Cardiovascular molecular imaging

Although there have been significant improvements in the treatment of cardiovascular diseases they still remain the main cause of morbidity and mortality globally. Currently available diagnostic approaches may not be adequate to detect pathologic changes during the early disease stages, which may be valuable for risk stratification and also to assess a response to a therapy. Therefore molecular imaging techniques such as Contrast Enhanced Ultrasound (CEU) molecular imaging to noninvasively image pathophysiologic processes on a molecular level have been developed. For CEU molecular imaging targeted microbubbles are used as contrast agent. However it has been shown that the targeting efficiency of microbubbles is low with retention of less than 5% of targeted microbubbles under high flow shear rate. Furthermore the application of CEU molecular imaging in assessing a response to therapeutic agents aiming at preventing cardiovascular events has not yet been studied. Therefore, in this PhD thesis first we investigated the influence of microbubble shell characteristics on targeting efficiency of microbubbles. Second we used ultrasound molecular imaging to assess the anti-inflammatory effects of statins in vascular inflammation in relevant murine models of atherosclerosis and finally we investigated the short-term effects of treatment with apocynin on endothelial inflammation and thrombogenicity in a murine model of early atherosclerosis by application of ultrasound molecular imaging.
In the first part of our study we demonstrated that microbubbles with a longer PEG spacer arm, which is used for the conjugation of the targeting ligand on the microbubble surface, yield a better targeting efficiency compared to the microbubbles with a shorter PEG spacer arm. The improvement of the targeting efficiency of microbubbles with the longer PEG spacer arm could be due to better accessibility of the conjugated ligands on the microbubble surface to their endothelial target and also due to more stable bonds formed between ligands presented by longer PEG spacer arms to their target under flow conditions. In addition the targeting efficiency of microbubbles was influenced by the degree to which the endothelial target was projected away from the endothelial surface. The attachment efficiency was low for short molecular targets, which were hidden in the glycocaylyx. This observation could have implications for the selection of potential molecular imaging targets. Moreover as the interaction of targeted microbubbles with their endothelial target occurs in the presence of the endothelial glycocalyx the thickness of the glycocalyx in the vessels may have an important influence on microbubble attachment efficiency.
In the second project we could show that CEU molecular imaging can assess the impact of therapy on endothelial inflammation in a murine model of early atherosclerosis where high frequency ultrasound imaging of the aortic wall was unable to show the effects of treatment on the atherosclerotic plaques. In atherosclerosis endothelial expression of the inflammatory cell adhesion molecule Vascular Cell Adhesion Molecule- 1 (VCAM-1) plays an important role in the initiation and progression of atherosclerosis. CEU molecular imaging by targeting microbubbles to VCAM-1 demonstrated a selective signal enhancement for these microbubbles compared to control microbubbles in non- treated animals but not in atorvastatin treated animals. Therefore given that phenotypic changes on the endothelial surface are responsible for the initiation of atherosclerosis, this method could be used for assessing treatment effects during the initial stages of atherosclerosis.
Lastly, We were able to demonstrate that short-term treatment with apocynin, an antioxidant, anti-inflammatory agent, in a mouse model of early atherosclerosis results in reduced aortic endothelial inflammation presented as a reduced expression of endothelial VCAM-1 and platelet adhesion. However, these anti-inflammatory effects were not associated with measurable reductions in vascular NADPH oxidase activity or superoxide content. Our findings from CEU molecular imaging demonstrated that there is a lower attachment of microbubbles targeted to endothelial VCAM-1 and also activated platelets to aortic endothelial cells after treatment with apocynin compared to the group which were imaged at baseline and a group which received saline treatment as a control group. However, apocynin therapy did not reduce ROS content or superoxide generation. These effects of apocynin are probably due to ROS- independent mechanisms in the early stages of atherosclerotic plaque development.
In conclusion CEU molecular imaging could be useful in the future for assessing the effects of drug treatment in individual patients, but also for screening of the effects of novel drug classes in preclinical field.