nature cardiovascular research
Review article
https://doi.org/10.1038/s44161-024-00569-y
Chronic inflammation and vascular cell
plasticity in atherosclerosis
Received: 27 September 2023
Alexander Lin1,2, Joseph M. Miano
, Edward A. Fisher
3
4,5
& Ashish Misra
1,6
Accepted: 10 October 2024
Published online: 9 December 2024
Check for updates
Vascular smooth muscle cells, endothelial cells and macrophages undergo
phenotypic conversions throughout atherosclerosis progression, both
as a consequence of chronic inflammation and as subsequent drivers of it.
The inflammatory hypothesis of atherosclerosis has been catapulted to
the forefront of cardiovascular research as clinical trials have shown that
anti-inflammatory therapy reduces adverse cardiovascular events. However,
no current therapies have been specifically designed to target the phenotype
of plaque cells. Fate mapping has revealed that plaque cells convert to
detrimental and beneficial cell phenotypes during atherosclerosis, with
cumulative evidence highlighting that vascular cell plasticity is intimately
linked with plaque inflammation, ultimately impacting lesion stability.
Here we review vascular cell plasticity during atherosclerosis in the context
of the chronic inflammatory plaque microenvironment. We highlight the
need to better understand how plaque cells behave during therapeutic
intervention. We then propose modulating plaque cell phenotype as an
unexplored therapeutic paradigm in the clinical setting.
Atherosclerotic cardiovascular disease, the primary cause of myocardial infarction and stroke, is the primary cause of mortality worldwide.
Ischemic heart disease and stroke are responsible for approximately
200 deaths per 100,000 each year1, and numerous individuals are left
living with disabilities2. This trend is expected to continue in the coming decades, placing substantial strain on global healthcare3. Recent
technological advancements in diagnostic and management options
have been countered by epidemiological shifts that have maintained
disease burden, such as increased life expectancy, widespread obesity
and an increased disease prevalence in lower-income countries where
healthcare is harder to access4. Thus, there is a need to better understand disease mechanisms and develop more efficacious treatments.
Excessive lipid accumulation within the intimal layer of arterial
beds initiates a decades-long process of atherogenesis, which results
in advanced atherosclerotic plaques that may cause myocardial infarction and stroke when they rupture. Plaque instability, and therefore the
likelihood of rupture, is dictated by the plaque microenvironment and
the cells that reside within it. Myofibroblasts compose the protective
fibrous cap and secrete collagen to promote mechanical stability of the
plaque. Immune cells constitute the thrombogenic plaque core and
generally destabilize plaques via proinflammatory cytokine secretion,
matrix metalloproteinase (MMP) secretion and various other processes. However, genetic fate mapping techniques have redefined our
understanding of plaque development, revealing extensive vascular
cell plasticity. This plasticity allows many cells to adopt phenotypes
that can be both protective and detrimental during the progression of
plaque development. The therapeutic targeting of such cell behavior
is likely to improve patient outcomes but has yet to be investigated in
a clinical setting.
Statins have remained the gold standard for medical management of atherosclerotic cardiovascular disease for decades. However,
while efforts to control plasma lipid levels have markedly improved
patient outcomes, many patients do not respond to intensive statin
therapy, and of those on this regimen, many still exhibit residual risk5.
Atherosclerosis and Vascular Remodelling Group, Heart Research Institute, Sydney, New South Wales, Australia. 2School of Biomedical Engineering,
Faculty of Engineering, The University of Sydney, Sydney, New South Wales, Australia. 3Vascular Biology Center, Medical College of Georgia at Augusta
University, Augusta, GA, USA. 4Division of Cardiology, Department of Medicine, New York University Grossman School of Medicine, New York, NY, USA.
5
Cardiovascular Research Center, New York University Grossman School of Medicine, New York, NY, USA. 6Faculty of Medicine and Health, The University
of Sydney, Sydney, New South Wales, Australia.
e-mail: ashish.misra@hri.org.au
1
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https://doi.org/10.1038/s44161-024-00569-y
Monocyte
adherence and
trans-migration
through the
endothelium
observed
(Poole & Florey)
Inflammation
suggested to
directly drive
atherosclerosis
(Virchow)
1858
1913
1950
1958
Foam cell
identification
(Anitschkow)
Morphological
suggestions of
EndoMT
(Altschul)
The CANTOS trial proves
anti-inflammatory
therapy improves patient
cardiovascular outcomes
independently of lipid
lowering (Ridker et al.)
The JUPITER trial
suggests lowering
inflammation in
normolipidemic
patients improves
patient outcomes
(Ridker et al.)
Light microscopy
suggests a VSMC
origin of foam
cells (Altschul)
Leukocyte
heterogeneity
in plaques is
established
(Jonasson et
al.)
1973
The
Response
to Injury
Hypothesis
is proposed
(Ross &
Glomset)
1985
1994
VSMC clonal
expansion and
trans-differentiation
demonstrated
using genetic fate
mapping (Feil et al.)
Ccl2 and Ccr2
deficiencies are
shown to impair
atherogenesis
(Gu et al.,
Boring et al.)
1998
CRP is shown
to predict
poor patient
outcomes
(Liuzzo et al.)
2003
2008
2010
2014
2015
The COLCOT study
shows anti-inflammatory
success with colchicine
(Tardif et al.)
2017
EndoMT
shown to drive
inflammation
(Chen et al.)
VSMCs shown
to adopt
macrophage
gene
expression
(Rong et al.)
The importance
of the NLRP3
inflammasome in
atherosclerosis is
established
(Duewell et al.)
The fibrous cap is
shown to be formed
from multiple cell
sources (Newman et al.)
2018
2019
IL-1 signaling
suggested to
be protective
for VSMC
transdifferentiation
(Gomez et al.)
2020
2021
A multipotent
VSMCderived
transitional
phenotype
discovered
(Alencar et
al., Pan et al.)
2023
Colchicine is
FDA approved
as the first antiinflammatory
therapy for
atherosclerosis
Inflammation
Vascular cell plasticity
Inflammation and plasticity
Fig. 1 | Historical timeline of key discoveries in studies investigating
inflammation and vascular cell plasticity in atherosclerosis. Inflammation has
long been considered a driver of atherogenesis, but therapeutic interventions
targeting inflammatory pathways have only recently emerged. Vascular cell
plasticity within the plaque has been suspected for decades and has recently
gained traction with genetic fate mapping techniques. Discoveries related to
inflammation6–8,126,174–180 are shown in blue; discoveries related to vascular cell
plasticity29,34,35,181,182 are shown in yellow; discoveries related to both inflammation
and vascular cell plasticity40,59,92,181 are shown in green. CRP, C-reactive protein.
This remaining risk profile is largely attributed to residual inflammatory risk, which has a larger association with adverse cardiovascular events than any residual dyslipidemia6. Thus, the recent
cardiovascular benefits associated with anti-inflammatory therapy,
hypothesized for decades, represent a new era in cardiovascular
therapy7–9 (Fig. 1).
In this Review, we explore the fate of vascular cells during atherogenesis in the context of the chronic inflammatory plaque milieu. We
propose that targeting cell fate is a clinically uncharted strategy that
may aid in resolving inflammation and improving patient outcomes.
Colchicine and a new indication allowed by the FDA
Inflammation in atherosclerosis
The CANTOS trial was a seminal proof of principle
Inflammation has emerged as a pivotal player throughout atherogenesis, from the earliest recruitment of leukocytes to the rupture
of unstable lesions10. However, despite extensive preclinical data,
clinical support for this inflammatory hypothesis of atherosclerosis has only recently been acquired. The seminal Canakinumab
Anti-inflammatory Thrombosis Outcomes Study (CANTOS) trial was
crucial in demonstrating that targeting inflammation could improve
cardiovascular outcomes—albeit modestly—beyond traditional secondary prevention measures (statins, aspirin and other medications)7.
Patients with myocardial infarction with residual inflammatory risk
(high-sensitivity C-reactive protein (hsCRP) ≥2 mg l−1) were treated
with varying doses of a monoclonal interleukin-1β (IL-1β) antibody
in addition to statin therapy. Suppression of the proinflammatory
cytokine IL-1β caused a reduction in hsCRP and IL-6 levels, as well
as a modest 15% reduction in major adverse cardiovascular events,
independently of any lipid-lowering effects. However, patients
experienced adverse side effects related to systemic inflammatory
suppression7. These modest benefits and significant adverse outcomes probably contributed to the US Food and Drug Administration
(FDA)’s rejection of canakinumab for patients with atherosclerotic
cardiovascular disease.
The microtubule-disrupting drug colchicine has recently received
FDA approval for atherosclerotic cardiovascular disease, marking a
significant milestone in anti-inflammatory therapy. Colchicine (brand
name Lodoco) is prescribed as a long-term treatment at an oral dose
of 0.5 mg per day, for patients with atherosclerosis or multiple cardiovascular disease risk factors. The COLCOT (Colchicine Cardiovascular
Outcomes Trial) and LoDoCo2 (Low-Dose Colchicine 2) clinical trials
were pivotal in demonstrating that colchicine could reduce adverse
cardiovascular events—within 30 days of myocardial infarction in the
COLCOT trial and for patients with stable coronary atherosclerosis
in the LoDoCo2 trial8,9. Interestingly, a meta-analysis of the earlier
colchicine trials found a 46% reduction in risk of stroke, compared
with a 22% reduction for myocardial infarction and a 23% reduction
for coronary revascularization11, which has prompted the hypothesis
that colchicine may be more effective in patients with stroke. However,
the recent CHANCE-3 (Colchicine in High-Risk Patients with Acute
Minor-to-Moderate Ischemic Stroke or Transient Ischemic Attack)
and CONVINCE (Colchicine for Prevention of Vascular Inflammation in
Non-Cardioembolic Stroke) trials found no reduction in in adverse cardiovascular events in the short term (~90 days) or long term (~3 years),
respectively12,13. It should be noted that, upon on-treatment analysis
in the CONVINCE trial, a decline in adverse events was observed in the
colchicine group, suggesting that longer-term colchicine treatment
is necessary to observe patient benefits. Furthermore, the subgroup
of patients known to have atherosclerosis demonstrated a greater
reduction in adverse cardiovascular events with colchicine, which may
indicate that colchicine has less efficacy in patients with stroke from
a nonatherosclerotic origin. More studies are needed to determine
which patient populations benefit most from colchicine intervention.
The mechanisms by which colchicine improves patient outcomes
are unclear. Colchicine inhibits microtubule polymerization, which
is thought to interfere with activation of the NLRP3 inflammasome14
and subsequent IL-1β production15, ultimately initiating a host of
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anti-atherosclerotic effects16. However, given that canakinumab also
suppresses IL-1β, the relative success of colchicine is possibly due to
pleiotropic atherosclerotic benefits beyond NLRP3 inhibition. Colchicine has been shown to suppress neutrophil activation17,18 and subsequent extracellular trap formation18, and to reduce foam cell formation
by attenuating the uptake of oxidized low-density lipoprotein (LDL)19.
It has also been suggested to contribute to the promotion of a stable
fibrous cap and a collagen-secreting phenotype in vascular smooth
muscle cells (VSMCs), independently of its typical anti-inflammatory
properties20. Further identification of the protective features of colchicine will thus enable the development of better targeted therapies
that may avoid unwanted side effects.
What should we be targeting with anti-inflammatory therapy?
Numerous anti-inflammatory clinical trials have not shown as much
promise as colchicine or canakinumab. Notably, methotrexate therapy in the CIRT (Cardiovascular Inflammation Reduction Trial) study
did not improve cardiovascular outcomes or reduce levels of hsCRP,
IL-1β or IL-621. However, patients had a baseline hsCRP concentration of
1.53 mg l−1, that is, they had no residual inflammatory risk, which may
explain the lack of anti-inflammatory effect. Notably, methotrexate has
shown cardiovascular benefits in patients with arthritis and psoriasis22—
patients with systemic inflammation, which suggests that further study
is needed in patients with atherosclerosis with hsCRP ≥2 mg ml−1.
The main question that arises is what makes some antiinflammatory therapies more effective than others. One likely possibility is that some inflammatory pathways are more important in atherosclerosis than others and that targeting these pathways will yield better
patient outcomes. Cardiovascular benefits have been seen with canakinumab and colchicine, which both interfere with the IL-1 inflammatory
axis. The NLRP3 small-molecule inhibitor MCC950 has shown promise
attenuating plaque burden in preclinical models23, but clinical trials
for patients with arthritis with this molecule have been halted owing
to liver toxicity. Downstream IL-6 pathway inhibition with the anti-IL-6
monoclonal antibody ziltivekimab has been shown to reduce levels of
inflammatory biomarkers in the RESCUE (Trial to Evaluate Reduction in
Inflammation in Patients with Advanced Chronic Renal Disease Utilizing
Antibody-Mediated IL-6 Inhibition) trial24. Thus, it will be interesting
to see the results of the ongoing ZEUS (Effects of Ziltivekimab versus
Placebo on Cardiovascular Outcomes in Participants with Established
Atherosclerotic Cardiovascular Disease, Chronic Kidney Disease and
Systemic Inflammation) trial investigating these effects in patients with
atherosclerosis and chronic kidney disease. This will help establish the
efficacy and safety of IL-6 inhibition in these patients with atherosclerosis and whether patients with a chronic kidney disease comorbidity
respond to anti-inflammatory therapy.
Other inflammatory pathways should not be overlooked as potential clinical targets. Atherogenesis still occurs even after genetically
suppressing NLRP3-mediated inflammatory pathways25, and it would be
interesting to investigate how inflammation changes in these plaques.
That is, how does cytokine production differ within these plaques and
do other proinflammatory cytokines, such as tumor necrosis factor
(TNF) or interferon-γ (IFNγ), compensate when the IL-1 axis is interrupted? This will provide insight into the relative importance of the IL-1
axis during atherogenesis, how cytokine production responds during
anti-inflammatory therapy and whether targeting multiple proinflammatory cytokines could be a better therapeutic approach in the future.
Fig. 2 | Inflammation and vascular cell plasticity during atherosclerosis
progression. a, In healthy arteries, ECs within regions with low shear stress in
the vasculature display signs of activation, including expression of adhesion
molecules. b, In early atherosclerosis, upon exposure to retained lipoproteins,
ECs undergo further expression of adhesion molecules and secretion of
cytokines. Monocytes adhere to the endothelium and transmigrate into the
plaque, where they differentiate into macrophages. These macrophages undergo
https://doi.org/10.1038/s44161-024-00569-y
Another possibility is that nonspecific compounds, such as colchicine and methotrexate, have pleiotropic effects that may promote or
hinder their therapeutic effects. Regardless of which anti-inflammatory
therapies have provided cardiovascular benefits, these large-scale
clinical trials have demonstrated that targeting inflammation in atherosclerosis is a therapeutic strategy. Identification of why some therapies are more successful than others, and how to limit their off-target
side effects, will enable better drug development. We need a better
understanding of how these treatments affect atherosclerosis at a
cellular level, which is expected to come from preclinical research.
This is especially necessary because most studies on inflammation
and anti-inflammatory therapy in atherosclerosis focus on immune
cell populations and the hematopoietic bone marrow, with minimal
regard to the fate of VSMCs and endothelial cells (ECs).
VSMC plasticity in atherosclerosis
VSMCs populate the medial wall and clonally expand to form
the fibrous cap
VSMCs, identified by various contractile apparatus proteins such as
smooth muscle myosin heavy chain 11 (MYH11) and smooth muscle α2
actin (ACTA2), populate the medial layer of healthy arteries and regulate
vascular tone (Fig. 2a). These cells display a remarkable capacity for
clonal expansion and transdifferentiation in a variety of cardiovascular
diseases, such as atherosclerosis and aneurysm26.
During the earliest stages of atherogenesis, a VSMC clonal progenitor proliferates and migrates from the medial wall into the intimal layer,
surrounding the accumulated macrophages and forming the fibrous
cap (Fig. 2b)27. The mechanisms underlying why VSMCs clonally expand
remain unknown. Nevertheless, once in the fibrous cap, VSMC-derived
cells express platelet-derived growth factor receptor-β (PDGFRβ)27, a
tyrosine kinase receptor involved in VSMC recruitment during vascular
development28. In a similar manner, platelet-derived growth factor
(PDGF) signaling regulates VSMC investment in and maintenance of
the fibrous cap29 as it promotes VSMC proliferation and synthesis
of extracellular matrix proteins30. These protective fibrous cap cells
display a myofibroblast-like phenotype in which they secrete collagen
for plaque stability and are generally identified by their expression of
ACTA2, although this occurs to a lesser extent than in mature medial
wall VSMCs.
ACTA2 is an actin isoform involved in muscular contraction that
is predominantly and abundantly expressed in VSMCs. Beyond its
role as a contractile protein, ACTA2 has also recently been shown to
translocate into the nucleus, where it binds to VSMC gene promoters,
enhancing contractile gene expression and propagating the contractile
phenotype31. In atherosclerosis, ACTA2 has been used extensively to
identify protective collagen-secreting cells in the fibrous cap, historically believed to be of VSMC origin based on staining for this marker.
However, recent lineage tracing experiments have revisited this notion,
identifying transdifferentiated ECs and macrophages as contributors
to the ACTA2+ fibrous cap29. The importance of this contractile protein
in VSMCs and atherosclerosis is highlighted by studies showing that
mutations in ACTA2 impair VSMC contractility and predispose individuals to multiple premature cardiovascular pathologies in the absence of
traditional risk factors32. For example, the heterozygous p.Arg149Cys
missense mutation promotes VSMC transdifferentiation and atherosclerotic plaque burden throughout the aorta despite comparable lipid
levels33. Further study is necessary to elucidate how these mutations
local proliferation and polarize to a variety of pro- or anti-inflammatory states.
VSMCs from the vascular wall clonally expand and migrate, forming the fibrous
cap of the plaque. c, In late atherosclerosis, as the plaque progresses, fibrous
cap VSMCs migrate into the core, transdifferentiating into a variety of different
phenotypes, including macrophage-like and osteogenic-like cells, which
destabilize the plaque. ECs undergo EndoMT, where they secrete collagen and
promote inflammation.
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a
Monocytes
Healthy artery
• Low shear stress in
atherosclerosis-prone regions
• Early EC activation
• Lipoprotein retention
Lipoproteins
Adhesion
molecules
ECs
VSMCs
b
Cytokine
secretion
Fibrous
cap
Early atherosclerosis
• EC dysfunction
• Monocyte recruitment
• Macrophage polarization
• Clonal expansion of VSMCs
to form fibrous cap
Collagen secretion
Foamy
macrophage
Inflammatory macrophage
Myofibroblast-like
VSMCs
Clonal
expansion
Macrophage
Resident
macrophage
c
Late atherosclerosis
• Fibrous cap thinning
• Extensive trans-differentiation
of VSMCs
• Many ECs undergo EndoMT
• Highly inflammatory
microenvironment
Myofibroblast-like
ECs
Collagen
secretion
Macrophage-like
ECs?
Osteogenic-like
ECs?
Stem cell-like
VSMCs
Macrophage-like VSMCs
Osteogenic-like
VSMCs
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affect fibrous cap formation and VSMC behavior, especially because
the mutation used in this model is present from birth and its effects
on blood flow probably confound its effects during atherogenesis.
VSMC cap to core migration
Following accumulation in the fibrous cap, clonally derived
myofibroblast-like cells migrate into the core of the lesion27, further
downregulating contractile gene expression and converting to a transitional stem cell-like phenotype34,35 (Fig. 2c). This transitional phenotype
has been defined as expressing markers of stem cells (Ly6a), activated
ECs (Vcam1) and macrophages (Ly6c1 and Lgals3) and exhibits multipotent capacity34,35. Indeed, a recent dual-lineage approach demonstrated that most VSMC-derived cells within the plaque transition via
this Lgals3+ cell state before contributing to multiple phenotypically
distinct single-cell RNA sequencing (scRNA-seq) clusters, many of
which no longer express this marker35.
These stem-like clones can convert to a variety of phenotypes,
including osteogenic-like and macrophage-like phenotypes34,35
(Fig. 2c), as well as having the potential to revert back to a beneficial
ACTA2+ phenotype34, making them a potential therapeutic target in
the future. The osteogenic-like transition of VSMCs is thought to be a
driving cause of vascular calcification. During this transition, VSMCs
begin to acquire the expression of proteins involved in bone formation, such as RUNX2 and SOX9 (refs. 35–37). These transdifferentiated
VSMCs begin depositing type 2 collagen, followed by hydroxyapatite, in a process mimicking endochondral ossification38. Microcalcifications within the plaque are thought to drive plaque rupture via
increases in locally concentrated tissue stress, while large macrocalcifications provide mechanical stability39. VSMCs also transition to a
phagocytic macrophage-like phenotype in which cells express many
canonical macrophage markers, such as CD68 and CD11b34–36,40–44.
These macrophage-like cells can engulf oxidized lipids and contribute to a large majority of foam cells within the plaque45,46. While these
transdifferentiated macrophage-like cells mimic many functions of
bona fide macrophages, they lack the same phagocytic capacity as
true macrophages41. Whether VSMC-derived foam cells behave similarly to macrophage-derived foam cells remains to be seen. Other
VSMC-derived phenotypes have been identified, such as T cell-like35,
adipocyte-like36 and pericyte-like43 cells, but their role within the plaque
is not yet known.
The signaling mechanisms underlying VSMC plasticity are not
completely understood, and different mechanistic pathways are
altered as VSMCs adopt different lineages. However, the transforming growth factor-β (TGFβ) pathway has a central role during VSMC
transdifferentiation, as it is well known to regulate VSMC identity. In
the context of atherosclerosis, genetic deletion of Tgfbr2 and Smad3
in mature VSMCs promotes aberrant transdifferentiation and plaque
burden36,37,44. Mechanistically, the downstream TGFβ mediators SMAD2
and SMAD3 directly bind to regulators of Krüppel-like factor 4 (KLF4),
a transcription factor involved in stem cell behavior, leading to KLF4
suppression36. KLF4 represses myocardin, the principal driver of
smooth muscle cell differentiation47, and disrupts myocardin–serum
response factor (SRF) binding to CArG elements, preventing activation of promoters regulating VSMC gene expression48. Klf4 deletion
in VSMCs improves plaque stability and reduces the proportion of
detrimental VSMC phenotypes35,36,49. However, therapeutic targeting of
this signaling pathway has not yet been achieved. It would be interesting to see how temporal deletion of Klf4 in the advanced plaque affects
VSMC behavior, which may somewhat mimic interventional targeting
in a clinical setting. Crucially, how inflammatory pathways affect KLF4
expression is not well understood, and how inflammation ultimately
influences the process of VSMC transdifferentiation will be discussed
in the subsequent section.
Fate mapping and single-cell transcriptomics have been used
to show that VSMCs have critical roles throughout mouse plaque
https://doi.org/10.1038/s44161-024-00569-y
pathogenesis, from initial clonal expansion to subsequent transdifferentiation. However, despite the clear importance of VSMCs throughout atherogenesis, VSMCs remain a clinically unexplored target for
improving patient outcomes. The various protective and detrimental
functions of different VSMC phenotypes and the varying functions
of targetable proteins and signaling pathways through VSMC phenotypic switching all probably contribute to difficulties in producing
viable VSMC-specific therapies. However, it is likely that targeting
VSMCs at specific times during phenotypic modulation or producing
therapies that are always beneficial in VSMCs will become prominent
in the future.
VSMC phenotype is influenced by inflammation
and anti-inflammatory therapy
IL-1β regulates the VSMC phenotype
VSMCs have important roles in shaping the inflammatory landscape of
atherosclerotic plaques. Under inflammatory conditions, VSMCs begin
expressing adhesion molecules, which aid in immune cell recruitment,
and secrete an array of proinflammatory cytokines, which propagate
the inflammatory cascade. For example, VSMCs express a variety of
chemokines that influence VSMC–monocyte/macrophage crosstalk,
including, but not limited to, CCL2 (ref. 50), CCL5 (ref. 51) and CXCL10
(ref. 51) CCL2 is an important molecule secreted by plaque VSMCs
(among other cells), which is classically known as a chemokine that
promotes monocyte infiltration into the plaque. While global CCL2
knockouts appear to reduce plaque formation52, VSMC-specific deficiencies in CCL2 appear to promote plaque burden50,53. Interestingly,
CCL2 deletion in Lgals3+ VSMC-derived cells promotes plaque stability
and reduces VSMC transdifferentiation, suggesting that CCL2 secretion
from VSMC-derived cells is detrimental after initial lesion formation53.
Research suggests that CCL2 is used by fibrous cap VSMC-derived cells
to keep macrophages localized to the fibrous cap region, where the
interplay between these cells promotes a beneficial macrophage phenotype and limits plaque inflammation50, supporting the idea of different effects of CCL2 at different stages of plaque progression. Further
study is needed to investigate whether CCL2 secretion from fibrous
cap cells helps prevent VSMC-derived cells from undergoing cap to
core migration, given that VSMCs also migrate in response to CCL2.
Interestingly, chemokine secretion from inflamed VSMCs influences not only the plaque milieu, but also the adventitia around the
atherosclerotic vessel. In atherosclerotic blood vessels, medial VSMCs
underneath the plaque begin to express CCL21 and CXCL13, which promote the aggregation of T cells and B cells and subsequent formation of
artery tertiary lymphoid organs (ATLOs) within the adventitia54. ATLOs
contain many different immune cell types, including both inflammatory and disease-protective cells54,55. High endothelial venules of ATLOs
appear to be connected to the medial wall via conduits, and the adjacent
medial wall displays breakages in elastin layers, which suggest crosstalk with the plaque54. However, how ATLOs influence atherosclerotic
disease progression is still not understood.
In addition to promoting inflammation in VSMCs, IL-1β has been
shown to suppress the expression of VSMC contractile genes, suggesting that IL-1β may alter the phenotype of VSMCs beyond the activation
of some inflammatory pathways56. Consistent with this notion, IL-1β
promotes VSMC proliferation and migration57, although probably
in a separate manner to PDGFRβ signaling56. While it is difficult to
attribute VSMC behavior within the plaque solely to IL-1β stimulation,
these in vitro studies support findings that inflammatory Itgb3−/− and
Tet2−/− macrophages regulate the clonal expansion and subsequent
transdifferentiation of VSMCs27,58.
Given the chronic inflammatory nature of atherosclerosis, the
results of the CANTOS trial and the impact of IL-1β on VSMC behavior,
it was hypothesized that suppressing IL-1β should improve VSMC phenotype. Surprisingly, Gomez et al.59 observed that IL-1β suppression in
advanced plaques caused a reduction in ACTA2+ fibrous cap thickness, a
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loss of VSMC-derived ACTA2+ myofibroblast-like cells and an associated
decline in collagen content, despite beneficial effects on inflammatory
pathways. Unexpectedly, treatment with anti-IL-1β antibody promoted
VSMC transdifferentiation to an osteogenic-like phenotype and abrogated any beneficial changes made by diet management60. These findings suggest that IL-1β signaling may have a role in maintaining fibrous
cap composition by VSMCs and that, while anti-inflammatory therapy
may be beneficial in the immune compartment, this may not negate
harmful effects in the VSMC population. Taken in context, the protective role of IL-1β in VSMC-derived cells may be a contributing factor
explaining why canakinumab has only led to modest cardiovascular
benefits in clinical trials7 and suggests that perhaps future IL-1β therapies should be targeted specifically to immune cells.
Interestingly, in atherosclerotic Jak2- or Tet2-mutant clonal
hematopoiesis models, IL-1β suppression promoted thickening of
the ACTA2+ fibrous cap, but VSMC-derived cells were not responsible
for this increase61. Consistent with the aforementioned studies59,60,
the proportion of VSMC-derived cells in the fibrous cap declined61,
highlighting the importance of non-VSMC-derived fibrous cap cells in
plaque stability29. These studies further highlight the need to establish
which patients will be responsive to anti-inflammatory therapy, as
these findings suggest that inhibiting IL-1β is beneficial only in patients
with intensely heightened inflammasome activity. It is also necessary
to determine from where the increases in ACTA2+ fibrous cap cells are
derived, as these cells could potentially be therapeutic targets in the
future. Deletion of Il1r1 in ECs29 or GLI1+ mesenchymal stem cells61 did
not promote their expansion or transdifferentiation into ACTA2+ cells
in the fibrous cap, which potentially suggests that these cells are not
responsible for the increases in fibrous cap thickness in IL-1β-inhibited
clonal hematopoiesis plaques61. However, anti-IL-1β antibody antagonism in a clinical setting is not cell type specific. Using these genetic
approaches may avoid the potential for IL-1β suppression to cause
paracrine effects from other cell types, which could lead to fibrous cap
stabilization by ECs or cells from other sources.
By contrast, colchicine has been suggested to promote the ACTA2+
myofibroblast-like phenotype in VSMC-derived cells, thereby improving fibrous cap thickness and increasing collagen secretion20. Given
that colchicine also suppresses IL-1β, albeit indirectly via the NLRP3
inflammasome, the comparison with previous findings suggests an
additional effect beyond IL-1β. Indeed, direct stimulation of VSMCs with
colchicine promoted the expression of VSMC genes in the absence of
any inflammatory stimulus, an effect that could not be mimicked using
MCC950 (ref. 20). Remarkably, the increase in ACTA2+ VSMC-derived
cells was suggested to be due to a regression in the abundance of
CD68+ macrophage-like and RUNX2+ osteogenic-like cells20. While
this should be further validated using dual-lineage tracing techniques,
previous supporting findings suggest that colchicine reduces macrophage foam cell formation via downregulation of the CD36 scavenger
receptor19, which could also be hypothesized to apply to VSMC-derived
macrophage-like foam cells62. It would also be very interesting to determine how much benefit can be attributed to anti-inflammatory effects
and to plasticity-altering effects during atherosclerotic plaque regression. Nevertheless, the combination of anti-inflammatory therapy
with beneficial effects on VSMC phenotype probably contributes to
colchicine’s relative success in the clinic.
Vascular smooth muscle-derived macrophage-like cells
VSMCs can accumulate lipids and transition to a macrophage-like
phenotype34,35,45,46,49, in which they are traditionally thought to promote plaque instability via proinflammatory activity. However, several
scRNA-seq studies have led to some controversy surrounding this phenotypic transition, with some groups not identifying this population
of cells29,63–65, whereas others suggest quite an abundance34,42. It has
been proposed that the cause of these inconsistencies in scRNA-seq
data is doublet incorporation and discrepancies in gating strategies,
https://doi.org/10.1038/s44161-024-00569-y
which result in an overestimation of VSMC-derived macrophage-like
cells65. Other possible contributors to this variability include potential
bias against macrophages and macrophage-like cells during sample
preparation66,67 and/or undersampling of plaque cells—and hence
transdifferentiated VSMC clusters—when the entire blood vessel is
digested, as opposed to just the atherosclerotic lesion. While there is
no doubt that VSMCs accumulate lipids and form foamy cells45,46,63, the
crux of this ambiguity seems to be surrounding the degree to which
VSMCs become ‘macrophage-like’. Clustering alongside bona fide
macrophages in scRNA-seq would suggest that these VSMC-derived
cells recapitulate macrophages across the transcriptome rather than
expressing only a few macrophage genes. These issues are potentially
further confounded by reports that tissue dissociation procedures
activate macrophages67. However, whether this is prevalent in other
vascular cells is not yet known. Further study is thus necessary to resolve
these ambiguities, which will probably involve the use of spatial transcriptomics to better understand the localization of these cells and
how they influence the plaque microenvironment.
Nevertheless, it has been suggested that, when VSMCs undergo
this macrophage-like conversion, they adopt multiple features of
macrophages, including presenting a degree of phenotypic heterogeneity seen in bona fide macrophages42. Indeed, in a meta-analysis
of VSMC fate mapping studies, VSMC-derived cells were found to
contribute to a large proportion of foamy macrophage clusters, half
of inflammatory macrophages and a significant proportion of resident macrophages42. It is generally accepted that improving plaque
stability relies on reverting macrophage-like (and other detrimental
phenotypic) VSMC-derived cells back into a protective ACTA2+ phenotype. However, it would also be interesting to determine whether
we can modulate these VSMC-derived macrophage-like polarization
states and promote proresolving macrophage-like phenotypes to
induce beneficial changes in plaques. For example, it is interesting to
note that foamy VSMC-derived cells express Trem2 (refs. 42,68), which
has a role in efferocytosis, a key pathway that is increasingly defective
during lesion development69. Apoptotic VSMCs and macrophages
contribute to intraplaque inflammation and lesion vulnerability70.
Restoring efferocytosis is thought to reduce plaque burden and
improve patient outcomes71–73, but whether VSMC-derived cells contribute during this process has not been well established. In this regard,
cholesterol-loaded VSMCs exhibit lower efferocytotic activity than
authentic macrophages41, but whether we can therapeutically promote
efferocytosis by VSMC-derived cells is not yet known.
Vascular smooth muscle-derived osteogenic-like cells
Both inflammation and plaque calcification are well correlated with
the degree of atherosclerotic plaque burden39,74. It has been shown that
inflammatory macrophages are dominant paracrine drivers of VSMC
transdifferentiation to osteogenic-like cells via IL-1β (and probably
other cytokines)74. As such, IL-1 receptor antagonism in developing
plaques reduced lesion size and plaque calcification74. By contrast,
IL-1β suppression in advanced lesions promoted the phenotypic conversion of VSMCs to osteogenic-like cells and increased the number
of calcified lesions60. To reconcile these differences, it is highly likely
that IL-1 inhibition is more effective at preventing calcification in earlier stages of lesion development, before VSMCs have transdifferentiated. Thus, further investigation is necessary to determine whether
anti-inflammatory intervention should be prescribed at specific times
during atherogenesis.
Given the clear importance of paracrine influences from macrophages on VSMCs and reports that IL-1β suppression may promote a plaque-resolving phenotype59, it would also be interesting to
investigate how VSMC transdifferentiation is affected by different
macrophage polarization states and whether they may promote beneficial macrocalcification. Indeed, M2 polarization has been shown
to promote the osteogenic-like differentiation of mesenchymal stem
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cells75 and MC3T3 cells76. In a similar paracrine manner, osteogenic-like
VSMCs have been suggested to promote the phenotypic switching of
bone marrow macrophages into an osteoclast-like phenotype, which
may help regulate plaque calcification77,78.
Taking these findings together, it has become clear that VSMCs
exhibit a vast degree of cellular plasticity during atherogenesis, contributing to both protective and detrimental populations within the
plaque. Inflammatory pathways appear to have a key role in regulating VSMC phenotype, which in turn further influences the inflammatory milieu. Current anti-inflammatory therapies appear to affect
VSMC plasticity in preclinical models, but whether this is applicable
in patients has yet to be seen. Nevertheless, while VSMC plasticity has
been underappreciated in a clinical context, mouse modeling suggests
that it should be targeted in the future.
EC plasticity in atherosclerosis
ECs line the intimal layer of blood vessels and mediate the
earliest stages of atherogenesis
The vascular endothelium is a monolayer structure covering the arterial intima and is intimately involved in blood vessel homeostasis, as
well as most vascular pathologies. In healthy arteries, ECs exhibit tight
cell–cell junctions, mediating the permeability of the vascular wall.
Indeed, two of the most common proteins used to identify mature ECs
are platelet/EC adhesion molecule 1 (PECAM1) and vascular endothelial
cadherin (CDH5), two adhesion molecules that have integral roles in
maintaining the junctional contacts between ECs.
The phenotypic heterogeneity of ECs commences long before the
onset of atherosclerosis. Hemodynamic flow regulates the mechanical
microenvironment of ECs throughout the vasculature, which in turn
mediates disease progression. For example, intercellular adhesion molecule 1 (ICAM-1) is expressed within the endothelium of nonatherogenic
mice and healthy people predominantly in regions of low shear stress,
which are more prone to plaque formation79 (Fig. 2a). This observation
may explain the accumulation of intimal resident macrophages in
regions of disturbed flow80.
During atherogenesis, modification of accumulating LDLs induces
endothelial dysfunction and the initiation of proatherogenic processes
(Fig. 2a,b). Modified LDLs increase the capacity for transportation of
macromolecules and leukocytes across the endothelium via alteration
of EC junctions81 and induction of apoptosis82,83. Leukocyte recruitment
is enhanced via modified LDL-induced EC chemokine secretion84,85 and
adhesion molecule presentation86–89 (Fig. 2b). These maladaptive processes are maintained throughout atherogenesis, contributing to the
continual recruitment of monocytes and propagation of the inflammatory cascade. As such, many of these pathways have been suggested as
potential targets for the therapeutic treatment of atherosclerosis90,91.
ECs transition to a mesenchymal-like phenotype in
advanced plaques
In advanced lesions, ECs undergo endothelial to mesenchymal transition (EndoMT), in which ECs acquire mesenchymal characteristics at
the expense of their canonical EC identity (Fig. 2c). Cells undergoing
EndoMT begin to lose their tight junctional contacts and typical cobblestone morphology, alongside suppression of EC marker proteins.
The transition to a mesenchymal-like phenotype is characterized by
increased migratory capacity and a more elongated VSMC-like morphology, with upregulation of many mesenchymal proteins, including
ACTA2, fibroblast-specific protein-1 and vimentin. This phenomenon is
well correlated with atherosclerotic plaque burden92–94, with estimates
using fate mapping approaches suggesting that over 30% of luminal ECs
undergo EndoMT in advanced mouse lesions92. Likewise, approximately
70% of ECs in severe human coronary plaques have been suggested to
be undergoing EndoMT92, which is probably an underestimate given
the lack of fate mapping techniques to analyze human pathology.
These discrepancies are probably due to the stable nature of mouse
https://doi.org/10.1038/s44161-024-00569-y
atherosclerotic plaques. Thus, it would be interesting to further investigate the extent and functional consequences of EndoMT in models
of more severe unstable plaques95. Nevertheless, EndoMT is highly
abundant throughout the later stages of atherogenesis, suggesting
key functional consequences in disease progression.
EndoMT is primarily thought to exacerbate plaque inflammation,
with transdifferentiated cells displaying increased expression of adhesion molecules and greater secretion of chemoattractants92,94. The
connection between EndoMT and the inflammatory response will be
further discussed in subsequent sections. EndoMT has also been suggested to increase MMP secretion and promote plaque vulnerability
via degradation of collagen93. Many cell types within atherosclerotic
lesions secrete MMPs, and it is thus necessary to further investigate
whether EndoMT is a significant source. Somewhat paradoxically,
the increased collagen secretion from transdifferentiated cells also
suggests a potential plaque-stabilizing role. In fact, it has been suggested that ~20% of collagen-secreting ACTA2+ myofibroblast-like
fibrous cap cells are derived from the endothelium29. Mice in which
VSMC investment in the plaque was prevented via deletion of Pdgfrb
in VSMCs or lethal irradiation still created collagen-rich lesions, which
were thought to be primarily derived from enhanced expansion of
EC-derived cells. Interestingly, lesional collagen content declined in
later-stage plaques29, which probably suggests that the MMP production and inflammation associated with EndoMT eventually overcome
any protective collagen secretion.
ECs may also transition to other phenotypes within the plaque,
but whether this is via EndoMT or an independent transition is not yet
known. scRNA-seq analysis has suggested that disturbed flow may
induce transitions to an immune cell-like phenotype or a hematopoietic
cell-like phenotype, which probably exacerbates inflammation96. It has
been suggested that ECs form lipid-laden foam cells in vitro97 and may
constitute a small proportion of foam cells within advanced plaques98.
Likewise, there is evidence to suggest that ECs transdifferentiate to an
osteogenic-like state to contribute to vascular calcification in mouse
Mgp−/− models99,100 and with an osteogenic stimulus in vitro101–103, but
evidence for this phenomenon in atherosclerosis is limited104.
Known inducers of EndoMT typically promote this transition
via the TGFβ pathway. For instance, disturbed shear stress, which is
observed in atherosclerosis-prone regions of the vasculature and further induced by plaque growth, is well known to promote EndoMT92,105.
Reduced flow has been shown to suppress production of tenascin X, an
extracellular matrix glycoprotein that directly binds TGFβ ligands and
suppresses their interaction with TGFβ receptors105. Low shear stress
also interferes with homeostatic autophagy106, which typically inhibits
EndoMT-induced inflammation and fibrosis107,108, at least in part via
TGFβ108. This reduced blood flow possibly also contributes to hypoxia
within the plaque, which also drives EndoMT93,109, probably via loss of
fibroblast growth factor signaling110,111 and subsequent downregulation of let-7 microRNAs111. let-7 miRNAs directly reduce the half-life of
TGFBR1 mRNA and suppress TGFβ signaling111. Further mechanistic
understanding of EndoMT is necessary if we are to better manipulate
this phenotypic transition to increase plaque stability.
EC plasticity and inflammation constitute a
proinflammatory cascade
ECs are a critical escalator of chronic inflammation throughout all
stages of atherogenesis. In response to proinflammatory cytokines
and oxidized lipids, ECs increase secretion of proatherogenic cytokines
alongside expression of leukocyte adhesion molecules. These proteins
not only are secreted directly into the extracellular space, but can also
be released and taken up via extracellular vesicles. For example, stimulated ECs can release extracellular vesicles containing ICAM-1, which
deliver ICAM-1 and also promote ICAM-1 expression in other ECs in a
feedforward mechanism to promote monocyte recruitment112. Interestingly, IL-1β, TNF and IFNγ all induce a small degree of EndoMT92,
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which has led to the suggestion that EndoMT is not a separate phenomenon, but rather represents the transition to a highly activated
EC phenotype113. Consistent with this notion, combinations of these
cytokines are more effective at inducing EndoMT than any cytokine
on its own92. Inflammation not only promotes EndoMT, but also has a
critical role in its induction29. EC-specific deletion of Il1r1 reduced the
proportion of EC-derived myofibroblast-like cells in the fibrous cap,
suggesting the importance of IL-1 signaling in EndoMT29. This probably
explains why compensatory EndoMT29 is not seen with antibody-based
IL-1β inhibition59. Nevertheless, the mesenchymal phenotype is highly
inflamed, with cells markedly upregulating proinflammatory cytokine
production. Studies in adipose tissue ECs have suggested that EndoMT
promotes the production of extracellular vesicles and increases
their inflammatory signature, although this has yet to be shown in
atherogenesis114. Extracellular vesicles generally protect their cargo
from degradation and allow communication over longer- distances,
which suggests that these vesicles should be therapeutically targeted
in the future.
It is interesting to note that TGFβ signaling promotes EndoMT
and its associated inflammation, despite largely being considered
an anti-inflammatory cytokine. Nuclear translocation of SMAD2 and
SMAD3 allows their binding to inflammatory regulators within the
genome, which appears to be promoted in ECs, but not in VSMCs94.
As such, EC-specific deletion of Tgfbr1 and Tgfbr2 strongly inhibits progression of atherosclerosis both before and during plaque
development. This was associated with a decline in macrophage and
T cell composition and a decreased inflammatory response to TNF
stimulation. The decline in immune cell composition is probably a
combination of reduced leukocyte recruitment and proliferation94,
which both have key roles in maintaining the intraplaque immune
cell population115–118. Remarkably, depletion of EC TGFβ signaling
led to regression of atherosclerosis when depletion was mediated
by both temporal genetic recombination and nanoparticle delivery
of short interfering RNA models94. It should also be noted that, while
the net effect of inhibiting endothelial TGFβ signaling is probably
beneficial, there were also declines in intraplaque collagen and
ACTA2+ cells.
Therefore, it is evident that ECs and their mesenchymal derivatives
contribute to a chronic inflammatory cascade throughout atherogenesis. Given the apparent contradictory increases in collagen production
and inflammation during phenotypic switching, EndoMT has been
suggested to have both protective and detrimental contributions to
lesion development. As future therapeutics are developed, it will be useful to manipulate EndoMT and only promote the collagen-producing
features in these cells.
Macrophage plasticity in atherosclerosis
Macrophages are phagocytic immune cells that predominantly lie
within the adventitial layer of healthy blood vessels, where they regulate
homeostatic immunity119 and collagen production by VSMCs120. A small
number of macrophages also reside within the intimal layer and help
regulate thrombosis alongside the endothelium80.
During atherogenesis, intimal macrophages form the earliest foam
cells within the nascent lesion121. Bone marrow-derived monocytes
from the circulation are recruited by the activated endothelium and
differentiate into macrophages as they transmigrate into the subendothelial space122 (Fig. 2b). Within the plaque, macrophages accumulate in number, largely, although not entirely, via self-proliferation118.
Lipid engulfment can lead to the formation of foam cells and activate
proinflammatory pathways via the NLRP3 inflammasome123. As such,
macrophages are a dominant source of immune regulation within the
plaque and are a common focus of anti-inflammatory therapy. The role
of macrophages in regulating inflammation has been well reviewed
by others124, and we will only briefly discuss macrophage plasticity in
atherosclerosis.
https://doi.org/10.1038/s44161-024-00569-y
Macrophages display multiple phenotypes that influence atherogenesis, which high-throughput transcriptomics has elucidated
beyond the traditional M1/M2 paradigm. Inflammatory macrophages
express a variety of proinflammatory transcripts, including Il1b, Tnf,
Cxcl1 and Ccl2, and are therefore likely to exacerbate chronic inflammation and promote plaque rupture125. Resident macrophages are
characterized by the expression of Lyve1 and Mrc1, which are traditionally thought to be expressed by yolk sac and tissue-resident
macrophages125. These are probably resident adventitial macrophages,
but whether macrophages become resident-like within the plaque
and their functional consequences remain unclear. Foamy macrophages are large constituents of atherosclerotic plaques, identified by high Trem2 expression and a foamy appearance125. Classical
dogma suggests that foam cells secrete proinflammatory cytokines
due to cholesterol-mediated activation of the NLRP3 inflammasome126.
However, findings in peritoneal foam cells127 and recent scRNA-seq
analysis of intraplaque foam cells98,128 suggest that they are not proinflammatory and instead have a plaque-resolving transcriptional signature. TREM2 has been suggested to regulate foam cell lipid uptake,
survival and efferocytosis, thereby protecting against necrotic core
formation68,128. The inflammatory nature of foam cells is probably
more nuanced than these contrasting viewpoints. For example, recent
transcriptomic analysis of human lesions discovered a PLIN2hiTREM1hi
foam cell cluster that exhibited a proinflammatory phenotype, and
the authors suggest that these cells are derived from TREM2hi foam
cells and postulate that they could correspond to foam cells undergoing apoptosis129. Plaques from mouse models appeared to lack this
apoptotic foam cell population, which may be due to the stable nature
of mouse plaques. Interferon-inducible macrophages express genes
commonly seen in response to type 1 interferons, such as Ifit3 and Irf7,
and are suspected to be proatherogenic in nature125. Further study is
necessary to determine how macrophage phenotypes are changed
with anti-inflammatory therapy, especially as macrophages have a
significant role in regression of atherosclerosis130.
Clinical prospects
Currently used therapies and effects on cellular plasticity and
inflammation
In the current climate of anti-inflammatory therapy built upon a foundation of lipid-lowering agents, no treatments used in the clinic have
been designed to specifically target vascular cell plasticity. However,
multiple treatments have been suggested to alter cellular plasticity,
which may limit or confound their clinical success (Fig. 3). Statins have
seen unmatched success in managing atherosclerotic cardiovascular
disease, primarily owing to the inhibitory regulation of cholesterol
biosynthesis and consequent upregulation of LDL receptors131. Of
note, statins display anti-inflammatory properties in multiple vascular cell types131. Interestingly, statins have been suggested to improve
endothelial dysfunction in diabetic mouse models by epigenetically
suppressing EndoMT132. Consistent with the beneficial effects of statins,
lowering apolipoprotein B levels with antisense oligonucleotides, which
mimic the major benefit of statin therapy, leads to a large reduction in
plaque size, which is accompanied by an increase in protective ACTA2+
VSMC-derived cells and a reduction in pathogenic VSMC-derived
osteogenic-like cells64. This was thought to be caused by decreased
NF-κB signaling. However, the mechanism by which reductions in
apolipoprotein B suppress NF-κB in VSMCs remains to be elucidated.
Colchicine is FDA approved as an anti-inflammatory therapy but
has also been suggested to induce a myofibroblast-like phenotype in
VSMC derivatives, thereby improving plaque stability20. These effects
are not mimicked by IL-1β inhibition59–61, which suggests that using
‘dirty’ pleiotropic therapies—drugs that interact with multiple targets—may offer some benefits. This highlights that resolving inflammation does not always lead to beneficial phenotypic transitions for
VSMCs or ECs.
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Stable plaque
Unstable plaque
↑ Inflammation
↑ Detrimental vascular
cell trans-differentiation
Therapies that may affect inflammation and vascular cell plasticity
Canakinumab
• Inhibits IL-1β
• Promotes VSMC trans-differentiation
Vitamin C
• Improves EC function
• Promotes VSMC contractility?
Rapamycin
• Inhibits mTOR
• Inhibits EC activation
• Promotes VSMC contractility?
ATRA
• Improves EC function
• Reduces platelet inflammation
• Maintains VSMC differentiated state
Colchicine
• Inhibits microtubules
• Inhibits NLRP3 inflammasome
• Inhibits neutrophil activation
• Reduces foam cell formation
• Promotes VSMC contractility?
Statins
• Reduce LDL
• Reduce inflammation
• Promote efferocytosis
• Suppress EndoMT?
Fig. 3 | Vascular cell plasticity is affected by some therapeutic treatments.
No current atherosclerosis therapies are designed specifically to improve
vascular cell plasticity. However, some FDA-approved atherosclerosis therapies
and some treatments to manage other diseases have been suggested to affect
inflammation and plaque cell plasticity in preclinical models.
Therapies for management of other diseases have been suggested
to influence inflammation and cellular plasticity in preclinical models
of atherosclerosis, although these therapies are not currently clinically
prescribed in the setting of atherosclerotic cardiovascular disease
(Fig. 3). All-trans-retinoic acid (ATRA) is a vitamin A derivative currently prescribed for the treatment of acute promyelocytic leukemia.
ATRA exhibits favorable effects on VSMC phenotype, presumably
through its interaction with nuclear receptors that direct changes
in gene expression133. In preclinical rabbit atherosclerosis models,
ATRA has been suggested to reduce plaque burden by attenuating
platelet inflammation134 and improving endothelial function135. In
atherosclerotic mouse models, ATRA appears to inhibit VSMC transdifferentiation, thereby ameliorating the formation of VSMC-derived
macrophage-like cells and reducing adhesion molecule presentation
on VSMC-derived cells34. This is thought to occur via nuclear retinoic acid receptor α binding and subsequent epigenetic regulation of
stem cell-like marker expression136, ultimately leading to reductions
in lesion size and increased fibrous cap thickness34,136. Similarly, vitamin C deficiencies have been linked to increased inflammation137 and
reduced collagen content of atherosclerotic lesions138. As such, vitamin
C administration has been suggested to improve EC function139 and
VSMC contractile gene expression140, which may explain the observations mentioned previously.
Rapamycin is an inhibitor of the mammalian target of rapamycin (mTOR) pathway, currently used as an immunosuppressant for
managing transplant rejection and to slow abnormal cell growth in
lymphangioleiomyomatosis. It is also commonly used in drug-eluting
coronary stents to prevent restenosis via inhibition of VSMC proliferation and migration. Rapamycin impairs leukocyte recruitment
into the plaque141,142, foam cell formation143 and inflammatory cytokine
secretion144. Within the medial wall, rapamycin induces VSMC contractility via TET2, which has an epigenetic influence on the expression of
VSMC genes145,146. As such, rapamycin appears to promote collagen
content and ACTA2 expression in plaques, suggesting improved plaque
stability147,148.
Together, evidence from these preclinical studies suggests that it
is possible to ameliorate inflammation and promote beneficial VSMC
phenotypic transitions to improve patient outcomes. Identification
of the mechanisms by which these therapies achieve these effects is
vital for the development of more effective treatments. Given that
inflammation and vascular cell phenotypic changes are an ongoing
process throughout atherogenesis, therapies targeting these factors
will probably be long-term treatments. The long-term effects and any
off-target side effects of these future treatments will need to be carefully taken into consideration.
What can we learn from targeting cellular plasticity in cancer?
Further insights into targeting cellular plasticity may come from the
cancer field, in which clinical trials are currently underway with therapies targeted against the epithelial-to-mesenchymal transition (EMT).
Netrin-1 is a chemotropic protein involved in axon guidance during
neuronal development. In cancer, netrin-1 overexpression drives disease progression by inhibiting apoptosis149,150 and promoting EMT151,152.
As such, there are currently ongoing cancer clinical trials with NP137,
an anti-netrin-1 monoclonal antibody. In mouse models, treatment
with NP137 has been shown to inhibit EMT, thereby reducing the number of metastases151,152. Likewise, human biopsies from patients with
endometrial cancer after NP137 therapy demonstrated a reduction
in the proportion of tumor cells, with a shift toward a more epithelial
phenotype152. Given the dual-action benefits of netrin-1 inhibition, it
will be useful to better understand how much of the observed phenotype is caused by inhibiting EMT and how much is due to induction
of apoptosis.
In the context of atherosclerosis, netrin-1 is thought to be secreted
by foam cells to reduce macrophage migration out of the plaque and
induce VSMC migration153. As such, bone marrow transplants from
Ntn1-deficient mice or mice with Ntn1 deletion in monocytes and macrophages promote the resolution of inflammation and thus reduced
plaque burden153,154. Given that netrin-1 expression was induced by
oxidized LDL loading of macrophages153, it would be interesting to
investigate whether transdifferentiated VSMC-derived foam cells also
produce netrin-1. It would also be interesting to investigate NP137 treatment in advanced plaques to establish how plaques respond in a more
clinically relevant model and to determine whether netrin-1 deficiency
affects VSMC and EC plasticity.
Cancer therapy has also benefited from multiple FDA-approved
histone deacetylase (HDAC) inhibitors, which help combat the epigenetic dysregulation observed in cancer cells. Epigenetic inhibition of
EMT may partially account for some of the benefits seen in patients
with cancer155. A growing body of evidence suggests that HDACs influence vascular cell plasticity and inflammation in atherosclerosis as
well. HDAC9 has been shown to promote EndoMT156 and a proinflammatory phenotype in macrophages157, which drive atherosclerotic
plaque burden. Multiple HDACs also regulate the transcription of
contractile genes in VSMCs in response to PDGF signaling158,159. HDAC6
may be particularly interesting for atherosclerosis in the context of
colchicine’s cardiovascular success, as HDAC6 also regulates microtubule dynamics160. These findings suggest that HDAC inhibitors
should be further investigated for regulation of vascular cell behavior
in atherosclerosis.
Another key target in cancer clinical trials is the TGFβ pathway, given its central involvement in EMT and the activation of
cancer-associated fibroblasts. While this target is backed by substantial
preclinical evidence, blocking TGFβ signaling has not had the desired
antitumorigenic effect in clinical trials161. It is possible that inhibiting
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a
VSMC
b
Myofibroblast-like
VSMC
Promote
collagen
secretion
EC
Prevent EndoMT
Prevent VSMC
transdifferentiation
Convert
VSMCs
back into
a protective
phenotype
Inhibit
proinflammatory
cytokines and
adhesion
molecule
presentation
Inhibit
proinflammatory
cytokines and
adhesion molecule
presentation
Stem-like
VSMC
Myofibroblast-like EC
c
Promote
efferocytosis
and protective
macrophage-like
phenotypes
Macrophage
Macrophage-like
VSMC
Unstable
Reverse
EndoMT
Promote
collagen
secretion
Reduce
macrophage
number
Osteogenic-like
VSMC
Promote
efferocytosis
and protective
macrophage
phenotypes
Stable
Inhibit
proinflammatory
cytokines and
detrimental MMPs
Macrophage phenotypes
↑ Protective phenotypes
↓ Inflammation
↑ Plaque stability
↑ Patient outcomes
Fig. 4 | Potential therapeutic strategies for resolving inflammation and
improving the vascular cell phenotype in atherosclerosis. Anti-inflammatory
therapy is only beginning to emerge for atherosclerotic cardiovascular disease.
Most research with anti-inflammatory agents has centered around immune cells,
with little research into how these agents affect the phenotype of VSMCs and ECs.
No therapies are currently designed to target vascular cell plasticity for plaque
stabilization. a–c, These therapies could include promoting VSMCs toward
collagen-secreting and/or efferocytic phenotypes (a), reversing EndoMT or
promoting collagen secretion from ECs (b) and/or promoting plaque-resolving
macrophage phenotypes (c). Resolving inflammation and improving plaque
cell phenotype (potential strategies shown in red) will probably be necessary
to regress unstable plaques (increase fibrous cap thickness, reduce necrotic
core size, decrease inflammation and induce other features of stable plaques),
ultimately improving patient outcomes in the future. The blue arrows show
cytokine secretion.
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TGFβ is only effective in specific cell types within tumors or at specific
stages during disease progression. For example, in atherosclerosis,
inhibiting TGFβ signaling may be beneficial in preventing EndoMT
but may promote the transdifferentiation of VSMCs. Further study is
required for targeting and manipulation of these signaling pathways,
which is becoming possible as therapies become more targeted.
Targeted therapy
Atherogenesis is a complex disease derived from a variety of risk factors
that affect how the disease progresses. However, therapies are currently prescribed when disease biomarkers reach a specific threshold—a
threshold that has largely been set on a population basis. The promise
and benefits of personalized medicine will only come to fruition with a
better understanding of how atherogenesis progresses and responds
to therapy based on varying genetic and environmental factors. Work
toward this future arises from large datasets that can be used to identify
how specific risk factors affect patient outcomes. Furthermore, patients
do not respond homogeneously to treatment, and therapies should be
specifically tailored to every individual for maximum benefit.
For example, the UK Biobank has been used to identify that
South Asian individuals have an increased risk of atherosclerotic cardiovascular disease compared with European individuals162. While the
underlying cause of this increased risk is multifaceted, one interesting
possible contributor is related to variation in gut microbiota across
ethnicities. South Asian individuals often lack the Christensenellaceae,
Methanobrevibacter and Ruminococcaceae network, whose presence
is inversely correlated with cardiovascular disease risk163. The gut
microbiome is an emerging field and is known to heavily influence
inflammation and atherosclerosis164. For example, the gut metabolite
trimethylamine-N-oxide increases inflammation and macrophage
foam cell formation, thereby promoting plaque burden165,166. However,
whether trimethylamine-N-oxide or other gut metabolites influence the
plasticity of vascular cells is not yet known. Given that the microbiota
composition in the gut is heavily influenced by genetic and lifestyle
factors, the gut microbiome is a possible target that could be modulated on an individual basis to reduce atherosclerotic plaque burden.
At a cellular level, a large pitfall with currently used therapies is
that they usually do not target specific cell populations. It is well known
that inhibiting signaling pathways may promote beneficial responses
in some cell types, but not in others. For example, KLF4 deficiency
in VSMC-derived cells is known to reduce VSMC phenotypic switching and promote plaque stabilization35,49. By contrast, loss of KLF4
in the myeloid population promotes macrophage recruitment and
polarization toward an inflammatory phenotype, exacerbating lesion
vulnerability167. As such, the future of atherosclerotic cardiovascular
disease treatment probably relies on therapeutically targeting different
cell types within the plaque.
One method of achieving this is via the use of nanoparticles that
can be targeted to specific locations and/or different cell types. For
example, peptide amphiphile micelles have been used to deliver
miR145 to CCR2+ plaque cells168,169. miR145 is an established regulator
of VSMC differentiation that mediates signaling from the myocardin–
SRF complex170. During atherogenesis, VSMCs lose their expression of
miR145, which accompanies a reduction in the contractility of VSMCs
as they proliferate and migrate170. Nanoparticle delivery of miR145
was shown to reduce plaque burden by promoting a contractile VSMC
phenotype168,169 and reduce serum levels of TNF and IL-6 (ref. 169). It
would also be interesting to investigate whether miR145 can induce a
myofibroblast-like phenotype in macrophages, given that many plaque
macrophages also express CCR2. In this manner, nanomedicine is a
quickly growing field that offers the promise of better targeted therapy
for the treatment of atherosclerosis.
While the target signaling pathway may be active in multiple cell
types, the ways in which signaling is regulated may be cell type specific
and may be therapeutically treated. The CXCL12–CXCR4 pathway
https://doi.org/10.1038/s44161-024-00569-y
has emerged as an atherosclerotic disease risk locus171,172. Cxcr4 deficiency in VSMCs promotes VSMC transdifferentiation and Cxcr4 deficiency in ECs increases endothelial leakiness, which both promote
atherosclerotic plaque burden172. It has been shown that miR206-3p
regulates the expression of CXCR4 in VSMCs and ECs but, crucially,
not in macrophages173. Blocking binding of miR206-3p to the CXCR4
3′ untranslated region has been shown to improve indices of plaque
stability by promoting CXCR4 signaling in VSMCs and ECs173. Thus,
cell-type-specific regulators are prime targets for therapeutically
suppressing or augmenting specific signaling pathways in distinct
cell types.
The future of patient care will require more therapeutic finesse.
Identification of which subsets of patients will respond to different
treatments will enable better therapeutic efficacy, and identification
of which cell types should be targeted will help maximize therapeutic benefits while minimizing off-target side effects. Targeting cell
type-exclusive pathways may be beneficial in allowing easily accessible
systemic drug delivery. Additionally, targeting specific locations and
specific cells will enable the use of lower drug doses and further reduce
unwanted side effects.
Concluding remarks
The chronic inflammation present during atherosclerosis is both a
consequence of pathogenic vascular cell plasticity and a cause of
further phenotypic switching that cascades plaque progression and
worsens patient outcomes. While targeting inflammation is emerging as a new criterion in disease management, there is still very little insight into how this influences the phenotype of plaque VSMCs
and ECs. For example, hsCRP is commonly used as a biomarker for
systemic inflammation and as a surrogate indicator of atherosclerotic inflammatory risk. However, this tells us very little about the
stability of the patient’s plaques, which is largely dictated by plaque
cellular composition and plasticity. Vascular cells are intricately
involved throughout atherogenesis and display remarkable potential for transdifferentiation into a variety of phenotypes, ultimately
affecting lesion stability. However, no current therapies are directly
designed to target VSMC and EC phenotypes. In this context, inspiration can be taken from the cancer field, where targeting EMT may
prevent metastasis. Given the complexity of atherosclerotic lesions,
more specific targeted approaches are necessary to limit unwanted
side effects. Favorable manipulation of vascular cell phenotypes at
the single-cell level alongside inflammation resolution is probably
essential for achieving better patient outcomes (Fig. 4).
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Acknowledgements
A.M. is supported by a Heart Foundation Vanguard grant (NHF1017)
and a New South Wales Cardiovascular Research Capacity Program
grant (DOH1024). E.A.F. is supported by a National Heart, Lung and
Blood Institute grant (National Institutes of Health R01HL084312).
A.L. is supported by an Australian Government Research Training
Program Scholarship.
Author contributions
A.L. drafted the manuscript with input from J.M.M., E.A.F. and A.M.
All authors reviewed and edited the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to
Ashish Misra.
Peer review information Nature Cardiovascular Research thanks Jason
Kovacic, Nathan Palpant and the other, anonymous, reviewer(s) for
their contribution to the peer review of this work.
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