Monitoring Endothelin-A Receptor Expression during the Progression of Atherosclerosis

Abstract

Cardiovascular disease remains the most frequent cause of death worldwide. Atherosclerosis, an underlying cause of cardiovascular disease, is an inflammatory disorder associated with endothelial dysfunction. The endothelin system plays a crucial role in the pathogenesis of endothelial dysfunction and is involved in the development of atherosclerosis. We aimed to reveal the expression levels of the endothelin-A receptor (ET_AR) in the course of atherogenesis to reveal possible time frames for targeted imaging and interventions. We used the ApoE^-/- mice model and human specimens and evaluated ET_AR expression by quantitative rtPCR (qPCR), histology and fluorescence molecular imaging. We found a significant upregulation of ET_AR after 22 weeks of high-fat diet in the aortae of ApoE^-/- mice. With regard to translation to human disease, we applied the fluorescent probe to fresh explants of human carotid and femoral artery specimens. The findings were correlated with qPCR and histology. While ET_AR is upregulated during the progression of early atherosclerosis in the ApoE^-/- mouse model, we found that ET_AR expression is substantially reduced in advanced human atherosclerotic plaques. Moreover, those expression changes were clearly depicted by fluorescence imaging using our in-house designed ET_AR-Cy 5.5 probe confirming its specificity and potential use in future studies.

Keywords: ApoE-KnockOut; atherosclerosis; carotid endarterectomy; endothelin receptor expression; endothelin system; fluorescence imaging; molecular imaging.

Figure 1

Histology and qPCR results indicate upregulation of ET_AR 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 ET_AR (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 ET_AR (arrowheads) from proliferating smooth muscle cells are co-resident. The prominent signal of ET_AR within the adventitia is possibly in part due to adventitial fibroblasts or macrophages infiltrating the diseased vascular wall. (D–F) qPCR comparison of ET_AR, 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 vs. t = 0 (asterisks indicate significance: ** p < 0.01, *** p < 0.005).

Figure 2

Fluorescence imaging of ApoE^−/− aortae with the ET_AR-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).

Figure 3

Expression of ET_AR in human atherosclerotic plaques is downregulated. (A–C) Histology of a human artery pudenda externa as healthy control stained with EvG (A) and for ET_AR (C) showing a strong ET_AR 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 ET_AR (F) showing advanced atherosclerotic lesions with locally reduced staining for ET_AR 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 ET_AR, 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 ET_AR 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).

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 ET_AR-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 ET_AR-Cy 5.5 for 30 min, indicating a significant reduction of ET_AR expression in atherosclerotic tissue (** p < 0.01).