P2252Hyperglycemia drives myeloid CD34+ stem cells differentiation towards pro-inflammatory and senescent monocyte subpopulations
EUROPEAN HEART JOURNAL(2019)
Abstract
Abstract Background/Introduction Diabetes is characterized by a chronic low-grade inflammatory status mediated by cellular senescence and alterations of the circulating cytokine profile and of innate immune system cell components. An abnormal expansion of intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations with highly inflammatory and senescent-associated secretory phenotype (SASP) have been observed in T2DM patients with cardiovascular (CV) complications. It is unknown whether CD34+ hematopoietic stem cells (HSCs), players of CV prognosis, are involved in this process. Purpose To assess whether hyperglycemia might induce a pro-inflammatory priming of hematopoietic CD34+ HSCs that through senescence and acquisition of SASP skew their myeloid differentiation into more aggressive monocyte populations. Methods CD34+ cells were purified from cord blood of healthy donors and expanded in normal-glucose (NG; with 30 mM mannitol for osmotic control) or high-glucose (HG; 30 mM) serum-free medium plus cytokines. The cell were counted after 10, 20 and 30 days. The expression of p27, p21 RELA/p65, IL6, TNFα genes and telomere length was assessed by qPCR and secreted cytokines by ELISA. Apoptosis, ROS and monocyte subpopulations were evaluated by flow cytometry after Annexin V, CellRox and CD14/CD16 staining respectively. Results CD34+ HSCs cultured in HG (HG-CD34+) displayed a significant proliferation impairment when compared to their osmotic control (NG-CD34+). This loss of glucose tolerance was associated with a significant increase in mitochondrial ROS production (n=6; p≤0.01) without induction of apoptosis as showed by flow cytometry analysis for Annexin V. Moreover, qPCR assay revealed a significant telomere shortening (n=4; p≤0.05) and up-regulation of cyclin-dependent kinase inhibitors p27 and p21 in HG-CD34+cells (n=13; p≤0.05) along with an enhanced expression and secretion of TNFα (n=9; p≤0.05) and IL6 (n=10; p≤0.05) in HG-CD34+ when compared with NG-CD34+. SASP phenotype in HG-CD34+ was associated to a significant up-regulation of RELA/p65 gene in HG-CD34+ when compared with NG-CD34+ (n=8; p≤0.05). Furthermore, we found that in vitro HG-CD34+ differentiation into myeloid lineage generated higher levels of pro-inflammatory intermediate (n=3; p≤0.05) and non-classical (n=3; p≤0.01) monocyte subsets when compared with the normoglycemic counterpart. Conclusion(s) These data suggest that HG exposure primes HSCs myeloid differentiation towards inflammatory and senescent monocyte subpopulations. Acknowledgement/Funding This work was supported by Ricerca Finalizzata, Ministero della Salute [PE-2011-02348537]
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Key words
stem cells,p2252hyperglycemia drives,senescent monocyte subpopulations,cells differentiation,pro-inflammatory
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