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Concerted effect of transforming growth factor-, cyclin inhibitor p21, and c-myc on smooth muscle cell proliferation

Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Submitted 20 May 2003 ; accepted in final form 4 November 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Increased aortic smooth muscle cell (SMC) proliferation is a key event in the pathogenesis of atherosclerosis. Transforming growth factor- (TGF-) is one of the potent inhibitors of SMC proliferation. The purpose of this study was 1) to explore the effect of TGF- inhibition on proliferation of SMC and expression of growth regulatory molecules like p21 and c-myc and 2) to determine whether restoration of cell cycle regulatory molecules normalizes the altered proliferation. To test the role of TGF- in SMC proliferation, using antisense plasmid DNA, we inhibited TGF- gene from aortic SMC, which resulted in a significant increase (P < 0.03) in proliferation (studied by quantifying new DNA synthesis with [³H]thymidine uptake assay). In TGF--altered SMC (TASMC), the mRNA expression (studied by RT-PCR) of c-myc was increased whereas that of the cyclin inhibitor p21 was completely inhibited. Using p21 sense plasmid DNA, we transfected p21 gene in TASMC, which restored p21 mRNA and protein expression and decreased proliferation (P < 0.002) in TASMC. Similar treatment with c-myc antisense oligonucleotides significantly (P < 0.001) decreased the proliferation of TASMC. TASMC also exhibited alteration in morphological changes in SMC but returned to normal with treatment of p21 and TGF- sense plasmid DNA. Two-dimensional gel electrophoresis analysis of SMC and TASMC demonstrated differential expression of proteins relevant to cellular proliferation and atherosclerosis. This study uniquely analyzes the effect of TGF- at the molecular level on proliferation of SMC and on cell cycle regulatory molecules, implicating their potential role in the pathogenesis of atherosclerosis.

atherosclerosis; cancer; gene overexpression; reverse transcriptase-polymerase chain reaction; antisense plasmid DNA; antisense oligonucleotide

In this study, we explored the effect of TGF- and related molecules in the process of VSMC proliferation. Using the anti-TGF- antisense plasmid DNA RLDN-AS, we successfully inhibited TGF- gene from A549 cells (human lung adenocarcinoma cells; Ref. 29). With the same method, we inhibited TGF- gene from aortic SMC and observed that the inhibition of TGF- gene altered new DNA synthesis (proliferation), expression of p21, c-myc, and inducible nitric oxide synthase (iNOS) and other TGF--regulated genes in these cells. Proteomic analysis also demonstrated differential expression of proteins relevant to cellular proliferation in these cells. With molecular manipulation techniques, we studied the interaction of these growth-regulating molecules in the proliferation of aortic SMC proliferation, which might impact the pathogenesis of atherosclerosis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Construction of aortic SMC lacking TGF- gene expression. With our study design described previously (29), SMC (obtained from Cambrex, East Rutherford, NJ) were cultured and transfected with TGF- antisense plasmid DNA. After 5 h of transfection, the cells were washed and allowed to grow in serum-containing medium for 24 h. These cells, which were between four and six passages, were then trypsinized and washed. Transfected cells were treated with 400 µg/ml of G418 and grown for 2–3 days. The cells that took up TGF- antisense DNA survived, whereas the others died. After propagating the survivors, we studied proliferation and gene expression for TGF-, p21, and c-myc by RT-PCR. The efficiency of transfection was also determined by RT-PCR of cells from individual wells. Aortic SMC with no detectable TGF- mRNA were called TGF--altered SMC (TASMC). Untransfected cells and cells transfected with empty plasmid DNA were used as controls.

Effect of gene alteration on cellular proliferation. The effect of the altered expression of different genes on cellular proliferation was studied by [³H]thymidine uptake assay (28). SMC and TASMC were grown and maintained in Opti-MEM (GIBCO, Long Island, NY) containing 5% fetal bovine serum at 37°C in a 5% CO₂-95% air atmosphere. After 72 h of culture, cells were trypsinized, washed with medium, and counted as described previously (28). Briefly, each cell type (100,000 cells) was added to each well of a 24-well plate (Primaria) and allowed to grow for 24 h at 37°C in a 5% CO₂-95% air atmosphere in the presence of [³H]thymidine during the last 4 h. The cells were washed twice with cold PBS, fixed with cold methanol (95%), and then washed once again with cold PBS. NaOH (0.2 M; 0.5 ml) was added to each well, and plates were stored at 4°C for 30 min. The contents of each well were transferred to liquid scintillation vials, and [³H]thymidine uptake was quantified as counts per minute with a liquid scintillation counter.

PCR. Total RNA was isolated from SMC, TASMC, and SMC transfected with control plasmid DNA as described previously (29), and the quality of RNA was verified by 260- to 280-nm ratio. One microgram of RNA was reverse-transcribed to cDNA with Ready-To-Go You-Prime first-strand beads and oligo(dT) primers (Pharmacia Biotech). Amplification by PCR was carried out with 10 µl of cDNA, Ready-To-Go PCR beads (Pharmacia Biotech), and 2 µl each of coding and noncoding oligonucleotide primers (2.5 µM). The primer sequences for TGF-, p21, and -actin were described previously (29) for c-myc: coding 5'-AAGGACTATCCTGCTGGCAA-3' and noncoding 5'-GGCCTTTTCATTGTTTTCCA-3' (4). The PCR amplification profile consisted of 95°C for 45 s, 60° for 45 s, and 72°C for 75 s. The amplification of -actin and other genes were achieved with 27 and 35 cycles, respectively. The PCR products were resolved in 1% agarose gel electrophoresis; ethidium bromide-stained specific bands were visualized under ultraviolet light and photographed. Quantitative analysis was not performed because amplification up to 45 cycles resulted in undetectable mRNA expression for these genes, except for -actin and c-myc.

Reexpression of p21 gene in TASMC. Using the p21 sense plasmid DNA described in our earlier study (30), we reintroduced p21 in TASMC. DNA from p21/pcDNA3-Zeo(+) plasmids was transfected into TASMC. After 5 h of transfection, cells were washed and allowed to grow in serum-containing medium for 24 h. Cells were treated with Zeocin (600 µg/ml) for 2–3 days, and the surviving cells were propagated. RT-PCR and Western blot analysis were used to confirm the expression of p21 after transfection with p21 sense plasmid DNA. The cells now expressing p21 genes were identified and used for the proliferation assay. Untransfected TASMC and TASMC transfected with the empty vector pcDNA3-Zeo(+) were used as controls.

Effect of c-myc antisense oligonucleotides on proliferation of TASMC. We attempted to inhibit the overexpressed c-myc gene in TASMC by using an antisense oligonucleotide. The sequence of the oligonucleotide is 5'-ACG TTG AGG GGC ATC GTC GC-3'. This is a 20-mer antisense oligonucleotide capable of inhibiting c-myc expression modified both at the 5' and 3' ends to make it stable and resistant against nuclease digestion. TASMC were plated into a six-well plate and transfected with 10 µM antisense oligonucleotide. After 5 h of transfection, cells were washed and allowed to grow in serum-containing medium for 24 h and proliferation was quantified as described previously (28).

Measurement of TGF- protein in culture supernatants. TGF- protein in the conditioned medium of SMC and TASMC was measured as previously described (28). TGF-1 protein was assayed on acid-activated samples by sandwich ELISA with a TGF-1-specific kit purchased from Promega (Madison, WI).

Morphology studies. SMC and TASMC transfected with p21, TGF- sense plasmid DNA, empty vector plasmid DNA, c-myc antisense oligonucleotides, and scrambled oligonucleotides were cultured overnight in six-well plates as described in our earlier study (31) and were washed with PBS and photographed with a Cohn high-performance charge-coupled device camera.

Differential expression of proteins in SMC and TASMC. We performed proteomic analysis of proteins in SMC and TASMC to identify proteins relevant to SMC proliferation and their relationship with TGF- expression. Differential expression of proteins in SMC and TASMC was studied by proteomic analysis with two-dimensional (2D) gel electrophoresis and sequencing.

Preparation of lysates and 2D gel electrophoresis of proteins. Twenty to one hundred micrograms of protein were solubilized in 2D sample buffer (8 M urea, 2% Nonidet P-40, 2% ampholytes, 100 mM dithiothreitol, 0.1% SDS, 12.5 mM Tris, pH 8.0). For separation of proteins based on their isoelectric pH in the first dimension, protein mixtures were applied to immobilized pH gradient (IPG) strips of defined pH range 3–10. First-dimension separation of proteins was carried out in an isoelectric focusing cell (Bio-Rad, Hercules, CA) with IPG buffer. The IPG strips were transferred to a minigel apparatus (Bio-Rad), in which pH-immobilized proteins were separated in the second dimension based on their molecular weight with SDS-PAGE (10% Tris-glycine gel). Gels were stained with a standard silver staining protocol. 2D polyacrylamide gels were analyzed with Phoretix 2D software purchased from Nonlinear Dynamics (Newcastle, UK).

Protein sequencing. The dried tryptic digests were dissolved in 0.1% trifluoracetic acid (TFA) in water and allowed to bind to C18 ZipTips, which were then washed 3 times with 0.1% TFA-water. The bound material was eluted in 10 mg/ml -cyano-4-hydroxycinnamic acid in 60% acetonitrile-0.1% TFA. Mass spectrometry was performed with an Applied Biosystems Voyager DE-PRO MALDI-TOF in positive ion reflector mode. Spectra were calibrated with trypsin autolytic peptides as internal standards. The obtained peptide sequences were then matched with the database by using Protein Prospector MS-Fit software and the National Center for Biotechnology Information nonredundant protein sequencing database.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of TGF- deficiency on proliferation and expression of growth-controlling genes in SMC. TASMC exhibited statistically significantly increased proliferation compared with unaltered SMC (Bonferroni P value <0.03), whereas no difference in proliferation was observed with SMC transfected with empty vector plasmid DNA (Fig. 1). Because a significant increase in proliferation of SMC was observed in TASMC, we studied mRNA expression of c-myc and p21, positive and negative regulators of cellular proliferation, respectively, to determine whether cell cycle regulatory genes were differentially expressed in these cells. As depicted in Fig. 2, both unaltered SMC and TASMC expressed similar -actin levels but TASMC did not express either TGF- or p21 mRNA. In sharp contrast, c-myc mRNA in TASMC expressed at least a fourfold increase compared with SMC. We did not study the expression of either TGF-2 or -3 in these cells because the expression of these isoforms did not change when cells were transfected with this plasmid DNA (61).

View larger version (14K): [in this window] [in a new window]   Fig. 1. Effect of the deletion of transforming growth factor- (TGF-) gene on new DNA synthesis/proliferation of aortic smooth muscle cells. Aortic smooth muscle cells [SMC; untreated or transfected with empty vector plasmid DNA (RLDN) or TGF- antisense plasmid DNA (RLDN/ASTGF-)] were cultured, and proliferation was quantified as new DNA synthesis by [3H]thymidine uptake assay and expressed as counts per minute (cpm). A significant increase of proliferation of TGF--altered SMC (TASMC) compared with untransfected cells was seen, and the proliferation of untransfected cells and cells transfected with control plasmid for TGF- were not different (n = 3). *P < 0.03, SMC vs. TASMC.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Differential expression of p21 and c-myc mRNA in TASMC. A representative photograph (n = 6) of ethidium bromide-stained agarose gel of the PCR products from RNA prepared from SMC and TASMC amplified for TGF-, p21, and c-myc mRNA expression is shown. No TGF- or p21 mRNA expression was observed in TASMC; in contrast, c-myc mRNA was significantly increased compared with SMC. Some extra bands may represent either primer dimers or unused cDNA. L, ladder.

Effect of TGF- deficiency on TGF- protein expression by SMC. We did not observe any TGF- protein in the conditioned medium of TASMCs; however, VSMC and VSMC transfected with empty vector plasmid (RLDN) DNA expressed 120 ± 18 and 135 ± 22 pg/ml (means ± SE from 3 experiments) of TGF- protein, respectively.

Effect of reexpression of p21 mRNA on proliferation of TASMC. Because TASMC lacked expression of p21, we hypothesized that reintroduction of p21 in TASMC would restore the normal proliferation of TASMCs. Therefore, using p21 sense plasmid DNA, we transfected p21 gene in TASMC. Cells transfected with empty vector plasmid DNA were used as controls. As shown in Fig. 3, transfection with p21 sense plasmid DNA, but not with control plasmid DNA, resulted in the reintroduction of p21 gene and protein expression in these cells. We compared the proliferation of normal SMC, TASMC, and TASMC transfected with p21 sense and empty vector plasmid DNA. The results shown in Fig. 4 demonstrate that after reintroduction of p21 expression, the proliferation/new DNA synthesis in p21-transfected TASMC was significantly less (P < 0.002) than in untransfected TASMC but was not statistically different from untransfected SMC (a nonsignificant Bonferroni P value >0.05).

View larger version (53K): [in this window] [in a new window]   Fig. 3. Restoration of p21 mRNA and protein expression in TASMC. TASMC were transfected with either p21 sense plasmid DNA or empty vector plasmid DNA. From one set of cells, RNA was prepared for mRNA expression, and from the other set of cells, lysates were prepared to study p21 protein expression. RNA was reverse-transcribed to cDNA and amplified for p21 mRNA, and the expression of p21 can be seen in 3 different clones (TASMC-p21 1, 2, and 3). The p21 mRNA expression from SMC as well as the absence of p21 mRNA from TASMC and TASMC transfected with empty vector plasmid DNA are also shown (bottom). Some extra bands may represent either primer dimers or unused cDNA. An equal amount of protein from lysates from these cells was electrophoresed, transferred to nitrocellulose paper, and probed with anti-p21 antibody, and the bands were visualized by chemiluminescence assay. The expression of p21 protein can be seen in 3 different clones (TASMC-p21 1, 2, and 3) of SMC; the absence of p21 protein from TASMC and TASMC transfected with empty vector plasmid DNA is also shown (top).

View larger version (16K): [in this window] [in a new window]   Fig. 4. Effect of the restoration of p21 gene on the proliferation/new DNA synthesis of TASMC. TASMC left untreated or transfected with p21 sense plasmid DNA or empty vector plasmid DNA were cultured, and the proliferation was quantified as new DNA synthesis by [3H]thymidine uptake assay and expressed as counts per minute. A significant decrease in proliferation (n = 3) in p21-transfected (TASMC-p21) compared with untreated TASMC (TASMC-C) is shown (means ± SE from 3 consecutive experiments; SMC, untransfected aortic SMC). Transfection with DNA from empty vector did not show any effect on proliferation compared with untreated TASMC, and the proliferation of p21-restored TASMC (TASMC-p21) was also not statistically different from that of untransfected SMC. *P < 0.03, SMC vs. TASMC; **P < 0.002, TASMC vs. TASMC-p21.

Effect of c-myc antisense oligonucleotides on proliferation/new DNA synthesis of TASMC. TASMC demonstrated significantly increased c-myc mRNA expression; therefore, we hypothesized that the transient inhibition of c-myc with antisense oligonucleotide would decrease their proliferation/new DNA synthesis. TASMC were treated with 10 µM anti-c-myc oligonucleotide and similar concentrations of a scrambled sequence of anti-c-myc oligonucleotide. The cells were incubated for 24 h with [³H]thymidine present during the last 4 h. The results depicted in Fig. 5 show a significant reduction in the proliferation/new DNA synthesis of TASMC in the presence of c-myc antisense oligonucleotide.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Effect of inhibition of c-myc by c-myc antisense oligonucleotide on the proliferation of TASMC. TASMC were treated with 10 µM c-myc antisense oligonucleotides or scrambled oligonucleotide, and the proliferation/new DNA synthesis of cells was quantified as described in MATERIALS AND METHODS. A statistically significant inhibition of proliferation with c-myc antisense oligonucleotides can be seen compared with the scrambled oligonucleotide-treated cells (n = 3).

Effect of restoration of altered genes on SMC morphology. We compared the differences in the morphology of SMC and TASMC. TASMC grew more densely and proliferated faster than SMCs. We hypothesized that these changes might be attributed to alterations in the cell cycle-regulating molecules TGF-, p21, and c-myc. Therefore, we transfected TASMC with p21 sense, TGF- sense, and c-myc antisense oligonucleotides and compared the transfected cells with normal SMC and TASMC. Appropriate controls of either empty vectors or scrambled oligonucleotides were used in each case. The morphology of TASMC when transfected with p21 sense, TGF- sense, and c-myc antisense oligonucleotides is similar to that of untransfected SMC, whereas the morphology of TASMC and cells transfered with empty vector plasmid DNA was not different. Figure 6 shows the morphological changes observed in SMC and TASMC. SMC cultures were predominantly composed of elongated, spindle-shaped cells that grew to 50–60% confluence. A close look demonstrated that these cells also formed the hill-and-valley pattern, whereas TASMC were slightly smaller and more rounded, consisted of densely packed elongated cells, and grew readily, suggestive of disregulated-growth TASMC, although occasionally spindle-shaped, thin elongated cells and epithelioid type cells were also seen. The cell densities observed in differently treated SMC were in accordance with the SMC proliferation data. Most of the SMCs, either untreated or TASMC treated with p21 sense plasmid DNA, TGF- sense plasmid DNA, or c-myc antisense oligonucleotide, appeared healthy and excluded Trypan blue, and no difference in morphology was observed among these cells. However, untreated TASMC and those treated with control plasmid DNA and scrambled c-myc oligonucleotide appeared grossly similar with identical morphological changes. These results suggest that the abnormal proliferation and morphological changes resulting after TGF- gene deletion could be corrected with either TGF- alone or by modulating the expression of p21 and c-myc genes, implicating their role in increased SMC proliferation in atherosclerosis.

View larger version (111K): [in this window] [in a new window]   Fig. 6. Effect of the deletion of TGF-, restoration of TGF- and p21 genes, and inhibition of c-myc expression on the morphology of aortic SMC. Aortic SMC left untreated or transfected with DNA from antisense TGF-, sense TGF-, sense p21, and empty vector plasmids as well as with c-myc antisense and scrambled oligonucleotides were grown in 6-well culture plates for 24 h and viewed and photographed with a Cohn High Performance CCD camera (x20). Untreated SMC, SMC transfected with empty vector RLDN plasmid DNA, SMC transfected with TGF- antisense plasmid DNA (TASMC), TASMC transfected with empty vector Zeo/+ plasmid DNA, TASMC transfected with TGF- sense plasmid DNA, TASMC transfected with p21 sense plasmid DNA, TASMC transfected with scrambled c-myc antisense oligonucleotide, and TASMC transfected with c-myc antisense oligonucleotide are shown. A similar, normal morphology can be seen in untreated SMC and SMC transfected with empty vector, TGF- sense plasmid DNA, p21 sense plasmid DNA, and c-myc antisense oligonucleotide, whereas TASMC and TASMC transfected with empty vector DNA and c-myc scrambled antisense oligonucleotide show the morphology of crowded/increased proliferated SMC, possibly similar to that observed in atherosclerosis.

Proteomic analysis of proteins from SMC and TASMC: high-resolution 2D gel electrophoresis and protein microsequencing to examine differences in protein expression of SMC and TASMC. As shown in Effect of restoration of altered genes on SMC morphology, modulation of TGF- genes in SMC resulted in not only aberrant proliferation but also differential expression of other genes relevant to cell cycle control and atherosclerosis. Therefore, we performed experiments to study whether these cells (SMC and TASMC) also exhibited differential expression of proteins relevant to atherosclerosis.

2D gel electrophoresis was carried out with 60 µg of protein solubilized in 2D sample buffer. Gels were fixed and silver stained as described in MATERIALS AND METHODS. Images were obtained to help identify spots different in SMC and TASMC. About 10 spots in each gel were identified, and protein microsequencing was performed in the protein and DNA sequencing core facility, as described in MATERIALS AND METHODS.

A differential expression of proteins relevant to smooth muscle proliferation and atherosclerosis was observed in SMC and TASMC. Proteins observed in protein extracts from TASMC included calreticulin (GenBank accession no. 6680836), a calcium-binding protein of the endoplasmic reticulum; calcineurin A (accession no. 14495188); calcineurin calmodulin-stimulated protein phosphatase (EC 3.1.3.16 [EC] ), the major calmodulin-binding protein in brain, whose activity is inhibited by binding of immunophilin ligand complex; myosin heavy light chain (accession no. 20555319), the motor protein of muscle thick filaments, which is involved in the modulation of the actin-activated ATPase activity of myosin cell division; control protein cdc21 (accession no. 1566539), a cell division control protein; MYB (accession no. 13278576), an oncogene and a positive regulator of cellular proliferation; extendin (accession no. 14389431), a group of peptide hormones related to the glucagon family; stress-induced phosphoprotein I (accession no. 13277819), a cochaperone that is homologous with the human heat shock cognate protein and involved in vascular diseases; and HSP70/HSP90 organizing protein (accession no. 21654757), an acute-phase response protein with cytoskeletal regulator activity. The protein expression of kinesin (accession no. 1764561), keratin 10 (accession no. 4557697), Krueppel-like zinc finger protein (accession no. 10334857), and myosin heavy light chain (accession no. 20555319) were observed in protein extracts from SMC. These results show that TGF- inhibition might have led to differential expression of proteins in SMC and TASMC. This differential expression of proteins may relate to the increased proliferation of TASMC compared with SMC.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The study results demonstrate in a unique way that TGF- regulates the proliferation/new DNA synthesis of SMC, because the inhibition of TGF- gene results in the abnormal proliferation of these cells. The increase in SMC proliferation is considered an important step in the pathogenesis of atherosclerosis (54, 55). The effect of TGF- on the proliferation of SMC has been reported in several studies. Magnier-Gaubil et al. (35) showed that TGF- inhibits PDGF-stimulated proliferation of SMC. The studies of Grainger and colleagues (17, 19, 42) also strongly support the role of TGF- in atherosclerosis, because they showed that a population of patients with advanced atherosclerosis all had fivefold less active TGF- in their sera than patients with normal coronary arteries. This correlation of TGF- with atherosclerosis was considered much stronger than for other known major risk factors; therefore, it may have important diagnostic and prognostic significance.

TGF- has been implicated in the process of SMC proliferation in several other ways as well (43). For example, it was hypothesized that lipoprotein(a) [Lp(a)] induces VSMC proliferation and inhibits the activation of plasminogen to plasmin. Plasmin proteolytically cleaves active TGF- from latent TGF-. In the absence of active TGF-, a potent inhibitor of SMC proliferation, hyperproliferation of VSMC occurs, possibly leading to atherosclerosis. Grainger and Metcalfe (18) earlier reported that activation of TGF- was inhibited in apolipoprotein(a) transgenic mice, thereby enhancing SMC proliferation and the process of atherosclerosis in these mice.

The results of this study, which suggest that the expression of p21 was abolished along with TGF-, are provocative and significant, because there is a very close relationship between TGF-, p21, and inhibition of proliferation. p21 is an effector protein that blocks cdk and is induced by TGF-. Li et al. (34) and Elbendary et al. (15) studied the effect of TGF- on different cell lines and observed that the induction of p21 by TGF- was altered only in TGF--sensitive cell lines. Datto et al. (14) observed that treatment with TGF- resulted in the stimulation of p21 promoter activity in HaCaT cells. We reported previously (29) that the CsA-mediated induction of p21 mRNA in lymphoid and nonlymphoid cells and stimulation of p21 promoter activity are mediated by TGF-. The increased expression of c-myc in TASMC is also important, because, in sharp contrast with the inhibitory effects of TGF- and p21, c-myc is known to augment the proliferation of VSMC. The role of c-myc in proliferation of SMC has been highlighted in several studies, including that of Kanse et al. (26), who noted that the urokinase-type plasminogen activator stimulated DNA synthesis and proliferation of VSMC, which was accompanied by the stimulation of c-myc mRNA. Similarly, the increase in cellular proliferation by Lp(a) and linoleic acids resulted in the upregulation of c-myc mRNA (3, 20). Therefore, these studies support the present findings that in the TGF- deficient SMC, the decreased proliferation was accompanied by a significant decrease in p21 and increase in c-myc mRNA expression. These results suggest that p21 and c-myc may play a key role in the proliferation of SMC, a key event in the pathogenesis of atherosclerosis.

These findings, which suggest that the restoration of p21 expression corrected the aberrant proliferation of TASMC, are significant and supported by other studies in which, with in vitro and in vivo transfection studies, an important role of p21 in the regulation of Retinoblastom a protein phosphorylation and cell cycle progression in VSMC has been demonstrated. These studies also featured a gene therapeutic approach for restenosis and vascular diseases associated with proliferative disorders. Fukui et al. (16) demonstrated that p21 inhibits both migration and proliferation of SMC. Chang et al. (8) showed that overexpression of adenovirus-mediated p21 gene expression in rat VSMC inhibits proliferation. In a porcine balloon arterial model using adenovirus-mediated transfer of the p21 gene, Yang et al. (63) showed a 35% reduction of in vivo cell proliferation and intimal thickening. Ihling et al. (24) observed the expression of p21 in atherosclerotic plaques. The recent study of Chen et al. (10) confirms the conclusions of the present study, demonstrating the p21-dependent inhibition of VSMC proliferation by blocking thrombospondin-1. Our own recent studies (30) demonstrated that in vitro and in vivo overexpression of p21 results in decreased lymphocyte proliferation. Condorelli et al. (13) also demonstrated that mutated p21 transgene overexpression reduces SMC proliferation and restenosis in hypercholesterolemic apolipoprotein E knockout mice.

The gene expression of c-myc, a protooncogene whose overexpression causes aberrant proliferation of cells, is inhibited by TGF-. Malliri et al. (36) observed that in squamous cell carcinoma cell lines TGF- inhibits c-myc and induces p21 gene expression. This is supported by several other studies (2, 9, 62) that, with HeCat, mink lung epithelial cell lines, and T cell lines, demonstrated the interplay of TGF-, p21, and c-myc during cellular proliferation and inhibition. Studies (11, 51) showing that the downregulation of c-myc correlates with the inhibition of vascular smooth muscle proliferation or growth arrest support our finding that the transient inhibition of c-myc by treatment with antisense oligonucleotides significantly inhibits the proliferation of TASMC. We previously demonstrated (32) the efficacy of this c-myc antisense oligonucleotide. Similarly, studies of Claassen and Hann (12) suggested that the inhibition of c-myc after TGF- signaling was significant for subsequent regulation of p21 and cell cycle inhibition, connecting these molecules during cellular proliferation/inhibition and supporting the conclusions of this study.

The morphological changes in TASMC compared with SMC were found to be similar to those reported for SMC isolated from experimental models of atherosclerosis and restenosis (5, 23, 49). These findings are suggestive of a role of TGF- in maintaining normal morphology of SMC, because in its absence the morphological changes of TASMC were similar to those observed in SMC isolated from experimental animals.

A careful analysis of the proteins that were differentially expressed in TASMC reveals their relationship with TGF- and SMC proliferation. There are studies supportive of these findings. Patton et al. (50) treated rat aortic VSMC with growth factors (10% fetal calf serum, PDGF, or angiotensin II) for 24 h and prepared cell lysates. High-resolution 2D gel electrophoresis of these lysates demonstrated changes in specific proteins associated with either hyperplastic or hypertrophic growth. The levels of HSP60 and HSP70, protein disulfide isomerase, protein disulfide isomerase isozyme Q-2, elongation factor-1, and calreticulin were increased. These results are similar to those of our study, in which expression of a few of these proteins was also increased in TASMC, e.g., calreticulin and HSPs. Calreticulin is a major calcium binding and putative molecular chaperone protein, and TGF- can inhibit its activity (48); therefore, it is likely that in the absence of TGF- its expression was increased in TASMC. Also, calreticulin participates in the inducible decay of AT₁ receptor mRNA, which is implicated in the pathogenesis of atherosclerosis (6). The expression of calcineurin, which also affects SMC proliferation and is regulated by TGF-, was also increased in TASMC. The studies of Ohkawa et al. (47) also demonstrated that the calcineurin-mediated pathway is critical for maintaining the differentiated phenotype of SMCs. Afroze et al. (1) showed that c-Myb and calcineurin influence Ca²⁺-mediated signals of cell cycle and c-Myb activity at the G₁/S transition increased at least threefold. These results demonstrate the roles of calcineurin and c-Myb in development, differentiation, and proliferation of aortic SMC. We also observed increased expression of MYB binding protein, and these protooncogenes, including c-Myb, are expressed early after vascular injury. c-Myb expression has been shown to be involved in the control of cell proliferation in a variety of cell types (21, 59). HSPs and stress-induced proteins were also differentially expressed in TASMC compared with SMC. HSP70 and HSP90 participate in mechanical stress-induced proliferation in SMC (60). Several studies have shown increased expression of HSPs, and stress proteins play a role in the initiation of proinflammatory events relevant to the pathogenesis of atherosclerosis and are considered as a marker for cellular mortal and immortal phenotypes (44, 52). Kanwar et al. (27) also showed that the expressions of HSP60 and the stress-inducible form of the cytoprotector HSP70 correlated with the development of atherosclerotic lesions in the aortic tree of apolipoprotein E-deficient [apoE(–/–)] mice. This description of the relationship of the proteins calreticulin, calcineurin A, MYB binding proteins, myosin heavy and light chain, HSP, etc. with TGF- and SMC proliferation supports our findings that these proteins may be regulated by TGF- and participate in events leading to SMC proliferation.

In summary, this study uniquely demonstrates the role of TGF- in aortic SMC proliferation and demonstrates that SMC deficient in TGF- gene proliferate abnormally. Alteration in TGF- gene expression results in the differential expression of the potent negative regulator p21 and the growth-promoting oncogene c-myc. We were able to reintroduce p21 mRNA and protein expression in TASMC, which restored the normal proliferation/new DNA synthesis of these cells. It was also possible to restore the normal proliferation of TASMC through the inhibition of c-myc expression by antisense oligonucleotides. Also, proteins with relevance to smooth muscle proliferation were differentially expressed in TASMC. This might prove that 1) TGF- is a potent antiproliferative molecule for SMC because its deletion results in the abnormal proliferation of SMC; 2) the inhibition of p21 and increased c-myc expression are a result of the increased proliferation/new DNA synthesis; and 3) restoration of p21, as well as inhibition of c-myc expression, can reverse the abnormal cellular proliferation. Therefore, as in cancer, the loss of p21 and the increased expression of oncogene c-myc can be a prodiagnostic marker for increased proliferation of SMC in atherosclerosis.

In conclusion, this study provides evidence that TGF- plays an important role in the control of SMC proliferation, one of the major causes of human atherosclerosis. Together, these results provide a possible association between advanced atherosclerosis and cell cycle control. Future studies will explore whether these molecules act alone or in concert to alter the proliferation of aortic SMC, leading to atherosclerosis. The development of agents capable of controlling one or all of these molecules may lead to the better management and treatment of atherosclerosis.

	ACKNOWLEDGMENTS

 
The author is grateful to Matthew Plummer for excellent technical help and Seema Sernovitz for editorial help.

GRANTS

This work was supported in part by National Institute of Allergy and Infectious Diseases Grant RO1-AI-41703.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. Khanna, Dept. of Medicine, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: akkhanna{at}mcw.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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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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