Liraglutide Lowers Endothelial Vascular Cell Adhesion Molecule-1 in Murine Atherosclerosis Independent of Glucose Levels.

The authors determined the effect of the GLP-1 receptor agonist liraglutide on endothelial surface expression of vascular cell adhesion molecule (VCAM)-1 in murine apolipoprotein E knockout atherosclerosis. Contrast-enhanced ultrasound molecular imaging using microbubbles targeted to VCAM-1 and control microbubbles showed a 3-fold increase in endothelial surface VCAM-1 signal in vehicle-treated animals, whereas in the liraglutide-treated animals the signal ratio remained around 1 throughout the study. Liraglutide had no influence on low-density lipoprotein cholesterol or glycated hemoglobin, but reduced TNF-α, IL-1ß, MCP-1, and OPN. Aortic plaque lesion area and luminal VCAM-1 expression on immunohistology were reduced under liraglutide treatment.

Designed Ankyrin Repeat Proteins as Novel Binders for Ultrasound Molecular Imaging.

Clinical translation of ultrasound molecular imaging will depend on the development of binders that can easily be generated, manufactured and coupled, and that are compatible with in vivo use. We describe targeted microbubbles (MBs) using designed ankyrin repeat proteins (DARPins) as a novel class of such translatable binders. Candidate DARPin binders for vascular cell adhesion molecule 1, an endothelial cell adhesion molecule involved in inflammatory processes, were selected using ribosome display and coupled to MBs. Flow-chamber assays of five MBs carrying high-affinity binders showed selective retention on endothelial cells activated by tumor necrosis factor-α for two binders compared with a MB carrying a control DARPin. In vivo ultrasound molecular imaging in a murine hind-limb inflammation model demonstrated up to a fourfold signal enhancement for three of the five MBs versus control. However, there was no correlation between results from flow-chamber assays and in vivo imaging. Thus, we conclude that ultrasound molecular imaging of inflammation using DARPin binders is feasible per se, but that screening of candidates cannot be accomplished with flow-chamber assays as used in our study.

Seeing the Invisible-Ultrasound Molecular Imaging.

Ultrasound molecular imaging has been developed in the past two decades with the goal of non-invasively imaging disease phenotypes on a cellular level not depicted on anatomic imaging. Such techniques already play a role in pre-clinical research for the assessment of disease mechanisms and drug effects, and are thought to in the future contribute to earlier diagnosis of disease, assessment of therapeutic effects and patient-tailored therapy in the clinical field. In this review, we first describe the chemical composition and structure as well as the in vivo behavior of the ultrasound contrast agents that have been developed for molecular imaging. We then discuss the strategies that are used for targeting of contrast agents to specific cellular targets and protocols used for imaging. Next we describe pre-clinical data on imaging of thrombosis, atherosclerosis and microvascular inflammation and in oncology, including the pathophysiological principles underlying the selection of targets in each area. Where applicable, we also discuss efforts that are currently underway for translation of this technique into the clinical arena.

Ultrasound Molecular Imaging of Atherosclerosis With Nanobodies: Translatable Microbubble Targeting Murine and Human VCAM (Vascular Cell Adhesion Molecule) 1.

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 (MBcAbVcam1-5) and microbubble with a control nanobody (MBVHH2E7) 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 MBcAbVcam1-5 and MBVHH2E7. Ex vivo CEUMI of human endarterectomy specimens was performed in a closed-loop circulation model. The surface density of the nanobody ligand was 3.5×105 per microbubble. Compared with MBVHH2E7, MBcAbVcam1-5 showed increased attachment under continuous flow with increasing shear stress of 1-8 dynes/cm2 while under pulsatile flow attachment occurred at higher shear stress. CEUMI in double knockout mice showed signal enhancement for MBcAbVcam1-5 in early (P=0.0003 versus MBVHH2E7) and late atherosclerosis (P=0.007 versus MBVHH2E7); in wild-type mice, there were no differences between MBcAbVcam1-5 and MBVHH2E7. CEUMI in human endarterectomy specimens showed a 100% increase in signal for MBcAbVcam1-5versus MBVHH2E7 (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.