Assessment of atherosclerotic plaques in the rabbit abdominal aorta with interleukin-8 monoclonal antibody-targeted ultrasound microbubbles

Abstract

In this study, we aimed to prepare a neovascularization-relevant inflammatory cytokine-targeted ultrasound contrast agent and apply it in the ultrasound imaging of atherosclerotic plaque. An interleukin-8 (IL-8) monoclonal antibody was conjugated to SonoVue microbubbles using the N-succinimidyl-3-(2-pyridyldithio)propionate cross-linking method. Then, a prepared IL-8-targeted contrast agent was used for contrast-enhanced ultrasound (CEU) to detect rabbit abdominal aorta atherosclerotic plaque and to investigate the imaging characteristics of atherosclerotic plaque with the contrast agent. We found that an IL-8 monoclonal antibody can be successfully coupled to SonoVue microbubbles with stable biological characteristics. CEU with this IL-8-targeted contrast agent can increase the atherosclerotic plaque detection sensitivity, with stronger echo, so that three more plaques were detected compared with using non-targeted SonoVue microbubbles. Thus, an inflammatory cytokine-targeting ultrasound contrast agent carrying IL-8 monoclonal antibody can provide unique advantages for researching the characteristics of atherosclerotic plaque.

Fig. 1

Light micrograph of SonoVue microbubbles. a Non-conjugated SonoVue microbubbles. b IL-8 monoclonal antibody-conjugated SonoVue microbubbles (×100)

Fig. 2

Light micrograph of the mixture of SonoVue microbubbles and goat anti-mouse serum. a Non-conjugated microbubbles (×100). b Non-conjugated microbubbles (×400). c IL-8 monoclonal antibody-conjugated microbubbles (×100). d IL-8 monoclonal antibody-conjugated microbubbles (×400)

Fig. 3

Immunofluorescence image of IgG-FITC-stained SonoVue microbubbles. a No fluorescence was observed on the surface of non-conjugated SonoVue microbubbles. b Bright-green fluorescence can be observed on the surface of IL-8-conjugated SonoVue microbubbles (×400). (Color figure online)

Fig. 4

Representative ultrasound images of the atherosclerotic plaques in the rabbit abdominal aorta. a Two-dimensional image showing a plaque (large arrow); a soft plaque was invisible (small arrow). b Contrast ultrasound imaging with normal microbubbles showing two plaques with stronger echo compared with the two-dimensional view. The enhancement of the echo was more apparent for the soft plaque (small arrow). c Contrast ultrasound imaging with IL-8-targeted microbubbles showing two plaques with a markedly stronger echo compared with normal contrast ultrasound

Fig. 5

Sequential ultrasound images of IL-8-targeted imaging for an atherosclerotic plaque of the rabbit abdominal aorta. a Conventional two-dimensional view. b–g Sequential targeted CEU view: b 3.8 s post-contrast agent injection, c: 4.3 s post-contrast agent injection, d 4.9 s post-contrast agent injection; e 9.8 s post-contrast agent injection, f 68 s post-contrast agent injection, g 176 s post-contrast agent injection

Fig. 6

The time-intensity curves of contrast ultrasound imaging with normal microbubbles (a) and IL-8-targeted microbubbles (b). The yellow, blue and red curves indicate the intensity of the lumen, intima and plaques, respectively. (Color figure online)

Fig. 7

Typical histological images of rabbit abdominal aortic atherosclerotic plaque (a×100, b×400)

Fig. 8

Immunohistochemical expression of IL-8 in atherosclerotic plaques in the rabbit abdominal aorta. Brown-stained area indicates positive IL-8 expression. (a ×40; b ×200). (Color figure online)

Fig. 9

The missed soft plaque in two-dimensional images was identified by CD34 immunohistochemistry by its greatly increased brown staining (CD34-positive) in the inner part and base of the plaque compared with adjacent healthy intima. This result indicates a high neovascular density for the plaque, which is consistent with enrichment of the targeted microbubbles in contrast imaging. (a ×40; b ×100). (Color figure online)