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| Status |
Public on Dec 21, 2015 |
| Title |
Genome wide promoter methylation analysis of mouse macrophages and endothelial cells isolated from ischemic hindlimbs using RRBS sequencing |
| Organism |
Mus musculus |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
Purpose: The goal of this study was to compare the genome-wide promoter methylation alterations in macrophages and endothelial cells during hindlimb ischemia among normal, hyperlipidemic and type-2 diabetic mice.
Methods: Unilateral hindlimb ischemia was induced by ligating femoral artery proximal to the bifurcation of superficial and deep femoral artery in mice deficient of LDL receptor and expressing only apolipoprotein B100 (LDLR-/-ApoB100/100, C57BL/6J background) (The Jackson Laboratory, Bar Harbor,USA) and mice with β-cell specific over-expression of insulin-like growth factor-2 in atherosclerotic background (IGF-II/LDLR-/-ApoB100/100, C57BL/6J background) with type 2 diabetic features on high-fat diet (TD 88173, Harlan Teklad: 42% of calories from fat and 0.15% from cholesterol, no sodium cholate) 8 weeks prior to surgery and continued throughout the study 1. C57BL/6J (WT) mice fed with regular chow-diet (R36, Lactamin) served as controls. All animals were aged between 20 to 24 weeks at the time of hindlimb operations. For sorting macrophages from ischemic muscles, ischemic gastrocnemius muscles were minced and enzymatically dissociated using a cocktail containing 450 U/mL Collagenase I, 125 U/mL Collagenase XI, 60 U/mL DNAseI, and 60 U/mL hyaluronidase (Sigma Aldrich) for 1 h at 37°C. The cells were then counted and divided into CD31+ve and CD31-vefractions using CD31 magnetic bead enrichment (Miltenyi Biotec). For macrophage sorting CD31-ve fraction was incubated for 15 minutes with rat anti-mouse CD16/32 mAb (Fc Block, BD-pharmingen) and stained with FITC conjugated rat anti-mouse F4/80 antibody (Serotec) for 20 minutes at 4˚C. For endothelial sorting CD31+ fraction was incubated for 15 minutes with rat anti-mouse CD16/32 mAb (Fc Block, BD-pharmingen) and stained with APC conjugated rat anti-mouse CD31 antibody (BD-pharmingen) and FITC conjugated rat anti-mouse CD45 ((BD-pharmingen) for 20 minutes at 4˚C. FACS sorting was performed on FACS AriaIII (BD Biosciences). Genomic DNA was isolated from FACS sorted macrophages and endothelial cells using AllPrep DNA/RNA/Protein Mini Kit (Qiagen Finland, Helsinki, Finland) according to manufacturer's instructions.
Results: The sample similarity as assessed by Pearson’s correlation matrix and Hierarchial clustering showed high correalation among macrophages, as well as endothelial cells. There was a clear clustering of macrophages and endothelial cells as evidence by their CpG methylation clustering, furthermore macrophages from HL and T2DM mice showed clear clustering compared to control macrophages. Differential methylation analysis of RRBS methylation data from macrophages and endothelial cells was performed using Methylkit. Using a threshold of adjusted p value (Q) <0.05 and percentage methylation difference of >5%, we identified 198 and 272 genes whose promoters were hypomethylated in HL and T2DM macrophages. Similarly, there were 102 and 136 gene promoters were hypermethylated in HL and T2DM macrophages, respectively compare to control macrophages. Thus, proximal promoter methylation suggested that HL and T2DM have convergent influences on the proximal promoter methylation of numerous macrophage specific genes. In order to find out whether these genes with differential methylated promoters were differentially expressed at mRNA expression level in purified macrophages, we further compared our data with the GEO datasets as above. Of the 198 genes with promoter hypomethylation in HL macrophages 72 genes were suggested to be upregulated in M1- Mϕs; whereas, of the 102 genes with promoter hypermethylation, 51 genes were suggested to be upregulated in M2- Mϕs. Similarly, out of 272 genes with differentially methylated promoters in T2DM macrophages 88 genes were suggested to be upregulated in M1-Mϕs; whereas, out of 136 genes with promoter hypermethylation 60 genes were suggested to be upregulated in M2- Mϕs. Thus a significant promoter hypomethylation of M1-Mϕ and hypermethylation of M2-Mϕ genes suggested the predominance of proinflammatory M1-Mϕs in ischemic muscles of HL and T2DM compared to M2-Mϕs in control mice.
Conclusions: We found significant promoter hypomethylation of genes typical for proinflammatory M1-Mϕs and hypermethylation of anti-inflammatory, proangiogenic M2-Mϕ associated genes in HL and T2DM ischemic muscles. Epigenetic alterations skewing Mϕ phenotype towards proinflammatory as opposed to anti-inflammatory, proangiogenic and tissue repair phenotype may contribute to impaired adaptive vascular growth in these pathological conditions.
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| Overall design |
Macrophages and endothelial whole genome DNA methylation was performed in triplicates (Each sample was pooled from 3-4 mice) by RRBS Sequencing approach using Illumina HiSeq 2500. qRT–PCR validation was performed using TaqMan assays.
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| Contributor(s) |
Ylä-Herttuala S, Babu M |
| Citation(s) |
26085133 |
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| Submission date |
Feb 09, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Mohan Babu |
| E-mail(s) |
babu.mohan@uef.fi
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| Phone |
+358403553685
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| Organization name |
A.I.Virtanen Institute, University of Eastern Finland
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| Department |
Biotechnology and Molecular Medicine
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| Lab |
Molecular Medicine
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| Street address |
Neulaniementie 2
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| City |
Kuopio |
| ZIP/Postal code |
70211 |
| Country |
Finland |
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| Platforms (1) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
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| Samples (18)
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| This SubSeries is part of SuperSeries: |
| GSE65803 |
Differential promoter methylation of macrophage genes correlates with impaired vascular growth in ischemic muscles of hyperlipidemic and type 2 diabetic mice |
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| Relations |
| BioProject |
PRJNA274974 |
| SRA |
SRP053380 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE65759_ControlEC-vs-DiabEC_Diff-all-promoter.txt.gz |
143.4 Kb |
(ftp)(http) |
TXT |
| GSE65759_ControlEC-vs-HypEC_Diff-all-promoter.txt.gz |
169.1 Kb |
(ftp)(http) |
TXT |
| GSE65759_ControlMac-vs-ControlEC_Diff-all-promoter.txt.gz |
162.5 Kb |
(ftp)(http) |
TXT |
| GSE65759_ControlMac-vs-DiabMac_Diff-all-promoter.txt.gz |
157.9 Kb |
(ftp)(http) |
TXT |
| GSE65759_ControlMac-vs-HypMac_Diff-all-promoter.txt.gz |
135.9 Kb |
(ftp)(http) |
TXT |
| GSE65759_DiabMac-vs-DiabEC_Diff-all-promoter.txt.gz |
139.8 Kb |
(ftp)(http) |
TXT |
| GSE65759_HypMac-vs-HypEC_Diff-all-promoter.txt.gz |
139.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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