doi: 10.1161/CIRCRESAHA.123.322858.
Epub 2023 Nov 10.
Disturbed Sleep Supports Neutrophil Activation and Promotes Atherosclerosis and Plaque Necrosis
Affiliations
- PMID: 37947098
- PMCID: PMC10842439
- DOI: 10.1161/CIRCRESAHA.123.322858
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Disturbed Sleep Supports Neutrophil Activation and Promotes Atherosclerosis and Plaque Necrosis
Circ Res.
.
No abstract available
Keywords: atherosclerosis; neutrophils; plaque necrosis; sleep fragmentation.
Conflict of interest statement
Figures
SF accelerated atherogenesis with increased plaque formation in a vulnerable area. 8–10 weeks-old Apoe−/− females were placed either in sleep fragmented (SF) or activity control (AC) or regular home cages (HC) and fed a High Fat Diet (Envigo, TD.88137) for 12 weeks. Histological images underwent double-blind analysis, followed by data processing and the selection of representative images reflecting the main conclusion. Representative images and lesion quantification of Oil Red O-stained aortas (n=7–21/per group, One-Way ANOVA-Turkey) and aortic arches with lesion grading (from Grade 0 – as no lesions; (n=17–21 per group). Here and below, each data point represents an animal in a figure. Normality of data was assessed using the Shapiro-Wilk test and visual inspection using a QQ plot in GraphPad Prism. Picrosirius Red-stained aortic roots were quantified for necrotic core (n=5–10/per group), fibrotic cap (cap-to-core ratio, n=3–5/per group, One-Way ANOVA), collagen fibers (n=8–14/per group, One-Way ANOVA or Brown-Forsythe ANOVA). Peripheral blood dsDNA and LPS levels were analyzed using PicoGreen and ELISA (n=4–8/per group, Kruskal-Wallis test). SF primes neutrophils towards a hyperactivated phenotype. Bone marrow Lin−Sca1−Kit+ cells (n=8–11/per group); WBC Count (n=8–11/per group), CD63+Ly6G+ and DCFDA+ROS+ neutrophils (n=4–7/per group) in peripheral blood of Apoe−/− mice; Brown-Forsyth-ANOVA. Representative images of blood neutrophils: BF: bright-field. IS Similarity Score quantifying the degree of nuclear translocation of MPO (~100cells/mouse, n=3–4/per group; One-way ANOVA-Fisher’s LSD). Gated neutrophils are analyzed for nuclear vs cell circularity. The images were obtained from events in each quadrant. Some neutrophils were stimulated with LPS. Activated neutrophils traffic to the aorta following SF. Enzymatically-digested aortas were analyzed for CD63+Ly6G+CD45+ neutrophils by FACS. Competitive homing: 10×106 of fluorescent dye-labeled Leukocytes from SF-, AC-, and HC-Apoe−/− mice were injected IV/tail vein into aged Apoe−/− recipients (n=4–5/per group; Kruskal-Wallis test). 14 hrs later, aortas were analyzed for Dye+CD45+Ly6G+ neutrophils. Data were normalized to the start population of injected cells. Activated VMSCs trigger NETosis in SF neutrophils. Collected supernatants from VSMCs treated with PDGF-BB (10 ng/mL/6 hrs) were incubated with neutrophils from SF- or HC- Apoe−/− mice - and stained with SytoxGreen/Ly6G (n=5/per group). Representative images and averages are shown. Neutrophil depletion improves plaque phenotype of SF Apoe−/−
mice. Scheme of neutrophil depletion, representative images of aortic arches with lesion grading (from Grade 0– as no lesions). Picrosirius Red-stained aortic roots were quantified for necrotic core and collagen fibers (n=4/per group; Matt-Whitney test).