
characteristic dyslipoproteinemia of the atherome-
tabolic syndrome increases plasma concentrations
of C-TRL remnants, not LDL. The major defect is
impaired hepatic removal of C-TRLs from plasma
&
[68 ]. Like LDL [9], C-TRL remnants become retained
&
within the arterial wall [6 ,11,33,69]. Mendelian
randomization studies have clearly demonstrated
their causal role in human ASCVD events [70]. Of
note, each C-TRL particle contains up to 40 times
more cholesterol compared with an LDL particle.
Unfortunately, patients treated with optimal statin
&& &&
therapy [71] and even PCSK9 inhibitors [3 ,4 ]
exhibit considerable residual risk for ASCVD events,
which may occur, in large part, because these agents
lower fasting or postprandial C-TRL levels by only
4–25% [72–74]. There is therefore a need to find
new therapeutic approaches to restore normal, rapid
hepatic removal of C-TRLs from plasma, to lower
residual ASCVD risk in obesity, the atherometabolic
syndrome, and type 2 diabetes.
www.co-lipidology.com 477
Atherosclerosis: cell biology and lipoproteins
Aggregation and modifications of retained
apolipoprotein-B-lipoproteins within the
artery wall
Following retention of cholesterol-rich apoB-
lipoproteins within the artery wall, the lipoproteins
have been shown to undergo several modifications
with important biological consequences. For
example, proteoglycan-bound LDL in vitro forms
aggregates that resemble material seen in vivo, and
retained lipoproteins are more susceptible to further
modifications. Key enzymes implicated in apoB-lip-
oprotein retention, aggregation, and atherogenesis
include the secretory sphingomyelinase (S-SMase),
lipoprotein lipase, and the nonpancreatic secretory
group V phospholipase-A2 (PLA2-V) [9,42–45,75]
(Fig. 1). These enzymes, particularly lipoprotein
lipase, bridge between LDL and proteoglycans inde-
pendently of the physical state of apoB and thereby
cause a shift in the retentive mechanism from a
low-affinity process (apoB-GAG binding) in the
pristine arterial wall to a high-affinity process (lip-
oprotein-GAG binding) in an established atheroma.
This shift in retentive mechanism has direct clinical
consequences: a lifetime plasma LDL cholesterol
concentration of 80 mg/dl almost always protects
from atherosclerosis [76] but pre-existing plaques
grow if the LDL-cholesterol is 80 mg/dl [77].
Acidity in deeper parts of normal intima and
within atherosclerotic plaques enhances lipolytic,
proteolytic, and other modifications of apoB-con-
taining lipoproteins that strongly increase their
affinity for proteoglycans and may therefore accel-
erate their retention and the ensuing maladaptive
&
cellular responses in the arterial intima [78 ]. In
a fascinating advance, Sneck et al. elucidated the
mechanism for SMase-induced aggregation of LDL.
The enzyme induces a massive, global confor-
mational change in apoB that exposes hydrophobic
motifs that apparently adhere from one digested
LDL particle to the next [79].
Several lines of evidence implicate aggregation
of apoB-lipoproteins within the arterial wall as a
major accelerator of lipoprotein retention and athe-
rogenesis. Once LDL and other apoB-lipoproteins in
the arterial wall have aggregated, diffusion back into
plasma becomes essentially impossible, due to the
large size of the aggregates and their greatly enhanced
affinity as multivalent ligands for arterial matrix,
as well as conformational changes that expose
additional positively charged domains on apoB. Lipid
hydrolysis and other modifications of the retained,
aggregated lipoproteins release biologically active
byproducts that recruit macrophages and other cells
into the developing lesion, while also blocking their
emigration, thereby accelerating disease progression
(Fig. 1; recently reviewed in [80]).
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Ruuth et al. [46] recently demonstrated that
susceptibility of plasma LDL to aggregation upon
exposure to SMase ex vivo varies considerably
amongst human patients, predicts future deaths
from ASCVD, and can be altered by diet. This work
adds to a growing body of data implicating SMase in
human atherosclerosis [9,75]. Crucially, the work
identifies LDL aggregability in the presence of SMase
as a new, potentially modifiable cardiovascular risk
factor. Aggregation-prone LDL – dubbed ‘unstable’
LDL – exhibits several compositional character-
istics, including an increased sphingomyelin : phos-
phatidylcholine (SM : PC) ratio [46]. Interestingly,
an increase in dietary polyunsaturated fatty acids
and fiber decreased the SM : PC ratio and stabilized
LDL, whereas an increase in dietary sucrose caused
the opposite. Consistent with these findings, a diet
rich in saturated fats has been shown to increase
serum SMase activity and, consequently, increase
LDL aggregation in mice [81]. These results further
emphasize the importance of LDL quality in human
ASCVD. Moreover, measurement of LDL instability
in the presence of SMase may serve as a predictive
biomarker for the identification of patients at unrec-
ognized risk of cardiovascular death who might
benefit from specific, targeted interventions.
Retained lipoproteins induce maladaptive
cellular responses in the arterial wall that
accelerate further apolipoprotein-B-
lipoprotein retention
Retained and aggregated lipoproteins provoke a series
of local biological responses that eventually include a
maladaptive reaction dominated by abnormally per-
sistent macrophages and T cells that accelerate lip-
oprotein retention and other features of plaque
development (Fig. 1). Aggregated apoB-lipoproteins
are avidly taken up by macrophages [9,82] and by
vascular SMCs [83] leading to foam-cell formation.
These processes stimulate the release of proathero-
genic factors that induce the synthesis of proteogly-
cans with enhanced affinity for atherogenic
lipoproteins [9,82]. In addition, monocyte/macro-
phages recruited into atheromata secrete proretentive
enzymes, notably lipoprotein lipase, sphingomyeli-
nase, and PLA2, that accelerate further retention of
atherogenic lipoproteins. In addition, persistent
macrophages in atherosclerotic plaques secrete pro-
teases that weaken the overlying fibrous cap, and
these cells release tissue factor, a potent procoagulant.
These maladaptive responses favor plaque rupture,
robust clot formation that often occludes the arterial
lumen, and downstream ischemia (Fig. 1).
A critical question is why retained apoB-lipopro-
teins induce this abnormal persistence of macro-
phages and T cells? It has been hypothesized that
Volume 27  Number 5  October 2016
 The pathogenic simplicity of atherosclerosis Borén and Williams
specific cellular and molecular programs that cause
immune cells to remain in place may have evolved as
a defense against Mycobacterium tuberculosis, an acid-
fast bacillus that is not killed after phagocytosis and
would therefore be spread throughout the body by
emigrating macrophages. Interestingly, retained and
modified apoB-containing lipoproteins within the
arterial wall elicit many of the same antiemigration
&
signals as M. tuberculosis does [6 ,11,84–86]. Unfortu-
nately, the result is a crippling of the reticuloendo-
thelial system, which otherwise has a huge capacity
that could easily handle a few grams of intramural
cholesterol and other debris.
New data regarding possible roles for HDL
in the response-to-retention model of
atherogenesis
The pathogenic importance in atherosclerosis of
plasma HDL-cholesterol (HDL-C) levels has come
into doubt, due to recent failures of clinical trials
of agents that raise plasma HDL-C concentrations
[87,88] and the lack of ASCVD protection from
human gene polymorphisms that raise plasma con-
centrations of HDL-C [89]. In animal models, how-
ever, injections of HDL, overexpression of apoA-I,
and strategies to enhance HDL function delay
atherosclerosis progression and even promote plaque
stabilization and regression [11,85,88]. In the
context of the response-to-retention hypothesis,
roles for HDL in every step have been hypothesized
[87] – namely, interfering with the irreversible bind-
ing of plasma LDL to arterial wall proteoglycans [90–
92], blocking SMase-induced aggregation of LDL
[79,93], removing toxic lipids, and resolving the
maladaptive inflammatory infiltrate [94–98]. Along
these lines, the apoA-I mimetic peptide 4F was
recently shown to block SMase-induced LDL aggre-
gation and the increase in binding of the modified
&
LDL particles to human aortic proteoglycans [99 ]. In
contrast, abnormal HDL or apoA-I within the arterial
&
wall may have adverse effects [96,100,101 ,102].
Atherogenicity of apolipoprotein-B-
lipoproteins
Based on our present understanding for how
cholesterol-rich apoB-lipoproteins induce athero-
sclerosis, the atherogenicity of apoB-containing
lipoproteins may depend on
(1) Their plasma concentrations, because high
levels of circulating apoB-lipoproteins increase
the probability that these particles will enter
and be retained in the subendothelium.
(2) Their ability to become retained in the artery
wall. This is influenced by their size (<70 nm)
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Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
and their affinity for arterial-wall proteoglycans.
The affinity is modulated by the lipid and
protein composition of the lipoprotein particles.
In particular, apoC-III on LDL increases its
proteoglycan binding.
(3) The instability of retained lipoproteins, that is,
how readily they aggregate in the presence of
arterial-wall enzymes, particularly S-SMase.
This property has been linked to LDL lipid com-
position and is affected by diet.
(4) The susceptibility of aggregated and retained
lipoprotein to further modifications within
the artery wall.
(5) The ability of the modified lipoproteins to
induce a maladaptive cellular response.
Thus, it can be hypothesized that patients with
atherogenic apoB-lipoproteins characterized by high
binding affinity to arterial proteoglycans, increased
susceptibility to aggregate, and features that promote
a hyperresponsive maladaptive cellular infiltrate may
develop atherosclerosis despite only moderately elev-
ated plasma levels of cholesterol. It remains to be
demonstrated whether assessments of these specific
qualities of LDL and C-TRLs will add predictive power
beyond conventional risk factors or help identify
groups of patients for targeted therapies.
Reclassification of risk factors for
atherosclerosis into causative agents,
exacerbators, and mere bystander
phenomena
Our understanding of the pathogenesis of ASCVD is
based on laboratory investigation, human epidemi-
ology, genetic studies, and clinical trials. Collec-
tively, these studies have demonstrated beyond
any doubt that cholesterol-rich apoB-lipoproteins
are causally linked to atherogenesis and that low-
ering the plasma concentration of LDL is the most
effective therapy to date against ASCVD. The critical
step is that cholesterol-rich apoB-lipoproteins from
plasma become pathogenic only after their reten-
tion within the arterial wall.
Using our extensive knowledge of the pathogen-
esis of atherosclerosis, we can now reclassify nearly all
epidemiologic risk factors into causative agents,
exacerbating factors, and bystander phenomena
(Table 1) [80]. Causative and exacerbating factors
are targets for therapy, bystander phenomena are not.
CONCLUSION
It is hard to unravel the root cause of a condition
from an end-stage lesion. Atherosclerosis was no
exception. For more than a century and a half, many
www.co-lipidology.com 479
Atherosclerosis: cell biology and lipoproteins

Table 1. Reclassification of human epidemiologic risk factors for ASCVD events into causative agents, exacerbating factors,
and mere bystander phenomena
Pathophysiologic
Epidemiologic risk factors for ASCVD reclassification Evidence from human RCTs or human MRS

Elevated plasma concentrations of total Causative LDL: proven in RCTs of statins and nonstatins and in MRS; Other
cholesterol, LDL-cholesterol, non-HDLc, apoB-lipoproteins: Proven in MRS
C-TRL remnants, and apoB
Tobacco smoking Exacerbating Lifetime low plasma LDL levels from genetics or unusual lifestyles
protect smokers from ASCVD events
Diabetes mellitus Exacerbating Likewise for diabetes: lifetime low LDL levels protect patients with
diabetes from ASCVD events
Hypertension Exacerbating Likewise for hypertension (but extreme hypertension still causes other
health problems)
Male sex (age) Exacerbating Likewise for elderly men, many of whom no longer develop clinically
significant ASCVD
Elevated plasma glucose levels per se Exacerbating Modest genetic elevations in plasma glucose in MRS detectably
increase ASCVD, but in the context of abundant plasma apoB-
lipoproteins
Low plasma HDLc concentrations Exacerbating or No benefit yet in RCTs of HDLc-raising treatments; no benefit of
bystander higher HDLc in MRS
Elevated plasma homocysteine Bystander Disproven in RCTs to lower plasma homocysteine levels
concentrations
Chlamydia pneumoniae infection; Bystander Disproven in RCTs of antibiotics
periodontal disease (gingivitis)
Elevated plasma C-reactive protein Bystander Disproven in MRS
Oxidized LDL Bystander Disproven in many large RCTs of antioxidant supplements.
Moreover, several components of the heart-healthy lifestyle are
pro-oxidant, such as exercise, dietary PUFAs, and moderate
alcohol consumption

ASCVD, atherosclerotic cardiovascular disease; C-TRL, cholesterol-rich and triglyceride-rich apoB-containing remnant lipoprotein; HDLc, HDL-cholesterol; MRS,
Mendelian randomization studies; RCTs, randomized controlled trials.

theories had been articulated to explain how a nor- (J.B. and K.J.W.), ALF-medel Västra Götalandsregionen
mal artery becomes atherosclerotic. As a testament (J.B. and K.J.W.), and the Ruth and Yonatan Ben-Avra-
to the importance of this area, most of these theories ham Fund (K.J.W.).
were tested in clinical trials. As a consequence,
it is now established that increased plasma Conflicts of interest
concentrations of cholesterol-rich apoB-containing Dr Williams reports purchasing stock in Gemphire
lipoproteins are causatively linked to ASCVD. Therapeutics, Inc., a company focused on the treatment
Accordingly, the most effective therapies against of dyslipidemias.
atherothrombotic cardiovascular disease – LDL-
lowering drugs – are based upon the principle that
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