Angiopoietin-1 promotes atherosclerosis by increasing the proportion of circulating Gr1⁺monocytes

Figures

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  Figure 1. Ang-1 enhances atherosclerotic plaque formation in ApoE-/- mice fed a Western diet. ApoE-/- mice fed a Western diet for 4 weeks were infected systemically with adenoviruses encoding Ang1 (AdAng-1) or a control virus (AdEV). The mice were euthanized 4 weeks after adenovirus treatment and effects on early to intermediate atherosclerotic lesions were investigated. (A) Representative sections stained with oil red O (top panel) and immunohistochemical staining of monocytes/macrophages and endothelium with the MOMA-2 monoclonal antibody (middle panel) and CD31 antibody (bottom panel) respectively in AdEV or AdAng1-treated ApoE-/- mice. Original magnification, ×40 (Oil red), ×100 (MOMA-2). Bars, 100 µm. (B) Atherosclerotic lesion area was quantified by oil red O staining of lesions from serial aortic sections using ImageJ image analysis software. Nine serial sections at 40 µm intervals were used from each animal for analysis. Results show the mean from nine cross-sectional lesion size (mm2) for each animal and the line indicates median value per treatment of mice. *P < 0.01. (C) Similarly, five mice were randomly picked from each treatment group, and tissue sections from each animal…

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  Figure 2. Ang-1 promotes early atherosclerotic plaque formation in ApoE-/- mice fed a normal chow diet. ApoE-/- mice fed a normal chow diet were infected systemically with adenoviruses encoding Ang-1 (AdAng-1), or control virus (AdEV). The mice were euthanized after 4 weeks of adenovirus treatment and effects on early to intermediate atherosclerotic lesions was investigated. (A) Representative picture of Sudan IV en face (top panel) (n = 4 per group). (B) Sudan IV en face staining was quantified from the beginning of aortic arch to the left common carotid artery using image analysis software and expressed as the percentage lesion area in each vessel. **P < 0.001. (C) Oil red O-positive atherosclerotic areas were calculated by using ImageJ image analysis software (n = 12–15 per group). Atherosclerotic plaques were quantified as described in the legend to Figure 1. *P<0.05. (D) Similarly, three sections from each animal were analysed for MOMA-2-positive lesion size (mm2) (n = 12–15 per group). *P<0.05. Original magnification ×2.5 (Sudan IV en face); bars, 1 mm (Sudan IV).

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  Figure 3. Ang-1 increases circulating numbers of Gr1+ monocytes. Whole-blood samples were stained with CD11b, Gr1 and Ly6C antibodies and analysed by flow cytometry. (A) CD11b-positive cells were gated (R1) according to CD11b expression and side scatter. R1 cells were further divided into SSClowCD11b+Gr1-Ly6Clow monocytes (R2) and SSClowCD11b+Gr1+Ly6Chigh monocytes (R3) and SSChighCD11b+ Gr1highLy6Cinte neutrophils (R4). (B) 10 days after virus administration, the proportions of Gr1- and Gr1+ moncoytes in each treatment group was calculated as the percentage of total white blood cells. (C) To correct for variability among individual experiments, the Gr1+/Gr1- ratio was calculated for each treatment group. (D) The correlation between MOMA-2-positive areas and Gr1+ and Gr1- monocytes on day 10 after AdAng-1 administration was analysed. Pooled data are presented from three separate independent experiments (n = 4–5 per experiment). **P<0.01.

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  Figure 4. Ang-1 induces Ly6C + CD11b+ monocytes accumulation in ApoE-/- mice fed a normal chow diet. Representative immunofluorescence staining of Ly6C and CD11b monoclonal antibodies in AdAng1-treated ApoE-/- mice. Red: Ly6C, green: CD11b, blue: 4’,6-diamidino-2-phenylindole (DAPI). Original magnification, ×40 (left), ×80 (right). White arrow is the border line between arterial wall and plaque judging by out layer of muscle filament. Yellow arrow is double staining mononuclear cell.

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  Figure 5. Ang-1 increases plasma levels of VEGF and MCP-1 ApoE-/- mice fed a normal chow diet. Whole blood was collected at 3 and 10 and 28 days after AdEV or AdAng-1 administration and plasma was isolated. ELISA was used to measure the level of VEGF (A) and MCP-1 (B). Pooled data are presented from four independent experiments (n = 17–19 per group). **P < 0.01.

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  Figure 6. Ang-1 induces the VEGF and MCP-1 release in conditioned medium from aortic ring. Thoratic artery from apoE-/- mouse was cut into 1 mm segments and aortic rings were stimulated with Ang-1 (400 ng/mL) with or without pre-incubation with Tie2 blocking peptide (NLLMAAS, 100 µM) for 30 min. Medium containing 0.01% DMSO was served as vehicle control. The levels of VEGF (A) and MCP-1 (B) in conditioned medium were measured by ELISA assay. Pooled data are presented from four independent experiments performed in duplicates.

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  Figure 7. Ang-1 reduces cell-surface CD11b expression on Gr1+ monocytes. Whole-blood samples were stained with anti-CD11b and anti-Gr1 antibodies, and analysed by flow cytometry as described in the legend to Figure 3. (A) CD11b expressions [median fluorescence intensity (MFI)] on circulating monocytes in control and AdAng-1 groups were compared 10 days after virus administration. (B) CD11b expression on Gr1- and Gr1+ monocytes in control and AdAng-1 groups were analysed and representative histograms were depicted. (C) The correlation between MOMA-2-positive areas and CD11b expression on Gr1+ monocytes on day 10 after AdAng-1 administration was analysed. CD11b expression was represented by MFI. Pooled data are presented from three independent experiments (n = 5 per experiment). **P<0.01.