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Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Virginia
ABSTRACT 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 1. CArG SRF-dependent regulation plays a key role in regulation of most SMC differentiation marker genes characterized to date A) REGULATION OF THE LEVEL OF SRF EXPRESSION. B) REGULATION OF SRF BINDING AFFINITY BY HOMEODOMAIN FACTORS AND/OR POSTTRANSLATIONAL MODIFICATIONS OF SRF. C) COOPERATIVE INTERACTION OF MULTIPLE CARG ELEMENTS. D) POSTTRANSCRIPTIONAL MODIFICATION OF SRF AS WELL AS CONTROL OF NUCLEAR LOCALIZATION. E) SMC-SPECIFIC/SELECTIVE SRF COACTIVATORS. 2. Cis-elements and trans-binding factors in addition to CArG-SRF also play a key role in regulating SMC-selective gene expression 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 Atherosclerotic 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 1. PDGF 2. TGF-{beta} 3. MMPs 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 ACKNOWLEDGMENTS REFERENCES
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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 |
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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) that have a lumen diameter in excess of 100 µm, 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 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.
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II. MOLECULAR REGULATION OF SMOOTH MUSCLE CELL DIFFERENTIATION AND MATURATION DURING VASCULAR DEVELOPMENT AND DISEASE |
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Before considering molecular mechanisms that control SMC differentiation in development and disease, it is important to first briefly review some general principles of 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 cell-selective/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 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?
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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 immunosuppression 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 hypercholesteremic 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 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 Differentiation-Maturation, 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 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.
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, 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 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 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 species-dependent 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 non-SMC, 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 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 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 LKLF-dependent 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 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 promoter-enhancer 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 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 cultures 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.
D. The SMC-Specific Transcriptional Regulation Is Dependent on Complex Combinatorial Interactions of Multiple Cis-Elements (Regulatory Modules) and Their Trans- Binding 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 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 cell-specific 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).
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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 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 rate-limiting 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 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 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 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.
