<i>A priori</i> test of CpG sites in SMC specific genes reveals a trend towards hypermethylation.

<p>A priori test was performed to select 6831 CpG sites associated with SMC specific genes from the Illumina 450 K array data, followed by t-test and Benjamini-Hochberg correction for multiple testing. Heat map demonstrates a global shift in hypermethylation in top 100 SMC-related CpG sites.</p

Differentially methylated CpG sites (T-test <0.01, adjusted p value <0.05, Benjamini-Hochberg) revealed after analysis of specific regions or all sites of the epigenome.

<p>Differentially methylated CpG sites (T-test <0.01, adjusted p value <0.05, Benjamini-Hochberg) revealed after analysis of specific regions or all sites of the epigenome.</p

Nuclear Localization of DNMT3A is dependent upon the time after plating and transcription.

<p>(A) Timecourse of intracellular DNMT3A expression/localization after plating cells on NC and DNC. DNC plated cells show stronger DNMT3A signals overall than NC plated cells. The 36 hour timepoint shows strong signal in the nucleus of DNC plated cells. At 48 hours there continues to be high expression in the DNC cells, though the nuclear stain was not as clear as the 36 hour timepoint. NC cells did not show nuclear staining. (B) DNMT3A nuclear localization is slightly affected by inhibitors of transcription (actinomycin D) and translation (cyclohexamide) on NC, but downregulation on DNC strongly depends on both functions. SMC were plated for 4 hours as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069089#pone-0069089-g001" target="_blank">Figure 1</a> and treated with cyclohexamide or actinomycin for the next 44 hours.</p

Damaged matrix induces DNMT3A nuclear expression in human bladder SMC and changes in methylation status in CpG sites of the Illumina 450K methylation array.

<p>(A) Human bladder smooth muscle cells were plated on native (NC) or denatured collagen (DNC) at low density (4×10⁴ cells/mL) for 6 hours in EMEM with 6% FCS, then media was changed to 2% FCS in EMEM. Nuclear expression of DNMT3A is increased in SMC cultured on DNC. By immunofluorescent staining, levels of DNMT3A and smooth muscle myosin heavy chain (MHC, smooth muscle-specific form) were examined by spinning disk microscopy using Volocity software, then analysed with Image J. DNMT3A and 3B were both examined by QPCR. While DNMT3A levels were not significantly increased by mRNA expression, protein expression of DNMT3A and DNMT3B levels were increased *, p<0.05. (B) Illumina 450 K CpG methylation array of human SMC plated onto NC and DNC show several significant changes at discrete hypomethylated and hypermethylated CpG sites on DNC compared to NC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069089#pone.0069089.s005" target="_blank">Table S2</a> for raw data). Red diamonds indicate significantly altered CpG methylation (adjusted p<0.05, by Benjamini-Hochberg).</p

<i>A priori</i> test of CpG sites in SMC specific genes reveals specific changes in DNA methylation.

<p>(A) Volcano plot of hypomethylated and hypermethylated CpG sites reveals a clear trend toward hypermethylation of sites in cells plated on DNC. 14 CpG sites have statistically significant increase in methylation. (B) Beta values (degree of methylation) in 14 CpG sites near 12 genes differed between cells cultured on NC and DNC. Differences between cells on native collagen and denatured collagen were significantly altered in all sites (adjusted p value <0.05).</p

Expression of DNMT3A is differentially regulated by proliferative state on tissue culture plastic.

<p>SMC were plated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069089#pone-0069089-g001" target="_blank">Figure 1</a> then either fixed for immunstaining or harvested for protein and analysis by western. (A) Immunofluorescent staining of DNMT3A and myosin in quiescent (serum-starved) or proliferative (10% serum) SMC on tissue culture plastic (TC). Proliferative SMC express more peri-nuclear/cytosolic DNMT3a compared to quiescent SMC. (B) Western blotting of DNMT3A1 in extractions of SMC show that proliferative vs. quiescent (serum starved) SMC (plated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069089#pone-0069089-g004" target="_blank">Figure 4</a>.A) expressed higher levels of DNMT3A1 protein (120 kDa) compared to quiescent SMC. (C) Western blotting of DNMT3A isoforms in SMC plated on DNC, TC and NC. DNMT3A1 shows more expression on DNC than on TC but is not expressed on NC. On the other hand, DNMT3A2 shows less expression on DNC compared to NC while showing the most protein expression on TC.</p

Phenotypic Switching Induced by Damaged Matrix Is Associated with DNA Methyltransferase 3A (DNMT3A) Activity and Nuclear Localization in Smooth Muscle Cells (SMC)

<div><p>Extracellular matrix changes are often crucial inciting events for fibroproliferative disease. Epigenetic changes, specifically DNA methylation, are critical factors underlying differentiated phenotypes. We examined the dependency of matrix-induced fibroproliferation and SMC phenotype on DNA methyltransferases. The cooperativity of matrix with growth factors, cell density and hypoxia was also examined. Primary rat visceral SMC of early passage (0–2) were plated on native collagen or damaged/heat-denatured collagen. Hypoxia was induced with 3% O₂ (balanced 5% CO₂ and 95% N₂) over 48 hours. Inhibitors were applied 2–3 hours after cells were plated on matrix, or immediately before hypoxia. Cells were fixed and stained for DNMT3A and smooth muscle actin (SMA) or smooth muscle myosin heavy chain. Illumina 450 K array of CpG sites was performed on bisulfite-converted DNA from smooth muscle cells on damaged matrix vs native collagen. Matrix exquisitely regulates DNMT3A localization and expression, and influences differentiation in SMCs exposed to denatured matrix +/− hypoxia. Analysis of DNA methylation signatures showed that Matrix caused significant DNA methylation alterations in a discrete number of CpG sites proximal to genes related to SMC differentiation. Matrix has a profound effect on the regulation of SMC phenotype, which is associated with altered expression, localization of DNMTs and discrete changes DNA methylation.</p></div

Hypoxia and damaged matrix increase DNMT3A nuclear expression in a cooperative fashion.

<p>SMCs were plated on native (NC) or denatured collagen (DNC) at low density (4×10⁴ cells/mL) for 6 hours in EMEM with 6% FCS, then media was changed to 2% FCS in EMEM. (A) SMC were plated on native (NC) or denatured collagen (DNC) and cultured under normoxia (21% O₂) or hypoxia (3% O₂). Hypoxia significantly enhanced the nuclear expression of DNMT3A and the down-regulation of myosin. By immunofluorescent staining, levels of DNMT3A and smooth muscle myosin heavy chain (MHC, smooth muscle-specific form) were examined by spinning disk microscopy using Volocity software, then analysed with Image J. *, p<0.05. (B) Expression of α-SMA was significantly decreased under the combined stimulation by hypoxia and damaged collagen, compared to native collagen. Both PCR and immunofluorescent staining with anti-smooth muscle actin antibody revealed a significant decrease in actin expression only on denatured collagen. (C) The expression of DNMT3A is upregulated in DNC compared to NC. Consistent with immunofluorescent staining data, the upregulation of DNMT3A mRNA expression on DNC is enhanced by hypoxia.</p

DNMT3A expression is regulated by cell-density, mitosis but not mitogenic growth factors.

<p>SMC were plated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069089#pone-0069089-g001" target="_blank">Figure 1</a>. (A) Cell density affects localization of DNMT3A to the nucleus. (B) Nuclear expression of DNMT3A is decreased by the mitotic inhibitor nocodazole in cells. (C) EGF (50 μg/mL) and FGF (10 μg/mL) fail to alter nuclear localization from patterns established on NC or DNC.</p

Proliferation and de-differentiation of SMC on denatured matrix depends on DNMT activity in visceral smooth muscle cells.

<p>(A) SMC were plated on native (NC) or denatured collagen (DNC) at low density (2×10⁴ cells/mL) for 6 hours in EMEM with 6% FCS, then treated with 5-aza-2′-deoxycytidine (DAC) or vehicle for another 42 hours in EMEM with 2% FCS. Six different fields per treatment for cells positive for DAPI were examined at 5× magnification and counted using Volocity analysis software, and averaged to obtain the # cells/field. * p<0.05 vs DAC treatments. (B) Loss of smooth muscle myosin could be reversed with rapamycin plus DAC. <b><u>Before treatment</u></b>, SMC were cultured for 48 hours <i>in vitro</i> on damaged collagen matrices (DNC), which suppressed expression of the differentiation marker Myosin (relative immunofluorescence expression  = 1.0). The mTOR inhibitor rapamycin alone showed only a trend in increasing Myosin expression (p = 0.11), but combined use of epigenetic inhibition (with DAC) + rapamycin significantly restored myosin expression (*p<0.04).</p