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. 2015 Oct 24:22:97.
doi: 10.1186/s12929-015-0201-8.

Reactive oxygen species contribute to dysfunction of bone marrow hematopoietic stem cells in aged C57BL/6 J mice

Marcella L Porto  1 , Bianca P Rodrigues  2 , Thiago N Menezes  3 , Sara L Ceschim  4 , Dulce E Casarini  5 , Agata L Gava  6 , Thiago Melo C Pereira  7   8 , Elisardo C Vasquez  9   10 , Bianca P Campagnaro  11 , Silvana S Meyrelles  12
Affiliations

Reactive oxygen species contribute to dysfunction of bone marrow hematopoietic stem cells in aged C57BL/6 J mice

Marcella L Porto et al. J Biomed Sci. .

Abstract

Background: Stem cells of intensely regenerative tissues are susceptible to cellular damage. Although the response to this process in hematopoietic stem cells (HSCs) is crucial, the mechanisms by which hematopoietic homeostasis is sustained are not completely understood. Aging increases reactive oxygen species (ROS) levels and inflammation, which contribute to increased proliferation, senescence and/or apoptosis, leading to self-renewal premature exhaustion. In this study, we assessed ROS production, DNA damage, apoptosis, senescence and plasticity in young, middle and aged (2-, 12- and 24-month-old, respectively) C57BL/6 J mice.

Results: Aged HSCs showed an increase in intracellular superoxide anion (1.4-fold), hydrogen peroxide (2-fold), nitric oxide (1.6-fold), peroxynitrite/hidroxil (2.6-fold) compared with young cells. We found that mitochondria and NADPHox were the major sources of ROS production in the three groups studied, whereas CYP450 contributed in middle and aged, and xanthine oxidase only in aged HSCs. In addition, we observed DNA damage and apoptosis in the middle (4.2- and 2-fold, respectively) and aged (6- and 4-fold, respectively) mice; aged mice also exhibited a significantly shorter telomere length (-1.8-fold) and a lower expression of plasticity markers.

Conclusion: These data suggest that aging impairs the functionality of HSCs and that these age-associated alterations may affect the efficacy of aged HSC recovery and transplantation.

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Figures

Fig. 1
Fig. 1
ROS generation and oxidative stress pathways are involved in aging. A- ROS production was assessed by DHE, DCF, DAF and HPF staining. Top panel shows representative images of HSCs; the aged group presented change in the number of cells that stained ROS-positive compared with young animals. Bar graph shows the difference on MFI of the ROS measured by flow cytometry among the groups. Values are means ± SEM, *p < 0.05 vs. young. # p < 0.05 vs. middle
Fig. 2
Fig. 2
Identification of the main sources of ROS production and antioxidant enzyme capacity by oxidative stress pathway analysis. Bar graphs show the effect of different oxidant proteins inhibitors on ROS production and antioxidant proteins inhibitors on ROS reduction. ROS levels were analyzed by DHE and DCF fluorescence intensity. Unshaded bars represent basal ROS levels, and shaded bars show ROS levels after blockade. Abbreviations: NADPH oxidase (NADPHox), xanthine oxidase (XO), cytochrome P450 (CYP450), superoxide dismutase (SOD), catalase (CAT), gluthatione peroxidase (GPx). Values are means ± SEM, *p < 0.05 vs. matched basal
Fig. 3
Fig. 3
Augmented ROS leads to DNA damage and apoptosis of HSCs during aging. a The top panel shows typical photographs of comets with higher DNA fragmentation in the middle and aged groups. Bar graphs show the percentage of DNA in the tail and the tail moment. b Table shows the average percentage of viable cells, early apoptosis, late apoptosis and necrosis detected by annexin-V and PI staining. c Correlation between intracellular ROS levels and apoptosis. Values are means ± SEM, *p < 0.05 vs. young. # p < 0.05 vs. middle
Fig. 4
Fig. 4
Aging leads to molecular and cellular senescence of HSCs. a The top panel shows typical senescent cells with blue cytoplasm (arrow) and the bar graph shows the percentage of positive cells quantified by cell counting (bottom panel). b Top panel shows the correlation between intracellular ROS levels and acridine orange fluorescence. The bar graph shows the median fluorescence intensity of acridine orange (bottom panel). c The top panel shows the correlation between the intracellular ROS levels and relative telomere length (RTL). Bottom panel show the percentage of RTL. Values are means ± SEM, *p < 0.05 vs. young
Fig. 5
Fig. 5
Pluripotency markers are downregulated by augmented ROS production during aging. The top panels show the correlation between intracellular ROS and ONS transcription factors. Bar graphs show the percentage of positive ONS cells. Values are the means ± SEM, *p < 0.05 vs. young. Abbreviation: Oct-3,4/Nanog/Sox-2 transcription factors (ONS)
Fig. 6
Fig. 6
Scheme showing multiple age-related factors regulating hematopoietic stem cell function. Alterations in HSC function observed in aging: generation and main types of damage induced by ROS, different mechanisms of oxidative cellular/molecular damage and the potential role of antioxidants on HSC fate during the aging process. Lines with arrowhead indicate stimulation and lines with crosshead indicate inhibition. A line with a double arrowhead indicates a normal condition. Abbreviations: hematopoietic stem cells (HSC), reactive oxygen species (ROS), NADPH oxidase (NADPHox), cytochrome P450 (CYP450), xanthine oxidase (XO), lymphoid cells (L), myeloid cells (M), tumoral necrosis factor (TNF), interleukin-12p70 (IL-12), interleukin-6 (IL-6)
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