Tribbles 3 deficiency promotes atherosclerotic fibrous cap thickening and macrophage-mediated extracellular matrix remodelling

Figures

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  FIGURE 1. TRIB3 expression is increased in human unstable carotid plaques, in unstable plaque regions and in macrophages from unstable plaques, relative to stable plaques. (A) H&E staining of human carotid plaques was used to identify plaque features, classified as stable or unstable using the Stary classification guidelines. Below, representative images of the shoulder region of these plaques with TRIB3 staining (red) with DAPI (blue) as a nuclear marker (scale bars represent 100 μm). (B) Quantification of TRIB3 expression, as a percentage of the plaque area, of stable (0.944 ± 0.38) and unstable (2.88 ± 0.72) plaques. (C) Percentage of the total TRIB3 staining in the shoulder region (stable: 43.71 ± 9%; Unstable: 56 ± 9%). (D) Representative images of TRIB3 and CD68 macrophage co-staining from stable and unstable plaques (scale bars represent 100 μm). (E) Quantification of percentage CD68 + macrophages co-expressing TRIB3 in stable and unstable plaques. Data is shown as the mean ± SEM; each data point represents one donor (N = 3–5 patients). Unpaired t-test, *p < 0.05, **p < 0.01.

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  FIGURE 2. Atherosclerosis model and weight gain in Trib3KO mice. (A) Schematic representation of atherosclerosis development model used in Trib3WT and Trib3KO mice. (B) Body weight measurements in Trib3WT and Trib3KO mice at 13 weeks.

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  FIGURE 3. Atherosclerotic lesion size, disease progression and features in Trib3KO mice. (A) Representative images and quantification (B) of atherosclerotic lesions stained with ORO in the inner lumen of the aorta and in the aortic arch. (C) Total lesion quantification in the aortic root. Graphs are shown as the mean ± SEM, each data point represents a single mouse, unpaired t-test. (D) Representative images of Elastic van Gieson [elastin, original, and inset (high power image)] and picosirius red (collagen) stained cross-sections of aortic root lesions. (E) Collagen content quantification using picrosirius staining shown as mean ± SEM where each data point represents a single lesion; un-paired t-test *p < 0.05. (F) Double-blind plaque grading, made according to classification in Supplementary Table 2, presented as percentage of the total number of lesions (n = 14–17 lesions) using a Chi-square test, ****p < 0.0001. The necrotic core relative to lumen area (G) and fibrous cap relative to lumen thickness (H) quantification shown as mean ± SEM where each data point represents a single lesion where more than one lesion was assessed per individual mouse for a N = 6 mice for each group;…

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  FIGURE 4. Genetic deletion of Trib3 has no effect on SMC content but increases macrophage presence in advanced atherosclerotic lesions with altered ECM degradative function. (A) Representative images of α-SMA (red) and MAC-3 (green) staining of aortic root lesions from Trib3KO and Trib3WT mice. DAPI (blue) was used as a marker for nuclear staining. Quantification of the SMC area (B) and macrophage area (C) immunostaining as a percentage of the total lesion area. Graphs are shown as the mean ± SEM; each data point represents a lesion (n = 9–10 lesions from N = 6 mice for each group). Unpaired t-test, *p < 0.05.

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  FIGURE 5. Loss of TRIB3 alters extracellular matrix remodelling pathways in human and mouse macrophages. (Ai) Microarray data of MDMs from 596 human donors was ranked based on TRIB3 expression and the top high and bottom low expressors were selected for further analysis. (ii) Under a low TRIB3 expression condition, the volcano plot shows the significant genes correlatively expressed with TRIB3, passing the cut off p-value < 0.05 and passing threshold fold change < 0.8 (red—downregulated, so correlatively expressed, with low TRIB3 expression; examples include P2 × 7, PTGES, IL-8, IL15, IL1A, SPIRE1, APOL6, SXSL2, TNF, TIMP1, IL1B) and > 1.2 (green—upregulated, so inversely correlated, with low TRIB3 expression; examples include LPL, TIMP3, FABP4, MMP19, MMP8, MMP7, APOC2, APOC1). (iii) Functional gene set enrichment analysis using Metascape for genes inversely correlated with low TRIB3 expression. (B) siTRIB3-knockdown mediated changes in mRNA expression levels of genes involved in lipid metabolism (LPL), cytoskeleton and ECM remodelling (SPIRE1, MMP7, and TIMP3) in human MDMs. Graphs are presented as individual connected points representing one donor, paired student’s T-test, *p < 0.05.…

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  FIGURE 6. Increased F-actin and size of macrophages with reduced TRIB3 expression. (A) F-actin staining and quantification of human MDMs with non-targeting or siTRIB3-knockdown; representative images of phalloidin (green) and Dapi (blue) staining (upper) with quantification of cell size (F-actin area), F-actin mean fluorescence intensity and shape descriptors (circularity and elongation), shown connected points for each donor, compared by paired student’s T-test, *p < 0.05, ***p < 0.001. (B) F-actin staining and quantification of Trib3WT and Trib3KO BMDMs; representative images of phalloidin (green) and Dapi (blue) staining (upper) with quantification of cell size (F-actin area), F-actin mean fluorescence intensity and shape descriptors (circularity and elongation). Shown as Mean ± SEM where each data point is the average of all cell measurements per individual mouse (3 fields of view per mouse N = 3–6) unpaired student’s t-test, *p < 0.05 (C) Trib3WT and Trib3KO BMDMs imaged by scanning EM; quantification of cell surface area, elongation and ridge area density shown as Mean ± SEM where each data point is from an individual cell (3 fields of view per mouse N = 3, 30–60 cells for surface…

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  FIGURE 7. Trib3KO BMDMs show altered gene expression after polarisation. qPCR gene expression analysis after macrophage polarisation shows changes in the expression levels of (Ai) Trib3 to confirm the knockout genotype, (ii) β-actin for changes in the cytoskeleton, (B) and pro-inflammatory markers (Nos2 and Cd11c) and (C) anti-inflammatory markers Arg1 and Il-10. Gapdh expression was used as an internal control and normalised to unpolarised controls to give 2–ΔΔCt values. Graphs are presented as Mean ± SEM, two-way ANOVA with Fisher’s post hoc comparisons, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.