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We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-beta (TGF-beta), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-beta inhibited serum-induced proliferation of rat aortic smooth muscle cells (ED50 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:7155-7160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-beta. TGF-beta concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47% increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-beta-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in G1 were hypertrophied with respect to the corresponding cells in vehicle-treated controls. Chronic treatment with TGF-beta (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-beta from these cultures was associated with the appearance of a significant fraction of cycling cells with greater than 4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-beta in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-beta.

We have previously shown that maximal expression of the rat smooth muscle myosin heavy chain (SM-MHC) gene in cultured rat aortic smooth muscle cells (SMCs) required the presence of a highly conserved domain (nucleotides -1321 and -1095) that contained two positive-acting serum response factor (SRF) binding elements (CArG boxes 1 and 2) and a negative-acting GC-rich element that was recognized by Sp1 (Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997) J. Biol. Chem. 272, 6332-6340). In this study, to better understand the functional role of these three cis elements, we created a series of SM-MHC reporter-gene constructs in which each element was mutated either alone or in combination with each other and tested them for activity in transient transfection assays using primary cultured rat aortic SMCs. Results demonstrated that the most proximal SRF binding element (CArG-box1) was active in the absence of CArG-box2, but only upon removal of the GC-rich repressor. In contrast, regardless of sequence context, CArG-box2 was active only when CArG-box1 was present. We further demonstrated using electrophoretic mobility shift assays that Sp1 binding to the GC-rich repressor element did not prevent SRF binding to the adjacent CArG-box2. Thus, unlike other proteins reported to inhibit SRF activity, the repressor activity associated with the GC-rich element does not appear to function through direct inhibition of SRF binding. As a first step toward understanding the importance of these elements in vivo, we performed in vivo footprinting on the intact rat aorta. We demonstrated that both CArG boxes and the GC-rich element were bound by protein within the animal. Additionally, using the rat carotid injury model we showed that Sp1 protein was significantly increased in SMCs located within the myointimal lesion, suggesting that increased expression of this putative repressor factor may contribute to the decreased SM MHC expression within SMCs found in myointimal lesions.

The goal of the present study was to determine the molecular mechanism whereby transforming growth factor beta (TGFbeta) increases smooth muscle (SM) alpha-actin expression. Confluent, growth-arrested rat aortic smooth muscle cells (SMC) were transiently transfected with various SM alpha-actin promoter/chloramphenicol acetyltransferase deletion mutants and stimulated with TGFbeta (2.5 ng/ml). Results demonstrated that the first 125 base pairs of the SM alpha-actin promoter were sufficient to confer TGFbeta responsiveness. Three cis elements were shown to be required for TGFbeta inducibility: two highly conserved CArG boxes, designated A (-62) and B (-112) and a novel TGFbeta control element (TCE) (-42). Mutation of any one of these elements completely abolished TGFbeta-induced reporter activity. Results of electrophoretic mobility shift assays demonstrated that nuclear extracts from TGFbeta-treated SMC enhanced binding activity of serum response factor to the CArG elements and binding of an as yet unidentified factor to the TCE. Northern analysis showed that TGFbeta also stimulated transcription of two other SM (SM myosin heavy chain) differentiation marker genes, SM myosin heavy chain and h1 calponin, whose promoters also contained a TCE-like element. In summary, we identified a TGFbeta response element in the SM alpha-actin promoter that may contribute to coordinate regulation of expression of multiple cell-type specific proteins during SMC differentiation.

To identify cis- and trans-acting factors that regulate smooth muscle-specific gene expression, we studied the smooth muscle myosin heavy chain gene, a rigorous marker of differentiated smooth muscle. A comparison of smooth muscle myosin heavy chain promoter sequences from multiple species revealed the presence of a highly conserved 227-base pair domain (nucleotides -1321 to -1095 in rat). Results of a deletion analysis of a 4.3-kilobase pair segment of the rat promoter (nucleotides -4220 to +88) demonstrated that this domain was necessary for maximal transcriptional activity in smooth muscle cells. Gel-shift analysis and site-directed mutagenesis demonstrated that one true CArG and another CArG-like element contained within this domain were both recognized by the serum response factor and were both required for the positive activity attributable to this domain. Additional studies demonstrated that mutation of a GC-rich sequence within the 227-base pair conserved domain resulted in a nearly 100% increase in transcriptional activity. Gel-shift analysis showed that this GC-rich repressor element was recognized by both Sp1 and Sp3. These data demonstrate that transcriptional control of the smooth muscle myosin heavy chain gene is highly complex, involving both negative and positive regulatory elements, including CArG sequences found in the promoters of multiple smooth muscle differentiation marker genes.

The relationship between growth and cytodifferentiation was studied in cultured rat aortic smooth muscle cells (SMCs) using expression of the smooth muscle (SM)-specific isoactins (Vanderkerckhove, J., and K. Weber, 1979, Differentiation, 14:123-133) as a marker for differentiation in these cells. Isoactin expression was evaluated by: (a) measurements of fractional isoactin content and synthesis ([35S]methionine incorporation) by densitometric evaluation of two-dimensional isoelectric focusing sodium dodecyl sulfate gels, and (b) immunocytological examination using SM-specific isoactin antibodies. Results showed the following: (a) Loss of alpha-SM isoactin was not a prerequisite for initiation of cellular proliferation in primary cultures of rat aortic SMCs. (b) alpha-SM isoactin synthesis and content were low in subconfluent log phase growth cells but increased nearly threefold in density-arrested postconfluent cells. Conversely, beta-nonmuscle actin synthesis and content were higher in rapidly dividing subconfluent cultures than in quiescent postconfluent cultures. These changes were observed in primary and subpassaged cultures. (c) alpha-SM actin synthesis was increased by growth arrest of sparse cultures in serum-free medium (SFM; Libby, P., and K. V. O'Brien, 1983, J. Cell. Physiol., 115:217-223) but reached levels equivalent to density-arrested cells only after extended periods in SFM (i.e., greater than 5 d). (d) SFM did not further augment alpha-SM actin synthesis in postconfluent SMC cultures. (e) Serum stimulation of cells that had been growth-arrested in SFM resulted in a dramatic decrease in alpha-SM actin synthesis that preceded the onset of cellular proliferation. These findings demonstrate that cultured vascular SMCs undergo differential expression of isoactins in relation to their growth state and indicate that growth arrest promotes cytodifferentiation in these cells.

We explored the hypothesis that discrepancies in the literature concerning the nature of myosin expression in cultured smooth muscle cells are due to the appearance of a new form of myosin heavy chain (MHC) in vitro. Previously, we used a very porous sodium dodecyl sulfate gel electrophoresis system to detect two MHCs in intact smooth muscles (SM1 and SM2) which differ by less than 2% in molecular weight (Rovner, A. S., Thompson, M. M., and Murphy, R. A. (1986) Am. J. Physiol. 250, C861-C870). Myosin-containing homogenates of rat aorta cells in primary culture were electrophoresed on this gel system, and Western blots were performed using smooth muscle-specific and nonmuscle-specific myosin antibodies. Subconfluent, rapidly proliferating cultures contained a form of heavy chain not found in rat aorta cells in vivo (NM) with electrophoretic mobility and antigenicity identical to the single unique heavy chain seen in nonmuscle cells. Moreover, these cultures expressed almost none of the smooth muscle heavy chains. In contrast, postconfluent growth-arrested cultures expressed increased levels of the two smooth muscle heavy chains, along with large amounts of NM. Analysis of cultures pulsed with [35S] methionine indicated that subconfluent cells were synthesizing almost exclusively NM, whereas postconfluent cells synthesized SM1 and SM2 as well as larger amounts of NM. Similar patterns of MHC content and synthesis were found in subconfluent and postconfluent passaged cells. These results show that cultured vascular smooth muscle cells undergo differential expression of smooth muscle- and nonmuscle-specific MHC forms with changes in their growth state, which appear to parallel changes in expression of the smooth muscle and nonmuscle forms of actin (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol. 102, 343-352). The reappearance of the smooth muscle MHCs in postconfluent cells suggests that density-related growth arrest promotes cytodifferentiation, but the continued expression of the nonmuscle MHC form in these smooth muscle cells indicates that other factors are required to induce the fully differentiated state while in culture.

We have previously demonstrated that both CC(A/T-rich)6GG (CArG) elements A and B of the smooth muscle (SM) alpha-actin promoter are required for smooth muscle cell (SMC)-specific expression and angiotensin II (AII)-induced stimulation. Moreover, results provided evidence that AII responsiveness of SM alpha-actin was at least partially dependent on modulation of serum response factor (SRF) binding to the SM alpha-actin CArGs by the homeodomain containing protein, MHox. The goal of the present study was to investigate whether the degeneracy of the SM alpha-actin CArGs (both contain a Gua or Cyt substitution in their A/T-rich center) and their reduced SRF binding activity as compared with c-fos serum response element (SRE) is important for conferring cell type-specific expression and AII responsiveness. Transient transfection assays using SM alpha-actin reporter gene constructs in which the endogenous SM alpha-actin CArGs were replaced by c-fos SREs demonstrated the following: 1) relaxation of cell-specific expression, 2) a 50% reduction in AII responsiveness, and 3) reduced ability to be transactivated by MHox. In addition, we also showed that the position of the SM alpha-actin CArGs was important in that interchanging them abolished both basal and AII-induced activities. Taken together, these results suggest that the reduced SRF binding activities of the SM alpha-actin CArGs and CArG positional context contribute to SMC-specific expression of SM alpha-actin as well as maximal AII responsiveness.

There is an inverse relationship between cellular proliferation and smooth muscle alpha-isoactin expression in cultured vascular smooth muscle cells (SMCs) (Owens, G.K., Loeb, A., Gordon, D., and Thompson, M.M. (1986) J. Cell Biol. 102, 343-352). In the present studies, changes in isoactin expression were studied during developmental growth of rat aortic SMCs (ages 1-180 days) to better understand interrelationships between growth and cytodifferentiation in these cells in vivo. Actin expression (i.e. content and synthesis) was evaluated by one- and two-dimensional gel electrophoresis and using isoactin-specific antibodies. The major actin present in cells from newborn rats was nonmuscle beta-actin (56% of total actin), whereas cells from adult animals contained principally smooth muscle alpha-actin (Sm-alpha-actin) (76% of total actin). Increases in Sm-alpha-actin content with increasing age were due, in part, to an increase in Sm-alpha-actin synthesis. However, in SMCs from 90- and 180-day-old rats, the fractional content of Sm-alpha-actin exceeded its fractional synthesis at a time when total Sm-alpha-actin content was increasing. This suggests that Sm-alpha-actin turns over more slowly in mature animals. Decreases in the frequency of SMCs undergoing DNA synthesis with age could not account for increases in Sm-alpha-actin expression with age. However, combined immunocytological and [3H]thymidine autoradiographic studies demonstrated that nearly 50% of the medial derived cells from newborn rat aortas did not show detectable staining with a monoclonal antibody to smooth muscle-specific isoactins, and the replicative frequency was much higher in these cells than in cells that contained Sm-alpha-isoactins. Taken together, the results of the present studies and previous studies in cultured SMCs support the hypothesis that cessation of proliferation during development is associated with the induction of Sm-alpha-actin expression, but that factors other than cellular growth state play an important role in determining the level of Sm-alpha-actin expression in fully differentiated SMCs.

Arteries of hypertensive animals have a greater mass of smooth muscle than those of normotensive controls. We examined the contribution of smooth muscle cell hypertrophy and hyperplasia to this increase in mass. Cell size measurements obtained by (i) image analysis of enzyme-dispersed cells, (ii) morphometric evaluation of tissue sections, and (iii) biochemical measures of protein/cell and actin/cell ratios on isolated cells showed that average cell size was greater in spontaneously hypertensive rats than in normotensive Wistar-Kyoto and Sprague-Dawley controls. Average DNA/cell ratios were also increased in spontaneously hypertensive rats while protein/DNA ratios were not different. Analysis of nuclear DNA content of individual cells by flow microfluorimetry and Feulgen-DNA microdensitometry measurements showed that greater than 20% of spontaneously hypertensive rats cells were polyploid while less than 10% of control cells were polyploid. Estimates of cell number per centimeter of aortic length, based on ploidy and DNA content, show no difference between control and hypertensive rats. Thus, smooth muscle hypertrophy alone accounts for the increased mass of smooth muscle in aortas of spontaneously hypertensive rats. Furthermore, this cellular hypertrophy is accompanied by a change in nuclear ploidy. This nuclear response in hypertension may represent a fixed change related to the establishment of a chronic hypertensive state.

Although polyploid nuclei have long been known to be present in many adult human tissues, the ploidy of smooth muscle cells in human artery wall has never been determined. We measured DNA content in individual smooth muscle cell nuclei of artery wall specimens by two means: Feulgen microdensitometry and flow microfluorimetry. A significant percentage of nuclei were polyploid; most of these were tetraploid, although higher levels were also found. The frequency of polyploidy varied with age from less than 1% at birth to a mean of 7% in adult aortic, carotid, and iliac vessels. Atherosclerotic plaques had a lower tetraploid content than the underlying media, whereas normal intima was similar to the corresponding media. The increase in frequency of hyperploid smooth muscle cell nuclei correlates with the normal growth, development, and aging of human artery wall. We suggest that the regular existence of a subset of polyploid smooth muscle cells may indicate an important functional role for this phenotype.

Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics.

Hypertrophy of vascular smooth muscle cells (VSMC) is an important adaptive response of hypertension. Drug intervention studies have implicated a role for angiotensin II (A-II) in the mediation of VSMC hypertrophy in vivo, and A-II is a potent hypertrophic agent for VSMC in culture. Our laboratory has previously shown that A-II-induced hypertrophy of cultured VSMC is due in part to generalized increases in protein synthesis and increased content of rRNA. The aim of the present study was to determine if A-II stimulates rRNA gene synthesis and whether the rRNA transcription factor, upstream binding factor (UBF), is involved. Nuclear run-on analysis demonstrated that A-II induced a greater than 5-fold increase in rRNA gene synthesis within 6 h of stimulation. A-II also stimulated a rapid increase in UBF phosphorylation as well as nucleolar localization, but no changes in the content of UBF. Phosphoamino acid analysis showed that phosphorylation occurred only on serine residue(s). Results demonstrate that increased transcription of ribosomal DNA contributes to the A-II-induced increase in protein synthesis and VSMC hypertrophy, and suggest that an important regulatory event in this pathway may be the phosphorylation and/or nucleolar localization of UBF.

To identify potential regulators of smooth muscle cell (SMC) differentiation, we studied the molecular mechanisms that control the tissue-specific transcriptional expression of SM alpha-actin, the most abundant protein in fully differentiated SMCs. A construct containing the region from -1 to -125 of the promoter (p125CAT) had high transcriptional activity in SMCs (57-fold > promoterless) and endothelial cells (ECs) (18-fold) but not in skeletal myoblasts or myotubes. Mutation of either of two highly conserved CC(AT-rich)6GG (CArG) motifs at -62 and -112 abolished the activity of p125CAT in SMCs but had no effect in ECs. In contrast, high transcriptional activity in skeletal myotubes, which also express SM alpha-actin, required at least 271 base pairs of the promoter (-1 to > or = -271). Constructs containing 547 base pairs or more of the promoter were transcriptionally active in SMCs and skeletal myotubes but had no activity in skeletal myoblasts or ECs, cell types that do not express SM alpha-actin. Electrophoretic mobility shift assays provided evidence for binding of a unique serum response factor-containing complex of factors to the CArG box elements in SMCs. Results indicate that: 1) transcriptional expression of SM alpha-actin in SMCs requires the interaction of the CArG boxes with SMC nucleoprotein(s); 2) expression of SM alpha-actin in skeletal myotubes requires different cis-elements and trans-factors than in SMCs; and 3) negative-acting cis-elements are important in restricting transcription in cells that do not express SM alpha-actin.

Previous studies have demonstrated that rat aortic smooth muscle cells (SMC) show marked changes in smooth muscle (SM) α-actin content and fractional synthesis as a function of cell density and growth (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol.102, 343–352; Blank, R., Thompson, M. M., and Owens, G. K. (1988) J. Cell Biol. 107, 299–306). Results of this study show that, although there is a 6-fold increase in SM α-actin content in postconfluent density arrested cultures as compared to proliferating subconfluent cultures, SM α-actin mRNA levels are not different between these cells. This suggests that the SM α-actin gene is constitutively active under both of these conditions and that accumulation of SM α-actin in postconfluent cells is due to translational and/or post-translational controls.