Mitochondrial Oxidative Stress Promotes Atherosclerosis and Neutrophil Extracellular Traps in Aged Mice

Abstract

Rationale: Mitochondrial oxidative stress (mitoOS) has been shown to be increased in various cell types in human atherosclerosis and with aging. However, the role of cell type-specific mitoOS in atherosclerosis in the setting of advanced age and the molecular mechanisms remains to be determined in vivo.

Objective: The aim of this study was to examine the role of myeloid cell mitoOS in atherosclerosis in aged mice.

Approach and results: Lethally irradiated low-density lipoprotein receptor-deficient mice (Ldlr^-/-) were reconstituted with bone marrow from either wild-type or mitochondrial catalase (mCAT) mice. mCAT transgenic mice contain ectopically expressed human catalase gene in mitochondria, which reduces mitoOS. Starting at the age of 36 weeks, mice were fed the Western-type diet for 16 weeks. We found that mitoOS in lesional myeloid cells was suppressed in aged mCAT→Ldlr^-/- chimeric mice compared with aged controls, and this led to a significant reduction in aortic root atherosclerotic lesion area despite higher plasma cholesterol levels. Neutrophil extracellular traps (NETs), a proinflammatory extracellular structure that contributes to atherosclerosis progression, were significantly increased in the lesions of aged mice compared with lesions of younger mice. Aged mCAT→Ldlr^-/- mice had less lesional neutrophils and decreased NETs compared with age-matched wild-type→Ldlr^-/- mice, whereas young mCAT→ and wild-type→Ldlr^-/- mice had comparable numbers of neutrophils and similar low levels of lesional NETs. Using cultured neutrophils, we showed that suppression of mitoOS reduced 7-ketocholesterol-induced NET release from neutrophils of aged but not younger mice.

Conclusions: MitoOS in lesional myeloid cells enhanced atherosclerosis development in aged mice, and this enhancement was associated with increased lesional NETs. Thus, mitoOS-induced NET formation is a potentially new therapeutic target to prevent atherosclerosis progression during aging.

Keywords: DNA, mitochondrial; atherosclerosis; extracellular trap; mitochondria; reactive oxygen species.

Figure 1. Suppression of myeloid cell mitochondrial oxidative stress in aged mCAT→Ldlr^−/− mice

36-week-old mCAT→Ldlr^−/− and WT →Ldlr^−/− (littermate control) chimeric mice were fed the Western-type diet for another 16 weeks and aortic root lesions were analyzed by confocal fluorescence microscopy. (A) Representative immunofluorescence staining of Ly6G (the marker of neutrophil, red), and non-nuclear 8OHDG staining (a marker of DNA oxidative damage, green) in the aortic root lesions from two groups of mice, with the intima outlined by the dotted line. In the merged images, the green 8OHDG signal locates either within the blue nuclei (arrowheads) or in juxtaposed with the blue nuclei (arrows). When mitochondrial DNA oxidative damage is suppressed, the green 8OHDG signal was exclusively restricted to nuclei region. The non-nuclei 8OHDG signal was abrogated and myeloid cells give red signals (arrowheads). The images in the 2^nd or 4^th rows are of higher magnifications of the boxed areas in the images above. Scale bar, 20μm (1^st and 3^rd rows) and 5μm (2^nd and 4^th rows). A, adventitia; I, intima; L,lumen. (B) Data were quantified as the percentage of non-nuclear 8OHDG⁺ cell among all Ly6G+ cells in intima per section, the absolute number of 8OHDG⁺Ly6G⁺ cells in intima per section, and number of 8OHDG⁺Ly6G⁺ cell normalized to lesion area. n = 9 WT vs. 5 mCAT mice; *P<0.05 using student’s t test.

Figure 2. Suppression of myeloid cell mitochondrial oxidative stress protects against atherosclerosis and reduces myeloid-cell accumulation in aged mice

(A) Shown are hematoxylin and eosin–stained aortic root lesions, with the intima marked by dotted lines, and total lesion area quantification (n= 9 WT vs. 8 mCAT; *P<0.05 using student’s t test). Scale bar, 40 μm. (B) Representative images of Ly6G staining (green) and counter-stained nuclei DAPI (blue) in the lesions from two groups of mice, with the intima outlined by the dotted line. The images in the 2^nd or 4^th rows are of higher magnifications of the boxed areas in the images above. Scale bar, 20 μm (1^st and 3^rd rows) and 10 μm (2^nd and 4^th rows). I, intima; A, adventitia. (C) Data were quantified and presented as No. of Ly6G⁺ cell in the intimal area per section or No. of Ly6G⁺ cell normalized to the intimal area. n= 9 WT vs. 5 mCAT; *P<0.05 using student’s t test.

Figure 3. The formation of neutrophil extracellular traps (NET) in atherosclerotic lesions was increased during aging, and mCAT expression limited NET formation only in the aged mice

(A) Aortic root lesions from 36-week-old (aged) and 14-week-old (young) mice were placed on WD for additional 16 weeks were stained for citrullinated histone H3 (Cit-H3, a NET marker, red), myeloperoxidase (MPO, green) and nuclei (DAPI, blue), with intima and tunica media outlined. The bottom row were the H&E of neighboring sections with intima outlined in red dotted lines and necrotic core area outlined in blue lines (the 5^th row). NET formed in both necrotic areas (the 1^st and 3^rd columns) and non-necrotic areas (the 2^nd and 4^th columns). White arrows depicted media erosion and white arrowheads depicted NET that extended to adventitia (the 1^st column). Red stars depicted the emergence of the coronary artery in the bottom row. The images in the 4^th row are of higher magnifications of the boxed areas in the images above. Scale bars, 40μm for rows #3 and 5; and 20μm for row #4. I, intima; NC, necrotic core; M, tunica media; A, adventitia. (B) NET formation was quantified as the areas that were covered by extracellular Cit-H3⁺ structures. Shown are the quantification of NET-covered areas per section and % of NET-covered areas per section normalized to lesion area. n = 9 WT vs. 5 mCAT aged mice; 6 WT vs. 7 mCAT young mice; * P < 0.05 using one-way ANOVA followed by Bonferroni post hoc test. (C) Aortic root lesions from two groups of aged mice were stained for Cit-H3 (magenta), Ly6G (red) and 8OHDG (green) and nuclei (blue). Shown are the co-localization of extracellular Cit-H3⁺structures with Ly6G and 8OHDG immunofluorescence signals in the arotic root lesions, which are depicted by white arrows. When mitoOS was suppressed, the extracellular Cit-H3 positive DNA structures was diminished, and thus did not overlap with 8OHDG signals. The images in the 2^nd and 4^th rows are of higher magnifications of the boxed areas in the 1^st and 3^rd rows respectively. Scale bars, 20 μm for rows #1 and 3, 10 μm for rows #2 and 4.

Figure 4. Inhibition of mitoOS by mitoTEMPOL reduced NET formation in neutrophils from aged mice

(A) Neutrophils isolated from 58-week-old (old) and 16-week-old (young) WT mice were stained with 5μM mitochondrial superoxide indicator MitoSOX. Cells were then incubated with vehicle or mitochondria-targeted antioxidant mitoTEMPOL (10μM) for 30 min followed by incubation with vehicle or 7KC for additional 4 hr. Mitochondrial ROS production was quantified as the mean fluorescence intensity (MFI) of MitoSOX by flow cytometry. (B) Isolated neutrophils were pre-incubated with vehicle or 10μM mitoTEMPOL for 30 min followed by incubation with 7KC for 4 hr. Cells were then fixed and stained with antibody against Cit-H3 (red), 8OHDG (reflective of mitochondrial DNA oxidative damage, green) and DAPI (blue), and visualized by fluorescence microscopy. Shown were the representative images. The images in the 5^th row are of higher magnifications of the boxed areas in the 4^th row. Scale bars, 5μm for the 1^st – 4^th rows, 1.25μm for the 5^th row. Arrows depicted oxidized mitochondrial DNA-bound NETs where antibodies against 8OHDG co-localized with Cit-H3 positive chromosome DNA. Data were quantified in the graphs below, as the percentage of neutrophils that release NETs (the upper graph) and the area of NET⁺ structure normalized to the number of NET⁺ cells (the lower graph). Each dot represented the average of 16 randomly picked-up areas in each well, quadruplets for each animal. n = 3 young and 3 old mice. * P <0.05 using one-way ANOVA followed by Bonferroni post hoc test. N.S., not significant.