Marlies Oostendorp

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.

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.

Vessel growth and function: depiction with contrast-enhanced MR imaging.

Magnetic resonance (MR) imaging is a versatile noninvasive diagnostic tool that can be applied to the entire human body to revealing morphologic, functional, and metabolic information. The authors review how MR imaging can depict both the established and the developing vasculature with techniques involving intravenously administered contrast agents. In addition to macrovascular morphology and flow, MR imaging is able to exploit microvascular properties, including vessel size distribution, hyperpermeability, flow heterogeneity, and possibly also upregulation of endothelial biomarkers. For each MR method, the basic principles, potential acquisition and interpretation pitfalls, solutions, and applications are described. Furthermore, discussion includes current shortcomings and the impact of future developments (eg, higher magnetic field strength systems, targeted macromolecular contrast agents) on the visualization of blood vessel growth and function with contrast-enhanced MR imaging.

MRI of renal oxygenation and function after normothermic ischemia-reperfusion injury

The in vivo assessment of renal damage after ischemia–reperfusion injury, such as in sepsis, hypovolemic shock or after transplantation, is a major challenge. This injury often results in temporary or permanent nonfunction. In order to improve the clinical outcome of the kidneys, novel therapies are currently being developed that limit renal ischemia–reperfusion injury. However, to fully address their therapeutic potential, noninvasive imaging methods are required which allow the in vivo visualization of different renal compartments and the evaluation of kidney function. In this study, MRI was applied to study kidney oxygenation and function in a murine model of renal ischemia–reperfusion injury at 7 T. During ischemia, there was a strongly decreased oxygenation, as measured using blood oxygen level‐dependent MRI, compared with the contralateral control, which persisted after reperfusion. Moreover, it was possible to visualize differences in oxygenation between the different functional regions of the injured kidney. Dynamic contrast‐enhanced MRI revealed a significantly reduced renal function, comprising perfusion and filtration, at 24 h after reperfusion. In conclusion, MRI is suitable for the noninvasive evaluation of renal oxygenation and function. Blood oxygen level‐dependent or dynamic contrast‐enhanced MRI may allow the early detection of renal pathology in patients with ischemia–reperfusion injury, such as in sepsis, hypovolemic shock or after transplantation, and consequently may lead to an earlier intervention or change of therapy to minimize kidney damage. Copyright

Reliability of Pharmacokinetic Parameters: Small vs. Medium-Sized Contrast Agents

Current clinical applications of dynamic contrast‐enhanced MRI (DCE‐MRI) are based on the extravasation of relatively small contrast agents (SCAs). SCAs are considered disadvantageous, as they require high image sampling rates. Medium‐sized contrast agents (MCAs) leak more slowly into tissue and allow longer dynamic acquisition times, enabling improved image quality. The influence of molecular size on the reliability of pharmacokinetic parameters, including the transfer constant Ktrans, was investigated. Computer simulations were performed, with in vivo measured arterial input functions (AIFs), to determine the bias and variance of pharmacokinetic parameters as a function of contrast agent size, sampling frequency, noise level, and acquisition time. Better reliability of all parameters was obtained for the MCA compared to the SCA. To obtain similar variance (10%) in Ktrans, the sampling frequency for the SCA (28 min−1) had to be 20 times faster than for the MCA (1.3 min−1). Optimal reliability in parameter estimation required longer acquisition times for MCAs (13 min for the fraction of the extravascular extracellular space into which the contrast agent distributes (ve) and 5 min for Ktrans) than for SCAs (1.7 min for Ktrans and ve). Reliable estimation of the fractional blood plasma volume (vp) was only achieved with MCAs. In conclusion, MCAs provided superior reliability for pharmacokinetic parameter estimation compared to SCAs. Magn Reson Med, 2009.

Optimized pharmacokinetic modeling for the detection of perfusion differences in skeletal muscle with DCE‐MRI: Effect of contrast agent size

PURPOSE The goal of this study was to optimize dynamic contrast-enhanced (DCE)-MRI analysis for differently sized contrast agents and to evaluate the sensitivity for microvascular differences in skeletal muscle. METHODS In rabbits, pathophysiological perfusion differences between hind limbs were induced by unilateral femoral artery ligation. On days 14 and 21, DCE-MRI was performed using a medium-sized contrast agent (MCA) (Gadomer) or a small contrast agent (SCA) (Gd-DTPA). Acquisition protocols were adapted to the pharmacokinetic properties of the contrast agent. Model-based data analysis was optimized by selecting the optimal model, considering fit error, estimation uncertainty, and parameter interdependency from three two-compartment pharmacokinetic models (normal and extended generalized kinetic models and Patlak model). Model-based parameters were compared to the model-free parameter area-under-curve (AUC). Finally, the sensitivity of transfer constant Krans and AUC for physiological and pathophysiological microvascular differences was evaluated. RESULTS For the MCA, the optimal model included Ktrans and plasma fraction nu(p). For the SCA, Ktrans and interstitial fraction nu(e) should be incorporated. For the MCA, Ktrans were (4.8 +/- 0.2) x 10(-3) min(-1) (mean standard error) and (3.6 +/- 0.1) x 10(-3) min(-1) for the red soleus and white tibialis muscle, respectively, p < 0.01. With the SCA, Ktrans were (81 +/- 5) x 10(-3) min(-1) (soleus) and (66 +/- 5) x 10(-3) min(-1) (tibialis) p < 0.01. In the ischemic limb, Ktrans was significantly decreased relative to the control limb (soleus: 15%-20%; tibialis: 5%-10%). Similar differences in AUC were found for both contrast agents. CONCLUSIONS For optimal estimation of microvascular parameters, both model-based and model-free analysis should be adapted to the pharmacokinetic properties of the contrast agent. The detection of microvascular differences based on both Ktrans and AUC was most sensitive when the analysis strategy was tailored to the contrast agent used. The MCA was equally sensitive for microvascular differences as the SCA, with the advantage of improved spatial resolution.

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.

MR Angiography of Collateral Arteries in a Hind Limb Ischemia Model: Comparison between Blood Pool Agent Gadomer and Small Contrast Agent Gd-DTPA

The objective of this study was to compare the blood pool agent Gadomer with a small contrast agent for the visualization of ultra-small, collateral arteries (diameter<1 mm) with high resolution steady-state MR angiography (SS-MRA) in a rabbit hind limb ischemia model. Ten rabbits underwent unilateral femoral artery ligation. On days 14 and 21, high resolution SS-MRA (voxel size 0.49×0.49×0.50 mm3) was performed on a 3 Tesla clinical system after administration of either Gadomer (dose: 0.10 mmol/kg) or a small contrast agent (gadopentetate dimeglumine (Gd-DTPA), dose: 0.20 mmol/kg). All animals received both contrast agents on separate days. Selective intra-arterial x-ray angiograms (XRAs) were obtained in the ligated limb as a reference. The number of collaterals was counted by two independent observers. Image quality was evaluated with the contrast-to-noise ratio (CNR) in the femoral artery and collateral arteries. CNR for Gadomer was higher in both the femoral artery (Gadomer: 73±5 (mean ± SE); Gd-DTPA: 40±3; p<0.01) and collateral arteries (Gadomer: 18±4; Gd-DTPA: 9±1; p = 0.04). Neither day of acquisition nor contrast agent used influenced the number of identified collateral arteries (p = 0.30 and p = 0.14, respectively). An average of 4.5±1.0 (day 14, mean ± SD) and 5.3±1.2 (day 21) collaterals was found, which was comparable to XRA (5.6±1.7, averaged over days 14 and 21; p>0.10). Inter-observer variation was 24% and 18% for Gadomer and Gd-DTPA, respectively. In conclusion, blood pool agent Gadomer improved vessel conspicuity compared to Gd-DTPA. Steady-state MRA can be considered as an excellent non-invasive alternative to intra-arterial XRA for the visualization of ultra-small collateral arteries.

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.

Pharmacokinetics of contrast agents targeted to the tumor vasculature in molecular magnetic resonance imaging

Molecular magnetic resonance imaging (MRI) is increasingly used to investigate tumor angiogenic activity non-invasively. However, the pharmacokinetic behavior and tumor penetration of the often large contrast agent particles is thus far unknown. Here, pharmacokinetic analysis of cyclic asparagine-glycine-arginine (cNGR) labeled paramagnetic quantum dots (pQDs) was developed to quantify the contrast agents homing efficacy to activated endothelial cells of angiogenic tumor vessels using dynamic contrast-enhanced (DCE) MRI. cNGR homes to CD13, an overexpressed aminopeptidase on angiogenic tumor endothelial cells. First, a two-compartment pharmacokinetic model, comprising the blood space and endothelial cell surface, was compared with a three-compartment model additionally including the extravascular-extracellular component. The resulting extravasation parameter was irrelevantly small and was therefore neglected. Next, the association constant K(a), the dissociation constant k(d) and the fractional plasma volume v(P) were determined from the time-series data using the two-compartment model. Magnitude and spatial distribution of the parameters were compared for cNGR-labeled and unlabeled pQDs. The tumor area with significant K(a) values was approximately twice as large for cNGR-pQDs compared with unlabeled pQDs (p < 0.05), indicating more contrast agent binding for cNGR-pQDs. Using cNGR-pQDs, a two-fold larger area with significant K(a) was also found for the angiogenic tumor rim compared with tumor core (p < 0.05). It was furthermore found that both contrast agents perfused the tumor at all depths, thereby providing unequivocal evidence that rim/core differences can indeed be ascribed to stronger angiogenic activity in the rim. Summarizing, molecular DCE-MRI with pharmacokinetic modeling provides unique information on contrast agent delivery and angiogenic activity in tumors.