Background-Angiogenesis is a critical feature of plaque development in atherosclerosis and might play a key role in both the initiation and later rupture of plaques that lead to myocardial infarction and stroke. The precursory molecular or cellular events that initiate plaque growth and that ultimately contribute to plaque instability, however, cannot be detected directly with any current diagnostic modality. Methods and Results-Atherosclerosis was induced in New Zealand White rabbits fed 1% cholesterol for Ϸ80 days.␣ v ␤ 3 -Integrin-targeted, paramagnetic nanoparticles were injected intravenously and provided specific detection of the neovasculature within 2 hours by routine magnetic resonance imaging (MRI) at a clinically relevant field strength (1.5 T). Increased angiogenesis was detected as a 47Ϯ5% enhancement in MRI signal averaged throughout the abdominal aortic wall among rabbits that received ␣ v ␤ 3 -targeted, paramagnetic nanoparticles. Pretreatment of atherosclerotic rabbits with ␣ v ␤ 3 -targeted, nonparamagnetic nanoparticles competitively blocked specific contrast enhancement of the ␣ v ␤ 3 -targeted paramagnetic agent. MRI revealed a pattern of increased ␣ v ␤ 3 -integrin distribution within the atherosclerotic wall that was spatially heterogeneous along both transverse and longitudinal planes of the abdominal aorta. Histology and immunohistochemistry confirmed marked proliferation of angiogenic vessels within the aortic adventitia, coincident with prominent, neointimal proliferation among cholesterol-fed, atherosclerotic rabbits in comparison with sparse incidence of neovasculature in the control animals. Conclusions-This molecular imaging approach might provide a method for defining the burden and evolution of atherosclerosis in susceptible individuals as well as responsiveness of individual patients to antiatherosclerotic therapies.

Background-Molecular imaging of thrombus within fissures of vulnerable atherosclerotic plaques requires sensitive detection of a robust thrombus-specific contrast agent. In this study, we report the development and characterization of a novel ligand-targeted paramagnetic molecular imaging agent with high avidity for fibrin and the potential to sensitively detect active vulnerable plaques. Methods and Results-The nanoparticles were formulated with 2.5 to 50 mol% Gd-DTPA-BOA, which corresponds to Ͼ50 000 Gd 3ϩ atoms/particle. Paramagnetic nanoparticles were characterized in vitro and evaluated in vivo. In contradistinction to traditional blood-pool agents, T1 relaxation rate as a function of paramagnetic nanoparticle number was increased monotonically with Gd-DTPA concentration from 0.18 mL ⅐ s Ϫ1 · pmol Ϫ1 (10% Gd-DTPA nanoparticles) to 0.54 mL ⅐ s Ϫ1 · pmol Ϫ1 for the 40 mol% Gd-DTPA formulations. Fibrin clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T1-weighted contrast enhancement that improved with increasing gadolinium level (0, 2.5, and 20 mol% Gd). Higher-resolution scans and scanning electron microscopy revealed that the nanoparticles were present as a thin layer over the clot surface. In vivo contrast enhancement under open-circulation conditions was assessed in dogs. The contrast-to-noise ratio between the targeted clot (20 mol% Gd-DTPA nanoparticles) and blood was Ϸ118Ϯ21, and that between the targeted clot and the control clot was 131Ϯ37. Conclusions-These

Objective-Angiogenic expansion of the vasa vasorum is a well-known feature of progressive atherosclerosis, suggesting that antiangiogenic therapies may stabilize or regress plaques. ␣ ␤ 3 Integrin-targeted paramagnetic nanoparticles were prepared for noninvasive assessment of angiogenesis in early atherosclerosis, for site-specific delivery of antiangiogenic drug, and for quantitative follow-up of response. Methods and Results-Expression of ␣ ␤ 3 integrin by vasa vasorum was imaged at 1.5 T in cholesterol-fed rabbit aortas using integrin-targeted paramagnetic nanoparticles that incorporated fumagillin at 0 g/kg or 30 g/kg. Both formulations produced similar MRI signal enhancement (16.7%Ϯ1.1%) when integrated across all aortic slices from the renal arteries to the diaphragm. Seven days after this single treatment, integrin-targeted paramagnetic nanoparticles were readministered and showed decreased MRI enhancement among fumagillin-treated rabbits (2.9%Ϯ1.6%) but not in untreated rabbits (18.1%Ϯ2.1%). In a third group of rabbits, nontargeted fumagillin nanoparticles did not alter vascular ␣ ␤ 3 -integrin expression (12.4%Ϯ0.9%; PϾ0.05) versus the no-drug control. In a second study focused on microscopic changes, fewer microvessels in the fumagillin-treated rabbit aorta were counted compared with control rabbits. Conclusions-This study illustrates the potential of combined molecular imaging and drug delivery with targeted nanoparticles to noninvasively define atherosclerotic burden, to deliver effective targeted drug at a fraction of previous levels, and to quantify local response to treatment. Key Words: magnetic resonance imaging Ⅲ atherosclerosis Ⅲ molecular imaging Ⅲ angiogenesis Ⅲ nanoparticles Ⅲ fumagillin A key feature of the atherosclerotic process is the angiogenic expansion of the vasa vasorum in the adventitia, which extends into the thickening intimal layer of the atheroma in concert with other neovessels originating from the primary arterial lumen. 1 Extensive neovascular proliferation within atherosclerotic plaques is prominent within "culprit" lesions clinically associated with unstable angina, myocardial infarction, and stroke. [2][3][4] Plaque angiogenesis has been suggested to promote plaque growth, intraplaque hemorrhage, 5 and lesion instability.Magnetic resonance (MR) molecular imaging of focal angiogenesis in vivo with integrin-targeted paramagnetic contrast agents was reported with perfluorocarbon nanoparticles 6 -8 and liposomes. 9 Subsequently, we have developed MRI and postprocessing techniques to permit molecular imaging of the diffuse proliferating neovasculature associated with atherosclerotic plaque development. 10,11 The widespread expression of ␣ ␤ 3 integrins observed by MR agreed with the diffuse nature of angiogenesis microscopically observed in the early atherosclerotic aortas of cholesterol-fed rabbits.The importance of angiogenesis in the progression of atherosclerotic plaque combined with the antiangiogenic impact of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase ...

These data provide the first in vivo demonstration of a site-specific ultrasonic contrast agent and have potential for improved sensitivity and specificity for noninvasive diagnosis of thrombi and other pathological diseases.