1. ATHEROSCLEROSIS
Atherosclerotic diseases, particularly coronary artery disease and stroke, are
leading causes of death (Mozaffarian et al., 2015). Atherosclerosis is a chronic
process that progresses in a clinically silent manner for decades. The devas-
tating consequence of lesion evolution is the abrupt formation of thrombi,
due to lesion rupture or erosion that occludes blood flow, with the attendant
overt clinical symptoms (Bentzon et al., 2014) (Figure 1). The slow progres-
sion of human atherosclerosis, combined with tissue inaccessibility and the
absence of effective imaging techniques, provides challenges for characteri-
sation of lesion evolution. Hence, the understanding of mechanisms underly-
ing the evolution of human atherosclerotic lesion progression is incomplete.
FIGURE 1 A section of a human atherosclerotic coronary artery. The lesion contains a large
lipid-rich core, containing cholesterol crystals, derived from apoptotic and/or necrotic foam cells.
A thin fibrous cap, consisting primarily of collagen underlies a compromised endothelial cell layer.
Smooth muscle cells have migrated from the media into the intima where they proliferate and can
become lipid-rich foam cells. Macrophage-derived and smooth muscle cell-derived foam cells
populate the edges of the growing lesion and the fibrous cap. The lesion reduces the artery lumen
diameter. A lesion with a thin fibrous cap covering a large lipid core is prone to rupture and rapid
thrombus formation resulting in an acute coronary event.
As a result, there has been a dependence on information gleaned from ani-
mal models of the disease (Getz and Reardon, 2012). One feature common
to all animal models is the need to induce hypercholesterolaemia for lesion
development, which is achieved by dietary and/or genetic manipulations. The
mechanisms described in this chapter are derived predominantly from stud-
ies in apolipoprotein E (ApoE−/−) or low-density lipoprotein (LDL) receptor
(Ldlr−/−)-deficient mice. While many factors influence the initiation, severity
and nature of lesions in hypercholesterolaemic animals, the basic tenet of this
chapter is that aberrant lipoprotein metabolism is a requirement for athero-
sclerosis development.
Atherosclerosis develops in specific regions of the arterial tree that are
characterised by turbulent blood flow. The initial morphological change is the
subendothelial (intimal) deposition of aggregated lipoproteins (Tabas et al.,
2007). These aggregates promote chemotaxis of leukocytes through several
mechanisms, including oxidation, and enzymatic and nonenzymatic prote-
olysis of apolipoproteins. In addition, monocyte/macrophage adhesion to the
arterial endothelium occurs in the initial stages of experimental atherosclero-
sis, followed by progressive subendothelial accumulation of macrophages in
both experimental and human lesions (Moore and Tabas, 2011). In addition to
recruiting more macrophages, these cells avidly take up native and modified
ipoproteins resulting in lipid droplets filled predominantly with cholesteryl
esters (CE), a process known as ‘foam cell’ formation. Macrophage uptake and
clearance of subendothelial lipoproteins are likely beneficial during the initial
stages of atherosclerosis.
The resulting dysregulation of lipid metabolism alters macrophage pheno-
type and compromises crucial functions of the cell (Moore et al., 2013). Foam
cells that accumulate in lesions have a decreased ability to migrate from the
lesion, which contributes to chronic inflammation and lesion progression. More
complex plaques involve other immune cells and vascular smooth muscle cells
(VSMCs). In advanced lesions, macrophages continue as major contributors to
lesion growth and the inflammatory response through secretion of proinflam-
matory mediators and reactive oxygen and nitrogen species, as well as matrix-
degrading proteases. These processes lead to eventual cell death by either
necrosis or apoptosis. Dead macrophages release their lipid contents and tissue
factors, resulting in the formation of a prothrombotic necrotic core, which is a
critical component of unstable plaques. Instability contributes to the rupture of a
thin fibrous cap that covers the plaque and leads to subsequent thrombus forma-
tion that underlies an acute myocardial infarction or stroke (Figure 1).
Although many cell types, including monocytes, dendritic cells, lympho-
cytes, eosinophils, mast cells, endothelial cells and smooth muscle cells, contrib-
ute to lesion formation and growth, foam cells are central to the pathophysiology
of atherosclerosis. Therefore, this chapter will focus on the relationship of
altered lipid and lipoprotein metabolism to the mechanisms underlying mono-
cyte recruitment, macrophage foam cell formation, the inflammatory response,
cell death, macrophage emigration and the resolution of inflammation (Moore
et al., 2013). Initially, we will review critical components and pathways in lipid
metabolism that contribute to atherosclerosis, followed by a review of emerging
mechanisms underlying the influence of lipid metabolism in atherosclerosis at
various stages of lesion development or regression. We also review the current
development of therapeutic strategies for treatment of atherosclerotic diseases.
2. LIPOPROTEIN TRANSPORT IN ATHEROSCLEROSIS
2.1 Low-Density Lipoprotein
The most atherogenic dyslipidaemia is hypercholesterolaemia, especially
familial hypercholesterolaemia (FH) (Sahebkar and Watts, 2013a,b). FH is
characterised by greatly increased plasma concentrations of LDL cholesterol,
primarily due to downregulation, deficiency or dysfunction of LDL receptors
(Chapter 17). Pathogenic mutations causing FH frequently occur in the LDL
receptor gene and less frequently in apolipoprotein B (apoB) and proprotein
convertase subtilisin/kexin type 9 (PCSK9) genes. As a consequence, plasma
LDL cholesterol concentrations increase, due to an inability of hepatic LDL
receptors to clear LDL particles. Overproduction of LDL also contributes to
elevated LDL. LDL penetrates the subendothelium and accumulates within the subendothelium and accumulates within
IGURE 2 A schematic of exogenous and endogenous lipoproteins involved in atherogen-
esis. Elevated low-density lipoproteins (LDL) promote atherosclerosis as typified by patients with
familial hypercholesterolaemia. Very-low-density lipoproteins (VLDL), their remnants (IDL) and
chylomicron remnants also amplify atherogenesis. Lp(a) readily enters the arterial intima and pro-
motes atherosclerosis. Low levels of HDL or dysfunctional HDL are unable to promote reverse
cholesterol transport from lesion macrophages, contributing to the formation of foam cells.
the intima of susceptible arteries (Figure 2). LDL then aggregates and/or is
readily oxidised, resulting in an inflammatory response as well as foam cell
formation in both macrophages and smooth muscle cells. This process leads
to premature atherosclerosis in patients with hypercholesterolaemia, particu-
larly those with FH.
2.2 Very-Low-Density Lipoprotein
Another dyslipidaemia that predisposes to premature atherosclerosis is famil-
ial combined hyperlipidaemia (FCH) (Sahebkar and Watts, 2013a). FCH is the
most common form of inherited dyslipidaemia and is genetically heteroge-
neous, resulting from variations in multiple genes involved in the metabolism
and clearance of plasma lipoproteins (Chapter 15). Combined hyperlipidaemia
is often accompanied by additional metabolic abnormalities, including adipose
tissue dysfunction, increased fatty acid flux to the liver and insulin resistance
(Chapter 19). The metabolic phenotype of this dyslipidaemia is hepatic overpro-
duction of very-low-density lipoprotein (VLDL), elevated plasma VLDL tria-
cylglycerol (TAG), apoB and small dense LDL (Figure 2). These lipoproteins
also readily accumulate within the arterial intima, eliciting inflammatory and
other cellular responses linked to atherogenesis.
Hypertriglyceridaemia is a dyslipidaemia characterised by increased plasma
VLDL TAG concentrations and in the more severe form, elevated chylomicrons
and chylomicron remnants (Hegele et al., 2014) (Chapter 16). Hypertriglyceri-
daemia is associated with increased risk of coronary heart disease. Heritability
accounts for more than 50% of the individual variations in plasma TAG concen-
trations. Genome-wide association studies identified gene variants, including
APOC3, LPL, TRIB1, GCKR and APOA5 that modulate plasma TAG and the
risk for atherosclerosis. Hypertriglyceridaemia results from hepatic overpro-
duction and/or decreased catabolism of TAG-rich lipoproteins, accumulation
of small dense LDL particles and low concentrations of high-density lipopro-
tein (HDL) (Figure 2). This dyslipidaemia is often linked to obesity, metabolic
syndrome and type 2 diabetes (Chapter 19). Although small dense LDL par-
ticles and dysfunctional HDL contribute, atherosclerosis associated with this
dyslipidaemia is primarily due to retention of TAG-rich lipoprotein remnants in
the artery wall. Remnants are retained by intimal extracellular matrix proteins,
and can be oxidised and readily induce foam cell formation. Fatty acids from
these particles readily elicit an inflammatory response and promote lipotoxicity
within cells involved in atherosclerosis.
2.3 Remnants of VLDL and Chylomicrons
The most compelling evidence that remnants of TAG-rich lipoproteins are ath-
erogenic comes from patients with dysbetalipoproteinaemia who have a very
high incidence of premature atherosclerosis (Brahm and Hegele, 2015). In this
rare disorder, remnants of VLDL and chylomicrons accumulate in plasma due
to a genetic variant of APOE (APOE2) bound to the surface of these particles
(Chapter 15). Normally, apoE is the ligand responsible for the clearance of chy-
lomicron remnants and VLDL remnants by hepatic LDL receptors; however,
apoE2 binds poorly to these receptors, resulting in delayed remnant clearance.
When coupled to an overproduction of intestinal or hepatic TAGs (Chapter 16),
remnants attain high plasma concentrations, thereby increasing their exposure
to the arterial intima (Figure 2).
2.4 Lipoprotein(a)
Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are strongly associ-
ated with increased risk of atherosclerosis, although the definitive function of
this lipoprotein is still unclear (Koschinsky and Boffa, 2014) (Figure 2). Lp(a)
is composed of LDL with an additional single molecule of apo(a) that is bound
covalently to apoB-100 (Chapter 16). Plasma Lp(a) concentrations are not mod-
ulated by diet, age, sex or physical activity, but are largely determined by varia-
tion at the LPA gene locus, making elevated Lp(a) highly heritable.
The apo(a) gene contains a variable number of tandem repeats that encode
a kringle moiety identical to kringle IV of plasminogen. This leads to Lp(a)
isoforms that differ in molecular mass. There is a strong and inverse association
between plasma Lp(a) concentration and molecular mass of apo(a). Smaller apo(a)
isoforms possess greater atherogenic potential. Similar to LDL, Lp(a) is retained
within the arterial intima through extracellular matrix interactions, and induces
chemokine secretion, inflammation and foam cell formation. Plasma Lp(a) carries
oxidised phospholipids, and its atherogenicity may be associated with delivery
of these proinflammatory lipids to lesions. Lp(a) can also induce apoptosis in
endoplasmic reticulum (ER)-stressed macrophages, thereby contributing to lesion
vulnerability and thrombosis. In addition, apo(a) homology to plasminogen may
inhibit the fibrinolytic pathway, making Lp(a) a thrombogenic lipoprotein.
2.5 High-Density Lipoprotein
HDL cholesterol concentrations are inversely related to the risk of cardiovas-
cular disease and death (Luscher et al., 2014). However, there is increasing
awareness that HDL function plays a more important role in protection from
atherosclerotic disease, a feature not captured in measures of plasma HDL con-
centration. Many dyslipidaemias are associated with low plasma concentrations
and/or dysfunctional HDL. Causes of low HDL cholesterol are not well under-
stood, but involve enhanced catabolism of the cholesteryl ester-depleted HDL
particle (Chapter 15).
HDL possesses anti-inflammatory and antioxidant properties, but its prin-
cipal antiatherogenic function is due to its ability to mediate cholesterol export
from cells involved in atherosclerosis and subsequent elimination of HDL
cholesterol through reverse cholesterol transport (Chapter 15) (Figure 2). In
experimental models of atherosclerosis, promotion of macrophage cholesterol
export attenuates progression or induces lesion regression. Other functions of
HDL impact atherogenesis. HDL-associated paraoxanase-1 (PON-1) normally
prevents HDL from oxidative modification. Reduced levels of HDL-associated
PON-1 activity lead to the generation of modified HDL that inhibits endothelial
cell nitric oxide synthase (eNOS) activation, thereby losing its anti-inflammatory
properties. These HDL are also defective in cholesterol export.
3. LIPOPROTEIN RECEPTORS AND LIPID TRANSPORTERS
Lipoproteins are complex macromolecules that are recognised by a variety of
receptors (Chapter 17) and function to facilitate physiological and pathological
processes. There is direct in vivo evidence for a role of lipoprotein receptors in
the development of atherosclerosis as summarised in Table 1.
3.1 LDL Receptors
LDL receptors were defined by the classic studies of Goldstein and Brown
in which they described a process for transporting large lipoprotein particles nsporting large lipoprotein particles
(∼24nm) across the cell membrane. The first step in this process is the interac-
tion of the apoB of LDL with the cysteine-rich receptor-binding domain of LDL
receptors. Engagement of apoB with LDL receptors results in endocytosis of the
entire LDL particle with subsequent delivery to the lysosome where the particle
is degraded to its components. Delivery of free (unesterified) cholesterol to the
ER induces homeostatic mechanisms that inhibit continuous exogenous delivery
or endogenous cholesterol synthesis (Chapters 17 and 11). Deficiency of LDL
receptors increases plasma cholesterol and accelerates atherosclerosis. Humans
with homozygous FH develop severe atherosclerotic disease within two decades
of life if untreated. Deficiency of LDL receptors in rabbits also leads to pro-
nounced hypercholesterolaemia and accelerated atherosclerosis. Genetic deple-
tion of LDL receptors in mice leads to a modest hypercholesterolaemia, which is
greatly augmented by feeding diets that are enriched in saturated fat and/or cho-
lesterol. LDL receptors can also be negatively regulated by PCSK9. Expression
of gain-of-function PCSK9 mutants leads to depletion of LDL receptors, hyper-
cholesterolaemia and atherosclerosis (Dadu and Ballantyne, 2014).
3.2 Scavenger Receptors
LDL receptors are downregulated by increased cellular cholesterol to pre-
vent sterol engorgement, which is a characteristic of cells in atherosclerotic
lesions. Therefore, it is unlikely that LDL promotes atherosclerosis through
this receptor. As an alternative, it was proposed that in hypercholesterolaemic
states, an increased residence time of LDL particles in the subendothelial
space leads to modifications, resulting in enhancement of macrophage uptake
that is not regulated by cellular sterol content. Acetylation of LDL was the
first modification identified that enhanced uptake in macrophages. Binding of
acetylated LDL to macrophages is saturable, consistent with a receptor-mediated
interaction. These recognitions led to the concept that hypercholesterolae-
mia promotes atherosclerosis through modified LDL uptake by ‘scavenger’
receptors (Chapter 17).
The subsequent effort to identify scavenger receptors led to the cloning of
a trimeric membrane glycoprotein that was expressed as two major splice vari-
ants. This receptor is now referred to as scavenger receptor class A or SR-A
(Canton et al., 2013). Although expression of the cloned protein demonstrated
the ability of acetylated LDL to bind to SR-A in cultured cells, studies in which
SR-A was genetically manipulated in atherosclerosis-susceptible mice demon-
strated a full spectrum of effects on lesion size from being increased, unaffected
or decreased (Canton et al., 2013) (Table 1). SR-A is now recognised as the
receptor for a wide array of ligands that influence atherosclerosis, and is also an
integrin-independent adhesion molecule (Chapter 17). Therefore, it is unclear
whether the multiple ligands of SR-A contribute to the variable effects observed
on atherosclerotic lesion formation.
There are now a large number of other receptors that are collectively classed
as scavenger receptors. Most of these receptors bind to modified LDL, in which
acetylation or oxidation is the most common modification. In addition to SR-A,
the major members of this class that have been studied for effects on experimen-
tal atherosclerosis are CD36, SR-B1 and LOX1 (Canton et al., 2013) (Chapter
17). CD36 was originally described as a fatty acid transporter and was subse-
quently identified as a receptor for copper-oxidised LDL. It is highly expressed
in cultured macrophages and atherosclerotic lesions. Like SR-A, the data on
the role of CD36 in atherosclerosis in either ApoE−/− or Ldlr−/− mice have been
inconsistent (Table 1).
SR-B1 was identified as a structural variant of CD36 with similar binding
characteristics for acetylated LDL. While many of the same ligands bind both
CD36 and SR-B1, LDL only binds to SR-B1. It was subsequently demonstrated
that SR-B1 is an HDL receptor that facilitates bidirectional movement of cho-
lesterol between cells and HDL particles (Canton et al., 2013) (Chapter 15).
Mice with deficiencies of both SR-B1 and apoE exhibit an array of cardiovas-
cular pathologies at an early age, including accelerated atherosclerosis. Lesions
in these mice are present in coronary arteries, which is uncommon in most small
animal models of atherosclerosis. Overexpression of SR-B1 in Ldlr−/− mice
decreases atherosclerosis with concomitant decreases in plasma HDL choles-
terol concentrations. Deleting SR-B1 increases atherosclerosis in Ldlr−/− mice
with increases in plasma cholesterol concentrations and very large HDL par-
ticles (Table 1). The effects of SR-B1 on plasma lipoprotein concentrations and
characteristics do not appear to affect atherosclerosis since deletion of SR-B1
in bone marrow-derived cells augments lesion size in mice, without discernible
effects on plasma cholesterol.
Although macrophages have been the major focus of expression for most
scavenger receptors, endothelial dysfunction is also crucial in atherosclerotic
lesions. A screen of an endothelial cell cDNA library identified a protein des-
ignated as lectin-like oxidised LDL (oxLDL) receptor or LOX-1 that binds
oxLDL (Chapter 17). LOX-1 is also expressed in macrophages and smooth
muscle cells of atherosclerotic lesions. LOX1 deficiency reduces atherosclero-
sis, whereas its overexpression in endothelial cells increases atherosclerosis in
hypercholesterolaemic mice, suggesting that this protein contributes to lesion
development (Table 1).
3.3 Low-Density Lipoprotein Receptor-Related Protein
LDL receptor-related protein (LRP1), a multifunctional protein in the LDL recep-
tor superfamily, is present on the surface of multiple cell types (Chapter 17). Since
its discovery by Herz and colleagues in 1988, more than 40 ligands have been
identified to interact with LRP1, implicating complex functions of this protein
(Chapter 17). This large protein contains a 515-kDa α chain (amino-terminal part)
and an 85-kDa β chain (membrane-bound) carboxyl terminal fragment. Deficiency
of LRP1 in macrophages reduces VLDL uptake, and enhances the secretion of
inflammatory mediators including monocyte chemoattractant protein-1 (MCP-1,
CCL2), interleukin (IL)-1, IL-6 and TNFα. Deficiency of LRP1 in macrophages
does not affect plasma cholesterol concentrations or lipoprotein–cholesterol
profiles, but augments atherosclerosis in hypercholesterolaemic mice (Table 1).
In addition to macrophages, LRP1 is abundant in VSMCs. Deficiency of LRP1
in VSMCs augments atherosclerosis in hypercholesterolaemic mice (Gonias and
Campana, 2014).
3.4 ATP-Binding Cassette Subfamily (ABCs)
Cells normally respond to excessive lipid accumulation by upregulation of path-
ways that promote export of cholesterol and other lipids (Chapter 15). Although
passive diffusion of cholesterol from the plasma membrane occurs, macrophage
foam cells possess several transporters that export lipids, including members
of the ATP-binding cassette subfamily (ABCs) ABCA1 and ABCG1. ABCA1
facilitates cholesterol export to lipid-poor apoA-I within pre-β-HDL, whereas
ABCG1 facilitates export to mature HDL particles (Figure 2). Macrophage
expression of ABCA1 and ABCG1 is upregulated in response to increased cel-
lular cholesterol concentrations by liver X receptors (LXR). This ligand-acti-
vated nuclear receptor acts as a sterol sensor and is transcriptionally activated by
metabolites of lipoprotein-derived cholesterol, including the oxysterols 27-OH-
cholesterol, 24-OH-cholesterol, 7-keto-cholesterol and desmosterol, a choles-
terol precursor. All of these metabolites accumulate in macrophage foam cells.
Activation of LXR also inhibits inflammatory responses. In experimental mod-
els, strategies that increase activation or expression of LXR or ABC transport-
ers attenuate atherosclerosis, whereas inhibition of their expression promotes
atherosclerosis.
4. CONTRIBUTIONS OF LIPOPROTEIN-MEDIATED
INFLAMMATION TO ATHEROSCLEROSIS
Multiple cell types are involved in the development of atherosclerosis. One
well-recognised mechanistic theory is that endothelial dysfunction of the arte-
rial wall is the initial step in provoking monocyte adhesion, followed by mac-
rophage infiltration into the subendothelial space (intima) to form lipid-laden
foam cells. Resident cells of the arterial wall together with leukocytes recruited
from peripheral blood orchestrate a series of inflammatory responses in the arte-
rial wall, thereby promoting the progression of atherosclerosis. The ‘response-
to-retention’ hypothesis (Tabas et al., 2007) proposes that lipoproteins normally
diffuse across the endothelial barrier, become trapped in the subendothelium,
stimulate inflammatory responses and promote accumulation of lipid-laden
macrophages. It is generally accepted that accumulated lipids within macro-
phages are derived from plasma lipoproteins through uptake by endocytic,
phagocytic, receptor-involved or nonreceptor-mediated processes. This section
updates the current knowledge regarding lipid and lipoprotein-stimulated cellu-
lar changes and cell-specific manipulations of components that have been stud-
ied in atherosclerotic animal models.
4.1 Cell Types Involved in Atherosclerotic Lesions
Associations between inflammation and atherosclerosis are complex, they
involve multiple cell types and a network of signalling pathways. A subtle
change in this complex system may lead to substantial changes in a spectrum
of signalling pathways. Hypercholesterolaemia induces leukocytosis (Murphy
et al., 2014), thereby increasing their likelihood of recruitment into the suben-
dothelial space. Modified lipoproteins can activate endothelial cells to secrete
adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1),
intracellular adhesion molecule 1 (ICAM-1) and selectins. These molecules, together with chemoattractant mediators including complement factors and
MCP-1, promote monocyte adhesion to the impaired endothelial barrier and
emigration into the intima. Monocytes become activated to macrophages, which
take up native and modified lipoproteins to form lipid-laden foam cells. Among
all leukocyte types, macrophages are the predominant cell type in atheroscle-
rosis and are major contributors to the development of lesions. Therefore, the
contributions of this cell type to atherosclerosis will be reviewed in detail in
Section 4.2. Lipoproteins accumulating in the intimal space (intima) and their
uptake by macrophages contribute to the secretion of multiple proatherogenic
cytokines such as IL-1, IL-6 and TNFα (Figure 3). FIGURE 3 Pathways for monocyte recruitment and macrophage retention, foam cell forma-
tion, apoptosis, lipid export and emigration in plaques. GR1+LY6Chi monocytes are recruited
to mouse atherosclerotic lesions using chemokine–chemokine receptor pairs (CCR2:CCL2 and
CCR5:CCL5) to infiltrate the intima, which is facilitated by endothelial adhesion molecules,
including P-selectin and P-selectin glycoprotein ligand 1 (PSGL1), intercellular adhesion molecule
1 (ICAM1) and vascular adhesion molecule 1 (VCAM1). Recruited monocytes differentiate into
macrophages in the intima, where they interact with atherogenic lipoproteins (see text) resulting
in foam cell formation. Foam cells secrete proinflammatory cytokines, chemokines and retention
factors that induce macrophage proliferation, promote foam cell accumulation within lesions and
amplify the inflammatory response. The inflammatory milieu and excess lipid accumulation induce
macrophage ER stress and apoptosis. Cell death, together with defective efferocytosis, results in
the formation of a necrotic core, which is characteristic of advanced lesions. The promotion of
lipid unloading by ABCA1 and cholesterol export to lipid-poor apoA-I reverses cellular lipid accu-
mulation and induces plaque regression. Events that stimulate efferocytosis lead to reverse trans-
migration to the lumen and macrophage emigration to adventitial lymphatics. LFA1, lymphocyte
function-associated antigen 1; VLA4, very late antigen 4. Adapted from Moore et al. (2013). In addition to macrophages, T lymphocytes are present in atherosclerotic
lesions and, like macrophages, accumulate in early lesions (Libby, 2012). LDL
stimulates T lymphocytes to produce interferon-γ (IFN-γ), which contributes
to further recruitment of T lymphocytes. In contrast to T lymphocytes, athero-
sclerotic lesions contain minimal B lymphocytes. oxLDL stimulates antibody
production from B lymphocytes that accumulate in atherosclerotic lesions. It
is generally accepted that T lymphocytes augment atherosclerosis, whereas
antibodies produced by a subclass of B lymphocytes, termed B1 cells, protect
against formation and development of atherosclerosis.
Neutrophils are the most numerous leukocyte type in blood. This cell type
is present in blood for only 1–2days until activated, which promotes migration
into tissues. In the innate immune system, neutrophils are the first line in acute
inflammatory responses. The role of neutrophils in atherosclerosis has not been
appreciated until recently. Neutrophils become activated and are recruited to
sites of atherosclerotic lesions, but the mechanisms by which neutrophils con-
tribute to atherosclerosis are unclear (Weber and Noels, 2011).
VSMCs are the predominant cell type in the normal arterial medial layer.
Vascular inflammation leads to VSMC proliferation, and their migration into
atherosclerotic lesions. Recent studies revealed that progenitor cells present in
the adventitia of the arterial wall have the capability to become VSMCs and
contribute to the development of atherosclerosis. VSMCs secrete extracellular
matrix that stabilises lesions through the formation of a fibrous cap. VSMCs
also have the capacity to transform into foam cells by accumulating lipoprotein-
derived lipids; however, VSMC foam cells lose their capacity to secrete extra-
cellular matrix. Lipoproteins, such as oxLDL, induce the proliferation of
smooth muscle cells. In human coronary lesions, recent studies found that over
50% of foam cells were derived from smooth muscle cells, and these cells
expressed the macrophage marker CD68 (Allahverdian et al., 2014). These
discoveries have made research on vascular smooth muscle-related mecha-
nisms of atherosclerosis more challenging (Tellides and Pober, 2015; Zhang
and Xu, 2014).
4.2 Foam Cells
4.2.1 Recruitment of Circulating Monocytes
Activation of endothelium promotes recruitment of circulating monocytes
(Moore et al., 2013) (Figure 3). Of significance, hypercholesterolaemia is
associated with increased numbers of circulating monocytes. Their precursors,
hematopoietic stem and progenitor cells, become enriched in cholesterol and
deficient in cholesterol export, leading to cell proliferation. Circulating mono-
cytes in mice consist of at least two major subsets, Ly6Chi and Ly6Clow mono-
cytes. Ly6Chi cells constitute the majority of cells recruited to the intima, and
represent precursors of M1 classically activated macrophages and thus are key
participants in foam cell formation and inflammatory responses Recruitment of circulating monocytes into atherosclerotic lesions requires
integration of at least three discrete processes. These are capture, rolling and
transmigration and each step is regulated by several molecular factors (Figure 3).
Molecules on the surface of activated endothelial cells mediate capture and
rolling of monocytes. This depends on their immobilisation by endothelial cell
chemokines, P-selectin, VCAM1 and ICAM1, which interact with chemokine
receptors and integrins on monocytes. Extravasation of monocytes across the
endothelium and into plaques is mediated by the interaction of monocyte che-
mokine receptors with the chemokines PECAM1 or VCAM1 that are secreted by
endothelial cells, macrophages and smooth muscle cells. In addition, neuronal
guidance cues expressed by endothelial cells, including ephrin B2, netrin 1 and
semaphorin 3A, can increase monocyte recruitment or inhibit the chemokine-
directed monocyte migration (Figure 3).
4.2.2 Foam Cell Formation
Macrophage foam cell formation represents one of the earliest stages of lesion
development, and continues throughout lesion evolution (Moore and Tabas,
2011) (Figure 4). Although macrophages can take up apoB-containing lipopro-
teins through the LDL receptor, this receptor is downregulated during foam
cell formation by increased cellular cholesterol. Modified lipoprotein uptake by
scavenger receptors, which is not regulated by intracellular cholesterol, contrib-
utes to foam cell formation. Multiple means of lipoprotein modification have
been described; however, the relevant pathways that promote foam cell forma-
tion in vivo remain to be fully elucidated.
Oxidative stress in the arterial intima promotes lipoprotein modification that
generates ‘damage’ signals that are recognised by scavenger receptors on macro-
phages and other cells of the innate immune system. oxLDL has been identified
in both human and mouse lesions and antibodies that recognise oxidation-
specific epitopes of LDL have also been discovered. Mediators of lipopro-
tein oxidation, including 12/15-lipoxygenase, myeloperoxidase, free radicals
including superoxide, hydrogen peroxide and nitric oxide, have been identified
in the artery wall. Lipoproteins modified through these mechanisms are readily
endocytosed by scavenger receptors in vitro (Figure 4). Scavenger receptors,
including SR-A1 (encoded by MSR1), macrophage receptor with collagenous
structure (MARCO; also known as SR-A2), CD36 (also known as platelet gly-
coprotein 4), SR-B1, LOX1, scavenger receptor expressed by endothelial cells
1 (SREC1) and scavenger receptor for phosphatidylserine and oxidised LDL
(SR-PSOX; also known as CXCL16) all can bind oxidised lipoproteins and pro-
mote unimpeded foam cell formation (Section 3.2). SR-A1 and CD36 mediate
the majority of uptake of oxidised lipoproteins by macrophages in vitro. These
receptors internalise lipoproteins into late endosomes and lysosomes, where
lysosomal acid lipase mediates the hydrolysis of lipoprotein CE to cholesterol
and fatty acids (Figure 4). Endolysosomal cholesterol is trafficked to the ER
or plasma membrane by NPC1 and NPC2. In the ER, cholesterol undergoes. FIGURE 4 Mechanisms controlling macrophage lipoprotein uptake, lipid export and intra-
cellular cholesterol trafficking. Macrophages internalise native LDL, VLDL and oxidised lipo-
proteins within the lesion by scavenger receptors outlined in Table 1. Internalised lipoproteins and
their lipids are digested in the lysosome. Cholesterol is transferred to the plasma membrane for
export from the cell or to the endoplasmic reticulum for esterified by acyl-coenzyme A:cholesterol
acyltransferase 1 (ACAT1) and ultimately stored as CE in cytosolic lipid droplets. Stored lipids
are released for export by either neutral cholesteryl ester hydrolase 1 (NCEH1)-mediated lipolysis
or via lipophagy (not shown). Cellular cholesterol accumulation increases oxysterol synthesis and
activation of LXR–retinoid X receptor (RXR) transcription factor complex that upregulates expres-
sion of ABCA1 and ABCG1. These transporters transfer cholesterol to lipid-poor apoA-I to form
nascent HDL or to lipidate mature HDL particles. Exported cholesterol is esterified by lecithin
cholesterol acyltransferase (LCAT). Excessive cellular cholesterol can interfere with the function of
the ER as well as induce cholesterol crystal formation in the lysosome, resulting in activation of the
inflammasome. If prolonged, this results in cell death by apoptosis. Increased cholesterol content of
membrane-associated lipid rafts increases proinflammatory Toll-like receptor 4 (TLR4) signalling
that activates nuclear factor-κB (NF-κB), resulting in the production of proinflammatory cytokines
and chemokines. Adapted from Moore et al. (2013). reesterification by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) to CE that
are stored within neutral lipid droplets that appear ‘foamy’ under the micro-
scope (Figure 4).
Although ApoE−/− mice deficient in both SR-A1 and CD36 have reduced
lesion inflammation, macrophage apoptosis and plaque necrosis, loss of these
receptors did not reduce foam cell formation or lesion size (Moore et al., 2005).
Together with the results of human clinical trials of antioxidant vitamins E and
C that failed to show a reduction of cardiovascular events, these studies have
prompted the field to consider alternative mechanisms for foam cell formation. Proteases and lipases within the arterial intima also mediate lipoprotein modi-
fications, particularly LDL aggregation (Moore et al., 2013). Extracellular matrix
glycoproteins within the intima contribute to this process by retaining apoB-con-
taining lipoproteins and by modulating the activity of enzymes, including group
IIA secretory phospholipase A2 (PLA2G2A), PLA2G5 and PLA2G10, as well as
secretory sphingomyelinase. These lipolytic enzymes produce modified forms of
LDL that are taken up by macrophages independent of scavenger receptors. Evi-
dence from mouse studies supports a function for PLA2 in atherosclerosis progres-
sion, and circulating PLA2 levels in humans correlate with risk for atherosclerosis.
Although foam cell formation by native apoB-containing lipoproteins was not orig-
inally considered a major pathway, recent studies have documented macrophage
uptake of LDL by pinocytosis in vitro and in the arterial intima leading to foam cell
formation (Figure 4). This receptor-independent endocytic pathway also delivers
cholesterol to the endolysosomal compartment and stimulates ACAT1-mediated
cholesterol esterification. Thus, in vivo, it is likely that atherogenic LDL and other
apoB-containing lipoproteins induce foam cell formation by multiple pathways.
Excessive uptake of lipoproteins eventually results in defective lipid metab-
olism within macrophages, ultimately leading to increased lesion complexity.
When retained within lipid droplets, CE is metabolically inert, whereas cho-
lesterol within cell membranes can be toxic. Enrichment of macrophage ER
membranes with cholesterol, which occurs with excessive lipoprotein uptake,
eventually suppresses its esterification by ACAT1, resulting in further cholesterol
accumulation. Furthermore, plasma membranes become cholesterol enriched,
leading to amplified inflammatory signalling through Toll-like receptors (TLR;
see Section 4.6) (Figure 4). In addition, trafficking of cholesterol from lyso-
somes becomes compromised, which inhibits macrophage cholesterol export
and further enhances the inflammatory response. This dysregulated lipid metab-
olism contributes to macrophage ER stress, and if prolonged, can ultimately
lead to apoptosis and cell death (Figure 4). Efficient clearance of apoptotic cells
from lesions by surrounding macrophages (known as efferocytosis) requires the
engulfing cells to metabolise lipids derived from the apoptotic cells (Figure 3).
Therefore, excess lipoprotein uptake by macrophages compromises cellular lipid
metabolism, promotes apoptosis and suppresses efferocytosis, which leads to
secondary necrosis and a necrotic core (Figure 4). A necrotic core, together with
a thinned fibrous cap, is characteristic of plaques more vulnerable to rupture.
4.3 Macrophage Polarisation
On entry into the arterial intima, monocytes are polarised to M1 macrophages,
known as classically activated inflammatory cells, or M2 macrophages, known
as alternately activated cells that resolve inflammation (Moore et al., 2013). It
is thought that M1 macrophages are derived from Ly6Chi monocytes, whereas
M2 macrophages are from Lys6Clow monocyte precursors. In vivo, it is likely
that macrophage phenotype is more complex. Furthermore, the factors in lesion
microenvironments that promote macrophage polarisation remain incompletely
defined. M1 macrophages represent foam cell precursors and in human plaques
they are enriched in lipids and localised to areas distinct from the less inflam-
matory M2 macrophages. In mouse models, M1 macrophages become predomi-
nant in complex lesions, whereas enrichment of M2 macrophages occurs in
lesions in which regression of atherosclerosis has been induced. Administration
of the M2-polarising cytokine IL-13 to Ldlr−/− mice drives lesion macrophages
to M2-like cells and inhibits lesion progression. Recent evidence indicates that
oxLDL induces a distinct macrophage phenotype that has been termed Mox,
characterised by the increased expression of NRF2, although the function of
these cells in atherosclerosis has not been defined.
4.4 Inflammatory Responses
Innate immune activation is considered a central feature of the pathogenesis of
atherosclerosis and is largely a consequence of dysregulated lipid metabolism
within developing lesions (Moore et al., 2013). Lipids, oxidised lipids and other
ligands that accumulate within lesions trigger macrophage receptors includ-
ing scavenger receptors, TLRs and nucleotide-binding oligomerisation domain
(NOD)-like receptors that in turn activate inflammatory responses (Figure 4).
Cholesterol crystals are detected in extracellular spaces and within macro-
phages in both early and more advanced lesions. Cholesterol crystals induce the
NLRP3 inflammasome, which leads to the processing and secretion of several
proinflammatory cytokines (Figure 4). The exposure of macrophages to fatty
acids derived from VLDL or VLDL remnants also activates the expression of
proinflammatory cytokines, independent of TLRs.
TLRs are a class of membrane-spanning, noncatalytic receptors that play
critical roles in innate immune responses. Multiple ligands, including oxLDL
and oxidised phospholipids, activate TLR2- and/or TLR4-related signalling
cascades to promote inflammation. In the presence of lipid ligands, CD36
and SRA interact with TLRs, resulting in activation of myeloid differentiation
factor (MyD) 88 and c-Jun N-terminal kinases (JNK), which promote apoptosis
(Figure 4). Their role in atherosclerosis is supported by studies in ApoE−/− mice
and Ldlr−/− mice, whereby genetic deficiency of TLR2, TLR 4 or the TLR
adapter protein MyD88 reduces lesion development. However, despite consistent
effects of global deficiency of TLRs and MyD88 in reducing atherosclerosis in
mice, their absence in bone marrow-derived cells has been conflicting. These
studies suggest that the primary impact of TLR signalling on atherosclerosis is
not through hematopoietic cells, including macrophages.
4.5 Atherosclerotic Lesion Macrophage Retention and Emigration
Atherogenesis is related to macrophage accumulation within lesions that
is determined by monocyte recruitment, macrophage proliferation, foam
cell formation and macrophage emigration and death (Moore et al., 2013)
(Figure 3). Atherogenic lipoproteins in the intima are primary determinants
of macrophage recruitment. Macrophage emigration occurs in early athero-
sclerotic plaques, but the rate of egress decreases with lesion progression.
In mouse models, emigration is an important factor linked to attenuation
of progression or the induction of lesion regression. Lesional macrophages
are subject to both retention and emigration signals. Macrophage foam cells
exhibit increased expression of the neuro-immune guidance cues, netrin 1 and
semaphorin 3E, both of which induce macrophage chemostasis (Figure 3).
Deficiency of netrin 1 attenuates lesion progression and increases mac-
rophage emigration. The signals that guide macrophages to exit lesions,
by transmigration through the endothelium to the lumen or by migrating
through the media to the adventitial lymphatics, are not well defined, but
likely involve macrophage expression of the CC-chemokine receptor CCR7
and the CC-chemokine ligands CCL19 and CCL21 that regulate cell homing
the lymph. As lesions progress, the continued presence of foam cells in a
lipid-rich lesion environment leads to cholesterol- and saturated fatty acid-
induced cytotoxicity, and ER stress, thereby increasing apoptosis (Figure 4).
Efferocytosis, the ability of macrophages to clear apoptotic cells through
receptors including tyrosine protein kinase MER (MERTK) and LRP1,
becomes compromised by cholesterol accumulation in the engulfing mac-
rophage. Defective efferocytosis contributes to secondary necrosis as well
as to formation and expansion of lipid-rich necrotic cores, which, in turn,
contribute to lesion rupture.
4.6 Atherosclerotic Lesion Regression
With the exception of early lesions, which are dominated by foam cells,
atherosclerosis was originally considered irreversible (Moore et al., 2013).
Recent studies demonstrating macrophage emigration from plaques in mice,
and that inflammation-resolving and tissue-remodelling M2 macrophages
are present in human and animal lesions, suggest that regression in humans
may be achievable. Some of the determinants of lesion regression have
recently emerged (Figure 3). Common to all mouse models of regression is
the requirement for aggressive lowering of plasma lipids and the observation
that within the regressing lesion there is a marked decrease in macrophage
number. Of those remaining, a majority display an M2 phenotype. In mac-
rophages from regressing lesions, the expression of genes including netrin
1, semaphorin 3E and members of the cadherin family is downregulated,
whereas CCR7 and cell motility factors are upregulated, thereby enhancing
the migratory ability of macrophages. Although other key factors that pro-
mote regression remain to be identified, improved lipid metabolism, includ-
ing marked attenuation of foam cell formation, is critical for regression to
be achieved. 5. NEW EMERGING MECHANISMS OF LIPID METABOLISM
INFLUENCING ATHEROSCLEROSIS
5.1 MicroRNAs
MicroRNAs (miRs) are a class of small noncoding RNAs that participate in a
diversity of biological and pathophysiological processes through posttranscrip-
tional regulation of gene expression. Many miRs regulate lipoprotein metabo-
lism in the circulation and/or local tissues (Novak et al., 2014). A major focus
of recent research is regulation of HDL metabolism by miRs. HDL particles are
carriers of miRs in plasma. Conversely, miRs also regulate expression of many
genes associated with HDL biosynthesis, uptake and metabolism.
miR-33 is the most intensely studied miR that regulates HDL metabolism.
miR-33 is embedded in intron 16 of the sterol regulatory element-binding fac-
tor (SREBP) gene, a transcription factor involved in cholesterol homeostasis
(Chapter 11). Although many studies reported that inhibition of miR-33 by anti-
sense oligonucleotides (ASO) in mice and monkeys or genetic deficiency of miR-
33 in mice increased plasma HDL cholesterol concentrations (Figure 5), other
studies have demonstrated that inhibition of miR-33 did not increase, or only
increased transiently, plasma HDL cholesterol concentrations. As with conflict-
ing findings in regulation of HDL cholesterol, effects of miR-33 on atherosclero-
sis are also inconsistent. In hypercholesterolaemic mice, an ASO against miR-33
or its genetic deficiency prevented, or had no effect on, the development of ath-
erosclerosis, or regressed preexisting atherosclerosis. These conflicting findings
admonish further studies before regulation of miR-33 can be used in human
studies. In addition to conflicting findings in mouse models, multiple differences
between mice and humans warrant careful interpretation. For example, miR-33
has differential effects in mice and humans on the expression of Niemann–Pick
C1 (NPC1) and ABCG1. These differences may be partially attributed to the
finding that mice only have miR-33a, whereas humans and primates have both
miR-33a and miR-33b (Fernandez-Hernando and Moore, 2011; Naar, 2013).
Several other miRs have also been shown to regulate lipoprotein metabo-
lism. We provide a brief review of those that have been studied in atherosclerotic
animal models. HDL carries more miRs than LDL particles. miR-155 is one of
the few exceptions and exhibits higher abundance in LDL than in HDL. miR-
155 regulates inflammatory responses. In addition, enhancement of miR-155
increases oxLDL uptake into cultured macrophages and impairs macrophage
cholesterol export to apoA-I, and suppression of miR-155 leads to increases
of cholesterol export to apoA-I in the absence of changes in the expression of
ABCA1 and ABCG1. Inhibition of miR-155 by an antagonist of a microRNA
(antagomiR) system reduced atherosclerotic lesion size in ApoE−/− mice fed a
high-fat, high-cholesterol diet without affecting plasma total cholesterol con-
centrations. miR-155 deficiency in bone marrow-derived cells reduced athero-
sclerotic lesions in ApoE−/− mice, but augmented atherosclerotic lesion size in
Ldlr−/− mice (Nazari-Jahantigh et al., 2015). It is unclear whether miR-155 has FIGURE 5 Existing and potential therapeutic targets for the treatment of lipid metabolism
and prevention or treatment of atherosclerosis. The therapeutic objective is to decrease the con-
centration of atherogenic lipoproteins or increase antiatherogenic lipoproteins in the circulation,
thereby improving lesion pathology or plaque regression. Statins inhibit the hepatic enzyme HMG-
CoA reductase, leading to increased expression of the LDLR and stimulation of the hepatic clearance
of LDL and VLDL remnants. Cholesterol-absorption inhibitors (ezetimibe) reduce the absorption
of cholesterol from the intestine by inhibiting the transporter NPC1L1 and are very effective when
combined with statins. Inhibition of PCSK9 with injected monoclonal antibodies blocks LDLR deg-
radation and increases hepatic cell surface LDLR, which results in an increased hepatic clearance of
LDL, VLDL remnants and Lp(a). Inhibition of microsomal TAG transfer protein (MTP) decreases
the assembly of VLDL in the liver and chylomicrons in the intestine by blocking the transfer of TAG
and CE to apoB-100 or apoB-48. CETP inhibitors increase HDL cholesterol and decrease LDL
cholesterol levels as well as Lp(a). CETP normally transfers CE from HDL to VLDL, IDL and LDL,
where theoretically this can promote the atherogenicity of these lipoproteins. ApoB antisense oligo-
nucleotides (ASO) inhibit the translation and synthesis of apoB and therefore decrease the secretion
of apoB-containing lipoproteins (VLDL) into plasma. An apoC-III ASO inhibits the synthesis and
secretion of apoC-III, a protein that normally delays the catabolism of TAG-rich lipoproteins. An
apo(a) ASO inhibits the hepatic synthesis of apo(a), therefore inhibiting the formation of Lp(a).
Inhibition of ACAT-2 with ASOs in mice inhibits the secretion of both chylomicrons and VLDL into
plasma and attenuates atherosclerosis in mice. Specific DGAT-2 inhibitors decrease hepatic VLDL
secretion and LDL cholesterol in experimental animals and are currently under development for
clinical use. MicroRNA-33 (miR-33) inhibits the expression of ABCA1 and ABCG1 and decreases
HDL formation. miR-33 ASOs or anti-miR-33 in mice increases cellular ABCA1 expression and
plasma HDL cholesterol concentrations and attenuate atherosclerosis in mice. differential effects between Ldlr−/− mice and ApoE−/− mice, leading to different
contributions of miR-155 to atherosclerosis in these two mouse strains.
miR-30c targets the 3′ untranslated region of microsomal triacylglyceride
transfer protein (MTP) mRNA. In addition to reducing MTP activity and apoB
secretion, miR-30c diminishes lipid synthesis in an MTP-independent manner. Lentiviral transduction of miR-30c reduced, and anti-miR-30c increased, plasma
cholesterol concentrations in C57BL/6 or ApoE−/− mice fed a Western diet, an
effect attributed to changes in secretion of plasma apoB-containing lipoproteins.
Consistent with modulation of plasma apoB-containing lipoproteins, transduc-
tion of miR-30c in female ApoE−/− mice fed a Western diet attenuated athero-
sclerosis, whereas inhibition of this miR augmented lesion development.
Given their multitargeting features, some circulating miRs might serve as
biomarkers to predict the development of atherosclerosis, and inhibiting or
enhancing certain miRs might be a good therapeutic strategy. However, the
most comprehensively studied miRs, such as miR-33, still require further char-
acterisation and thorough preclinical testing prior to applying therapeutic strate-
gies to humans (Naar, 2013).
5.2 Inflammasomes
Cholesterol crystal accumulation in macrophages is a distinguishing feature
of atherosclerosis from initiation through advanced stages of lesion develop-
ment, and is both a consequence of lipid overloading in cells and a critical
contributor to the progression of atherosclerosis (Figures 1 and 4). Choles-
terol crystals activate immune cells and induce inflammation through multiple
mechanisms. One proposed mechanism is that cholesterol crystals activate
nucleotide-binding oligomerisation domain receptors (NLRP)3 inflamma-
somes (Figure 4) (Lu and Kakkar, 2014).
The inflammasome is a cytosolic caspase-activating molecular complex that
contributes to inflammatory responses. Uptake of oxLDL leads to activation of
NLRP3 inflammasomes in cultured macrophages, and increases IL-1β release.
As a consequence, NLRP3 deficiency in bone marrow-derived cells reduces
hypercholesterolaemia-induced atherosclerosis in mice. This is a leukocyte-
specific effect of NLRP3 inflammasomes because whole body deficiency of
NLRP3 inflammasome components such as NLRP3, apoptosis-associated
speck-like protein or caspase-1 have no effect on atherosclerosis development
in hypercholesterolaemic mice.
oxLDL uptake increases IL-1β secretion in macrophages, an important
cytokine representing NLRP3 inflammasome activation. However, global defi-
ciency of IL-1β, but not its deficiency in bone marrow-derived cells, affects ath-
erosclerosis in hypercholesterolaemic mice, implicating macrophage NLRP3
inflammasomes in promotion of atherosclerosis through an IL-1β-independent
mechanism. Activation of NLRP3 inflammasomes also results in release of
IL-1α. Comparable to effects of IL-1β, global deficiency of IL-1α reduces
hypercholesterolaemia-induced atherosclerosis in mice. Fatty acids, especially
oleic acid, increase secretion of IL-1α in macrophages independent of NLRP3
inflammasome activation. Deficiency of IL-1α in bone marrow-derived cells
reduces atherosclerosis. Therefore, although activation of NLRP3 inflamma-
somes increases both IL-1α and IL-1β, these two cytokines contribute to athero-
sclerosis through different mechanisms. Currently, a clinical trial, CANTOS (NCT01327846), is ongoing to deter-
mine whether inhibition of IL-1β has beneficial effects on coronary artery
disease. Despite the conflicting findings in animal models, completion of this
clinical trial will provide insights into the effects and mechanisms of this IL-1
subtype in the development of atherosclerosis in humans (Lu and Kakkar, 2014).
5.3 Trimethylamine and Trimethylamine-N-oxide
For decades, physicians and research investigators have noted that consumption
of food abundant in saturated fat and cholesterol increases risk for atheroscle-
rosis. These foods are also rich in trimethylamine (TMA). Recently, potential
mechanisms by which a TMA-enriched diet promotes atherosclerosis have been
examined. In addition to food, TMA is produced by intestinal microbiota diges-
tion of choline and phosphatidylcholine. On absorption, TMA is oxidised by
hepatic flavin monooxygenases to generate trimethylamine-N-oxide (TMAO).
TMAO has been shown to augment atherosclerosis in mice through the reg-
ulation of multiple components of lipid metabolism. For example, TMAO
decreases mRNA abundance of ABCA1 and ABCG1 in cultured mouse perito-
neal macrophages, resulting in attenuated cholesterol export to apoA-I (Chapter
15). TMAO increases CD36 and SR-A in macrophages, modulates bile acid
metabolism and sterol transporters in both the liver and the intestine, and pro-
motes cholesterol delivery but diminishes reverse cholesterol transport in vivo
(Tang and Hazen, 2014). However, TMAO does not influence mRNA levels of
LDL receptor or cholesterol synthesis genes (Chapters 11 and 17). Effects of
TMAO on atherosclerosis development are also supported by findings in human
cohort studies. Plasma concentrations of TMAO are associated with common
events of atherosclerosis such as myocardial infarction, stroke or death (Tang
and Hazen, 2014; Micha et al., 2010).
6. TRADITIONAL AND EVOLVING LIPID-LOWERING
THERAPIES FOR THE TREATMENT OF ATHEROSCLEROSIS
In addition to lifestyle changes, lipid-regulating agents are widely used to
improve dyslipidaemia in high-risk patients (Chan et al., 2014). Current guide-
lines recommend statins as first-line lipid regulators. Inhibitors of cholesterol
absorption or bile acid sequestrants represent additional or alternative agents
for patients who are intolerant to statins or require optimal reduction in plasma
LDL cholesterol. However, many statin-treated patients, including those with
metabolic syndrome or type 2 diabetes, have significant residual atherosclerosis
risk, due in part to persistent abnormalities in TAG-rich lipoproteins and HDL
(Chapter 19). Treatment approaches involve the additional use of other lipid-
regulating agents to treat atherogenic dyslipidaemia by harnessing their comple-
mentary mechanisms of action. Increased understanding of the biological and
molecular mechanisms underlying the atherosclerotic process at its interface with lipoprotein metabolism, together with progress in our comprehension of
the molecular genetics of lipid disorders, has highlighted several new targets for
therapeutic intervention, as well as novel treatment options (Figure 5).
6.1 Statins
Inhibition of HMG-CoA reductase, a rate-limiting enzyme in hepatic cholesterol
synthesis by statins, results in the reduction of intracellular cholesterol content
that in turn induces an increase in SREBP-2-mediated hepatic LDL receptor
synthesis (Sahebkar and Watts, 2013b) (Chapter 11). This increases the clear-
ance of atherogenic lipoproteins, particularly LDL, as well as chylomicron rem-
nants and VLDL remnants (Figure 5). Statins are the most efficacious agents for
lowering the plasma concentration of LDL cholesterol and apoB-100. Statins
also decrease chylomicron remnants and VLDL TAGs, reduce small dense
LDL particles and modestly increase HDL cholesterol. Statins decrease hepatic
apoB-100 production, although results are inconsistent. Divergent results have
also been reported on the effects of statins on HDL metabolism. However, there
is no evidence that statin-induced HDL effects contribute to cardiovascular ben-
efit. Irrespective of its effects, clinical trials have consistently demonstrated that
statin therapy reduces cardiovascular events.
6.2 Fibrates
Several trials demonstrate that fibrates decrease atherosclerotic events in
patients with the metabolic syndrome and type 2 diabetes, which is character-
ised by increased plasma VLDL TAG and reduced plasma HDL cholesterol (Do
et al., 2014). Fibrates are agonists of the nuclear hormone receptor, peroxisome
proliferator-activated receptor-α (PPAR-α). Fibrates decrease plasma TAG up
to 50%, LDL cholesterol up to 20%, increase HDL cholesterol up to 20% and
decrease small dense LDL particles. The mechanistic action of fibrates is not
fully understood, but they decrease TAG substrate availability to liver by stimu-
lating fatty acid oxidation, thereby decreasing hepatic VLDL secretion. Fibrates
also promote VLDL lipolysis by activating lipoprotein lipase (LPL) and reduc-
ing apoC-III gene expression. Fibrates increase the expression of apoA-I and
ABCA1 by activating LXR, thereby promoting reverse cholesterol transport.
6.3 Niacin
Niacin has the theoretical potential to be an optimal drug for the treatment of ath-
erosclerotic diseases (Do et al., 2014). Niacin lowers plasma TAG concentrations
by up to 30%, LDL cholesterol up to 15%, and increases HDL cholesterol by up
to 25% and causes a shift of small dense LDL to large buoyant LDL particles.
Niacin is one of the few agents that can decrease plasma Lp(a). The mechanism
by which niacin modulates lipid metabolism has not been elucidated. Niacin decreases hepatic secretion of VLDL TAG by inhibiting fatty acid mobilisation
from peripheral adipocytes, thus decreasing the hepatic synthesis and secretion
of VLDL and the concentrations of its products, intermediate-density lipopro-
tein (IDL) and LDL. Niacin also increases plasma HDL cholesterol and apoA-I
concentrations by decreasing clearance of HDL apoA-I. Despite this desirable
profile of action on plasma lipoproteins, and positive results from early clini-
cal trials, two recent clinical trials failed to demonstrate significant benefits of
niacin on cardiovascular events in statin-treated patients (Rached et al., 2014).
6.4 Cholesterol Absorption Inhibitors
Ezetimibe selectively lowers plasma LDL cholesterol concentrations (Do
et al., 2014). Ezetimibe inhibits the function of Niemann–Pick 1-Like 1 pro-
tein (NPC1L1) resulting in reduced intestinal cholesterol absorption, attenuated
cholesterol transport in chylomicron remnants and reduced hepatic cholesterol
content (Figure 5). SREBP-2-mediated upregulation of hepatic LDL recep-
tors stimulates hepatic uptake of plasma apoB-100-containing lipoproteins and
accounts for up to 20% decreases in plasma LDL cholesterol. Ezetimibe also
significantly decreases LDL particle number, but has an inconsistent effect on
LDL particle size. Ezetimibe decreases intrahepatic TAGs, independent of body
weight, visceral fat and insulin sensitivity, although the mechanism remains
unclear. Inhibition of cholesterol absorption and hepatic cholesterol synthe-
sis with ezetimibe in combination with a statin, has complementary effects
on the fractional catabolism of apoB-containing lipoproteins. Accordingly, a
combination of ezetimibe and a low-dose statin can more effectively decrease
LDL cholesterol than higher doses of statin alone. Although some short-term
imaging studies have reported little or no benefit of ezetimibe treatment, the
recent Improved Reduction of Outcomes: Vytorin Efficacy International Trial
(IMPROVE-IT) study demonstrated significant benefits of ezetimibe for the
reduction of cardiovascular events.
6.5 ω-3 Polyunsaturated Fatty Acids
Fish oils are a rich source of ω-3 polyunsaturated fatty acids (PUFAs), mainly
eicosapentenoic acid (EPA) and docosahexenoic acid (DHA). Fish oils are rec-
ommended as an adjunct to diet for the reduction of VLDL–TAG and prevention
of acute pancreatitis (Rached et al., 2014). Human studies demonstrate that ω-3
PUFA supplementation decreases hepatic VLDL production and accordingly
reduces VLDL–TAG. There are no consistent effects on plasma concentra-
tions of LDL cholesterol, HDL cholesterol or HDL–apoA-I. The mechanistic
action of ω-3 PUFAs on plasma TAGs includes inhibition of diacylglycerol
acyltransferase (DGAT), fatty acid synthase and acyl-CoA carboxylase enzyme
activities, resulting in decreased TAG and fatty acid synthesis. ω-3 PUFAs also
enhance fatty acid β-oxidation by stimulating PPAR-α. However, they are much
weaker PPAR-α agonists than fibrates. Although some early trials have shown
cardiovascular benefits, a recent meta-analysis has cast doubts on the therapeu-
tic efficacy of ω-3 PUFA and concluded that its supplementation is not associ-
ated with decreased risk of atherosclerosis-related events.
6.6 Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitors
PCSK9 targets the LDL receptor for degradation and was discovered as the third
gene involved in autosomal dominant hypercholesterolaemia. Some mutations
of PCSK9 enhance its function and cause hypercholesterolaemia, whereas loss-
of-function mutations are associated with hypocholesterolaemia and reduced
cardiovascular risk. By enhancing hepatic LDL receptor degradation, PCSK9
increases plasma LDL cholesterol. PCSK9 is primarily regulated transcription-
ally by SREBP2 and the secreted protein binds the epidermal growth factor-like
repeat A domain of the LDLR, which results in attenuated LDLR recycling
and its targeting to lysosomes for degradation. While statins effectively reduce
plasma LDL cholesterol in many patients, statins also upregulate PCSK9 via
SREBP2, thereby limiting statin’s efficacy in reducing plasma LDL cholesterol.
Antibodies against PCSK9 have been developed that increase the numbers of
LDL receptors available at the cell surface and reduce plasma LDL cholesterol
(Figure 5) (Rached et al., 2014). These include human mononclonal antibod-
ies or humanised antibodies that are injected intravenously or subcutaneously.
In phase II and III trials, anti-PCSK9 therapy of patients lowers LDL choles-
terol by 40–65% in both heterozygous FH and non-FH patients, with or without
statin and ezetimibe treatment. Lp(a) concentrations were also decreased sig-
nificantly. Other strategies to inhibit PCSK9, including ASO therapies, small
interfering RNAs and recombinant adnectins are under development. PCSK9
inhibition is a promising new approach for treatment of hypercholesterolaemia
and potentially atherosclerosis.
6.7 ASO Therapies
ASOs are short, single-stranded, synthetic analogues of natural nucleic acids
designed to bind to a target mRNA in a sequence-specific manner. Injection
of a short complementary ASO sequence binds to mRNA and induces either
selective degradation of the complex by endogenous nucleases or inhibition of
mRNA processing and/or function. The most advanced ASO is mipomersen
that is designed to inhibit hepatic apoB-100 synthesis (Rached et al., 2014; Do
et al., 2014) (Figure 5). Phase II and III trials revealed that mipomersen reduced
plasma concentrations of apoB, LDL cholesterol, non-HDL cholesterol and
TAGs by 20–65% in patients with different forms of hyperlipidaemia, includ-
ing heterozygous and homozygous FH, and in patients treated with statins and
ezetimibe. Mipomersen reduced Lp(a) levels by 25% by inhibiting its synthesis.
The main side effects involve injection-site reactions and hepatic steatosis. Beyond apoB and PCSK9, several other potential ASO targets for treat-
ment of hyperlipidaemia have been identified, including apoC-III and Lp(a)
(Figure 5). ApoC-III normally inhibits LPL-mediated lipolysis of chylomicrons
and VLDL, and attenuates hepatic clearance of their remnants. Human genetic
studies have associated decreased plasma concentrations of apoC-III with lower
TAG concentrations and diminished cardiovascular events. In animal models
and Phase I human trials, an apoC-III ASO inhibitor produced potent, selec-
tive reductions in plasma apoC-III and TAG concentrations as well as marked
increases in HDL-C. ASOs with the potential to lower PCSK9 and Lp(a) plasma
concentrations are in early stages of development.
6.8 Microsomal Triacylglyceride Transfer Protein Inhibitors
MTP is critical for the formation and secretion of apoB-containing lipopro-
teins from the liver and intestine (Do et al., 2014) (Chapter 16). MTP trans-
fers TAG, CE and phospholipid to apoB within the cell during the lipoprotein
assembly process. Mutations in the MTP gene lead to a rare condition known
as abetalipoproteinaemia, in which plasma apoB-containing lipoproteins are
undetectable. In animal models, MTP inhibition results in profound reductions
in plasma TAG and cholesterol concentrations (Figure 5). Although early MTP
inhibitors reduced LDL cholesterol, further development of most inhibitors
has been discontinued due primarily to hepatic fat accumulation. Lomitapide
is the only systemic MTP inhibitor currently in development. Lomitapide sub-
stantially reduced levels of LDL cholesterol in homozygous FH and in clini-
cal trials proved very effective in reducing LDL cholesterol in patients with
homozygous FH as well as in patients with moderate hypercholesterolaemia,
either as monotherapy or when combined with ezetimibe. However, variable
gastrointestinal side effects, minor elevations in liver transaminase levels and
increases in hepatic fat were reported. Lomitapide was recently approved for
treatment of homozygous FH, as this drug was considered to provide the ben-
efits of cardiovascular protection that outweighed risks of increased hepatic fat.
An intestine-targeted MTP inhibitor has been shown to decrease both VLDL
and chylomicron production and resulted in weight loss without elevations of
liver enzymes or increases in hepatic fat, suggesting that this approach may
be more applicable to treatment of a broader range of lipid disorders (Rached
et al., 2014).
6.9 ACAT and DGAT Inhibitors
One of the important steps in atherogenesis and cholesterol accumulation in the
arterial wall is esterification of cholesterol in macrophages, which promotes
foam cell formation. This provided a rationale for the development of ACAT
inhibitors; however, early versions inhibited both ACAT isoforms (ACAT-1 and -2)
(Rached et al., 2014). Imaging based clinical trials failed to demonstrate reduced atherosclerosis progression in patients with FH. By contrast, an ACAT-
1-selective inhibitor K-604 is under development by Kowa Pharmaceuticals.
ACAT-2 is the isoform responsible for cholesterol esterification in the liver and
intestine and the provision of CE for lipoprotein synthesis (Figure 5). Genetic
deletion of ACAT-2 or pharmacological inhibition of ACAT-2 with ASOs in
mice inhibits the secretion of both chylomicrons and VLDL into plasma and
attenuates atherosclerosis in Ldlr−/− mice. As a result, specific inhibitors of
ACAT-2 are currently under development.
DGATs esterify the third fatty acid to DAG to form TAG in adipose tissue, the
intestine and the liver. Studies in mice with DGAT-1 deficiency or mice treated
with DGAT-1 inhibitors demonstrated reduced plasma TAGs, hepatic steatosis and
obesity, which were paralleled by improved insulin resistance. In patients with
severe hypertriglyceridaemia, with LPL deficiency, a DGAT-1 inhibitor decreased
fasting plasma TAG levels. Clinical trials are ongoing in patients with coronary
artery disease to further substantiate these findings. DGAT-2 is the isoform respon-
sible for TAG synthesis in liver and intestine and the provision of CE for lipopro-
tein synthesis (Figure 5). Specific DGAT-2 inhibitors decrease plasma and liver
TAG in experimental animals and are currently under development for clinical use.
6.10 High-Density Lipoprotein Modulating Drugs
Epidemiological evidence demonstrates that low HDL cholesterol concentra-
tions predict cardiovascular events across multiple populations. However, thera-
peutic strategies to target HDL have not yet achieved success (Rached et al.,
2014). Three HDL cholesterol-raising drugs, two early cholesteryl ester trans-
fer protein (CETP) inhibitors and niacin, have failed in prospective interven-
tion trials due to off-target side effects or lack of efficacy. Current therapeutic
approaches are focussed on improved HDL metabolism, increased HDL particle
number and enhanced HDL function. Inhibition of CETP lipid transfer activ-
ity elevates plasma HDL cholesterol concentrations by 30–140% and reduces
concentrations of LDL cholesterol and Lp(a) by up to 40% (Figure 5). Large
cardiovascular end-point trials of two recently developed CETP inhibitors are
ongoing. Other approaches that transiently increase HDL particle numbers and
enhance HDL functionality include infusions of reconstituted HDL, administra-
tion of apoA-I mimetic peptides, antagonists of miR33 (antimiR33) (Figure 5)
and injection of delipidated HDL. These strategies involve structural remodel-
ling of endogenous HDL particles and are targeted towards export of cholesterol
from the atherosclerotic plaque (Figure 5). The impact of these agents on cardio-
vascular events remains to be evaluated.
7. FUTURE DIRECTIONS
The pathogenesis and mechanisms of atherosclerosis are complex. In the
past several decades, lipid and lipoprotein-related mechanisms have been studied extensively, and medical treatments of atherosclerosis targeting
lipid metabolism have been improved. However, many questions remain
unresolved: (1) Although inflammation is recognised as a critical mechanism in
hypercholesterolaemia-induced atherosclerosis, the therapeutic effects of directly
targeting inflammation have not been translated into human use. (2) There is
substantial experimental evidence that HDL reduces atherosclerosis; however,
efforts to increase HDL cholesterol were hampered by either off-target effects or
lack of beneficial effects on atherosclerotic diseases. (3) Currently, there is only
weak evidence that drugs regress preexisting atherosclerosis in humans. Several
animal studies have demonstrated that therapeutic intervention can induce regres-
sion of atherosclerosis, but only in the setting of significant lowering of plasma
cholesterol concentrations. (4) Therapeutic approaches that directly influence
mechanisms of atherogenesis within the arterial wall, including those that spe-
cifically target cellular lipid metabolism, have yet to be developed or evaluated.
In future studies, in addition to continuously exploring the complex mechanisms
of lipid/lipoprotein-related atherosclerosis in animal models, it will be important
to apply convincing findings from animal models into human clinical trials.