HDL and Atherosclerosis Regression: Insights from Animal Models and Their
Clinical Relevance
Jonathan E. Feig MD, PhD
Abstract
In epidemiologic studies, plasma high-density lipoprotein cholesterol (HDL-C)
levels have an inverse relationship to the risk of atherosclerotic coronary artery disease
(CAD). It has been assumed that by raising HDL-C, not only would the progression of
plaques slow, but that regression, the ultimate therapy for CAD, would ensue. Clinical
intervention studies to causally link plasma HDL-C levels to decreased progression or to
the regression of atherosclerotic plaques, however, are relatively few because of the lack
of therapeutic agents to selectively and potently raise HDL-C. These will be discussed in
relation to the pre-clinical literature that has established the promotion of plaque
regression by HDL in a number of animal models, and in more recent work, has pointed
to multiple mechanisms by which this has been achieved.
Condensed abstract: Epidemiologic studies have repeatedly shown an inverse
relationship between plasma HDL cholesterol levels and the risk of coronary artery
disease. Pre-clinical studies have recently shown that HDL can not only delay
atherosclerosis progression, but also promote regression. The mechanisms for this will be
discussed and linked to the clinical literature.
2
Introduction
Epidemiologic studies have demonstrated that there exists a strong negative correlation
between plasma HDL cholesterol (HDL-C) and the risk of cardiovascular disease [1-4].
Recent insights have added to the potential mechanisms, which include the stimulation of
reverse cholesterol transport (RCT) from foam cells in coronary plaques to the liver,
protection of the endothelium (by activation of the eNOS pathway), and inhibition of
LDL oxidation [5-8]. While pre-clinical research on HDL has been conducted in a variety
of animal models, a number of genetically modified mouse lines have been developed to
more deeply explore these mechanisms and to discover additional ones at the molecular
level. It is important to note that murine HDL metabolism has three major differences
compared to that in humans: HDL, not LDL, is the principal carrier of circulating
cholesterol in mouse plasma, mouse HDL is a mono-disperse population (i.e., without
discrete density sub-classes), and the activity of cholesterol-ester transfer protein (CETP)
is absent [7-8]. In this review, we will focus mainly on the mouse models, but will also
link them to findings in other systems, including clinical settings.
HDL and Reverse Cholesterol Transport
Lipid-free apoAI and lipid poor pre-β-HDL particles are produced in the liver and
intestine. Cholesterol becomes associated with these HDL particles, and is then esterified
by lecithin-cholesterol acyltransferase (LCAT). Cholesteryl ester (CE) moves to the
developing core of the HDL particles, which converts them to spheres and also allows
their surfaces to accept more free cholesterol. In human plasma, there is a reciprocal
exchange of HDL-CE for triglycerides carried on apoB-containing lipoproteins, which is
mediated by cholesteryl ester transfer protein (CETP). At the same time, the HDL that is
3
becoming enriched in triglycerides is a substrate for hepatic lipase. The CE are
subsequently cleared by the liver when the apoB-lipoproteins undergo hepatic uptake
through LDL-receptor dependent and independent mechanisms. The activities of CETP
and hepatic lipase help to remodel HDL particles to become a preferred binding partner
for scavenger receptor type BI (SR-BI), the major HDL receptor on hepatocytes. Hence,
reverse cholesterol transport (RCT) can be considered a cycle in which acceptors of
cholesterol are continuously regenerated to undertake their function of promoting
cholesterol efflux from the peripheral tissues to the liver (Figure 1) [7, 9-11].
Experiments with transgenic animals suggest that disruption of one or more steps
in RCT results in accelerated atherosclerosis, whereas overexpression of pivotal proteins
in RCT, such as apoAI, LCAT, and SR-BI is atheroprotective [7-8, 12-13]. Traditionally,
the anti-atherogenic role of HDL has been attributed to the presence of apoAI. For
example, transgenic mice with high plasma human apolipoprotein AI and HDL plasma
levels were protected from development of fatty streak lesions when fed an atherogenic
diet [14]. Furthermore, overexpression of human apoAI in apoE-/- mice resulted in
greatly retarded progression of atherosclerosis [15-16].
While it is clear that HDL is a key player in RCT, it has important cellular
partners. We will only discuss the major players due to space constraints. The ATPbinding cassette transporters ABCA1 and ABCG1 are increased by liver X receptor
transcription factors, key regulators of cholesterol homeostasis [17-19]. Although
ABCA1 promotes cholesterol efflux to cholesterol-deficient and phospholipid-depleted
apoA-I, ABCG1 promotes efflux to HDL particles [17, 20-21]. Recently, insights into the
coordinated participation of ABCA1 and ABCG1 in mediating macrophage cholesterol
4
efflux have been reported. Interestingly, a single deficiency of ABCA1 in mice results in
a moderate increase in lesion development, and deficiency of ABCG1 has no effect;
however, combined deficiency resulted in markedly accelerated atherosclerosis [22].
Double-knockout macrophages showed markedly defective cholesterol efflux to HDL
and apoA-I as well as increased inflammatory responses when treated with
lipopolysaccharide [23].
Beyond Reverse Cholesterol Transport
Endothelial dysfunction is one of the early hallmarks in the pathogenesis of
atherosclerosis [24]. It was shown that oxidized LDL-induced displacement of
endothelial nitric oxide synthase (eNOS) from caveolae and impairment of NO
production was prevented in the presence of HDL [25]. It has also been demonstrated that
the apoAI mimetics, L-4F and D-4F, protect endothelial cell function in mice by
inhibiting native and oxidized LDL’s uncoupling of eNOS activity, thereby preventing
superoxide production from overtaking that of NO [26]. Independent of the ability to
counteract adverse effects of LDL and oxidized LDL, it also has been shown that HDL
promotes eNOS activation and NO release, with resulting vasorelaxation [27-28].
Interestingly, apoE-/- mice, which have naturally low HDL levels, exhibit attenuated NOmediated vasodilation. In fact, these mice have more rapid progression of atherosclerosis
when subjected to either long term NOS antagonism or genetic eNOS deficiency [29-31].
Experiments in vivo that support a positive role for HDL in promoting endothelial health
include a study in which carotid artery re-endothelialization following perivascular
electric injury was diminished in apoAI-null mice, but was normalized by the restoration
of apoAI [32-33]. HDL also inhibits the interaction of monocytes with endothelial cells
5
and smooth muscle cells, as well as the adhesion of monocytes to endothelial cells
induced by oxidized LDL. Theilmeier and colleagues showed that overexpression of
human apoAI in apoE-/- mice reduced endothelial adhesion molecule expression and
macrophage homing to the endothelium [34].
In addition to the studies on endothelium, a growing body of research suggests
that HDL counteracts a number of the adverse effects of LDL oxidation. Current thinking
attributes some of this protection to anti-oxidant properties of HDL, particularly related
to its content of α-tocopherol and other lipophilic anti-oxidants, as well as enzymes with
antioxidant-like activities (platelet activating factor acetylhydrolase (PAF-AH) and
paraoxonase (PON)). In addition, apoAI, which possesses several methionine groups,
may act directly as an anti-oxidant [7, 35-36]. Anti-oxidant effects would be expected to
prevent the formation of lipid hydroperoxides (LOOX), oxidized cholesteryl esters, and
oxidized phospholipids (such as 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine
(PGPC) and 1-palmitoyl-2(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC)).
These lipid species are generated from LDL lipids, and are thought to be responsible for
several deleterious effects on all cell types of the arterial wall. For example,POVPC
stimulates the production of cytokines like MCP-1 and M-CSF, as well as induces
adhesion of monocytes to the endothelium [35]. The oxidized lipid species are generated
in a process that requires the presence of “seeding molecules,”(e.g.,
hydroperoxyoctadecadienoic acid [HPODE]) as catalysts,which are generated by 12lipoxygenase. In fact, HDL was demonstrated to limit the levels of these seeding
molecules and to degrade them in an enzymatic process catalyzed by PON and PAF-AH
[35-39].
6
To identify new athero-modulating factors associated with HDL, a shotgun
proteomic approach has been employed to test the hypothesis that HDL carries previously
unappreciated proteins that make contributions to its cardioprotective and antiinflammatory activities. The authors identified multiple complement-regulatory proteins
and a diverse array of distinct serpins with serine-type endopeptidase inhibitor activity.
Many acute-phase response proteins were also detected. It was suggested that HDL has a
role in regulating the complement system and protecting tissue from proteolysis, and that
the protein cargo of HDL contributes to its anti-inflammatory and anti-atherogenic
properties [7, 40].
HDL and Atherosclerosis Regression: Pre-clinical Studies
The idea that human atheromata can regress at all is something that met
considerable resistance over the years. The reason for this may have been that advanced
atherosclerotic lesions in humans and in animal models contain calcification and fibrosis,
characteristics that seem irreversible [41-42]. Nonetheless, a number of studies beginning
decades ago argue that the impression of irreversibility is misguided. The first
interventional study demonstrating substantial shrinkage of atherosclerotic lesions was
performed in cholesterol-fed rabbits over fifty years ago [43]. Animals received
intravenous bolus injections of phosphatidylcholine (PC). After less than a week and a
half of treatment, the remaining plaques were fewer and much smaller than initially with
approximately 75% of the arterial cholesterol stores being removed. Using a variety of
atherosclerotic animal models, other groups showed similar arterial benefits from the
injection of dispersed phospholipids (e.g., [44]).
7
Armstrong and colleagues found that advanced arterial lesions in cholesterol-fed
rhesus monkeys underwent shrinkage and remodeling during long-term follow-up after a
switch to low-fat or linoleate-rich diets [45-46]. The subsequent regression period (lasting
40 months) resulted in the loss of approximately two-thirds of coronary artery
cholesterol, substantial reduction in necrosis, improvement in extracellular lipid levels
and fibrosis, and lesion shrinkage. Success in atherosclerosis regression was again
achieved in rabbits in 1976, following reversion to a normal-chow diet in combination
with the administration of hypolipidemic agents [47]. Decades later, a series of studies
achieved shrinkage of atheromata in rabbits via injections of HDL [48] as well as
demonstrating the anti-atherogenic effects of apoAI in cholesterol-fed rabbits [49].
In spite of the clinical desirability to achieve regression, further research into the
molecular factors that may be mediating this process has been hampered by the scarcity
of appropriate animal models. The relative ease of progression studies, using apoE-/- or
LDLR-/- mice, has led to an emphasis on this phase of the disease process. The
similarities between atherosclerosis in humans and mice deficient either in apoE [50-53]
or the LDL receptor [54] suggest that molecular mechanisms underlying regression in
these mouse models could be relevant to the reduction in plaque burden in the human
population [41-42].
Plaque regression in mouse models of atherosclerosis has previously been
demonstrated primarily by somatic adenoviral gene transfer [55-56]. Such approaches
have been limited mainly because of the eventual loss of transgene expression, even with
second-generation viral vectors, likely caused by immune responses directed against both
the transgene product and adenoviral proteins. A new regression model was introduced by
8
our group which involves transplantation of either a thoracic aortic segment [57] or an
aortic arch segment [58] from apoE-/- mice to wild-type (WT) recipient mice. Under the
conditions of the WT mouse in which the dyslipidemia is corrected, regression is rapidly
apparent (as judged by plaque content of CD68+ monocyte derived cells, which are
primarily macrophages), whereas when the recipient is an apoE-/- mouse, further
progression is evident [58-61]. Using laser capture microdissection of CD68+ cells in the
plaque, it was demonstrated that regression was characterized by the decrease in
expression of inflammation-related genes such as VCAM-1, ICAM-1, MCP-1, and TNFα [61]. Notably, there were also quantitative changes in macrophage content, which was
attributed to emigration of these cells from plaques to regional and systemic lymph nodes
under regression, but not progression, conditions [59, 61]. The emigrating cells
expressed markers of macrophages (such as CD68 and CD115) and dendritic cells (DCs)
(such as CD11c)- cell types that are derived from monocytes [59, 61]. Since migration of
DCs to lymph nodes absolutely requires the chemokine receptor CCR7 [62], we
hypothesized that it became induced in foam cells under regression conditions. Indeed,
we found an increase in CCR7 mRNA and protein expression only in foam cells from the
regression environment [61] and went on to show the functional requirement of CCR7 for
regression in our transplant model [61], though recent results have also shown that
decreased recruitment of monocytes may contribute to palque regression as well [63].
At least three plasma parameters are changed in the transplantation model when
regression was observed: (1) non-HDL levels decreased; (2) HDL levels were restored
from ~33% of normal to wild type levels; (3) apoE was now present. For the purpose of
this review, we will focus on the HDL change. To selectively test this as a regression
9
factor, we adopted the transplant approach by using as recipients human apoAI
transgenic/apoE-/- mice (hAI/EKO) [64] or apoAI-/- mice [65]. Briefly, plaque-bearing
aortic arches from apoE-/- mice (low HDL-C, high non-HDL-C) were transplanted into
recipient mice with differing levels of HDL-C and non-HDL-C: C57BL/6 mice (normal
HDL-C, low non-HDL-C), apoAI-/- mice (low HDL-C, low non-HDL-C), or hAI/EKO
mice (normal HDL-C, high non-HDL-C). Remarkably, despite persistent elevated nonHDL-C in hAI/apoEKO recipients, plaque CD68(+) cell content decreased by >50% by
one week after transplantation, whereas there was little change in apoAI-/- recipient mice
despite hypolipidemia. Interestingly, the decreased content of plaque CD68+ cells was
associated with their emigration and induction of their chemokine receptor CCR7 [66].
These data are consistent with a recent meta-analysis of clinical studies in which it was
shown that atherosclerosis regression (assessed by IVUS) after LDL lowering was most
likely to be achieved when HDL was also significantly increased [67].
The induction of CCR7 is likely related to changes in the sterol content of foam
cells when they are placed in a regression environment, given that its promoter has a
putative sterol regulatory element (SRE). This idea is in agreement with a report that
demonstrated that loading THP-1 human monocytes with oxidized LDL suppresses the
expression of this gene [68]. Notably, we have found that statins, potent regulators of
SRE-dependent transcription can induce CCR7 expression in vivo and promote regression
via emigration of CD68+ cells in a CCR7 dependent manner [69]. Recently, it was
reported that both atorvastatin and rosuvastatin can promote regression of atherosclerosis
as assessed by IVUS [70]. Our data, therefore, suggest that activation of the CCR7
pathway may be one contributing mechanism.
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Another aspect of interest has been the effect of HDL on the inflammatory state of
CD68+ cells in plaques. A number of benefits from this can be envisioned such as a
reduced production of monocyte attracting chemokines and plaque “healing” by
macrophages prodded to become tissue re-modelers (M2 macrophages). There are
multiple reasons for HDL to have anti-inflammatory effects on plaques, including the
anti-oxidant properties of its enzymatic and non-enzymatic components, the ability to
remove normal and toxic lipid species from cells, and the dampening of TLR signaling
by regulating plasma membrane cholesterol content [7, 23, 71]. It is important to note
that in CD68+ cells laser-captured from the plaques, normalization of HDL-C led to
decreased expression of inflammatory factors and enrichment of markers of the M2
macrophage state [66, 72]. Macrophage heterogeneity in human atherosclerotic plaques is
widely recognized, with both M1 (activated) and M2 markers being detectable in lesions
[73-74], but little is known about the factors that regulate M2 marker expression in
plaques in vivo.
Cholesterol homeostasis has also recently been investigated with microRNAs
(miRNA), which are small endogenous non–protein-coding RNAs that are
posttranscriptional regulators of genes involved in physiological processes. MiR-33, an
intronic miRNA located within the gene encoding sterol-regulatory element binding
protein-2, inhibits hepatic expression of both ABCA1 and ABCG1, reducing HDL-C
concentrations, as well as ABCA1 expression in macrophages, thus resulting in decreased
cholesterol efflux [75]. Interestingly, enrichment of M2 markers in plaque CD68+ cells
was observed in LDLR-/- mice treated with an antagamir of miR-33 [75]. The treated
mice also exhibited plaque regression (fewer macrophages and intimal area). The
11
therapeutic potential of miR-33 antagmirs to cause similar benefits in people was
suggested by plasma levels of HDL being raised in treated non-human primates [76].
Thus, antagonism of miR-33 may represent a novel approach to enhancing macrophage
cholesterol efflux and raising HDL-C levels in the future.
HDL and Atherosclerosis Regression: Clinical Studies
In addition to the pre-clinical studies reviewed above, there are a limited number
of human studies in which HDL levels have been manipulated by infusion, and the
effects on plaques assessed. In the first [77], patients at high risk for cardiovascular
disease were infused with either an artificial form of HDL (apoAI milano/phospholipid
complexes) or saline (placebo) once a week for 5 weeks. By intravascular ultrasound
(IVUS), there was a significant reduction in atheroma volume (-4.2%) in the combined
(high and low dose) treatment group, though no dose response was observed of a higher
vs. lower dose of the artificial HDL. There was no signifcant difference in atheroma
volume compared to the placebo group, but the study was not powered for a direct
comparison. In the second infusion study, high-risk patients received 4 weekly infusion
with reconstituted HDL (rHDL; containing wild type apoAI) or saline (placebo) [78].
Similar to the previous study, there was a signficant decrease in atheroma volume (3.4%) (as assessed by IVUS) after treatment with rHDL compared to baseline, but not
compared to placebo (which the study was not powered for). However, the rHDL group
had statistically significant improvements in plaque characterization index and in a
coronary stenosis score on quantitative coronary angiography compared to the placebo
group. In the third infusion trial [79], a single dose of reconstituted human HDL was
infused into patients undergoing femoral atherectomies, with the procedure performed 5-
12
7 days later. Compared to the control group (receiving saline solution), in the excised
plaque samples in the HDL infusion group, macrophage activation state (i.e. diminished
VCAM-1 expression) as well as cell size (due to diminished lipid content) were reduced.
In addition to the aforementioned meta-analysis of statin trials in which the
relationships among LDL, HDL, and plaque regression were analyzed, there are also a
number of other drug studies in which effects on plaques were ascribed to the raising of
HDL levels. This includes the VA-HIT study, in which coronary events were reduced by
11% with gemfibrozil for every 5-mg/dL increase in HDL-C [80]. In a more recent series
of studies (“ARBITER” [81-84]), high-risk patients were placed on either statins or
statins plus niacin. Over a 18-24 month observation periods, carotid intimal-medial
thickness (cIMT) measurements were obtained as a surrogate for coronary artery plaque
burden. As expected, when niacin was part of the treatment, HDL-C levels were
increased (by 18.4%), and the authors attributed the improvement in cIMT particularly to
this change. It is important to note that niacin does more than just raise HDL-C levels; it
also decreases plasma triglyceride levels, makes LDL size increase, and possesses antiinflammatory properties all of which have the potential to limit plaque progression [8587]. These pleiotropic effects obviously confound the interpretation of both the
ARBITER and another statin-niacin clinical trial- the HATS study [88]. In the latter
study, the addition of niacin to statin treatment resulted not only in a reduction in
coronary artery stenosis, but also in events. The encouraging results with niacin,
however, were recently called into question by the early termination of the
Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High
Triglycerides [AIM-HIGH] study, which failed to show a benefit in the treatment group
13
[89]. This study has been criticized, however, as being underpowered and for the fact that
both the treatment group and the control group in the study received statin therapy,
making additional benefits harder to detect, as well as for the placebo that the control
patients received was actually a low dose of niacin [90]. The lack of efficacy was also
observed in the Heart Protection Study 2- Treatment of HDL to Reduce the Incidence of
Vascular Events [HPS2-Thrive] was also an unexpected surprise.
Recently, cholesteryl ester transfer protein (CETP) inhibitors have been
investigated as pharmacological agents to raise HDL levels. Surprisingly, torcetrapib, the
first CETP inhibitor tested in a clinical trial, increased the all-cause mortality and
cardiovascular events, which led to the premature ending of the ILLUMINATE trial [91].
Subsequent studies indicated that the observed off-target effects of torcetrapib (increased
blood pressure and low serum potassium by stimulation of aldosterone production) were
rather molecule specific, unrelated to CETP inhibition and thereby might have
overshadowed the beneficial effects of the raised HDL-C levels. Importantly, posthoc
analysis of ILLUMINATE showed that subjects with greater increases of HDL-C or
apoAI levels had a lower rate of major cardiovascular events within the torcetrapib group
[92]. Despite the general failure of torcetrapib, in the posthoc analysis of the
ILLUSTRATE (Investigation of Lipid Level Management Using Coronary Ultrasound to
Assess Reduction of Atherosclerosis by CETP Inhibition and HDL Elevation) study,
regression of coronary atherosclerosis (as assessed by IVUS) was observed in patients
who achieved the highest HDL-C levels with torcetrapib treatment [93]. In vitro studies
showed an improved functionality of HDL-C particles under CETP inhibition, as HDL-C
isolated from patients treated with torcetrapib and anacetrapib exhibited an increased
14
ability to promote cholesterol efflux from macrophages [94-95]. Indeed, the CETP
inhibitors anacetrapib, dalcetrapib and evacetrapib increase HDL-C levels between 30138%, and have not shown the off-target effects of torcetrapib in recent clinical phase II
trials, confirming the premise of a non-class related toxicity of torcetrapib [96-99]. Thus,
raising HDL-C by CETP inhibition or modulation remains a potential therapeutic
approach for atherosclerotic cardiovascular disease. Large clinical outcome trials were
initiated for dalcetrapib (dal-OUTCOMES) and anacetrapib (REVEAL) including a total
of approximatley 45,000 patients. Surprisingly, in May 2012 Roche stopped the complete
dal-HEART program for dalcetrapib after an interims anaylsis of dal-OUTCOMES due
to a lack of clinically meaningful efficacy (Roche Inc., Roche provides update on Phase
III study of dalcetrapib; media release). The failure of dal-OUTCOMES might have been
a result of the rather moderate increases in HDL-C levels (30%) and minor impact on
LDL-C levels induced by dalcetrapib, a fate that does not necessarly apply for
anacetrapib which has been shown to incease HDL-C levels by 138% accompanied by
more robust reductions in LDL-C levels [100]. Whether the failure of dal-OUTCOMES
challenges the benefits of raising HDL-C, in general, or rather the underlying mechanims
of how HDL-C is to be raised needs to be determined in future studies. More conclusive
answers on the concept of raising HDL-C by CETP inhibition can be expected from the
final analysis of the dal-OUTCOMES and REVEAL studies over the next few years.
HDL and Imaging Modalities
Although it is well known that HDL-C levels are inversely related with the risk
for CAD, it is the composition and the inflammatory activity of the atheromatous plaque
that have important implications for clinical events. Imaging modalities has allowed for
15
the analysis of structural changes in the vessel wall which include parameters such as
vessel wall thickness, specific measures of plaque size, composition, and inflammatory
status [101-104]. Interestingly, it has been demonstrated that modified HDL is
macrophage specific and therefore can provide macrophage density information via
noninvasive MRI. As such, modified HDL can be a valuable contrast agent for probing
preclinical atherosclerosis [105-107].
Noninvasive imaging of atherosclerosis can be used to assess efficacy and to
provide additional information regarding the action of HDL-C–raising drugs. The effects
of various HDL-C raising therapies on cIMT have been examined in multiple clinical
trials [108]. For example, niacin therapy has been accompanied by decreased rates of
atherosclerosis progression [81-84, 109-110]. In the ARBITER-6 study, the addition of
niacin resulted in decreased cIMT [84]. The CETP inhibitor torcetrapib has been
investigated in two cIMT trials (Rating Atherosclerotic Disease Change by Imaging with
a New CETP Inhibitor [RADIANCE] 1 and 2 [111-112]); however, treatment with this
potent HDL-C raising agent either did not change cIMT (RADIANCE 2) or increased it
(RADIANCE 1). These findings were in agreement with the outcome of the morbidity
and mortality study (ILLUMINATE) in which an increase in all-cause mortality was
observed in the torcetrapib group [91].
MRI has emerged as one of the leading in vivo imaging non-invasive techniques
for atherosclerosis, because it enables visualization of all stages of atherosclerosis. For
example, vessel wall thickening by MRI reflects the accumulation of lipids in the intima
of the vessel wall as seen in the earlier stages of atherosclerosis, whereas identification of
lipid-rich necrotic core depicts advanced atherosclerosis [103-104]. MRI of vessel wall
16
thickness has also been used to investigate the efficacy of HDL-C–raising therapies
[108]. The effect of niacin on MRI quantified carotid atherosclerosis progression was
investigated in a double-blind, randomized placebo-controlled in statin-treated subjects
[113]. For example, MRI scans were performed at baseline and after one year follow-up
in patients with low HDL-C (<40 mg/dL) and either type 2 diabetes with CAD or
carotid/peripheral atherosclerosis and showed that niacin significantly reduced
progression of carotid wall thickness. In addition, the dal-PLAQUE imaging study
investigated the efficacy of the CETP inhibitor dalcetrapib on carotid plaque progression
in a multicenter, randomized placebo-controlled trial. MRI scans was performed at
baseline and after 6, 12, and 24 months of dalcetrapib therapy [101, 114]. By MRI, there
was evidence of a significant reduction in carotid total vessel area, after 24 months, with
dalcetrapib compared to placebo [101, 114].
18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) is a highly
sensitive modality that can be utilized as a surrogate-imaging marker for vessel wall
inflammation [115]. The FDG-PET/CT data showed a reduction in carotid inflammation
with dalcetrapib compared to placebo. Increases in HDL-C correlated with reductions in
carotid inflammation and reductions in carotid inflammation by FDG-PET appeared to be
associated with subsequent attenuation of structural changes on MRI [101, 114].
Interestingly, markers of systemic inflammation did not decrease in the dalcetrapib
group, underscoring the poor correlation between systemic and vessel wall inflammation.
This suggests that it is crucial to assess the effect of drugs on vessel wall inflammation
and cardiovascular outcome.
17
Conclusions
The cardioprotective effects of HDL are strongly suggested by the consistent inverse
relationship between plasma HDL-C levels and CAD risk in observational studies. In
contrast to the intervention studies that have directly established LDL as a causative
factor in atherosclerosis progression and CAD risk, comparable clinical studies for HDL
have been hampered by the lack of potent and specific drugs akin to statins. Based on the
pre-clinical and human HDL studies to date (summarized in Table 1), a general pattern is
emerging that when HDL levels are increased, either by stimulating its production or by
providing it by infusion, benefits on plaques result. Research has revealed that HDL has
multiple functions including playing a pivotal role in RCT, endothelial protection,
bearing anti-inflammatory and anti-oxidant properties, as well as promoting the
regression of lesions (Figure 2, Figure 3). Yet, several recent pharmacological and
genetic studies have failed to demonstrate that increased plasma levels of HDL-C resulted
in decreased cardiovascular disease risk, giving rise to a controversy regarding whether
plasma levels of HDL-C reflect HDL function, or that HDL is even as protective as
assumed. The evidence from preclinical and (limited) clinical studies shows that HDL
can promote the regression of atherosclerosis when the levels of functional particles are
increased from endogenous or exogenous sources. The data show that regression results
from a combination of reduced plaque lipid and macrophage contents, as well as from a
reduction in its inflammatory state. Although more research will be needed regarding
basic mechanisms and to establish that these changes translate clinically to reduced
cardiovascular disease events, that HDL can regress plaques suggests that the recent trial
failures do not eliminate HDL from consideration as an atheroprotective agent but rather
18
emphasizes the important distinction between HDL function and plasma levels of HDLC. With the availability of a number of animal models, including those reported by us
and the others described in this article, it is hoped that the mechanistic bases for the
effects of HDL on plaque size, composition, and inflammatory state can be unraveled at
progressively deeper levels.
Acknowledgments
This work was supported by NIH grants HL-084312, HL-098055 (EAF) and NIH
fellowship AG-029748 (JEF).
19
Author
Species
Badimon et al.
Rabbit
1990 [48]
(New
Approach/drug
Dosage
Administration
Plaque site
Main findings in plaques
HDL-VHDL
50 mg
i.v., weekly over 4
Total aorta surface
Extent of fatty streaks 
Aortic lipid accumulation (TC, FC and PL) 
weeks
Zealandw
hite
rabbits)
Parolini et al. 2008
[116]
Shah et al. 2001
[117]
Cho et al. 2009
[118]
Rabbit
(New
apoA-IMilano ( ETC-
1) 5 mg/kg
5 i.v. injections,
Carotid arteries
Atheroma volume  with 3 highest dosages
216)
2) 10 mg/kg
every 4 days
(assessed by IVUS and
Significant regression after 2 nd administration of 150
MRI)
mg/kg
Aortic root (48 hours
Lipid content 
post injection)
Macrophage content 
Aortic root
Lipid content: rHDL, V156K , R173C 
2) V156K-rHDL
(24h and 48h post
Macrophage content: rHDL , V156K , R173C 
3) R173C-rHDL (apoA-
injection)
Zealandw
3) 20 mg/kg
hite
4) 40 mg/kg
rabbits)
5) 150 mg/kg
Mouse
apoA-IMilano
400 mg/kg
Single i.v. injection
(apoE-/-)
Mouse
(apoE-/-)
1) rHDL
120 mg/kg
Single i.v. injection
IMilano)
Feig et al. 2011
[66]
Mouse
(apoE-/-)
Aortic arch transplant
Aortic arch (7 days post
Plaque size 
into apoE-/- mice
transplant)
Macrophage content 
transgenic for human
M1 macrophages 
apoA-I
M2 macrophages 
CCR7  in plaque macrophages
Rayner et al. 2012
[75]
Mouse
(LDLr-/-)
Anti-miR33
10 mg/kg
1st week: 2 s.c.
Aortic root (after 4
Plaque size 
injections, followed
weeks of treatment)
Macrophage content 
by weekly
Lipid content 
injections
Collagen content 
20
M1 macrophages 
M2 macrophages 
Table 1: Selected HDL and apoA-I regression studies
ACS = acute coronary syndrome; FC = free cholesterol; i.v. = intravenous; IVUS = intravascular ultrasound; n.s.= non significant; PAD = peripheral artery disease; PL
= phospholipids; reconstituted high-density lipoprotein = rHDL; s.c. = subcutaneous; SFA = superficial femoral artery; TC = total cholesterol; ERASE = Effect of rHDL
on Atherosclerosis - Safety and Efficacy.
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