Monitoring Endothelin-A Receptor Expression during the Progression of Atherosclerosis

Figures

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  Figure 1. Histology and qPCR results indicate upregulation of ETAR in atherosclerotic tissue of ApoE−/− mice. (A) Elastica van Gieson (EvG) staining of an explanted healthy aortic arch of a control C57Bl/6 mouse without high-fat diet. The magnification showing the bifurcation of the left carotid artery. (B) EvG staining of an aorta from an ApoE−/− mouse after 22 weeks of high-fat diet showing extensive plaque deposits. The magnification shows both affected (arrows) and unaffected vessel walls (arrowheads) around the bifurcation. (C) Immunofluorescence staining, depicting ETAR (red) and SMA (green) expression in a vessel from an ApoE−/− mouse after 12 weeks of high-fat diet (+ DAPI nuclear stain, blue). A developing plaque can be localized at the left vessel wall. Inside the plaque SMA (asterisk) and ETAR (arrowheads) from proliferating smooth muscle cells are co-resident. The prominent signal of ETAR within the adventitia is possibly in part due to adventitial fibroblasts or macrophages infiltrating the diseased vascular wall. (D–F) qPCR comparison of ETAR, SMA and MMP-9 expression in aortic tissue of ApoE−/− mice (n = 6–11) and C57Bl/6 mice (n = 3–11) after indicated time of high-fat diet…

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  Figure 2. Fluorescence imaging of ApoE−/− aortae with the ETAR-targeted probe show enhanced signal intensity after 22 weeks of high-fat diet. (A,B) Images of aortic arches of a C57Bl6 mice (A) and an ApoE−/− mouse (B) after 22 weeks of high-fat diet prior to extraction. A high amount of plaque lesions/calcifications can be observed in the aortae of ApoE−/− mice. (C,D) Fluorescence reflectance images of aortic arches of C57Bl6 (left) and ApoE−/− mice (right) before (0 weeks, (C)) and after 22 weeks of high-fat diet (D). Images were captured directly after extraction of the aortic arch, 24 h after injection of 2.0 nmol of the probe. (E,F) Graphs showing the detected fluorescence intensities (means ± SEM, all data points indicated) of ex vivo imaged aortae of C57Bl/6 (triangles, (E)) and ApoE−/− mice (squares, (F)) before (0 weeks, n = 8 vs. n = 8) and after 6 weeks (n = 7 vs. n = 8), 12 weeks (n = 8 vs. n = 7) and 22 weeks (n = 12 vs. n = 9) of high-fat diet. Significant differences could be identified by one-way ANOVA after the longest period of diet in both strains (* p < 0.05, *** p < 0.005, **** p < 0.001).

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  Figure 3. Expression of ETAR in human atherosclerotic plaques is downregulated. (A–C) Histology of a human artery pudenda externa as healthy control stained with EvG (A) and for ETAR (C) showing a strong ETAR expression within the media ((B) negative control w/o first antibody). (D–F) Histology of paraffin-embedded human carotid artery specimen after endarterectomy stained with EvG (D) and for ETAR (F) showing advanced atherosclerotic lesions with locally reduced staining for ETAR within the lesion (red arrowheads in magnification), compared to putatively healthy tissue (black arrow. (E) negative control w/o first antibody). (G,H) Box plots with min-to-max whiskers of qPCR data showing the expression ratios of ETAR, SMA (left) and MMP-9 (right) in atherosclerotic carotid specimen ((G) n = 8) versus healthy arteries (n = 7) and atherosclerotic femoral specimen ((H) n = 8) versus healthy arteries (n = 7). Significant reductions in ETAR expression were found in both lesions, while SMA is only significantly reduced in carotid tissue. MMP-9 was significantly elevated in specimen from carotid arteries, but not from femoral arteries (* p < 0.05; ** p < 0.01; *** p < 0.005).

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  Figure 4. Fluorescent imaging of human specimen shows reduced signal intensities in atherosclerotic carotid and femoral tissue. (A,B) Human carotid specimen after incubation in 1.0 µM ETAR-Cy 5.5 for 30 min. (A) Color photograph. (B) Fluorescent image. (C,D) Human femoral tissue after extraction and incubation. (C) Color photograph. (D) Fluorescent image. (E,F) Piece of the arteria pudenda externa. (E) Color photograph. (F) Fluorescent image. (G) Graphical analysis of fluorescence intensities from human specimen after incubation in 1.0 µM ETAR-Cy 5.5 for 30 min, indicating a significant reduction of ETAR expression in atherosclerotic tissue (** p < 0.01).