Specific detection and imaging of enzyme activity by signal-amplifiable self-assembling (19)F MRI probes.

Specific turn-on detection of enzyme activities is of fundamental importance in drug discovery research, as well as medical diagnostics. Although magnetic resonance imaging (MRI) is one of the most powerful techniques for noninvasive visualization of enzyme activity, both in vivo and ex vivo, promising strategies for imaging specific enzymes with high contrast have been very limited to date. We report herein a novel signal-amplifiable self-assembling (19) F NMR/MRI probe for turn-on detection and imaging of specific enzymatic activity. In NMR spectroscopy, these designed probes are "silent" when aggregated, but exhibit a disassembly driven turn-on signal change upon cleavage of the substrate part by the catalytic enzyme. Using these (19) F probes, nanomolar levels of two different target enzymes, nitroreductase (NTR) and matrix metalloproteinase (MMP), could be detected and visualized by (19) F NMR spectroscopy and MRI. Furthermore, we have succeeded in imaging the activity of endogenously secreted MMP in cultured media of tumor cells by (19) F MRI, depending on the cell lines and the cellular conditions. These results clearly demonstrate that our turn-on (19) F probes may serve as a screening platform for the activity of MMPs.

Constitutive SIRT1 overexpression impairs mitochondria and reduces cardiac function in mice.

Heart failure is associated with a change in cardiac energy metabolism. SIRT1 is a NAD(+)-dependent protein deacetylase, and important in the regulation of cellular energy metabolism. To examine the role of SIRT1 in cardiac energy metabolism, we created transgenic mice overexpressing SIRT1 in a cardiac-specific manner, and investigated cardiac functional reserve, energy reserve, substrate uptake, and markers of mitochondrial function. High overexpression of SIRT1 caused dilated cardiomyopathy. Moderate overexpression of SIRT1 impaired cardiac diastolic function, but did not cause heart failure. Fatty acid uptake was decreased and the number of degenerated mitochondria was increased dependent on SIRT1 gene dosage. Markers of reactive oxygen species were decreased. Changes in morphology and reactive oxygen species were associated with the reduced expression of genes related to mitochondrial function and autophagy. In addition, the respiration of isolated mitochondria was decreased. Cardiac function was normal in transgenic mice expressing a low level of SIRT1 at baseline, but the mice developed cardiac dysfunction upon pressure overload. In summary, the constitutive overexpression of SIRT1 reduced cardiac function associated with impaired mitochondria in mice.

Systematic study of protein detection mechanism of self-assembling 19F NMR/MRI nanoprobes toward rational design and improved sensitivity.

(19)F NMR/MRI probe is expected to be a powerful tool for selective sensing of biologically active agents owing to its high sensitivity and no background signals in live bodies. We have recently reported a unique supramolecular strategy for specific protein detection using a protein ligand-tethered self-assembling (19)F probe. This method is based on a recognition-driven disassembly of the nanoprobes, which induced a clear turn-on signal of (19)F NMR/MRI. In the present study, we conducted a systematic investigation of the relationship between structure and properties of the probe to elucidate the mechanism of this turn-on (19)F NMR sensing in detail. Newly synthesized (19)F probes showed three distinct behaviors in response to the target protein: off/on, always-on, and always-off modes. We clearly demonstrated that these differences in protein response could be explained by differences in the stability of the probe aggregates and that "moderate stability" of the aggregates produced an ideal turn-on response in protein detection. We also successfully controlled the aggregate stability by changing the hydrophobicity/hydrophilicity balance of the probes. The detailed understanding of the detection mechanism allowed us to rationally design a turn-on (19)F NMR probe with improved sensitivity, giving a higher image intensity for the target protein in (19)F MRI.

Construction of a 19F-lectin biosensor for glycoprotein imaging by using affinity-guided DMAP chemistry.

In this study, assisted by affinity-guided DMAP strategy, we developed a novel (19)F-modified lectin as a biosensor for specific detection and imaging of glycoproteins. Exploited the large chemical shift anisotropy property of (19)F nuclei, glycoproteins detected by our (19)F-biosensor are signatured by broadened peaks in (19)F NMR, hence enabled the distinction between glycoproteins and small molecule saccharides. Such signal on/off switching was also applied to glycoprotein imaging by (19)F MRI.

Monitoring of biological one-electron reduction by (19)F NMR using hypoxia selective activation of an (19)F-labeled indolequinone derivative.

Biological reduction of fluorine-labeled indolequinone derivative (IQ-F) was characterized by (19)F NMR for quantitative molecular understanding. The chemical shift change in (19)F NMR allowed monitoring of the enzymatic reduction of IQ-F. Upon hypoxic treatment of IQ-F with NADPH:cytochrome P450 reductase, IQ-F was activated via catalytic one-electron reduction to release nonafluoro-tert-butyl alcohol (F-OH), while the formation of F-OH was significantly suppressed under aerobic conditions. Similar hypoxia-selective reduction of IQ-F occurred within A549 cells, which expresses NADPH:cytochrome P450 reductase. The kinetic analysis was also performed to propose a reaction mechanism. The molecular oxygen slightly prevents the binding of IQ-F to reductase, while the rate of net reaction was decreased due to oxidation of a semiquinone anion radical intermediate generated by one-electron reduction of IQ-F. The disappearance of IQ-F and appearance of F-OH were imaged by (19)F fast spin echo, thus visualizing the hypoxia-selective reduction of IQ-F by means of MR imaging.

Modification of nitroxyl contrast agents with multiple spins and their proton T(1) relaxivity.

The purpose of this study is to test the performance of multispin nitroxyl contrast agents in improving the sensitivity of MR detection for nitroxyl contrast agents. The relation between T(1) relaxivity and the number of paramagnetic centers in a molecule was investigated. Compound 1 is a single molecule of methoxycarbonyl-PROXYL (MC-PROXYL). Two and three MC-PROXYL molecules were chemically coupled to obtain Compounds 2 and 3, which have two and three nitroxyl spins in the molecule, respectively. A good linear relation, the slope of which increased depending on the number of nitroxyl spins in the molecule, was obtained between T(1)-weighted (fast low-angle shot) MR image contrast enhancement at 7 T and the concentration of nitroxyl contrast agents. T(1)-weighted MR image contrast enhancement and T(1) relaxivity levels of nitroxyl contrast agents were increased depending on the number of nitroxyl spins in the molecule. Multicoupling nitroxyl molecules can enhance the T(1)-weighted contrast effect while maintaining the quantitative behavior of the molecule for up to three spins.

Hyperpolarized 129Xe phase-selective imaging of mouse lung at 9.4T using a continuous-flow hyperpolarizing system.

The use of hyperpolarized (HP) 129Xe magnetic resonance (MR) imaging to regionally evaluate gas diffusion and perfusion processes as well as ventilation in the lung has been expected. In this study, we used a continuous-flow hyperpolarizing (CF-HP) system to acquire gas- and dissolved-phase 129Xe images from mouse lung, employing standard gradient echo sequence equipped with chemical shift selective excitation and 90 degrees flip angle. The character of non-recoverable HP magnetization enabled the use of a phase (frequency)-selective 90 degrees pulse for direct visualization of only a given-phase 129Xe magnetization replenished into the slice during repetition time (TR). We combined gas- and dissolved-phase 129Xe images to map the ratio of dissolved- to gas-phase 129Xe replenished into the slice during TR (Mdissolved/Mgas) and found it to be approximately 0.05 to 0.08 in the peripheral regions of mouse lungs. This result suggested that replenishment of dissolved-phase 129Xe magnetization by gas diffusion and pulmonary perfusion would be faster than that of gas-phase by ventilation. The use of a CF-HP system that allows the application of relatively long TR to HP 129Xe imaging using a phase-selective 90 degrees pulse would be useful in evaluating gas transport mechanisms in the lung.

Comparisons of EPR imaging and T1-weighted MRI for efficient imaging of nitroxyl contrast agents.

The resolution and signal to noise ratio of EPR imaging and T(1)-weighted MRI were compared using an identical phantom. Several solutions of nitroxyl contrast agents with different EPR spectral shapes were tested. The feasibility of T(1)-weighted MRI to detect nitroxyl contrast agents was described. T(1)-weighted MRI can detect nitroxyl contrast agents with a complicated EPR spectrum easier and quicker; however, T(1)-weighted MRI has less quantitative ability especially for lipophilic nitroxyl contrast agents, because T(1)-relaxivity, i.e. accessibility to water, is affected by the hydrophilic/hydrophobic micro-environment of a nitroxyl contrast agent. The less quantitative ability of T(1)-weighted MRI may not be a disadvantage of redox imaging, which obtains reduction rate of a nitroxyl contrast. Therefore, T(1)-weighted MRI has a great advantage to check the pharmacokinetics of newly modified and/or designed nitroxyl contrast agents.

Analysis of hyperpolarized 129Xe dynamics in mouse lungs under spontaneous respiration for separate determination of lung functional parameters and relaxation time.

Magnetic resonance spectroscopy (MRS) was used to investigate the dynamics of hyperpolarized (HP) 129Xe respiration in the chests of mice under spontaneous respiration. The washout curve was analyzed using Kety's exchange model of inert gases, and the 3 factors that affect the slope of the washout curve, i.e., the RF flip angle, respiratory parameters, and apparent relaxation time (which comprises terms including the relaxation time in alveoli, T1air, and perfusion), were determined separately. Flip angle was determined precisely using the dual flip angle method, and ventilation volume was determined using SF6 gas at thermal equilibrium. Furthermore, an attempt was made to separate out the terms of T1air and perfusion from the apparent relaxation time after exploiting the ventilation model of lungs in steady state. Values of relaxation time T1air=30.5 s and perfusion term lambdaQ/VA=0.016 s-1 were obtained, supporting the applicability of the ventilation model proposed.

MR imaging of mouse lung using hyperpolarized 3He: image acquisition and T1 estimation under spontaneous respiration.

PURPOSE: The purpose of this study was to acquire a hyperpolarized (HP) (3)He image of the mouse lung and to estimate 3He T1 in the lung from wash-out curve analysis under spontaneous respiration. MATERIALS AND METHODS: We first tested the K-Rb hybrid method for the spin-exchange optical pumping (SEOP) of 3He using a home-built noble gas polarizer operated at atmospheric pressure and then applied it to MR imaging and spectroscopy of the mouse lung. The longitudinal relaxation time (T1) of 3He in the mouse lung was estimated under spontaneous respiration by exploiting a novel method in which SF6 gas at thermal equilibrium was utilized in combination with the HP 3He gas in the quantitative wash-out curve analysis. This method utilizes the difference in the profile of the wash-out curve of HP 3He and SF6 at thermal equilibrium. That is, the slope of the 3He wash-out curve in the semi-logarithmic plots is affected by 3 factors, including RF pulse angle, respiration, and T1, whereas the slope of the SF6 wash-out curve is only the function of respiratory term. RESULTS: A 3He lung image was obtained successfully, and we were able to estimate successfully 3He T1 in the mouse lung under spontaneous respiration using a novel method; the estimated T1 value was 68+/-25 s, which was reasonable compared with the value calculated from the literature data measured during breath-hold. CONCLUSION: We succeeded in acquiring the first 3He image of mouse lung in vivo in this country, and our proposed method of estimating 3He T1 in the lung under spontaneous respiration is noninvasive and readily applied to animals and would be useful to evaluate the alveolar gas exchange function as well as oxygen partial pressure (pO2) in lungs of animals.

Design and synthesis of a novel magnetic resonance imaging contrast agent for selective sensing of zinc ion.

A series of new diethylenetriaminepentaacetic acid (DTPA)-bisamide chelators has been prepared and characterized for application as zinc sensors. We have designed and synthesized (GdL(a))(2-), which contains a DTPA-bisamide moiety. The R(1) relaxivity of (GdL(a))(2-) solution decreased monotonically on the addition of Zn(2+). Moreover, (GdL(a))(2-) showed high selectivity for Zn(2+) against Ca(2+) and Mg(2+). We also measured the UV-visible spectra and the coldspray ionization (CSI) MS spectra and concluded that the 1-to-1 Zn(2+) complex of (GdL(a))(2-) is stable at higher concentrations of Zn(2+). These complexes should provide the basis for creating a superior Zn(2+)-sensitive MRI contrast agent and are excellent candidates for incorporation into sensors designed for selective detection of Zn(2+) in biological applications.

A motion tracking method that applies a spread spectrum communication technique to tagging MR imaging.

PURPOSE: We propose a new motion tracking method that encodes an object's position information using tagging magnetic resonance (MR) images as digital codes, and we assess the method's feasibility in stationary and moving phantoms. The encoding and decoding of tag patterns employ principles of spread spectrum communication. METHODS: We used a segmented fast low angle shot cine sequence (FLASH) with spatial modulation of magnetization (SPAMM) preparation pulses to encode position information as 7-bit code words and used this spread code to decode the position information. To make 7-bit code words using tag images, we adjusted the flip angle and phase of the 4 composite radiofrequency (RF) pulses and the gradient magnetic field strength in the SPAMM pulse to generate 7 types of tag patterns. The proposed method required 7 acquisitions with 7 types of tag patterns. We compared results with images obtained by conventional tagging in stationary and moving phantom experiments. RESULTS: In a stationary phantom, the proposed method showed the same ability to identify pixel position as conventional tagging method using improved SNR images with the average of 7 acquisitions. In a moving phantom, pixel position was successfully decoded by the proposed method on a pixel-by-pixel basis. In this method, the motion of the phantom was detected by the simple calculation of the correlation coefficient of the code words. CONCLUSION: We introduced a spread spectrum communication technique to tagging MR imaging that regards tag patterns as digital codes, and we demonstrated the method's potential to detect pixel position in sub-pixel resolution.

Measurement of technetium-99m sestamibi signals in rats administered a mitochondrial uncoupler and in a rat model of heart failure.

BACKGROUND: Many methods have been used to assess mitochondrial function. Technetium-99m sestamibi ((99m)Tc-MIBI), a lipophilic cation, is rapidly incorporated into myocardial cells by diffusion and mainly localizes to the mitochondria. The purpose of this study was to investigate whether measurement of (99m)Tc-MIBI signals in animal models could be used as a tool to quantify mitochondrial membrane potential at the organ level. METHODS AND RESULTS: We analyzed (99m)Tc-MIBI signals in Sprague-Dawley (SD) rat hearts perfused with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler known to reduce the mitochondrial membrane potential. (99m)Tc-MIBI signals could be used to detect changes in the mitochondrial membrane potential with sensitivity comparable to that obtained by two-photon laser microscopy with the cationic probe tetramethylrhodamine ethyl ester (TMRE). We also measured (99m)Tc-MIBI signals in the hearts of SD rats administered CCCP (4 mg/kg intraperitoneally) or vehicle. (99m)Tc-MIBI signals decreased in rat hearts administered CCCP, and the ATP content, as measured by (31)P magnetic resonance spectroscopy, decreased simultaneously. Next, we administered (99m)Tc-MIBI to Dahl salt-sensitive rats fed a high-salt diet, which leads to hypertension and heart failure. The (99m)Tc-MIBI signal per heart tissue weight was inversely correlated with heart weight, cardiac function, and the expression of atrial natriuretic factor, a marker of heart failure, and positively correlated with the accumulation of labeled fatty acid analog. The (99m)Tc-MIBI signal per liver tissue weight was lower than that per heart tissue weight. CONCLUSION: Measurement of (99m)Tc-MIBI signals can be an effective tool for semiquantitative investigation of cardiac mitochondrial membrane potential in the SD rat model by using a chemical to decrease the mitochondrial membrane potential. The (99m)Tc-MIBI signal per heart tissue weight was inversely correlated with the severity of heart failure in the Dahl rat model.

Potential for differential diagnosis with gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid-enhanced magnetic resonance imaging in experimental hepatic tumors.

RATIONALE AND OBJECTIVES: To investigate whether dynamic and delayed magnetic resonance imaging (MRI) with gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA), a hepatobiliary MRI contrast agent, has potential for the differential diagnosis of experimental hepatic tumors. METHODS: Twelve male rats received N-nitrosomorpholine solution as drinking water to induce hepatic tumors. After injection of Gd-EOB-DTPA, rats were subjected to dynamic and delayed MRI. The relative enhancement (RE) was calculated, and the time of the maximum RE (Tmax) was evaluated. After MRI, liver was histologically analyzed. RESULTS: One hundred sixty-three hepatic tumors 3-12 mm in diameter were induced after 18 weeks of treatment with 0.01 wt/vol% of N-nitrosomorpholine, and 81 of them were evaluated. The RE in hyperplastic nodules (HPNs) was significantly higher than that in moderately or poorly differentiated hepatocellular carcinomas (HCCs) in the late phase, whereas there was no significant difference in RE between well-differentiated HCCs and HPNs. The average Tmax in HPNs was about 13 minutes, whereas that of each differentiated HCCs was about 1 minute. CONCLUSIONS: It was possible to differentiate benign HPNs and malignant HCCs (especially well-differentiated HCCs) by evaluating the change of RE or comparison of Tmax with Gd-EOB-DTPA-enhanced MRI.

19F Magnetic resonance imaging of perfluorooctanoic acid encapsulated in liposome for biodistribution measurement.

The tissue distribution of perfluorooctanoic acid (PFOA), which is known to show unique biological responses, has been visualized in female mice by (19)F magnetic resonance imaging (MRI) incorporated with the recent advances in microimaging technique. The chemical shift selected fast spin-echo method was applied to acquire in vivo (19)F MR images of PFOA. The in vivo T(1) and T(2) relaxation times of PFOA were proven to be extremely short, which were 140 (+/- 20) ms and 6.3 (+/- 2.2) ms, respectively. To acquire the in vivo (19)F MR images of PFOA, it was necessary to optimize the parameters of signal selection and echo train length. The chemical shift selection was effectively performed by using the (19)F NMR signal of CF(3) group of PFOA without the signal overlapping because the chemical shift difference between the CF(3) and neighbor signals reaches to 14 kHz. The most optimal echo train length to obtain (19)F images efficiently was determined so that the maximum echo time (TE) value in the fast spin-echo sequence was comparable to the in vivo T(2) value. By optimizing these parameters, the in vivo (19)F MR image of PFOA was enabled to obtain efficiently in 12 minutes. As a result, the time course of the accumulation of PFOA into the mouse liver was clearly pursued in the (19)F MR images. Thus, it was concluded that the (19)F MRI becomes the effective method toward the future pharmacological and toxicological studies of perfluorocarboxilic acids.

Improvement of T1 determination of hyperpolarized 129Xe in mouse brain under controlled-flow.

The method of determining the longitudinal relaxation time of hyperpolarized 129Xe in the mouse brain has been established in vivo with the ventilation technique under controlled-flow conditions. The uptake and washout processes for nine mice were traced through observation of time-dependent changes in NMR (nuclear magnetic resonance) signal amplitudes and analyzed by means of a two-compartment model, thus providing the quantitative value of 14.1+/-1.6 s as the relaxation time.

Cardiac-specific inhibition of kinase activity in calcium/calmodulin-dependent protein kinase kinase-ß leads to accelerated left ventricular remodeling and heart failure after transverse aortic constriction in mice.

BACKGROUND: The mechanism of cardiac energy production against sustained pressure overload remains to be elucidated. METHODS AND RESULTS: We generated cardiac-specific kinase-dead (kd) calcium/calmodulin-dependent protein kinase kinase-ß (CaMKKß) transgenic (α-MHC CaMKKßkd TG) mice using α-myosin heavy chain (α-MHC) promoter. Although CaMKKß activity was significantly reduced, these mice had normal cardiac function and morphology at baseline. Here, we show that transverse aortic binding (TAC) in α-MHC CaMKKßkd TG mice led to accelerated death and left ventricular (LV) dilatation and dysfunction, which was accompanied by significant clinical signs of heart failure. CaMKKß downstream signaling molecules, including adenosine monophosphate-activated protein kinase (AMPK), were also suppressed in α-MHC CaMKKßkd TG mice compared with wild-type (WT) mice. The expression levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, which is a downstream target of both of CaMKKß and calcium/calmodulin kinases, were also significantly reduced in α-MHC CaMKKßkd TG mice compared with WT mice after TAC. In accordance with these findings, mitochondrial morphogenesis was damaged and creatine phosphate/ß-ATP ratios assessed by magnetic resonance spectroscopy were suppressed in α-MHC CaMKKßkd TG mice compared with WT mice after TAC. CONCLUSIONS: These data indicate that CaMKKß exerts protective effects on cardiac adaptive energy pooling against pressure-overload possibly through phosphorylation of AMPK and by upregulation of PGC-1α. Thus, CaMKKß may be a therapeutic target for the treatment of heart failure.

Quantitative 17O imaging towards oxygen consumption study in tumor bearing mice at 7 T.

(17)O magnetic resonance imaging (MRI) using a conventional pulse sequence was explored as a method of quantitative imaging towards regional oxygen consumption rate measurement for tumor evaluation in mice. At 7 T, fast imaging with steady state (FISP) was the best among gradient echo, fast spin echo and FISP for the purpose. The distribution of natural abundance H2(17)O in mice was visualized under spatial resolution of 2.5 × 2.5mm(2) by FISP in 10 min. The signal intensity by FISP showed a linear relationship with (17)O quantity both in phantom and mice. Following the injection of 5% (17)O enriched saline, (17)O re-distribution was monitored in temporal resolution down to 5 sec with an image quality sufficient to distinguish each organ. The image of labeled water produced from inhaled (17)O2 gas was also obtained. The present method provides quantitative (17)O images under sufficient temporal and spatial resolution for the evaluation of oxygen consumption rate in each organ. Experiments using various model compounds of R-OH type clarified that the signal contribution of body constituents other than water in the present in vivo(17)O FISP image was negligible.

Persistent overexpression of phosphoglycerate mutase, a glycolytic enzyme, modifies energy metabolism and reduces stress resistance of heart in mice.

BACKGROUND: Heart failure is associated with changes in cardiac energy metabolism. Glucose metabolism in particular is thought to be important in the pathogenesis of heart failure. We examined the effects of persistent overexpression of phosphoglycerate mutase 2 (Pgam2), a glycolytic enzyme, on cardiac energy metabolism and function. METHODS AND RESULTS: Transgenic mice constitutively overexpressing Pgam2 in a heart-specific manner were generated, and cardiac energy metabolism and function were analyzed. Cardiac function at rest was normal. The uptake of analogs of glucose or fatty acids and the phosphocreatine/ßATP ratio at rest were normal. A comprehensive metabolomic analysis revealed an increase in the levels of a few metabolites immediately upstream and downstream of Pgam2 in the glycolytic pathway, whereas the levels of metabolites in the initial few steps of glycolysis and lactate remained unchanged. The levels of metabolites in the tricarboxylic acid (TCA) cycle were altered. The capacity for respiration by isolated mitochondria in vitro was decreased, and that for the generation of reactive oxygen species (ROS) in vitro was increased. Impaired cardiac function was observed in response to dobutamine. Mice developed systolic dysfunction upon pressure overload. CONCLUSIONS: Constitutive overexpression of Pgam2 modified energy metabolism and reduced stress resistance of heart in mice.

Size-controlled and biocompatible Gd2 O3 nanoparticles for dual photoacoustic and MR imaging.

The synthesis, characterization, and functional evaluation of new size-controlled and biocompatible Gd(2) O(3) nanoparticles as a bimodal contrast agent for use in photoacoustic (PA) and magnetic resonance (MR) imaging are reported. These nanoparticles show a clear PA image by themselves, without conjugation with gold, rare earth metals, or dyes. Relaxivity measurement by MR imaging clearly shows that their relaxivity, r(1) , is twice that of clinically available Gd-DTPA.