Selective On/Off-Nitroxides as Radical Probes to Investigate Non-radical Enzymatic Activity by Electron Paramagnetic Resonance.

A nitroxide carrying a peptide specific to the binding pocket of the serine proteases chymotrypsin and cathepsin G is prepared. This peptide is attached as an enol ester to the nitroxide. Upon enzymatic hydrolysis of the peptide, the enol ester moiety is transformed into a ketone moiety. This transformation affords a difference of 5 G in phosphorus hyperfine coupling constant between the electronic paramagnetic resonance (EPR) signals of each nitroxide. This property is used to monitor the enzymatic activity of chymotrypsin and cathepsin G by EPR. Michaelis constants were determined and match those reported for conventional optical probes.

The ß-phosphorus hyperfine coupling constant in nitroxide: part 3: titration of water by electron paramagnetic resonance.

Recently, we showed that the phosphorus hyperfine coupling constant aPß of persistent cyclic nitroxides decreased with the normalized polarity Reichardt's constant E. Thus, we investigated the changes in aPß in binary mixtures of solvents. The sensitivity of aPß to the solvent was high enough to allow us to perform water titration in THF, 1,4-dioxane, and acetonitrile by EPR. Accuracies of a few percent were achieved.

Enzymatically Shifting Nitroxides for EPR Spectroscopy and Overhauser-Enhanced Magnetic Resonance Imaging.

In vivo investigations of enzymatic processes using non-invasive approaches are a long-lasting challenge. Recently, we showed that Overhauser-enhanced MRI is suitable to such a purpose. A ß-phosphorylated nitroxide substrate prototype exhibiting keto-enol equilibrium upon enzymatic activity has been prepared. Upon enzymatic hydrolysis, a large variation of the phosphorus hyperfine coupling constant (Δa(P)=4 G) was observed. The enzymatic activities of several enzymes were conveniently monitored by electronic paramagnetic resonance (EPR). Using a 0.2 T MRI machine, in vitro and in vivo OMRI experiments were successfully performed, affording a 1200% enhanced MRI signal in vitro, and a 600% enhanced signal in vivo. These results highlight the enhanced imaging potential of these nitroxides upon specific enzymatic substrate-to-product conversion.

Alkoxyamines: toward a new family of theranostic agents against cancer.

Theranostics combines therapeutic and diagnostic or drug deposition monitoring abilities of suitable molecules. Here we describe the first steps of building an alkoxyamine-based theranostic agent against cancer. The labile alkoxyamine ALK-1 (t(1/2) = 50 min at 37 °C) cleaves spontaneously to generate (1) a highly reactive free alkyl radical used as therapeutic agents to induce cell damages leading to cell death and (2) a stable nitroxide used as contrast agent for Overhauser-enhanced magnetic resonance imaging (OMRI). The ALK-1 toxicity was studied extensively in vitro on the glioblastoma cell line U87-MG. Cell viability appeared to be dependent on ALK-1 concentration and on the time of the observation following alkoxyamine treatment. For instance, the LC50 at 72 h was 250 µM. Data showed that cell toxicity was specifically due to the in situ released alkyl radical. This radical induced oxidative stress, mitochondrial changes, and ultimately the U87 cell apoptosis. The nitroxide production, during the alkoxyamine homolysis, was monitored by OMRI, showing a progressive MRI signal enhancement to 6-fold concomitant to the ALK-1 homolysis. In conclusion, we have demonstrated for the first time that the alkoxyamines are promising molecules to build theranostic tools against solid tumors.

Alkoxyamines: a new family of pro-drugs against cancer. Concept for theranostics.

Development of anti-cancerous theranostic agents is a vivid field. This article describes a theranostic approach that relies on the triggering of cancer cell death by generation of alkyl radicals at the right place and at the right time using the presence of active proteases in the tumour environment. Alkoxyamines (R(1)R(2)NOR(3)) are labile molecules that homolyze into nitroxides (R(1)R(2)NO˙) and reactive alkyl radicals (R(3)˙). They are used as a source of active alkyl radicals for curing and nitroxides for monitoring by Overhauser-enhanced magnetic resonance imaging (OMRI). Herein, the requirements needed for applying alkoxyamines are described: (i) highly selective activation of the alkoxyamine by specific proteases; (ii) fast homolysis of the alkoxyamine C-ON bond at physiological temperature; (iii) activation of cell death processes through an increase of the local oxidative stress or potential re-activation of the immune system due to short-lived alkyl radicals; and (iv) imaging of the tumor and the drug release by sensing the nitroxide by OMRI.

Comprehensive phenotyping of salt-induced hypertensive heart disease in living mice using cardiac magnetic resonance.

OBJECTIVES: To characterise the effects of high-salt diet (HSD) on left ventricular (LV) mass, systolic function and coronary reserve in living mice using cardiac magnetic resonance imaging (MRI). METHODS: Thirty C57BL/6 1-month-old female mice were fed either a control (n = 15) or an HSD (n = 15). After 3 months, LV volumes, ejection fraction and mass were assessed using time-resolved three-dimensional (3D) black-blood manganese-enhanced MRI, and coronary flow velocity reserve (CFVR) was assessed using dynamic MR angiography at rest and during adenosine-induced hyperaemia. Hearts were excised to assess LV wet mass and micro-vascular remodelling at histology. RESULTS: Micro-vascular remodelling was found at histology in all investigated hearts from the HSD group and none from the control group. No difference between the HSD and control groups was found in terms of heart weight, LV volumes and ejection fraction. Heart to body weight ratio was higher in the HSD group (4.39 ± 0.24 vs 4.02 ± 0.16 mg/g, P < 0.001), because of lower body weight (22.3 ± 0.9 vs 24.0 ± 1.4 g, P < 0.001). CFVR was lower in the HSD group (1.73 ± 0.11 vs 1.94 ± 0.12, P < 0.001). CONCLUSIONS: Phenotyping of hypertensive heart disease is feasible in living mice using dynamic MR angiography and time-resolved 3D black-blood manganese-enhanced MRI. HSD is associated with early impairment of coronary reserve, before the onset of significant hypertrophy.

4-Amino-TEMPO loaded liposomes as sensitive EPR and OMRI probes for the detection of phospholipase A2 activity.

This work aims at developing a diagnostic method based on Electron Paramagnetic Resonance (EPR) measurements of stable nitroxide radicals released from "EPR silent" liposomes. The liposome destabilisation and consequent radical release is enzymatically triggered by the action of phospholipase A2 (PLA2) present in the biological sample of interest. PLA2 are involved in a broad range of processes, and changes in their activity may be considered as a unique valuable biomarker for early diagnoses. The minimum amount of PLA2 measured "in vitro" was 0.09 U/mL. Moreover, the liposomes were successfully used to perform Overhauser-enhanced Magnetic Resonance Imaging (OMRI) in vitro at 0.2 T. The amount of radicals released by PLA2 driven liposome destabilization was sufficient to generate a well detectable contrast enhancement in the corresponding OMRI image.

A system for in vivo on-demand ultra-low field Overhauser-enhanced 3D-Magnetic resonance imaging.

Development of very-low field MRI is an active area of research. It aims at reducing operating costs and improve portability. However, the signal-to-noise issue becomes prominent at ultra-low field (<1 mT), especially for molecular imaging purposes that addresses specific biochemical events. In the context of preclinical molecular MRI of abnormal proteolysis the paper describes a MRI system able to produce Overhauser-enhanced MR images in living rats through in situ Dynamic Nuclear Polarization at 206 µT using stable and non-toxic nitroxides. In parallel conventional images are generated at 206 µT following pre-polarization at 20 mT. Results show that nitroxides are visualized in 3D within a few minutes in the lungs, kidneys and bladder post-administration. This system will be used for molecular imaging of inflammation using protease-specific nitroxide probes.

Effect of chronic hypoxia on pulmonary artery blood velocity in rats as assessed by electrocardiography-triggered three-dimensional time-resolved MR angiography.

Pulmonary arterial hypertension (PAH) is a severe disease that leads to increased pulmonary vascular resistance and right heart failure. Noninvasive methods are needed to detect changes in the pulmonary artery circulation during PAH establishment and/or treatment. Pulmonary blood flow velocity can be evaluated by dynamic MR angiography, although the relevance of such data in the context of PAH remains to be demonstrated. A novel dynamic MR angiography technique was used in this work to measure blood flow velocity in the pulmonary arteries of the same living animals, before and after the establishment of chronic hypoxia-induced PAH. Chronic hypoxia decreased significantly the blood flow velocity (43.8 ± 4.9 vs 24.3 ± 8.7 cm/s) on electrocardiography-triggered time-resolved angiograms. In parallel, chronic hypoxia-induced PAH was confirmed from invasive measurements of the mean pulmonary arterial pressure (32.1 ± 4.8 vs 12.5 ± 2.2 mmHg) and the ratio of the right ventricle weight to the left ventricle plus septum weight (Fulton index: 0.54 ± 0.06 vs 0.27 ± 0.04). This study demonstrates the potential interest of dynamic MR angiography for the investigation of experimental models and for the evaluation of treatment efficacy.

Magnetic Resonance Imaging of Protease-Mediated Lung Tissue Inflammation and Injury.

Pulmonary inflammation usually involves strong neutrophil recruitment with a marked release of proteases such as neutrophil elastase (NE). Noninvasive in vivo assessment of unregulated elastase activity in the lungs would provide a valuable diagnostic tool. Here, it is proposed to use Overhauser-enhanced magnetic resonance imaging (OMRI) in mice where inflammation was induced by the instillation of lipopolysaccharide (LPS). OMRI contrast in the lungs was generated by a dedicated NE free radical substrate. The free radical decayed more rapidly in LPS-treated mouse lungs than in control mice, indicating the occurrence of increased proteolysis under inflammation. Preclinical detection of abnormal proteolysis opens the way for new diagnosis modality and antiprotease testing in vivo.

In vivo MR angiography and velocity measurement in mice coronary arteries at 9.4 T: assessment of coronary flow velocity reserve.

PURPOSE: To demonstrate the feasibility of coronary magnetic resonance (MR) angiography in living mice and to evaluate a dynamic MR angiographic method for coronary flow measurement at 9.4-T field strength. MATERIALS AND METHODS: This study was conducted according to European law and was in full compliance with National Institutes of Health recommendations for animal care and a local institutional animal care committee. Mice were anesthetized by using isoflurane. First, time-of-flight MR angiography was performed in 10 mice to measure coronary diameters at 80-mum isotropic resolution. Second, left coronary artery (LCA) velocity measurements were performed at seven cardiac phases in nine other mice to assess the velocity curve profile. Third, coronary velocities were measured at the middiastolic phase in 13 mice at rest and during adenosine-induced hyperemia to calculate coronary flow velocity reserve (CFVR). The Pearson coefficient compared the correlation between isoflurane dose and CFVR. Paired t tests compared R-R intervals and respiratory rates between rest and hyperemia. RESULTS: Proximal diameters were, respectively, 404 mum +/- 34 [standard deviation] and 259 mum +/- 22 for the LCAs and the right coronary arteries, which were in accordance with reported values. The velocity curve profile throughout the cardiac cycle was similar to values from the literature. Baseline and hyperemic velocities were, respectively, 19.0 cm/sec +/- 4.4 and 33.7 cm/sec +/- 4.7 (P<.001), resulting in a CFVR of 1.77 +/- 0.19. CFVR did not correlate with isoflurane dose (r = 0.05, P = .88). R-R intervals shortened by 2.5% during hyperemia (P = .04). Respiratory rates showed no difference between rest and hyperemia (P = .39). CONCLUSION: High-spatial-resolution three-dimensional coronary MR angiography is feasible in living mice. Dynamic MR angiography depicts coronary velocity changes throughout the cardiac cycle and between rest and maximum hyperemia, providing a tool for CFVR assessment.

4D retrospective black blood trueFISP imaging of mouse heart.

The purpose of this study was to demonstrate the feasibility of steady-state True fast imaging with steady precession (TrueFISP) four-dimensional imaging of mouse heart at high resolution and its efficiency for cardiac volumetry. Three-dimensional cine-imaging of control and hypoxic mice was carried out at 4.7 T without magnetization preparation or ECG-triggering. The k-space lines were acquired with the TrueFISP sequence (pulse repetition time/echo time = 4/2 ms) in a repeated sequential manner. Retrospective reordering of raw data allowed the reconstruction of 10 three-dimensional images per cardiac cycle. The acquisition scheme used an alternating radiofrequency phase and sum-of-square reconstruction method. Black-blood three-dimensional images at around 200 mum resolution were produced without banding artifact throughout the cardiac cycle. High contrast to noise made it possible to estimate cavity volumes during diastole and systole. Right and left ventricular stroke volume was significantly higher in hypoxic mice vs controls (20.2 +/- 2 vs 15.1 +/- 2; P < 0.05, 24.9 +/- 2 vs 20.4 +/- 2; P < 0.05, respectively). In conclusion, four-dimensional black-blood TrueFISP imaging in living mice is a method of choice to investigate cardiac abnormalities in mouse models.

In vivo quantification of blood velocity in mouse carotid and pulmonary arteries by ECG-triggered 3D time-resolved magnetic resonance angiography.

Blood flow velocity is a functional parameter of fundamental importance in diagnosis and follow-up of various vascular diseases. Vascular pathologies can be efficiently studied in animal models, especially in small rodents. ECG-gated magnetic resonance imaging (MRI) assessment of blood velocity in small animals is a challenge because of limited spatial resolution and high-frequency physiological parameters. Here it is shown that a bright-blood cine-3D-MRI method can be used to measure blood velocity at specific times of the cardiac cycle in mouse pulmonary and carotid arteries. The method used a series of time-of-flight (TOF) acquisitions in a volume of interest at different times after signal cancellation in the same volume. This scheme was repeated at different periods of the cardiac cycle by varying the delay between the ECG R-wave peak and signal cancellation. Velocity values in mouse pulmonary artery varied from 35 cm/s in systole to 0-10 cm/s in diastole. A similar pattern was displayed in carotid arteries (18 and 2.5 cm/s, in systole and diastole, respectively). Results are discussed in terms of efficiency, limitation, and comparison with other methods.

An elastase activity reporter for Electronic Paramagnetic Resonance (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI) as a line-shifting nitroxide.

Pulmonary inflammatory diseases are a major burden worldwide. They have in common an influx of neutrophils. Neutrophils secrete unchecked proteases at inflammation sites consequently leading to a protease/inhibitor imbalance. Among these proteases, neutrophil elastase is responsible for the degradation of the lung structure via elastin fragmentation. Therefore, monitoring the protease/inhibitor status in lungs non-invasively would be an important diagnostic tool. Herein we present the synthesis of a MeO-Suc-(Ala)2-Pro-Val-nitroxide, a line-shifting elastase activity probe suitable for Electron Paramagnetic Resonance spectroscopy (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI). It is a fast and sensitive neutrophil elastase substrate with Km = 15 ±â€¯2.9 µM, kcat/Km = 930,000 s-1 M-1 and Km = 25 ±â€¯5.4 µM, kcat/Km = 640,000 s-1 M-1 for the R and S isomers, respectively. These properties are suitable to detect accurately concentrations of neutrophil elastase as low as 1 nM. The substrate was assessed with broncho-alveolar lavages samples derived from a mouse model of Pseudomonas pneumonia. Using EPR spectroscopy we observed a clear-cut difference between wild type animals and animals deficient in neutrophil elastase or deprived of neutrophil Elastase, Cathepsin G and Proteinase 3 or non-infected animals. These results provide new preclinical ex vivo and in vivo diagnostic methods. They can lead to clinical methods to promote in time lung protection.

Ultrafast multiplanar determination of left ventricular hypertrophy in spontaneously hypertensive rats with single-shot spin-echo nuclear magnetic resonance imaging.

OBJECTIVE: To determine whether left ventricular hypertrophy can be correctly evaluated in hypertensive rats with a new nuclear magnetic resonance (NMR) imaging modality that is relatively simple to operate and provides results of constant quality while offering a high signal-to-noise ratio. DESIGN Left ventricular mass as calculated from the NMR imaging analysis was compared with the actual left ventricular mass measured by gravimetry. METHODS: Single-shot ultrafast spin-echo (SSFSE) imaging of hearts of Wistar-Kyoto rats and spontaneously hypertensive rats was performed at 4 T. Left ventricular mass was determined by using Simpson's rule on stacks of images acquired in systole and diastole. RESULTS: SSFSE NMR imaging performed in systole or in diastole evaluated and quantified left ventricular hypertrophy in hearts of spontaneously hypertensive rats very similarly to gravimetry. The left ventricular mass as determined by NMR was in good accordance with the actual left ventricular weight (SEE: 30.39 and 35.86 mg for the systolic and diastolic NMR acquisitions, respectively). CONCLUSION: Using an SSFSE sequence, high-quality NMR images of the rat heart can be generated very reliably with sufficient contrast and temporal and spatial resolution, and allow precise, non-invasive and fast characterization of left ventricular hypertrophy in a hypertensive rat model.

In vivo Overhauser-enhanced MRI of proteolytic activity.

There is an increasing interest in developing novel imaging strategies for sensing proteolytic activities in intact organisms in vivo. Overhauser-enhanced MRI (OMRI) offers the possibility to reveal the proteolysis of nitroxide-labeled macromolecules thanks to a sharp decrease of the rotational correlation time of the nitroxide moiety upon cleavage. In this paper, this concept is illustrated in vivo at 0.2 T using nitroxide-labeled elastin orally administered in mice. In vitro, this elastin derivative was OMRI-visible and gave rise to high Overhauser enhancements (19-fold at 18 mm nitroxide) upon proteolysis by pancreatic porcine elastase. In vivo three-dimensional OMRI detection of proteolysis was carried out. A keyhole fully balanced steady-state free precession sequence was used, which allowed 3D OMRI acquisition within 20 s at 0.125 mm(3) resolution. About 30 min after mouse gavage, proteolysis was detected in the duodenum, where Overhauser enhancements were 7.2 ± 2.4 (n = 7) and was not observed in the stomach. Conversely, orally administered free nitroxides or pre-digested nitroxide-labeled elastin were detected in the mouse's stomach by OMRI. Combined with specific molecular probes, this Overhauser-enhanced MRI technique can be used to evaluate unregulated proteolytic activities in various models of experimental diseases and for drug testing.

Overhauser-enhanced MRI of elastase activity from in vitro human neutrophil degranulation.

BACKGROUND: Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research. METHODOLOGY/PRINCIPAL FINDINGS: We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI) contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4) cells per milliliter, well under the physiological neutrophils concentrations. CONCLUSIONS/SIGNIFICANCE: These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis) by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

In vivo high-resolution 3D overhauser-enhanced MRI in mice at 0.2 T.

Overhauser-enhanced MRI (OMRI) offers the potentiality of detecting low-concentrated species generated by specific biological processes. However molecular imaging applications of OMRI need significant improvement in spatial localization. Here it is shown that 3D-OMRI of a free radical injected in tumor-bearing mice can be performed at high anatomical resolution at a constant field. A 30 mm cavity operating at 5.43 GHz was inserted in a C-shaped magnet for proton MRI at 0.194 T. Nude mice with or without brain-implanted C6 rat glioma were positioned in the cavity and injected with TOPCA (1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-carboxylic acid). OMRI was performed in 3D within several minutes in the brain region without high overheating of the animals. Voxel size was 0.5 × 0.5 × 1 mm³ , providing good delineation of brain regions. Signal amplifications ranged from 2 in tumors to 10 in vessels several minutes after TOPCA injection. Time-course of signal enhancement could be measured by 2D OMRI at 15 s time intervals in a localized thin slice. The method opens the way for molecular imaging of biological activities able to generate OMRI-visible free radicals.

Refinement of cardiac NMR imaging in awake hamsters: proof of feasibility and characterization of cardiomyopathy.

The goal of this study was to demonstrate the feasibility of cardiac NMR imaging in conscious hamsters and its usefulness in evaluating cardiac abnormalities in a small-animal model of cardiomyopathy. Awake hamsters, controls and cardiomyopathic ones (CHF 147), were immobilized in a dedicated holder. Half-Fourier single-shot FSE imaging, with outer-volume suppression and 'black-blood' contrast provided images free from motion artifact with good visualization of cardiac anatomy at any point in the cardiac cycle. Series of double-oblique views were acquired with or without electrocardiograph gating. Image acquisition time was 55 ms, with an in-plane resolution of 470 x 625 microm2. Left ventricular volumes, ejection fraction, and myocardium NMR signal heterogeneity were compared in CHF 147 and control hearts. Left ventricles of CHF 147 hamsters were dilated, as indicated by the increase in end-diastolic cavity volume (299 +/- 79 mm3 compared with the controls (141 +/- 39 mm3; P = 0.0002). Left ventricular ejection fraction was largely reduced (45 +/- 9% vs 86 +/- 4%; P < 0.0001). The NMR signal distribution at an effective echo time of 41 ms was more heterogeneous in the myocardial wall of CHF 147 hamsters than in controls (1.87 +/- 0.37 a.u. vs 0.98 +/- 0.12 a.u., respectively; P = 0.0002). This study is a refinement of animal experimentation, as it demonstrates for the first time that characteristic features of cardiac pathology can be evaluated with ultra-fast NMR imaging in conscious small rodents.

New insight into abnormal muscle vasodilatory responses in aged hypertensive rats by in vivo nuclear magnetic resonance imaging of perfusion.

OBJECTIVE: Increased peripheral arterial resistances and decreased maximum vasodilation are characteristic features of chronic hypertension. However, little data are available in the literature regarding the possible alterations in the temporal patterns of vasodilatory responses elicited by various stimuli. DESIGN: This question was addressed by measuring skeletal muscle perfusion using nuclear magnetic resonance imaging combined with arterial spin labeling. METHODS: Ninety-week-old male spontaneously hypertensive (SHR; n = 7) and normotensive Wistar Kyoto (WKY; n = 8) rats were studied, and calf muscle perfusion was measured at rest and during reactive hyperemia following total ischemia of 5 and 30 min duration. RESULTS: Reactive hyperemia profiles differed according to duration of ischemia. In WKY rats, 5 min of ischemia induced a short peak of hyperemia lasting no more than 63 s, while 30 min of ischemia were followed by a prolonged hyperemic response of 261 s. In SHRs, after 5 min of ischemia, peak muscle arterial conductance was decreased to 0.5 +/- 0.3 versus 0.9 +/- 0.3 ml.min(-1).100 g(-1).mm Hg(-1) in the WKY rats (p < 0.05), as expected. After 30 min of ischemia, there was, in addition, a shortening of the hyperemic response duration. Time to post-ischemic half normalization of arterial conductance was 38 +/- 24 s in the SHRs versus 149 +/- 58 s in the WKY rats (p < 0.001). CONCLUSION: In vivo perfusion measurement not only confirmed the existence of a reduced maximum peripheral vasodilation in chronically hypertensive rats, it revealed a blunted hyperemic response after prolonged ischemia in the SHRs, which might be an important contributing factor to the increased sensitivity to ischemia in hypertension.