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 trimethylation 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.

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ObjectiveElevated Reactive Oxygen Species (ROS) in Endothelial Cells (EC's) have been implicated in the pathogenesis of cardiovascular diseases, and much research has gone into elucidating the mechanisms through which oxidative stress leads to these diseases. Research in the last decade, however, has uncovered the central role lower levels of ROS play in physiological intracellular signaling pathways, such as those stimulated by Vascular Endothelial Growth Factor (VEGF) and Tumour Necrosis Factor‐α (TNF‐α) in EC's. Our objective in this study was to determine the effect of a lack of ROS in EC's on the intracellular signaling pathways stimulated by VEGF and TNF‐α.MethodsThe activity of these pathways can be measured by the expression of key downstream proteins that are common to these two pathways such as Akt, ERK‐1/2, IKKα/B, AMPK, eNOS, mTOR and FOXO1. To investigate these pathways in vitro, Bovine Endothelial Aortic Cells (BAEC's) were treated with ROS inhibitors (NAC, DPI) and then exposed to VEGF or TNF‐α to stimulate their respective pathways. Protein was then extracted and transferred to a membrane by Western Blotting then visualized by immunoflourescence.ResultsWestern blots demonstrated that reduction of ROS levels by NAC inhibited VEGF‐induced Akt phosphorylation but not phosphorylation of ERK1/2 in BAEC. The findings of TNF‐induced signaling activation were not conclusive.ConclusionWhereas VEGF‐mediated activation of PI3K‐Akt activation requires ROS, ERK1/2 activation is independent of ROS in ECs.This research was funded by National Institutes of Health (NIH) and American Heart Association (AHA) grants.