Physiol Rev
84: 767– 801, 2004; 10.1152/physrev.00041.2003.
Molecular Regulation of Vascular Smooth Muscle Cell
Differentiation in Development and Disease
GARY K. OWENS, MEENA S. KUMAR, AND BRIAN R. WAMHOFF
Department of Molecular Physiology and Biological Physics, University of Virginia
School of Medicine, Charlottesville, Virginia
I. Introduction: Phenotypic Modulation/Switching of the Smooth Muscle Cell Plays a Key Role in a
Number of Major Diseases in Humans
II. Molecular Regulation of Smooth Muscle Cell Differentiation and Maturation During Vascular
Development and Disease
A. Vascular SMCs are multifunctional and their functions vary during different stages of vascular
development
B. SMCs express multiple markers indicative of their relative state of differentiation-maturation, but
no single marker may exclusively identify SMCs to the exclusion of all other cell types
C. Environmental cues important in control of SMC differentiation
D. The SMC-specific transcriptional regulation is dependent on complex combinatorial interactions
of multiple cis-elements (regulatory modules) and their trans-binding factors
III. Characteristics and Role of Phenotypic Modulation of Smooth Muscle Cells in the Development,
Progression, and End-Stage Clinical Sequelae of Atherosclerosis
A. Origin of intimal SMCs in atheroclerotic lesions: media derived or blood derived?
B. Characterization of the SMC within atherosclerotic lesions of human and experimental animal
models
IV. Mechanisms That Contribute to Phenotypic Modulation of Smooth Muscle Cells Associated With
Vascular Injury and Experimental Atherosclerosis
A. Summary of environmental factors thought to be important
B. Molecular mechanisms of decreased SMC differentiation marker expression associated with
atherosclerosis: a novel experimental approach to studying SMC phenotypic modulation
V. Summary, Conclusions, and Future Directions/Challenges
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Owens, Gary K., Meena S. Kumar, and Brian R. Wamhoff. Molecular Regulation of Vascular Smooth Muscle Cell
Differentiation in Development and Disease. Physiol Rev 84: 767– 801, 2004; 10.1152/physrev.00041.2003.—The focus
of this review is to provide an overview of the current state of knowledge of molecular mechanisms/processes that
control differentiation of vascular smooth muscle cells (SMC) during normal development and maturation of the
vasculature, as well as how these mechanisms/processes are altered in vascular injury or disease. A major challenge
in understanding differentiation of the vascular SMC is that this cell can exhibit a wide range of different phenotypes
at different stages of development, and even in adult organisms the cell is not terminally differentiated. Indeed, the
SMC is capable of major changes in its phenotype in response to changes in local environmental cues including
growth factors/inhibitors, mechanical influences, cell-cell and cell-matrix interactions, and various inflammatory
mediators. There has been much progress in recent years to identify mechanisms that control expression of the
repertoire of genes that are specific or selective for the vascular SMC and required for its differentiated function. One
of the most exciting recent discoveries was the identification of the serum response factor (SRF) coactivator gene
myocardin that appears to be required for expression of many SMC differentiation marker genes, and for initial
differentiation of SMC during development. However, it is critical to recognize that overall control of SMC
differentiation/maturation, and regulation of its responses to changing environmental cues, is extremely complex
and involves the cooperative interaction of many factors and signaling pathways that are just beginning to be
understood. There is also relatively recent evidence that circulating stem cell populations can give rise to smooth
muscle-like cells in association with vascular injury and atherosclerotic lesion development, although the exact role
and properties of these cells remain to be clearly elucidated. The goal of this review is to summarize the current state
of our knowledge in this area and to attempt to identify some of the key unresolved challenges and questions that
require further study.
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0031-9333/04 $15.00 Copyright © 2004 the American Physiological Society
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I. INTRODUCTION: PHENOTYPIC MODULATION/
SWITCHING OF THE SMOOTH MUSCLE CELL
PLAYS A KEY ROLE IN A NUMBER OF
MAJOR DISEASES IN HUMANS
The vascular smooth muscle cell (SMC) in mature animals is a highly specialized cell whose principal function is
contraction and regulation of blood vessel tone-diameter,
blood pressure, and blood flow distribution. SMCs within
adult blood vessels proliferate at an extremely low rate,
exhibit very low synthetic activity, and express a unique
repertoire of contractile proteins, ion channels, and signaling molecules required for the cell’s contractile function that
is clearly unique compared with any other cell type (191)
(see also Ref. 238). Unlike either skeletal or cardiac muscle
that are terminally differentiated, SMCs within adult animals
retain remarkable plasticity and can undergo rather profound and reversible changes in phenotype in response to
changes in local environmental cues that normally regulate
phenotype (191). However, it is important to appreciate that
the SMC can also undergo much more subtle changes in
phenotype including alterations in calcium sensitivity/handling as exemplified by comparisons of different SMC subtypes that undergo phasic versus tonic contraction and
switching between these phenotypes (239). Striking examples of SMC plasticity can be seen during vascular development when the SMC plays a key role in morphogenesis of
the blood vessel and exhibits high rates of proliferation,
migration, and production of extracellular matrix components such as collagen, elastin, and proteoglycans that make
up a major portion of the blood vessel wall while at the same
time acquiring contractile capabilities. Similarly, in response
to vascular injury, the SMC dramatically increases its rate of
cell proliferation, migration, and synthetic capacity and
plays a critical role in vascular repair. The extensive plasticity exhibited by the fully mature SMC is an inherent property
of the differentiated phenotype of the SMC that likely
evolved in higher organisms because it conferred a survival
advantage; that is, mutations that compromised the ability of
the SMC to participate in vascular repair were likely detrimental to the organism and did not persist. However, an
unfortunate consequence of the high degree of plasticity
exhibited by the SMC is that it predisposes the cell to abnormal environmental cues/signals that can lead to adverse
phenotypic switching and acquisition of characteristics that
can contribute to development and/or progression of vascular disease. Indeed, there is strong evidence that phenotypic
switching of the SMC, which we define as any change in the
normal structure or function of the differentiated SMC, plays
a major role in a number of major diseases in humans
including atherosclerosis, cancer, and hypertension. Of
these, the changes that occur in atherosclerosis are perhaps
the most profound and will be used throughout this review
to illustrate key principles regarding how phenotypic switching of SMC might contribute to development of vascular
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disease. However, before considering how SMC differentiation is regulated under normal conditions, it is noteworthy to
first briefly consider several other less notable examples of
disease states in humans that are characterized by SMC
phenotypic switching to illustrate the general importance of
this phenomenon for human health.
Systemic hypertension is a widespread cardiovascular disease clinically defined as a sustained diastolic pressure of ⬎90 mmHg or a systolic blood pressure ⬎140
mmHg (122a). Although the etiology is extremely complex and undoubtedly varies between individuals, a common feature in the majority of cases of hypertension is an
increase in peripheral resistance as a result of increased
vascular tone/SMC contractility and vascular remodeling
(62, 184) that are each complex processes that involve
phenotypic switching of the SMC. Although controversial,
the changes in contractility have been attributed to many
different factors including alterations in intracellular calcium handling/release (94) and alterations in membrane
potential (270). These changes in SMC function appear to
be much more subtle than those associated with developmental processes and several other diseases but nonetheless serve as good examples of the wide plethora of
phenotypic changes that SMC can undergo in response to
changing environmental cues (see sect. IIC). In contrast,
alterations in vascular structure are associated with extensive remodeling of the resistance vessels as a consequence of more profound changes in SMC phenotype
including increased growth, synthesis of matrix materials,
reorganization of cell-cell and cell-matrix contacts, apoptosis associated with vessel rarefaction, and many other
changes (117, 184).
Additional examples of diseases associated with alternations in SMC function include asthma (195), obstructive bladder disease (142), and numerous gastrointestinal
and reproductive disorders (76). Although the precise role
of the SMC in the initial cause of these diseases is controversial, there is clear evidence that the plethora of
changes that occur play a key role in the clinical consequences of these diseases.
An extremely important but underappreciated example illustrating defective SMC differentiation in human
disease is seen in many forms of cancer. Although it is
widely recognized that growth of solid tumors is dependent on development of a circulatory supply (26), what is
much less well recognized is that the blood vessels that
form within many tumors are often immature or defective
in that they show very poor investment with SMCs or
pericytes and are greatly enlarged and extremely leaky
(56, 182). In addition, in many cases there appears to be
some investment by presumptive SMC, but the phenotype
of the SMC is abnormal with gross alterations in morphology and the failure to express the appropriate repertoire
of SMC differentiation marker genes (182). Indeed, it is
not uncommon to find “capillaries” (no SMC investment)
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MOLECULAR REGULATION OF SMC DIFFERENTIATION
that have a lumen diameter in excess of 100 ␮m, and the
prevalence of these so-called “giant capillaries” is equated
by pathologists with a high propensity for tumor cell
shedding and possible metastasis, since these vessels are
readily penetrated by tumor cells. In contrast, mature
blood vessels (SMC invested) appear to be highly resistant to tumor cell penetration and show very low rates of
tumor cell shedding. Whereas the mechanisms responsible for defective SMC-pericyte investment of tumor vessels and/or failed SMC differentiation/maturation are very
poorly understood, in the final analysis the problem relates at least in part to abnormal recruitment and/or differentiation of SMCs and/or SMC precursor cells.
The most widely acknowledged example of a disease
in which SMC phenotypic switching is believed to play a
key role is atherosclerosis, a disease that is responsible
for over 55% of all deaths in Western civilization. Atherosclerosis is an extremely complex disease involving many
cell types including macrophages, lymphocytes, neutrophils, endothelial cells, and vascular SMC (211). In addition, relatively recent evidence has implicated possible
involvement of circulating multipotential stem cells derived from bone marrow, which may give rise to a variety
of lesion cells including SMCs or SMC-like cells (84, 221,
230), although the contribution of theses cells in human
disease remains quite controversial (75, 111) (see sect. III).
Of interest, the role of the SMC appears to vary depending
on the stage of the disease, with it playing a maladaptive
role in lesion development and progression (191, 211), but
likely playing a beneficial adaptive role within the fibrous
cap in stabilizing plaques before activation of protease
cascades that may contribute to end-stage disease events
such as plaque rupture (69, 70). What has also become
clear is that the contributions of the SMC are not a simple
function of alterations in its growth state but rather are a
function of very complex changes in the differentiated
state of the SMC including increased matrix production
(148, 246, 255), production of various proteases (70), participation in chronic inflammatory responses including
production of inflammatory cytokines and expression of
at least some inflammatory cell markers (85, 208, 209),
altered contractility and expression of contractile proteins (129, 130), and a variety of other changes that have
collectively been referred to as “phenotypic modulation”
(reviewed in Ref. 191), a very useful descriptive term
originally coined by Julie Chamley-Campbell et al. (29)
nearly 30 years ago (see sect. III).
Although the term phenotypic modulation (or the
synonomous term of phenotypic switching) was originally based largely on morphological criteria, over the
past several decades its definition has been expanded by
the vascular biology field to encompass the full range of
possible alterations in functional and structural properties that can be exhibited by the SMC in response to
changing environmental cues, including both profound
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but also subtle changes in gene expression patterns, signaling mechanisms, contractility, etc. It is equally important to recognize that the process of phenotypic modulation or switching, as applied herein, is applicable to all
SMCs or SMC-like cells irrespective of their origins or
location in the body; that is, it is not a phenomenon
restricted to consideration of intimal SMC within atherosclerotic lesions but rather applies to the process by
which environmental cues influence the behavior of all
SMC under all circumstances. As such, although there is
evidence suggesting that SMCs or SMC-like cells within an
injured blood vessel in animal models, or human atherosclerotic lesion may be derived from a variety of sources
including medial SMCs (212), transdifferentiation of endothelial cells (47, 63), adventitial fibroblasts (217), or
circulating “stem” cells (24, 221, 247), the principal of
local environmental cues impacting the patterns of gene
expression and behavior of these cells applies.
The focus of this review is to provide an overview of
the current state of knowledge of molecular mechanisms/
processes that control differentiation of vascular SMC
during normal development and maturation of the vasculature, as well as how these mechanisms/processes are
altered with vascular injury or disease. We do not review
the topic of origins of vascular SMC during vascular development, since there are already several excellent relatively recent reviews on this topic (52, 113). In addition,
we do not focus on providing a comprehensive review of
the function of various SMC differentiation marker genes
or mechanisms that regulate SMC contractility, since this
topic was elegantly reviewed very recently by Somlyo and
Somlyo (239). Rather, we update information provided in
our 1995 Physiological Reviews article (191) with respect
to identification of novel SMC selective genes and gene
products that have helped advance our knowledge of
molecular mechanisms that control SMC differentiation
in development and disease. Finally, we wish to apologize
in advance to our many outstanding colleagues whose
work we may have either inadvertently overlooked or
been unable to discuss in sufficient detail due to space
constraints.
II. MOLECULAR REGULATION OF SMOOTH
MUSCLE CELL DIFFERENTIATION AND
MATURATION DURING VASCULAR
DEVELOPMENT AND DISEASE
A. Vascular SMCs Are Multifunctional and Their
Functions Vary During Different Stages of
Vascular Development
Before considering molecular mechanisms that control SMC differentiation in development and disease, it is
important to first briefly review some general principles of
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regulation of normal cellular differentiation, as well as
some unique aspects of control of differentiation of vascular SMCs. We will start with a simple definition of
cellular differentiation. Although this may be obvious to
the majority of readers, we nonetheless review it here
since we continually encounter statements in the literature that convey some confusion in this area.
Cellular differentiation is simply the process by
which multipotential cells in the developing organism
acquire those cell-specific characteristics that distinguish
them from other cell types. Although the process of cellular differentiation is quite complex, in the final analysis
it can be subdivided into the following three major regulatory components: 1) selective activation of the subset of
genes required for the cell’s differentiated function or
functions; 2) coordinate control of expression of cellselective/specific genes at precise times and stochiometries; and 3) continuous regulation of gene expression
through effects of local environmental cues on the genetic
program that determines cell lineage, including control of
chromatin structure or epigenetic programming that can
influence the ability of transcription factors to access
regulatory regions of genes. In addition, it is important to
recognize that an understanding of the differentiation of
any cell type not only involves elucidating cell autonomous mechanisms that control gene expression patterns
and functional properties (i.e., specialization of inividual
cells), but also must encompass how the cell interacts
with its environment (i.e., other cells, matrix, etc.) and the
complex processes that control overall tissue and organ
morphogenesis.
A major challenge in understanding differentiation of
the SMC is that it can exhibit a wide range of different
phenotypes at different stages of development, and even
in adult organisms the cell is not terminally differentiated
and is capable of major changes in its phenotype in response to changes in its local environment (see reviews in
Refs. 191, 225) (Fig. 1.) For example, during early stages
of vasculogenesis SMCs are highly migratory and undergo
very rapid cell proliferation. Indeed, recent live videos of
vascular development, the SMC investment process, and
vascular remodeling in zebrafish (118) and avian systems
(45) indicate that there is a remarkable amount of movement of SMCs and SMC progenitor cells as part of the
complex morphogenic events that result in formation of
the cardiovascular system. During vascular development,
SMCs also exhibit very high rates of synthesis of extracellular matrix components including collagen, elastin,
proteoglycans, cadherins, and integrins that comprise a
major portion of the blood vessel mass. At this stage of
development, SMCs form abundant gap junctions with
endothelial cells, and the process of investment of endothelial tubes with SMCs or pericytes is critical for vascular
maturation and vessel remodeling (113). In contrast, in
adult blood vessels the SMC shows an exceedingly low
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rate of proliferation/turnover, is largely nonmigratory,
shows a very low rate of synthesis of extracellular matrix
components, and is a cell virtually completely committed
to carrying out its contractile function. Indeed, the mature
fully differentiated SMC expresses a repertoire of appropriate receptors, ion channels, signal transduction molecules, calcium regulatory proteins, and contractile proteins necessary for the unique contractile properties of
the SMC (191). However, upon vascular injury, “contractile” SMCs are capable of undergoing transient modification of their phenotype to a highly “synthetic” phenotype
(see sect. III), and they play a critical role in repair of the
vascular injury. Upon resolution of the injury, the local
environmental cues within the vessel return to normal,
and SMCs reacquire their contractile phenotype/properties. Taken together, the model that has emerged is that
SMCs within adult mammals are highly plastic cells that
are capable of rather profound alterations in their phenotype in response to changes in local environmental cues
important for their differentiation (Fig. 1). Key questions
are thus, 1) What genes and gene products serve as appropriate markers with which to study SMC differentiation/maturation? 2) What are the key environmental cues
or signals that control the expression of these SMC-specific/selective marker genes?
Before considering these questions, we wish to
briefly consider several lines of evidence challenging the
dogma that repair of vascular injury is carried out principally (or exclusively) by reversible phenotypic modulation of preexisting SMCs. Two alternative mechanisms
have been proposed, although in reality none is mutually
exclusive. The first line of evidence is that circulating
bone marrow-derived SMC progenitor cells play a major
role in normal vascular injury repair (84, 221, 230) (see
also sect. III). Note that we are excluding consideration of
the possible role of recipient-derived stem cells in normal
or transplant atherosclerosis (24, 102) in the present discussion, although we will consider this very interesting
topic in section III. The second line of evidence is that
SMC populations within blood vessels are extremely heterogeneous with resident stable populations of preexisting SMCs that are phenotypically distinct from the classical definition of a contractile SMCs (64, 86) and that these
cells carry out injury repair. We will briefly consider each
of these issues in the next two paragraphs.
A number of relatively recent studies have provided
evidence showing that circulating cells, presumably derived from bone marrow, can contribute to neointima
formation and repair following vascular injury (84, 221,
230). However, for the most part, studies in animal models have either involved very extensive damage to medial
SMCs (indeed, nearly complete destruction of the media
and SMC death), and/or transplantation-associated immunological injury due to genetic mismatch of host and
donor tissues combined with lack of adequate immuno-
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FIG. 1. The differentiation state of the vascular smooth muscle cell (SMC) is highly plastic and dependent on
integration of multiple local environmental cues. This figure summarizes many of the extrinsic factors or local
environmental cues that are either known or believed to be important in influencing the differentiation/maturation state
of the vascular SMC. The figure is intended to emphasize that SMC differentiation/maturation and phenotypic modulation/switching is dependent on the complex interaction of a multitude of local environmental cues, not any single factor,
and that a change in any one of these may lead to alterations in the phenotypic state of the SMC (i.e., phenotypic
switching); that is, the SMC must have evolved mechanisms whereby there is constant integration of signals present in
the local environment that in aggregate determine the pattern of gene expression appropriate for that circumstance.
Importantly, there appear to be a broad range of phenotypic states that can be exhibited by the SMC, depending on the
variable expression of SMC-selective differentiation markers. This includes a spectrum of phenotypes ranging from the
highly synthetic/proliferative SMC depicted on the left to the highly contractile fully differentiated/mature SMC shown
on the right. However, these should only be viewed as useful generic terms to describe the wide spectrum of different
phenotypes that can be exhibited by vascular SMC. The multiple arrows connecting the cell types are meant to illustrate
the complexity of steps involved in transitions between the different phenotypes and the fact that changes appear to be
reversible. Two separate pathways are depicted rather than a single reversible pathway, since it is not at all clear that
transitions in phenotype follow the same pathway. For example, the transient phenotypic switching associated with
repair of vascular injury is unlikely to recapitulate regulatory steps involved in controlling SMC differentiation during
initial vascular development, although there may be at least some common components. Likewise, environmental cues
that stimulate phenotypic switching associated with atherosclerosis are unlikely to recapitulate events that occur during
vascular development. Although not depicted, there may also be completely distinct developmental pathways for
different subsets of SMC within different SMC tissues, and/or within a given SMC tissue with changing environmental
cues. The top arrows in the diagram increase in size from left to right and the bottom arrows from right to left to depict
the relative increase in markers that typify a fully differentiated mature SMC on the right (i.e., smooth muscle ␣-actin
and smooth muscle myosin heavy chain) versus an increase in markers that typify a more immature SMC on the left (i.e.,
Smemb). It should not be implied that the SMC on the left necessarily does not express any SMC differentiation marker
genes and that the differentiated cell on the right does not express markers typical of the immature SMC. As discussed
at length in the text, there is considerable controversy as to the relative contribution of bone marrow-derived progenitor
cells (BMC, the dashed cell) in the developing neointima and whether these cells are capable of becoming fully
differentiated SMCs (as indicated by the “?” and the dashed arrows). PDGF, platelet-derived growth factor; ET,
endothelin; TGF, transforming growth factor; ROS, reactive oxygen species; NO, nitric oxide; EC, endothelial cells.
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OWENS, KUMAR, AND WAMHOFF
suppression therapies. As such, the very high frequencies
of investment of circulating cells may not accurately reflect what normally occurs with more subtle forms of
injury. In addition, a limitation in the field is that no
studies to date in the severe mechanical injury models
have provided compelling evidence that bone marrow
cells within lesions express definitive SMC markers such
as smooth muscle (SM) myosin heavy chain (MHC) and
smoothelin. Moreover, no studies have adequately addressed the possibility of fusion of circulating progenitor
cells with resident SMC. These issues as well as a discussion of the relevance of these animal studies to development of transplant atherosclerosis in humans are considered in greater detail in section III.
There are a number of reports in the literature demonstrating that there are heterogeneities between SMCs
within a given blood vessel with retention of a resident
stable population of cells that have a “synthetic phenotype” (64, 86). For example, Frid et al. (64) used a panel of
antibodies specific for different markers of SMC differentiation including SM ␣-actin, SM MHC, calponin, desmin,
and meta-vinculin to perform immunofluorescence labeling studies on cryosections of adult and fetal bovine main
pulmonary arteries. In addition, they performed Western
analyses of these marker genes in the three different
layers of the adult bovine pulmonary artery. Due to space
constraints, we cannot review their results in detail. However, they reported the presence of what they categorized
as four distinct populations or clusters of SMC based on
morphology, cell orientation, pattern of elastic lamellae,
and immunostaining patterns and speculated that these
distinct populations may represent unique lineages that
may serve different functions within the arterial media,
and respond differently to pathophysiological stimuli.
Whereas there is without question overwhelming evidence for the existence of heterogeneous populations of
SMC in vivo, no studies have shown that these represent
distinct stable SMC lineages that play a preferential role in
carrying out repair of vascular injury in vivo. Indeed, the
seminal studies of Clowes and co-workers (37– 41) would
seem to refute such a possibility in that they showed SMC
growth fractions (i.e., the fraction of medial SMC at time
0 that leave G0 and reenter the cell cycle) of up to 60%
following balloon injury of the rat carotid artery, indicating that the majority of SMC within the media retain the
capacity to reenter the cell cycle and contribute to vessel
repair in adult animals. As such, the preexisting “subpopulation” of SMC capable of phenotypic switching is far
greater than the frequencies observed by Frid et al. (64)
and indeed would have to represent a large fraction of
SMC in the vessel wall. Consistent with these results,
classic studies by Thomas et al. (251) involving generation
of complex SMC ancestor tables for the entire SMC population within the thoracic and abdominal aorta based on
pulse-chase labeling with [3H]thymidine in hypercholesPhysiol Rev • VOL
teremic swine models of atherosclerosis provided evidence that intimal lesions were polyclonal and derived
from multiple histologically discrete medial SMC that initiated DNA replication and subsequently underwent several rounds of DNA replication. Taken together, the preceding observations appear to be inconsistent with a
model in which only a small fraction of medial SMC
contribute to lesion formation. However, data are by no
means definitive, and further studies are needed to determine if the well-defined SMC subpopulations identified by
Frid et al. (64) represent distinct SMC lineages using
classical lineage tracing and transplantation methodologies; that is, do the various SMC populations observed
retain their unique properties following transplantation to
the locus of another putative lineage? Significantly, Bochaton-Piallat et al. (17) observed that distinct populations of rat cultured SMCs (adult and embryonic) retained
at least some phenotypic differences when implanted into
a rat carotid artery in vivo. These results suggest that
there is considerable stability in the phenotype of these
cells, but it is possible that the stable epigenetic reprogramming of these cells was a function of their extensive
growth in culture. In addition, since relatively large numbers of cells were transplanted, it is possible that transplanted cells may have created their own “microenvironmental domain or milieu” and that autocrine and paracrine effects contributed to the retention of phenotypic
differences. Of interest, the observations of Frid et al. (64)
were made in large arteries from large species where the
possibility of the existence of microenvironmental domains
would be much greater than in very small vessels in rats and
mice used in the majority of growth fraction studies. In any
case, it is clear that there is a critical need for rigorous
lineage tracing studies to clearly identify the origins and
functions of different SMC subpopulations in vivo.
Finally, we want to briefly comment on one study in
the literature that suggests the existence of a subpopulation of terminally differentiated SMC that is incapable of
cell cycle reentry (227). Whereas such an idea is intriguing, the evidence for this is based solely on studies showing the failure of a subpopulation of SMC derived from
dog aorta to proliferate in culture. However, this result
may simply represent the lack of appropriate culture reagents and/or conditions necessary to support growth of
these cells, and at present, there is no compelling evidence for the existence of a distinct terminally differentiated SMC population in vivo.
In summary, we feel that there is irrefutable evidence
that the principal source of SMCs responsible for repair of
vascular injury under “normal” circumstances are preexisting SMCs that undergo transient and reversible phenotypic modulation (see Fig. 1). However, it is also likely
that circulating bone marrow cells, cells derived from the
adventitia, and/or preexisting subpopulations of phenotypically modulated SMC can participate to some extent
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as well. As will be discussed in greater detail in section III,
the relative role of these different populations may vary as
a function of the nature of the vascular injury (e.g., the
degree of damage and whether it is induced through
mechanical or immunological means), or the disease
state.
B. SMCs Express Multiple Markers Indicative
of Their Relative State of DifferentiationMaturation, but No Single Marker May
Exclusively Identify SMCs to the Exclusion
of All Other Cell Types
The initial step in studying cellular differentiation is
to identify a set of cell-specific/selective target genes that
contribute to the differentiated function or functions of
the cell. This task has proven particularly challenging for
the SMC field because of the diverse functions of the SMC
and because most (if not all) SMC markers, although
selective for SMCs in adult animals, are expressed, at
least transiently, in other cell types during development,
tissue repair, or disease states. Nevertheless, a variety of
SMC-selective or specific genes and gene products have
been identified that serve as useful markers of the relative
state of differentiation-maturation of the SMC. These include the smooth muscle isoforms of contractile apparatus proteins: SM ␣-actin (67, 114, 156), SM MHC (8, 9, 65,
160, 177), h1-calponin (55, 176, 243), SM22␣ (55, 126, 144),
aortic carboxypeptidase-like protein (ACLP) (138, 267),
desmin (18, 174), h-caldesmon (66, 237, 278), metavinculin (74), telokin (96), and smoothelin (258). A detailed
description of several of these proteins and other potentially useful SMC differentiation markers and their expression patterns have been reviewed previously (113,
191) and will not be repeated here. The goals in this
section will be to very briefly summarize the various SMC
markers that are useful for assessing SMC differentiation/
maturation with a particular focus on identifying issues of
importance for assessing phenotypic switching of the
SMC in atherosclerosis and vascular injury.
One of the major deficiencies in studies investigating
the role of the SMC phenotypic switching in vascular
disease has been the failure of most studies to adequately
distinguish “differentiation markers” that serve as indices
of the relative state of differentiation of the SMC versus
“lineage markers” that can serve to identify SMCs to the
exclusion of all other cell types. As a consequence, many
cells may have either been misidentified as SMCs because
of assessment of the wrong or more often an inadequate
number of markers. Alternatively, many SMCs may not
have been identified as such, because of the inability to
recognize phenotypically modified SMCs due to (temporary) loss of expression of normal markers of SMCs as
part of the injury/disease process. The problem is further
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confounded by the fact that virtually all known SMC
differentiation markers, with the possible exception of
SM MHC, have been shown to be expressed, at least
transiently, in other cell types either during development
or in response to pathophysiological stimuli (see Table 1
and reviews in Refs. 113, 191). For example, the most
widely used SMC marker by far is SM ␣-actin in part
because of the commercial availability of a number of
very high-affinity and highly selective antibodies for this
protein (235). Indeed, SM ␣-actin is an excellent SMC
differentiation marker in that it is the first known protein
expressed during differentiation of the SMC during development (66, 114), and it is highly selective for SMC or
SMC-like cells in adult animals under normal circumstances. Moreover, it is required for the high force development properties of fully differentiated SMCs and is by
far the single most abundant protein in differentiated
SMCs making up to 40% of total cell protein (59). However, by no means is it a definitive SMC lineage marker in
that it is known to be expressed in a wide variety of
non-SMC cell types under certain circumstances including 1) skeletal and cardiac muscle during normal development (274), 2) in adult cardiomyocytes in association
with various cardiomyopathies (1), 3) in fibroblasts (or
so-called myofibroblasts) in a wide range of circumstances including wound repair (reviewed in Ref. 217), 4)
in endothelial cells during vascular remodeling and/or in
response to transforming growth factor (TGF)-␤ stimulation (7, 10), and 5) in numerous tumor cells (34, 35).
Despite this fact, there are literally hundreds of papers in
the literature in some of the highest quality journals that
have inappropriately equated expression of SM ␣-actin as
sufficient evidence for identification of the SMC lineage.
Similarly, virtually all the remaining SMC differentiation marker genes are expressed in a variety of circumstances in other cell types. SM22␣, a calponin-like protein
of unknown function (281), exhibits an expression pattern very similar to SM ␣-actin. It is expressed in skeletal
and cardiac muscle during development (Table 1), and
within activated fibroblasts under a variety of conditions
(55, 126, 144, 217). H1 calponin, a calcium regulatory
protein, is expressed in cardiac myocytes, myofibroblasts,
and a variety of tumor cells (179, 214, 215, 271). Metavinculin is an intracellular protein localized at sites of insertion of microfilament bundles into cell membranes and is
expressed in cardiac muscle and myofibroblasts in addition to SMC (14, 57, 73). ACLP is widely expressed in
many tissues (138). Telokin, a gene contained within the
myosin light-chain kinase gene and believed to play a role
in SMC relaxation/Ca2⫹ sensitivity (275), appears to be
relatively specific for SMCs (96) but unfortunately is expressed at very low levels in many vascular SMCs of
interest in terms of vascular disease including the aorta
and coronary arteries (Table 1). Smoothelin appears to be
selectively expressed in differentiated SMCs as two
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OWENS, KUMAR, AND WAMHOFF
TABLE
1. cis-Regulatory elements required for SMC promoter expression in vivo
Embryonic
Promoter
Wild type
SM MHC
(160,167,169)
SM ␣-actin
(151,156)
SM22␣ (2,106,
126,145)
Telokin (106)
Desmin (174)
CArG B mutant
SM MHC (167)
SM ␣-actin
(156)
SM22␣
(127,145,244)
Telokin‡ (106)
Desmin§ (174)
GC box mutant
SM MHC (159)
SM ␣-actin
SM22␣ (201)
Telokin
Desmin
CArG A mutant
SM MHC (167)
SM ␣-actin
(156)
SM22␣
(27,145,244)
Telokin‡ (106)
Desmin§ (174)
TCE mutant
SM MHC (87)
SM ␣-actin
(151)
SM22␣ (2)
Telokin
Desmin
Intronic CArG
mutant
SM MHC
(167)
SM ␣-actin
(156)
SM22␣
Telokin
Desmin
SM
Adult
CRD SKM Aorta
Coronary
artery
Mesentaric
artery
Vena
cava Airways
Stomach
Intestine
Bladder
Injured
non-SM Carotid
Experimental
Atherosclerosis
4
1*
0
4
4
4
4
4
4
4
4
0
0
NT
4
4
4
4
4
4
4
4
4
4
4
0
0
NT
4
4
4
4
0
4
4
0
4
4
0
4
4
0
4
4
0
4
0
0
0
4
4
0
0
4
0
0
4
0
4
4
0
0
0
Heart
0
NT
NT
NT
NT
NT
2–3†
0
0
0
0
2
0
2
3
3
3
0
NT
NT
0
0
0
0
0
0
0
0
0
0
0
0
NT
NT
0
0
0
0
Present but not tested
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
Present but not tested
Unknown
4
4
4
4
Unknown
Unknown
4
4
0
4
0
0
4
0
NT
NT
4
NT
NT
0
0
0
0
0
0
0
0
0
0
0
0
NT
NT
0
0
0
0
0
0
0
0
0
0
0
0
NT
NT
0
0
0
0
Present but not tested
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
NT
NT
NT
NT
NT
NT
NT
NT
Present but not tested
0
0
Unknown
Unknown
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
NT
NT
NT
NT
NT
NT
NT
NT
2–4†
0
0
0
2
4
4
4
4
4
4
0
NT
NT
0
Unknown
Unknown
Unknown
0
0
0
0
0
0
0
0
0
0
0
NT
NT
NT
NT
NT
NT
NT
NT
This table supports a model wherein smooth muscle cell (SMC) subtypes employ different modular regulatory regions for expression including
selective CArG-dependent transcription regulatory schemes in vivo. What is evident from this table is a regulatory scheme that is consistent with
the high degree of plasticity of the SMC, and presumably is at least in part a function of differences in activation of selected promoter
regions/cis-elements by unique extrinsic local environmental cues in one SMC tissue versus another. In contrast, it is also apparent that there exists
a paucity of studies testing the molecular mechanisms involved in regulating SM-selective genes in the context of vascular injury (injured carotid)
and experimental models of experimental atherosclerosis (ApoE or Ldl-R deficient mice). Given space constraints, the cis-modular regulatory
regions described herein directly parallel the scheme shown in Figure 1 including (5⬘ to 3⬘): CArG B (5⬘ CArG far or CArG2), GC box, CArG A (5⬘
CArG near or CArG 1), TCE (TGF-␤ control element), and the intronic CArG. Only SMC promoter-reporter constructs that have been tested in
transgenic mice are described. These promoter-reporter constructs include: SM MHC (rat ⫺4,216 to ⫹11,795) (167), SM ␣-actin (rat ⫺2,560 to
⫹2,784) (156), SM22␣ (rat ⫺445) (127, 145), telokin (mouse ⫺190 to ⫹180) (106), and desmin (mouse ⫺4,005) (174). For simplicity, the level of
␤-galactosidase expression is shown on a scale of 0 to 4, with 0 equalling no expression and 4 equalling the wild-type expression. SM, smooth muscle
containing tissue; CRD, cardiac muscle; SKM, skeletal muscle; NT, not tested. Reference numbers are given in parentheses.
* Wild-type SM MHC
expression is only weakly detectable in a very small population of cells within the right atrium at embryonic day 8.5 that may represent a population
of epicardial cells that may transiently differentiated into SMCs during the process of epithelial-mesenchymal transformation of proepicardial cells
and formation of coronary vessels (202).
† The expression patterns of the SM MHC CArG B and intronic CArG mutants at embryonic day 19.5
closely mimic those in adult mutants (167).
‡ The telokin promoter (mouse ⫺190 to ⫹180) only contains 1 CArG (106).
§ The desmin
promoter (mouse ⫺4,005) contains 6 CArG-like elements and mutation of CArG4 results in loss of tissue expression (174).
Physiol Rev • VOL
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MOLECULAR REGULATION OF SMC DIFFERENTIATION
known isoforms: a 55-kDa (type A) and 120-kDa (type B)
isoform that are expressed selectively in visceral SMCs
and vascular SMCs, respectively (204, 258). However, as
acknowledged in the initial papers identifying smoothelin
(204, 258), this gene is expressed at relatively high levels
in a variety of organs and tissues including heart, kidney,
and brain (e.g., see Fig. 4 of Ref. 204). Whereas the
authors concluded that this signal was derived from blood
vessels contained within these tissues, we feel that there
has not yet been sufficient scrutiny of smoothelin expression to unequivocally establish it as a legitimate SMC
lineage marker, particularly since there is at least one
report showing that it is expressed in prostate stromal
cells in culture in response to basic fibroblast growth
factor (bFGF) or TGF-␤2 stimulation (248), and as yet no
studies have been reported examining its expression in
many cell lineages such as myofibroblasts and tumor cells
that have been shown to express many other putative
SMC lineage markers.
Expression of SM MHC has been extensively scrutinized by a large number of different laboratories (5, 160,
177, 202, 218). Of particular note, Miano et al. (177) carried out very detailed in situ hybridization analyses of
expression of SM MHC throughout development and maturation of whole mouse embryos and found no evidence
for expression of this marker in cell types other than
SMCs. Consistent with these results, we found a high
degree of SMC specificity of expression of a ⫺4.2 to 11.7
SM MHC promoter enhancer reporter gene in transgenic
mice either using direct measurement of a lacZ reporter
transgene (160) (Table 1), or a highly sensitive cre recombinase inducible system (202). Importantly, the latter involved crossing mice containing a SM MHC promoter
enhancer cre recombinase gene to an indicator mouse
strain that shows cre-inducible (permanent) activation of
a LacZ gene in any cell that has ever expressed this
promoter-enhancer throughout development and maturation. Results showed complete specificity of expression in
SMCs with the exception of a small population of cells
within the right atrium at embryonic day 8.5 that may
represent a population of epicardial cells that may have
transiently differentiated into SMCs during the process of
epithelial-mesenchymal transformation of proepicardial
cells and formation of coronary vessels. Moreover, we
found no evidence of activation of this gene in myofibroblasts in a dermal wound healing model (J. J. Tomasek, D.
Raines, and G. K. Owens, unpublished observations). As
such, in many laboratories, expression of SM MHC seems
to be highly restricted to SMCs. There are, however, a
number of reports of expression of SM MHC within myofibroblasts, endothelial cells, and tumor cells (19, 139).
However, we have found that the primary antibody employed in these studies cross reacts with a nonmuscle
isoform of MHC, i.e., SMemb or nonmuscle (NM)-B MHC,
at least in some species, thus raising some uncertainty
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regarding reports of expression of SM MHC outside of
SMCs. Importantly, confirmation of specificity of putative
SM MHC antibodies must include high-resolution Western
analyses sufficient to resolve the multiple SM and NM
isoforms of MHC that differ in mass by only 5% (213). In
addition, very careful attention must be given to the species of animal employed, since there appear to be speciesdependent variations in reactivity of different SM MHC
antibodies that may confound interpretation of experimental results. In summary, whereas we cannot rule out
that SM MHC may be expressed at least transiently under
certain circumstances in cells other than SMCs in vivo, to
our knowledge, conclusive evidence of SM MHC expression (by immunostaining with SM MHC specific antibodies or RT-PCR followed by sequencing) in a non-SMC in
vivo does not exist, and at present, it is the most discriminating marker for the SMC identified to date. However,
we would not be surprised if SM MHC is found in nonSMC, and as such, it is recommended that identification of
vascular SMCs use SM MHC as well as several other
markers such as SM ␣-actin, smoothelin, SM22␣, and
h1-calponin. In addition, one should not ignore use of
classical histological and ultrastructural methods such as
the presence of a basement membrane, proximity to endothelial cells or epithelial cells, an abundance of contractile myofilaments, and a spindle shape. However, it
must be recognized that these criteria are very qualitative
in nature and do not distinguish the SMC to the exclusion
of other cell types including activated myofibroblasts, a
cell type that shares many features in common with the
SMC that we will consider in further detail at the end of
this section.
The preceding studies have focused on identification
of markers of the differentiated contractile SMC. However, as outlined in section IIA, the SMC has a multitude of
different functions that vary during development/maturation in different blood vessel types, and as part of the
response to vascular injury. As such, rather than focusing
on markers that are suppressed during transition of contractile SMC to an alternative state, a number of laboratories have focused on identifying genes that serve as
markers of these states and have investigated mechanisms that regulate coordinate expression of theses
genes. In our opinion, some of the most exciting work in
this area has been carried out by Nagai and co-workers
(166, 228, 268) who have shown that one of the most
useful definitive “positive” markers of the phenotypically
modulated SMC is the nonmuscle MHC isoform designated NM-B MHC, or SMemb (SM MHC embryonic). Of
interest, this marker appears to be relatively specific for
phenotypically modified or embryonic SMCs, although it
also is expressed in neuronal lines (119). Its expression is
induced in association with vascular injury and within
intimal SMC of atherosclerotic lesions. These investigators have also extensively characterized mechanisms that
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OWENS, KUMAR, AND WAMHOFF
control transcription of this gene (228, 268), and as will be
noted in section IID, there appear to be some reciprocal
control processes that positively regulate these genes
while simultaneously repressing expression of genes indicative of the contractile state of SMCs. Geary et al. (72)
have recently completed a very comprehensive analyses
of markers of intimal SMCs using array analyses, although
it is not clear to what extent the differences reported
reflect phenotypic switching of the SMC per se as opposed to characteristics of intimal versus medial SMCs.
Nonetheless, it will be interesting to ascertain which of
the many differentially expressed genes they have identified might serve as specific markers of phenotypically
modified SMC.
As noted in the preceding sections, there is compelling evidence that activated myofibroblasts and SMCs
express a number of common marker genes including SM
␣-actin, SM22␣, h1-calponin, metavinculin, and possibly
SM MHC (179, 217, 248). These observations are not
surprising given that these cell types also share a number
of common functional properties including force development/contraction and extensive production of extracellular matrix proteins. Indeed, it has been hypothesized that
the fibroblast/myofibroblast represents an alternative
phenotype of the SMC and/or a progenitor/precursor of
fully differentiated mature SMC. However, at present
there is no direct evidence in support of this hypothesis,
including indisputable evidence for the interconversion of
the two cell types based on transplantation and lineage
tracing studies. Indeed, since each cell type manifests a
broad range of different phenotypes, there is no general
agreement as to the functional, morphological, and molecular distinctions between phenotypically modulated
SMCs and activated myofibroblasts and no widely accepted in vitro models with which to adequately resolve
these issues. Of interest, we have recently demonstrated
that although TGF-␤1 treatment of 10T1/2 embryonic fibroblasts resulted in activation of expression of SM ␣-actin, SM22␣, and several other SMC differentiation markers, we saw no evidence of activation of expression of SM
MHC or the highly potent and cardiac/SMC-selective SRF
coactivator myocardin (280). As such, in so much that
TGF-␤-treated 10T1/2 cells represent a model of myofibroblast activation, these results suggest that one may be
able to distinguish myofibroblasts from SMC on the basis
of these two marker genes. However, to our knowledge,
no studies have as yet assessed myocardin expression
within activated myofibroblasts in vivo, and further studies of this nature are clearly warranted. One must also
consider the distinct possibility that activated myofibroblasts, phenotypically modulated SMCs, and fully differentiated SMCs may not be easily distinguished on the
basis of qualitative differences in the patterns of gene
expression but rather may differ primarily by virtue of the
level of expression of known marker genes; that is, fully
Physiol Rev • VOL
differentiated/mature SMCs undoubtedly express far
higher levels of many of these marker genes including SM
␣-actin and SM MHC than myofibroblasts, and indeed, this
is a fundamental reason for the quantitative differences in
force-generating capabilities between these cell types. In
any case, there are still many unresolved but important
questions regarding both the properties that distinguish
the SMC versus the activated fibroblast, as well as the
origins/lineage relationship between these two cell types.
Indeed, as with studies of SMC subpopulations, clear
resolution of these issues is likely to be dependent on
definitive in vivo lineage tracing and cell transfer studies.
C. Environmental Cues Important in Control
of SMC Differentiation
Despite extensive evidence indicating that phenotypic modulation/switching of the SMC plays a key role in
the etiology of a number of major vascular diseases and
injury repair, very little is known regarding the specific
environmental cues and mechanisms that regulate SMC
differentiation/maturation in vivo. Results of gene knockout studies in mice have implicated a number of factors/
pathways, but results are equivocal due to uncertainties
regarding whether loss of the gene in question had a
direct or indirect effect on SMC differentiation. For example, knockout of the Krupple-like transcription factor
LKLF (or KLF2) was shown to be embryonic lethal at day
12.5 due in part to defective vascular maturation and
hemorrhage (135). However, based on observations that
LKLF was expressed in endothelial cells but not SMCs,
investigators speculated that defective SMC investment/
differentiation was likely the consequence of an indirect
effect mediated through some as yet unidentified LKLFdependent process in endothelial cells. Similarly, knockout of the type I TGF-␤ receptor alk1 (189), the TGF-␤
receptor II (190), the TGF-␤ signaling molecule SMAD5
(277), TGF-␤1 (48), Edg-1 the G protein-coupled receptor
for sphingosine-1-phosphate, (152), or the thrombin receptor PAR1 (80) are all associated with early embryonic
lethality due at least in part to defective vascular maturation and/or SMC investment/differentiation, although in
some cases there is incomplete penetrance in individual
animals. However, in no case has it been clearly shown
that the primary defect was the result of a direct effect on
the SMC. Indeed, a major difficulty in identifying factors
that regulate SMC differentiation in vivo is that most
candidate factors identified based on studies in cultured
SMCs are also involved in regulating differentiation of
other cell types during embryogenesis, and when knocked
out result in embryonic lethality before SMC differentiation normally occurs, and/or alter SMC differentiation
through secondary mechanisms.
Whereas very little is known regarding factors that
regulate SMC differentiation in vivo, results of studies in
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MOLECULAR REGULATION OF SMC DIFFERENTIATION
cultured SMC have implicated a large number of factors
including mechanical forces (205), contractile agonists
(71, 88, 100), extracellular matrix components such as
laminin and type I and IV collagens (25, 51, 90, 196, 252),
neuronal factors (28), reactive oxygen species (245), endothelial-SMC interactions (98, 103), thrombin (194), and
TGF-␤1 (2, 87, 120, 229) (reviewed in Refs. 29, 191), all
which have been shown to promote expression of at least
some SMC marker genes in cultured cell systems (Fig. 1).
However, these results, including the studies from our
own laboratory, need to be interpreted with caution, since
there is unequivocal evidence showing that that cultured
SMC systems employed often fail to adequately recapitulate regulatory pathways that are critical in vivo (see a
detailed discussion of this topic in Ref. 194). Particularly
informative are studies in our own lab showing that regions of the SM ␣-actin promoter or SM MHC genes that
are sufficient to drive cell-specific expression in cultured
SMC were completely inactive in vivo in transgenic mice
(156, 160). For example, we found that 547 bp of the
5⬘-region of the SM ␣-actin promoter had ⬎50-fold greater
activity than control constructs in multiple independent
lines of cultured SMC but was inactive in endothelial
cells, 10T1/2 cells, 3T3 fibroblasts, adventitial fibroblasts,
and rat2 fibroblasts in culture (231), suggesting that we
had defined sufficient regions of the promoter to control
cell-specific expression of this gene. However, this same
547-bp promoter construct was completely inactive in
SMCs in vivo in over 12 independent founder lines, although it was sufficient to drive very high expression in
cardiac and skeletal muscle during development in a manner similar to the endogenous SM ␣-actin gene (156). Our
conclusion from these studies is that cultured SMCs,
while expressing very high levels of their endogenous SM
␣-actin gene, do not fully recapitulate cell-specific gene
regulatory pathways critical in vivo. Although space does
not permit, our lab has now identified at least 15 similar
cases of major differences in expression of SM promoterenhancer reporter genes in cultured SMCs versus in vivo.
Although sometimes results are similar, in many cases
they are not. As such, unless one validates a putative gene
regulatory mechanism in vivo in transgenic mice, we are
dubious of its validity.
Surprisingly, despite the fact that cultured SMC lines
are highly modulated, and that phenotypic modulation is
a critical process in atherogenesis and vascular injury
repair, very few factors/pathways have been identified
that selectively and directly promote phenotypic modulation of the SMC with the exception of platelet-derived
growth factor-BB (PDGF-BB) (16, 43, 108, 147), whose
effects will be discussed in detail later in this section, and
an as yet unidentified factor produced by cultured endothelial cells (260) that has several characteristics similar
to connective tissue growth factor (CTGF) (22). The reason for the paucity of studies in this area is likely due to
Physiol Rev • VOL
777
two factors: 1) the incorrect belief that SMC phenotypic
modulation is simply secondary to growth stimulation,
i.e., the old and incorrect adage that differentiation and
proliferation are mutually exclusive processes in all cell
types; and 2) the untested assumption by many that phenotypic modulation of SMCs is a passive rather than
active process and is due simply to loss of positive SMC
differentiation factors.
It is now well established that differentiation and
proliferation are not necessarily mutually exclusive processes and that many factors other than the SMC’s proliferation status influences its differentiation state. This
topic has been reviewed extensively (191) and thus we
will only briefly summarize several relevant observations
here that substantiate this point. First, during late embryogenesis and postnatal development, SMCs are known to
have an extremely high rate of proliferation (42), yet at
this time they undergo the most rapid rate of induction of
expression of multiple SMC differentiation marker genes
(193). Second, SMC within advanced atherosclerotic lesions show a very low rate of proliferation that approaches that of fully differentiated SMC yet are highly
phenotypically modulated as evidenced by marked reductions in expression of SMC marker genes (188, 273).
These results show that cessation of proliferation alone is
not sufficient to promote SMC differentiation and suggest
that other SMC differentiation cues are absent and/or that
there are active repressors of SMC differentiation present.
Consistent with the hypothesis that phenotypic modulation of the SMC may be controlled actively and not
simply by loss of positive differentiation signals, we (16,
43, 108) and others (147, 238, 253) have shown that treatment of postconfluent cultures of rat aortic SMCs with
PDGF-BB is associated with rapid downregulation of expression of multiple SMC differentiation marker genes. Of
particular significance, under the conditions of our experiments, we found that PDGF-BB elicited only a transient
mitogenic effect with cell proliferation returning to control values within 36 h, despite repeated daily pulsing with
PDGF-BB (16). However, suppression of SMC marker
gene expression, including SM ␣-actin and SM MHC, persisted as long as PDGF-BB was present. Indeed, we found
that cultured SMC could be sustained in a highly dedifferentiated state with virtually no detectable expression
of SM ␣-actin indefinitely by treatment with PDGF-BB.
However, upon removal of PDGF-BB, SMC marker genes
were rapidly reinduced. Of further interest, we also
showed that the concentration of PDGF-BB required for
inducing SMC phenotypic modulation was 10-fold lower
than that required to elicit a growth response under these
experimental conditions; that is, we could induce downregulation of SM ␣-actin expression in the absence of cell
cycle entry. In contrast, we found that bFGF and fetal
bovine serum (FBS) had little or no effect on SMC differentiation marker gene expression in postconfluent cul-
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OWENS, KUMAR, AND WAMHOFF
tures despite eliciting nearly identical proliferative responses, and thrombin-induced proliferation was associated with increased not decreased expression of SMC
marker genes (16, 43, 261). Taken together, these results
indicate that PDGF-BB is a highly efficacious and selective negative regulator of SMC differentiation, and that its
effects on differentiation are not secondary to growth
stimulation. Whereas the results of these culture studies
are extremely interesting, as yet there is no definitive
evidence that PDGF-BB is a potent negative regulator of
SMC differentiation in vivo, although conventional PDGF
␤-receptor knockout mice do show reduced investment of
arterioles with SMCs (149) (as discussed in sect. IV). However, it is unclear whether this represents 1) a direct or
indirect effect of loss of PDGF ␤-receptor signaling in
SMCs, 2) loss of PDGF ␤-receptor-dependent amplification of cells that give rise to SMC, and/or 3) impaired SMC
investment secondary to reduced migration. In addition,
there is paradoxical evidence that PDGF-BB treatment of
proepicardial organ cells (137) or mononuclear circulating progenitor cells isolated from the blood buffy coat
plated on collagen (234) can enhance rather than repress
SMC gene expression in these systems. There are several
possibilities to explain these differences. 1) PDGF-BB
may have opposite effects in adult versus embryonic
SMC, and/or 2) increased expression of SMC markers in
proepicardial and circulating progenitor cells in vitro may
reflect selective amplification of PDGF ␤-receptor positive SMC progenitor cells in these systems rather than a
direct effect on SMC gene expression per se.
In summary, although there is extensive evidence
showing that SMCs are highly plastic and can respond to
changes in environmental cues by changing their phenotype, the precise factors and mechanisms that regulate
both normal and abnormal differentiation of SMCs in vivo
are very poorly understood. However, the model that has
evolved is that differentiation/maturation of the vascular
SMC is dependent on constant integration of a large number of local environment cues that in aggregate determine
the pattern of gene expression appropriate for that circumstance (Fig. 1). It is also important to recognize that
there may be many different SMC phenotypes possible as
required to carry out the multitude of functions necessary
during development, maturation, vascular remodeling,
and disease (Fig. 1). Clearly, extensive investigation is
needed to test many of the factors/pathways that have
been implicated based on studies in cultured SMC systems. However, due to the fact that many of these pathways and factors are also involved in regulating a wide
variety of other processes in other cell types, definitive
studies in this area are likely to be dependent on use of
sophisticated SMC- and SMC-conditional gene targeting
systems.
Physiol Rev • VOL
D. The SMC-Specific Transcriptional Regulation
Is Dependent on Complex Combinatorial
Interactions of Multiple Cis-Elements
(Regulatory Modules) and Their TransBinding Factors
Tremendous progress has been made in the past
decade in identifying mechanisms that contribute to transcriptional regulation of SMC marker genes [see reviews
by Owens and co-workers (134, 194), Firulli and Olson
(61), Majesky (162), and Miano (175)]. Due to the massive
amount of work in this area and previous reviews in this
area we cannot discuss each of these regulatory pathways
in detail; rather, we will focus on briefly reviewing several
examples that illustrate general paradigms of SMC-specific gene regulation. In addition, we will briefly consider
several regulatory pathways that act to suppress expression of SMC marker genes, since these may play a role in
phenotypic modulation of SMCs in response to vascular
injury or atherosclerosis, as discussed in section IV.
1. CArG SRF-dependent regulation plays a key role
in regulation of most SMC differentiation marker
genes characterized to date
Site-directed mutagenesis studies in transgenic mice
have shown that expression of virtually all SMC marker
genes identified to date are dependent on one or more
CArG elements [i.e., a CC(AT)6GG motif] found within
their promoter and/or intronic sequences (126, 144, 156,
167, 174, 278) (Table 1). For example, we demonstrated
that the region of the SM ␣-actin promoter from ⫺2,560 to
⫹2,784 completely recapitulated expression patterns of
the endogenous SM ␣-actin gene in vivo in transgenic
mice. However, expression of this nearly 6,000-bp promoter enhancer was completely abolished by a 4-bp mutation of any one of three highly conserved CArG elements contained within it (156). Similarly, mutation of
conserved CArG elements within the SM MHC (167),
SM22␣ (126, 145), and desmin (174) promoter enhancers
also virtually abolished expression in vivo in transgenic
mice, although of interest we found that mutation of each
of the three conserved SM MHC CArG elements had differential effects on different SMC subsets (167). For example, mutation of CArG1, which is the most proximal
CArG element in the 5⬘-region of the SM MHC promoter,
completely abolished expression in all SMC subtypes,
whereas mutation of the intronic CArG element completely abrogated expression in large-conduit arteries and
the coronary circulation but had no effect in muscular
arteries, pulmonary airway SMC, or gastrointestinal SMC.
Furthermore, we found that deletion of regions of the
large 17-kb SM MHC promoter enhancer (based on DNase
mapping studies) resulted in selective loss of transcription in subsets of SMC (169). Similarly, a 445-bp region of
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MOLECULAR REGULATION OF SMC DIFFERENTIATION
the SM22␣ promoter region has been shown to be sufficient to drive expression in SMCs within large arteries
and arterioles, but not within gastrointestinal or other
SMC tissues in adult mice that do express their endogenous SM22␣ gene, suggesting that additional regulatory
sequences are required for expression in these tissues but
are dispensable for expression in arterial SMCs (126, 145).
Taken together, the results of the preceding studies
support a model wherein SMC subtypes employ different modular regulatory regions for expression including
selective CArG-dependent transcription regulatory schemes.
Such a regulatory scheme is consistent with the high
degree of plasticity of the SMC, and presumably is at least
in part a function of differences in activation of selected
promoter regions/cis-elements by unique extrinsic local
environmental cues in one SMC tissue versus another.
CArG elements bind the transcription factor SRF, a
MADS (MCM1, Agamous, Deficiens, SRF) box transcription factor, that was first identified and named because of
its ability to confer serum inducibility to the growth responsive gene c-fos through binding to a sequence known
as the serum response element (SRE) (or CArG box). SRF
binds CArG boxes as a dimer, with dimerization and DNA
binding occurring through the MADS box domain [reviewed in Shore and Sharrocks (232)]. In addition to
regulating growth responsive genes such as c-fos, and
multiple SMC marker genes (137, 158), SRF binding to
CArG boxes has also been shown to regulate numerous
skeletal and cardiac muscle specific genes (219).
A long-standing paradox and unresolved issue has been
to determine how SRF, a ubiquitously expressed transcription factor, can regulate both growth responsive and cellspecific genes in SMCs and muscle and nonmuscle cell
types. Several possible mechanisms have been proposed
including a number that appear unique to SMCs. Importantly, these mechanisms are not mutually exclusive, and it
is extremely likely that SMC selectivity is the result of some
combination of the following mechanisms and/or others yet
to be discovered (Fig. 2)(134, 175).
A) REGULATION OF THE LEVEL OF SRF EXPRESSION. SRF levels
appear to be higher in SMCs (and all muscle cells) compared
with most other tissues (12, 44), although as yet there is a
lack of compelling evidence that this is a critical determinant of SMC-specific expression. Moreover, as will be further discussed later, at least in a number of SMC differentiation systems examined, SRF levels per se do not appear to
be the major rate-limiting factor in induction of SMC genes
during early stages of SMC differentiation but rather this is
a function of regulation of the ability of SRF to bind to CArG
regions of SMC genes (168).
B) REGULATION OF SRF BINDING AFFINITY BY HOMEODOMAIN
FACTORS AND/OR POSTTRANSLATIONAL MODIFICATIONS OF SRF. Interestingly, CArG boxes within many SMC promoter-enhancer regions have a reduced binding affinity for SRF
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compared with CArG boxes from growth responsive
genes such as c-fos and egr1 (30, 89). For example, the SM
␣-actin 5⬘-CArG elements each have a single G or C substitution within the central A/T-rich region of the CArG
box, which substantially lowers SRF binding affinity (89).
Of major significance, these G/C base substitutions are
completely conserved across hundreds of millions of
years of evolution, strongly suggesting that they play a
critical regulatory role. It has been hypothesized that the
decreased affinity of SMC promoter CArG boxes serves to
restrict SRF binding, thereby restricting expression of
SMC marker genes to cells that express higher levels of
SRF, and/or cells that have evolved mechanisms to enhance SRF binding affinity to the degenerate CArG elements. In support of this hypothesis, Chang et al. (30)
demonstrated that in embryonic day 11.5 mouse embryos,
a transgene containing multimerized c-fos CArG boxes
upstream of a minimal promoter showed widespread activity while a transgene containing multimerized SM22␣
CArG-near boxes upstream of a minimal promoter was
active primarily in smooth, cardiac, and skeletal muscle;
that is, the muscle-restricted activity of the SM22␣ gene
was lost when its CArG elements were replaced by a
consensus SRE that binds SRF with very high affinity.
However, they reported that at later developmental time
points and postnatally, the SM22␣ CArG-near box reporter was also active throughout the embryo, suggesting
that SRF levels/SRF binding affinity may only be ratelimiting very early in embryonic development and that
other mechanisms subsequently take over to restrict muscle gene expression. While this study used artificial multimerized CArG box reporters, several studies have actually replaced one or more muscle CArG boxes with c-fos
CArG boxes and studied the effect of this substitution in
the context of the intact SMC marker gene promoter. The
results of these types of studies have shown either no
effect on muscle specificity (250), increased basal expression with reduced cell specificity (89), or lack of expression (244). Results of studies in our lab (89) showed that
substitution of SM ␣-actin CArG elements with c-fos SREs
resulted in complete loss of the SMC specificity of this
promoter in cultured cell systems. However, in transgenic
mice the SRE substitution mutant SM ␣-actin promoter
constructs retained appropriate tissue specificity (J. Hendrix and G. K. Owens, unpublished observations). As
such, there is a lack of compelling evidence that the
reduced SRF binding affinity of SMC gene promoters is a
primary determinant of SMC specificity, at least during
normal development and maturation of SMC.
An alternative possibility is that SMCs have evolved
mechanisms that regulate the affinity of SRF binding to
CArG elements and that these mechanisms regulate the
overall rate of gene transcription but not cell selectivity
per se. Consistent with this hypothesis, we demonstrated
that SMCs express a homeodomain containing protein
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FIG. 2. SMC-specific/selective gene expression is dependent on complex combinatorial interaction of multiple
cis-elements and trans-binding factors. This figure presents a schematic model illustrating some of the complex
protein:protein and DNA:protein interactions that are hypothesized to be important in determining cell-selective
expression of multiple SMC differentiation marker genes with a focus on the importance of cooperative interactions of
multiple CArG elements [CC(AT)6GG] located within both 5⬘ and intronic promoter regions, serum response factor
(SRF), and SRF accessory proteins (SAPs) such as myocardin. In this model, transcription by the TATA binding protein
(TBP)-containing RNA polymerase II complex is dependent on cooperative interactions between multiple cis-elements
and trans-binding factors including the CArG elements, as well as the TCE, G/C repressor, and other elements such as
E boxes (CANNTG) (not shown). In addition, the figure depicts mechanisms whereby environmental cues such as ANG
II and TGF-␤ can alter expression of SMC differentiation marker genes. For example, there is evidence that ANG II
stimulates SMC promoter activity at least in part through increased expression of Mhox, a homeodomain protein that
enhances formation of the CArG-SRF-myocardin higher order complex. Similarly, there is evidence that TGF-␤ regulates
SMC gene expression, at least in part, through enhanced formation of the CArG-SRF-myocardin complex and increased
expression of BTEB2/IKLF (KLF5) a KLF transcription factor shown to SMC gene expression by binding to the TGF-␤
control element (TCE). In contrast, TGF-␤ downregulates expression of the repressor TCE binding factor GKLF (KLF4).
A G/C element, which is positioned between the 5⬘-CArG regions of many SMC genes, has been shown to function as a
repressor possibly by inhibiting cooperative interactions between CArG elements Sp1 or Sp1-like protein dependent
mechanisms. Moreover, as discussed in detail in section IV, this element has been shown to be required for downregulation of SM22␣ gene expression in vivo in response to vascular injury. Finally, although PDGF-BB has been shown to
be the most potent negative regulator of SM-selective gene expression and implicated in the pathogenesis of atherogenesis, molecular mechanisms for this process remain unknown (as denoted by the “?”).
designated Mhox that can dramatically enhance binding
of SRF to the degenerate SM ␣-actin 5⬘-CArG elements
(88). Of further interest, we showed that overexpression
of Mhox transactivated the SM ␣-actin promoter in cultured SMCs and that this effect was at least partially
dependent on a homeodomain binding site located in
close proximity to the SM ␣-actin CArG B element. We
also demonstrated that ANG II, which stimulates SM ␣-actin expression, increased expression of Mhox, and dramatically increased SRF binding activity to the degenerate SM ␣-actin CArG A and B elements in gel shift assays
in the absence of any detectable change in SRF levels.
Results thus support a model in which ANG II-dependent
increases in SMC gene expression are mediated at least in
part by increased expression of Mhox and subsequent
enhanced CArG-dependent gene transcription (Fig. 2).
However, as yet, there is no loss of function data to
support this model, and one must also consider the
possibility that other SMC homeodomain proteins such
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as the Nkx factors (27, 78, 95, 178, 187) may have
similar activity.
C) COOPERATIVE INTERACTION OF MULTIPLE CARG ELEMENTS. In
contrast to the c-fos promoter and many other ubiquitously expressed promoters that contain a single CArG
box, many of the SMC marker gene promoter-enhancers
characterized to date contain two or more CArG elements
that are required for transcriptional activity in vivo in all
or at least a subset of SMCs (126, 144, 156, 167, 174, 176,
278). These observations raise the possibility that cooperative interactions between multiple CArG elements as
well as their spatial relationship to one another contribute
to SMC-selective expression of CArG-dependent SMC
genes. Consistent with this idea, Hautman et al. (89)
demonstrated that the position of the two CArG boxes in
the 5⬘-promoter region of the SM ␣-actin gene were not
interchangeable in that this mutant promoter was completely inactive in cultured SMCs. Moreover, Mack et al.
(158) demonstrated that introduction of mutations that
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altered the spacing or phasing of the two 5⬘-CArG elements of the SM ␣-actin promoter that are designated
CArG A and CArG B was associated with significant effects on gene expression in cultured SMCs. Of interest,
not only are the 5⬘ CArG elements of the SM ␣-actin
promoter completely conserved between species but so is
their spacing. Indeed, CArG A and B are separated by
exactly 40 bp in all species, and since the DNA helix has
a complete turn every 10 bp, the CArG elements normally
lie on the same face of the DNA molecule (i.e., they are in
phase). We found that introduction of a 5- or 15-bp spacer
between CArG A and B, which destroyed CArG phasing,
completely abrogated activity of the promoter in cultured
SMC. In contrast, insertion of a 10-bp spacer that retained
CArG phasing had no effect on activity, insertion of a
20-bp spacer resulted in a 40% decrease in promoter activity, and a 40-bp insert abolished promoter activity.
Taken together, results support the hypothesis that cooperative interaction of multiple CArG elements contributes
to SMC-selective gene expression and that changes in the
spacing/phasing of these two CArG boxes profoundly affect the activity of this promoter, at least in cultured
SMCs (Fig. 2). However, it is also important to note that a
number of genes that are expressed selectively in SMC
contain either a single CArG element (telokin) (97) or no
CArG elements (ACLP) (138), thus indicating that cooperative interaction of multiple CArG elements is not required for expression of all SMC differentiation marker
genes. However, the significance of the latter observation
in terms of understanding SMC selective gene expression
is questionable, since ACLP is also expressed in many
non-SMC tissues.
D) POSTTRANSCRIPTIONAL MODIFICATION OF SRF AS WELL AS
CONTROL OF NUCLEAR LOCALIZATION. There is evidence suggesting that SRF-dependent gene transcription may also be
regulated by a variety of posttranscriptional mechanisms
including production of alternative splice forms that may
function as naturally occurring dominant negatives (12,
13, 124), phosphorylation events that may alter CArG
binding affinity and/or its interaction with cofactors (170,
207), and rhoA/rho kinase (ROK)-dependent SRF nuclear
translocation and/or activation (23, 157, 181). With respect to the latter studies, we demonstrated that overexpression of constitutively active RhoA stimulated transcription of multiple SMC genes including SM ␣-actin, SM
MHC, and SM22␣ (158). In contrast, administration of C3
transferase, which ADP ribosylates and irreversibly inhibits RhoA, or Y-27632, a selective rho kinase inhibitor,
decreased expression of multiple CArG-dependent SMC
genes. Importantly, these effects were selective in that no
effects were seen on the c-fos gene promoter that contains only one CArG/SRE element, and for rhoA compared
with other members of the rho family in that overexpression of constitutively active Rac and cdc42 had no effect
on SMC gene expression. Of particular interest, we prePhysiol Rev • VOL
781
sented evidence that the effects of rho were mediated
through regulation of the actin cytoskeleton and specifically through alterations in the concentration of monomeric or G-actin; that is, we found that treatment with
agents that increased formation of filamentous (F)-actin
and decreased G-actin profoundly increased SMC gene
transcription, whereas treatment with agents that increased G-actin markedly inhibited SMC gene expression.
However, the mechanisms linking changes in G-actin to
SMC gene transcription were unknown. Of major interest,
recent studies by Miralles et al. (181) showed rho-actin
signaling regulated the subcellular localization of the
myocardin-related SRF coactivator MAL/MRTF-A in NIH
3T3 fibroblasts. They also found that MAL was found
predominantly in the cytoplasm in serum-starved 3T3
cells but accumulated in the nucleus following serum
stimulation. In addition, they showed that MAL associated
with unpolymerized actin through two RPEL motifs contained in its NH2-terminal sequences. Although studies of
MAL translocation and activity in SMC have not yet been
reported, Solway and co-workers (23, 150) have provided
very interesting results showing serum-induced translocation of SRF into the nucleus in cultured tracheal SMC and
that this process was rho kinase dependent. As such, it is
interesting to postulate a model in which agonist-induced
activation of rhoA and subsequent translocation of SRF
and/or myocardin/MAL (see sect. IIID1E) may play an
important role in activation of CArG-dependent SMC
genes. However, at present, this model is highly speculative and further studies are needed to fully clarify the
intracellular signaling pathways whereby rhoA and agonists that activate rho stimulate SMC gene expression,
and to determine the potential contribution of this signaling pathway to control of SMC gene expression in vivo.
E) SMC-SPECIFIC/SELECTIVE SRF COACTIVATORS. The c-fos
CArG box is adjacent to a binding site for a subfamily of
ETS domain transcription factors known as ternary complex factors (TCFs), which, as their name suggests, form
a ternary complex with SRF and the CArG box to activate
transcription of the c-fos gene (232, 254). TCFs including
SRF accessory proteins 1 and 2 (or SAP-1, SAP-2) have
been shown to play a key role in mediating responsiveness of immediate early response genes such as c-fos to
various growth factors at least in part through mitogenactivated protein kinase (MAPK)-dependent signaling
pathways. However, the majority of SMC marker gene
promoters are not flanked by such TCF binding sites, and
despite extensive effort, no one has been successful in
showing binding of classical TCFs to SMC CArG elements
in association with SRF, although one can readily detect
TCF-SRF higher order complexes in these same cells with
the c-fos SRE (126, 126, 143, 158, 244). These results
suggest that classical TCFs/SAPs are not involved in regulation of SMC marker genes. However, it has long been
hypothesized that cell-specific or selective SRF coactiva-
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OWENS, KUMAR, AND WAMHOFF
tors are likely to exist in SMCs and play a critical role in
regulating SMC-specific gene expression. Consistent with
this hypothesis, several years ago we presented evidence
that the SM ␣-actin 5⬘-CArG elements formed a unique
higher order CArG-SRF complex with SMC nuclear extracts, whereas this complex was not observed with nuclear extracts from skeletal myoblasts or myotubes, endothelial cells, or fibroblasts (158). Of interest, formation
of the higher order SRF containing complex was dependent on both CArGs A and B but not on any specific CArG
flanking sequence, suggesting that recruitment of the SRF
interactive protein occurred primarily through proteinprotein interactions rather than binding to DNA. However, we were unable to identify the SMC-selective SRF
cofactor. Interestingly, the homeodomain proteins
Barx1b and Nkx3.2 and the zinc finger protein GATA6
have each been shown to form a stable, detectable ternary
complex with SRF-CArG DNA (185, 187), and a recent
interesting study from Nishida et al. (187) showed that the
triad of SRF, Nkx3.2, and GATA6 cooperatively activated
several SMC marker genes, including SM22␣ and caldesmon, but not c-fos. Thus this combination of factors provides a mechanism by which SMC-specific genes can be
regulated selectively and independently of growth responsive genes. Moreover, although each individual factor was
not SMC specific, this triad of factors was found to be
coexpressed only in vascular SMCs, illustrating an important paradigm whereby SMC-specific gene transcription
can be achieved in the absence of SMC-specific transcription factors through combinatorial interactions between
multiple transcription factors whose presence, at the appropriate levels and stoichiometry, may be SMC specific.
One of the most significant and exciting advances for
the field of SMC differentiation in recent years was the
discovery of myocardin by Olson and co-workers (263).
Myocardin is an extremely potent SRF coactivator that is
selectively expressed in cardiac (263) and differentiated
SMC in vivo (31, 54) (Fig. 2). Moreover, mouse embryos
homozygous for a myocardin loss-of-function mutation
die by embryonic day 10.5 and show no evidence of
vascular SMC differentiation based on detailed in situ
analysis that revealed lack of SMC-selective gene expression (146), although a confounding aspect of this study
was that authors also found gross abnormalities in yolk
sac vaculogenesis suggesting that effects on SMC development could have been indirect. Myocardin has been
shown to selectively induce expression of all CArG-dependent SMC marker genes (Fig. 2) tested to date including SM ␣-actin, SM MHC, SM22␣, and calponin (31, 54,
263, 280). Of interest, myocardin appears to be most
efficacious in activating those genes that contain multiple
CArG elements, and Olson and colleagues (266) have
presented evidence for a model whereby the leucine zipper motif of myocardin may bridge adjacent CArG elements and unmask myocardin’s activation domain. This
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model is intriguing and may provide a mechanism to
explain the effects of varying the phasing and spacing of
the 5⬘ SM ␣-actin CArG elements observed in our previous
studies (158) (see sect. IID1C). We (280) and others (266)
have shown that adenoviral-mediated overexpression of
myocardin was sufficient to activate multiple SMC differentiation markers in NIH 3T3 or 10T1/2 cell fibroblasts
including the definitive marker SM MHC, suggesting that
myocardin is sufficient to activate the SMC lineage program in fibroblasts. Significantly, administration of a myocardin siRNA (54, 280), or adenovirus-mediated overexpression of a myocardin dominant negative construct
(280), significantly reduced expression of multiple SMC
marker genes including SM MHC, SM22␣, and SM ␣-actin
by up to 80% in cultured SMCs, thus providing direct
evidence that endogenous myocardin plays a key role in
regulation of expression of multiple SMC marker genes.
However, as yet it is not clear that myocardin can activate
all SMC genes or give rise to fully differentiated SMC.
Indeed, major unresolved issues include the following: 1)
Can myocardin fully activate the full SMC differentiation
program given the fact that at least a number of SMC
marker genes such as telokin and ACLP should not be
directly activated by myocardin? 2) Does suppression of
myocardin expression or activity contribute to phenotypic modulation of SMC in response to vascular injury or
disease? 3) Do all SMC subtypes express myocardin, and
what role if any is played by the myocardin-related transcription factors (MRTF) MRTF-A (MAL) and MRTF-B
that are expressed in numerous embryonic and adult
tissues (264)? 4) Are additional myocardin-related gene
products or functional homologs important in control of
SMC differentiation?
Of particular interest, we found that myocardin was
expressed in a unique SMC progenitor line, designated
A404 as previously described by our laboratory (168), in
the absence of detectable expression of any other known
SMC marker including the earliest known markers SM
␣-actin and SM22␣ (280). In contrast, myocardin expression was absent from P19 embryonal carcinoma stem
cells, the parental line from which A404 cells were derived. Treatment of A404 cells with all trans-retinoic acid,
which induced expression of all known SMC marker
genes including SM MHC and smoothelin (168), was associated with further increases in myocardin expression.
In addition, we showed that although SRF was highly
expressed in A404 cells, it was unable to bind to the CArG
containing regions of SMC genes, although it did bind to
the constitutively expressed c-fos CArG promoter region.
Treatment of A404 cells with retinoic acid resulted in
association of SRF with CArG containing regions of SMC
promoters, as well as hyperacetylation of histones associated with these regions. Taken together, these results
support a model in which myocardin and SRF are expressed in A404 progenitor cells but are unable to bind to
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CArG-containing regions of SMC genes because of spatial
restrictions associated with chromatin structure that are
selective for SMC promoter regions. However, evidence
suggests that upon treatment with retinoic acid the chromatin organization within SMC promoter regions is relaxed at least in part by histone acetylation; SRF then
binds, recruits myocardin and other possible coactivators, and activates expression of multiple CArG-dependent SMC genes. Consistent with these results, Qui et al.
(197) presented evidence that CREB-CArG-dependent expression of the SM22 gene in cultured SMC was dependent on histone acetyltransferase (HAT) activity. In brief,
they found that treatment of cells with trichostatin A, a
histone deacetylase (HDAC) inhibitor, increased whereas
overexpression of HDACs decreased SM22 promoter activity. Although the results of the latter studies clearly
implicated histone acetylation in SMC gene expression,
an inherent limitation of such studies is that the experimental modifications in histone acetylation were genome
wide and thus provided no direct evidence that modification of the SM22 locus per se regulates its activity. Nevertheless, taken together the evidence thus far indicates
that regulation of chromatin structure or “epigenetic programming” (206) plays a key role in control of SMCspecific gene expression. However, as yet, the specific
molecular mechanisms that contribute to selective modification of chromatin structure within CArG-containing
regions of SMC genes are poorly understood.
2. Cis-elements and trans-binding factors in addition
to CArG-SRF also play a key role in regulating
SMC-selective gene expression
Although the SRF-CArG-dependent pathway has
been shown to be critically important in the regulation of
multiple SMC marker genes, CArG elements are clearly
not sufficient for SMC-specific gene regulation (134, 175).
For example, we found that a transgene construct consisting of three tandem copies of the SM MHC intronic
CArG region coupled to a minimal thymidine kinase promoter was expressed in SMCs in transgenic mice but was
also promiscuously expressed in a variety of other cell
types (133, 167). Similarly, Strobeck et al. (244) found that
a promoter consisting of a multimerized copy of the c-fos
SRE coupled to the SM22␣ minimal promoter, or the
SM22␣ promoter where the SM22␣ CArG box regions are
replaced with the corresponding region of the c-fos gene
were completely inactive in vivo in transgenic mice (244).
Moreover, even more importantly, we (2, 133, 151) have
shown that mutation of non-CArG elements including a
TGF-␤ control element (TCE) (Table 1) and E-box elements also abrogate expression of SMC marker genes in
the setting of transgenic mice. Among these, the TGF-␤
control element or TCE (2, 87) is of particular interest in
that it may play a key role in regulating alterations in
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expression of SMC genes in the context of vascular injury
and/or atherogenesis.
TGF-␤ has been shown to increase the expression of
most SMC differentiation marker genes including SM
␣-actin, SM MHC, h1-calponin, and SM22␣ in cultured
SMCs (2, 87). TGF-␤ has also been shown to induce
expression of a variety of SMC markers in 10T1/2 fibroblasts (104, 105), although we do not find induction of
either SM MHC or myocardin (280), raising questions as
to whether this model reflects activated myofibroblasts or
SMC. Studies on the SM ␣-actin promoter revealed that
three cis-elements, the two 5⬘ CArG boxes and a novel
TGF-␤ control element (TCE), were required for TGF-␤
inducibility (87) (Fig. 2). Of interest, TGF-␤ dramatically
enhanced SRF binding to the CArG boxes and enhanced
formation of the SMC-selective SRF-CArG complex at
least partly due to increased SRF expression. The TCE
element, G(A/C)GT(T/G)GG(T/G)GA, is found in several
SMC-selective promoters, and mutation of this element in
the SM22␣ promoter (2) or SM ␣-actin promoter (151)
completely inactivated these promoters in vivo in transgenic mice. TGF-␤ induced the binding of a factor or
factors to the TCE element of which appeared to be
members of the Kruppel-like transcription factor family
(or KLF family) of zinc finger proteins that includes
GKLF, BTEB2/KLF5, and Sp1 (2). Interestingly, GKLF
repressed expression of the SM ␣-actin, SM MHC, and
SM22␣ promoters (151), whereas another related KLF
member, KLF5 (or IKLF/BTEB2), activated transcription
(2, 151). As such, results suggest that the TCE can play a
reciprocal role in control of SMC differentiation through
binding of different KLF factors (Fig. 2). However, further
work is needed to identify the KLF members that activate
or repress SMC marker gene expression in vivo and to
determine the possible role of the TCE in control of
phenotypic modulation of SMC in response to vascular
injury or atherosclerosis.
E-boxes (CANNTG motifs) bind to homo- or heterodimers of basic helix-loop-helix (bHLH) proteins (171)
and have been found in many cell-specific promoters
including the SMC-specific/selective promoters, SM MHC
(272), SM22␣ (125), Crp2/SmLim (279), APEG-1 (110), and
SM ␣-actin (122, 231). The discovery in 1989 that MyoD, a
bHLH protein, functions as a master regulatory gene for
skeletal myogenesis, sufficient to activate the skeletal
muscle differentiation program in a variety of nonmuscle
cell types (269), led to intense interest in identifying similar master regulatory bHLH proteins in other cells. While
this search has resulted in the identification of bHLH
proteins critical to the differentiation of numerous cell
types including neurons, pancreatic ␤-cells, hematopoietic cells, and cardiac muscle [reviewed in Massari and
Murre (171)], no SMC-specific master regulatory bHLH
protein has yet been identified. Although several bHLH
proteins have now been found in SMCs including class I
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bHLHs such as E2A and E2–2 (116, 173) that are not cell
selective, and class II cell selective bHLHs such as capsulin, eHAND, and dHand (99, 107, 276), their role in SMC
differentiation is still largely undefined.
We recently demonstrated that mutation of two Eboxes in the 5⬘-region of the SM ␣-actin promoter (designated E1 and E2) nearly abolished expression of this gene
in vivo in transgenic mice (133). Moreover, we found that
1) several class I bHLH factors including E12, HEB, and
E2–2 transactivated the SM ␣-actin promoter in BALBc
3T3 fibroblasts and that activation by E12 was E-box
dependent; 2) that E2A proteins (E12/E47) were associated with the E-box-containing region of the SM ␣-actin
promoter in the context of intact chromatin as determined by chromatin immunoprecipitation assays; 3) E2–2
or HEB and SRF cooperatively activated the SM ␣-actin
promoter; 4) none of the known class II bHLHs in SMCs
including capsulin, dHand, or eHand activated the SM
␣-actin promoter in cotransfection studies; and 5) overexpression of the naturally occurring dominant negative/
inhibitory bHLH factors, Id and twist, inhibited both the
E12 and SRF-induced activation of the SM ␣-actin promoter in BALBc 3T3 cells. Taken together, these results
clearly demonstrate the importance of E-box elements in
the expression of SM ␣-actin in vivo. Moreover, results of
studies in cultured cells implicate an important role for
class I bHLH factors such as E2–2 or E12. However, at
present, there is no clear evidence for involvement of a
long sought SMC-specific/selective class II bHLH factor.
Of interest, because myogenic bHLH proteins have been
shown to interact with SRF (81), it is also tempting to
speculate that interactions between E-box bound bHLH
proteins and SRF will prove to be important in the regulation of SMC marker genes.
A final cis-element that will be briefly reviewed in this
section is referred to as a G/C repressor and was originally described by our laboratory as an element located
between the SM MHC 5⬘-CArG1 and CArG 2 elements that
could mediate transcriptional repression in cultured SMC
(159, 161) (Fig. 2). Of interest, G/C-like elements are also
found in close proximity to CArG elements within most
SMC marker gene promoters including SM22␣ (201). The
G/C repressor region binds to members of the Sp1 transcription factor family in gel shift assays (159, 201). As
will be discussed in detail in section IV, we found that this
G/C repressor element was required for injury-induced
downregulation of a SM22-LacZ transgene in vivo (201)
(Table 1, Fig. 3.)
In summary, there is extensive evidence supporting a
model in which the differentiated state of the SMC is
dependent on integration of multiple environmental cues
that determine the pattern of gene expression appropriate
for that particular circumstance. This property is important in all cell types but is particularly critical in nonterminally differentiated cells like the SMC that must necesPhysiol Rev • VOL
sarily retain considerable plasticity even in adult animals.
As will be discussed in detail in section III, this property is
important for vascular repair following injury and should
probably be viewed as a unique and inherent property of
the differentiated SMC that has been conserved through
evolution because it conferred a survival advantage; that
is, mutations that compromised SMC plasticity and thus
the ability to carry out vascular repair would likely have
been detrimental to survival of the organism and been
selected against during millions of years of evolution of
higher organisms, including humans.
III. CHARACTERISTICS AND ROLE OF
PHENOTYPIC MODULATION OF SMOOTH
MUSCLE CELLS IN THE DEVELOPMENT,
PROGRESSION, AND END-STAGE CLINICAL
SEQUELAE OF ATHEROSCLEROSIS
The focus in this section will be to briefly summarize
specific changes that occur in SMCs within atherosclerotic
lesions with a particular focus on consideration of the possible contributions of phenotypic modulation/switching of
SMC to lesion development, progression, and clinical sequelae including plaque rupture. We feel that changes in
SMC phenotype associated with vascular injury and atherosclerosis provide a general paradigm for understanding SMC
phenotypic switching in many pathophysiological states, although as has been emphasized throughout this review, the
precise mechanisms are undoubtedly different as a function
of the nature of changes in local environmental cues associated with that particular circumstance. The key points that
we hope to convey are the following: 1) a broad range of
changes in the phenotype of the SMC have been described
that vary as a function of disease stage and location within
the atherosclerotic lesion (e.g., cells within the fibrous cap
differ from those in the base of the lesion or the necrotic
core); 2) it is often difficult to identify whether lesion cells
were or were not derived from preexisting SMC, since phenotypically modified SMC no longer express most of the
markers that allow us to identify them; 3) the repertoire of
environmental cues that exist within atherosclerotic lesions
are no doubt very different from those that exist within a
normal healthy blood vessel (or within a non-atherosclerotic
injured blood vessel) and these change at different stages of
lesion development and progression and thereby are likely
to contribute to continued phenotypic switching of SMC or
SMC like cells within the lesion; and 4) the phenotypically
modified SMC itself is likely to contribute to alterations in
the extracellular milieu within the intima through production of matrix components and other secreted products that
in turn influence the state of differentiation of the SMC.
However, before considering these issues, we will first consider recent evidence that has challenged the long-standing
dogma that intimal SMCs are derived primarily from preexisting medial SMC (212).
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FIG. 3. Molecular mechanisms of decreased SMC differentiation marker expression associated with vascular injury:
a novel experimental approach to study SMC phenotypic modulation. Light micrographic images showing ␤-galactosidase staining of mouse carotid arteries from SM ␣-actin-LacZ (A–D), SM MHC-LacZ (E–H), SM22␣-LacZ (I–L), and
SM22␣gc-LacZ (M–P) transgenic mice 7 or 14 days after injury or in uninjured contralateral carotid vessels. Of
significance, 7 days after injury, LacZ activity was undetectable in SM ␣-actin, SM MHC, and SM22␣ transgenic mice,
indicating transcriptional suppression of the transgene. Although not shown here, the decrease in transgene expression
corresponded to a decrease in SM ␣-actin and SM MHC protein expression and SM ␣-actin, SM MHC and SM22␣ mRNA.
By 14 days after injury, transgene activity was observed to increase within isolated subpopulations of SMCs with staining
for the SM ␣-actin transgene greater than that for either the SM MHC or SM22␣ transgenes. These data show the dynamic
nature of phenotypic switching of SMC genes during injury-induced phenotypic switching. Mutation of the G/C-rich
cis-regulatory element positioned between 2 CArGs in the SM22␣ promoter (SM22␣gc-LacZ) completely abrogated
suppression of SM22␣ transgene at both 7 and 14 days after injury (compare LacZ staining in J vs. N and L vs. P), thus
providing direct evidence for a role for the G/C repressor and some unknown transcription factor(s), possibly Sp1 or
Sp1-like transcription factor(s), in injury-induced suppression of SM22␣ and the concomitant phenotypic modulation of
the SMC. [From Regan et al. (201), copyright 2000 by the Journal of Clinical Investigation.]
A. Origin of Intimal SMCs in Atherosclerotic
Lesions: Media Derived or Blood Derived?
The long-standing dogma in the field has been that
the majority of intimal SMC are derived from preexisting
medial SMC that undergo migration into the intima and
phenotypic modulation (212). Indeed, there is extensive
indirect evidence in support of this hypothesis (see sect.
IIIB), including classic ultrastructural studies by Schwartz
et al. (226) showing the presence of morphologically identifiable SMC in the process of migrating through the internal elastic lamina following vascular injury. However,
it is unclear from such studies what proportion of intimal
cells are derived from medial SMCs, due primarily to the
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lack of direct and reliable methods to quantitatively measure the proportion of intimal SMCs derived from preexisting medial SMCs. The situation is further confounded
by two additional factors: 1) the fact that intimal SMCs
downregulate expression of those markers that allow us
to define them as SMCs and 2) many SMC markers are not
unique to this cell type including SM ␣-actin, which is the
marker that has been used most frequently in studies in
this area. As a consequence, there is a distinct possibility
of both false-positive and false-negative identification of
SMC lineage. Indeed, there is clear evidence that cultured
endothelial cells (EC) and adventitial fibroblasts can be
induced to express multiple markers of SMC lineage including SM ␣-actin, calponin, and SM myosin (63). More-
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over, there is also evidence suggesting that ECs (47) and
adventitial fibroblasts (217, 242) in vivo may give rise to
SMC-like cells within the intima and contribute to repair
of vascular injury. However, the relative contribution of
these cells versus preexisting SMC to formation of intimal
SMC or SMC-like cells is still unclear.
In contrast, there is recent compelling evidence that
bone marrow-derived cells resident in the circulating
blood invest in the neointima following vascular injury
and can give rise to cells that express at least some
properties of vascular SMCs (203, 234). Alternatively,
these circulating cells may fuse with resident SMCs and
thus show colocalization of SMC markers and bone marrow lineage markers, although to date, no direct evidence
for cell fusion in the vasculature has been shown. For
example, Sata et al. (221) lethally irradiated wild-type
mice and reconstituted with bone marrow cells from a
ROSA26 mouse that ubiquitously express a ␤-galactosidase gene, thus allowing lineage tracing. They then injured the femoral artery with a large wire that caused
severe damage of the vessel including complete loss of
endothelium and extensive medial SMC apoptosis. They
then examined for investment by LacZ-positive cells at 1
and 4 wk as well as expression of the SMC differentiation
marker gene SM ␣-actin by immunostaining. Of interest,
at 4 wk, a significant fraction of neointimal (63.0 ⫾ 9.3%)
and medial cells (45.9 ⫾ 6.9%) were LacZ⫹ as determined
by X-gal staining, whereas no LacZ⫹ cells were present in
uninjured femoral arteries. Moreover, at least some of the
LacZ⫹ cells also expressed either SM ␣-actin (a marker
for SMC as well as myofibroblasts) or CD31 (a marker for
endothelial cells). The authors interpreted these studies
as evidence that bone marrow-derived cells can give rise
to vascular cells following mechanical injury and contribute to vessel repair (221). Consistent with the preceding
results, Han et al. (84) had previously conducted similar
experiments in which they lethally irradiated female
C57Bl/6 mice and reconstituted with bone marrow cells
from congenic (Ly5.1) male donors and characterized the
contributions of the bone marrow cells to neointima formation and medial repair by in situ hybridization with a
Y-chromosome-specific probe. Of particular significance,
they examined a scratch injury, or silk suture models of
vascular injury that evoked injuries of varying severity.
Results demonstrated the presence of abundant bone
marrow cells within the media and intima in cases of
severe injury of medial SMC, whereas no bone marrow
cells were present in cases where there was denudation of
endothelial cells but minimal damage to medial SMC (84).
These results suggest that bone marrow cells are recruited in vascular healing to complement resident SMC.
However, the relative proportion of these cells that contribute varies as a function of the severity of medial
damage and only in cases of severe medial damage when
few resident SMC are available to effect repair was the
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bone marrow contribution extensive, as recently described in several mouse arterial injury models by Tanaka
et al. (247). Importantly, a limitation in studies exploring
the possible contribution of bone marrow cells to neointimal SMCs is the failure to provide compelling evidence
regarding expression of definitive markers of SMC lineage
such as SM MHC. As stated clearly by Han et al. (84), the
use of SM ␣-actin to further characterize bone marrowderived neointimal cells shows that “these marrow-derived cells resemble fetal/immature vascular SMC or myofibroblasts” at best.
An additional limitation of most bone marrow transfer
experiments described thus far is the failure to provide
high-resolution confocal analysis of expression of lineage
tracer markers and SMC markers within single cells to
clearly establish that bone marrow cells and not adjacent
cells express the SMC markers/bone marrow lineage markers and/or to rule out possible fusion of circulating stem
cells with resident vascular cells. The latter issue is critical
to resolve, since there is clear evidence for fusion of stem
cells with resident differentiated cells in a variety of in vivo
models including liver regeneration (259, 265). As such, the
presence of SM ␣-actin-positive bone marrow-derived cells
within the vasculature reported in many recent studies may
have represented simple fusion of bone marrow cells with
resident SMCs as opposed to de novo differentiation of
these stem cells into SMC lineages. However, it should also
be noted that contrary to opinions in a number of editorials
that have proclaimed that all stem cell investment represents fusion, there is also clear evidence that under some
circumstances circulating stem cells can give rise to differentiated cell populations without cell fusion, including glucose-competent pancreatic endocrine cells (115) and skeletal muscle satellite cells (136).
An additional limitation of studies suggesting that
bone marrow stem cells give rise to SMC lineages within
injured blood vessels is that there has not yet been a
comprehensive evaluation of the time course of expression of definitive SMC markers to rule out confounding
effects of transient downregulation of expression of SMC
markers secondary to phenotypic modulation. For example, we have shown using a mouse carotid wire injury
model that SM ␣-actin, SM22␣, and SM MHC gene expression and protein are nearly undetectable in the media and
neointima 7 days after injury and that by 14 days, these
genes begin to increase, with gene expression of SM
␣-actin greater than SM MHC or SM22␣ (Fig. 3, Table 1)
(201). Thus it is possible that bone marrow-derived cells
may indeed have the potential to fully differentiate into
SMC lineages but may not do so due to the lack of
appropriate environmental cues within the injured blood
vessel to promote expression of the full repertoire of
marker genes characteristic of mature SMC; that is, examination of bone marrow cells that invest the neointima
within a carotid injury model using SM MHC as a SMC
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marker could potentially yield a false negative at 14 days,
a time point during which SM MHC expression is just
beginning to increase (Fig. 3).
There have also been a number of studies showing
that bone marrow cells contribute to intimal lesion formation in mouse or rat models of transplant arteriosclerosis (102, 111, 221, 230). Blood vessel allograft arteriosclerosis typically results in the accumulation of mononuclear cell types and enhanced SMC proliferation that
ultimately culminates in vascular stenosis and ischemic
graft failure. Until recently, the source of SMC investment
in allograft blood vessel stenosis was unknown. Using
MHC class I haplotype-specific immunohistochemical
staining and single-cell PCR analyses, Hillebrands et al.
(102) demonstrated that neointimal ␣-actin-positive cells
in rat aortic or cardiac allografts were of recipient and not
donor origin. Of interest, treatment with the immunosuppressant cyclosporine prevented neointima formation and
preserved the vascular media in allografts. In two separate studies involving lethal irradiation followed by reconstitution with bone marrow cells from mice expressing
LacZ, Sata et al. (221) and Shimizu et al. (230) found that
⬃10 –20% of SM ␣-actin-positive cells in the neointimal
lesions of aortic allografts were colocalized or in close
proximity with LacZ⫹ stained cells. Authors concluded
that bone marrow-derived cells gave rise to SMC-like cells
within the neointima, although studies lacked presentation of compelling evidence of expression of definitive
SMC markers like SM MHC and failed to clearly resolve
whether SM ␣-actin/LacZ positive cells were in fact one
cell or merely two adjacent cells. To overcome this limitation, Hu et al. (111) carried out a clever series of studies
using bone marrow cells derived from a SM22␣-LacZ
transgenic mouse to allow simultaneous lineage tracing
and characterization of expression of this SMC differentiation marker within the same cell. These workers found
that there were no LacZ⫹ cells in the neointima of mice
that received bone marrow from the SM22␣-LacZ mice 6
wk after allograft implantation, although consistent with
studies of others they did see investment of bone marrow
cells from ROSA LacZ mice within the neointima. To
confirm that the SM22␣-LacZ promoter can be potentially
active at 6 wk in the allograft neointima, a BALB/c to
SM22␣-LacZ aortic transplant was performed. SM22␣LacZ⫹ cells were identified in the allograft neointima,
indicating that SM22␣-LacZ⫹ cells migrated from the host
vessel to the neointima, and that the SM22␣-LacZ promoter is active in the neointima at 6 wk in this model
(111). Taken together, these studies seem to refute earlier
studies in that although bone marrow-derived cells clearly
invest the neointima, these cells failed to express even the
early SMC differentiation marker SM22␣ as detected by
LacZ staining. One way to reconcile these results is that SM
␣-actin staining may have been more sensitive than detection of the SM22␣ LacZ. However, in any case, the lack of
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demonstration of expression of multiple definitive markers
of SMC lineage raises doubts as to whether bone marrow
cells can undergo differentiation into mature SMC.
A critical question is whether there is compelling
evidence that “SMC or SMC-like cells” within human atherosclerotic lesions are derived from bone marrow, although relatively few studies have been published in this
area due to inherent limitations underlying the use of
human subjects. However, of interest, several studies
have analyzed the contribution of host versus recipient
contributions to intimal lesions (24, 75, 109, 198). In each
study, the possibility of investment of cells from the heart
or bone marrow transplant donor was determined by Y- or
X-chromosome labeling in blood vessels of recipients of
the opposite gender. For example, detection of Y-chromosome-positive cells within blood vessels of a male patient
receiving a heart from a female donor must have come
through the systemic circulation from undifferentiated
cells in the male recipient’s bone marrow, from cells that
entered the circulation from non-bone marrow sources,
or from the recipient cardiac remnant. Results from several of these studies (24, 75, 198) differed greatly from the
others (109), and none of the studies provides definitive
insight as to the precise role or frequency of investment of
blood-derived cells into the developing lesion. For example, Quaini et al. (198) estimated that as many as 60% of
cells investing in newly developed arterioles in the collateral circulation of male patients who had received a female heart were Y-chromosome/SM ␣-actin positive. In
contrast, Glaser et al. (75) showed that at most, 6% (the
observed frequency was actually 2.7% but authors appropriately allowed for possible false negatives) of the cells
were Y-chromosome/SM ␣-actin positive in medium and
small arteries, whereas collateral arterioles were not examined. Consistent with the latter findings, Hruban et al.
(109) found no evidence of bone marrow-derived SMCs
within coronary lesions.
In what appears to be one of the most complete
studies reported to date, Caplice et al. (24) used 13 sexmismatched bone marrow transplant human subjects to
examine the contribution of bone marrow-derived cells to
diseased segments of coronary arteries. Of interest, they
observed frequencies of bone marrow-derived cells that
expressed SM ␣-actin ranging from 5.9 to 10.4% within
diseased vessel segments compared with values ⬃100fold less than this in undiseased coronary segments. In
addition, these workers performed chromosomal ploidy
analyses using markers for chromosome 18 and found no
evidence that coexpression of SMC markers and bone
marrow markers was the result of cell fusion. However, as
with animal studies, the major limitation in human studies
reported to date is the failure to present rigorous evidence
of colocalization of expression of multiple definitive
markers of SMC lineage such as SM MHC and markers of
bone marrow cells within a single cell by high-resolution
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confocal analyses. Indeed, in general, there has been an
over-reliance on use of SM ␣-actin as a SMC-specific
marker since, as discussed in section IIB, this gene can be
expressed in many cell types in conjunction with injury/
inflammation. Although several studies claim to have examined SM MHC expression in blood-derived cells, only
in one case have these data actually been shown (24), but
in this case no evidence was presented documenting that
the antibodies employed were completely specific for SM
MHC (see sect. IIB). Nevertheless, despite the controversy
in this area, there appears to be clear evidence that significant numbers of blood-derived cells can invest within
developing human atherosclerotic lesions and give rise to
cells that express at least some markers characteristic of
early stages of SMC differentiation. However, a critical
question is whether results observed in nonautologous
organ and bone marrow transplant patients reflect what
happens in the normal course of atherogenesis in humans,
and whether these blood-derived cells ever truly become
fully differentiated SMC; that is, it is still uncertain
whether blood-derived cells give rise to SMCs that play a
major role in the developing atherosclerotic lesions in
humans, and extensive additional work is warranted in
this area. Nevertheless, irrespective of whether bone marrow-derived cells normally contribute to lesion formation,
the results of these studies are very exciting in that they
demonstrate that bone marrow-derived cells have the
capacity to invest injured blood vessels, raising the possibility of exploiting this process for stem cell-based gene
therapies.
In summary, whereas it is certainly plausible that
bone marrow-derived SMCs play a major role in development of atherosclerosis, further extensive studies will be
necessary to prove this is the case. In contrast, there is
compelling evidence that phenotypic modulation/switching of SMC, irrespective of their original source, plays a
key role in the development and/or progression of atherosclerotic disease, and section IIIB briefly reviews studies
in this area as well as possible mechanisms that control
this process.
B. Characterization of the SMC Within
Atherosclerotic Lesions of Human and
Experimental Animal Models
The terms contractile and synthetic phenotype have
been used extensively in the literature to describe the
possible phenotypic states of the vascular SMC. Indeed,
these terms have proven to be useful (if for no other
reason than economy of language) for describing distinct
generic spectrums of phenotypes available to the SMC.
However, we now recognize that a simple two-state
model is inadequate to explain the diverse range of phenotypes that can be exhibited by the SMC under different
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physiological and pathological circumstances (Fig. 1) (reviewed in Ref. 191). Not surprisingly, as the repertoire of
SMC markers has expanded, the picture that has emerged
is that there is likely a wide spectrum of possible SMC
phenotypes that might exist such that it may be very
artificial to assign cells to distinct subcategories. If one
extends this logic to consideration of quantitative measures of large numbers of markers that represent the
“contractile” and “synthetic” state of the SMC, the distinctions between these subtypes become very difficult to
distinguish, as in the case of vascular development when
SMCs are first acquiring their contractile properties yet
also simultaneously participating in vessel growth and
remodeling. Similarly, the complexity of different phenotypes that may be manifested by SMC is clearly also
evident within atherosclerotic lesions, where there is very
clear evidence that the morphological, biochemical, physiological, and molecular properties of the SMC vary at
different stages of atherosclerosis, within different lesion
types, and between SMCs located in different regions
within a given lesion. With this in mind, we will not
attempt to review the literally thousands of papers that
have been published on this topic and unequivocally established that intimal SMCs (irrespective of the original
sources of these cells) are altered with respect to normal
medial SMCs following vascular injury, or in association
with experimental or human atherosclerosis (reviewed in
Ref. 211). Rather, we briefly describe just a few representative examples of SMC phenotypes that appear to exist
within atherosclerotic lesions, beginning with what we
believe were several seminal studies by Aikawa and coworkers (4 – 6) that focused on analysis of the definitive
SMC differentiation marker SM MHC as well as nonmuscle-type MHC (NM-B MHC or SMemb) in human and
rabbit lesions.
Aikawa and colleagues (4, 5) found evidence for
altered expression of SM MHC isoforms (SM-1 an SM-2)
and SM ␣-actin in tissue samples obtained from autopsied
patients and atherectomy specimens from patients undergoing percutaneous transluminal coronary angioplasty
(PTCA). Medial SMCs were positive for SM-1, SM-2, and
SM ␣-actin. However, 16 –20 days after PTCA, neointimal
cells contained SM ␣-actin but little or no SM-1 or SM-2,
indicating partial SMC phenotypic modulation to an immature state. In contrast, 6 mo after PTCA, SMCs sequentially recovered SM-1 and then SM-2 expression and, consequently, a more mature phenotype. Increased expression of SMemb, a marker of fetal SMCs or immature adult
SMCs (4), was found throughout the intima with no apparent relationship to the time after PTCA. These findings
were extended in a separate study in a rabbit hypercholesterolemic angioplasty model of atherosclerosis (6).
Whereas expression of SM-1 and SM-2 was significantly
reduced in the media and outermost edge of the atheroma, animals undergoing the same treatment followed by
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extensive lipid lowering for 16 mo showed a time-dependent increase in SM-1 and SM-2 expression in the media
and neointima, indicating that SMCs exhibit a more mature phenotype after lipid lowering. Perhaps of even
greater interest, they observed that intimal SMCs in the
group undergoing lipid lowering showed marked reductions in expression of matrix metalloproteinase (MMP)-2
and MMP-9, reduced expression of PDGF-BB, and acquired the morphological appearance of a mature/contractile SMC (6).
Intimal SMCs have been shown to undergo many
additional changes including 1) increased DNA synthesis
and expression of proliferation markers and cyclins such
as proliferating cell nuclear antigen (PCNA) (77); 2) decreased expression of proteins characteristic of differentiated SMCs including SM MHCs (SM-1, SM-2), SM ␣-actin,
SM22␣, smoothelin, h-caldesmin, desmin, calponin, and
vinculin and increased ACLP and SMemb (4, 129, 130, 138,
183, 257); 3) alterations in calcium handling and contractility (49, 101, 262); and 4) alterations in cell ultrastructure, including a general loss of myofilaments, which is
replaced largely by synthetic organelles such as Golgi and
rough endoplasmic reticulum, rounding of the cell from
its typical elongated contractile morphology, and alterations in basement membrane (129, 130, 183). The preceding studies have been extended by Geary et al. (72),
who completed microarray-based profiling of gene expression patterns of SMCs in the neointima formed 4 wk
after aortic grafting compared with those from the normal
aorta in primates. A total of 147 genes were differentially
expressed in neointimal SMCs versus normal aorta SMCs,
most genes underscoring the importance of matrix production during neointimal formation. However, as elegant
and informative as these gene profiling studies are, we
feel they fail to capture the spatial-temporal patterns and
dynamic aspects of SMC phenotypic modulation in the
naturally occurring lesion, and clearly represent an oversimplification. For example, although the rate of SMC
proliferation may be elevated during early stages of lesion
formation, or after balloon injury of an advanced lesion as
part of an angioplasty procedure, the proliferation index
of SMC within mature advanced lesions is not elevated
(77). Similarly, depending on the nature of the lesion,
intimal SMCs may or may not show loss of many SMC
marker genes (273). Finally, aortic grafting and vessel
injury in healthy animals develop a predominantly SMCrich neointima over a relatively short time period compared with the naturally occurring multicellular atherosclerotic lesion that takes decades to develop.
In summary, the preceding studies support a model
in which the SMC can exhibit a wide range of different
phenotypes depending on the stage of lesion development
(or regression), and/or location of the SMC within a specific region of the atherosclerotic lesion. As such, the
functional role of phenotypic modulation of the SMC
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likewise is likely to vary depending on the stage of the
disease. For example, this process presumably plays a
maladaptive role in early lesion development and progression (210, 211), but may have a beneficial adaptive role in
stabilizing plaques in mature eccentric atherosclerotic
lesions. However, it may later contribute to plaque destabilization through apoptosis and/or activation of various
protease cascades (69, 70). In any case, these few examples illustrate the fact that regulation of transitions in the
phenotypic state of the SMC is likely to be extremely
complex as is the functional role of these changes, a topic
we address in section IV.
IV. MECHANISMS THAT CONTRIBUTE TO
PHENOTYPIC MODULATION OF SMOOTH
MUSCLE CELLS ASSOCIATED WITH
VASCULAR INJURY AND EXPERIMENTAL
ATHEROSCLEROSIS
A. Summary of Environmental Factors Thought
to Be Important
It is not possible to consider all environmental factors thought to be important in SMC phenotypic modulation within the space constraints of this review. Moreover,
this task is made even more daunting by the fact that it is
almost certain that very different factors may be involved
in initial recruitment of SMCs into lesions as opposed to
activation of proteases or apoptosis in end-stage disease
and plaque rupture. Many factors have been implicated
based on the morphological and biochemical characteristics of human lesions as well as numerous studies in
cultured cell systems and animal models. Suffice it to say
that we do not know what factors are important in human
lesions. However, our synopsis of many studies is that
phenotypic modulation of SMC within lesions is very
complex and likely to involve many factors including
growth factors and cytokines, inflammatory cell mediators, lipids, lipid peroxidation products, reactive oxygen
species, etc. (for reviews, see Refs. 153, 211). Rather than
trying to do justice to these studies, we will focus on
summarizing evidence for a number of factors where
there is in vivo evidence implicating the SMC in a particular stage of lesion formation including the following:
1) PDGF, which as summarized in section II is a highly
efficacious negative regulator of SMC gene expression in
cultured SMC, for initial SMC recruitment and migration,
proliferation, and modulation; 2) TGF-␤ as a candidate for
molecule that may have multiple roles including SMC
redifferentiation and concomitant plaque stabilization; and
3) MMPs which are differentially expressed in SMCs
throughout the development of the atherosclerotic lesion
and in the unstable plaque. We will focus on reviewing
studies in genetically modified mouse models of atheroscle-
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rosis that we believe illustrate general paradigms that may
be operative in atherosclerotic lesion formation in humans,
although recognizing there are likely to be many important
differences.
Although the rat model of vascular injury was initially
used by investigators to characterize the complex interaction between potential growth factors involved in phenotypic SMC modulation associated with neointimal hyperplasia, this model more closely mimics vascular repair
or at best restenosis in humans and not the natural progression of atherosclerosis which occurs over decades
(224). However, this being said, the initial observations
regarding growth factor expression that were made in the
rat (163, 180, 220) were very important in guiding subsequent studies in mice and ultimately were the foundation
of many human clinical restenosis trials. In mice, a detailed analysis of lesion development throughout the arterial tree by Nakashima et al. (186) and Zhang et al. (282)
showed that atherosclerotic lesions in the ApoE-deficient
mouse (ApoE ⫺/⫺) contained a spectrum of lesions similar to those observed during atherogenesis in humans,
including 1) monocyte attachment to endothelial cells
and subendothelial infiltration and macrophage investment, 2) foam cell lesion formation and fatty streaks, and
3) complex fibrous plaque formation including recruitment of SMCs to the cap, processes that are enhanced by
a high-fat/cholesterol-enriched diet as in humans.
1. PDGF
PDGF is a potent chemoattractant produced by activated platelets and lesion macrophages (91) that induces
rapid downregulation of SM-selective markers in cultured
SMC (16, 43, 108, 238) and stimulates SMC proliferation
and migration in arterial injury models (60, 121). PDGF
exists as a disulfide-linked dimer and is composed of two
chains, A and B (92). There are two PDGF receptors,
PDGFR-␣ and PDGFR-␤, whose intrinsic tyrosine kinase
activity is activated by PDGF-A alone, or PDGF-A and
PDGF-B, respectively (36). The role of PDGF receptors
has been described in several postinjury models (46), and
it is reported that both PDGF chains and their receptors
are detected in human coronary arteries following balloon angioplasty (249, 256). Conventional knockout of
PDGF-A (20), PDGF-B (141), PDGFR-␣ (241), and
PDGFR-␤ (240) has been shown to result in early embryonic or perinatal lethality, thereby prohibiting use of these
mice to investigate the role of PDGF signaling in vascular
injury responses or experimental atherogenesis. However, in ApoE ⫺/⫺ mice fed a Western diet for 12–18 wk,
Sano et al. (216) showed a 67% reduction in atherosclerotic lesion size and an 80% reduction in SMC cell investment of the neointima (as identified by SM ␣-actin-positive cells) following injection of rat monoclonal antibodies directed against both the PDGF ␣- and ␤-receptors
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compared with IgG-injected control mice. Blockade of
PDGFR-␣ alone had no effect on lesion size or smooth
muscle investment compared with controls. Thus regulating signal transduction via the PDGFR-␤ receptor in SMC
may play a key role in SMC migration and proliferation.
Elegant work by Kozaki et al. (131) tested a similar hypothesis but studied atherosclerosis development for up
to 50 wk. PDGF-B-deficient embryonic liver cells were
used to reconstitute circulating blood cells in lethally
irradiated ApoE ⫺/⫺ mice, where no PDGF-B was detected in circulating platelets and macrophages. At 35 wk,
lesions in the PDGF-B ⫺/⫺ mice contained mostly macrophages, appeared less mature, and had a reduced frequency of a fibrous cap. However, after 45 wk, SMC
accumulation in the fibrous cap was indistinguishable
from controls. The delayed onset of SMC proliferation
and migration was also observed in the same study by
dosing ApoE ⫺/⫺ mice with a PDGFR-␣/␤ antagonist
CT52923. Thus results show that inhibition of PDGF signaling or elimination of PDGF-B from circulating cells
does not appear sufficient to prevent SMC accumulation
in advanced lesions and suggest that PDGF-BB is not
required for lesion formation at this stage, or that other
growth factor signaling pathways mediate these effects
when PDGF signaling is blocked. However, it is also
possible that non-blood cell-derived PDGF-BB is important, i.e., endothelial cell and SMC PDGF production (91),
and/or that PDGF receptor blockade was incomplete. In
addition, it is not possible to deduce from the preceding
studies whether effects were the result of PDGF-dependent signaling in SMC versus being secondary to alterations in SMC phenotype per se. Indeed, resolution of
these important issues will be extremely difficult, and
likely will be dependent on development of SMC-specific
and/or conditional gene knockout mice that show selective abrogation of PDGF-dependent signaling pathways in
SMC, as discussed at the end of this section.
2. TGF-␤
TGF-␤ has been shown to promote SMC differentiation in cell culture by coordinately upregulating SM-selective markers such as SM ␣-actin and SM MHC (2, 87, 192).
Consistent with these results, knockout studies in mice of
the type I TGF-␤ receptor (189), TGF-␤ receptor II (190),
TGF-␤1 (48), or the TGF-␤ signaling molecules SMADs
(277) result in early embryonic lethality. Interestingly,
targeted overexpression of TGF-␤1 under control of the
SM22␣ promoter also results in embryonic lethality (3).
TGF-␤ levels are rapidly increased within 6 –24 h in experimental balloon injury models (164). Neointima formation, matrix deposition, and smooth muscle proliferation
are increased by overexpressing TGF-␤ and decreased by
inhibition in balloon injury models (222, 236). Whereas
the preceding results clearly implicate a possible role for
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MOLECULAR REGULATION OF SMC DIFFERENTIATION
TGF-␤ signaling in lesion formation, it is unclear what its
precise role is in vivo and even whether it has a beneficial
or detrimental effect. Of interest, McCaffery et al. (172)
showed that advanced human plaque SMCs contain mutations in the TGF-␤ type II receptor that decrease the
sensitivity of these cells to TGF-␤, and patients with
unstable angina typically have decreased plasma levels of
TGF-␤ (79), suggesting that TGF-␤ signaling may have an
overall protective effect. However, the most convincing
evidence for a protective role of TGF-␤ comes from two
studies in the ApoE ⫺/⫺ mouse that suggest that TGF-␤
may be critical for SMC matrix production and development of a stable fibrotic plaque (154, 165). Neutralizing
antibodies to TGF-␤1, TGF-␤2, and TGF-␤3 were shown
to accelerate the development of atherosclerosis at 15 wk,
and the lesions displayed increased inflammatory cells
and decreased collagen content (165). The latter findings
suggest that TGF-␤ may contribute to matrix production
within lesions but also acts to reduce inflammation. Consistent with this hypothesis, Lutgens et al. (154) observed
that 12-wk treatment of ApoE ⫺/⫺ mice with a soluble
TGF-␤ receptor II protein (TGF␤RII:Fc) that inhibits
TGF-␤ signaling resulted in an increased frequency of
CD3- and CD45-positive cells in the atherosclerotic lesions at 17 wk. More profound effects were found when
the 12-wk treatment was started 17 wk into the development of atherosclerosis. In these mice, lipid cores were
65% larger, the inflammatory cell content had increased
2.7-fold, fibrosis decreased 50%, and intraplaque hemorrhages were observed frequently. Thus, although not directly addressed in either study, these results suggest that
TGF-␤ may be essential for SMC matrix production and
fibrotic stabilization of the developing atherosclerotic
plaque.
3. MMPs
MMPs are endopeptidases produced by SMCs and
macrophages that are believed to contribute significantly
to the degradation and remodeling of the plaque extracellular matrix (69). Thus MMPs that are induced to be
expressed by environmental cues present within the lesion can in turn actively modify the matrix in which SMCs
reside and actively contribute to further phenotypic
switching of the SMC. Of interest, a number of factors that
have been shown to modify SMC phenotype, including
PDGF (32), TGF-␤ (155), nitric oxide (82), and reactive
oxygen species (83), have been shown to modify MMP
production in cultured SMCs, although as yet there is no
definitive evidence as to mechanisms and factors that
control expression of MMPs in vivo. Importantly, ultimately it is the balance between production of matrix
degrading MMPs, the inhibitors of MMPs or tissue inhibitor of metalloproteinase (TIMPs), and matrix production
by SMC that is a major determinant of plaque stability (58,
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140). In nondiseased human and experimental animal
arteries, MMP-2 (72-kDa gelatinase) and TIMP-1 and
TIMP-2 are constitutively expressed at levels providing a
stable balance between endogenous matrix production
and matrix degradation, and a healthy MMP-to-TIMP ratio
(69). This ratio is tipped towards MMPs in the developing
lesion as described in part by an increase in MMP-3
(stromelysin) and MMP-9 (92-kDa gelatinase), increased
MMP-to-TIMP ratio (69). Presumably, MMP overexpression would be required for SMC migration and formation
of a SMC-rich fibrous plaque and subsequent plaque stabilization. However, a potentially very significant study by
Galis et al. (70) showed that SMCs in the shoulder region
of human atherosclerotic plaques can also express MMP-3
and MMP-9, possibly leading to plaque destabilization and
rupture. Furthermore, as discussed in detail in section
IIIB, Aikawa et al. (6) showed that decreased SM-1 and
SM-2 marker expression correlated with increased PDGF
and MMP-3/9 expression in the rabbit neointima. Thus far,
knockout gene studies for MMP-9 have shown decreased
intimal SMC hyperplasia and reduced late lumen loss in
the mouse carotid artery flow cessation model (68) and
similar results in the carotid wire injury model (33). To
our knowledge, there are no studies in the MMP-9/ApoEdeficient mouse; however, in ApoE ⫺/⫺ mice, differential
expression of MMP-9 increases with time in lesional areas
versus nonlesion areas during the natural progression of
atherosclerosis (123). Indeed, TIMP-1 ⫺/⫺ mice crossed
to ApoE ⫺/⫺ mice showed increased aortic medial ruptures compared with control mice after 10 wk of Western
diet feeding, indicating that an imbalance between MMP
expression and TIMP expression can ultimately lead to
lesion instability (140). However, a critical unresolved
question is what environmental cues within the lesion
differentially regulate SMC and macrophage MMP production, as well as production of various TIMPs, thereby
influencing plaque formation and/or stability (see Ref. 69
for an excellent review of this critically important topic).
In summary, although the preceding studies give
some insight into the potential factors that might control
SMC phenotypic switching during atherogenesis, at
present there is a lack of direct evidence for involvement
of these factors in vivo, and in general, our understanding
of factors and mechanisms that control SMC phenotypic
switching in vivo is very poor. There is a need for further
detailed kinetic studies of candidate factors such as
PDGF and TGF-␤ that have been implicated in control of
this process. However, at best, such studies would only
provide correlative evidence regarding the large plethora
of signals present within an atherosclerotic lesion, and
more direct evidence using conditional knockout and/or
transgenic approaches are needed to significantly advance our understanding in this area, and to identify
specific factors and signaling pathways that contribute to
SMC phenotypic switching in vivo. In section IVB, we
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OWENS, KUMAR, AND WAMHOFF
briefly describe an experimental approach that we have
employed that we believe provides a powerful means to
advance our understanding in this important area.
B. Molecular Mechanisms of Decreased SMC
Differentiation Marker Expression Associated
With Atherosclerosis: A Novel Experimental
Approach to Studying SMC Phenotypic
Modulation
As an alternative to attempting to individually dissect
which of the wide plethora of altered environmental cues
associated with vascular injury (or experimental atherogenesis) directly contribute to phenotypic modulation of
the SMC phenotype in vivo, we developed what we refer
to as the “inside-out” approach; that is, since we know
that a signature feature of the phenotypically modified
SMC is reduced expression of SMC marker genes such as
SM ␣-actin and SM MHC, we first asked the very simple
questions. 1) Is injury-induced downregulation of SMC
marker genes mediated at the transcriptional level? 2) If
so, what specific cis-elements and trans-factors are required for this effect? The rationale is that this information could then be used to identify environmental cues
that alter the activity of these cis-elements and transfactors and thereby identify candidate environmental
cues/factors responsible for phenotypic switching that
could then be studied further using conditional cell targeted knockout and overexpression systems.
Whereas it may seem intuitive that repression of SMC
marker expression in lesions would be transcriptionally
mediated, surprisingly there was no direct evidence that
this was the case until relatively recently, despite the fact
that phenotypic modulation of SMC had been recognized
to be a key feature of atherosclerotic lesions for over 25
years (223). Indeed, observations in cultured SMCs that
PDGF-BB-induced transcriptional repression was mediated in large part by selective destabilization of transcripts for SMC marker genes (16, 43, 108, 147) raised the
distinct possibility that SMC phenotypic modulation in
vivo might not include a transcriptional component. To
directly address this question, we carried out a series of
vascular injury experiments in SM ␣-actin, SM MHC, and
SM22␣ LacZ transgenic mice. As shown in Figure 3 (see
top 3 panels) and Table 1, the SM ␣-actin, SM MHC, and
SM22␣ LacZ transgenes were highly expressed throughout the media of the uninjured mouse carotid artery.
However, after vascular injury, we observed nearly complete loss of expression of all three transgenes, thus demonstrating for the first time that SMC phenotypic modulation in vivo was mediated at least in part by transcriptional repression. Of interest, we also observed that after
14 days of injury, subpopulations of cells began to show
redifferentiation as evidenced by increased expression of
Physiol Rev • VOL
SM ␣-actin and SM MHC, further documenting the dynamic nature of SMC phenotypic modulation in vivo as
emphasized in section III.
Previous studies from our lab had identified a G/Crich cis-regulatory element in the SM MHC promoter that
functioned as a repressor in vitro and was shown to bind
the transcription factors Sp1 and Sp3, both of which are
increased in vascular models of injury (159, 161). Of major
significance, we showed that mutation of a similar G/Crich cis-regulatory element (positioned between both
CArGs) in the SM22␣ promoter had no effect on the
normal pattern of expression of this transgene during
development, but completely abrogated injury-induced
downregulation (see Fig. 3, compare the lower 2 rows of
images, and Table 1). These results thus indicate that the
G/C repressor element is dispensable for normal developmental regulation of this gene but is required for injuryinduced downregulation. Moreover, we showed that expression of the G/C repressor element binding factor Sp1
was dramatically upregulated by vascular injury (159).
Because G/C repressor elements are found between CArG
elements of many SMC marker gene promoters, results
support the possibility that phenotypic modulation of the
SMC (or at least repression of SMC marker genes) may be
regulated by injury-induced increases in expression of
Sp1 or Sp1-like transcription factors that bind to the G/C
repressor element, disrupt required cooperative interactions between CArG elements, and thereby inhibit SMC
gene expression (for a review of Sp1 and Sp1-like transcription factors, see Ref. 15). Of further interest, there is
evidence that Sp1-dependent mechanisms may also be
responsible for activation of genes characteristic of the
phenotypically modulated SMC, such as the constitutive
expression of PDGF-BB (199, 200) and SMemb (268); that
is, there may be common factors/pathways that turn off
certain subsets of genes while simultaneously turning on
others during SMC phenotypic switching. Whereas much
additional work needs to be done to thoroughly test this
hypothesis, this work clearly provides a foundation of
information with which to now begin to identify specific
environmental cues and mechanisms that control phenotypic switching of SMC. One can simply identify which of
the plethora of factors present in the lesion activate transcriptional control pathways are directly linked to the
end-stage markers of phenotypic modulation. In addition,
one can begin to dissect possible posttranslational controls of phenotypic switching in SMC, an area that is likely
to be extremely important but largely unexplored.
We feel that this field is in its infancy and that similar
“inside-out” approaches need to be applied to elucidating
mechanisms that activate expression of many of the genes
that exemplify the many phenotypic states of the SMC in
atherosclerotic lesions, including switching the balance of
production of gene products that stabilize the fibrous cap
versus those that result in destabilization.
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MOLECULAR REGULATION OF SMC DIFFERENTIATION
Finally, we would like to emphasize two additional
points in this section: 1) based on studies conducted thus
far, it has not been possible to distinguish whether phenotypic switching of the SMC is an initial cause or an
effect of atherosclerosis; and 2) although there are many
studies that have shown atheroprotective effects upon
deletion of a particular candidate gene, the underlying
mechanisms responsible for the observed effects are usually unclear. With respect to the first issue, we would
argue that in terms of the long-term consequences of
atherosclerosis, it probably does not matter; that is,
whether phenotypic modulation/switching of the SMC
plays a role in initial development of atherosclerosis, or
whether atherosclerosis develops and leads to SMC modulation, in the final analysis this process is a key part of
the progression and final clinical sequelae of the disease.
With respect to the second issue, a major rate-limiting
factor in conventional gene knockout studies is that the
gene of interest is absent from all cells throughout the
entire developmental history of the animal such that there
are likely to be a wide range of secondary compensatory
changes in these mice that confound data interpretation.
For example, knockout of intracellular adhesion molecule-1 (ICAM-1) resulted in the attenuation of atherosclerosis when crossed to the ApoE /⫺ mouse (128). However, an unpredicted result in the ICAM-1 ⫺/⫺ knockout
mouse was the development of severe obesity compared
with control mice, suggesting that there are a large number of undefined secondary metabolic changes in these
mice and that any observed phenotypic differences with
wild-type mice cannot be simply ascribed to the absence
of the ICAM-1 receptor on leukocytes or endothelial cells
as has often been assumed (50). Likewise, many candidate pathways that may play a role in regulating SMC
phenotypic switching are also involved in regulating responses in many different cell types during development
and maturation, and when knocked out may result in
embryonic lethality (e.g., as was the case with PDGF-BB
and TGF-␤ knockout mice discussed above). As such,
further progress in this area is likely to be highly dependent on development of both SMC-specific and/or conditional gene knockout mice that permit selective abrogation of candidate pathways of interest. One of the most
powerful approaches is to make use of cre-recombinase
gene targeting systems such as the SM MHC cre mouse
developed by our laboratory (202) or the SM22␣-CreER(T2) system that utilizes a mutated estrogen receptor
bound to cre-recombinase under control of the SM22␣
promoter to allow for tissue-selective cre-induction after
administration of tamoxifen to activate CreER(T2) (132).
However, as yet, these approaches are only beginning to
be utilized to investigate SMC phenotypic modulation.
In summary, due to inherent limitations in human
studies, further progress in this field will likely be dependent on use of animal models, albeit ones that are unlikely
Physiol Rev • VOL
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to completely recapitulate many of the regulatory processes involved in humans. Nevertheless, studies of these
simple animal models have already yielded and will continue to yield some very interesting potential insights into
SMC phenotypic modulation and particularly mechanisms
that regulate this process during the natural development
of the atherosclerotic lesion.
V. SUMMARY, CONCLUSIONS, AND FUTURE
DIRECTIONS/CHALLENGES
In this review we have attempted to summarize what
is known regarding mechanisms that regulate the differentiated state of the vascular SMC under normal circumstances and how these regulatory processes are altered
during formation of intimal lesions associated with vascular injury, or atherogenesis. The model that has evolved
is that regulation of SMC differentiation is extremely complex and involves constant interplay between environmental cues and the genetic program that controls the
coordinate expression of genes characteristic of the SMC
lineage (Fig. 1). Whereas studies in animal models have
clearly demonstrated that circulating bone marrow-derived cells can contribute to formation of the intimal
lesions under some circumstances involving extensive
medial necrosis or transplant rejection, the role of these
circulating cells in formation of human atherosclerotic
lesions is unclear. Indeed, preliminary studies suggest
they do not play a major role in most human lesions;
rather, the majority of SMCs within lesions appear to be
derived from phenotypic modulation of preexisting SMCs
in response to the plethora of alterations in environmental
cues present within the atherosclerotic lesion and/or site
of vascular injury including increased lipids, lipid peroxidation products, inflammatory cytokines, altered cell-cell
and cell matrix contacts, exposure to circulating blood
products, platelet-derived products, growth factors, and
perhaps specific negative regulators of SMC differentiation. Indeed, it is likely that our susceptibility to development of atherosclerosis may be, at least in part, a consequence of the necessity of the fully differentiated SMC to
retain extensive plasticity, a property that is essential for
vascular wound repair and survival but which unfortunately makes us susceptible to a plethora of atherogenic
stimuli prevalent in modern society.
Despite compelling evidence that phenotypic modulation of the SMC plays a key role in vascular injury repair
and in the development and/or progression of atherosclerosis, relatively little is known about how this process is
regulated in vivo. In fact, we are just beginning to understand some of the molecular mechanisms and factors that
control transitions in the phenotypic state of the SMC
associated with vascular injury, and we know almost
nothing regarding what controls these transitions during
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OWENS, KUMAR, AND WAMHOFF
different stages of development of atherosclerosis in humans. Key unresolved questions include the following: 1)
What are the key environmental cues/factors that induce
phenotypic modulation/switching of SMC following vessel injury or during different stages of atherogenesis? 2)
What are the molecular mechanisms by which these environmental cues/factors induce phenotypic modulation
of the SMC? 3) What factors and mechanisms convert a
SMC to a phenotype that promotes plaque stabilization
versus plaque destabilization? 4) Can the phenotypic state
of the SMC within lesions be manipulated for therapeutic
purposes? 5) What are mechanisms/adaptations that may
prevent SMC phenotypic switching in pathological setting, e.g., cardioprotective mechanisms related to exercise or a nonsedentary life-style (21)? 6) What is the
relationship of the SMC and activated myofibroblast, can
they be interconverted, and how can they be distinguished? 7) Can one exploit the fact that circulating stem
cells appear to be capable of at least partial differentiation
into SMC lineages to develop therapeutic approaches for
preventing atherosclerosis or restenosis using autologous
somatic stem cell-derived SMC as gene delivery vehicles?
8) What is the role of posttranscriptional regulatory mechanisms in control of phenotypic switching of SMC, an area
that is grossly understudied particularly given the large
number of SMC-selective alternative splice products important for the differentiated function of the SMC? 9)
What factors and mechanisms control SMC phenotypic
switching in other disease states including cancer, pulmonary and systemic hypertension, and diabetes?
Clearly, there has been much exciting progress in our
understanding of molecular mechanisms that regulate
SMC differentiation in recent years, but much additional
work is needed. Given that many of the factors that
appear to play an important role in control of SMC differentiation are also involved in regulating many other cellular processes, it is likely that major progress in this area
is going to be dependent on development of sophisticated
loss of function approaches including SMC-specific conditional gene knockout models and/or local inhibition of
candidate regulatory factors/pathways specifically at sites
of lesion formation. Finally, much additional work is also
needed to identify the precise origins of intimal SMCs and
to clearly define the specific contributions of the SMC
versus other cell types within the lesion, such as macrophages and endothelial cells, to the end-stage clinical
sequelae of atherosclerosis including plaque rupture,
thrombosis, infarction, vasospasm, myocardial ischemia,
and death.
We acknowledge the outstanding support of the University
of Virginia Cardiovascular Research Center and our many good
friends and colleagues in the field for their many outstanding
studies and creative ideas that we hope have been captured in
this review.
Physiol Rev • VOL
This work was supported by National Heart, Lung, and
Blood Institute Grants P01-HL-19242, R01-HL-38854, R21-HL71976, and R37-HL-57353 (to G. K. Owens); by the University of
Virginia Cardiovascular Training Grant HL-07284 (to M. S. Kumar and B. R. Wamhoff); by Mid-Atlantic Affiliate of the American Heart Association Fellowship Grant 0110071U (to M. S.
Kumar); and American Physiological Society Functional Genomics Fellowship GF10641 (to B. R. Wamhoff).
Address for reprint requests and other correspondence:
G. K. Owens, Dept. of Molecular Physiology and Biological
Physics, Univ. of Virginia School of Medicine, 415 Lane Rd.,
Medical Research Building 5, Rm. 1220, PO Box 801394, Charlottesville, VA 22908 (E-mail: gko@virginia.edu).
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