Hyperglycemia epigenetically propels senescence-associated secretory phenotype of CD34 hematopoietic stem cells and their myeloid expansion into inflammatory monocyte subpopulations

Abstract Background The "leitmotif "underlying the cardiovascular disease initiation and progression in diabetic patients is the chronic inflammation in which monocytes/macrophages are the key players [1]. Diabetes mellitus (DM) showed to alter hematopoiesis, by promoting myelopoiesis and the generation of monocyte subsets with inflammatory and atherogenic phenotype [2-4]. Epigenetic modifications have been described in the inflammation and in control of monocyte-derived macrophage polarization [5,6]. However, there is no notion of when these epigenetic modifications take place and whether can be transferred to more differentiated cells. Purpose The aims of the study were 1) to assess in vitro if high glucose (HG) exposure promotes epigenetic changes and alteration in myeloid differentiation of hematopoietic stem/progenitor cells HSPCs; 2) if such epigenetic modifications are transferred to cell progeny and 3) if similar mechanisms are at work in bone marrow (BM)-derived HSPCs of DM patients. Methods HSPCs were isolated form the cord blood (CB) and from BM of DM patients. CB-HSPCs were grown in normal-glucose (NG;30 mM mannitol) or HG (30 mM) conditions and counted after 10, 20 and 30 days. The expression of p27, p21, IL6, TNFα, RELA/p65, KAT2B/PCAF, SetD7 genes and telomere length was assessed by qPCR; cytokines by ELISA. Western Blot was used to evaluate NFkB-p65 expression and acetylation at lysine-310. H3K9me3, H3K4me1 modifications and SetD7, RNA polymerase II at RELA/p65 gene promoter were assessed by ChIP-qPCR assay. Flow cytometry was used to assess ROS, CD14/CD16 monocyte subpopulations and NFkB-p65 nuclear translocation. Results HG-exposure of CB-HSPCs induced a senescent-associated secretory phenotype characterized by cell proliferation lowering, ROS production, telomere shortening, up-regulation of p21 and p27 genes, increased secretion of TNFα and IL6 cytokines, and upregulation of NFkB-p65 transcription factor (fig.1). NFkB-p65 gene promoter analysis showed H3K9me3 and H3K4me1 histone mark alterations associated with increased SetD7 and RNA polymerase II recruitment. Importantly, the upregulation of KAT2B gene linked with increased lysine-310 acetylation, nuclear translocation and binding activity of NFkB-p65 (fig.1). HG-HSPCs, once differentiated into myeloid lineage, generated higher level of intermediate (CD14++CD16+) proinflammatory monocyte subpopulations (Fig.2). Similarly, BM-HSPCs from DM patients displayed a senescent-associated secretory phenotype and abnormal intermediate (CD14++CD16+) monocyte output when differentiated into myeloid lineage. Importantly, epigenetic changes at the level of NFkB-p65 gene promoter persisted in both HG-HSPC- and DM-HSPC-derived monocytes populations (fig.2). Conclusions Overall, our data show that hyperglycemia elicits epigenetic changes in HSPCs that persist in monocyte progeny and are potentially involved in their inflammatory phenotype.fig.1fig.2