Acute mental stress drives vascular inflammation and promotes plaque destabilization in mouse atherosclerosis

Figures

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  Figure 1. Acute mental stress exposure promotes inflammatory blood leucocyte uptake into tissues. (A) Visual analogue scales for stress intensity in soccer spectators either 24 h before or immediately after watching a key soccer match (n = 35 healthy individuals, aged 26.1 ± 1.3, 46% female, Wilcoxon matched-pairs test). (B, C) Gating and quantification of human blood leucocytes and subsets in soccer spectators (paired t-test). (D, E) Gating and quantification of murine blood leucocytes and subsets in non-stressed vs. stressed wild-type mice (immediately after a single 3 h episode of restraint stress, n = 12 per group, Mann–Whitney U-test). (F, G) Gating and quantification of green fluorescent protein (GFP)high myeloid cells in various organs after adoptive transfer of GFPhigh monocytes and neutrophils into non-stressed vs. stressed wild-type mice (21 h after a single 3 h episode of restraint stress, n = 11–18 per group, Student’s t-test for lung, bone marrow, and blood; Mann–Whitney U-test for skin, heart, spleen, liver, kidney, and lymph node). Numbers next to gates indicate frequencies (%). Data are presented as mean ± SD.

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  Figure 2. Acute mental stress exposure aggravates inflammatory blood leucocyte migration into atherosclerotic plaques. (A, B) Gating and quantification of GFPhigh myeloid cells in atherosclerotic aortas after adoptive transfer of GFPhigh monocytes and neutrophils into non-stressed vs. stressed ApoE−/− mice (21 h after a single 3 h episode of restraint stress, n = 5 per group, Student’s t-test). (C, D) Gating and quantification of aortic myeloid cells from non-stressed vs. stressed ApoE−/− mice (n = 6 per group, Mann–Whitney U-test for Ly6Chigh monocytes, Student’s t-test for neutrophils and macrophages). (E, F) Representative images (myeloid cells, CD11bhigh) and quantification of adherent myeloid cells in non-stressed vs. stressed ApoE−/− mice using intravital microscopy on carotid arteries (immediately after a single 1 h episode of restraint stress, n = 7–10 per group, Student’s t-test, Mann–Whitney U-test for neutrophils). Scale bars represent 100 µm. (G, H) Representative immunohistochemical staining of aortic roots from non-stressed vs. stressed ApoE−/− mice (after three episodes of 3 h restraint stress once daily) for myeloid cells (CD11b). Scale bars represent 200 µm. Bar graphs…

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  Figure 3. Acute mental stress exposure promotes leucocyte recruitment by modulating endothelial cells. (A) Protein levels as mean fluorescent intensities of adhesion molecules expressed by aortic endothelial cells from non-stressed vs. stressed wild-type mice (immediately after 30 min of restraint stress, n = 9–10 per group, Student’s t-test). (B) Pathway analysis of up-regulated genes as assessed by RNA sequencing from fluorescence-activated cell sorting-isolated aortic endothelial cells from non-stressed vs. stressed ApoE−/− mice (after three episodes of 3 h restraint stress once daily, n = 3 per group). Percentage of up-regulated genes per respective pathway and q-values as assessed by Benjamini–Hochberg correction are shown for the top 10 pathways as defined by P-values. (C) Quantification of plasma chemokine levels measured by ELISA in non-stressed vs. stressed wild-type mice (before, during, and immediately after 3 h of restraint stress, n = 6–18 per group, one-way ANOVA for CCL7, Kruskal–Wallis test for CXCL1, only P-values for comparisons with a control group are annotated). (D) Quantification of stress hormones measured by ELISA in plasma and aortic arches from non-stressed vs.…

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  Figure 4. Depleting norepinephrine signalling reverts stress-induced endothelial cell activation. (A) Quantification of GFPhigh myeloid cells in atherosclerotic aortas after adoptive transfer of GFPhigh monocytes and neutrophils into stressed ApoE−/− mice with or without prior chemical norepinephrine depletion (21 h after a single 3 h episode of restraint stress, n = 10–11 per group, Mann–Whitney U-test). 6-OHDA, 6-Hydroxydopamine. (B) Quantification of GFPhigh myeloid cells in atherosclerotic aortas after adoptive transfer of GFPhigh monocytes and neutrophils into stressed ApoE−/− mice with or without prior surgical norepinephrine depletion (21 h after a single 3 h episode of restraint stress, n = 5 per group, Student’s t-test). (C, D) Representative immunohistochemical staining and quantification of aortic roots from stressed ApoE−/− mice with or without prior surgical norepinephrine depletion (after three episodes of 3 h restraint stress once daily) for myeloid cells (CD11b). Scale bars represent 200 µm. Bar graphs show the percentage of positive area per plaque area (n = 5–6 per group, Students t-test). (E) Experimental setup for panels F–H. (F, G) Gating and quantification of GFPhigh…