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Kristof Graf
Professor of Medicine
Head
Cardiovascular Research Unit
Eike Nagel
Director CMR Academy
Assistant Professor of Medicine
Eckart Fleck
Professor of Medicine
Director
Department of Cardiology
Deutsches
Herzzentrum
Berlin
Germany
Several retrospective autopsy series and cross-sectional clinical studies have suggested that thrombotic coronary death and acute coronary syndromes are caused by plaque features and associated factors, including plaque rupture, plaque erosion, calcified nodules and hypercoagulate state. Despite major achievements in risk assessment in patients with cardiovascular risk – especially those with diagnoses of diabetes, obesity and hypertension – and in the management of patients with acute coronary syndromes, early identification of rupture-prone atherosclerotic lesions remains a challenge. It is a problem since nonstenotic lesions are far more frequent than stenotic plaques and account for the majority of ruptured vulnerable plaques.
The identification of these lesions in patients with a risk for cardiovascular disease by new imaging modalities identifying inflammatory “vulnerable” plaque lesions would open a field for novel diagnostic concepts and treatment strategies, which could potentially include specific targeting of genes and proteins that govern cellular mechanisms in atherosclerosis. In recent years researchers at the Department of Medicine/Cardiology, Berlin, have combined methods for identifying cellular mechanisms in plaque progression by biochemical and morphological means, with the use of innovative imaging modalities such as high-resolution MRI to identify new targets.
With the support of internal and external funds (eg, the Philip Morris External Research Program) the work has led to the identification of key factors that regulate cellular functions in atherosclerosis. This includes the identification of cell surface adhesion receptors, such as CD40, and the characterisation of specific extracellular matrix proteins expressed in plaques such as the fibronectin splice variant ED-B. CD40 is involved in inflammatory pathways signalling. ED-B was found to be highly expressed in lesions of atherosclerotic ApoE(-/-) mice.
One of the preferred imaging modalities for in-vivo experiments is near-infrared optical imaging (NIR) using a cooled charged-couple detector (CCD) camera providing a rapid, sensitive and cost-effective option – especially in small animal studies. Furthermore, conjugation of antibodies or modified single-chain antibodies with NIR dyes is a well-established technique with high yield and high success rates. Using this technique, the feasibility of ED-B-targeted antibodies (NIR-fluorochromes) could be shown for lesion detection, demonstrating a close correlation with plaque formation as confirmed by histology (Figure 1).
[[HHE06_fig1_Ca27]]
Moreover, we have recently characterised furin and PC5, two enzymes of the group of proprotein convertases as central regulators of vascular smooth muscle cell and macrophage function in atherosclerosis. These enzymes are required for endoproteolytic activation of precursor proteins, an ancient mechanism that leads to biological active molecules in a highly selective manner. We used gene-silencing methods, specific small interfering RNA against furin to reduce the intracellular protein expression of furin by up to 80%, to investigate the functional role of this proprotein convertases. We identified furin/PC5 as key regulators of vascular smooth muscle cell – macrophage cooperation in vitro. Furin/PC5 activates matrix metalloproteinases in macrophages – these are the major enzymes involved in the degradation of the plaque cap and plaque destabilisation leading to acute coronary events. Furthermore, their increased expression in vulnerable human lesions indicates that they are suitable imaging targets as well.
Several methods are available for more clinically oriented noninvasive imaging such as optical (NIR), ultrasound, CT and cardiac magnetic resonance imaging (MRI). Tremendous technical progress and improvements in recent years have made first studies possible using MRI or CT for identification and analysis of atherosclerotic lesions, preferably of the carotid and coronary arteries.
Our group has developed new MRI techniques for atherosclerotic plaque imaging. By introducing new motion compensation techniques in a high-resolution 3T MRI we have established imaging sequences for coronary plaque detection in patients with coronary artery disease. In ex-vivo experiments using high-field MRI (7T), we have developed MRI sequences suitable for detecting and analysing atherosclerotic lesions in human arteries, demonstrating close correlation between histological plaque stages and especially T2-weighted images. For molecular imaging we have produced stable antibody-contrast agent conjugates using iron oxide nanoparticles, which can be used for MRI. The first in-vivo experiments with antibody contrast-conjugates have been performed using CD40 antibodies and have demonstrated specific target-binding using high-resolution MRI of the aortic arch in ApoE(-/-) mice.
[[HHE06_fig2_Ca27]]
We believe that this combinational approach, which brings together expertise in biochemical methods with novel imaging modalities, might provide the substantial background to develop specific noninvasive imaging methods for identifying patients at a high risk of plaque rupture.
