Leucine-Rich Alpha-2 Glycoprotein 1 Accumulates in Complicated Atherosclerosis and Promotes Calcification

Figures

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  Figure 1. LRG1 in calcified atherosclerotic plaques in a mouse model of advanced atherosclerosis. (A) Body weights in male (n = 9–11/group) and female (n = 18/group) ApoE−/− mice after 27 weeks of Western diet (WD) or normal chow diet (CD). (B) Quantification of the plaque area and representative images of en face aortas for CD and WD-fed male and female mice (n = 9–11/group). (C–E) Representative histological images and quantitative analysis in serial sections of aortic sinus from CD and WD-fed male and female mice showing plaque lipid content with Oil Red O coloration (n = 9–11/group) ((C) red staining), plaque calcification with the Alizarin Red staining (n = 16–18/group) ((D) dark red staining) and LRG1 expression (n = 16–18/group) ((E) brown staining). Scoring for Alizarin Red and LRG1 was performed with 3 scores: 0 (no staining), 1 (weak staining), and 2 (strong localized staining). ** p < 0.01, *** p < 0.001, **** p < 0.0001 via Mann–Whitney U test for (A,C,D) and Student’s t-test for (B,E). Normally distributed data are presented as mean ± SEM, and non-normally distributed data are presented as median with lower and upper quartiles.

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  Figure 2. LRG1 in calcified atherosclerotic plaques in humans. Histological images of human carotid plaque serial sections showing (A) plaque calcification with the Alizarin Red staining (dark red staining) and (B) LRG1 expression (brown staining).

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  Figure 3. LRG1 expression in endothelial cells and VSMC in response to inflammatory stimuli. Quantification of LRG1-relative mRNA expression in (A,B) human umbilical vein endothelial cells HUVECs, (C,D) murine vascular smooth muscle cells MOVAS, and (E,F) human vascular smooth muscle cells hASMCs stimulated for 24 h and 48 h with 30 ng/mL of TNFα or 4 ng/mL of IL1β. The expression levels of LRG1 mRNA were corrected with Rpl13 expression. N = 3–5 independent experiments/group; AU: Arbitrary Unit; * p < 0.05, ** p < 0.01 by Kruskal–Wallis with Dunn’s multiple comparison test. Data are presented as median with lower and upper quartiles.

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  Figure 4. Effect of LRG1 in VSMC trans-differentiation and calcification. (A,B) Representative images and quantification of Alizarin Red staining (A) and quantification of calcium deposition (B) in MOVAS treated with control (−Pi) or calcification medium (+Pi) with or without 20 μg/mL LRG1 (+LRG1) for 6 days (n = 15 replicates/group from 5 independent experiments). (C) Quantification of osteoblast-like cells phenotype markers relative mRNA expression Runx2, Spp1, and Ankh in MOVAS treated with control (−Pi) or calcification medium (+Pi) with or without 20 μg/mL LRG1 (+LRG1) for 6 days (n = 4 independent experiments/group). * p < 0.05, *** p < 0.001 by Mann–Whitney U test for (A,C) and Student’s t-test for (B). Normally distributed data are presented as mean ± SEM and non-normally distributed data are presented as median with lower and upper quartiles.

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  Figure 5. Effect of LRG1 on TGFβ-induced SMAD signaling. (A,B) Quantification and representative capillary Western blots of pSMAD1-5/SMAD1 and (B) pSMAD2-3/SMAD2 in MOVAS treated with 20 μg/mL LRG1 with or without 10 ng/mL TGFβ for 1 h. n = 3–5 independent experiments/group; ns: non significative; ** p < 0.01 via Mann–Whitney U test. Data are presented as median with lower and upper quartiles.