Kim Douma

Quantitative Molecular Magnetic Resonance Imaging of Tumor Angiogenesis Using cNGR-Labeled Paramagnetic Quantum Dots

The objective of this study was to develop and apply cyclic Asn-Gly-Arg (cNGR)-labeled paramagnetic quantum dots (cNGR-pQDs) for the noninvasive assessment of tumor angiogenic activity using quantitative in vivo molecular magnetic resonance imaging (MRI). cNGR was previously shown to colocalize with CD13, an aminopeptidase that is highly overexpressed on angiogenic tumor endothelium. Because angiogenesis is important for tumor growth and metastatization, its in vivo detection and quantification may allow objective diagnosis of tumor status and evaluation of treatment response. I.v. injection of cNGR-pQDs in tumor-bearing mice resulted in increased quantitative contrast, comprising increased longitudinal relaxation rate and decreased proton visibility, in the tumor rim but not in tumor core or muscle tissue. This showed that cNGR-pQDs allow in vivo quantification and accurate localization of angiogenic activity. MRI results were validated using ex vivo two-photon laser scanning microscopy (TPLSM), which showed that cNGR-pQDs were primarily located on the surface of tumor endothelial cells and to a lesser extent in the vessel lumen. In contrast, unlabeled pQDs were not or only sparsely detected with both MRI and TPLSM, supporting a high specificity of cNGR-pQDs for angiogenic tumor vasculature.

Liposome-enhanced MRI of neointimal lesions in the ApoE-KO mouse.

Conventional high‐resolution MRI is capable of detecting lipid‐rich atherosclerotic plaques in both human atherosclerosis and animal models of atherosclerosis. In this study we induced neointimal lesions in ApoE‐KO mice by placing a constrictive collar around the right carotid artery. The model was imaged with conventional multispectral MRI, and the thickened wall could not be distinguished from surrounding tissue. We then tested paramagnetic liposomes (mean size = 90 nm) for their ability to improve MRI visualization of induced thickening, using Gd‐DTPA as a control. T1‐weighted (T1‐w), black‐blood MRI of the neck area of the mice was performed before and 15 min, 45 min, and 24 hr after intravenous injection of either paramagnetic liposomes or Gd‐DTPA. The collared vessel wall of mice that were injected with liposomes showed a pronounced signal enhancement of ∼100% immediately after injection, which was sustained largely until 24 hr postinjection. In contrast, the vessel wall of all controls (left carotid artery and animals injected with Gd‐DTPA) did not show significant contrast enhancement at those time points. This study demonstrates that intimal thickening in ApoE‐KO mice can be effectively detected by contrast‐enhanced (CE)‐MRI upon injection of paramagnetic liposomes. Magn Reson Med, 2006.

CD36 inhibition prevents lipid accumulation and contractile dysfunction in rat cardiomyocytes.

An increased cardiac fatty acid supply and increased sarcolemmal presence of the long-chain fatty acid transporter CD36 are associated with and contribute to impaired cardiac insulin sensitivity and function. In the present study we aimed at preventing the development of insulin resistance and contractile dysfunction in cardiomyocytes by blocking CD36-mediated palmitate uptake. Insulin resistance and contractile dysfunction were induced in primary cardiomyocytes by 48 h incubation in media containing either 100 nM insulin (high insulin; HI) or 200 μM palmitate (high palmitate; HP). Under both culture conditions, insulin-stimulated glucose uptake and Akt phosphorylation were abrogated or markedly reduced. Furthermore, cardiomyocytes cultured in each medium displayed elevated sarcolemmal CD36 content, increased basal palmitate uptake, lipid accumulation and decreased sarcomere shortening. Immunochemical CD36 inhibition enhanced basal glucose uptake and prevented elevated basal palmitate uptake, triacylglycerol accumulation and contractile dysfunction in cardiomyocytes cultured in either medium. Additionally, CD36 inhibition prevented loss of insulin signalling in cells cultured in HP, but not in HI medium. In conclusion, CD36 inhibition prevents lipid accumulation and lipid-induced contractile dysfunction in cardiomyocytes, but probably independently of effects on insulin signalling. Nonetheless, pharmacological CD36 inhibition may be considered as a treatment strategy to counteract impaired functioning of the lipid-loaded heart.

Molecular Magnetic Resonance Imaging of Myocardial Angiogenesis After Acute Myocardial Infarction

Background— Angiogenesis is a natural mechanism to restore perfusion to the ischemic myocardium after acute myocardial infarction (MI). Therapeutic angiogenesis is being explored as a novel treatment for MI patients; however, sensitive, noninvasive in vivo measures of therapeutic efficacy are lacking and need to be developed. Here, a molecular magnetic resonance imaging method is presented to noninvasively image angiogenic activity in vivo in a murine model of MI with cyclic Asn-Gly-Arg (cNGR)–labeled paramagnetic quantum dots (pQDs). The tripeptide cNGR homes specifically to CD13, an aminopeptidase that is strongly upregulated during myocardial angiogenesis. Methods and Results— Acute MI was induced in male Swiss mice via permanent ligation of the left anterior descending coronary artery. Molecular magnetic resonance imaging was performed 7 days after surgery and up to 2 hours after intravenous contrast agent administration. Injection of cNGR-pQDs resulted in a strong negative contrast that was located mainly in the infarcted myocardium. This negative contrast was significantly less in MI mice injected with unlabeled pQDs and in sham-operated mice injected with cNGR-pQDs. Validation with ex vivo 2-photon laser scanning microscopy revealed a strong colocalization of cNGR-pQDs with vascular endothelial cells, whereas unlabeled pQDs were mostly extravasated and diffused through the tissue. Additionally, 2-photon laser scanning microscopy demonstrated significant microvascular remodeling in the infarct/border zones compared with remote myocardium. Conclusions— cNGR-pQDs allow selective, noninvasive detection of angiogenic activity in the infarcted heart with the use of in vivo molecular magnetic resonance imaging and ex vivo 2-photon laser scanning microscopy.

Nanoparticles for Optical Molecular Imaging of Atherosclerosis

Molecular imaging contributes to future personalized medicine dedicated to the treatment of cardiovascular disease, the leading cause of mortality in industrialized countries. Endoscope-compatible optical imaging techniques would offer a stand-alone alternative and high spatial resolution validation technique to clinically accepted imaging techniques in the (intravascular) assessment of vulnerable atherosclerotic lesions, which are predisposed to initiate acute clinical events. Efficient optical visualization of molecular epitopes specific for vulnerable atherosclerotic lesions requires targeting of high-quality optical-contrast-enhancing particles. In this review, we provide an overview of both current optical nanoparticles and targeting ligands for optical molecular imaging of atherosclerotic lesions and speculate on their applicability in the clinical setting.

Plaque-Associated Vasa Vasorum in Aged Apolipoprotein E–Deficient Mice Exhibit Proatherogenic Functional Features In Vivo

Objective—Neovascularization of human atherosclerotic plaques is implicated in plaque progression and destabilization, although its functional implications are yet unresolved. Here, we aimed to elucidate functional and morphological properties of plaque microvessels in mice in vivo. Methods and Results—Atherosclerotic carotid arteries from aged (>40 weeks) apolipoprotein E–deficient mice were imaged in vivo using multiphoton laser scanning microscopy. Two distinct groups of vasa vasorum microvessels were observed at sites of atherosclerosis development (median diameters of 18.5 and 5.9 &mgr;m, respectively), whereas microvessels within the plaque could only rarely be found. In vivo imaging showed ongoing angiogenic activity and injection of fluorescein isothiocyanate-dextran confirmed active perfusion. Plaque vasa vasorum showed increased microvascular leakage, combined with a loss of endothelial glycocalyx. Mean blood flow velocity in plaque-associated vasa vasorum was reduced by ±50% compared with diameter-matched control capillaries, whereas mean blood flow was reduced 8-fold. Leukocyte adhesion and extravasation were increased 6-fold in vasa vasorum versus control capillaries. Conclusion—Using a novel in vivo functional imaging strategy, we showed that plaque-associated vasa vasorum were angiogenically active and, albeit poorly, perfused. Moreover, plaque-associated vasa vasorum showed increased permeability, reduced blood flow, and increased leukocyte adhesion and extravasation (ie, characteristics that could contribute to plaque progression and destabilization).

Optical molecular imaging of atherosclerosis using nanoparticles: shedding new light on the darkness

The application of optical nanoparticles in cardiovascular research is increasing because of the high spatiotemporal resolution and high sensitivity of optical techniques as compared with other imaging platforms. The major cause of cardiovascular events is atherosclerosis, which is a chronic inflammation of the arterial wall. Interestingly, the composition rather than the size of nonstenotic atherosclerotic plaques and severe plaques with >90% stenosis are indicators for high-risk vulnerability to rupture and acute cardiovascular events. Optical techniques may be highly suitable for discriminating, at subcellular resolution, the different stages of plaque progression by targeting bright and nontoxic optical nanoparticles toward distinct molecular epitopes in order to distinguish vulnerable from stable atherosclerotic plaques. Several optical techniques including two-photon laser scanning microscopy (TPLSM), optical coherence tomography (OCT), and photoacoustic imaging (PAI) have been applied for (in vivo) characterization of atherosclerotic plaques, in addition to investigate their feasibility in the clinical setting. Optical nanoparticles, however, have predominantly been used in optical molecular imaging of tumors, but their application in cardiovascular research is increasing. In this review, we first describe shortly the basics of the mentioned optical techniques. Then, we detail on the most-extensively studied optical nanoparticles and relatively new optical nanoparticles that hold promise for in vivo applications in atherosclerosis research.

Evaluation of Magnetic Resonance Vessel Size Imaging by Two-Photon Laser Scanning Microscopy

MR vessel size imaging (MR‐VSI) is increasingly applied to noninvasively assess microvascular properties of tumors and to evaluate tumor response to antiangiogenic treatment. MR‐VSI provides measures for the microvessel radius and fractional blood volume of tumor tissue. However, data have not yet been evaluated with three‐dimensional microscopy techniques. Therefore, three‐dimensional two‐photon laser scanning microscopy (TPLSM) was performed to assess microvascular radius and fractional vessel volume in tumor and muscle tissue. TPLSM data displayed a mazelike architecture of the tumor microvasculature and mainly parallel oriented muscle microvessels. For both MR‐VSI and TPLSM, a larger vessel radius and fractional blood volume were found in the tumor rim than in the core. The microvessel radius was approximately six times larger in tumor and muscle for MR‐VSI than for TPLSM. The tumor blood volume was 4‐fold lower with MR‐VSI than with TPLSM, whereas muscle blood volume was comparable for both techniques. Differences between the tumor rim, core, and muscle tissue showed similar trends for both MR‐VSI and TPLSM parameters. These results indicate that MR‐VSI does not provide absolute measures of microvascular morphology; however, it does reflect heterogeneity in microvascular morphology. Hence, MR‐VSI may be used to assess differences in microvascular morphology. Magn Reson Med 63:930–939, 2010.

In Vivo Molecular Imaging of Apoptosisand Necrosis in Atherosclerotic PlaquesUsing MicroSPECT-CT and MicroPET-CT Imaging

PurposeThe purpose of this paper is to study molecular imaging of apoptosis and necrosis, two key players in atherosclerosis instability, using a multimodal imaging approach combining single photon emission computed tomography (SPECT), positron emission tomography (PET), and computed tomography (CT).ProceduresCollar-induced carotid atherosclerosis ApoE knockout mice were imaged with 99mTc-AnxAF568 SPECT-CT to study apoptosis and sequentially with PET-CT following 124I-Hypericin (124I-Hyp) injection to visualize necrosis.ResultsSPECT depicted increased 99mTc-AnxAF568 uptake in both atherosclerotic carotid arteries, whereas our data suggest that this uptake is not merely apoptosis related. Although PET of 124I-Hyp was hampered by the slow blood clearance in atherosclerotic mice, 124I-Hyp was able to target necrosis in the atherosclerotic plaque.ConclusionBoth 99mTc-AnxAF568 and 124I-Hyp uptake are increased in atherosclerotic carotid vasculature compared to control arteries. While apoptosis imaging remains challenging, necrosis imaging can be feasible after improving the biodistribution characteristics of the probe.

Gadolinium-labeled quantum dots for molecular magnetic resonance imaging: R1 versus R2 mapping

Quantum dots labeled with paramagnetic gadolinium chelates can be applied as contrast agent for preclinical molecular MRI combined with fluorescence microscopy. Besides increasing the longitudinal relaxation rate, gadolinium‐labeled quantum dots may increase the transverse relaxation rate, which might be related to their magnetic properties. Furthermore, molecular MRI experiments are primarily conducted at high magnetic fields, where longitudinal relaxation rate becomes less effective, and the use of transverse relaxation rate as a source of contrast may become attractive. Consequently, the optimal method of contrast enhancement using gadolinium‐labeled quantum dots is a priori unknown. The objective of this study was to compare longitudinal relaxation rate– and transverse relaxation rate–based contrast enhancement, proton visibility, and changes thereof induced by gadolinium‐labeled quantum dots targeted to the angiogenic vasculature of murine tumors, using in vivo longitudinal and transverse relaxation rate mapping. At a field strength of 7 T, longitudinal relaxation rate–based measures were superior to transverse relaxation rate–based measures in detecting both the level and spatial extent of contrast agent–induced relaxation rate changes. Magn Reson Med, 2010.