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.

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 ...

MRI has been employed to elucidate the migratory behavior of stem/progenitor cells noninvasively in vivo with traditional proton (1H) imaging of iron oxide nanoparticle-labeled cells. Alternatively, we demonstrate that fluorine (19F) MRI of cells labeled with different types of liquid perfluorocarbon (PFC) nanoparticles produces unique and sensitive cell markers distinct from any tissue background signal. To define the utility for cell tracking, mononuclear cells harvested from human umbilical cord blood were grown under proendothelial conditions and labeled with nanoparticles composed of two distinct PFC cores (perfluorooctylbromide and perfluoro-15-crown-5 ether). The sensitivity for detecting and imaging labeled cells was defined on 11.7T (research) and 1.5T (clinical) scanners. Stem/progenitor cells (CD34+ CD133+ CD31+) readily internalized PFC nanoparticles without aid of adjunctive labeling techniques, and cells remained functional in vivo. PFC-labeled cells exhibited distinct 19F signals and were readily detected after both local and intravenous injection. PFC nanoparticles provide an unequivocal and unique signature for stem/progenitor cells, enable spatial cell localization with 19F MRI, and permit quantification and detection of multiple fluorine signatures via 19F MR spectroscopy. This method should facilitate longitudinal investigation of cellular events in vivo for multiple cell types simultaneously.

Gray matter T2 hypointensity in MS is associated with brain atrophy and is a stronger predictor of disability and clinical course than are conventional MRI findings. While longitudinal studies are warranted, these results suggest that pathologic iron deposition is a surrogate marker of the destructive disease process.

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Before molecular imaging with MRI can be applied clinically, certain problems, such as the potential sparseness of molecular epitopes on targeted cell surfaces, and the relative weakness of conventional targeted MR contrast agents, must be overcome. Accordingly, the conditions for diagnostic conspicuity that apply to any paramagnetic MRI contrast agent with known intrinsic relaxivity were examined in this study. A highly potent paramagnetic liquid perfluorocarbon nanoparticle contrast agent (ϳ250 nm diameter, >90000 Gd 3؉ /particle) was imaged at 1.5 T and used to successfully predict a range of sparse concentrations in experimental phantoms with the use of standard MR signal models. Additionally, we cultured and targeted the smooth muscle cell (SMC) monolayers that express "tissue factor," a glycoprotein of crucial significance to hemostasis and response to vascular injury, by conjugating an anti-tissue factor antibody fragment to the nanoparticles to effect specific bind-

Neovascularization is a critical component in the progression of malignant melanoma. The objective of this study was to determine whether ␣ ␤ 3 -targeted paramagnetic nanoparticles can detect and characterize sparse ␣ ␤ integrin expression on neovasculature induced by nascent melanoma xenografts (ϳ30 mm

OBJECTIVE Studies were performed to develop a prolonged antiangiogenesis therapy regimen based on theranostic ανβ3–targeted nanoparticles. BACKGROUND Antiangiogenesis therapy may normalize atherosclerotic plaque vasculature and promote plaque stabilization.ανβ3–targeted paramagnetic nanoparticles can quantify atherosclerotic angiogenesis and incorporate fumagillin to elicit acute antiangiogenic effects. METHODS In the first experiment, hyperlipidemic rabbits received ανβ3–targeted fumagillin nanoparticles (0, 30, or 90 μg/kg) with either a continued high fat diet or conversion to standard chow. The antiangiogenic response was followed for 4 weeks by MR molecular imaging with ανβ3–targeted paramagnetic nanoparticles. In a second 8-week study, atherosclerotic rabbits received atorvastatin (0 or 44 mg/kg diet) alone or with ανβ3–targeted fumagillin nanoparticles (only week 0 vs. weeks 0 and 4), and angiogenesis was monitored with MR molecular imaging. Histology was performed to determine the location of bound nanoparticles and to correlate the level of MRI enhancement with the density of angiogenic vessels. RESULTS ανβ3–targeted fumagillin nanoparticles reduced the neovascular signal by 50 to 75% at 1-week, and maintained this effect for 3 weeks regardless of diet and drug dose. In the second study, atherosclerotic rabbits receiving statin alone had no antineovascular benefit over 8 weeks. ανβ3–targeted fumagillin nanoparticles decreased aortic angiogenesis for 3 weeks as in study one and readministration on week 4 reproduced the 3-week antineovascular response with no carry over benefit. However, atorvastatin and two doses of ανβ3–targeted fumagillin nanoparticles (0 and 4 weeks) achieved marked and sustainable antiangiogenesis. Microscopic studies corroborated the high correlation between MR signal and neovessel counts and confirmed that the ανβ3-targeted nanoparticles were constrained to the vasculature of the aortic adventia. CONCLUSION MR molecular imaging with ανβ3–targeted paramagnetic nanoparticles demonstrated that the acute antiangiogenic effects of ανβ3–targeted fumagillin nanoparticles could be prolonged when combined with atorvastatin, representing a potential strategy to evaluate antiangiogenic treatment and plaque stability.