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Cardiovascular Research 2000 46(1):172-179; doi:10.1016/S0008-6363(00)00004-3
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Copyright © 2000, European Society of Cardiology

Methylation of the estrogen receptor-{alpha} gene promoter is selectively increased in proliferating human aortic smooth muscle cells

Anita K. Yinga, Hamdy H. Hassanaina, Christine M. Roosa, Dominic J. Smiragliab, Jean-Pierre J. Issac, Robert E. Michlera, Michael Caligiurib, Christoph Plassb and Pascal J. Goldschmidt-Clermonta,*

aHeart and Lung Institute, 514 Medical Research Facility, 420 W. 12th Street, Columbus, OH 43210, USA
bComprehensive Cancer Center, College of Medicine and Public Health, The Ohio State University, Columbus, OH, USA
cCancer Biology, Johns Hopkins University, Baltimore, MD, USA

* Corresponding author. Fax: +1-614-688-5779 goldschmidt-1{at}medctr.osu.edu

Received 25 May 1999; accepted 20 December 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Atherosclerosis is a multigenic process leading to the progressive occlusion of arteries of mid to large caliber. A key step of the atherogenic process is the proliferation and migration of vascular smooth muscle cells into the intimal layer of the arterial conduit. The phenotype of smooth muscle cells, once within the intima, is known to switch from contractile to de-differentiated, yet the regulation of this switch at the genomic level is unknown. Estrogen has been shown to regulate cell proliferation both for cancer cells and for vascular cells. However, methylation of the estrogen receptor-{alpha} gene (ER{alpha}) promoter blocks the expression of ER{alpha}, and thereby can antagonize the regulatory effect of estrogen on cell proliferation. We sought to determine whether methylation of the ER{alpha} is differentially and selectively regulated in contractile versus de-differentiated arterial smooth muscle cells. Methods: We used Southern blot assay, combined bisulfite restriction analysis (Cobra) and restriction landmark genome scanning (RLGS-M) to determine the methylation status of ER{alpha} in human aortic smooth muscle cells, either in situ (normal aortic tissue, contractile phenotype), or the same cells explanted from the aorta and cultured in vitro (de-differentiated phenotype). Results: We provide evidence that methylation of the ER{alpha} in smooth muscle cells that display a proliferative phenotype is altered relative to the same cells studied within the media of non-atherosclerotic aortas. Thus, the ER{alpha} promoter does not appear to be methylated in situ (normal aorta), but becomes methylated in proliferating aortic smooth muscle cells. Using a screening technique, RLGS-M, we show that alteration in methylation associated with the smooth muscle cell phenotypic switch does not seem to require heightened activity of the methyltransferase enzyme, and appears to be selective for the ER{alpha} and a limited pool of genes whose CpG island becomes either demethylated or de novo methylated. Conclusions: Our data support the concept that the genome of aortic smooth muscle cells is responsive to environmental conditions, and that DNA methylation, in particular methylation of the ER{alpha}, could contribute to the switch in phenotype observed in these cells.

KEYWORDS Artherosclerosis; Receptors; Smooth muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
DNA methylation is a naturally occurring epigenetic process leading to the chemical alteration of cellular DNA. The addition of methyl groups to cytosine located 5' to guanine in CpG nucleotides pairs represents one of the mechanisms by which the genome can behave as a ‘responsive organ’ in cells. This modification can play an important role in regulating gene expression, especially in CpG rich areas known as CpG islands [1–3]. Approximately 60% of the promoter regions of human genes are located within CpG islands [1]. In normal adult tissue, CpG islands are usually unmethylated with the exception of genes located on the inactive X-chromosome and a few imprinted genes [4,5]. In neoplastic tissue, however, there is aberrant promoter methylation of defined tumor suppressor genes [6–9]. The mechanism for the increased methylation is not known, but methylation has been correlated with inactivation of these genes, suggesting that methylation is an alternative to classical mutation in this pathological process.

A gene of common interest to both cancer and cardiovascular research is the estrogen receptor alpha (ER{alpha}) gene. There is loss of ER{alpha} expression in certain neoplastic [10,11] and atherosclerotic lesions [12], suggesting that ER{alpha} helps to maintain a homeostatic balance between proliferation and quiescence of cells. ER{alpha}, as a potential growth suppressor gene, is 100% methylated in colo–rectal cancer and most hematopoietic, lung, and hormone-insensitive breast cancers [10,11,13], with associated loss of gene expression. In various forms of occlusive arterial diseases, including atherosclerosis and restenosis, a state of excessive proliferation can be observed, like in neoplasia, in which smooth muscle cells (SMCs) proliferate to form an intimal SMC lesion [14–18]. Intimal SMCs are phenotypically distinct from those found in the media. Characteristically, medial SMCs are differentiated while intimal SMCs acquire a proliferating, or dedifferentiated, phenotype. In vivo and in vitro models of vascular disease have found estrogen to be protective against SMC proliferation and neointima formation [19–22]. Strong clinical evidence suggests that estrogen replacement therapy in post-menopausal women might help prevent cardiovascular disease. However, the preventive effect of estrogen might be limited in women with established atherosclerotic lesions [23], perhaps, as a result of the loss of estrogen responsiveness within affected arteries [12].

We have reported previously that methylation of the promoter region of the ER{alpha} gene is readily detectable in cardiovascular tissues obtained from patients undergoing revascularization procedures [24]. In the atrium, we have found that methylation of the ER{alpha} gene increases linearly with age. In macroscopically normal vessel samples obtained from these patients, methylation of the ER{alpha} gene remained low (4–7% of the promoters surveyed were methylated). However, methylation of the ER{alpha} gene was increased in atherosclerotic plaques obtained by atherectomy, relative to macroscopically normal arterial tissue. We have also determined which cells in the vessel wall contributed the methylated ER{alpha} promoters. We have studied endothelial cells (ECs) and SMCs explanted from aortic tissue. ECs consistently displayed low levels of methylation. In contrast, SMCs were found to have a higher degree of ER{alpha} gene methylation, suggesting that these cells were mainly responsible for the heightened level of ER{alpha} gene methylation found in atherosclerotic tissues [24].

Based on these findings, we hypothesized that DNA methylation of genes involved in mediating proliferation of SMCs, specifically ER{alpha}, may represent a missing link in understanding the mechanism of neointima formation, whether in primary or restenotic lesions. We designed the current study to investigate the level of ER{alpha} methylation in human aortic smooth muscle cells (HASMCs) in differentiated medial tissue compared to proliferating cells in culture derived from the same tissue. Since medial, or differentiated, SMCs placed into culture assume many of the characteristics of intimal SMCs in vivo [16], our model enables us to make comparisons between the two cell populations obtained from the same individuals. Based on evidence that estrogen is anti-proliferative and that DNA methylation is associated with inhibition of ER{alpha} transcription, we would predict that cells that can proliferate are protected from estrogen's inhibitory effects. Hence, methylation of the ER{alpha} might be selectively advantageous for proliferating SMCs. Using three different methylation detection techniques, our results show that higher levels of ER{alpha} methylation are found in proliferating, intimal-like SMCs versus medial SMCs in situ.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
Normal human vascular SMCs were derived from explants of thoracic aorta from patients donating organs for cardiac transplantation using procedures previously described. Briefly, we dissected away the adventitia, cut the remaining tissue into 1x1 cm pieces, and digested the endothelial side of the aorta with an enzyme mix (1 mg/ml collagenase type I and 1 mg/ml bovine serum albumin in 1X phenol-red free Hank's balanced salt solution) for 15 min at 37°C. The residual endothelial layer was removed by rinsing with Hank's. Then, a portion of the isolated media was snap frozen to preserve differentiated medial SMCs, while another portion was placed onto six-well plates, under coverslips to prevent dislodgment, in order to generate a proliferating SMC phenotype from the same specimen. Cultures from individual patients were maintained as separate cultures and not pooled. At passage 3, cultures were analyzed by flow cytometry to confirm smooth muscle cell origin (>85% SMC {alpha}-actin positive). Cultures were expanded and grown in SMGM-2 (Clonetics) per manufacturer's recommendations at 37°C, 5% CO2 in air. Studies were performed on cells passage 4–7 for all experiments, except for data presented in Fig. 1c.


Figure 1
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  		Fig. 1 Methylation of the ER{alpha} CpG island in differentiated HASMC tissue (a) and proliferating SMCs (b) from the same patients by Southern blot of genomic DNA digested with NotI/EcoRI. (a) Differentiated medial SMCs: Lanes: 1, 100% methylation shown by an uncut EcoRI fragment seen in HT-29, a human colonic carcinoma cell line control; 2, fully unmethylated pattern seen in MCF-7 cells, an estrogen receptor-positive breast carcinoma cell line; 3–5, differentiated SMCs from an 18-year-old male, 24-year-old male, and a 37-year-old female showing no methylation; (b) Southern blot of controls (lanes 1 and 2) and proliferating SMCs from the same patients as (a); (c) Southern blot of proliferating aortic SMCs showing successive passages for two representative cell populations (2708 and 2542). Note that methylation tends to increase with passages (7–9, 2708), and the absence of regression of methylation with further passages in cells displaying nearly 100% methylation (2542).

 
The MCF-7 cell line was obtained from American Type Culture Collection and maintained in DMEM/F12 phenol-red free (Gibco BRL Life Technologies, Gaithersburg, MD, USA) with 10% FBS, 2 mM L-glutamine, and 20 mg/l gentamycin. HT-29 cells were obtained from Dr. Paul Bray (Division of Hematology, The John Hopkins University School of Medicine) and maintained in DMEM with 10% FBS, 1% penicillin/streptomycin, and 10 µg/ml human apo-transferrin. All cell lines were incubated in low estrogen media, phenol-red free EBM (Clonetics) with 10% castrated horse serum and 1% penicillin and streptomycin, for 48 h prior to experiments. For experiments presented in Fig. 1c (late passages), HASMCs were purchased from Clonetics, and expanded in SMGM-2 with growth factors and antibiotics as recommended by the manufacturer.

2.2 DNA isolation
DNA was extracted from cell cultures using DNAzol reagent (Gibco BRL) per manufacturer's recommendations. DNA from primary snap frozen tissue was extracted using standard methods [24].

2.3 Southern blot analysis [24]
A 10-µg amount of genomic DNA was digested with 100 U of EcoRI and 500 U of the methylation-sensitive enzyme NotI for 16 h as specified by the manufacturer (New England Biolabs, Beverly, MA, USA). The NotI site is located at the heart of the CpG island, which covers exon1, a small domain 5' of exon 1 and a small domain 3' within intron 1. It is 190 bp downstream of the first ATG and 442 bp downstream of the TATA box. The NotI-treated DNA was precipitated, electrophoresed on a 0.8% agarose gel, transferred to a Hybond-N membrane (Amersham, Arlington Heights, IL, USA) with the VacuGene System (Pharmacia), and hybridized with a 0.3-kb fragment of the first exon of the ERa. The blots were exposed in a PhosphorImager (Molecular Dynamics) for 2 days. Using IMAGE-QUANT software from Molecular Dynamics, the percentage methylation was measured as the density of methylated NotI site (3.1-kb band) over total density of the bands in each lane. DNA from MCF-7 cells, 0% methylated in the ER{alpha} CpG island, and HT-29 cells, 100% methylated, were used as controls. The probe was obtained by PCR amplification of genomic DNA using primers 5'-AAGGGTCTATCTACTTTGGGAGCATT-3', upper and 5'-AACTTTACTTTACTTGTCGTCGCTGC-3', lower.

2.4 Combined bisulfite restriction analysis (COBRA)
COBRA consists of sodium bisulfite treatment of 1 µg of genomic DNA followed by PCR amplification of the region of interest, restriction digestion, and methylation quantitation [25]. Sodium bisulfite and PCR treatment converts unmethylated cytosine residues to thymidine, whereas methylated cytosine residues remain as cytosine. This modification creates unique restriction sites in the unmethylated DNA. The sodium bisulfite treatment was performed as previously described [26]. Primer pairs residing within the CpG island of the ER{alpha} and complementary to converted DNA sequences were purchased from Gibco (5'-GGTTTTTGAGTTTTTTGTTTTG-3', upper; 5' AACTTACTACTATCCAAATACACCTC-3', lower). The COBRA primers amplify a ~200-bp region that corresponds to –250 to –50 relative to the NotI site. The Bstu1 site used is 206 bp upstream of NotI. Therefore, it is 16 bp upstream of the first ATG and 236 bp downstream of the TATA box. Hence, the methylation site within the ER{alpha} studied by COBRA did not overlap with the one studied by Southern blot. A 2-µl volume of treated DNA was amplified in a 50-µl reaction containing 5 µl 10X PCR buffer (16.6 mM ammonium sulfate, 67 mM Tris–HCl (pH 8.8), 6.7 mM MgCl2, and 10 mM β-mercaptoethanol), 1.25 mM each dNTP, and 0.5 µl (5 U/µl) platinum Taq polymerase (Gibco). Reactions were carried out in a PE Thermal Cycler 9600 with a touchdown procedure: 2 min at 95°C; 30 s at 95°C, 30 s at 60°Cxthree cycles; 30 s at 95°C, 30 s at 57°Cxfour cycles; 30 s at 95°C, 30 s at 54°Cxfive cycles; 30 s at 95°C, 30 s at 51°Cxtwenty-five cycles. The PCR product was purified in a 50-µl volume (PCR purification kit, Qiagen, Valencia, CA, USA) and 15 µl of the PCR product was then digested with 20 U of BstUI (New England Biolabs) in a 30-µl reaction for 4 h at 60°C. The entire reaction was separated on a 3% high-resolution agarose gel (Amresco, Solon, OH, USA) with ethidium bromide. The BstUI site (CGCG) is present in methylated DNA but lost in unmethylated DNA due to sodium bisulfate treatment. The percentage of fully methylated BstUI sites in the ER{alpha} CpG island was calculated from the relative density of BstUI-cleaved PCR to total PCR product as determined by densitometry. A control digest with Hsp92II, whose restriction site (CATG) should be destroyed by bisulfite treatment, was also performed. Cleavage by Hsp92II would indicate incomplete sodium bisulfite conversion or non-CpG DNA methylation.

2.5 Restriction landmark genome scanning-M
RLGS-M for detection of methylation changes was performed according to our published protocol [27–29]. Briefly, high-molecular-weight genomic DNA was isolated as described [29]. To prevent nonspecific labeling, the sheared ends of the sample DNA were blocked by the addition of nucleotide analogs (aS-dGTP, aS-dCTP, aS-dATP, aS-dTTP). The DNA was then digested with 20 U NotI (Promega) and the resulting restriction sites were end-labeled in a fill-in reaction. The labeled DNA was digested with 20 U EcoRV (Promega) and electrophoresed through a 0.8% agarose tube gel (first dimension separation). The DNA was digested in the gel with 750 U HinfI (Promega) and electrophoresed in a 5% polyacrylamide gel (second dimension separation). The gel was next dried and exposed to Kodak X-Omat AR film in the presence of one Quanta III, intensifying screen (DuPont, Boston, MA, USA), for 2–14 days.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 ER{alpha} methylation in differentiated and proliferating medial SMCs
ER{alpha} has a CpG island in its promoter and first exon that contains several methylation-sensitive restriction enzyme target sites, including a single NotI site. Previous studies on methylation in neoplasms have shown that methylation at the NotI site in the ER{alpha} CpG island correlates with subsequent alteration in expression [10,11,13]. Aortic tissue samples from three heart donors produced three separate aortic SMC cultures for this study. We compared the methylation of ER{alpha} in the proliferating SMCs in culture to the original differentiated in situ SMCs from the same individuals. We measured methylation by digesting genomic DNA with NotI followed by Southern blotting and probing with a fragment specific for the ER{alpha} promoter region. If unmethylated, two fragments are produced at 1.2 and 1.9 kb; conversely, if the NotI site is methylated, a single uncut 3.1-kb fragment is observed, which corresponds to the intact EcoRI fragment. Since the probe hybridizes within the 1.2-kb fragment, we determined the percentage methylation by measuring the relative density of the methylated band (3.1 kb) to the total density (1.2- plus 3.1-kb bands) in each lane (Fig. 1a). Our results show a marked increase in methylation of the proliferating SMCs over the original differentiated medial SMCs of three different patients (Fig. 1b), with 60.0±3.3% of the proliferating SMCs displaying methylation of their ER{alpha}, versus 2.5±2.4% of the medial SMCs. We confirmed that the NotI site was present by amplifying the NotI region of each sample by PCR in the absence of methyltransferase, and by digesting with NotI (data not shown). Upon studying successive passages for individual cell lines, we observed that methylation increased with passages, and the absence of regression of methylation with further passages in cells displaying nearly 100% methylation (Fig. 1c).

In order to measure the level of ER{alpha} methylation at more than one site in the CpG island, we also performed a sodium bisulfite based methylation assay entitled COBRA [30]. Sodium bisulfite treatment of DNA changes all cytosines to uracils. Those that are methylated (5-methylcytosine), however, are resistant to this modification and remain cytosine [25]. Subsequent PCR-based amplification results in conversion of unmethylated cytosine residues into thymidine with methylated residues remaining cytosine [25,26,30–32]. This process can lead to the methylation-dependent creation of new restriction sites, or, in this case, the methylation-dependent retention of a pre-existing site, BstUI (CGCG), which allows for quantitation of methylation [30]. We measured the percentage methylation by measuring the relative density of the digested band (methylated BstUI site) to the total density of the lane.

This procedure differs from the methylation sensitive enzyme analysis by Southern blot in that it does not rely on absence of cleavage to detect methylation but rather on difference in restriction sites produced by methylation sensitive DNA modification. In order to ensure complete bisulfite modification and specific CpG methylation, we also digested all samples with Hsp92II, an enzyme whose restriction site (CATG) should be completely eliminated by the treatment (data not shown). We also performed COBRA analysis using DNA previously shown to be completely methylated or completely unmethylated at the ER{alpha} CpG island and found our method of quantification accurate and reliable (data not shown). Our results support the findings by Southern blot of increased levels of ER{alpha} methylation in the proliferating SMCs compared to the in situ SMCs of the same individuals (Fig. 2). The overall lower extent of DNA methylation detected with COBRA relative to Southern blot analysis is likely due to two reasons. First, the unmethylated band (upper band in Fig. 2) could be preferentially amplified by PCR since there are less GC rich areas after the bisulfite treatment [31]. Second, the BstUI site lies in an area in the CpG island that is methylated less frequently in pathologic tissue [32].


Figure 2
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  		Fig. 2 Combined bisulfite restriction analysis (COBRA): the same samples as for Fig. 1 were analyzed for DNA methylation on COBRA gels. Lanes: 1, DNA-ladder; 2, HT-29 cells; 3, MCF-7 cells; 4–6, differentiated SMCs; 7–9, proliferating SMCs from same patients.

 
3.2 Global DNA methylation in differentiated and proliferating medial SMCs
A possible mechanism for the increased methylation that we observed in proliferating SMCs is an increase in overall methylating activity in these cells. Methylation studies on the DNA of transformed cells has demonstrated increased methyltransferase activity, hypomethylation of normally methylated CpG dinucleotides not associated with CpG islands, and hypermethylation of normally unmethylated islands [33–35]. We assessed the degree of aberrant CpG island methylation between the two types of SMC phenotypes using a powerful methylation sensitive genome scanning technique: RLGS-M. When using the methylation sensitive NotI restriction enzyme in this assay, RLGS-M is uniquely suited to assess the methylation status of CpG islands, as 89% of all NotI sites are found in CpG islands.

We show in Fig. 3 a representative two-dimensional gel electrophoresis of end-labeled NotI/EcoRV fragments separated along the first dimension followed by in situ digestion with HinfI and subsequent second dimension separation. By comparing the RLGS-M profiles obtained using DNA from the two different SMC phenotypes for individual patients, we were able to identify changes in the methylation status of CpG islands by detecting the presence or absence of spots on the 2D-gels. The cause of such change in a spot is most likely due to alteration of the methylation status of the NotI site, as methylation of the NotI site prevents cutting, subsequent end labeling, and ultimately results in loss of that spot on the profile. Other possible explanations, including a point mutation in the recognition sequences of any of the three restriction enzymes used or homozygous deletion, are unlikely given the polyclonal character and the shared origin of the cells under testing.


Figure 3
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  		Fig. 3 Restriction landmark genome scanning-M: (a) entire 2-D gel for proliferating smooth muscle cells; (b) methylation within differentiated (top panel) vs. proliferating (lower panel) smooth muscle cells, the marks indicate the location of the ER{alpha} gene.

 
One spot found to be present at normal diploid intensity in the RLGS-M profile from differentiated SMC DNA, but nearly vanished from the RLGS-M profile from the proliferating SMC DNA of the same patient, is shown in Fig. 3b. This spot has been identified as representing the NotI site of the ER{alpha} gene based on previous work [36]. The degree of spot loss in this patient is consistent with the 68% methylation of the ER{alpha} gene NotI site indicated by the Southern blot analysis described in Fig. 1. In addition, after analysis of more than 1200 spots, we found a total of 12 alterations (eight spots newly identified and four spots lost from the proliferating SMC DNA profiles), likely to be due to changes in the methylation status of CpG islands. This represents ~1% of the CpG islands screened and therefore indicates a rather selective change in CpG island methylation as compared to what would be expected for a large general increase or decrease in methylation activity. This observation correlates with similar observations that have been made concerning the methylation changes detected in certain neoplasia [37].


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The genome is a responsive organ in the cell [38]. Its structure can adapt to changing conditions in ways that impact upon the phenotypic characteristics of the concerned cells. For example, genes can ‘jump’ [38], and their promoters can be chemically modified by methylation. For vascular SMCs, residence within the media of blood vessels corresponds to a privileged, ‘cocoon’-like environment. There, SMCs are sheltered from growth factors, cytokines, and other potentially noxious stimuli by the endothelium on the luminal side, and by the adventitia on the abluminal side. Moreover, they are surrounded by a supportive network of extracellular matrix proteins. Once exposed to atherosclerotic conditions, where the endothelium may be damaged and inflammatory cells can invade the inner layers of the vessel wall, one would expect to detect signs within SMCs of genomic adaptation to their new environmental conditions.

Although there have been numerous investigations into defining the origin of intimal SMCs, there is still extensive debate and many unanswered questions. One view is that intimal cells arise from clonal expansion of a subpopulation of phenotypically, and, perhaps, genotypically, different SMCs residing in the vascular media, or the adventitia [18,39,40]. Immunohistochemical data support heterogeneity of the vessel media with at least two distinct cell populations: a majority of differentiated SMCs and few intimal-like SMCs [41–43]. The alternative hypothesis, which is not mutually exclusive, is that intimal SMCs are derived from differentiated medial SMCs that undergo phenotypic modulation upon migrating to the intima. Campbell et al. described this phenomenon as a shift from a ‘contractile’ to a ‘synthetic’ phenotype [44]. Their study showed that adult vascular SMCs placed into culture undergo a phenotypic switch and assume many of the characteristics of intimal SMCs in vivo. An intertwining thread through both hypotheses is that neointima formation involves molecular modulation of the machinery regulating migration/proliferation/differentiation of cells in the media. The findings in this report suggest that CpG methylation may play an important role in either the clonal expansion of a selected group of SMCs displaying an altered CpG island methylation pattern, or that pathophysiological changes may trigger the reprogramming of gene activity mediated by changes in promoter methylation.

While we recognize that cultured SMCs merely reflect the phenotype of intimal SMCs in situ [16,45], our results show that the majority of SMCs that were able to proliferate from medial explants displayed methylation of the ER{alpha} gene and that ER{alpha} methylation tends to increase with further passages. We found very little methylation of ER{alpha} in the differentiated SMCs in situ. The increased methylation of the cells that proliferated into culture could be due to either an increase in activity of the enzyme responsible for methylation, DNA methyltransferase, or to the presence of a few medial SMCs in situ with the selective advantage of increased ER{alpha} methylation. Using RLGS-M to assess the level of CpG island methylation in proliferating SMCs indicates that there is not a large general increase in CpG island methylation. It is estimated that <10% of all CpG islands within the human genome contain a NotI site (~5000 CpG islands) [46]. Hence, <0.5% of all CpG islands screened by RLGS-M assay (those containing a NotI site) underwent a detectable change in methylation status. It is possible, however, than ten times as many genes are affected by a change in methylation status across the entire genome, if we extrapolate our RLGS-M assay findings to the entire genome (all CpG islands). This then suggests two possibilities: (i) that there is a more selective change occurring in CpG island methylation of differentiated medial SMCs contributing to their phenotypic switch to proliferating intimal SMCs; (ii) that there is, within the media, a small number of SMCs with altered CpG island methylation (including the ER{alpha} CpG island), and therefore possessing a selective growth advantage over other SMCs of the media. Why would methylation provide a selective advantage for growth? As mentioned previously, estrogen has an anti-proliferative effect [46]. Therefore, under conditions that support proliferation (i.e. exposure to growth factors and serum in a vessel wall or culture), only those SMCs with ER{alpha} methylation, and thus low levels of expression [46], can proliferate. This explanation is supported by findings that estrogen inhibits the proliferation of SMCs in vitro and in vivo [19–22].

The findings of this study bring forward important questions that warrant investigation, such as whether there are other growth-related genes affected by methylation. Perhaps, methylation of the ER{alpha} is not causative for the growth advantage, but instead reflects the altered methylation of the proliferating cells, with another gene responsible for the selective advantage. We are currently in the process of identifying genes altered by methylation in the proliferating cell as detected by RLGS-M. Another important question is defining the conditions that support or induce the selective expansion of methylated SMCs. Moreover, it is conceivable that gene methylation or absence thereof may contribute to susceptibility of SMCs to mutations [47,48]. Since mutation of SMC genes has been linked to plaque expansion, our findings are not inconsistent with other hypotheses proposed to explain the monoclonal origin of the atherosclerotic lesion [39,48].

DNA methylation is an epigenetic phenomenon that is present in unchecked proliferative states, such as neoplasms and restenotic lesions following angioplasty. Methylation has also been suggested as an adaptive mechanism to stress. While recognizing the limitations of our study, the fact that most CpG islands, which are associated with transcription start sites in some 40 000 human genes [46], are stringently protected from methylation, points to the importance of our findings of increased ER{alpha} methylation in a model of neointimal vs. medial cell type.

Time for primary review 22 days.


   Acknowledgements
 
We thank Julie Popovich and Julia Eisel for technical assistance. AKY was supported in part by the Stanley J. Sarnoff Endowment for Cardiovascular Science, CP by NIH grant GM58269, and DJS by NCI T32 CA09338. Support for this work was also provided by the Scleroderma Research Foundation and by NIH grant P30 CA16058.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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