Carotid artery ligation induced intimal thickening and proliferation is unaffected by ageing

Figures

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  Fig. 1. Wnt4 protein induced proliferation in VSMCs from young but not old mice. Proliferation was quantified in aortic VSMCs isolated from young and old mice and stimulated with 400 ng/mL recombinant Wnt4 protein for 24 h using immunofluorescence detection of EdU incorporation. Representative images are shown (a and b for young and old VSMCs, respectively). The red arrow indicates a positive cell. The scale bar represents 25 μm and applies to all images. The number of EdU positive cells (green) was counted and expressed as a percentage of the total number of cells (blue nuclei: hoechst) (c, d). * indicates p < 0.05 vs. control, NS indicates not significant, paired Student’s t-test, N = 6

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  Fig. 2. Neointimal area and percentage occlusion were unaffected by age. Left carotid arteries were taken from young and old mice 21 days after ligation and transverse sections were taken immediately adjacent to the ligature. Representative images of EVG-stained left carotid arteries are shown (a) and the neointimal area (b) and percentage occlusion of the lumen (c) were analysed. No significant differences (NS) were observed with age, unpaired Student’s t-test, N = 12. The scale bar represents 150 μm and applies to both images

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  Fig. 3. Neointimal Wnt4 protein expression was unaffected by age. Left carotid arteries were taken from young and old mice 21 days after ligation and immunohistochemistry for Wnt4 was performed on transverse sections taken adjacent to the ligature. a Representative images of Wnt4 immunofluorescence (green) with DAPI stained nuclei (blue). The top row shows representative images including a yellow dashed line indicating the media-intima boundary (internal elastic lamina, IEL) plus red arrows pointing to examples of Wnt4 positive cells. The bottom row shows corresponding non-immune rabbit IgG negative control images. The scale bar represents 50 μm and applies to all images. b Wnt4 positive pixels (green) in the neointima of left carotid arteries were quantified and expressed as a percentage of the neointimal area. No significant difference (NS) was observed with age, unpaired Student’s t-test, N = 12

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  Fig. 4. Neointimal cell number and density were unaffected by age. Left carotid arteries were taken from young and old mice 21 days after ligation and DAPI staining was performed on transverse sections taken adjacent to the ligature. Representative images with DAPI stained nuclei (blue) including a purple dashed line indicating the media-adventitia boundary (external elastic lamina, EEL) and a yellow dashed line indicating the media-intima boundary (internal elastic lamina, IEL) (a). The scale bar represents 150 μm and applies to all images. Neointimal cell counts were performed (b) and cell density was calculated by normalising the cell number to neointimal area (c). No significant differences (NS) were observed with age, unpaired Student’s t-test, N = 12 young and N = 11 old

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  Fig. 5. Neointimal cell proliferation was unaffected by age. Proliferation was quantified in left carotid arteries from young and old mice 21 days after ligation by proliferating cell nuclear antigen (PCNA) immunohistochemistry on transverse sections taken adjacent to the ligature. a Representative images are shown including a purple dashed line indicating the media-adventitia boundary (external elastic lamina, EEL), and a green dashed line indicating the media-intima boundary (internal elastic lamina, IEL) plus red arrows pointing to examples of PCNA positive nuclei. The bottom row shows the corresponding non-immune rabbit IgG negative control images. The scale bar represents 50 μm and applies to all images. b The proportion of nuclei in the intima staining positive for PCNA (brown nuclei) was expressed as a percentage of the total number of intimal nuclei viewed (blue nuclei: hematoxylin). No significant difference (NS) was observed with age, unpaired Student’s t-test, N = 12

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  Fig. 6. Neointimal cell apoptosis was negligible and unaffected by age. Apoptosis was assessed by in situ DNA end labelling (ISEL) in left carotid arteries from young and old mice 21 days after ligation. ISEL staining was performed on transverse sections taken adjacent to the ligature (N = 5). Negligible levels of ISEL positive cells were observed. Representative images of ISEL-stained ligated left carotid arteries are shown along with the positive control of a murine brachiocephalic atherosclerotic plaque from a male apolipoprotein-E deficient mouse fed high fat diet for 12 weeks (protocol previously described by Johnson et al. 2005). ISEL positive cells have brown shrunken nuclei, negative cells have blue nuclei (hematoxylin). The top row shows representative images including a purple dashed line indicating the media-adventitia boundary (external elastic lamina, EEL), and a green dashed line indicating the media-intima boundary (internal elastic lamina, IEL) plus red arrows pointing to an ISEL-positive nuclei in the positive control. The bottom row shows the corresponding negative control images (omission of DNA polymerase). The scale bar represents 50 μm and applies to the all images…