Feasibility of ex vivo fluorescence imaging of angiogenesis in (non-) culprit human carotid atherosclerotic plaques using bevacizumab-800CW

Lydian A Huisman,Gooitzen M Van Dam,Clark J Zeebregts,Hendrikus H Boersma,Jan-Luuk Hillebrands,Riemer H J A Slart,Pieter J Steinkamp,Matthijs D Linssen,Annelies Jorritsma-Smit

doi:10.1038/s41598-021-82568-8

Abstract

Vascular endothelial growth factor-A (VEGF-A) is assumed to play a crucial role in the development and rupture of vulnerable plaques in the atherosclerotic process. We used a VEGF-A targeted fluorescent antibody (bevacizumab-IRDye800CW [bevacizumab-800CW]) to image and visualize the distribution of VEGF-A in (non-)culprit carotid plaques ex vivo. Freshly endarterectomized human plaques (n = 15) were incubated in bevacizumab-800CW ex vivo. Subsequent NIRF imaging showed a more intense fluorescent signal in the culprit plaques (n = 11) than in the non-culprit plaques (n = 3). A plaque received from an asymptomatic patient showed pathologic features similar to the culprit plaques. Cross-correlation with VEGF-A immunohistochemistry showed co-localization of VEGF-A over-expression in 91% of the fluorescent culprit plaques, while no VEGF-A expression was found in the non-culprit plaques (p < 0.0001). VEGF-A expression was co-localized with CD34, a marker for angiogenesis (p < 0.001). Ex vivo near-infrared fluorescence (NIRF) imaging by incubation with bevacizumab-800CW shows promise for visualizing VEGF-A overexpression in culprit atherosclerotic plaques in vivo.

Highlights

Vascular endothelial growth factor-A (VEGF-A) is assumed to play a crucial role in the development and rupture of vulnerable plaques in the atherosclerotic process
The aim of this study was to determine whether the optical tracer bevacizumab-800CW is suitable for ex vivo visualization of intraplaque angiogenesis by means of identifying VEGF-A presence, and thereby able to distinguish between culprit and non-culprit atherosclerotic plaques
The optimal dose was used in 15 plaques: 11 plaques in the culprit group and three in the non-culprit group, based on the period of time between symptom onset and carotid endarterectomy (CEA), and one plaque was retrieved from an asymptomatic patient and was analyzed separately

Summary

Introduction

Vascular endothelial growth factor-A (VEGF-A) is assumed to play a crucial role in the development and rupture of vulnerable plaques in the atherosclerotic process. We used a VEGF-A targeted fluorescent antibody (bevacizumab-IRDye800CW [bevacizumab-800CW]) to image and visualize the distribution of VEGF-A in (non-)culprit carotid plaques ex vivo. Ex vivo near-infrared fluorescence (NIRF) imaging by incubation with bevacizumab-800CW shows promise for visualizing VEGF-A overexpression in culprit atherosclerotic plaques in vivo. There is no imaging modality available to stratify the risk of rupture in vivo. This leads to unnecessary surgical intervention in many patients, which is not desirable as the mortality rate is approximately 3% for this type of operation[6]. Imaging techniques might enable reliable selection for CEA surgery before symptom onset, in for example a high-risk patient population

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