Review
Lineage of Bone Marrow–Derived Cells in Atherosclerosis
Hiroshi Iwata, Ichiro Manabe, Ryozo Nagai
Abstract: Atherosclerosis is a chronic inflammatory disease driven by lipids and other atherogenic factors. It is
characterized by a dynamic and complex pathological process of bone marrow–derived cells playing divergent
roles. Recent studies have begun unraveling the contribution of growing varieties of subsets of immune cells and
other bone marrow–derived cells to atherogenesis. For example, bone marrow–derived vascular progenitor cells
have been shown to play an important role in the pathogenesis of atherosclerosis. This review provides an overview
of the current understanding of contributions of bone marrow–derived cells to atherosclerosis. Particular focus
is placed on myeloid cells and vascular progenitor cells. We also summarize the uncertainty surrounding cellular
lineage identity and functions. Expansion of our understanding of pathological roles of various subsets of bone
marrow–derived cells in atherosclerosis may lead to identification of novel cellular and molecular targets for
development of therapeutic strategies. (Circ Res. 2013;112:1634-1647.)
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Key Words: atherosclerosis
■
bone marrow–derived cells
L
■
inflammation
■
progenitors
to atherogenesis are still undergoing debate. This review
summarizes the current understanding of the functional
role of BM-derived leukocytes and progenitor cells in the
pathological processes of atherosclerosis and neointima
formation. Lineages, differentiation, and functions of BMderived cells in atherosclerosis are summarized in the Figure
and Table 1.
In this review, we primarily focus on the functions of cells
present within the arterial wall. However, recent studies have
shown that atherosclerosis is affected by inflammation and
immune responses in distant organs. Atherosclerosis may be
considered more appropriately as a manifestation of chronic
systemic inflammatory processes that are regulated by organ
networks. We advise readers to refer to recent excellent reviews on these matters.9,10
eukocyte infiltration into atherosclerotic lesions was first
described in the middle 19th century. Rudolf Virchow
suggested that inflammation or infiltrated leukocytes
directly contribute to the pathogenesis of atherosclerosis,
whereas Carl von Rokitansky considered these changes
secondary responses.1 After these early observations,
numerous studies have worked to unravel the role of
immune cells in atherogenesis. Now, it is widely accepted
that atherosclerosis is a chronic inflammatory disease.2,3
Chronic inflammatory processes alter the 3-dimensional
structure of the vascular wall (vascular modeling), leading
to the atherosclerotic plaque formation.4 Various bone
marrow (BM)–derived immune cells, including monocytes/
macrophages and lymphocytes, are known to be actively
involved in the processes. In addition to leukocytes, recent
studies have demonstrated that BM cells also may serve
as sources of vascular cell lineages, including endothelial
progenitor cells (EPCs) and smooth muscle progenitor
cells (SMPCs).5,6 Although these vascular progenitors
originally had been thought to differentiate into mature and
functional endothelial and smooth muscle cells (SMCs)
in physiological and pathological settings,5,7 subsequent
studies have reported conflicting results.8 Therefore, it is still
unclear whether these cells acquire definitive endothelial and
SMC identities. Accordingly, their functional contributions
Monocytes and Macrophages
Macrophages have received particular attention in atherogenesis because of early observations that macrophage-derived
foam cells centrally contribute to the formation of necrotic
core and lesion development in atherosclerosis. Infiltration of
circulating monocytes into the subendothelial space via adhesion molecules on activated endothelial cells (ECs) is one
of the key processes of plaque formation.11 Previous studies
have shown that monocytes that enter the vessel wall become
cells with macrophage-like features in Apoe−/− mice, although
Original received March 15, 2013; revision received May 8, 2013; accepted May 9, 2013. In April 2013, the average time from submission to first
decision for all original research papers submitted to Circulation Research was 13.5 days.
From the Center for Interdisciplinary Cardiovascular Sciences, Harvard Medical School, Cardiovascular Division, Department of Medicine, Brigham
and Women’s Hospital, Boston, Massachusetts (H.I.); Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine,
Bunkyo, Tokyo, Japan (H.I., I.M., R.N.); and Jichi Medical University, Yakushiji, Shimotsuke-shi, Tochigi Prefecture, Japan (R.N.).
Correspondence to Hiroshi Iwata, MD, PhD, Center for Interdisciplinary Cardiovascular Sciences, Harvard Medical School, Cardiovascular Division,
Department of Medicine, Brigham and Women’s Hospital, 3 Blackfan St, 17th Floor, Boston, MA 02115 (e-mail [email protected]); or Ichiro
Manabe, MD, PhD, Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo, Tokyo
113–8655, Japan (e-mail [email protected]).
© 2013 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.113.301384
1634
Iwata et al Bone Marrow Cells in Atherosclerosis 1635
Nonstandard Abbreviations and Acronyms
EPCs
IL
ILCs
KDR
LDL
MMPs
NK
NKT
SM-MHC
SMPCs
TLOs
Tregs
vWF
endothelial progenitor cells
interleukin
innate lymphoid cells
kinase insert domain–containing receptor
low-density lipoprotein
matrix metalloproteinases
natural killer
natural killer T
smooth muscle-myosin heavy chain
smooth muscle progenitor cells
tertiary lymphoid organs
regulatory T cells
von Willebrand factor
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the precise rates at which the recruited monocytes are retained
and differentiate into macrophages are not clear.12–14
Several monocyte subsets have been identified in mouse and
human circulation. In mice, 2 distinct monocyte subsets are
identified based on differential expression of Ly-6C, CX3CR1,
and CCR2.15 One subset exhibiting Ly-6ChighCX3CR1lowCCR2+
is designated inflammatory monocytes, and the other subset
with Ly-6ClowCX-3CR1highCCR2− is designated resident or patrolling monocytes.15–18 In humans, 3 monocyte subsets can be
identified mainly based on CD14 and CD16 expression.19 The
classical subset exhibiting CD14++CD16−CCR2high is thought
to correspond to mouse Ly-6Chigh monocytes, whereas the
nonclassical subset with CD14+CD16++CCR2low corresponds
to Ly-6Clow monocytes.20 The intermediate CD14++CD16+ subset also was identified.21
The pathological roles of each monocyte subset are still
undergoing investigation. Previous studies showed that in
mice both Ly-6Chigh and Ly-6Clow monocytes can accumulate
in atherosclerotic lesions, whereas Ly-6Clow monocytes
less frequently infiltrate into atheroma than Ly-6Chigh.13,14
Some fractions of Ly-6Chigh monocytes seem to differentiate
into macrophages. Although it has been suggested that
Ly-6Chigh monocytes primarily differentiate into M1type inflammatory macrophages, they may also acquire
M2-type.22,23 It is still unclear what phenotypes Ly-6Chigh
monocyte–derived macrophages acquire in atheroma. Recent
studies have demonstrated that, depending on environmental
cues, macrophages may dynamically alter their phenotypes
and that they cannot be easily classified within the M1/M2
notation.24–26 It is possible that macrophages derived from
the same Ly-6Chigh monocyte population spatiotemporally
change their phenotypes in lesions during development of
atheroma. In this regard, it is noteworthy that the induction
of foam cell formation unexpectedly deactivated the
proinflammatory phenotype of peritoneal macrophages.25
It is also well-known that phagocytosis of apoptotic cells
deactivates macrophages.27
In mice, hypercholesterolemia was shown to increase
Ly-6Chigh monocytes in peripheral circulation.13,14,28
Similarly, elevated CD14++CD16− monocyte levels are associated with an increased risk of cardiovascular events in
human.29 Hyperlipidemia-induced monocytosis is partly
caused by generation of monocytes in BM (medullary hematopoiesis). Recently, it also was shown that hematopoietic stem and progenitor cells relocate from the BM to
splenic red pulp, in which they expand and differentiate into
Ly-6Chigh.28,30,31 This additional pool of monocytes may promote further accumulation of monocytes/macrophages in
plaque and accelerate plaque development.
Less is known about the fate of Ly-6Clow monocytes within
the atheroma. However, these cells were shown to patrol the
endothelium for injury and infection by long-range crawling.32 Ly-6Clow monocytes also were shown to be recruited at
a late phase to the infarcted myocardium and were suggested
to promote wound healing.32 Their functional role in atheroma
remains to be elucidated.
Intimal macrophages may internalize oxidized low-density lipoproteins (LDLs) through scavenger receptors and
become foam cells, the main components of the fatty streak.
After these early events, the complex interplay between
ECs, SMCs, and immune cells, including macrophages and
T lymphocytes, further drive lesion progression through
Figure. Overview of lineages and differentiation of bone
marrow–derived cells in atherosclerosis. Solid arrows
indicate established or previously reported differentiation
and transdifferentiation in bone marrow–derived cells
and vascular smooth muscle cells (SMCs). Dotted arrows
indicate possible transdifferentiation among synthetic
smooth muscle cells, macrophages, and smooth muscle–
like cells (SMLCs; bone marrow–derived SMα-actin+ cells).71
Macrophages (Mφ) include subpopulations other than M1
and M2, such as Mox, Mhem, and M4.30–33 M2 macrophages
express scavenger receptors for internalize modified lowdensity lipoprotein (LDL; eg, oxidized LDL). Dendritic cells
(DCs) present various types of antigens for recognition
by T cells in lymphoid organs. Plasma cells produce
antibodies. EPC indicates endothelial progenitor cells;
HSC, hematopoietic stem cell; and SMPC, smooth muscle
progenitor cell.
1636 Circulation Research June 7, 2013
Table 1. Summary of Functions of Bone Marrow–Derived
Cells in Atherosclerosis
Function in
Atherosclerosis
Positive
Marker(s)
Lineages
Myeloid Monocyte/ M1
lineage macrophage
M2
CD86, CD11c,
TNFα, iNOS,
IL-1β
↑
↓
CD206, CD301,
Arg1
↓
↑
↑
↓
Others Foam cell
(M2-like?)
M4
CCL18, CCL22,
MMP7
↑?
↓?
Mox
HO-1
↓
↑?
CD163, HO-1
↓
↑?
Ly6c(m),
CD14(h), CD31,
KDR, CD34,
CD133
↓?
↑?
Ly6c(m), CD14(h),
SMα-actin,
SM22α, CD34,
CD133
↑
↓
Circulating
fibrocyte
CD34, Col1,
CD14, CD45,
SMα-actin,
SM22α
↑?
↑ or ↓
Neutrophil
LY6G(m), CD69(h)
↑
↓
↑ or ↓
↑ or ↓
↑
↓
↑ or ↓
↑ or ↓
Mhem
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Vascular
progenitor
EPC
SMPC/
SMLC
DC
Lymphoid
lineage
Athero­ Plaque
genesis Stability
T cell
CD11c, CD83
Th1
IL12Rβ2, CCR5,
CXCR3
Th2
CXCR4, CCR3,
CCR4
Treg
FoxP3
↓
↑
Th17
CD4, IL-17A/F
↑
↓?
NK T
cell
CD1d
↑
↓?
↑ or ↓
↑
↑ or ↓
↑ or ↓
B cell
CD19, CD20,
IL-10
Plasma
cell
NK cell
CD38
CD16, CD56(h),
NK1.1(m)
DC indicates dendritic cell; EPC, endothelial progenitor cell; (h), human; IL,
interleukin; HO-1, heme oxgenase-1; iNOS, inducible nitric oxide synthase;
KDR, kinase insert domain–containing receptor; (m), mouse; MMP, matrix
metalloproteinase; NK, natural killer; SMLC, smooth muscle-like cell; SMPC,
smooth muscle progenitor cell; and TNF, tumor necrosis factor.
↑ denotes progressive; ↓ denotes suppressive.
cytokine signaling networks that mediate the dynamic cellular interactions.
Development of atherosclerotic plaques may involve 2
scenarios that are not necessarily mutually exclusive.33 When
the balance between proinflammatory and anti-inflammatory
populations of accumulating BM-derived cells in the cellular
processes is tipped toward anti-inflammatory reactions that
promote healing, the plaque may become stabilized by a thick
and stable fibrous cap. Alternatively, active inflammation is
thought to result in instability of the plaque by promoting
thinning of the fibrous cap that is then prone to plaque rupture
and sudden lumen occlusion by thrombus. Unstable plaques
typically exhibit active inflammatory processes that are
characterized by constant infiltration of inflammatory cells
and production of inflammatory cytokines. The prolonged
accumulation of apoptotic cells, cell debris, and cholesterol
crystals promotes progression of the necrotic core, in which
apoptotic macrophages are the major constituent.34,35 Moreover,
macrophages produce inflammatory cytokines and matrix
metalloproteinases (MMPs), which promote inflammation and
plaque vulnerability.36 As such, macrophages are the major
active player in determining the evolution of atherosclerotic
plaque.
An important function of lesion macrophages is to remove
excess lipid, and the lipid-laden macrophages that appear
within the plaque are referred to as foam cells.12 Macrophages
may take-up natural and modified lipoproteins, such as acetylated LDL, oxidized LDL, and glycolaldehyde-LDL, as well
as LDL enzymatically altered by trypsin, cholesterol esterase,
and neuraminidase.37 These modified forms of LDL have been
shown to promote foam cell formation and atherogenesis by
stimulating secretion of various cytokines.38 This leads to the
further recruitment of inflammatory cells, resulting in formation of necrotic core.
Excess cholesterol ester accumulation in macrophages of
the vascular lesions occurs as a result of an imbalance between
delivery and removal. Macrophages are incapable of limiting
the uptake of lipids and, therefore, largely depend on cholesterol efflux pathways to maintain cellular lipid homeostasis.39
In early atherogenesis, macrophage apoptosis is associated
with reduced atherosclerosis progression40,41 attributable to
effective efferocytosis by neighboring phagocytes, which, in
turn, reduces proinflammatory mediators present in the lesion.42 However, as atherosclerosis progresses, efferocytosis
becomes impaired.43 Potential contributing mechanisms for
impaired efferocytosis include oxidative stress–induced death
of efferocytes attributable to the overabundance of apoptotic
cells within the plaque and dysfunction of the efferocytosis
receptor MerTK.44,45 A failure of effective efferocytosis leads
to secondary necrosis, in which macrophages die and release
their cellular contents, including debris, oxidized lipids, and
proinflammatory mediators. Secondary necrosis amplifies the
inflammatory response and accelerates the development of a
necrotic core in the plaque.35
In response to various local environmental cues, macrophages
are now known to assume divergent phenotypes. In vitro studies
have shown that Th1 cytokines alone, or in concert with microbial products, elicit classical M1 activation of macrophages,
whereas Th2 cytokines (interleukin [IL]-4 and IL-13) elicit an
alternative form of activation designated M2.46 Previous studies have implied that the balance of macrophage polarization
may affect plaque outcome.47 After the progression of atherosclerosis, it was shown that plaque macrophages in early lesions
expressed arginase-1–indicative M2 polarization, whereas at
later time points M1 macrophages predominated in the plaque.48
Iwata et al Bone Marrow Cells in Atherosclerosis 1637
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Phenotypic switching in macrophages from M2 to M1 also was
found in atherosclerosis on induction of disease regression.49
Although the M1 and M2 classifications highlight the 2
extremes of macrophage activation, it is becoming increasingly clear that macrophages can exhibit highly divergent
phenotypes and functions in vivo. In particular, M2 may include various forms of macrophage activation.50 Other types
of macrophage polarization have been described. Mox phenotype develops in response to atherogenic phospholipids via
expression of the redox-regulated transcription factor Nrf2
and has a lower phagocytotic and chemotactic capacity than
the conventional M1 and M2 macrophages.26 Moreover, it
also has been proposed that Mhem, which is activated by intraplaque hemorrhage, is often associated with atherothrombotic complications.51,52 M4 is a recently proposed subtype of
atherogenic macrophages induced by CXCL4.53 As such, the
classification of macrophages in atherosclerosis does not seem
to be as simple as previously proposed. Furthermore, there is
a significant gap in our understanding between macrophage
polarization in vitro and functional roles in vivo. Therefore,
future studies will need to address the functional contributions
of differentially activated macrophages to the physiology and
pathology of the blood vessel.54
Vascular Progenitor Cells
After the discovery of EPCs in circulation in mice,5 various
circulating progenitor cell populations that can acquire EClike or SMC-like phenotypes have been identified in mouse
and human.7,55 These cells have been shown to exhibit some
similarities in surface markers and functions with myeloid
cells.8,56,57 However, their origin, identity, and function are
still poorly understood, and a unified definition of EPCs and
SMPCs has not yet been established. It is, in fact, still undergoing debate which populations are bona fide progenitor cells
and whether these cells are even capable of differentiating into
definitive vascular lineages in in vivo settings. Moreover, physiological and pathological functions of EPCs and SMPCs are
not well-­understood, partly because they are assumed, a priori,
to acquire EC or SMC functions. Irrespective of whether they
are bona fide progenitor cells, numerous studies have identified BM-derived cells that express some of the endothelial or
SM genes within vascular lesions. Therefore, it is important
to assess whether and how they contribute to vascular disease.
Endothelial Progenitor Cells
Asahara et al5 published a landmark article in 1997 showing
that BM-derived CD34+VEGFR-2+ peripheral mononuclear
cells (MNCs), isolated from human blood and grown in culture are able to differentiate into cells expressing endothelial
markers, including CD31, E-selectin, and endothelial nitric oxide synthase. Subsequently, CD34+ cell–enriched MNCs transplanted into mouse and rabbit models of hindlimb ischemia
were shown to promote neovascularization and incorporate in
the capillary walls, suggesting they contribute to blood vessel
formation in adult.58 Based on these findings, the cells were
designated as EPCs.
Although other sources of EPCs, such as resident
progenitor cells in blood vessel wall,59 adipose tissue,60 and
spleen,61 were proposed, BM has been considered as the
main reservoir of these specialized cells. Various methods
to identify EPCs have been developed: methods of isolation,
cell surface markers, and functions in angiogenesis and in
atherosclerosis of EPCs are summarized in Table 2. Currently,
the most commonly used method to identify and distinguish
EPCs from mature ECs is flow cytometric analysis of
expression of cell surface antigens, such as CD34, kinase
insert domain–containing receptor (KDR), VE-cadherin,
von Willebrand factor, E-selectin, and CD133.62 Different
combinations of cell surface markers may identify different
cell populations.
The second commonly used identification method for
EPCs involves culturing circulating MNCs and monitoring
phenotypic changes over time. One subtype of EPCs arises 3 to 5 days after seeding of MNCs (early EPCs63,64 and
colony forming unit ECs).65 Another type of EPCs appear
after 2 to 3 weeks of culture (late EPCs or outgrowth ECs
[OECs]).64,66 These 2 subtypes seem to be substantially distinct in multiple respects. First, late EPCs or OECs have more
similarities to mature ECs in not only expression of cell surface antigens, such as von Willebrand factor, VE-cadherin,
and KDR, but also functional aspects, including high levels of endothelial nitric oxide synthase synthase production
and effective participation in tube-forming assays in vitro as
compared with early EPCs. Moreover, late EPCs or OECs
are potent sources of several growth factors, cytokines, and
chemokines, such as vascular endothelial growth factor, IL8, hepatocyte growth factor, granulocyte colony–stimulating
factor, and granulocyte macrophage colony–stimulating factor.67 Finally, early EPCs are mostly derived from the CD14+
population of MNCs, whereas OECs derive mostly from the
CD14− population.68
Functionally, early EPCs are suggested to participate in angiogenesis in a paracrine fashion like endothelial-like macrophages, rather than highly differentiated ECs.69 Late EPCs
or OECs are suggested to directly participate in angiogenesis
like cells composing blood vessels.70,71 Although these timedependent culture methods have demonstrated that ex vivo
culture of circulating MNCs produce cell populations that resemble ECs, these results may not necessarily indicate that
the same subsets of MNCs differentiate into ECs in vivo.
Long-term culture has been shown to greatly modulate epigenome72 and may induce aberrant gene expression that would
not occur in in vivo settings. Purhon et al73 demonstrated that
no BM-derived cells incorporated into vascular ECs in tumor
angiogenesis as well as matrigel assay. As such, whether BMderived cells acquire a definitive EC lineage is still undergoing
debate. In fact, many phenotypic characteristics and behaviors that are attributed to ECs are shared by myeloid cells.74
Moreover, monocytes/macrophages support and promote angiogenesis during development and in pathologies.75 Further
lineage tracing experiments are needed to clarify the cell fates
of circulating MNCs in vivo.
Circulating EPCs are generally considered protective in
the pathogenesis of atherosclerosis as well as neointima
hyperplasia after vascular injury. Observational clinical
studies of patients with coronary artery disease showed a
significantly lower incidence of adverse clinical events in
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individuals with higher numbers of circulating CD34+KDR+
cells.76,77 HMG-CoA reductase inhibitors (statins) and
physical exercise, which are known to improve EC
function, were shown to increase the number of circulating
EPCs.78–80 A beneficial effect of EPCs also was shown in
animal studies. For instance, in a rabbit balloon injury
model, the transfer of ex vivo cultured EPCs significantly
suppressed neointima hyperplasia.81 Although these studies
suggest beneficial roles of EPCs, some evidence indicated
unfavorable effect of EPCs, such as promoting plaque
progression and instability.82,83 We also previously reported
that the presence of a higher population of CD34+ cells in
coronary thrombi of patients with myocardial infarction had
higher incidence of restenosis after coronary intervention.84
These findings suggested an increased incidence of
restenosis after successful coronary intervention in these
patients, indicating that CD34+ cells in thrombi might
promote neointimal hyperplasia after angioplasty. A
potential reason for these discrepant roles of EPCs may arise
from the fact that each study used differently identified cell
populations than EPCs. In fact, the unequivocal definition
and isolation method of circulating EPCs have not been
established.85 Most cell surface antigens that are currently
used to identify EPCs can be expressed by other cell types,
including hematopoietic stem cells.86 Moreover, several
studies suggest that myeloid progenitors have the capacity
to differentiate into vascular ECs,87 whereas others propose
that BM-derived cells are recruited to a perivascular position
and act as paracrine cells that promote angiogenesis but
do not differentiate into definitive ECs.67,88 As described,
early EPCs and late EPCs seem to be functionally different.
Accordingly, the impact of EPCs on physiology and
pathology of the blood vessel remains unclear. Because
current identification methods may isolate cells that do not
have potential for differentiating into definitive ECs in vivo,
all EPCs cannot be uniformly considered EPCs. At least
some of them may have different functions than definitive
ECs. As such, it may be important to carefully reevaluate
the functional roles of each EPC subset in vascular disease
and physiology.
SMPCs
Circulating SMPCs were identified as circulating BMderived cells that are recruited to blood vessels and acquire
phenotypes expressing SMC marker genes, particularly
SMα-actin.7,89–91 These SMPC-derived cells are often called
SM-like cells (SMLCs). In humans, donor-derived SMLCs
were detected throughout atherosclerotic vessels in patients
who received sex-mismatched BM transplantation, suggesting the involvement of circulating SMPCs in intimal proliferation.92 To date, no specific cell surface or intracellular
markers that differentiate SMPCs and SMLCs from SMCs
have been established. Therefore, these cells are largely identified as circulating or resident cells within the BM, vascular
wall, or perivascular area that acquires phenotypes with some
similarities to SMCs.
There are a number of SMC differentiation marker genes
with different specificity, including SMα-actin, SM22α,
SM-calponin, smoothelin, and SM-myosin heavy chain (SMMHC).93 One of the difficulties in identification of SMC
lineage is the plasticity in phenotypes of SMCs. SMCs can
greatly alter their phenotypes during development and vascular disease. Another problem is that all SMC differentiation
markers can be expressed by non-SMCs. Among SMC markers, SM-MHC is the most stringent marker for SMC lineage.94
The most commonly used SMC marker, SMα-actin, can be
expressed by various non-SMCs, including ECs, fibroblasts,
and macrophages. Therefore, using SMα-actin as a sole marker is insufficient to identify SMC lineage. Additionally, it also
was shown that phenotypically modulated SMCs can express
macrophage markers.95 As such, it is not trivial to unequivocally identify SMC lineages in vascular lesions.
Using multiple SMC differentiation markers, including SM-MHC, we carefully analyzed phenotypes of BMderived SMLCs.8 We found that BM-derived SMα-actin+
cells in vascular lesions in mice do not express the definitive
SMC lineage marker, SM-MHC, even under conditions in
which resident SMCs expressed SM-MHC. Because SMMHC expression in resident SMCs is downregulated in the
neointima, we analyzed lesions 12 months after vascular
injury. Even at this late stage during which resident SMCs
Table 2. Summary of Isolation Methods of EPCs and Their Functions in Angiogenesis and Atherosclerosis
Adherent/
Nonadherent in
Initial Culture
Culture Period, d
Selection of
Subpopulation
Endothelial Markers
(CD31, CD144, KDR,
vWF)
Myeloid Markers
(CD14, CD45)
Adherent
<5
−
+
+
Promote
angiogenesis in
paracrine manner5
Late EPC (outgrowth Adherent
EPC)
>14
−
++
−
Differentiate into
endothelium42
CFU-EC (CFU-Hill)
Nonadherent
<10
+
+
+
Promote
angiogenesis in
paracrine manner45
ECFC
Adherent
>14
+
++
−
Differentiate into
endothelium,
high proliferation
capacity37
Early EPC
Angiogenesis
Atherosclerosis
Generally
considered as
atheroprotective56
but several
reports indicate
proatherogenic
properties of
EPCs57,58
CFU-EC indicates colony-forming unit–endothelial cell; ECFC, endothelial colony–forming cells; EPC, endothelial progenitor cells; KDR, kinase insert domain–
containing receptor; and vWF, von Willebrand factor.
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fully recover SM-MHC expression, the BM-derived cells
did not express SM-MHC. Instead, the BM-derived SMαactin+ cells express markers of monocytes/macrophages.
Moreover, we found that adoptively transferred CD11b+Ly6C+ BM monocytes expressed SMα-actin in the injured artery. These results strongly suggest that a subset of myeloid
lineage cells can acquire SMC-like phenotype characterized
by SMα-actin expression. However, they do not acquire the
definitive phenotype of SMC lineage in the models we used,
including wire injury, heart transplantation, and Apoe−/−
mice.8 Expression of inflammatory genes and MMPs in
these SMα-actin–positive cells suggests that they contribute
to the remodeling processes.
Our results indicate that myeloid cells from BM can acquire phenotypes partly resembling SMCs. Interestingly, it
also was reported that SMCs can express genes that are expressed in macrophages. Cholesterol or oxidized LDL loading of SMCs resulted in a marked suppression in expression
of SMC markers in cultured SMCs.95,96 This SMC dedifferentiation was followed by activated expression of multiple
macrophage markers, including CD68, Mac-2, and ABCA1,
and macrophage-like phagocytic activity. These results suggest that a part of macrophage marker–positive cells within
vascular lesions may be of the SMC origin. These findings
point to the need to reevaluate the cell lineages of SMCs and
macrophages in vascular lesions.97 We need to clarify which
cells within atherosclerotic lesions are of BM or SMC origin,
and what roles these cells might play in lesion development,
progression, and plaque instability or rupture.
Functions of SMPCs in the process of atherogenesis are not
well-understood. Several reports suggest that SMPCs promote
inflammation and plaque instability by producing cytokines
and MMPs.8,98 It also was suggested that SMPCs are involved
in pathological angiogenesis56 in the atherogenic plaque. In
contrast, atheroprotective effects of SMPCs have been rarely reported, although in theory they might contribute to the
formation of fibrous cap. Clearly, further studies are needed
to better-understand phenotypic and functional features of
SMPCs in the vascular disease.
Circulating Fibrocytes
Fibrocytes99–103 are BM-derived mesenchymal progenitors104,105 that coexpress markers of hematopoietic stem cells,
the monocyte-lineage, and fibroblasts.106 They produce extracellular matrix (ECM) components as well as ECM-modifying
enzymes and can further differentiate into myofibroblast-like
cells.107 Fibrocytes are suggested to promote local inflammation via antigen presentation, cytokine and chemokine secretion, and production of MMPs.108 A gene expression profile
study of human fibrocytes identified signature genes, including TLR4, IL-1β, CCL2, CCL3, CCL7, CCL22, and C5aR,
suggesting that fibrocytes participate in an inflammatory
response.109
In vitro studies showed that fibrocytes are derived from
CD14+ BM–derived monocytes in humans109 and from the
Gr1+CD115+CD11b+ monocyte population in mice.110 Therefore,
it was proposed that circulating fibrocytes are a transitional cell
population between monocytes and fibroblasts.108 Interestingly,
they also were shown to have potential to dedifferentiate
back to monocytes or dendritic cells (DCs).108 Moreover,
human fibrocytes were shown to be capable of differentiating
into cells with characteristics of adipocytes, chondrocytes,
myofibroblasts, and osteoblasts.111,112 As such, fibrocytes may
differentiate along multiple mesenchymal lineages, but it is
less clear which differentiation paths are applicable in vivo.
More importantly, their lineage identity, particularly relative
to monocytes, is not clear. Clearly, further work is needed to
better-define the lineage of fibrocytes.
In human peripheral blood, 0.1% to 0.5% of nucleated cells
were identified as circulating fibrocytes that express type 1
and 2 collagens, vimentin, and, occasionally, SMα-actin.99
Because no single marker that unequivocally identifies fibrocytes has been established, combinations of expression of
collagen and other surface markers, including CD34, CD45,
and CD68, have been used. More recently, a combination of
CD45RO, 25F9, and S100A8/A9 or CD49 also was used as a
marker. However, it also was reported that there are considerable overlaps in the gene expression profiles among human
monocytes, macrophages, fibrocytes, and fibroblasts.113
In atherosclerotic lesions, fibrocytes expressing procollagen
I and CD34 have been identified in the fibrous cap of human
atherosclerotic lesions.114 Subendothelial SMα-actin–positive
myofibroblasts expressing the monocyte marker CD68 have
been found in lipid-rich areas of the atherosclerotic intima in
human aorta.115 One of the subsets of monocytes that are preferentially recruited to sites of atherosclerosis is the inflammatory subset of CD14+CD16− MNCs expressing CCR2,116,117
suggesting that the CD14+CD16−CCR2+ subpopulation may
be involved in atherogenesis and may contain precursors of fibrocytes that contribute to the development of atherosclerosis.
Marker expression profiles suggest that there may be considerable overlaps between fibrocytes and SMPCs/SMLCs;
therefore, it may be important to clarify the relationship in
lineages and functions between fibrocytes and SMPCs to
­better-understand the common physiological and pathological
roles of BM-derived SMC-like and fibroblast-like cells in the
response of the body to insults.
The expressions of collagen and other ECM proteins by
fibrocytes suggest that accumulation of these cells in the fibrous cap might stabilize the plaque. However, fibrocytes also
are known to express inflammatory cytokines and MMPs. As
such, future studies need to clarify functional roles of fibrocytes in the course of atherogenesis and plaque destabilization.
Identification of BM-derived circulating EPCs, SMPCs,
and fibrocytes has suggested more divergent roles of BMderived cells than previously considered. However, it is also
clear that our understanding of their lineages and functions
is still limited. In particular, it would be important to further
analyze their functional contribution to lesion evolution.
DCs
DCs are specialized antigen-presenting cells. Classical
DCs differentiate from common DC precursors in BM and
are p­resent in both lymphoid and nonlymphoid tissues at
steady-state.75 Classical DCs in nonlymphoid tissues are
highly migratory and can move from peripheral tissues to
the draining lymph nodes where they interact with T cells.
Plasmacytoid DCs also arise from common DC precursors
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and are characterized by rapid production of type I interferons
(IFNs) in response to viral infection. They are present in all
peripheral organs, including the aorta.75 Although monocytes
are not thought to differentiate into classical DCs, subpopulations of DCs have been suggested to arise from monocytes.118
However, the relationship between DCs and monocytes and
macrophages, particularly under conditions of inflammation,
is still undergoing debate.119,120 Surface expression of CD11c
often is used to identify DCs. However, CD11c is also expressed in a variety of tissue macrophages. Likewise, the
markers that have been used to distinguish macrophages from
DCs, such as F4/80, CD11c, CD11b, and MHC II, are not specific.119 Moreover, macrophages can present antigens.
In mouse and human aortae, classical DCs are present in
steady-state.121,122 In mouse, 2 subpopulations of DCs were
identified: CD103+CD11b−F4/80− classical DCs, whose
presence depends on Flt3 and Flt3 ligand signaling, and
CD103−CD11b+F4/80+DC-SIGN+ monocyte–derived DCs.121
Both populations were increased in atherosclerosis in the aortae of Ldlr−/− mice. In early Ldlr−/− atherosclerotic lesions, the
majority of intimal proliferating cells were positive for DC
markers, including CD11c, MHC clsss II, and 33D1, but were
low in CD11b.123 These results demonstrate that classical DCs
are present in atherosclerotic lesions.
Whether a DC will activate or inhibit T cells depends on
its pattern of cytokine release and expression of cell surface
costimulatory molecules. DCs therefore are a crucial link
between innate and adaptive immune responses.124 The functional role of DCs in atherosclerosis is not fully understood but
they are proposed to play an important role in T-cell activation
by presenting plaque-derived antigens. This is supported by
findings that deletion of antigen-presenting cell costimulatory
molecules CD80 and CD86 reduces atherosclerosis,125 and that
CD11c+ antigen–presenting cells interact with CD4+ T cells in
the aortic wall,126 although plaque-derived autoantigens are not
well-characterized. DCs also may be located within tertiary
lymphoid organs, which would facilitate antigen presentation.
Flt3 deletion markedly decreased aortic classical DCs and
increased lesion size in Ldlr−/− background, suggesting classical DCs are atheroprotective in this setting.121 In contrast,
diphtheria toxin–induced depletion of CD11c+ cells in Ldlr−/−
mice carrying the CD11c-DTR transgene that were fed a highcholesterol diet for 5 days reduced intimal lipid accumulation
and foam cells.127 In this study, the majority of foam cells was
positive for CD11c and 33D1 in Ldlr−/− mice, suggesting that
foam cells are derived mainly from resident intimal CD11c+
DCs at this early time point. The origin and lineage of these
intimal CD11c+ cells and their relationship with foam cells
in advanced atherosclerosis seem to need further investigation because, as discussed, clear discrimination of monocytes,
macrophages, and DCs has not been established.
It recently has been shown that the DC-derived chemokine
CCL17 is present in both human and mouse atherosclerotic
plaques, and that CCL17 deficiency in hypercholesterolemic
mice reduces disease development possibly through increased
activation of regulatory T cells (Tregs).128 Although more
studies are needed to clarify the role of DCs in atherosclerosis,
modulation of the plaque DC phenotype represents a potential
therapeutic target to decrease vascular inflammation and
atherosclerosis development.
Neutrophils
Despite being the most abundant leukocytes, the neutrophils
have received little attention in atherosclerosis, mainly because they have been less frequently found in atherosclerotic
lesions. Nevertheless, recent studies suggest a contributory
role of neutrophils in the development and the progression
of atherogenesis, as well as plaque destabilization by using
antibodies recognizing Ly6g and myeloperoxidase, which
enabled immunohistochemical detection of these cells in atherosclerotic lesions.129 A series of histological and intravital
studies revealed the localization of neutrophils in early130 and
advanced murine atherosclerotic lesions.129,131 In humans, recent analyses showed the association of intraplaque neutrophil numbers and histopathologic features of rupture-prone
atherosclerotic lesions.132 Additionally, activated neutrophils
have been identified in human atherosclerotic lesions with
histological identification of oxidant generation and esterase activity.133 As such, recent progress in discovering the
pathological role of neutrophils in atherogenesis may present
novel strategies for the prevention and the treatment of atherosclerosis. At the same time, these findings also suggest that
some of the pleiotropic effects of existing medications may
be attributable to interference with neutrophil activity, such as
statins. Recently, it was proposed that neutrophils, similarly to
macrophages, can polarize to antitumor N1 or protumor N2
phenotypes in lung.134 Tumor-associated neutrophils acquire
N2 phenotype largely driven by transforming growth factor-β.
The significance of understanding the roles of these subsets in
atherosclerosis is yet to be addressed.
Mast Cells
Mast cells are widely distributed throughout the vascularized
tissues and are well-positioned to be one of the first cell types
to interact with environmental antigens and allergens. These
cells also are localized in the vessel walls, particularly within
the adventitia. Clinical studies demonstrated the abundant presence of mast cells in the intima and adventitia of atherosclerotic plaques in the diseased coronary arteries.135–137 In human
coronary arteries, colocalization of activated mast cells with
microvessels in the plaque suggested their role in intraplaque
hemorrhage and plaque destabilization.138,139 Furthermore,
oxidized LDLs induce mast cell degranulation and release
of cathepsins, heparin, histamine, and a variety of inflammatory cytokines, resulting in exacerbation of atherosclerosis.140
Mast cell activation in Apoe−/− mice causes plaque instability141,142 via release of proteolytic enzymes, such as tryptase,
and metalloproteinases.141 Collectively, current evidence indicates that mast cells are generally proatherogenic and also
promote plaque instability.141 In addition to their function as
effector cells, growing evidence indicates that mast cells can
positively or negatively modulate immune responses.143 Mast
cells have been shown to produce proinflammatory cytokines,
including IL-6 and IFN-γ, and promote atherosclerosis.144 In
contrast, their negative regulatory role has not been addressed
in atherosclerosis.
Iwata et al Bone Marrow Cells in Atherosclerosis 1641
Lymphocytes
Lymphocytes also have been shown to play important roles in
the complex inflammatory processes in the vascular wall.145 A
simplified view is that proinflammatory Th1 and cytotoxic T
cells accelerate atherogenesis, whereas regulatory T and Th2
cells are protective.146 However, as shown in other fields, each
T-cell subset may have different roles in different phases of
atherosclerosis.
Helper T-Cell Subsets
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CD4+ Th cells have been shown to play key roles in regulating
the atherosclerotic processes. In Apoe−/− mouse on a severe
combined immunodeficiency background, supplementation
of CD4+ T cells aggravated atherosclerosis.147 Conversely, absence of CD4+ cells reduced plaque burden in Apoe−/− mice.148
In contrast, Elhage et al149 demonstrated that absence of CD4+
population significantly aggravated atherogenic change in
descending thoracic and abdominal aorta in Apoe−/− mice,
whereas it did not affect lesions in the aortic root. These results
suggested that the different populations of CD4+ T cells may
have different roles in atherosclerosis. A number of studies
have shown that Th1 cells play a major role in atherosclerotic
immune responses.150,151 Th1 cells produce proinflammatory
cytokines, such as IFNγ and tumor necrosis factor-α, which
have been shown to promote atherogenesis.152,153 Deficiency
of the transcription factor T-box expressed in T cells, which
is important for Th1 and Th17 differentiation, reduced atherosclerosis in Ldlr−/− mice.150 These results indicate that Th1
cells are proinflammatory and proatherogenic.
Th2 cells represent a lineage of CD4+ T cells that primarily interact with B cells. The role of Th2 cells in atherosclerosis seems to be variable, with studies indicating that these
cells promote,154 protect against,155 or have no effect154 on
atherosclerosis depending on experimental models. In addition, IL-4, the signature cytokine of Th2, has been shown to
be atheroprotective and is not frequently observed in human
plaques. As such, the role of Th2 immune responses in atherosclerosis is still undergoing debate.156
Another subpopulation in CD4+ Th cells that was recently
suggested to be involved in atherogenesis is IL-17–producing
T cells (Th17 cells). However, their role in the atherosclerosis
process remains controversial.157,158 Although several studies
showed their proatherosclerotic role in mice,156 Th17 cells
were not observed in human plaques.159
Tregs
Tregs are a heterogeneous group of immune-inhibitory cells
that play a role in suppressing pathogenic immune responses.
In atherosclerotic plaques, Tregs are found in very low numbers
compared with other chronically inflamed tissues, suggesting
that local immune tolerance is impaired in the plaques.160
The atheroprotective role of Tregs has been demonstrated
using immunologic deletion of Treg cells in Ldlr−/−156 and
Apoe−/− mice.161,162 Anti-inflammatory cytokines, IL-10, and
transforming growth factor-β are considered to be the major
protective mediators produced by Tregs.163,164 However, these
cytokines are known to be produced by other cell types. A
recent study suggested a novel antiatherosclerotic function
of Tregs.165 In this article, Klingenberg et al165 showed that
deletion of Foxp3+ Tregs increased atherosclerotic lesions
without enhancing vascular inflammation in Ldlr−/− mice.
The mice also exhibited increased plasma cholesterol
levels attributable to reduced clearance of very-low-density
lipoprotein and chylomicron remnants, suggesting that Foxp3+
Tregs modulate lipoprotein metabolism. Results of studies on
clinical significance of Tregs in atherosclerosis in human are
conflicting.166,167 More studies are needed to elucidate the role
of Tregs in atherosclerosis in human.
CD8+ T Cells
CD8+ T cells are present within atherosclerotic plaques.168,169
However, the impact of CD8+ T cells on atherosclerosis development is less established than that of Th1 cells.149,170 For
instance, CD8+ T cell–deficiency did not affect plaque burden
in Apoe−/− mice.149 CD8+ T cell populations in atherosclerosis include cytotoxic lymphocytes and possibly Tregs (CD8+
Tregs). A potential pathological role of cytotoxic CD8+ T
cells is to aggravate local inflammation by attacking vascular cells, such as SMCs, promoting both lesion progression171
and plaque instability.172 On the contrary, the CD8+ suppressor population (CD8+ Treg) has been shown to have a protective role in atherosclerosis.173 Therefore, the accumulating
evidence suggests that, in addition to CD4+ T cells, CD8+ T
cells also play an important role in modulating arterial injury
responses.
Natural Killer T Cells
Natural killer T (NKT) cells are a distinct subset of T lymphocytes coexpressing cell surface molecules of natural killer
(NK) cells (eg, NK1.1 and Ly49) and T-cell receptors. NKT
cells are unique in their ability to respond to glycolipid antigens presented by the major histocompatibility complex class
I–like CD1d molecule.174 After activation, NKT cells are able
to rapidly and robustly secrete large amounts of both proinflammatory and anti-inflammatory cytokines, thereby playing an important regulatory role in a number of pathological
states.175 Because the atherosclerotic lesion is characterized by
the retention and modification of lipids in the vascular wall,
their ability to recognize lipid antigens suggests NKT cells
may be involved in the vascular inflammatory response. There
are 2 classes of NKT cells. The majority of NKT cells express
a semi-invariant T-cell receptor and are designated invariant
NKT cells. Smaller numbers of NKT cell express more diverse T-cell receptors and are referred to as variant NKT cells.
In humans, NKT cells were found at the shoulder regions of
carotid artery plaques as well as in atherosclerotic tissue in
abdominal aortic aneurysms.176 Experimental mouse models
demonstrated a proatherogenic role of NKT cells. Invariant
NKT cell–deficiency attenuated atherosclerosis in Apoe−/−
mice.177,178 In addition, the exogenous administration of the
glycolipid, α-galactosylceramide, that activates NKT cells
resulted in a marked increase in plaque burden in Apoe−/− and
Ldlr−/− mice.179
B Cells
B cells are present in atherosclerotic lesions, although they are
less frequent than T cells. Although B cells seem to initially
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accumulate in the intima of early atherosclerotic lesions, B
cells are more prevalent in the adventitia than in the intima in
advanced atherosclerotic lesions.180 B cells with other immune
cells may form tertiary lymphoid organs, which promote
lymphocyte recruitment and facilitate the immune response
against stimulating antigens locally in adventitia in humans
and in mice.181,182 Interestingly, adventitial inflammation has
been shown to associate with intimal inflammation and was
suggested to affect intimal inflammation.183,184 Adventitial B
cells and tertiary lymphoid organs therefore may affect inflammatory responses in not only adventitia but also intima.
B cells have been suggested to be either atheroprotective
or proatherogenic. The atheroprotective effect of B cells was
originally suggested by a study demonstrating significant attenuation of atherosclerosis in Apoe−/− and Ldlr−/− mice by
adoptive transfer of B cells.185,186 Mechanistically, autoantibodies produced by B cells, antioxidized LDL IgM,187 for
example, may inhibit oxidized LDL uptake by macrophages
and prevent foam cell formation.188 On the contrary, immunologic depletion of B cells was shown to decrease atherosclerotic development in mice.189,190 This apparent discrepancy in
B-cell function in atherosclerosis may be explained partly by
the unique roles of specific B-cell subsets in atherogenesis,
such as B1a B cells (atheroprotective),191 B2 B cells (proatherogenic),190 and regulatory B cell (atheroprotective).192–194 It is
also possible that specific antibodies produced by B cells may
either promote or suppress atherogenesis.189,195 Regulatory B
cells are a B-cell subset that produces IL-10 and have been
suggested to regulate inflammation. Their contribution to atherosclerosis remains to be addressed.
Innate Lymphoid Cells
Innate lymphoid cells (ILCs) are newly identified members of
the lymphoid lineage and have emerged as important effectors
of innate immunity and tissue remodeling. ILCs are characterized by 3 main features: the lack of rearrangement of antigen
receptors; the lack of myeloid and DC markers; and lymphoid
morphology.196 They are subdivided into 3 subsets, group 1
ILCs (ILC1 and NK cells), group 2 ILCs (ILC2 cells), and
group 3 ILCs (ILC3 and lymphoid tissue–inducer cells), based
on their ability to produce type 1 (eg, IFN-γ), type 2 (IL-5 and
IL-13), and Th17 cell–associated cytokines (IL-17 and IL-22),
respectively. NK cells are the prototypical member of group 1
ILCs. Group 2 ILCs have been shown to be important for host
defense against nematodes, and also have been shown to be involved in the repair of damaged respiratory tissue.197 Group 3
ILCs include lymphoid tissue–inducer cells that are essential
for lymph node development and other ILC3 cells. Except for
NK cells, functions of newly identified ILCs in atherosclerosis
are unknown.
NK Cells
NK cells, a key cellular component of innate immunity,
participate in the defense against viral function, as well as
pathogenesis of autoimmune diseases by their cytolytic
activity and cytokine production.198 The role of NK cells
in atherosclerosis is not well-understood; however, their
production of proinflammatory cytokines, including IFNγ, tumor necrosis factor-α, and granulocyte macrophage
colony–stimulating factor, suggests proatherosclerotic
functions.198–200 Accordingly, inhibition of NK cells
attenuated atherosclerosis in Ldlr−/− mice,201 and the shoulder
region of human plaques was shown to contain modest
numbers of CD56+ NK cells.202 However, a clinical study
found a significant reduction of circulating NK cell number
in patients with unstable or stable coronary artery disease
in comparison with healthy controls, suggesting a potential
atheroprotective role of NK cell.203
Conclusions
Many studies in animals and in humans have demonstrated
that various lineages of BM-derived cells in the circulation
infiltrate vascular lesions. In addition to vascular progenitor
cells, new subsets of immune cells are emerging as important effectors and regulators of immune responses. Moreover,
recent studies have unraveled dynamic plasticity in their phenotypes and functions. Accordingly, we can expect rapid expansion of knowledge of the physiological and pathological
roles of these new players. However, the limited surface markers and other lineage markers available often hinder unequivocal identification of cellular identities. This is particularly the
case for myeloid lineage cells, which may include EPCs and
SMPCs as well. Further assessment of lineages is important
not only for clarifying cell identity but also for better understanding of complex interactions between different cell types
and the microenvironment. These factors may further affect
the recruitment of other cells to the atherosclerotic plaque and
play a role in altering their phenotypes. Another important issue is the correspondence between mouse and human cells.
In particular, human counterparts of newly identified mouse
immune cell subsets may not have been well-characterized.
More importantly, human atherosclerosis develops differently from mouse atherosclerotic models in many aspects. For
instance, human atherosclerosis begins as the deposition of
extracellular lipid within the deep layer of intima of diffuse
intimal thickening that consists of SMCs, elastin, and proteoglycans.204 Macrophages infiltrate into the intima, and a part of
them becomes foam cells at the middle layer of the intima. In
contrast, the proliferation of SMCs and accumulation of extracellular matrices and lipid droplets occur after the accumulation of foamy macrophages in mouse atherosclerosis, and foam
cell macrophages often occupy the whole thickness of the intima as a predominant component of the lesion. Accordingly,
interplays between immune cells, vascular cells, and microenvironment are likely to be different in humans compared with
mice. In addition, atherosclerotic lesions in the mouse models
seldom cause plaque disruption with thrombosis. Needless to
say, mechanisms identified in mouse models need to be evaluated in human atherosclerosis. In this regard, rapid advances in
experimental technologies (eg, proteomics, metabolomics, and
genomics) and diagnostic modalities may facilitate development of biomarkers that bridge animal experiments and human
diseases.23 Future studies of the molecular control of the networks of BM-derived cells and vascular cells in atherosclerosis
also may lead to identification of novel therapeutic targets.
Acknowledgments
The authors thank Dr Joshua Hutcheson for his editorial expertise.
Iwata et al Bone Marrow Cells in Atherosclerosis 1643
Sources of Funding
This study was supported by the Japan Society for the Promotion
of Science (JSPS) through its Funding Program for World-Leading
Innovative R&D on Science and Technology (FIRST Program), and
by grant-in-aid for Scientific Research (S), (B), and (C) from JSPS (to
R. Nagai, I. Manabe, and H. Iwata).
Disclosures
None.
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Lineage of Bone Marrow−Derived Cells in Atherosclerosis
Hiroshi Iwata, Ichiro Manabe and Ryozo Nagai
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Circ Res. 2013;112:1634-1647
doi: 10.1161/CIRCRESAHA.113.301384
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