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Cardiovascular imaging has traditionally focused on anatomy. Over the last few decades, we have made great strides in understanding the basic biology of cardiac disease and vascular disease. Several groups have recently attempted to exploit the newly discovered molecular and cellular mechanisms that have emerged regarding cardiovascular disease, to extend imaging beyond anatomy. In regard to atherosclerosis in particular, we have learned that the degree of stenosis isn't the only characteristic of an atherosclerotic plaque that can determine its clinical destiny.1 Indeed, qualitative aspects of a plaque can indicate whether the plaque might cause thrombotic complications.2,3 Various aspects of inflammation furnish novel targets for molecular imaging of atherosclerosis (Table I). Inflammation participates from the very inception of this disease through its progression, up to and including thrombotic complications.4
Recruitment of inflammatory cells from the blood is one of the first steps in the transition from the normal artery to the early atherosclerotic plaque. Risk factors associated with atherogenesis induce the expression, on the surface of endothelial cells, of adhesion molecules that capture blood leukocytes. One such molecule, vascular cell adhesion molecule-1 (VCAM-1), appears very early after rabbits or mice begin to consume an atherogenic diet. We have developed and evaluated as imaging agents sequential generations of peptide-based ligands that can bind to VCAM-1. This approach aims to enable imaging of a biological property of inflammatory activation of endothelial cells (Fig. 1).5,6
Once bound to adhesion molecules such as VCAM-1, leukocytes can penetrate the intima and take up residence in response to pro-inflammatory chemokines, which direct their migration. The success of the therapeutic strategy of targeting inflammatory mediators such as the chemokines depends on ongoing leukocyte recruitment. Quantitative tracking of labeled leukocytes has shown that monocyte accumulation in mouse atheromata is progressive and proportional to the extent of disease.7 Data gleaned from such tracking support the targeting of mononuclear cell uptake to mitigate atherosclerosis. This example illustrates how one can harness this technology not only to image, but also to learn more about the biology of atherosclerosis and the validation of therapeutic targets (Fig. 2).8
Once monocytes have accumulated in the artery wall, they mature into phagocytically active macrophages that can accumulate lipid; lipid-laden macrophages constitute the hallmark of the atherosclerotic lesion. Phagocytosis provides one way to probe a relevant pro-inflammatory function of mononuclear cells in the artery wall. Several groups are using nanotechnology approaches to image phagocytosis. We have used ultra-small particulate oxide (USPIO) particles for this purpose. These nanoparticles consist of a superparamagnetic substance, in this case iron oxide, with a dextran coating to provide biocompatibility. The development of nanoparticles with appropriate properties for use in vivo requires much optimization to make them biocompatible and to prolong their half-life enough to enable circulating nanoparticles to accumulate in target tissues. After the blood pool clears, magnetic resonance imaging (MRI) can visualize the site accumulation of such nanoparticles. We have tested, in experimental atherosclerosis in rabbits, whether we could track macrophage accumulation in plaques. These studies used high-resolution magnetic resonance with a three-Tesla (3T) magnet. Kinetic studies in vivo ascertained the optimum time after injection for imaging (allowing clearance of the agent from the blood pool). Exposure of cultured human monocyte-derived macrophages to such nanoparticles yielded a dose-dependent increase in iron accumulation. In vivo administration of the nanoparticles led to suppression of the signal in T2-weighted images at sites of macrophage accumulation, as was subsequently validated histologically. These analyses showed a good correlation between T2 signal reduction and macrophage content. Treatment of cholesterol-fed rabbits with a statin produced a striking decrease in lesional macrophage content and T2 signal suppression in the aorta. These results indicate that high-resolution 3T MRI using a phagocytosable paramagnetic imaging probe can detect therapeutic effects on macrophage-rich atherosclerotic plaques that are often the culprits in acute coronary syndromes. Positron-emission tomography–computed tomography can also visualize leukocyte accumulation in experimental atheromata (Fig. 3).9
We know from advances in our understanding of the molecular and cellular mechanisms of plaque disruption that overexpression of proteases can promote degradation of macromolecules of the arterial extracellular matrix, thereby rendering them susceptible to rupture—an event that can trigger thrombosis.10-12 Mediators of inflammation induce protease expression by lesional leukocytes. We have now begun to visualize inflammation in atherosclerosis by looking at the activity of such proteolytic enzymes as the matrix metalloproteases (MMPs) and certain cysteinyl cathepsins. For example, probes for protease activity that emit fluorescent signals when cleaved can report on the biochemical action of these molecular mediators of plaque disruption. Proof-of-principle experiments in atherosclerotic animals show that optical techniques can noninvasively visualize MMPs, such as MMPs 2 and 9, as well as cathepsins B and K.13,14
One future challenge is the translation of results from animal studies, such as those discussed above, to human beings. Nuclear imaging provides a relatively straightforward pathway to the clinical use of molecular imaging probes. Magnetic resonance imaging provides greater spatial resolution, but lower sensitivity, than does nuclear imaging. We, and others, are currently developing angioscopic platforms for visualizing near-infrared fluorescence, to ease the extension of animal studies to the clinic.
Molecular imaging may not see routine clinical application as a screening tool for so-called “vulnerable plaques” in unselected populations anytime soon. Yet, to move forward in cardiovascular drug development, we need approaches that will give us early signals of efficacy and help us to choose doses of new agents for the design of endpoint trials. We must exercise great care in the use of novel imaging strategies, especially in this time of concern about cost containment. We must ask some very important questions—not just about molecular imaging, but about the added value and cost-effectiveness of all our evolving imaging methods—and we should be as rigorous in their evaluation as we are, or should be, in evaluating all biomarkers. Imaging strategies will probably be part of a tiered management strategy that first applies to populations of unknown risk using tools such as the Framingham algorithm, or the Framingham algorithm enhanced with an inflammatory marker and family history (as in the Reynolds risk score). In a second tier, a subpopulation that shows evidence of increased risk through a combination of traditional and novel simple, non-imaging biomarkers would then be selected for targeted, advanced screening that might involve imaging. This tiered pathway might enable us to use molecular imaging in a cost-effective way in actual clinical practice.
We thank our colleagues in the Cardiovascular Division of the Brigham & Women's Hospital, the Center for Systems Biology at the Massachusetts General Hospital, and the D.W. Reynolds Clinical Cardiovascular Research Center at Harvard Medical School, for their contributions to this work. We thank Ms Sara Karwacki for editorial support.
Address for reprints: Peter Libby, MD, Chief, Cardiovascular Medicine, Brigham & Women's Hospital, 77 Ave. Louis Pasteur, Boston, MA 02115
Presented at the 9th Texas Update in Cardiovascular Advancements; Houston, Texas; 4–5 December 2009
Program Director: James T. Willerson, MD
This work was funded in part by the Donald W. Reynolds Foundation, Translational Program of Excellence in Nanotechnology grant U01HL080731, and NIH Grant HL080472.