Ultrasound Molecular Imaging of Atherosclerosis With Nanobodies

Abstract

Objective: Contrast-enhanced ultrasound molecular imaging (CEUMI) of endothelial expression of VCAM (vascular cell adhesion molecule)-1 could improve risk stratification for atherosclerosis. The microbubble contrast agents developed for preclinical studies are not suitable for clinical translation. Our aim was to characterize and validate a microbubble contrast agent using a clinically translatable single-variable domain immunoglobulin (nanobody) ligand. Approach and Results: Microbubble with a nanobody targeting VCAM-1 (MB cAbVcam1-5 ) and microbubble with a control nanobody (MB VHH2E7 ) were prepared and characterized in vitro. Attachment efficiency to VCAM-1 under continuous and pulsatile flow was investigated using activated murine endothelial cells. In vivo CEUMI of the aorta was performed in atherosclerotic double knockout and wild-type mice after injection of MB cAbVcam1-5 and MB VHH2E7 . Ex vivo CEUMI of human endarterectomy specimens was performed in a closed-loop circulation model. The surface density of the nanobody ligand was 3.5×10 5 per microbubble. Compared with MB VHH2E7 , MB cAbVcam1-5 showed increased attachment under continuous flow with increasing shear stress of 1-8 dynes/cm 2 while under pulsatile flow attachment occurred at higher shear stress. CEUMI in double knockout mice showed signal enhancement for MB cAbVcam1-5 in early ( P =0.0003 versus MB VHH2E7 ) and late atherosclerosis ( P =0.007 versus MB VHH2E7 ); in wild-type mice, there were no differences between MB cAbVcam1-5 and MB VHH2E7 . CEUMI in human endarterectomy specimens showed a 100% increase in signal for MB cAbVcam1-5 versus MB VHH2E7 (20.6±27.7 versus 9.6±14.7, P =0.0156). Conclusions: CEUMI of the expression of VCAM-1 is feasible in murine models of atherosclerosis and on human tissue using a clinically translatable microbubble bearing a VCAM-1 targeted nanobody.

VCAM-1 Target in Non-Invasive Imaging for the Detection of Atherosclerotic Plaques

“…Recently, cAbVCAM-1-5 has also been studied in CEU: Punjabi et al coupled anti-VCAM-1 nanobodies with microbubbles (MBcAbVCAM-1-5) to detect VCAM-1 expression in an atherosclerotic mouse model and ex vivo in human endarterectomy specimens using non-invasive ultrasound imaging. This study revealed that MBcAbVCAM-1-5 detected VCAM-1 expression in early and established atherosclerotic lesions in vivo and ex vivo, but it is not yet ready for clinical translation because the atherosclerosis developed in the mouse model studied is not similar to human atherosclerosis [106].…”

“…Detection of atherosclerotic lesions in the mouse aortic arch (Ao), near the heart (H), by in vivo β-CUBE imaging system after administration of [ 18 In summary, VCAM-1 imaging enables us to detect atherosclerotic plaques, stratify the risk and evaluate a new therapy in atherosclerosis in animal studies. Only a few studies [106,117] have demonstrated the efficiency of VCAM-1 targeting agents on ex vivo samples of human arteries.…”

Nanobodies for Medical Imaging: About Ready for Prime Time?

“…An example of such a cardiovascular complication that can be detected from VCAM-1 expression is atherosclerotic disease, which can lead to stroke and myocardial infarction. While there has been some recent progress made in treating the acute or fatal complications of atherosclerosis, it has remained difficult to assess patient risk for the purpose of detecting abnormalities early on [ 131 ]. By detecting a clearly atherogenic phenotype before the disease progresses into its later stages, treatment protocols can be better catered towards reversing or stopping plaque formation, which is currently the leading reason for atherosclerotic mortality.…”

“…Anti-VCAM-1 nanobodies were developed and conjugated to microbubbles to detect the VCAM-1 expression levels, both in mouse models of atherosclerosis and ex vivo human endarterectomy specimens [ 131 ]. These nanobodies were cross-reactive towards both murine and human VCAM-1, and the purpose of the microbubbles was to be used as a contrast agent for ultrasound imaging.…”

“…These nanobodies were cross-reactive towards both murine and human VCAM-1, and the purpose of the microbubbles was to be used as a contrast agent for ultrasound imaging. This tracing was done noninvasively using CEUMI, and the results of the anti-VCAM-1 nanobodies (MB cAbVcam1-5 ) were compared to those of the control nanobody (MB VHH2E7 ) [ 131 ]. As with the many nanobody imaging agents targeting other markers, the binding specificity of MB cAbVcam1-5 to VCAM-1 was also verified using IHC, autoradiography, and in vivo experiments.…”