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Circulation Research. 1996;78:196-204

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(Circulation Research. 1996;78:196-204.)
© 1996 American Heart Association, Inc.

Articles

Myocyte Enhancer Binding Factor-2 Expression and Activity in Vascular Smooth Muscle Cells

Association With the Activated Phenotype

Anthony B. Firulli, Joseph M. Miano, Weizhen Bi, A. Daniel Johnson, Ward Casscells, Eric N. Olson, John J. Schwarz

From the Department of Biochemistry and Molecular Biology (A.B.F., J.M.M., E.N.O.), University of Texas M.D. Anderson Cancer Center, Houston; the Division of Cardiology (W.B., W.C, J.J.S.), Department of Internal Medicine, University of Texas Medical School, Houston; and the Texas Heart Institute (A.D.J., W.C.), Houston.

Correspondence to Dr John J. Schwarz, Division of Cardiology, Department of Internal Medicine, University of Texas Medical School, Houston, TX 77030.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
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Abstract Proliferation and phenotypic modulation of smooth muscle cells (SMCs) are major components of the vessel's response to injury in experimental models of restenosis. Some of the growth factors involved in restenosis have been identified, but to date little is known about the transcription factors that ultimately regulate this process. We examined the expression of the four members of the myocyte enhancer binding factor-2 (MEF2) family of transcription factors in cultured rat aortic SMCs (RASMCs) and a rat model of restenosis because of their known importance in regulating the differentiated phenotype of skeletal and cardiac muscle. In skeletal and cardiac muscle, the MEF2s are believed to be important for activating the expression of contractile protein and other muscle-specific genes. Therefore, we anticipated that the MEF2s would be expressed at high levels in medial SMCs that are producing contractile proteins and that they would be downregulated along with the contractile protein genes in neointimal SMCs. On the contrary, we observe that MEF2A, MEF2B, and MEF2D mRNAs are upregulated in the neointima, with the highest levels in the layer of cells nearest to the lumen, whereas MEF2C mRNA levels do not appreciably increase. Moreover, few cells in the media are making MEF2 proteins detectable by immunohistochemistry, whereas large numbers of neointimal cells are positive for all four MEF2s. These data suggest that the MEF2s are involved in the activated smooth muscle phenotype and not in the maintenance of contractile protein gene expression.


Key Words: MEF2 • smooth muscle • transcription factor • balloon injury • mRNA


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
The inappropriate proliferation and phenotypic modulation of vascular SMCs are major components of atherosclerosis and the restenosis that can occur after balloon angioplasty. The rat model of restenosis does not mirror all aspects of human restenosis, but it does provide a reproducible and pharmacologically manipulatable system in which to study particular aspects of this disease. Extensive research since this model was introduced has established that the disease can be considered as progressing through three phases after deendothelialization of the artery.1 In the first phase, there is a significant proliferation of medial SMCs that begins on the first day and peaks 2 days after injury.2 3 During the next phase, SMCs in the media of the vessel wall migrate out to form a neointima.2 3 In the third phase, there is continued proliferation of these neointimal SMCs. In addition to being proliferative, these cells also undergo a phenotypic modulation in which contractile protein genes are downregulated, and there is increased synthesis of extracellular matrix proteins. This state has been termed activated or synthetic.4 5 Cultured RASMCs mirror some aspects of this process by becoming activated and more proliferative through successive passages.6 Presumably, modulation of the smooth muscle phenotype is regulated by transcription factors that control smooth muscle–specific gene expression. However, to date, no transcription factors that function in modulation of the smooth muscle cell phenotype have been identified, with the possible exception of the early response genes that are upregulated in vivo after balloon injury.7 8 9 Because these early response genes are not cell type–restricted and are induced by diverse stimuli, they are likely controlling cellular functions that are generalized and not smooth muscle specific. Thus, a large gap remains in our understanding of the molecular mechanisms that govern the phenotype of SMCs in vascular disease.

The transcription factors that regulate sarcomeric muscle growth and differentiation are more clearly understood than are those for smooth muscle and provide possible paradigms for understanding regulation in smooth muscle. They also could provide candidate genes to test for control of smooth muscle differentiation. Because there are several genes that are expressed in all three muscle types, it is possible that some of the transcription factors important for regulating the differentiated phenotype in skeletal and cardiac muscle will also be important for smooth muscle. For example, smooth muscle {alpha}-actin is expressed in skeletal and cardiac muscle early in development, and the intermediate filament desmin is expressed in all three muscle types and is therefore considered a general marker for muscle.10 11 Skeletal muscle is the best understood muscle cell type with respect to the genes that control differentiation. Terminal differentiation of skeletal muscle requires members of the MyoD family of transcription factors. Different members of this family act at distinct stages of the differentiation pathway with either MyoD or myf5 being essential for initiating myogenesis and myogenin being necessary for proper maturation of the myocyte into muscle fibers.12 13 14 Remarkably, these transcription factors are also each capable of initiating the skeletal muscle differentiation program in a number of nonmuscle cell lines and are therefore postulated to function as master regulatory genes.15 16 MyoD and other members of this family are not expressed in cardiac or smooth muscle, nor have any closely related genes been found that are expressed in these types.17 Therefore, other factors should be considered for modulation of the differentiated phenotype in SMCs. The genes controlling cardiac muscle differentiation are less well understood than for skeletal muscle; nonetheless, there are several transcription factors expressed at the earliest stages of cardiac development that might be involved in controlling its differentiation.18 19 20 21 Among these, only the members of the MEF2 family of transcription factors are expressed in sarcomeric and smooth muscle and are thus candidates for regulating differentiation in all muscle types.18

MEF2 was originally identified as a muscle-specific factor that binds an A/T-rich consensus sequence associated with a large number of genes expressed in skeletal and cardiac muscle. The MEF2 family has now been shown to consist of four members, termed MEF2A, MEF2B, MEF2C, and MEF2D.22 23 24 25 26 27 28 These factors belong to a larger class of factors that share homology within a DNA binding and dimerization motif known as a MADS box, which is named after the originally identified members: MCM1, agamous, deficiens, and serum response factor. Mutagenesis experiments have established the importance of MEF2 sites within the promoters of skeletal and cardiac muscle–specific genes. An interesting example of a MEF2-responsive promoter is that of myogenin, in which the MEF2 site is important for transcription in both tissue culture and transgenic mice.29 30 Since myogenin is essential for skeletal muscle maturation, this requirement for MEF2 provides evidence that the MEF2s may be near the top of a regulatory cascade for skeletal muscle and perhaps all muscle differentiation.29 30 The strongest support for MEF2 being important in all three muscle types is from the fruit fly Drosophila melanogaster, in which disruption of the single MEF2 gene, D-MEF2, causes lethal abnormality in the cardiac, skeletal, and visceral muscle lineages of the fly.31 32 33 34 Whether the MEF2 genes are equally important in higher organisms is currently under investigation.

Since MEF2 clearly plays an important role in skeletal and cardiac muscle gene regulation, we investigated the potential role of MEF2 in smooth muscle gene regulation by examining the expression and transcriptional activity of the four family members in cultured RASMCs and in medial and neointimal cells of balloon-injured rat carotid arteries. We find that MEF2A, MEF2B, and MEF2D mRNAs and proteins are expressed in cultured RASMCs as well as in the neointima of balloon-injured vessels, but very little MEF2A, MEF2B, or MEF2D expression is observed in the medial cells. MEF2C mRNA is present at low levels in cultured RASMCs and the neointima of injured vessels. Remarkably, even though MEF2C mRNA is not increased in the neointima, a large number of cells positive for MEF2C protein are detected in the neointima compared with the media. The presence of MEF2 proteins in cultures of proliferating RASMCs is in striking contrast to its expression in skeletal muscle, where MEF2 expression is associated with exit from the cell cycle and terminal differentiation. Taken together, these data indicate that contrary to expectations from skeletal muscle, MEF2 expression in smooth muscle is associated with phenotypically activated RASMCs of the neointima and not with the more differentiated cells of the media. The MEF2s may therefore play a role in regulating gene expression for activated smooth muscle.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Cell Culture
RASMCs were isolated from 250-g Sprague-Dawley rats by the explant method.35 The rats were anesthetized with sodium pentobarbital (50 mg/kg body wt). Aortas were taken from just below the aortic arch to the bifurcation by the kidney and were stripped of adventitia and endothelium, cut into 1- to 2-mm pieces, and placed in 60-mm tissue culture dishes (Corning) in DMEM supplemented with 10% FBS for 4 to 5 days. The nearly confluent cells were then split into 100-mm tissue culture dishes and allowed to reach confluence. All subsequent passages were at a 1:3 dilution. For cell treatments, the cells were first growth-arrested by 3 days of culture in DMEM/F-12 supplemented with insulin-transferrin-selenium (catalog number I-1884, Sigma Chemical Co) and then treated with either DMEM with 20% FBS or 10 ng/mL PDGF-BB (R&D Systems Inc) for 24 hours before harvest. The efficacy of the treatments was assessed by measuring thymidine uptake.

Thymidine Uptake
DNA synthesis was assayed using tritiated thymidine uptake. For these experiments, RASMCs at P44 were plated on 24-well plates (Corning) at 2.5x104 cells per well and made quiescent as described above. The cells were then stimulated with either 20% FBS or 10 ng/mL PDGF-BB. After 18 hours of stimulation, the cells were treated to a 6-hour pulse of 2 µCi/mL [methyl,1,2-3H]thymidine (specific activity, 121 Ci/mmol; Amersham). Cells were then washed and solubilized, and radioactivity was determined in a scintillation counter according to the method of Sudhir et al.36

Transfections and CAT Assays
RASMCs (P16, P30, and P44) of three different isolations were transfected using a Ca3(PO4)2 protocol as described previously.37 Briefly, 10 µg of the appropriate DNA was incorporated into a Ca3(PO4)2/HEPES-buffered saline precipitate, incubated for 20 minutes at room temperature, and placed onto 100-mm dishes of RASMCs for 14 to 18 hours. The plates were then washed with fresh media and grown in DMEM with 10% FBS for 48 hours before harvesting. CAT assays were performed and then quantified on a Phosphorimager (Molecular Dynamics) as described previously.37 All transfections and CAT assays were carried out in duplicate with a minimum of two experiments.

Northern Analysis and RNase Protections
Total RNA from cultured RASMCs (P44) and tissue was isolated as described previously.7 Approximately 20 µg of total RNA was electrophoresed through a 1.2% agarose gel containing 0.66 mol/L formaldehyde, transferred to nylon membrane (Zeta Probe, Bio-Rad Laboratories), and hybridized as described previously.7 The probes were as described previously,18 with the exception of the MEF2B probe, which was a 141-nt fragment of the murine MEF2B gene encompassing exon 4 and corresponding to nucleotides 1063 to 1204 of the human cDNA sequence (B. Black, J. Martin, E. Olson, unpublished data, 1994). Equal RNA loading was verified by hybridization to a 157-nt 3' UTR probe to the murine smooth muscle {alpha}-actin gene (data not shown).38 For RNase protection assays, 25 µg of total RNA was hybridized to a 180-nt fragment of the rat MEF2C cDNA encompassing the carboxy terminal and 3' UTR (EcoRI to Nsi I) sites as recommended by the manufacturer (RPA II kit, Ambion). Samples were treated with RNase A and T, separated through a 7 mol/L urea 5% polyacrylamide gel, dried, and exposed to x-ray film (Kodak XAR). All RNase analyses were confirmed by a minimum of two experiments with different isolations of RASMCs. To verify the identity of our cultured RASMCs, RNA protections were performed using nucleotides 60 to 285 of smooth muscle calponin and nucleotides 1921 to 2229 of the 3' UTR of smooth muscle myosin heavy chain probes.39 40

Antibodies
An affinity-purified rabbit antibody raised to a peptide corresponding to codons 487 to 507 of human MEF2A was purchased from Santa Cruz Biotechnology, Inc. This peptide has some homology to the carboxy terminal of MEF2C, and we detected a slight cross-reactivity with in vitro–translated MEF2C after long exposure. The proteins are of easily distinguishable sizes, and this slight cross-reactivity has not been detected in Western blots of smooth muscle lysates. Another rabbit antiserum raised against a peptide corresponding to codons 207 to 223 of human MEF2A was also used in immunohistochemistry to ensure that MEF2A was actually the isoform being detected. This second antiserum does not cross-react with the other MEF2s but does have a higher nonspecific background in Western blots and immunohistochemistry (data not shown). Antibodies to both MEF2A epitopes gave equivalent results, and only the immunohistochemistry with the antibody from Santa Cruz was used in Fig 6ADown. Antibody to MEF2C was raised against an isoform-specific peptide representing codons 300 to 316.25 27 The peptides for MEF2A and MEF2C were conjugated to keyhole limpet hemocyanin with EDC, using a kit from Pierce Chemical Co for inoculation of rabbits. An affinity column for purification of anti-MEF2C antibody was made by first conjugating 20 mg of MEF2C peptide to 50 mg of bovine serum albumin with 250 mg of EDC in 3 mL of 0.1 mol/L MES (pH 4.5) for 2 hours at room temperature. The bovine serum albumin/peptide conjugate was then dialyzed into 0.1 mol/L MOPS (pH 7.5) and linked to 1 mL of an activated gel (Affi-Gel 15, Bio-Rad Laboratories) in 0.1 mol/L MOPS (pH 7.5) at 4°C for 6 hours according to the manufacturer's instructions. Antibody was purified by loading 1 mL of antiserum diluted with 9 mL of 10 mmol/L Tris (pH 7.5) onto a 1-mL affinity column, washing with 20 mL of 10 mmol/L Tris (pH 7.5), and eluting with 10 mL of 100 mmol/L glycine (pH 2.5). The eluate was collected in a tube containing 1 mL of 1 mol/L Tris (pH 8.0). Antibody was concentrated to 1.2 mL and dialyzed in PBS. Purification was monitored by enzyme-linked immunosorbent assay, and nearly all of the MEF2C-specific antibody was eluted by 100 mmol/L glycine (pH 2.5). The rabbit antisera to MEF2B and MEF2D were provided by Ron Prywes (Columbia University). They were prepared against polyhistidine fusion proteins composed of codons 234 to 365 of human MEF2B and codons 292 to 514 of mouse MEF2D, respectively, and have been previously described.41 Only the antibody from Santa Cruz Biotechnology exhibits any cross-reactivity when tested against in vitro–translated proteins (data not shown). Antibody to {alpha}-tubulin was purchased from Sigma (catalog number T-9026).



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  		Figure 6. Detection of MEF2 proteins in the neointima of the rat carotid artery 14 days after balloon injury. Immunohistochemistry with antisera specific to each MEF2 isoform was carried out on paraffin-embedded sections as described in "Materials and Methods." Arrows indicate the inner elastic lamina. Panels are as follows: A, MEF2A; B, MEF2B; C, MEF2C; and D, MEF2D.

Western Blots
For Western blots, equivalent quantities of whole-cell extracts from P44 RASMCs were separated through a 10% SDS-polyacrylamide gel as described previously.42 Proteins were then transferred to nitrocellulose, incubated with one of the MEF2 polyclonal antibodies or {alpha}-tubulin at 1:1000 dilutions, and detected with an enhanced chemiluminescence kit (ECL, Amersham).

Balloon Catheter Injury of Rat Carotid Arteries
Rat carotid arteries subject to balloon catheter injury were prepared by Zivic-Miller Laboratories. Fourteen days after injury, the injured left and the uninjured right carotid arteries were perfusion-fixed in 4% paraformaldehyde and embedded in paraffin, and 6-µm sections were cut and mounted on ProBond slides (Fisher).

In Situ Hybridization
Mouse cDNAs of MEF2A, MEF2C, and MEF2D and the method used to generate riboprobes have been previously described.18 The MEF2B probe is described in the Northern analysis section. The MEF2B plasmid was linearized with EcoRI, and 35S-UTP (Amersham) was incorporated using T7 polymerase to prepare the antisense probe. A rat MEF2C cDNA consisting of codons 157 to 302 cloned into pBSSK was also used. It was linearized with Not I, and antisense transcripts were made with T7 polymerase. Unincorporated nucleotides were separated on a G-50 spin column, and the probes were precipitated. Riboprobes were resuspended in hybridization buffer at 50 000 cpm/µL. In situ hybridizations were performed as previously described.18 Sense strand riboprobes were also used as controls and yielded only low level background hybridizations (data not shown). Exposures were for 10 days.

Immunohistochemistry
Paraffin-embedded sections were deparaffinized and rehydrated as described for in situ hybridizations.18 The sections were then incubated in 3% H2O2 in water for 5 minutes to block endogenous peroxidase activity. Blocking of nonspecific binding was done with a 1-hour incubation in 1.5% goat serum and 1% bovine serum albumin in PBS; after which, the slides were incubated overnight in 1:1000 dilutions of the antiserum to the MEF2A, MEF2B, or MEF2D or 1:250 dilution for the affinity-purified antiserum to MEF2C. Binding of the primary antibody was detected with a biotinylated goat anti-rabbit antibody using the avidin-biotin complex with horseradish peroxidase technique (Vectostain Elite ABC kit, Vector Laboratories). Diaminobenzidine tetrahydrochloride was used as the peroxidase substrate. Preimmune serum was used as a control and produced only a diffuse background level of staining that was greatest in the adventitia and was not nuclear-localized (data not shown).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
MEF2 mRNA and Protein Are Present in Cultured RASMCs
We initiated the present study by examining the expression of MEF2 mRNAs in cultured RASMCs. Northern blots were performed with murine cDNA probes derived from regions unique to each MEF2 transcript. As shown in Fig 1ADown, MEF2A, MEF2B, and MEF2D mRNAs are present in RASMCs. Multiple transcripts for MEF2A, MEF2B, and MEF2D are observed and represent alternate splices within the genes that have been reported previously.24 25 26 27 28 MEF2C mRNA could not be detected by Northern analysis, so we used the more sensitive RNase protection assay to detect its expression. As shown in Fig 1BDown, MEF2C mRNA is present at low levels in these cells. It is important to note that two of the most smooth muscle–specific markers (smooth muscle myosin heavy chain and calponin)43 44 are present in the RASMCs, indicating that they are indeed SMCs (Fig 1BDown).



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  		Figure 1. A, Northern blots showing MEF2 mRNA expression in RASMCs. Total RNA was harvested, and 20 µg was loaded in each lane. Hybridization conditions and probe descriptions are found in "Materials and Methods." Multiple bands show the major alternately spliced transcripts for MEF2A, MEF2B, and MEF2D. Arrows denote the positions of 18S and 28S ribosomal RNAs. B, RNase protection assay showing MEF2C expression in RASMCs. Total RNA (25 µg) was hybridized, digested, and electrophoresed as described in "Materials and Methods." Smooth muscle myosin heavy chain (MHC) and smooth muscle calponin probes confirm the identity of these cells as smooth muscle.

MEF2A expression is partially regulated posttranscriptionally, as its mRNA is found in cell lines in which neither MEF2 protein nor binding activity can be detected.24 Moreover, MEF2A induction by serum stimulation has recently been shown to be translationally regulated in human SMCs.45 To determine if MEF2 proteins are also regulated by serum stimulation in RASMCs, we immunoblotted cell lysates from RASMCs that were either made quiescent by 3 days in serum-free media or treated with 20% FBS or 10 ng/mL PDGF-BB for 24 hours after being made quiescent. Tritiated thymidine uptake was used to determine if the cultures were quiescent and if the treatments induced the expected increase in DNA synthesis. As shown in Fig 2Down, treatment with PDGF-BB and serum resulted in an 8.9- and 19.3-fold increase, respectively, in thymidine uptake. Immunoblots with lysates from these cells were performed against each of the MEF2s. Consistent with the results of Suzuki et al,45 immunoblots showed a slight increase in MEF2A levels in the serum-treated cells compared with the lysates from quiescent cells. In addition, a slight increase from PDGF-BB treatment is seen (Fig 3Down). Interestingly, there is no change in the levels of MEF2B or MEF2D, suggesting that MEF2A may be regulated differently from these MEF2s. MEF2C protein could not be detected in these cells by Western blotting (Fig 3Down).



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  		Figure 2. DNA synthesis as measured by incorporation of tritiated thymidine. RASMCs were made quiescent and treated with serum or PDGF-BB for 18 hours, as stated in "Materials and Methods." Cells were treated with a 6-hour pulse of tritiated thymidine, and incorporation was measured as described in "Materials and Methods." The graph represents the means of six experiments, with the error bars denoting the standard errors of the means.



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  		Figure 3. Immunoblots of RASMCs showing MEF2 expression under various treatments. Cells were treated as described in Fig 2Up. Cells were then harvested, and total cell lysates were electrophoresed, blotted, and reacted with the indicated antibody as described in "Materials and Methods." Numbers at the sides of the immunoblots denote molecular weight standards in kilodaltons. Immunoreactivity to {alpha}-tubulin indicates equal protein loading. High molecular weight bands are immunoreactive material that does not migrate into the separating gel and are observed with preimmune serum and are therefore artifactual (data not shown).

MEF2s in RASMCs Can Activate Transcription
To determine if the MEF2s are transcriptionally active in the RASMCs, we transfected the MEF2 reporter construct (MEF2)4tkCAT25 into three different RASMC isolates and measured CAT activity. In the RASMCs, (MEF2)4tkCAT is expressed at a 5- to 12-fold higher level than tkCAT, which lacks MEF2 sites (Fig 4Down). These data indicate that MEF2 protein in these cells is functional.



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  		Figure 4. MEF2 transcriptional activity in RASMCs. RASMCs were split 24 hours before transfection and were transfected with 10 µg of tkCAT or (MEF2)4tkCAT, and cells were harvested and assayed as described in "Materials and Methods." The graph represents three independent experiments done in duplicate with three independent isolations of cells. Error bars denote standard deviation. Activity is compared relative to the tkCAT of RASMCs, which has been given the value of 1.

MEF2A, MEF2B, and MEF2D mRNA Levels Are Significantly Higher in Neointimal Cells Than in Medial SMCs
Our observations that MEF2 factors are expressed in proliferating SMCs in culture led us to test whether MEF2s were also present in the neointimal cells, which arise as a consequence of deendothelialization in the rat model of restenosis. In situ hybridizations were therefore performed on rat carotid arteries 14 days after balloon catheter injury. As shown in Fig 5Down, these experiments reveal a strong hybridization signal for MEF2A, MEF2B, and MEF2D mRNAs in the neointimal cells closest to the lumen. Expression declined further from the lumen but still remained stronger than in the medial layer. The signal from the medial cells is at or near background levels for MEF2A and MEF2D but appears to still be substantial for MEF2B. MEF2C hybridizations reproducibly produced a very different pattern in which the overall signal is relatively low, and there is not an obvious difference in the levels between the neointima and the media (Fig 5CDown). Similar results were obtained using a rat MEF2C cDNA as a probe (data not shown). From these experiments, we conclude that either the transcription or stability of MEF2A, MEF2B, and MEF2D mRNAs is increased in neointimal SMCs, with the highest levels being in the layer of cells nearest the lumen, which has been shown to be the most proliferative and to express PDGF-A and the PDGF ß-receptor.3 46 We further conclude that the MEF2C mRNA level is low in vascular SMCs of the carotid artery, and there does not appear to be any increase in expression for the cells that populate the neointima. This low level of MEF2C mRNA in the vessels in relation to the other MEF2s is consistent with the relatively low levels in culture (Fig 1BUp).



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  		Figure 5. mRNA expression of the MEF2s in the rat carotid artery 14 days after balloon injury as detected by in situ hybridization. 35S-labeled antisense riboprobes specific to each of the MEF2s were hybridized to sections overnight and were then washed and dipped in photographic emulsion. These were exposed for 10 days before development. The arrows indicate the inner elastic lamina. Panels are as follows: A, MEF2A; B, MEF2B; C, MEF2C; and D, MEF2D.

Proteins for All Four MEF2s Are Detected in the Nuclei of Neointimal SMCs
We next examined the injured vessels for production of MEF2 proteins by immunohistochemistry using the antibodies described for the Western blots. Immunohistochemistry for MEF2A was done with the antiserum used for the Western blots and with a second antiserum raised against a different peptide epitope. This antiserum was used as a control for the MEF2A antisera from Santa Cruz because of its slight cross-reactivity for MEF2C. From the results in Fig 6Up, it can be seen that as with the MEF2 mRNAs, the cells positive for MEF2 protein are almost entirely restricted to the neointima. It should be noted, however, that they represent only a subset of the neointimal cells. For MEF2A, MEF2B, and MEF2D, it is clear from Fig 6Up that most of the cells in the few layers closest to the lumen are positive for MEF2 proteins in their nuclei and that the number of positive cells decreases further from the lumen. There are also a few positive cells in the first layer of the media but not in deeper layers. In addition, although there are proportionally fewer positive cells further from the lumen, many of these have more intense signals than cells closer to the lumen. Surprisingly, the affinity-purified antiserum to a MEF2C peptide clearly detects a nuclear localized antigen in cells of the neointima (Fig 6CUp). The pattern of expression is similar to the patterns exhibited by the other MEF2s, suggesting that the signal is not from an arbitrary cross-reacting antigen. We are confident that this antibody is not cross-reacting with the other MEF2s, as we observed no cross-reactivity of the MEF2C antibody against in vitro–translated MEF2A, MEF2B, or MEF2D in Western analysis, confirming that the antibody is specific for MEF2C (data not shown).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
The importance of the MEF2s in contractile protein gene transcription has been deduced from the analysis of cardiac-specific and skeletal muscle–specific promoters, implying a central role for MEF2s in sarcomeric muscle differentiation.22 23 29 30 33 34 In the present study, we have examined the expression and activity of the MEF2 family of transcription factors in cultured RASMCs and their expression in balloon catheter–injured carotid arteries. In cultured RASMCs, MEF2A, MEF2B, and MEF2D mRNAs and protein are expressed and can activate transcription of an MEF2-responsive promoter. MEF2C mRNA is produced at low levels, and its protein could not be detected. Examination of the in vivo expression of the MEF2 factors in carotid arteries 14 days after injury produces the unexpected result that the highest levels of MEF2A, MEF2B, and MEF2D mRNA and all four MEF2 proteins are observed in the neointima, where levels of injury-induced genes and proliferation are at the highest levels, and not in the media, where contractile proteins are predominantly expressed.46 47 48 49 50 Their expression therefore correlates with the activated smooth muscle phenotype and not the differentiated phenotype. These findings imply that MEF2 function in smooth muscle is different from the function in cardiac and skeletal muscle. They further imply that the maintenance of contractile protein transcription does not require MEF2 expression, as the vast majority of medial SMCs do not express detectable levels of the MEF2 proteins.

Some MEF2s Are Regulated Posttranscriptionally in Neointimal Cells
Interestingly, the MEF2C protein is clearly upregulated in the neointima in a pattern similar to the other MEF2s; however, we could not detect any differences in MEF2C mRNA levels between the media and the neointima. We considered the possibility that we are detecting cross-reactivity between the MEF2C antibody and another MEF2 protein but think that this is unlikely for the following reasons: First, the antibody was raised against a region of MEF2C that is not conserved in the other MEF2s and was affinity-purified with this peptide. Second, we detected no cross-reactivity with the other MEF2s in Western blots of in vitro–translated products (data not shown). Moreover, immunoblots of injured carotid arteries with anti-MEF2C antibody have detected a protein of the same mobility as MEF2C (data not shown). Finally, immunohistochemistry of injured vessels using the MEF2C peptide as a specific competitor has shown that the MEF2C signal is competed away with increasing amounts of the peptide (data not shown). The simplest explanation for the discrepancy between the upregulation of protein but not the mRNA is that MEF2C expression is regulated posttranscriptionally in the neointimal SMCs. Although the evidence is not as strong for the other MEF2s as it is for MEF2C, posttranscriptional regulation in the neointima may also apply to other MEF2s as well. The evidence for this is seen most dramatically with MEF2B, where there is a major difference in distribution between the mRNA and protein (compare Fig 5BUp and Fig 6BUp) in that MEF2B mRNA is intensely expressed at the luminal edge of the neointima, but protein expression shows very little MEF2B in this region. Posttranscriptional regulation is also observed in other systems. For instance, MEF2A mRNA is observed in many cell lines and tissues, but the protein is only produced in differentiated muscle cells.24 It has also recently been reported that serum induction of MEF2A in human SMCs is regulated posttranscriptionally, wherein the MEF2A protein level is increased by serum stimulation but the mRNA level remains unchanged.45 Although mRNA and protein turnover data for the other MEF2s are lacking, these mechanisms are likely to play a role in gene regulation of all four MEF2s.

MEF2 Expression Pattern in the Neointima Resembles Those of Several Other Genes, Suggesting Possible Regulatory Relationships
The role that the MEF2s play in modulation of the smooth muscle phenotype has yet to be determined, and possible upstream regulators of MEF2 expression and downstream targets can only be speculative at this time. One possible target of the MEF2s in the neointima is c-jun, which is upregulated after balloon injury and whose serum induction has recently been shown to be controlled by MEF2D.7 41 In addition, the similarity of the MEF2 expression pattern to those of other genes that are upregulated in the neointima provides some candidates for further investigation. Of these, there are several cytokines and cytokine receptor genes that are upregulated in the neointima and are postulated to be involved in the restenosis process. These include PDGF-A, PDGF-B, basic fibroblast growth factor, transforming growth factor-ß1, thrombin, PDGF receptor ß-subunit, and fibroblast growth factor receptor-1.47 48 49 50 PDGF receptor ß-subunit, the high affinity receptor for PDGF-BB, has an expression pattern very similar to the pattern of the MEF2s in the neointima 14 days after injury.46 Therefore, it is tempting to speculate that this receptor may be involved in the regulation of MEF2s, and we are looking into this possibility. In our culture conditions, treatment with PDGF-BB had only a slight effect on MEF2A protein levels and no effect on the levels of MEF2B or MEF2D. This lack of an effect may not relate to their actual regulation in vivo, because the MEF2s are already being expressed at relatively high levels in cultured RASMCs even in the quiescent state, so further induction may be difficult to produce. In this regard, SMC culture conditions that downregulate MEF2 expression may be useful in defining inductive signals controlling SMC growth.

Another similarity between the expression of MEF2s and other upregulated genes is that they are expressed in only a subset of the neointimal cells. It is well established that vascular SMCs can be very heterogeneous in morphology and in the array of proteins that they produce in the developing vessel and in clonal isolates of cultured cells.51 Whether the MEF2s and these other genes are expressed in the same subset of cells or different cells is an important question with respect to this heterogeneity that has not been examined. Also unknown is whether MEF2 expression represents a temporary state of the cells or is a stable phenotype that will be maintained in culture. In this regard, it is interesting that cell lines that we obtain from explants of normal vessels express the MEF2s, even though the number of cells expressing detectable levels of the MEF2 proteins in a normal medium is low. Therefore, either the MEF2s are induced as an adaptation to culture, or only the small subset of cells producing the MEF2s in the vessel is able to grow in culture and is therefore selected.

SMC Replication and MEF2 Expression
SMC proliferation is an important component of neointimal formation and has been the target of many novel gene therapies for restenosis.52 53 Therefore, we were interested in the relationship between SMC proliferation and MEF2 expression. In other cell types, the relationship between proliferation and MEF2 expression is complex, with no clear general relationship apparent.18 54 We have not examined the relationship between replication and the MEF2 expression in vivo, but our results with cultured RASMCs suggest that there is little or no relationship between replication and the MEF2s. Specifically, treatment of quiescent cells with either 20% serum or the potent mitogen PDGF-BB caused a substantial increase in DNA synthesis (Fig 2Up) but only a slight increase in MEF2A protein and no increase in MEF2B or MEF2D protein levels (Fig 3Up).

In conclusion, contrary to our expectations based on their role in skeletal and cardiac muscle, expression of the MEF2s in smooth muscle is associated with the activated phenotype of neointimal cells. MEF2 expression in the activated neointimal SMCs may make them intriguing targets for gene therapies that could be exploited. For example, MEF2-responsive promoters could be used to restrict transcription of introduced genes to only those neointimal cells in the vessel expressing the MEF2s. Moreover, if the activity of the MEF2s proves to be necessary for the activated smooth muscle phenotype, then they can be targeted directly for inactivation through antisense oligonucleotides, dominant-negative mutants, or transcription factor binding site decoys. Regardless of their eventual utility for therapies, understanding MEF2 function in neointimal SMCs should expand our knowledge of the molecular mechanisms underlying the modulation of smooth muscle phenotypes in vascular disease.


*    Selected Abbreviations and Acronyms
 
EDC = 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride
MEF2 = myocyte enhancer binding factor-2
P (associated = passage number
with number)
PDGF = platelet-derived growth factor
RASMC = rat aortic SMC
SMC = smooth muscle cell
UTR = untranslated region


*    Acknowledgments
 
This study was supported by grants from the National Institutes of Health (NIH), the Muscular Dystrophy Association, and the Robert A. Welch Foundation to Dr Olson. Dr Firulli was supported by a Muscular Dystrophy Association postdoctoral fellowship. Dr Miano was funded by an NIH National Research Service Award postdoctoral fellowship. Dr Casscells was supported by NIH grant R01 HL-50260 and an American Heart Association Grant-in-Aid. Dr Schwarz was funded by a Grant-in-Aid from the American Heart Association, Texas Affiliate, Inc, and NIH grant R01 HL-53223. We would like to acknowledge Cathy Guo, Alishia Tizenor, and Kathy Tucker for excellent technical assistance, Ron Prywes for antibodies to MEF2B and MEF2D, and Tim Scott-Burden and Mark Majesky for many insightful conversations.

Received August 17, 1995; accepted November 22, 1995.


*    References
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*References
 

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