Risk assessment for atherosclerotis relies on established clinical risk factors. This approach places a large proportion of individuals in an intermediate risk category, where the value of interventions to reduce the risk for events is uncertain. Therefore, tools to better assess the risk in these patients are needed. It is generally thought that noninvasive imaging of molecular events associated with atherosclerotic disease may serve this purpose. Previous studies have shown that contrast enhanced ultrasound (CEU) molecular imaging using microbubble contrast agents directed against vascular cell adhesion molecule 1 (VCAM-1), which is involved in inflammatory processes in atherosclerosis, is feasible in murine disease models. However, the ultrasound contrast agents used in these studies are not suitable for clinical translation, and there is a need for the development of microbubbles employing (a) clinically translatable strategies for conjugation of targeting moieties, and (b) targeting ligands that can readily be used in the clinical field. Nanobodies are small antibody fragments (10-15kDa) derived from heavy-chain-only antibodies. They are attractive for applications in molecular imaging, as they are highly specific, non-immunogenic and thus offer the potential for clinical translation. Likewise, Designed Ankyrin Repeat Proteins (DARPins) are potential candidates for clinical ultrasound molecular imaging given their easy production and selection, high affinity and low immunogenicity.The overall aim of this application will therefore be to develop and validate novel VCAM-1 targeted microbubbles with clinically translatable binders coupled to the microbubble surface using maleimide covalent bonding. Preliminary data from our laboratory show that a VCAM-1 targeted nanobody can be conjugated to microbubbles using maleimide bonding. Preliminary in vitro and in vivo experiments show that maleimide microbubbles carrying a VCAM-1 targeted nanobody specifically attach to VCAM-1 under flow conditions, and to the aortic endothelium of mice with advanced atherosclerosis. Thus, we propose the following three study aims:Specific aim 1. To test whether CEU molecular imaging with two novel microbubbles carrying either nanobodies or DARPins targeting VCAM-1 can detect vascular inflammation in advanced atherosclerosis. CEU molecular imaging will be performed in 40 weeks old mice with established atherosclerosis and control animals. Specific aim 2. To test whether CEU molecular imaging with two novel microbubbles carrying either nanobodies or DARPins targeting VCAM-1 can detect vascular inflammation in early atherosclerosis. CEU molecular imaging will be performed in 10 weeks old mice with early atherosclerotic plaques and control animals.Specific aim 3: To test whether CEU molecular imaging with two novel microbubbles carrying either nanobodies or DARPins targeting VCAM-1 can detect vascular inflammation in human endarterectomy specimens. CEU molecular imaging will be performed on perfused human thrombendarterectomy specimens with VCAM-1 targeted and control microbubbles.Significance. Better noninvasive imaging tools for risk stratification in large patient populations with an intermediate risk for atherosclerotic complications are a clinical need. CEU molecular imaging can detect vascular inflammation even at very early stages of atherosclerosis, and could possibly contribute to risk stratification. However, the approaches that have been used in these studies are not readily translatable into the clinical practice. In this proposal, we will address these issues and will design targeted microbubbles that will be ready for clinical translation.