Activation of Bone Marrow-Derived Cells Angiotensin (Ang) II Type 1 Receptor by Ang II Promotes Atherosclerotic Plaque Vulnerability

Figures

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  Figure 1. Effect of AT1aR deficiency in BM-derived cells on genes involved in cholesterol metabolism. mRNA levels of Hmgcr, Ldlr, Srebf2, ApoB, and Acat2 were determined in liver tissue by real-time RT-PCR analysis. Data were normalized to 36B4 and expressed as fold change over controls which were set at 1. n = 9 animals per group.

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  Figure 2. Effect of AT1aR deficiency in BM-derived cells on Ang II-mediated atherosclerosis. (A) Representative photographs of pinned-out aortas en face stained with Oil red O (lipid staining) from 2K1C ApoE−/− mice transplanted with either AT1aR+/+ or AT1aR−/− BM; (B) quantitative morphometric analysis of aortic plaque surface expressed as % of lipid deposition of the total aortic area analyzed (n = 7 to 11 animals per group); (C) representative photographs of aortic sinus cross-sections stained with Movats pentachrome (scale bar = 200 μm); (D) quantitative morphometric analysis of aortic sinus plaque area expressed in µm2 (n = 13 to 16 animals per group). Circles represent individual mice, horizontal lines indicate the mean, and error bars represent SEM.

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  Figure 3. Effect of AT1aR deficiency in BM-derived cells on Ang II-mediated plaque morphology and phenotype. (A,B), Representative photographs of Movats pentachrome staining of aortic sinus cross-sections showing different plaque phenotype in 2K1C ApoE−/− mice transplanted with either AT1aR+/+ or AT1aR−/− BM (scale bar = 100 μm). Vulnerable plaque in 2K1C ApoE−/− mice transplanted with AT1aR+/+ BM characterized by the absence of fibrous cap (black solid arrows), presence of a large lipid core (in blue) containing a vast number of cholesterol clefts, buried cap (red dotted arrow), and extensive adventitia inflammation (black triangle). Note the presence of a plaque in a coronary artery at the bottom of the photograph (A) Stable plaque in 2K1C ApoE−/− transplanted with AT1aR−/− BM demonstrating continuous and thick fibrous cap (black solid arrow), presence of small lipid core (in blue) containing a low number of cholesterol clefts, absence of buried cap and of extensive adventitia inflammation (black triangle). (B) Presence of media degeneration is present in plaques of both groups (red solid arrows). (C) Semi-quantitative analysis of markers of plaque vulnerability (the data represent the…

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  Figure 4. AT1aR deficiency in BM-derived cells prevents Ang II-mediated vulnerable plaques containing a high number of macrophages, and low content of SM cells and collagen. (A) Representative photographs of aortic sinus cross-sections immunostained for Mac-2 (top panel) and α-SM actin (middle panel) for macrophage and SM cells determination, respectively, or stained with Sirius Red (bottom panel) for collagen determination in 2K1C ApoE−/− mice transplanted with either AT1aR+/+ or AT1aR−/− BM (scale bar = 100 μm). (B) Quantification of Mac-2, α-SM actin, and Sirius Red positive staining over the total area of plaque. (C) Quantification of Mac-2 to α-SM actin ratio. Data are mean ± SEM. n = 12 to 16 animals per group.

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  Figure 5. Effect of AT1aR deficiency in BM-derived cells on vascular expression of molecules involved in monocyte recruitment. mRNA levels of ICAM-1 and MIF were determined in aortic tissue by real-time RT-PCR analysis. Data were normalized to 36B4 and expressed as fold change over controls which were set at 1. Error bars are the SEM of the ∆CT values. n = 4 to 7 animals per group.

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  Figure 6. Effect of AT1aR deficiency in BM-derived cells on vascular expression of cytokines and macrophage phenotype markers. (A) mRNA levels of pro-inflammatory cytokines IL-1β, IL12p35, TNF-α, IL-6 and IFN-γ as well as anti-inflammatory cytokine IL-10; (B) mRNA levels of M1-associated marker CD11c as well as M2-associated marker CD206. mRNA levels were determined in aortic tissue by real-time RT-PCR analysis. Data were normalized to 36B4 and expressed as fold change over controls which were set at 1. Error bars are the SEM of the ∆CT values; (C) Quantification of CD11c to CD206 ratio (M1/M2 balance marker). n = 3 to 10 animals per group.

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  Figure 7. Effect of AT1aR deficiency in BM-derived cells on systemic expression of CD4+ T cells phenotype markers. mRNA levels of pro-inflammatory Th1 markers T-bet and IL-2 (A), anti-inflammatory Th2 markers GATA3 and IL-13 (B), Treg markers IL-10 and TGF-β (C), and Th17 marker IL-17 (D). mRNA levels were determined in splenic tissue by real-time RT-PCR analysis. Data were normalized to 36B4 and expressed as fold change over controls which were set at 1. Error bars are the SEM of the ∆CT values. Quantification of T-bet to GATA3 and IL-2 to IL-13 ratios (Th1/Th2 balance markers) (E), and of IL-10 to IL-17 and TGF-β to IL-17 ratios (Treg/Th17 balance markers) (F). n = 10–12 animals per group.

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  Figure 8. Effect of AT1aR deficiency in BM-derived cells on the systemic expression of macrophage phenotype markers. (A) mRNA levels of pro-inflammatory M1 macrophage marker CD11c, and anti-inflammatory M2 macrophage marker CD206. (B) Quantification of CD11c to CD206 ratio. (C) mRNA levels of M1-related pro-inflammatory cytokines IL-1β, TNF-α, IL-18 and of M2-related anti-inflammatory cytokine IL-1ra. mRNA levels were determined in splenic tissue by real-time RT-PCR analysis. Data were normalized to 36B4 and expressed as fold change over controls which were set at 1. (D) Plasma levels of IL-1β and TNF-α as determined by ELISA. n = 10–12 animals per group for real-time RT-PCR analysis. n = 4–8 animals per group for ELISAs; ND = Non detected.