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. 2013 Sep;33(9):2187-92.
doi: 10.1161/ATVBAHA.113.301710. Epub 2013 Aug 1.

Molecular imaging reveals rapid reduction of endothelial activation in early atherosclerosis with apocynin independent of antioxidative properties

Elham Khanicheh  1 , Yue Qi, Aris Xie, Martina Mitterhuber, Lifen Xu, Michika Mochizuki, Youssef Daali, Vincent Jaquet, Karl-Heinz Krause, Zaverio M Ruggeri, Gabriela M Kuster, Jonathan R Lindner, Beat A Kaufmann
Affiliations

Molecular imaging reveals rapid reduction of endothelial activation in early atherosclerosis with apocynin independent of antioxidative properties

Elham Khanicheh et al. Arterioscler Thromb Vasc Biol. 2013 Sep.

Abstract

Objective: Antioxidative drugs continue to be developed for the treatment of atherosclerosis. Apocynin is an nicotinamide adenine dinucleotide phosphate oxidase inhibitor with anti-inflammatory properties. We used contrast-enhanced ultrasound molecular imaging to assess whether short-term apocynin therapy in atherosclerosis reduces vascular oxidative stress and endothelial activation

Approach and results: Genetically modified mice with early atherosclerosis were studied at baseline and after 7 days of therapy with apocynin (4 mg/kg per day IP) or saline. Contrast-enhanced ultrasound molecular imaging of the aorta was performed with microbubbles targeted to vascular cell adhesion molecule 1 (VCAM-1; MB(V)), to platelet glycoprotein Ibα (MB(Pl)), and control microbubbles (MB(Ctr)). Aortic vascular cell adhesion molecule 1 was measured using Western blot. Aortic reactive oxygen species generation was measured using a lucigenin assay. Hydroethidine oxidation was used to assess aortic superoxide generation. Baseline signal for MBV (1.3 ± 0.3 AU) and MB(Pl )(1.5 ± 0.5 AU) was higher than for MBCtr (0.5 ± 0.2 AU; P<0.01). In saline-treated animals, signal did not significantly change for any microbubble agent, whereas short-term apocynin significantly (P<0.05) reduced vascular cell adhesion molecule 1 and platelet signal (MBV: 0.3 ± 0.1; MBPl: 0.4 ± 0.1; MBCtr: 0.3 ± 0.2 AU; P=0.6 between agents). Apocynin reduced aortic vascular cell adhesion molecule 1 expression by 50% (P<0.05). However, apocynin therapy did not reduce reactive oxygen species content, superoxide generation, or macrophage content.

Conclusions: Short-term treatment with apocynin in atherosclerosis reduces endothelial cell adhesion molecule expression. This change in endothelial phenotype can be detected by molecular imaging before any measurable decrease in macrophage content and is not associated with a detectable change in oxidative burden.

Keywords: acetovanillone; atherosclerosis; microbubbles; molecular imaging; oxidative stress.

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Figures

Figure 1
Figure 1
Assessment of vascular cell adhesion molecule 1 (VCAM-1) expression after 7 days of saline or apocynin treatment. A, VCAM-1 protein expression in the ascending aorta assessed by Western blot in nontreated (lanes 1–4) vs apocynin-treated (lanes 5–8) animals, n=4 per group, *P<0.05 vs nontreated animals. Representative examples of fluorescent immunohistochemistry images of the base of the aorta demonstrating endothelial VCAM-1 expression (red fluorescence) in a nontreated animal at 10-fold magnification (B), in the same animal at 40-fold magnification (C), and reduced VCAM-1 expression in an apocynin-treated animal (D and E). Autofluorescence delineating vessel anatomy is shown in green, 4´,6-diamidino-2-phenylindole staining of the nuclei in blue.
Figure 2
Figure 2
Assessment of plaque macrophage content after 7 days of saline or apocynin treatment. A, Percentage of the plaque area covered with macrophages (Mac-2 staining) at the base of the aorta and in the ascending aorta (n=8 in each group; P=nonsignificant vs non-treated). B, Example of trans-illumination image used for plaque delineation. C, Example of Mac-2 staining used for quantification of macrophage content in the plaque.
Figure 3
Figure 3
Assessment of platelet adhesion on the aortic endothelial surface after 7 days of saline or apocynin treatment. A, Percentage of the endothelial surface of the ascending aorta covered with platelet/leukocyte aggregates (n=5 in each group), *P<0.05 vs nontreated animals. Examples of en face fluorescence microscopy demonstrating 2 platelet/leukocyte aggregates on normal appearing endothelial surface in a nontreated animal (B) and absence of platelet/leukocyte aggregates in an apocynin-treated animal (C). Scale bar, 25 µm.
Figure 4
Figure 4
Assessment of reactive oxygen species generation after 7 days of saline or apocynin treatment. A, Superoxide-generating activity of whole aortic rings after the addition of 100 µmol/L NADPH at 4 minutes. Measurements represent relative light units (RLU).P=nonsignificant (ns) between saline-treated and apocynin-treated animals (n=10 in each group). B, High pressure liquid chromatography analysis of 2-hydroxyethidium generated in vascular rings exposed to 50 µmol/L hydroethidine. P=ns between saline-treated and apocynin-treated animals (n=8 in each group).
Figure 5
Figure 5
Molecular imaging of the ascending aorta before the start of treatment.A, Mean±SEM background-subtracted signal intensity for microbubbles targeted to vascular cell adhesion molecule 1 (VCAM-1; MBVCAM), to glycoprotein Ibα on activated thrombocytes (MBPl), and control microbubbles (MBCtr). *P<0.01 vs MBctr, #P<0.05 vs MBctr(n=12). Examples of color-coded contrast-enhanced ultrasound (CEU) images after injection of MBCtr (B), of MBVCAM (C), and of MBPl (D). The color scale for the CEU images is shown at the bottom of each frame.
Figure 6
Figure 6
Molecular imaging of the ascending aorta after 7 days of saline or apocynin treatment. A, Mean±SEM background-subtracted signal intensity in saline-treated (n=9) and apocynin-treated (n=10) animals for microbubbles targeted to vascular cell adhesion molecule 1 (VCAM-1; MBVCAM), to glycoprotein Ibα on activated thrombocytes (MBPl), and control microbubbles (MBCtr). *P<0.01 vs MBctr, #P<0.01 vs MBctr. ¶P<0.05 vs the same microbubble in apocynin-treated animals. Examples of color-coded contrast-enhanced ultrasound (CEU) images after injection of MBCtr (B), of MBVCAM (C), and of MBPl (D) in saline-treated animals. In the same order, examples of color-coded CEU images after injection of MBCtr (E), of MBVCAM (F), and of MBPl (G) in apocynin-treated animals are shown.
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