Global epigenetic alterations of mesenchymal stem cells in obesity: the role of vitamin C reprogramming

Abstract

Obesity promotes dysfunction and impairs the reparative capacity of mesenchymal stem/stromal cells (MSCs), and alters their transcription, protein content, and paracrine function. Whether these adverse effects are mediated by chromatin-modifying epigenetic changes remains unclear. We tested the hypothesis that obesity imposes global DNA hydroxymethylation and histone tri-methylation alterations in obese swine abdominal adipose tissue-derived MSCs compared to lean pig MSCs. MSCs from female lean (n = 7) and high-fat-diet fed obese (n = 7) domestic pigs were assessed using global epigenetic assays, before and after in-vitro co-incubation with the epigenetic modulator vitamin-C (VIT-C) (50 μg/ml). Dot blotting was used to measure across the whole genome 5-hydroxyemthycytosine (5hmC) residues, and Western blotting to quantify in genomic histone-3 protein tri-methylated lysine-4 (H3K4me3), lysine-9 (H3K9me3), and lysine-27 (H3K27me3) residues. MSC migration and proliferation were studied in-vitro. Obese MSCs displayed reduced global 5hmC and H3K4m3 levels, but comparable H3K9me3 and H3K27me3, compared to lean MSCs. Global 5hmC, H3K4me3, and HK9me3 marks correlated with MSC migration and reduced proliferation, as well as clinical and metabolic characteristics of obesity. Co-incubation of obese MSCs with VIT-C enhanced 5hmC marks, and reduced their global levels of H3K9me3 and H3K27me3. Contrarily, VIT-C did not affect 5hmC, and decreased H3K4me3 in lean MSCs. Obesity induces global genomic epigenetic alterations in swine MSCs, involving primarily genomic transcriptional repression, which are associated with MSC function and clinical features of obesity. Some of these alterations might be reversible using the epigenetic modulator VIT-C, suggesting epigenetic modifications as therapeutic targets in obesity.

Keywords: DNA hydroxymethylation; Epigenetics; histone tri-methylation; obesity; vitamin C.

Figure 1.

A) proliferation capacity of Lean (n = 5) and Obese (n = 5) mesenchymal stem cells (MSCs) per group; obese MSCs (dashed lines) had reduced proliferative capacity compared to Lean MSCs (solid lines) over time, and at 8 hours (p < 0.001). Error bars represent standard errors of proliferation for MSC samples at each time point. b) migration capacity of Lean (n = 5) and Obese MSCs (n = 5); obese and Lean MSCs had comparable migration capacity (p = 0.569). Comparison of mRNAs in Obese and Lean MSCs with heat maps showed 40 mRNAs upregulated (c) and 20 mRNAs downregulated (d) in Obese MSCs

Figure 2.

Global epigenetic alterations in Obese compared to Lean MSCs demonstrated by: a) dot-blotting of DNA hydroxymethylation measuring genomic 5-hydroxymethyl-cytosine (5hmC), b) and Western blotting of histone-3 protein measuring genomic tri-methylation on lysine-4 (H3K4me3), 9 (H3K9me3), and 27 (H3K27me3) residues. Genomic 5hmC and H3K4me3 were reduced in Obese compared to Lean MSCs, but there were no different in Global H3K9me3 and H3K27me3 between Lean and Obese MSCs. *p ≤ 0.05 vs. Lean MSC

Figure 3.

Effects of the epigenetic modulator vitamin C (VIT-C) on genomic 5hmC in Lean (solid lines) and Obese (dashed lines) MSCs. VIT-C increased genomic 5hmC in Obese MSCs, but did not change 5hmC in Lean MSCs. *p ≤ 0.05 vs. baseline

Figure 4.

Effects of VIT-C on protein expression of genomic histone H3K4me3, H3K9me3, and H3K27me3 in Lean (solid lines) and Obese (dashed lines) MSCs. VIT-C reduced H3K4me3 in Lean MSCs, and H3K9me3 and H3K27me3 in Obese MSCs. *p ≤ 0.05 vs. baseline

Figure 5.

Gene expression of epigenetic enzymes in Obese compared to Lean MSCs. The epigenetic enzymes EZH2, HAT1, TET1, TET2, and LSD2 had increased expression in Obese MSCs relative to GAPDH on RT-qPCR. *p ≤ 0.05 vs. Lean MSC