Effects of Aging and Hypercholesterolemia on Oxidative Stress and DNA Damage in Bone Marrow Mononuclear Cells in Apolipoprotein E-deficient Mice

Abstract

Recent evidence from apolipoprotein E-deficient (apoE-/-) mice shows that aging and atherosclerosis are closely associated with increased oxidative stress and DNA damage in some cells and tissues. However, bone marrow cells, which are physiologically involved in tissue repair have not yet been investigated. In the present study, we evaluated the influence of aging and hypercholesterolemia on oxidative stress, DNA damage and apoptosis in bone marrow cells from young and aged apoE-/- mice compared with age-matched wild-type C57BL/6 (C57) mice, using the comet assay and flow cytometry. The production of both superoxide and hydrogen peroxide in bone marrow cells was higher in young apoE-/- mice than in age-matched C57 mice, and reactive oxygen species were increased in aged C57 and apoE-/- mice. Similar results were observed when we analyzed the DNA damage and apoptosis. Our data showed that both aging and hypercholesterolemia induce the increased production of oxidative stress and consequently DNA damage and apoptosis in bone marrow cells. This study is the first to demonstrate a functionality decrease of the bone marrow, which is a fundamental extra-arterial source of the cells involved in vascular injury repair.

Figure 1

Total serum cholesterol levels in young and aged apoE^−/− mice compared with age-matched C57 mice fed a regular chow diet. The values are presented as means ± SEM. ** p< 0.01 vs. the age-matched C57 group.

Figure 2

Vascular lipid deposition and senescence. Representative aorta en face macrographs of (a) senescence (X-gal, pH 6.0) and (c) lipid deposition (Oil-Red-O staining) in young and aged C57 mice compared with age-matched apoE^−/− mice. A remarkable large area of vascular senescence and lipid deposition was observed in aged apoE^−/− mice. Bar graphs show average vascular senescence (b) and lipid deposition (d) areas. The values are presented as means ± SEM. * p < 0.05 vs. the respective young group, ^#p < 0.05 vs. the age-matched C57 group.

Figure 3

Production of superoxide anions. Representative histograms from flow cytometric analysis using dihydroethidium (DHE) in young and aged C57 mice (a) compared with age-matched apoE^−/− mice (b); the log fluorescence (X axis) illustrates the intensity of fluorescence for the number of cells counted (note the higher scales in the apoE histogram). A remarkable increase in the level of superoxide anions was observed in aged mice mainly in apoE^−/− mice. (c) Bar graph showing mean fluorescence intensity (MFI). The values are presented as means ± SEM. * p < 0.05 vs. the respective young group, ^#p < 0.05 vs. the age-matched C57 group.

Figure 4

Production of hydrogen peroxide. Representative histogram from flow cytometric analysis with 2′,7′-dichlorofluorescein (DCF) in young and aged C57 mice (a) compared with age-matched apoE^−/− mice (b); the log fluorescence (X-axis) illustrates the intensity of fluorescence for the number of cells counted (note the higher scales in the apoE^−/− histogram). Increased levels of hydrogen peroxide were observed in aged mice mainly in apoE^−/− mice; (c) Bar graph showing mean fluorescence intensity (MFI). The values are presented as the means ± SEM. * p < 0.05 vs. the respective young group, ^#p < 0.05 vs. the age-matched C57 group.

Figure 5

Detection of DNA damage in individual bone marrow MNC assessed by comet assay. (a) Typical comets showing higher DNA fragmentation in aged mice compared to young mice mainly in apoE^−/− mice; (b) Line graphs of average percentages of cells for each of the genotoxicity levels, showing higher percentage of cells at higher level of genotoxicity in aged mice, which was aggravated in hypercholesterolemic mice; (c) Bar graph showing the total score of MNC with DNA damage. Values are means ± SEM. * p < 0.05 vs. respective young group, ^#p < 0.05 vs. age-matched C57 group.

Figure 6

Flow cytometric analysis of apoptosis. Typical dot plots showing apoptosis ratio comparing C57 mice (a) with age-matched apoE^−/− mice (b) using propidium iodide (PI) and FITC-annexin V. Q1 quadrant represents damaged cells (PI positive and annexin negative). Q2 quadrant represents cell that are in late apoptosis or already dead (necrotic cells), i.e., are both annexin and PI positive). Q3 quadrant represents viable cells, i.e., cells that are both annexin and PI negative. Q4 quadrant represents cells in early apoptosis (cell apoptosis) annexin positive and PI negative. Note a remarkable increase in apoptotic cells number (Q2 + Q4) in the aged animals. (c) Bar graph shows average percentage of apoptotic cells (Q2 + Q4). Values are means ± SEM. * p < 0.05 vs. the respective young group.

Figure 7

Flow cytometric histogram used to analysis of mononuclear cells (MNC). Typical dot plot representing (in linear scale) the forward scatter (FSC, correlates with the cell volume) vs. side scatter (SSC, correlates with granularity of the cell). The square (red) gate indicates the selected portion of data which matches with MNC and that was used in the analysis of oxidative stress (Figure 3 and Figure 4). A Total of 10,000 events were acquired for analysis.