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We recently reported that the first detectable expression of SMC-specific proteins during coronary smooth muscle cell (CoSMC) differentiation from isolated proepicardial cells was restricted to cells undergoing epithelial-to-mesenchymal transformation (EMT). The objectives of this study were to examine more closely the relation between actin cytoskeletal rearrangements and serum response factor (SRF)-dependent transcription, and to specifically test whether rhoA-GTPase signaling is required for CoSMC differentiation. We report here that PDGF-BB stimulates EMT and promotes SRF-dependent expression of SMC marker genes calponin, SM22alpha, and SMgamma(actin) (SMgammaA) in proepicardial cells. C3 exoenzyme or rhoGDI, inhibitors of rhoA signaling, blocked PDGF-BB-induced EMT, prevented actin reorganization into stress fibers, and inhibited CoSMC differentiation. Incubation with the selective p160 rho-kinase (p160RhoK) inhibitor Y27632 (RKI) blocked EMT, prevented the appearance of calponin and SMgammaA-positive cells, and abolished expression and nuclear localization of SRF. To test the role of RhoK signaling for CoSMC differentiation in vivo, quail proepicardial organs (PEOs) were pretreated with RKI or vehicle and then grafted into age-matched host chick embryos to produce a chimeric epicardium. The ability of grafted cells to participate in coronary vessel formation was monitored by staining with antibodies for quail cell nuclear antigen and SMC marker proteins. Proepicardial cells pretreated with RKI failed to form CoSMCs in vivo. Time course studies traced this deficiency to a failure of epicardial-derived mesenchymal cells to migrate into or survive within the myocardium. In summary, these data point to important roles for rhoA-RhoK signaling in molecular pathways controlling cytoskeletal reorganization, SRF-dependent transcription, and cell survival that are required to produce CoSMCs from proepicardial cells.

Myocyte loss in the ischemically injured mammalian heart often leads to irreversible deficits in cardiac function. To identify a source of stem cells capable of restoring damaged cardiac tissue, we transplanted highly enriched hematopoietic stem cells, the so-called side population (SP) cells, into lethally irradiated mice subsequently rendered ischemic by coronary artery occlusion for 60 minutes followed by reperfusion. The engrafted SP cells (CD34(-)/low, c-Kit(+), Sca-1(+)) or their progeny migrated into ischemic cardiac muscle and blood vessels, differentiated to cardiomyocytes and endothelial cells, and contributed to the formation of functional tissue. SP cells were purified from Rosa26 transgenic mice, which express lacZ widely. Donor-derived cardiomyocytes were found primarily in the peri-infarct region at a prevalence of around 0.02% and were identified by expression of lacZ and alpha-actinin, and lack of expression of CD45. Donor-derived endothelial cells were identified by expression of lacZ and Flt-1, an endothelial marker shown to be absent on SP cells. Endothelial engraftment was found at a prevalence of around 3.3%, primarily in small vessels adjacent to the infarct. Our results demonstrate the cardiomyogenic potential of hematopoietic stem cells and suggest a therapeutic strategy that eventually could benefit patients with myocardial infarction.

Primary transcripts encoding the MADS box superfamily of proteins, such as MEF2 in animals and ZEMa in plants, are alternatively spliced, producing several isoformic species. We show here that murine serum response factor (SRF) primary RNA transcripts are alternatively spliced at the fifth exon, deleting approximately one-third of the C-terminal activation domain. Among the different muscle types examined, visceral smooth muscles have a very low ratio of SRFDelta5 to SRF. Increased levels of SRFDelta5 correlates well with reduced smooth muscle contractile gene activity within the elastic aortic arch, suggesting important biological roles for differential expression of SRFDelta5 variant relative to wild-type SRF. SRFDelta5 forms DNA binding-competent homodimers and heterodimers. SRFDelta5 acts as a naturally occurring dominant negative regulatory mutant that blocks SRF-dependent skeletal alpha-actin, cardiac alpha-actin, smooth alpha-actin, SM22alpha, and SRF promoter-luciferase reporter activities. Expression of SRFDelta5 interferes with differentiation of myogenic C2C12 cells and the appearance of skeletal alpha-actin and myogenin mRNAs. SRFDelta5 repressed the serum-induced activity of the c-fos serum response element. SRFDelta5 fused to the yeast Gal4 DNA binding domain displayed low transcriptional activity, which was complemented by overexpression of the coactivator ATF6. These results indicate that the absence of exon 5 might be bypassed through recruitment of transcription factors that interact with extra-exon 5 regions in the transcriptional activating domain. The novel alternatively spliced isoform of SRF, SRFDelta5, may play an important regulatory role in modulating SRF-dependent gene expression.

Fibroblast growth factor (FGF)-1 and FGF-2 are potent angiogenic factors and vascular smooth muscle cell (SMC) mitogens in vivo. They function via binding to a family of structurally related cell surface receptors that possess intrinsic tyrosine kinase activity. Several studies have indicated that increased FGF and/or FGF receptor (FGFR) expression may correlate with adult SMC proliferation in vivo. In this study, we used Northern blot hybridization and reverse transcription-polymerase chain reaction assays to compare the FGF and FGFR mRNA levels in newborn rat aorta, where SMCs have a high replication index, to those in adult rat aorta, where SMCs are relatively quiescent. We found that FGF-2 and FGFR-2 mRNA expression was elevated 8.2- and 5.6-fold, respectively, in adult aorta. Increased FGF-2 protein expression in the adult aorta was confirmed by Western blot analysis. We also examined FGF and FGFR mRNA expression levels in SMC cultures derived from newborn or adult rat aorta. FGF-1 transcripts were more abundant in newborn SMCs whereas FGF-2 and FGFR-1 mRNA expression was higher in adult SMCs. Furthermore, FGF-1 and FGF-2 mRNA expression levels were altered by cell culture density and by serum treatment. We conclude that elevated FGF ligand and receptor expression does not always correlate with a high SMC proliferative index, that FGF-1 or FGF-2 may not be the primary mitogens responsible for newborn SMC growth in vivo, and that FGF-1 and FGF-2 may serve nonmitogenic functions within the mature, adult vessel wall.

Lineage analysis studies in the avian embryo have identified two types of smooth muscle cells (SMCs) in the tunica media of large elastic arteries; one that originates within the cardiac neural crest and is ectoderm in origin (Ect) and another that arises from local mesenchyme of mesodermal origin (Mes). To determine if differences in primary embryonic lineage can give rise to SMCs with stable differences in growth and differentiation properties, we isolated Ect and Mes SMCs from the Day 14 chick embryo aorta. We report that despite different primary embryonic origins, Ect and Mes SMCs express nearly identical levels of seven SMC differentiation markers in vitro, consistent with their common smooth muscle developmental fates in vivo. By contrast, Ect SMCs displayed a greater capacity for growth in serum-free medium than Mes SMCs, but only under conditions permitting short-range cell-cell interactions. Most of the peptide growth factors tested that might account for serum-independent growth (PDGF-AA, PDGF-BB, basic FGF, EGF, or activin) stimulated DNA synthesis to similar extents in Ect and Mes SMCs. However, we found dramatic, lineage-dependent differences in SMC responses to transforming growth factor-beta (TGF-beta). Exposure to TGF-beta 1 (0.4 to 400 pmole/liter) consistently increased DNA synthesis in Ect SMCs, whereas in paired cultures of Mes SMCs, TGF-beta 1 was growth inhibitory. In SMC cultures transfected with p3TP-lux, a luciferase reporter controlled by the TGF-beta 1-response elements of the human PAI-1 promoter, TGF-beta 1 (120 pM) produced 12 +/- 2-fold increases in luciferase activity in Ect SMCs and only 3 +/- 1.5-fold increases in Mes SMCs. Analysis of TGF-beta receptor phenotypes by Northern blot, radioligand binding, and crosslinking assays showed that Ect and Mes SMCs expressed similar levels of types I, II, and III TGF-beta receptors. However, using a polyclonal antibody specific for the chick type II TGF-beta receptor subunit, we demonstrate that Mes SMCs produce a fully glycosylated form of this protein while Ect SMCs elaborate only an unglycosylated type II TGF-beta receptor. These results show that Ect and Mes SMCs exhibit lineage-dependent differences in growth and receptor-mediated transcriptional responses to at least one important class of SMC morphogens and growth modifiers, e.g., the TGF-betas. Our findings suggest that different SMC populations within a common vessel wall may respond in lineage-dependent ways to signals that direct formation of the tunica media in the embryo and to factors involved in the progression of vascular disease later in life.

Clear differences exist in the incidence and severity of atherosclerotic plaques that arise in different segments of the arterial tree. Aortic homograft transplant experiments in dogs showed that the greater incidence of plaque formation in the abdominal versus the thoracic aorta was due to intrinsic differences in the cell populations in these two segments rather than to hemodynamic factors. What is the basis for SMC diversity within a common vessel wall? Recent lineage analysis studies in the avian and mammalian embryo indicate that two distinct SMC lineages contribute to the formation of the major elastic outflow arteries including the aorta. A mixture of unique SMC types of diverse developmental lineages within a common vessel wall raises new questions about the potential for SMC type-specific responses to growth factors and cytokines involved in human atherosclerosis and restenosis.

Background: Retinoids have been used in the successful treatment of a variety of human hyperproliferative diseases. Their role in smooth muscle cell (SMC) growth control, however, has not been clearly established. The present study was designed to assess the retinoid receptor mRNA expression profile in SMCs and to determine whether retinoids exert a growth-inhibitory effect in these cells., Methods and Results: Five of the six retinoid receptors were expressed in both cultured SMCs and aorta as determined by Northern blotting or reverse transcriptase-polymerase chain reaction. Receptor activity was demonstrated in SMCs with the use of a reporter assay with a retinoid receptor DNA binding sequence linked to a chloramphenicol acetyltransferase reporter gene. DNA synthesis and cell proliferation assays were performed to show that all-trans retinoic acid (atRA) antagonized platelet-derived growth factor-BB and serum-stimulated SMC growth. Growth inhibition was distal to early growth-signaling events because induction of c-fos, c-jun, and egr-1 mRNA was unaffected by atRA. However, with an activated protein-1-linked chloramphenicol acetyltransferase reporter, atRA was shown to inhibit the activity of activated protein-1-dependent transcription in a transient transfection assay., Conclusions: These results establish the presence of functional retinoid receptors in SMCs and document the growth-inhibitory action of atRA on these cells. Retinoid compounds, already in clinical use as antiproliferative agents for nonvascular indications, should be assessed further in in vivo models of intimal disease.

Restenosis currently limits the long-term beneficial effects of balloon coronary angioplasty. Two important cellular events in the development of clinically significant luminal narrowing after angioplasty are 1) increased production of extracellular matrix proteins and 2) acquisition of a motile phenotype by vascular smooth muscle cells. In this paper, smooth muscle cell responses that produce a fibrocellular neointima after acute vascular injury are reviewed. Particular emphasis is placed on specialized extracellular matrix proteins implicated in cell movement and tissue repair. Tenascin and thrombospondin are large, modular extracellular matrix glycoproteins; they possess both adhesive and counteradhesive domains and are expressed at high levels during smooth muscle cell migration and neointima formation after balloon injury to rat carotid artery. The ability of both tenascin and thrombospondin to down-regulate the assembly and activity of focal adhesions (points of cell-extracellular matrix adhesive interactions) may be important in the conversion of stationary, quiescent smooth muscle cells to cells that are able to move and divide within the strongly adhesive vessel wall. Moreover, tenascin is present in the extracellular matrix as a large 6-armed oligomer (a hexabrachion) that contains both cell-binding and matrix protein-binding domains in each of the hexabrachion arms. The large size and multidomain structure of tenascin and thrombospondin suggest that these proteins may be particularly well suited to form a nascent provisional matrix at sites of 1) neointima formation after acute vascular injury, 2) new growth and expansion within primary atherosclerotic plaques, and 3) intimal repair and luminal narrowing in restenosis after angioplasty.

Sympathetic nerves and catecholamines exert growth-promoting trophic influences on arterial smooth muscle in vivo, but the molecular signals mediating these trophic effects are unknown. We report here that the alpha-adrenergic agonist phenylephrine (PE) produced dose-dependent stimulation of platelet-derived growth factor A-chain (PDGF-A) gene expression in rat thoracic aorta via agonist occupancy of alpha 1-adrenergic receptors. Increases in aortic PDGF-A mRNA levels were rapid (maximal at 6 h, 10-fold) and transient. Among seven different tissues studied, PE evoked significant increases in PDGF-A mRNA levels only in the aorta. When periaortic fatty/connective tissues normally adherent to thoracic aorta were examined separately from the remaining aortic vessel wall (endothelium removed), stimulated PDGF-A gene expression was found only in vessel wall (presumably smooth muscle). The physiological alpha-adrenergic agonist norepinephrine also increased aortic PDGF-A mRNA levels. Angiotensin II or endothelin, despite producing blood pressure increases similar to PE, had little or no effect on PDGF-A mRNA abundance in rat aorta. PE-stimulated PDGF-A gene expression was accompanied by increased expression of other growth-related genes including c-fos, c-myc, and ornithine decarboxylase but not DNA synthesis. These results suggest a mechanism for previously described trophic effects of sympathetic nerves and catecholamines on arterial smooth muscle mass, i.e. regulation of growth-related gene expression via alpha 1-adrenergic receptors.

Repair of arterial injury results in formation of a new structure, a neointima, that causes luminal narrowing. Smooth muscle cell (SMC) properties required for neointima formation are also found in nascent SMCs of developing blood vessels in the embryo (e.g., proliferation, extracellular matrix synthesis, cell migration). We isolated 2 distinct types of SMC from aortic media of newborn rats that were distinguished by cell shape, secretion of platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1), and expression of PDGF-B and PDGF alpha-receptor genes. These two SMC types did not interconvert over many cell generations in vitro. Adult rat aorta yields only one SMC type, suggesting that the "pup" SMC variant is developmentally regulated. However, SMC with the "pup" phenotype reappear in the adult artery wall during neointima formation after balloon catheter injury. These observations raise the possibility that SMC proliferation and arterial remodeling during development, repair and disease of the artery wall might depend upon a SMC subpopulation with special properties.