Background-Restenosis is a serious complication of coronary angioplasty that involves the proliferation and migration of vascular smooth muscle cells (VSMCs) from the media to the intima, synthesis of extracellular matrix, and remodeling. We have previously demonstrated that tissue factor-targeted nanoparticles can penetrate and bind stretch-activated vascular smooth muscles in the media after balloon injury. In the present study, the concept of VSMC-targeted nanoparticles as a drug-delivery platform for the prevention of restenosis after angioplasty is studied. Methods and Results-Tissue factor-targeted nanoparticles containing doxorubicin or paclitaxel at 0, 0.2, or 2.0 mole% of the outer lipid layer were targeted for 30 minutes to VSMCs and significantly inhibited their proliferation in culture over the next 3 days. Targeting of the nanoparticles to VSMC surface epitopes significantly increased nanoparticle antiproliferative effectiveness, particularly for paclitaxel. In vitro dissolution studies revealed that nanoparticle drug release persisted over one week. Targeted antiproliferative results were dependent on the hydrophobic nature of the drug and noncovalent interactions with other surfactant components. Molecular imaging of nanoparticles adherent to the VSMC was demonstrated with high-resolution T 1 -weighted MRI at 4.7T. MRI 19 F spectroscopy of the nanoparticle core provided a quantifiable approach for noninvasive dosimetry of targeted drug payloads. Conclusions-These data suggest that targeted paramagnetic nanoparticles may provide a novel, MRI-visualizable, and quantifiable drug delivery system for the prevention of restenosis after angioplasty.

. Regional ventricular wall thickening reflects changes in cardiac fiber and sheet structure during contraction: quantification with diffusion tensor MRI. Am J Physiol Heart Circ Physiol 289: H1898 -H1907, 2005. doi:10.1152/ajpheart.00041.2005.-Dynamic changes of myocardial fiber and sheet structure are key determinants of regional ventricular function. However, quantitative characterization of the contractionrelated changes in fiber and sheet structure has not been reported. The objective of this study was to quantify cardiac fiber and sheet structure at selected phases of the cardiac cycle. Diffusion tensor MRI was performed on isolated, perfused Sprague-Dawley rat hearts arrested or fixed in three states as follows: 1) potassium arrested (PA), which represents end diastole; 2) barium-induced contracture with volume (BVϩ), which represents isovolumic contraction or early systole; and 3) barium-induced contracture without volume (BVϪ), which represents end systole. Myocardial fiber orientations at the base, midventricle, and apex were determined from the primary eigenvectors of the diffusion tensor. Sheet structure was determined from the secondary and tertiary eigenvectors at the same locations. We observed that the transmural distribution of the myofiber helix angle remained unchanged as contraction proceeded from PA to BVϩ, but endocardial and epicardial fibers became more longitudinally orientated in the BVϪ group. Although sheet structure exhibited significant regional variations, changes in sheet structure during myocardial contraction were relatively uniform across regions. The magnitude of the sheet angle, which is an index of local sheet slope, decreased by 23 and 44% in BVϩ and BVϪ groups, respectively, which suggests more radial orientation of the sheet. In summary, we have shown for the first time that geometric changes in both sheet and fiber orientation provide a substantial mechanism for radial wall thickening independent of active components due to myofiber shortening. Our results provide direct evidence that sheet reorientation is a primary determinant of myocardial wall thickening. magnetic resonance imaging; potassium arrest; barium contracture; myofiber; reorientation; myocyte shortening THE VENTRICULAR EXCITATION-contraction process elicits transmural wall thickening that serves as the mechanism for ejection of blood. Although myocyte contraction provides the cellular basis for regional myocardial wall thickening, previous studies of dog hearts (27, 32) suggest that the typical 14% myocyte shortening leads to only an ϳ8% increase in myocyte diameter, which cannot fully account for the observed 28 -50% increase in average wall thickness (4, 28, 36). Apex-to-base (longitudinal) shortening represents another important mechanism that has recently been recognized as a significant contributor to the ejection fraction that necessitates complex myofiber rearrangements during systole. However, the microscopic structural changes that occur during myocardial contraction remain poorly defined because...

Unstable atherosclerotic plaques exhibit microdeposits of fibrin that may indicate the potential for a future rupture. However, current methods for evaluating the stage of an atherosclerotic lesion only involve characterizing the level of vessel stenosis, without delineating which lesions are beginning to rupture. Previous work has shown that fibrin-targeted, liquid perfluorocarbon nanoparticles, which carry a high payload of gadolinium, have a high sensitivity and specificity for detecting fibrin with clinical 1 H MRI. In this work, the perfluorocarbon content of the targeted nanoparticles is exploited for the purposes of 19 F imaging and spectroscopy to demonstrate a method for quantifiable molecular imaging of fibrin in vitro at 4.7 T. Additionally, the quantity of bound nanoparticles formulated with different perfluorocarbon species was calculated using spectroscopy. Results indicate that the high degree of nanoparticle binding to fibrin clots and the lack of background 19 F signal allow accurate quantification using spectroscopy at 4.7 T, as corroborated with proton relaxation rate measurements at 1.5 T and trace element (gadolinium) analysis. Finally, the extension of these techniques to a clinically relevant application, the evaluation of the fibrin burden within an ex vivo human carotid endarterectomy sample, demonstrates the potential use of these particles for uniquely identifying unstable atherosclerotic lesions in vivo.

The underlying cause of major cardiovascular events, such as myocardial infarctions and strokes, is atherosclerosis. For accurate diagnosis of this inflammatory disease, molecular imaging is required. Toward this goal, we sought to develop a nanoparticle-based, high aspect ratio, molecularly targeted magnetic resonance MR imaging contrast agent. Specifically, we engineered the plant viral nanoparticle platform tobacco mosaic virus (TMV) to target vascular cell adhesion molecule (VCAM)-1, which is highly expressed on activated endothelial cells at atherosclerotic plaques. To achieve dual optical and MR imaging in an atherosclerotic ApoE−/− mouse model, TMV was modified to carry near-infrared dyes and chelated Gd ions. Our results indicate molecular targeting of atherosclerotic plaques. On the basis of the multivalency and multifunctionality, the targeted TMV-based MR probe increased the detection limit significantly; the injected dose of Gd ions could be further reduced by 3 orders of magnitude compared to the suggested clinical use, demonstrating the utility of targeted nanoparticle cargo delivery.

Molecular imaging of microthrombus within fissures of unstable atherosclerotic plaques requires sensitive detection with a thrombus-specific agent. Effective molecular imaging has been previously demonstrated with fibrin-targeted Gd-DTPA-bisoleate (BOA) nanoparticles. In this study, the relaxivity of an improved fibrin-targeted paramagnetic formulation, Gd-DTPAphosphatidylethanolamine (PE), was compared with Gd-DTPA-BOA at 0.05-4.7 T. Ion-and particle-based r 1 relaxivities (1.5 T) for Gd-DTPA-PE (33.7 (s*mM) -1 and 2.48 ؋ 10 6 (s*mM) -1 , respectively) were about twofold higher than for Gd-DTPA-BOA, perhaps due to faster water exchange with surface gadolinium. Gd-DTPA-PE nanoparticles bound to thrombus surfaces via anti-fibrin antibodies (1H10) induced 72% ؎ 5% higher change in R 1 values at 1.5 T (⌬R 1 ‫؍‬ 0.77 ؎ 0.02 1/s) relative to Gd-DTPA-BOA (⌬R 1 ‫؍‬ 0.45 ؎ 0.02 1/s). These studies demonstrate marked improvement in a fibrin-specific molecular imaging agent that might allow sensitive, early detection of vascular microthrombi, the antecedent to stroke and heart attack. The acute formation of thrombus on ruptured atherosclerotic plaques is well recognized as the source of unstable angina, myocardial infarction, transient ischemic attacks, and stroke (1). Although myriad medical advances in the detection and treatment of advanced carotid and coronary artery disease have emerged, early detection of the most common source of thromboembolism-rupturing atherosclerotic plaques in arteries with modest 40-60% stenosis (2)-remains diagnostically elusive with the use of routine angiography or duplex ultrasound techniques.A variety of approaches have been proposed to improve detection of vulnerable plaques, including intravascular ultrasound elastography (3), radionuclide imaging (4), and thermography (5). Magnetic resonance imaging (MRI) also is emerging as a particularly sensitive modality to noninvasively visualize thromboses within the carotid artery (6). However, the proximate cause of heart attacks and strokes-rupture of the vulnerable plaque-cannot be reliably detected with any nondestructive imaging modality.Recent studies reveal that vulnerable plaque rupture and microthrombus formation precedes acute myocardial infarction by days to months (7), providing a window of opportunity to intercede and prevent serious sequelae. Sensitive molecular imaging and detection of microthrombi along the intimal surface of vulnerable plaques will require a high-avidity, target-specific probe with robust signal amplification compatible with a sensitive, high-resolution imaging modality. Until recently, the signal amplification required to detect and visualize important molecular or cellular moieties present in nano-and picomolar concentrations in vivo was obtainable only with nuclear imaging modalities. However, more recently, molecular imaging with magnetic resonance has shown promise (8,9).A new approach entails the use of a fibrin-specific paramagnetic molecular imaging agent to improve detection and quantification of these...

To compensate for the low sensitivity of magnetic resonance imaging (MRI), nanoparticles have been developed to deliver high payloads of contrast agents to sites of disease. Here, we report the development of supramolecular MRI contrast agents using the plant viral nanoparticle tobacco mosaic virus (TMV). Rod-shaped TMV nanoparticles measuring 300×18 nm were loaded with up to 3,500 or 2,000 chelated paramagnetic gadolinium (III) ions selectively at the interior (iGd-TMV) or exterior (eGd-TMV) surface, respectively. Spatial control is achieved through targeting either tyrosine or carboxylic acid side chains on the solvent exposed exterior or interior TMV surface. The ionic T1 relaxivity per Gd ion (at 60 MHz) increases from 4.9 mM−1s−1 for free Gd(DOTA) to 18.4 mM−1s−1 for eGd-TMV and 10.7 mM−1s−1 for iGd-TMV. This equates to T1 values of ~ 30,000 mM−1s−1 and ~ 35,000 mM−1s−1 per eGd-TMV and iGd-TMV nanoparticle. Further, we show that interior-labeled TMV rods can undergo thermal transition to form 170 nm-sized spherical nanoparticles containing ~ 25,000 Gd chelates and a per particle relaxivity of almost 400,000 mM−1s−1 (15.2 mM−1s−1 per Gd). This work lays the foundation for the use of TMV as a contrast agent for MRI.

Background-Altered electrical activation of the heart by pacing or disease induces profound ventricular electrical remodeling (VER), manifested electrocardiographically as T-wave memory and ultimately as deleterious mechanical remodeling from heterogeneous strain. Although T-wave memory is associated with altered expression of sarcolemmal ion channels, the biophysical mechanisms responsible for triggering remodeling of cardiac ion channels are unknown. Methods and Results-To test the hypothesis that mechanoelectrical feedback triggered by regional strain is a mechanism for VER, dogs (nϭ6) underwent 4 weeks of ventricular pacing to induce VER. Multisegment transmural optical action potential imaging of left ventricular wedges revealed profound and selective prolongation of action potential duration in late-activated (288Ϯ29 ms) compared with early-activated (250Ϯ9 ms) myocardial segments (PϽ0.05), providing the first experimental evidence that amplification of repolarization gradients between segments of left ventricle is the electrophysiological basis for T-wave memory. In vivo tagged magnetic resonance imaging revealed a 2-fold and preferential increase in circumferential strain in late-activated segments of myocardium, which exactly coincided with segments undergoing VER. VER could not be attributed to structural remodeling because it occurred without any histological evidence of cellular hypertrophy. Conclusions-The mechanism responsible for triggering remodeling of ion channel function in VER was locally enhanced circumferential strain. These data suggest a novel mechanoelectrical feedback mechanism for inducing physiological and potentially deleterious electrical heterogeneities in the heart. (Circulation. 2007;115:3145-3155.)

Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.