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Am J Physiol Cell Physiol 291: C50-C58, 2006. First published February 8, 2006; doi:10.1152/ajpcell.00524.2005
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VASCULAR BIOLOGY

Serum deprivation results in redifferentiation of human umbilical vascular smooth muscle cells

Mei Han,1 Jin-Kun Wen,1 Bin Zheng,1 Yunhui Cheng,1 and Chunxiang Zhang2

1Department of Biochemistry and Molecular Biology, Hebei Medical University, Shijiazhuang, China; and 2Vascular Biology Center of Excellence and Department of Surgery, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 19 October 2005 ; accepted in final form 3 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Phenotypic change of vascular smooth muscle cells (VSMCs) from a differentiated to a dedifferentiated state accompanies the early stage of atherosclerosis and restenosis. Although much progress has been made in determining the molecular mechanisms involved in VSMC dedifferentiation, research on VSMC redifferentiation is hindered by the lack of an appropriate complete redifferentiation model. We established an in vitro model of redifferentiation by using postconfluent VSMCs from human umbilical artery. We demonstrated that serum-deprived VSMCs are capable of complete redifferentiation. After serum deprivation, postconfluent cultured human umbilical VSMCs became elongated and spindle shaped, with elevation of myofilament density, and reacquired contraction. Expressions of VSMC-specific contractile proteins, such as smooth muscle (SM) {alpha}-actin, SM-myosin heavy chain, calponin, and SM 22{alpha}, were increased and reached the levels in differentiated cells after serum deprivation. To determine the molecular mechanism of the phenotypic reversion, the levels of expression, phosphorylation, and binding activity of serum response factor (SRF), a key phenotypic modulator for VSMCs, were measured. The results showed that SRF binding activity with CArG motif was significantly increased after serum deprivation, whereas no changes were found in SRF expression and phosphorylation. The increased SRF binding activity was accompanied by an increase in expression of its coactivators such as myocardin. Furthermore, the phenotypic reversion was markedly inhibited by decoy double-strand oligodeoxynucleotides containing SM {alpha}-actin CArG motif, which was able to competitively bind to SRF. The results suggested that serum deprivation results in redifferentiation of human umbilical VSMCs. This novel model of VSMC phenotypic reversion should be valuable for research on vascular disease.

phenotype reversion; gene expression; serum response factor

VASCULAR SMOOTH MUSCLE CELLS (VSMCs) can exhibit a wide range of different phenotypes at different stages of development, and even in the adult vessel the cells are not terminally differentiated and are capable of major changes in their phenotypes in response to changes in local environment (19). In response to injury, VSMCs undergo a phenotypic change from a differentiated to a dedifferentiated state, reminiscent of embryonic smooth muscle cells (14, 17). Concomitant with this phenotypic conversion, expression of numerous genes is altered. It is well established that phenotypic change of VSMCs from the differentiated to the dedifferentiated state accompanies their migration and proliferation. Dedifferentiated VSMCs are able to change back into the differentiated phenotype, termed redifferentiation, a common cellular event in the recovery from vascular injury and regression of atherosclerosis in vivo. However, study of the molecular mechanism of VSMC redifferentiation is somewhat hindered by the lack of an appropriate complete redifferentiation model in a cell culture system. Indeed, adult mammalian dedifferentiated VSMCs are resistant to complete redifferentiation; the ability to contract is especially difficult to reacquire, even under serum deprivation conditions (13). To overcome this pitfall, several VSMC clones were selected and some special culture substrates were added to the culture system to retain the ability to reacquire contractibility (1, 12, 13, 24). Obviously, these cell lines and special substrate-cultured VSMCs are capable of complete redifferentiation and suitable for redifferentiation research. However, drawbacks still exist in these in vitro models. For example, they may be less related to in vivo conditions. First, VSMCs may be multiple clones in the vascular wall; second, the special culture substrates are not representative of VSMC living conditions in vivo. When endothelial cells are injured or denuded, the circulating blood serum may contact directly with VSMCs. After endothelial cell recovery, the endothelial cells will separate serum and VSMCs from direct contact, and VSMC phenotypic reversion will then occur. Our serum deprivation model may mimic the above disease recovery and phenotypic reversion in vivo.

Using postconfluent human umbilical arterial VSMCs, we established a model of complete VSMC redifferentiation induced by serum deprivation. We demonstrated that serum-deprived human umbilical arterial VSMCs are capable of complete redifferentiation as shown by changes in histological features, functional characteristics, and expression of VSMC-specific genes. We also found that increased serum response factor (SRF) binding activity onto the VSMC CArG motif, but not the levels of SRF expression and phosphorylation, is associated with the expression of VSMC marker genes and VSMC redifferentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Human umbilical arterial VSMCs from full-term deliveries were grown in medium 199 (M199) supplemented with 10% heat-inactivated FBS, 1 mM nonessential amino acids, 20 mM L-glutamine, 50 mg/ml penicillin, 50 mg/ml streptomycin, and 10 mg/ml neomycin (GIBCO) in a humidified 5% CO2 atmosphere at 37°C. Cells were passaged after reaching confluence with 0.25% trypsin-3 mM EDTA (Sigma). Medium was changed every 3 days. VSMCs were identified by positive immunostaining for smooth muscle (SM) {alpha}-actin. Cells used in experiments were from passages 5–7. Confluent cultures were incubated in M199 containing serum for 3 days to achieve the postconfluent state. Serum was then withdrawn to induce redifferentiation. VSMCs enzymatically isolated directly from human umbilical artery were used as controls of the differentiated phenotype. The study protocol was approved by the medical ethics committee of Hebei Medical University.

Transcription analysis of VSMC marker genes. Total RNA was isolated from human VSMCs with RNA Stat 60 (Tel Test "B"). mRNA of myocardin, myocardin-related transcription factor (MRTF)-A, MRTF-B, and Gax was measured by quantitative RT-PCR normalized to the expression of internal GAPDH mRNA as described in our previous study (29). The specific primer sets for PCR were GAPDH: sense 5'-GAAGGTGAAGGTCGGAGTCA-3' and antisense 5'-GAAGATGGTGATGGGATTTC-3'; myocardin: sense 5'-CTGTGTGGAGTCCTCAGGTCAAACC-3' and antisense 5'-GATGTGTTGCGGGCTCTTCAG-3'; MRTF-A: sense 5'-CGAAGGAGGCGGTTA-3' and antisense 5'-GGACAGCTCCTGCAGTTC3-'; MRTF-B: sense 5'-CCAGACCGCTCTGAGCTTG-3' and antisense 5'-TCCTTGACACTCGAATCCAC-3'; and Gax: sense 5'-AAGACTTCGCAGGATATTAT-3' and antisense 5'-AGGCTATGCCGAGCCGCACT-3'.

For Northern blot analyses (28), 30 µg of total RNA was denatured with 50% formamide, separated by electrophoresis on 1% agarose-formaldehyde gels, and blotted on Hybond-N filters (Amersham). After UV fixation, prehybridization and hybridization were performed for 3 and 16 h, respectively, at 42°C in a solution of 50% formamide, 5x SSC, 5x Denhardt solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA), 0.01% SDS, and 400 µg/ml salmon sperm DNA. The probes were generated by RT-PCR, using mRNA from adult rat aorta or human umbilical artery. The primers used for amplification were SM {alpha}-actin: sense 5'-CCTGAAGTATCCGATAGAAC-3' and antisense 5'-GCCGACTCCATTCCAATGAA-3'; SM-myosin heavy chain (MHC), sense 5'-TGCAGAGACAGCTTCACGAGTATG-3' and antisense 5'-CGATGGTGGACTTGAACTTGGACT-3'; SmLIM: sense 5'-AGAACCGTGTACCACGCTGAA-3' and antisense 5'-ACACCACTACTGAGCATGAAC-3'; h1-calponin: sense 5'-CAAGTTCAGTCTACTCTCT-3' and antisense 5'-CAATAGTGTTCCTGCCTTC-3'; and SM 22{alpha}: sense 5'-GTGGAGTGGATCATAGTGCAGTGT-3' and antisense 5'-TAACTGATGATCTGCCGAGGTCGT-3'. PCR products were sequenced using the dideoxy method to verify that the products completely matched DNA sequences in GenBank.

Synthesis of oligodeoxynucleotides. The sequences of phosphorothioate double-stranded oligodeoxynucleotides (ODNs) containing SM {alpha}-actin CArG motif (–120 bp) (16) and mismatched ODNs used in this study were CArG decoy ODNs (consensus sequences are underlined): 5'-GGGCTGAGGTCCCTATATGGTTGTGT-3', 3'-ACTCCAGGGATATACCAACACAGGGG5'; mismatched decoy ODNs: 5'-GGGCTGAGGTCTCCCGATGGTTGTGT-3', 3'-ACTCCAGAGGGCTACCAACACAGGGG-5'. ODNs were annealed for 2 h while the temperature slowly descended from 80°C to 25°C in a solution containing (mM) 10 Tris·HCl (pH 7.4), 45 NaCl, and 1 EDTA.

Electrophoretic mobility shift assay. Preparation of nuclear extracts was done as previously described (6). 32P-labeled ODNs containing SM {alpha}-actin CArG motif, whose sequence was the same as described in Synthesis of oligodeoxynucleotides, were used as probes and incubated with nuclear extracts (5 µg) for 30 min at room temperature in a volume of 20 µl of binding buffer and 2 µg of poly(dI-dC). Competition experiments were performed with 100-fold molar excess of unlabeled SM {alpha}-actin CArG ODNs. The reaction mixtures were immediately loaded onto a 5% nondenaturing polyacrylamide gel containing 0.5x Tris-borate-EDTA buffer and electrophoresed at 120 V for 2 h. The gel was then autoradiographed. To specifically identify SRF protein in binding complexes, 2 µg of rabbit anti-SRF polyclonal antibody (Santa Cruz Biotechnology) was added to the binding reaction mix and incubated for 30 min at room temperature before the 32P-labeled CArG ODNs were added.

Chromatin immunoprecipitation assay. VSMCs in 100-mm dishes were fixed directly by adding formaldehyde to culture medium and incubating at 37°C for 10 min. The fixed cells were harvested and prepared for immunoprecipitation, using the protocol for chromatin immunoprecipitation (ChIP) assay described previously (15) with minor modifications. A quarter of the sample was precleared with salmon sperm DNA-protein A agarose (Santa Cruz Biotechnology) and subsequently incubated with either 2 µl of anti-SRF antibody (Santa Cruz Biotechnology) or no antibody at 4°C overnight. Chromatin samples were immunoprecipitated with salmon sperm DNA-protein A (Upstate Biotechnology). Immune complexes were eluted and subsequently reverse cross-linked and purified by phenol-chloroform extraction. The supernatant of an immunoprecipitation reaction in the absence of SRF antibody was purified and used as a control to show total input DNA. The PCR of supernatant DNA was carried out with primers from intron 1 regions of the SM-MHC gene and promoter regions of SM {alpha}-actin and c-fos. The sequences of the PCR primers were SM {alpha}-actin CArG: forward 5'-AGCAGAAACAGAGGAATGCAGTGGAAGAGAC-3' and reverse 5'-CCCAGAACTCAAGCCAGTCAGGCTGCATCG-3'; SM-MHC intronic CArG: forward 5'-GGCCAAGCCACCCTGGAGAAACCTGGAC-3' and reverse 5'-CCCAGAACTCAAGCCAGTCAGGCTGCATCG-3'; and c-fos CArG: forward 5'-CCTCCCTCCTTTACACAGGA-3' and reverse 5'-CTCCTGGACCCTCCGCATGT-3'.

Transfection of ODNs. Liposome complexes containing CArG ODNs at 14 µg/ml were prepared, and quiescent cultures of human VSMCs at 70% confluence were transfected with these complexes according to the manufacturer's specifications (Sigma). After transfection, cells were maintained in fresh medium with 10% FBS for 24–48 h to postconfluence and then cultured with serum-free medium.

Western blot analysis. Analysis of protein expression was performed as previously described (30). Equal amounts of protein were separated on 12% SDS-PAGE. Proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Millipore), which were then blocked overnight at 4°C with 5% nonfat dry milk in Tris-buffered saline containing 0.2% Tween 20. Blots were then incubated for 2 h at room temperature with specific antibody at a dilution of 1:500. All of the primary antibodies used for the experiments were purchased from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated anti-mouse, anti-goat, and anti-rabbit IgG (Amersham) were used as secondary antibodies. Blots were developed with enhanced chemiluminescence detection reagents (Boster Biotechnology). Contractile proteins SM {alpha}-actin, SM-MHC, and SM 22{alpha} were detected on the same blot membrane.

Analysis of SRF protein distribution and phosphorylation. VSMC nuclear extracts containing equal amounts of protein were precleared for 30 min with 50 µl of 10% protein A Sepharose. The precleared extracts were centrifuged at 12,000 g for 2 min. The extracts were incubated with antibody to phosphothreonine (Santa Cruz Biotechnology) overnight. Immunocomplexes were washed four times in a buffer containing 50 mM Tris·HCl (pH 7.6), 150 mM NaCl, and 0.1% Triton X-100 and then were subjected to Western blot.

Myofilament density assays. Fluorescence staining for F-actin was used to measure myofilament content changes. After fixation in 3.7% fresh paraformaldehyde in PBS for 15 min, the cells were washed twice with PBS, excess aldehyde was quenched with 50 mM NH4Cl for 15 min, and then the cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min. After treatment with blocking solution (1% BSA and 0.1% Triton X-100 in PBS) for 10 min, the cells were stained with FITC-phalloidin (1 µg/ml) in blocking solution for 20 min in a dark room at room temperature to localize F-actin. Actin was visualized with a CKX 41 fluorescence microscope (Olympus, Tokyo, Japan). The fluorescence intensity of FITC-phalloidin was simultaneously calculated from a view containing >15 cells. The measurements were taken from three fields for each treatment and averaged for a single data point. The excitation and emission wavelengths for FITC-phalloidin were 490 and 525 nm, respectively. Fluorescence intensity for FITC-phalloidin was recorded with Image-Pro Plus software (Medica Cybernetics, Silver Spring, MD).

Analysis of F-actin-to-G-actin ratio. The concentration of F-actin and G-actin in VSMCs was measured as described previously (25). Briefly, each group of VSMCs was homogenized in 200 µl of F-actin stabilization buffer (50 mM PIPES, pH 6.9, 50 mM NaCl, 5 mM MgCl2, 5 mM EGTA, 5% glycerol, 0.1% Triton X-100, 0.1% Nonidet P-40, 0.1% Tween 20, 0.1% beta-mercaptoethanol, 0.0011% antifoam, 1 mM ATP, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 10 µg/ml benzamidine, 500 µg/ml tosyl arginine methyl ester). The supernatants of protein extracts were collected after centrifugation at 15,000 g for 20 min at 4°C. The pellets were resuspended in ice-cold distilled H2O plus 1 µM cytochalasin D and then incubated on ice for 1 h to dissociate F-actin. The resuspended pellets were gently mixed every 15 min. The supernatant of the resuspended pellets was collected after centrifugation. Equal volumes of the first supernatant (G-actin) or second supernatant (F-actin) were subjected to analysis by immunoblot using anti-actin antibody. The amount of F-actin and G-actin was determined by scanning densitometry.

VSMC contractility measurements. Contraction of VSMCs was evaluated after stimulation by carbachol (CCh) at room temperature with a phase-contrast microscope. One milliliter of CCh (100 µM) was added to the cells cultured in M199 with 10% FBS or in serum-free medium, and images were recorded within 30 s after agonist administration. The images were digitized, and the length of individual cells was analyzed with a software package (Image-Pro Express). The extent of contraction was calculated as the ratio of the change of relative length to the initial value of the parameter.

Zymography. Matrix metalloproteinase (MMP)-2 activity in the medium was analyzed by nonreducing SDS-PAGE in 10% gels containing 0.1% (wt/vol) gelatin (8). Samples were denatured at room temperature in an equal volume of 0.25 M Tris·HCl (pH 6.8), 20% glycerol, 2% SDS, and 10 µg/ml bromophenol blue. After electrophoresis, MMP-2 was renatured by incubating the gel at room temperature for 30 min in 2.5% (vol/vol) Triton X-100 and then at 37°C for 18 h in 50 mM Tris·HCl (pH 7.6) containing 0.2 M NaCl, 5 mM CaCl2, and 0.02% Brij 35 (wt/vol). The gels were stained for 90 min with 0.5% Coomassie brilliant blue R250 and destained with 10% acetic acid in 40% methanol. MMP-2 activity was evident as a clear band against the blue background of stained gelatin.

Flow cytometry. VSMCs grown in M199 with 10% FBS or serum-free medium were harvested by trypsinization, fixed, and stained with propidium iodide. Cells demonstrating less than the diploid content of DNA were excluded from the measurement of the percentages of cells in each cell cycle phase. The cell cycle profile was determined with a Becton Dickinson Vantage flow cytometer and Lysis II cell cycle analysis software.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Serum deprivation elicits morphological changes in VSMCs. When cultured in M199 containing 10% FBS, human VSMCs grew exponentially and appeared as spindle-shaped cells that were well spread over the plates. The appearance of dedifferentiated VSMCs was similar to that of primary cultures, although they were somewhat larger. After serum withdrawal, VSMCs dramatically changed to highly elongated cells. The cells in the serum-starved state had less cytoplasmic granularity and a smoother plasma membrane surface than those in a serum-supplemented condition (Fig. 1). These changes were evident after 24 h and were complete by 72 h. In addition to the morphological changes of individual cells, we also observed a change in cell reorganization. At 48 h after serum withdrawal, the elongated VSMCs formed dense, multilayered cell bundles that appeared as well-aligned ridges, resembling the organization of VSMCs in blood vessels in vivo. However, after prolonged culture in 10% FBS or serum-free culture for >5 days, the postconfluent VSMCs began to shed and die.


Figure 1
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  		Fig. 1. Phase-contrast photomicrographs of human umbilical arterial vascular smooth muscle cells (VSMCs). Images show postconfluent human cells cultured with 10% FBS (A) and at 12, 24, 48, and 72 h after serum deprivation (B–E, respectively).

 
Serum deprivation induces expression of VSMC-specific genes. Expression of VSMC marker genes is a key feature of differentiated VSMCs. To determine whether serum deprivation could revert dedifferentiated human VSMCs to the differentiated state, we first assessed the mRNA level of VSMC-specific genes, including contractile proteins and transcription factors, which are regulators of cellular differentiation. As shown in Fig. 2, A and C, the mRNAs of SM {alpha}-actin, SM-MHC, and calponin were present in dedifferentiated VSMCs before serum withdrawal. After serum withdrawal, their expression gradually increased and peaked by 48–72 h. The expression of the SM2 isoform of MHC appeared to increase to a greater extent than that of the SM1 isoform. The ratio of SM2 to SM1 was increased from 5.5- to 14.5-fold after 12–48 h of serum deprivation (Fig. 2C). SM 22{alpha} mRNA could be detected after 24 h of serum deprivation and maintained a higher level thereafter. Expression of SmLIM, which could not be detected in postconfluent VSMCs growing in 10% FBS, peaked at 24–48 h after serum deprivation and declined thereafter. By quantitative PCR, Gax expression could not be detected before serum withdrawal but was remarkably induced after serum withdrawal. The recently discovered myocardin and MRTF-A and -B, which act as SRF coactivators, revealed an increase in expression at 12–24 h after serum deprivation and then made a quick recovery (Fig. 2B). Second, the level of contractile proteins was tested by Western blot. The results showed that the level of SM {alpha}-actin, SM-MHC, and SM 22{alpha} in the redifferentiated VSMCs cultured without serum was increased and was close to that in the differentiated VSMCs enzymatically isolated from human umbilical artery (Fig. 2, D and E). However, VSMCs cultured with serum had decreased expression of these contractile proteins. Together, these results suggest that serum deprivation can induce an increase in expression of VSMC-specific genes and achieve contractile protein profiles similar to differentiated VSMCs.


Figure 2
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  		Fig. 2. Expression of VSMC differentiation marker genes in human VSMCs after serum deprivation at 0, 12, 24, 48, and 72 h. A: Northern blot analysis showing the expression of smooth muscle (SM) {alpha}-actin, SM-myosin heavy chain (MHC), calponin, SM 22{alpha}, and SmLIM. Probe for SM-MHC detected both SM1 and SM2 isoforms. 28S rRNA was used as a control for equal loading of RNA. B: quantitative RT-PCR analysis of expression of myocardin, myocardin-related transcription factor (MRTF)-A, MRTF-B, Gax, and GAPDH in human VSMCs before and after serum deprivation. C: semiquantitative results of gene expression. D: Western blot of contractile proteins in VSMC cultured with or without serum for 48 h (lanes 1 and 2) and in differentiated VSMCs enzymatically isolated from human umbilical artery (lane 3). All experiments were repeated 3 times.

 
Increase in SRF binding activity is involved in expression of VSMC-specific genes induced by serum deprivation. SRF activates VSMC-specific genes through the CArG element, which is located in regulatory regions of all known VSMC differentiation marker genes, such as SM {alpha}-actin, SM-MHC, calponin, and SM 22{alpha} (20). To explore the molecular mechanism of VSMC-specific gene expression induced by serum deprivation, SRF binding activity was detected by EMSA. The 32P-labeled 30-bp oligonucleotide probe containing SM {alpha}-actin CArG motif, designated as SRF binding sequence, was incubated with nuclear extracts from VSMCs before and after serum withdrawal, and then the mixtures were subjected to EMSA. As shown in Fig. 3A, a shifted band of DNA-protein complex was formed after 24–48 h of serum deprivation. The band was completely abolished by adding 100-fold molar excess of mismatched probe. Antibody to SRF produced a significant supershift of the complex, indicating that SRF was activated and bound to the CArG element after serum deprivation. However, the binding activity of SRF from VSMCs cultured in 10% FBS was undetected in this assay, which was in accord with the lower level of contractile proteins and their mRNA in the dedifferentiated VSMCs.


Figure 3
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  		Fig. 3. Serum response factor (SRF)-CArG binding activity, but not protein and phosphorylation levels, increased in serum-deprived VSMC. A: EMSA for binding of VSMC nuclear proteins to the CArG element from SM{alpha}-actin promoter. Nuclear extracts were prepared from human postconfluent VSMCs cultured with 10% FBS (lane 2) or after serum deprivation for 12 (lane 3), 24 (lane 4), 48 (lane 5), and 72 (lane 6) h. Nuclear extracts (5 µg) were incubated with 32P-labeled CArG oligodeoxynucleotides (ODNs) from SM {alpha}-actin gene promoter (lanes 2–6). Lane 7 shows the supershifted band with antibody to SRF. Self-competitions at 100 molar excess are displayed in lane 8. Lane 1 represents free probes. B: chromatin immunoprecipitation analysis of SRF binding to the endogenous CArG regions: c-fos (a), SM {alpha}-actin (b), and SM-MHC (c). PCR was carried out to detect the endogenous CArG regions in immunoprecipitated chromatin fragments. Lanes 1 and 2 show amplification of 1:100 dilution samples of total input DNA for immunoprecipitation. Lanes 3 and 4 show amplification of target sequences in immunoprecipitated chromatin fragments with anti-SRF antibody (Ab). Lanes 5 and 6 show PCR amplification of control precipitation samples with no Ab. C: Western blot showing expression of SRF protein in VSMCs before (0 h) and after serum withdrawal (12, 24, 48, and 72 h). Nuclear and cytoplasmic extracts were resolved on SDS-polyacrylamide gel, and Western blot analysis was performed with anti-SRF polyclonal antibody. D: threonine phosphorylation of SRF immunoprecipitates before (0 h) and after (48 h) serum withdrawal.

 
To test whether the increase in SRF binding to CArG elements in VSMC-specific genes is associated with the level of SRF expression and/or the level of SRF phosphorylation, cytoplasmic and nuclear extracts were made from VSMCs before and after serum deprivation. Western blot analysis identified three SRF species: 67, 62, and 48 kDa. Only a 67-kDa band of SRF was detected in cytoplasm; however, all three SRF species were found in nuclear extracts (Fig. 3C). Unexpectedly, both cytoplasmic and nuclear SRF levels were unchanged after serum deprivation (Fig. 3C). In addition, no changes were found in the level of SRF phosphorylation before and after serum deprivation (Fig. 3D).

Although the above results suggested that SRF bound to CArG element in vitro, it is unknown whether SRF is able to bind CArG elements of the endogenous VSMC-specific differentiation marker genes because of the closed state of its nucleosomal target sites. To directly test this, we performed ChIP assays, which detect binding of SRF to target sites in chromatin in living cells. The cells were treated with formaldehyde before and after serum withdrawal, and cross-linked chromatin was subjected to ChIP with anti-SRF antibody. Neither SM {alpha}-actin nor SM-MHC CArG regions were amplified from anti-SRF chromatin immunoprecipitates derived from serum-cultured VSMCs (Fig. 3B, b, c, lane 3), whereas the c-fos promoter, which has been shown to be constitutively occupied by SRF in cells (7), was highly enriched in the anti-SRF chromatin immunoprecipitates from serum-cultured VSMCs (Fig. 3B, a, lane 3). In contrast, both SM {alpha}-actin and SM-MHC intron CArG regions were enriched in immunoprecipitates from serum-deprived VSMCs (Fig. 3B, lane 4). ChIP assays provided clear evidence showing that redifferentiation of VSMCs is associated with increased SRF binding to CArG elements of VSMC-specific genes within intact chromatin. These results did not conflict with the increase in SRF binding to VSMC-specific CArG in EMSA, suggesting that serum deprivation results in a shift of SRF from the regulatory elements of proliferation genes to those of differentiation genes in the absence of any detectable change in SRF expression and phosphorylation.

To further confirm that increased SRF binding activity onto the CArG element is necessary for contractile protein expression induced by serum deprivation, the CArG decoy ODNs, which are able to competitively bind to endogenous SRF, were transfected into VSMCs. As shown in Fig. 4, transfection of CArG decoy ODNs resulted in marked attenuation of transcription of VSMC-specific genes SM1/SM2, SM {alpha}-actin, calponin, and SM 22{alpha}. Thus an increase in SRF binding activity may be the mechanism promoting VSMC marker expression and redifferentiation.


Figure 4
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  		Fig. 4. Effect of CArG decoy element ODNs on expression of contractile protein genes induced by serum deprivation. (-), Untreated; (+), treated with CArG decoy ODNs. Thirty micrograms of total RNA was subjected to Northern blotting. 28S rRNA was used as control for equal loading. The experiment was repeated 3 times.

 
Change of myofilament in serum-deprived VSMCs. After serum deprivation for 48 h, the number and density of myofilaments in VSMCs were close to those found in the differentiated VSMCs in primary culture. Although the myofilament structure could not fully return to the differentiated state, contraction occurred. After transfecting CArG decoy ODNs, myofilament formation induced by serum starvation was disrupted, the number of myofilaments was reduced, and the arrangement of myofilaments became loose. Quantitative analysis of myofilament density also confirmed that serum deprivation resulted in an increase in the FITC-phalloidin staining intensity of F-actin compared with that in the VSMCs cultured in 10% FBS (Fig. 5A). F-actin was arranged as stress fibers in serum-free postconfluent VSMCs. We further determined the effect of serum withdrawal on actin polymerization by assessing the effects of serum withdrawal on the F-actin-to-G-actin ratio in VSMCs. VSMCs cultured in 10% FBS were deprived for 0, 24, 48, and 72 h for the analysis of F-actin and G-actin. The ratio of F-actin to G-actin significantly increased after serum withdrawal (Fig. 5B). These results indicated that serum deprivation led to actin polymerization and myofilament bundling. Interestingly, although the subconfluent cells were able to express SM {alpha}-actin under serum-free conditions, F-actin was arranged as a multilateral distribution, and VSMCs were unable to contract after agonist stimulation (results not shown).


Figure 5
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  		Fig. 5. Effect of serum deprivation on actin polymerization in VSMC. A: fluorescence staining and fluorescence intensity analysis of F-actin fibers. Cells were deprived for 0 and 48 h. F-actin fibers were stained with FITC-phalloidin. *P < 0.05 vs. VSMCs in the presence of 10% FBS (0 h). B: effects of serum withdrawal on F-actin-to-G-actin ratio in VSMC. Cells were deprived for 0, 24, 48, and 72 h. The relative concentrations of F-actin and G-actin were determined with anti-actin Ab by Western blot. F-actin/G-actin in serum-free VSMCs is significantly different from serum-cultured VSMCs (n = 3, *P < 0.05).

 
Serum-deprived VSMCs attain contractile function. The ability to contract in response to a physiological stimulus is the only unequivocal criterion for a differentiated VSMC phenotype. To determine whether expression of VSMC-specific genes is parallel with cell contraction, cells in M199 either with or without serum were stimulated with agonists. Administration of CCh (100 µM) to postconfluent VSMCs with serum withdrawal caused significant reduction in cell length. Maximal shortening of cell length was observed 48 h after serum withdrawal. However, no cell length alteration was observed in VSMCs in the serum-cultured condition or in serum-deprived subconfluent cells (Fig. 6, A–D). Quantitative analysis of cell shortening confirmed these observations. The changes in cell length normalized to resting length after agonists were added to the medium are depicted in Fig. 6E. The postconfluent cells cultured in 10% FBS did not undergo any marked change in length after agonist stimulation. In contrast, the cells grown in serum-free medium shortened 28% in response to CCh (P < 0.05). Similar contractile response was also found in these cells stimulated with another agonist, ACh (100 µM; data not shown).


Figure 6
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  		Fig. 6. Phase-contrast photomicrographs of agonist-induced contraction of human VSMCs. VSMCs cultured in 10% FBS (A) showed no cell length change with carbachol stimulation (100 µmol/l; B). After serum deprivation for 48 h (C), VSMC showed cell length shortening with carbachol stimulation (D). The comparison of relative length of VSMCs is shown in E. *P < 0.05 vs. VSMCs in the presence of 10% FBS (0 h).

 
Serum withdrawal is associated with growth arrest and decreased degradation of extracellular matrix. Lower growth rate and extracellular matrix turnover rate are another fundamental characteristic of differentiated, contractile VSMCs in the vessel wall. VSMC redifferentiation induced by serum deprivation could be the result of growth arrest. To discriminate this possibility, we examined the alteration of the cell cycle in response to serum deprivation by flow cytometry: 74.31% of serum-cultured cells were distributed in G0/G1 phase (Fig. 7A); after serum deprivation for 24 h, the percentage of cells in G0/G1 phase increased to 92.75%.


Figure 7
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  		Fig. 7. Effect of serum deprivation on cell cycle of human VSMCs. Cell cycle analysis for subconfluent VSMCs cultured in 10% FBS (a) and postconfluent VSMCs treated by serum deprivation for 0, 1, 2, 3, 4, and 5 days (b–g, respectively) is shown. Serum deprivation was associated with a decrease in S phase and a corresponding increase in cells in G0/G1 phase (n = 3).

 
To investigate whether serum deprivation affects degradation of extracellular matrix, both MMP-2 and tissue inhibitor of metalloproteinase (TIMP)-2 expression and activity were determined. As shown in the gelatin zymography in Fig. 8A, a major 70-kDa band of MMP-2, which is the activated form of MMP-2, was detected in VSMCs cultured with or without serum. In addition, there was a gradual reduction of MMP-2 activity in prolonged serum-deprived VSMCs. Western blot with monoclonal antibody to MMP-2 also demonstrated a gradual reduction of 68-kDa and 70-kDa bands of MMP-2 in prolonged periods of serum starvation. By Western blot, pro-MMP-2 was revealed in medium of serum-deprived VSMCs and was gradually increased. TIMP-2 expression was also detected by Western blot after 3 days of serum deprivation. Altogether, serum deprivation has little effect on the level of total MMP-2 (including 72, 70, and 68 kDa) but inhibits conversion of 72-kDa MMP-2 into 70- and 68-kDa active forms. Serum stimulation is necessary for MMP-2 activation.


Figure 8
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  		Fig. 8. Effect of serum deprivation on matrix metalloproteinase (MMP)-2 and tissue inhibitor of metalloproteinase (TIMP)-2 levels in medium of VSMCs. A: zymograph of MMP-2. B: Western blot of pro-MMP-2, MMP-2, and TIMP-2. The medium of cells was collected after serum deprivation for 0, 1, 2, 3, 4, and 5 days (lanes 1–6). All experiments were repeated 3 times.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
A number of clonal VSMC lines have been developed over the past several years in an effort to generate long-lived differentiated VSMCs. These include a cell line derived from rat embryonic thoracic aorta (10), a large T-transformed rat aortic SM cell (SMC) line (21), a rat pulmonary artery SMC line (22), and a human internal thoracic artery SMC clone (20). However, none of these VSMC lines has been shown to display complete phenotypic reversion properties, and the mechanism of regulatory phenotype is unclear, although they can be induced to express some VSMC-specific markers and have the ability to contract. In our study, we established an in vitro phenotype reversion system using human umbilical arterial VSMCs. Our model showed that postconfluent VSMCs rather than subconfluent cultures in serum-free medium reacquire characteristics of the contractile phenotype. These characteristics included growth arrest, morphological change, increase in F-actin-to-G-actin ratio, contractile protein profiles similar to differentiated phenotype, and contraction by agonist induction. We also demonstrated that VSMC redifferentiation induced by serum deprivation was meditated by increased SRF binding activity to CArG.

The expression of contractile proteins, including SM {alpha}-actin, SM-MHC, and SM 22{alpha}, is the molecular basis of VSMC redifferentiation in response to agonists and attainment of contraction. Our study showed that VSMC contraction was consistent with increased expression of contractile proteins induced by serum withdrawal. The mRNA expression of SM {alpha}-actin, SM 22{alpha}, and calponin and the ratio of MHC SM2 to SM1 (from 5.5:1 to 14.5:1) gradually increased in serum-deprived VSMCs. Meanwhile, increased SM {alpha}-actin was mainly distributed in the F-actin fraction, which correlated closely with increasing contractility of the VSMCs. We observed maximal VSMC shortening and myofilament formation when VSMC-specific gene expression peaked and the contractile proteins reached the profiles of differentiated VSMCs at 48 h after serum withdrawal. Hence, increases in VSMC-specific gene expression provide the molecular basis of functional and morphological changes during VSMC redifferentiated in our model of phenotype reversion.

The CArG cis-acting element, which contains the CC(A/T)6 GG consensus sequences, exists in the regulatory regions of almost all known VSMC marker genes. Although mechanisms of SRF regulating SMC-specific genes in embryonic development have been proposed, including SRF level, SRF phosphorylation modification, and redistribution, SRF binding activity plays a key role in regulation of VSMC marker genes (20). The CArG-SRF interaction is critical for tissue-specific gene expression of VSMC markers. To determine whether CArG-SRF activation is involved in the expression of VSMC-specific genes induced by serum withdrawal, we first tested the binding activity of nuclear extracts from VSMCs with CArG element by EMSA. Our results showed that the binding of ODNs containing SM {alpha}-actin CArG motif to VSMC nuclear extracts was increased after serum withdrawal. This result was consistent with the activation of VSMC-specific genes. ChIP assay further demonstrated that SRF binding to the CArG elements of VSMC-specific genes was increased within intact chromatin in serum-deprived cells. Involvement of SRF binding to CArG element was further confirmed by the result of a decrease in VSMC-specific gene expression and loose myofilaments in cells transfected by CArG decoy ODNs. In contrast, decreased CArG binding activity was found in nuclear extracts from serum-cultured VSMCs by EMSA. As for the undetected SRF binding to CArG of contractile protein genes in serum-cultured cells, we thought that the binding activity of SRF was so lowered as to be unable to be tested under these experimental conditions. However, SM {alpha}-actin was expressed in serum-cultured cells, suggesting that these cells retain a VSMC nature. SRF expression has been found to correlate closely with VSMC marker expression during development (20). Induction of SRF expression is a prerequisite for CArG-dependent gene expression in VSMCs. To explore whether levels of SRF expression and phosphorylation reflect the binding activity of nuclear extracts from VSMCs, we tested SRF expression, distribution in cytoplasmic/nuclear extracts, and phosphorylation by Western blot. However, although VSMC marker expression was increased, SRF protein expression and phosphorylation did not vary in redifferentiation of VSMCs induced by serum deprivation. Translocation of SRF from the cytoplasm into the nucleus was not increased with increased SRF binding to CArG element of VSMC marker genes. One mechanism to explain the increased expression of VSMC-specific genes in serum deprivation may simply be a mechanism involving redistribution of SRF within the nuclear compartment from growth-related loci to differentiation-related loci (16). Our results were different from reports from Nemenoff's group (9), which showed that PDGF stimulation of VSMCs reduced SRF binding to SM-specific CArG box, accompanying partial redistribution of SRF from the nucleus into the cytoplasm. One possibility is that the serum-containing component is complex; FBS stimulation could result in a complex set of positive and negative interactions resulting in no net cytoplasmic-nuclear redistribution. Similarly, PDGF treatment of quiescent VSMCs caused a marked decrease in SM {alpha}-actin mRNA in cultured aortic SMCs, whereas FBS did not (5). In addition, subconfluent and long-term serum-deprived (>7 days) canine tracheal SMCs exhibited reduction in nuclear SRF protein and nuclear SRF binding to consensus CArG sequences through extranuclear redistribution of SRF (2). These findings differed from those in human umbilical VSMCs in the present study. We thought that various factors, including species- and tissue-specific SMC, SMC growth density, and serum deprivation time, resulted in this difference between these studies. Both postconfluence and short-term serum deprivation (<5 days) were necessary for human umbilical VSMC redifferentiation and reacquisition of contraction in our study. The study found that there was an increase in SRF binding to CArG of SM {alpha}-actin or SM-MHC and a decrease in c-fos CArG-SRF interaction in VSMCs cultured in serum withdrawal by ChIP assay. Indeed, there may be some transcriptional factors, involving GATA and NK families, influencing SRF binding to the CArG element (18). These factors may also control the SRF redistribution in the nuclear compartment and SRF recruitment to SMC differentiation genes during serum deprivation. Recently, evidence has been provided for alterations in SRF localization and DNA binding after balloon injury at a time when SMC differentiation markers begin to decrease (11). These findings suggest that redifferentiation programs in VSMC phenotype remodeling are different from differentiation during VSMC development.

Coactivation of transcription factors may also play an important role in directing VSMC differentiation. Myocardin, as a SRF coactivator, stimulates SRF by forming a ternary complex with SRF on DNA and providing its strong transcriptional activation domain to SRF. Two additional members of the myocardin family, referred to as MRTF-A and MRTF-B, are involved in activation of endogenous SMC genes. Myocardin, MRTF-A, and MRTF-B are necessary and sufficient for SMC differentiation (19). We found that the expression peak of three SRF coactivators, as well as Gax (27) and SmLIM (4), which are developmentally regulated and preferentially expressed in quiescent VSMCs, was earlier than that of SMC markers such as SM {alpha}-actin, SM-MHC, and SM 22{alpha}, suggesting that these coactivators may be required for SRF-dependent activation of SMC genes.

Serum withdrawal resulted in not only a growth arrest but also a decline in rate of extracellular matrix turnover. Cultured VSMCs secrete MMPs, such as MMP-2, which is a factor necessary to degrade extracellular matrix complex. Formation of MMP-2 and TIMP-2 complex inhibits the activity of MMP-2. The balance between MMP-2 and TIMP-2 controls the level of net extracellular matrix proteolysis. In our study, we found that serum withdrawal resulted in reduction of MMP-2 expression and activity. Conversion of pro-MMP-2 into MMP-2 was markedly declined in medium from serum-deprived VSMCs. Pro-MMP-2 was not detected in medium of serum-cultured VSMCs; however, there was a significant increase in pro-MMP-2 in medium of serum-free VSMCs. Induction of TIMP-2 expression was observed after 3 days of serum deprivation. It has been reported that plasmin and thrombin contribute to conversion of pro-MMP-2 to MMP-2 and MMP-2 activation (26). Therefore, although pro-MMP-2 can be released by human VSMCs cultured without serum, MMP-2 activation is promoted only in the medium of serum-cultured VSMCs and not that of serum-deprived VSMCs. This might be one of the mechanisms by which VSMCs maintain a contractile phenotype in normal vessel wall. These data suggest that serum withdrawal preferentially inhibits MMP-2 activation through decline in conversion of pro-MMP-2 into MMP-2 and increase in the level of TIMP-2. We speculate that some factors that exist in serum may be necessary for the MMP-2 activation. MMP-2, which is the major MMP in blood vessels, regulates the extracellular matrix microenvironment and affects cellular signaling and functions by pericellular proteolysis. A decrease in proteolysis by MMP-2 after serum deprivation may block bioavailable growth factors and provide a relatively quiescent microenvironment for VSMC reversion to contractile phenotype.

The present study also adds to our understanding of VSMC multiphenotype and multifunctionality by establishing a VSMC reversion model from the synthetic to the contractile state that is a hallmark of some VSMCs. Several studies have shown that VSMC dedifferentiation obtained after passaging cannot revert to a differentiated phenotype, even under quiescent culture conditions (3, 23). It is thus possible that the experimental conditions in culture might erroneously be adopted to induce redifferentiation.

In summary, we have demonstrated for the first time that serum-deprived human umbilical VSMCs are capable of spontaneous redifferentiation. The morphological and biological properties of this novel in vitro model were characterized. Increase in SRF binding activity without increased SRF expression and phosphorylation was involved in promoting VSMC marker expression in the redifferentiation in this model. Our study may provide further insight into the molecular mechanisms underlying the development of vascular diseases.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-72902, American Heart Association Grant 0530106N, and American Diabetes Association Grant 105JF60 (to C. Zhang). This work was also supported by a grant from the National Natural Science Foundation of the People's Republic of China (No. 30270499; 90208014; 30472167; 2005CCA03100) and a grant from Hebei Province Natural Science Foundation of the People's Republic of China (No. 303454; 303455; C2005000722). J.-K. Wen was supported by a grant from the Major State Basic Research Development Program of the People's Republic of China (G2000056905). M. Han was supported by a grant from the National Plan Special Research Programs of the People's Republic of China (No. 2002BA755C).


   ACKNOWLEDGMENTS
 
The authors thank Dr. David Armbruster from the University of Tennessee Health Center for editing assistance.


   FOOTNOTES
 

J.-K. Wen, Dept. of Biochemistry and Molecular Biology, Hebei Medical Univ., No. 361, Zhongshan East Road, Shijiazhuang, 050017, China (e-mail: wjk{at}hebmu.edu.cn)


Address for reprint requests and other correspondence: C. Zhang, Vascular Biology Center of Excellence and Dept. of Surgery, Coll. of Medicine, Univ. of Tennessee Health Science Center, 956 Court Ave., Coleman Bldg., H300, Memphis, TN 38163 (e-mail: czhang{at}utmem.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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