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Arterial endothelial methylome: differential DNA methylation in athero-susceptible disturbed flow regions in vivo.

Jiang YZ, Manduchi E, Stoeckert CJ, Davies PF - BMC Genomics (2015)

Bottom Line: Gender-specific DMRs associated with ciliogenesis that may be linked to defects in cilia development were also identified in AA DMRs.An endothelial methylome analysis identifies epigenetic DMR characteristics associated with transcriptional regulation in regions of atherosusceptibility in swine aorta in vivo.The data represent the first methylome blueprint for spatio-temporal analyses of lesion susceptibility predisposing to endothelial dysfunction in complex flow environments in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Laboratory Medicine and Institute for Medicine & Engineering, Perelman School of Medicine, University of Pennsylvania, 1010 Vagelos Building, 3340 Smith Walk, Philadelphia, PA, 19104, USA. jyizhou@mail.med.upenn.edu.

ABSTRACT

Background: Atherosclerosis is a heterogeneously distributed disease of arteries in which the endothelium plays an important central role. Spatial transcriptome profiling of endothelium in pre-lesional arteries has demonstrated differential phenotypes primed for athero-susceptibility at hemodynamic sites associated with disturbed blood flow. DNA methylation is a powerful epigenetic regulator of endothelial transcription recently associated with flow characteristics. We investigated differential DNA methylation in flow region-specific aortic endothelial cells in vivo in adult domestic male and female swine.

Results: Genome-wide DNA methylation was profiled in endothelial cells (EC) isolated from two robust locations of differing patho-susceptibility:--an athero-susceptible site located at the inner curvature of the aortic arch (AA) and an athero-protected region in the descending thoracic (DT) aorta. Complete methylated DNA immunoprecipitation sequencing (MeDIP-seq) identified over 5500 endothelial differentially methylated regions (DMRs). DMR density was significantly enriched in exons and 5'UTR sequences of annotated genes, 60 of which are linked to cardiovascular disease. The set of DMR-associated genes was enriched in transcriptional regulation, pattern specification HOX loci, oxidative stress and the ER stress adaptive pathway, all categories linked to athero-susceptible endothelium. Examination of the relationship between DMR and mRNA in HOXA genes demonstrated a significant inverse relationship between CpG island promoter methylation and gene expression. Methylation-specific PCR (MSP) confirmed differential CpG methylation of HOXA genes, the ER stress gene ATF4, inflammatory regulator microRNA-10a and ARHGAP25 that encodes a negative regulator of Rho GTPases involved in cytoskeleton remodeling. Gender-specific DMRs associated with ciliogenesis that may be linked to defects in cilia development were also identified in AA DMRs.

Conclusions: An endothelial methylome analysis identifies epigenetic DMR characteristics associated with transcriptional regulation in regions of atherosusceptibility in swine aorta in vivo. The data represent the first methylome blueprint for spatio-temporal analyses of lesion susceptibility predisposing to endothelial dysfunction in complex flow environments in vivo.

No MeSH data available.


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MSP analyses of selected DMRs relative to gene expression in athero-susceptible endothelia. Methylation scores and mRNA levels of a ATF4, b microRNA-10a, c HOXA5, d HOXD4 and e ARHGAP25 were measured. The methylation scores of CpG sites were calculated by BALM. 1 = 100 % methylated, 0 = unmethylated. The average methylation scores of AA and DT are shown as black and grey areas respectively (upper panel, n = 12 animals). Distance to the TSS is shown on the x-axis. Relative methylation levels of regions within the DMRs were analyzed by MSP (bottom left). mRNA levels were quantified by qPCR (bottom right). MSP and qPCR data were normalized (See Methods for further details) and are expressed as fold of DT. *indicates significant difference between AA and DT (n = 6, p < 0.05). chr, chromosome. TSS, transcription start site. TTS, transcription termination site. mRNA data for ATF4 and microRNA-10a were adapted from Civelek et al. [6]. and Fang et al. [14]; reprinted with permission.
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Fig7: MSP analyses of selected DMRs relative to gene expression in athero-susceptible endothelia. Methylation scores and mRNA levels of a ATF4, b microRNA-10a, c HOXA5, d HOXD4 and e ARHGAP25 were measured. The methylation scores of CpG sites were calculated by BALM. 1 = 100 % methylated, 0 = unmethylated. The average methylation scores of AA and DT are shown as black and grey areas respectively (upper panel, n = 12 animals). Distance to the TSS is shown on the x-axis. Relative methylation levels of regions within the DMRs were analyzed by MSP (bottom left). mRNA levels were quantified by qPCR (bottom right). MSP and qPCR data were normalized (See Methods for further details) and are expressed as fold of DT. *indicates significant difference between AA and DT (n = 6, p < 0.05). chr, chromosome. TSS, transcription start site. TTS, transcription termination site. mRNA data for ATF4 and microRNA-10a were adapted from Civelek et al. [6]. and Fang et al. [14]; reprinted with permission.

Mentions: Transcript mRNA levels of annotated HOXA genes were measured by qPCR in endothelium from atherosusceptible AA and athero-protected DT sites to determine the relationship between AA/DT transcript expression (mRNA) and AA/DT DMR methylation score for 9 HOXA genes: − HOXA1-A7, HOXA10 and HOXA13 (Fig. 6). HOXA DMRs expressed a significant inverse relationship to transcript expression (Fig. 6b; r = −0.73, p = 0.027). AA hypermethylation predicted AA transcript suppression and hypomethylation predicted enhanced mRNA. However an exception to the inverse relationship is HOXA10 which is both hypomethylated in AA and transcriptionally suppressed in AA. This occurs because the DMR is located in the gene body (similar to HOXD4 in Fig. 7d below). Notably, HOXA5 transcription in AA was the most suppressed of the HOXA genes, consistent with its identification in disturbed flow regions of mouse carotid artery [20].Fig. 7


Arterial endothelial methylome: differential DNA methylation in athero-susceptible disturbed flow regions in vivo.

Jiang YZ, Manduchi E, Stoeckert CJ, Davies PF - BMC Genomics (2015)

MSP analyses of selected DMRs relative to gene expression in athero-susceptible endothelia. Methylation scores and mRNA levels of a ATF4, b microRNA-10a, c HOXA5, d HOXD4 and e ARHGAP25 were measured. The methylation scores of CpG sites were calculated by BALM. 1 = 100 % methylated, 0 = unmethylated. The average methylation scores of AA and DT are shown as black and grey areas respectively (upper panel, n = 12 animals). Distance to the TSS is shown on the x-axis. Relative methylation levels of regions within the DMRs were analyzed by MSP (bottom left). mRNA levels were quantified by qPCR (bottom right). MSP and qPCR data were normalized (See Methods for further details) and are expressed as fold of DT. *indicates significant difference between AA and DT (n = 6, p < 0.05). chr, chromosome. TSS, transcription start site. TTS, transcription termination site. mRNA data for ATF4 and microRNA-10a were adapted from Civelek et al. [6]. and Fang et al. [14]; reprinted with permission.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4492093&req=5

Fig7: MSP analyses of selected DMRs relative to gene expression in athero-susceptible endothelia. Methylation scores and mRNA levels of a ATF4, b microRNA-10a, c HOXA5, d HOXD4 and e ARHGAP25 were measured. The methylation scores of CpG sites were calculated by BALM. 1 = 100 % methylated, 0 = unmethylated. The average methylation scores of AA and DT are shown as black and grey areas respectively (upper panel, n = 12 animals). Distance to the TSS is shown on the x-axis. Relative methylation levels of regions within the DMRs were analyzed by MSP (bottom left). mRNA levels were quantified by qPCR (bottom right). MSP and qPCR data were normalized (See Methods for further details) and are expressed as fold of DT. *indicates significant difference between AA and DT (n = 6, p < 0.05). chr, chromosome. TSS, transcription start site. TTS, transcription termination site. mRNA data for ATF4 and microRNA-10a were adapted from Civelek et al. [6]. and Fang et al. [14]; reprinted with permission.
Mentions: Transcript mRNA levels of annotated HOXA genes were measured by qPCR in endothelium from atherosusceptible AA and athero-protected DT sites to determine the relationship between AA/DT transcript expression (mRNA) and AA/DT DMR methylation score for 9 HOXA genes: − HOXA1-A7, HOXA10 and HOXA13 (Fig. 6). HOXA DMRs expressed a significant inverse relationship to transcript expression (Fig. 6b; r = −0.73, p = 0.027). AA hypermethylation predicted AA transcript suppression and hypomethylation predicted enhanced mRNA. However an exception to the inverse relationship is HOXA10 which is both hypomethylated in AA and transcriptionally suppressed in AA. This occurs because the DMR is located in the gene body (similar to HOXD4 in Fig. 7d below). Notably, HOXA5 transcription in AA was the most suppressed of the HOXA genes, consistent with its identification in disturbed flow regions of mouse carotid artery [20].Fig. 7

Bottom Line: Gender-specific DMRs associated with ciliogenesis that may be linked to defects in cilia development were also identified in AA DMRs.An endothelial methylome analysis identifies epigenetic DMR characteristics associated with transcriptional regulation in regions of atherosusceptibility in swine aorta in vivo.The data represent the first methylome blueprint for spatio-temporal analyses of lesion susceptibility predisposing to endothelial dysfunction in complex flow environments in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Laboratory Medicine and Institute for Medicine & Engineering, Perelman School of Medicine, University of Pennsylvania, 1010 Vagelos Building, 3340 Smith Walk, Philadelphia, PA, 19104, USA. jyizhou@mail.med.upenn.edu.

ABSTRACT

Background: Atherosclerosis is a heterogeneously distributed disease of arteries in which the endothelium plays an important central role. Spatial transcriptome profiling of endothelium in pre-lesional arteries has demonstrated differential phenotypes primed for athero-susceptibility at hemodynamic sites associated with disturbed blood flow. DNA methylation is a powerful epigenetic regulator of endothelial transcription recently associated with flow characteristics. We investigated differential DNA methylation in flow region-specific aortic endothelial cells in vivo in adult domestic male and female swine.

Results: Genome-wide DNA methylation was profiled in endothelial cells (EC) isolated from two robust locations of differing patho-susceptibility:--an athero-susceptible site located at the inner curvature of the aortic arch (AA) and an athero-protected region in the descending thoracic (DT) aorta. Complete methylated DNA immunoprecipitation sequencing (MeDIP-seq) identified over 5500 endothelial differentially methylated regions (DMRs). DMR density was significantly enriched in exons and 5'UTR sequences of annotated genes, 60 of which are linked to cardiovascular disease. The set of DMR-associated genes was enriched in transcriptional regulation, pattern specification HOX loci, oxidative stress and the ER stress adaptive pathway, all categories linked to athero-susceptible endothelium. Examination of the relationship between DMR and mRNA in HOXA genes demonstrated a significant inverse relationship between CpG island promoter methylation and gene expression. Methylation-specific PCR (MSP) confirmed differential CpG methylation of HOXA genes, the ER stress gene ATF4, inflammatory regulator microRNA-10a and ARHGAP25 that encodes a negative regulator of Rho GTPases involved in cytoskeleton remodeling. Gender-specific DMRs associated with ciliogenesis that may be linked to defects in cilia development were also identified in AA DMRs.

Conclusions: An endothelial methylome analysis identifies epigenetic DMR characteristics associated with transcriptional regulation in regions of atherosusceptibility in swine aorta in vivo. The data represent the first methylome blueprint for spatio-temporal analyses of lesion susceptibility predisposing to endothelial dysfunction in complex flow environments in vivo.

No MeSH data available.


Related in: MedlinePlus