Chad R. Haney

Lung 18F-Fluorodeoxyglucose Positron Emission Tomography for Diagnosis and Monitoring of Pulmonary Arterial Hypertension

RATIONALE Pulmonary arterial hypertension (PAH) is a proliferative arteriopathy associated with glucose transporter-1 (Glut1) up-regulation and a glycolytic shift in lung metabolism. Glycolytic metabolism can be detected with the positron emission tomography (PET) tracer (18)F-fluorodeoxyglucose (FDG). OBJECTIVES The precise cell type in which glycolytic abnormalities occur in PAH is unknown. Moreover, whether FDG-PET is sufficiently sensitive to monitor PAH progression and detect therapeutic regression is untested. We hypothesized that increased lung FDG-PET reflects enhanced glycolysis in vascular cells and is reversible in response to effective therapies. METHODS PAH was induced in Sprague-Dawley rats by monocrotaline or chronic hypoxia (10% oxygen) in combination with Sugen 5416. Monocrotaline rats were treated with oral dichloroacetate or daily imatinib injections. FDG-PET scans and pulmonary artery acceleration times were obtained weekly. The origin of the PET signal was assessed by laser capture microdissection of airway versus vascular tissue. Metabolism was measured in pulmonary artery smooth muscle cell (PASMC) cultures, using a Seahorse extracellular flux analyzer. MEASUREMENTS AND MAIN RESULTS Lung FDG increases 1-2 weeks after monocrotaline (when PAH is mild) and is normalized by dichloroacetate and imatinib, which both also regress medial hypertrophy. Glut1 mRNA is up-regulated in both endothelium and PASMCs, but not airway cells or macrophages. PASMCs from monocrotaline rats are hyperproliferative and display normoxic activation of hypoxia-inducible factor-1α (HIF-1α), which underlies their glycolytic phenotype. CONCLUSIONS HIF-1α-mediated Glut1 up-regulation in proliferating vascular cells in PAH accounts for increased lung FDG-PET uptake. FDG-PET is sensitive to mild PAH and can monitor therapeutic changes in the vasculature.

Electron paramagnetic resonance oxygen images correlate spatially and quantitatively with Oxylite oxygen measurements.

Tumor oxygenation predicts cancer therapy response and malignant phenotype. This has spawned a number of oxymetries. Comparison of different oxymetries is crucial for the validation and understanding of these techniques. Electron paramagnetic resonance (EPR) imaging is a novel technique for providing quantitative high-resolution images of tumor and tissue oxygenation. This work compares sequences of tumor pO2 values from EPR oxygen images with sequences of oxygen measurements made along a track with an Oxylite oxygen probe. Four-dimensional (three spatial and one spectral) EPR oxygen images used spectroscopic imaging techniques to measure the width of a spectral line in each image voxel from a trityl spin probe (OX063, Amersham Health R&D) in the tissues and tumor of mice after spin probe injection. A simple calibration allows direct, quantitative translation of each line width to an oxygen concentration. These four-dimensional EPR images, obtained in 45 minutes from FSa fibrosarcomas grown in the legs of C3H mice, have a spatial resolution of ∼1 mm and oxygen resolution of ∼3 Torr. The position of the Oxylite track was measured within a 2-mm accuracy using a custom stereotactic positioning device. A total of nine images that involve 17 tracks were obtained. Of these, most showed good correlation between the Oxylite measured pO2 and a track located in the tumor within the uncertainties of the Oxylite localizability. The correlation was good both in terms of spatial distribution pattern and pO2 magnitude. The strong correlation of the two modalities corroborates EPR imaging as a useful tool for the study of tumor oxygenation.

PGC1α-mediated Mitofusin-2 Deficiency in Female Rats and Humans with Pulmonary Arterial Hypertension

RATIONALE Pulmonary arterial hypertension (PAH) is a lethal, female-predominant, vascular disease. Pathologic changes in PA smooth muscle cells (PASMC) include excessive proliferation, apoptosis-resistance, and mitochondrial fragmentation. Activation of dynamin-related protein increases mitotic fission and promotes this proliferation-apoptosis imbalance. The contribution of decreased fusion and reduced mitofusin-2 (MFN2) expression to PAH is unknown. OBJECTIVES We hypothesize that decreased MFN2 expression promotes mitochondrial fragmentation, increases proliferation, and impairs apoptosis. The role of MFN2s transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), was assessed. MFN2 therapy was tested in PAH PASMC and in models of PAH. METHODS Fusion and fission mediators were measured in lungs and PASMC from patients with PAH and female rats with monocrotaline or chronic hypoxia+Sugen-5416 (CH+SU) PAH. The effects of adenoviral mitofusin-2 (Ad-MFN2) overexpression were measured in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS In normal PASMC, siMFN2 reduced expression of MFN2 and PGC1α; conversely, siPGC1α reduced PGC1α and MFN2 expression. Both interventions caused mitochondrial fragmentation. siMFN2 increased proliferation. In rodent and human PAH PASMC, MFN2 and PGC1α were decreased and mitochondria were fragmented. Ad-MFN2 increased fusion, reduced proliferation, and increased apoptosis in human PAH and CH+SU. In CH+SU, Ad-MFN2 improved walking distance (381 ± 35 vs. 245 ± 39 m; P < 0.05); decreased pulmonary vascular resistance (0.18 ± 0.02 vs. 0.38 ± 0.14 mm Hg/ml/min; P < 0.05); and decreased PA medial thickness (14.5 ± 0.8 vs. 19 ± 1.7%; P < 0.05). Lung vascularity was increased by MFN2. CONCLUSIONS Decreased expression of MFN2 and PGC1α contribute to mitochondrial fragmentation and a proliferation-apoptosis imbalance in human and experimental PAH. Augmenting MFN2 has therapeutic benefit in human and experimental PAH.

Purification and chemical modifications of hemoglobin in developing hemoglobin based oxygen carriers.

The efficacy of blood substitutes, as a whole, has been readily demonstrated, in animals as well as clinical studies. It is well known that stroma free hemoglobin (SF-Hb) is very toxic, due to effects on renal and coagulation functions and vascular tone. Several modifications have been made to the hemoglobin tetramer in an attempt to eliminate its toxicity. Conjugation, cross-linking, polymerization, and recombinant technology have all been used to reduce toxicity, while aiming to optimize the therapeutic value of hemoglobin based blood substitutes. The remaining issue seems to be the hypertensive response seen in many hemoglobin solutions. The cause of the hypertensive response, and hence what chemical modifications are suitable to alleviate it are still under debate.

ELECTRON PARAMAGNETIC RESONANCE OXYGEN IMAGE HYPOXIC FRACTION PLUS RADIATION DOSE STRONGLY CORRELATES WITH TUMOR CURE IN FSA FIBROSARCOMAS

PURPOSE Tumor hypoxia has long been known to produce resistance to radiation. In this study, electron paramagnetic resonance (EPR) oxygen imaging was investigated for its power to predict the success of tumor control according to tumor oxygenation level and radiation dose. METHODS AND MATERIALS A total of 34 EPR oxygen images were obtained from the legs of C3H mice bearing 0.5-cm(3) FSa fibrosarcomas under both normal (air breathing) and clamped tumor conditions. Under the same conditions as those during which the images were obtained, the tumors were irradiated to a variety of doses near the FSa dose at which 50% of tumors were cured. Tumor tissue was distinguished from normal tissue using co-registration of the EPR oxygen images with spin-echo magnetic resonance imaging of the tumor and/or stereotactic localization. The tumor voxel statistics in the EPR oxygen image included the mean and median partial pressure of oxygen and the fraction of tumor voxels below the specified partial pressure of oxygen values of 3, 6, and 10 mm Hg. Bivariate logistic regression analysis using the radiation dose and each of the EPR oxygen image statistics to determine which best separated treatment failure from success. RESULTS The measurements of the dose at which 50% of tumors were cured were similar to those found in published data for this syngeneic tumor. Bivariate analysis of 34 tumors demonstrated that tumor cure correlated with dose (p = 0.004) and with a <10 mm Hg hypoxic fraction (p = 0.023). CONCLUSION Our results have shown that, together, radiation dose and EPR image hypoxic fraction separate the population of FSa fibrosarcomas that are cured from those that fail, thus predicting curability.

Gd(III)-labeled peptide nanofibers for reporting on biomaterial localization in vivo.

Bioactive supramolecular nanostructures are of great importance in regenerative medicine and the development of novel targeted therapies. In order to use supramolecular chemistry to design such nanostructures, it is extremely important to track their fate in vivo through the use of molecular imaging strategies. Peptide amphiphiles (PAs) are known to generate a wide array of supramolecular nanostructures, and there is extensive literature on their use in areas such as tissue regeneration and therapies for disease. We report here on a series of PA molecules based on the well-established β-sheet amino acid sequence V3A3 conjugated to macrocyclic Gd(III) labels for magnetic resonance imaging (MRI). These conjugates were shown to form cylindrical supramolecular assemblies using cryogenic transmission electron microscopy and small-angle X-ray scattering. Using nuclear magnetic relaxation dispersion analysis, we observed that thermal annealing of the nanostructures led to a decrease in water exchange lifetime (τm) of hundreds of nanoseconds only for molecules that self-assemble into nanofibers of high aspect ratio. We interpret this decrease to indicate more solvent exposure to the paramagnetic moiety on annealing, resulting in faster water exchange within angstroms of the macrocycle. We hypothesize that faster water exchange in the nanofiber-forming PAs arises from the dehydration and increase in packing density on annealing. Two of the self-assembling conjugates were selected for imaging PAs after intramuscular injections of the PA C16V3A3E3-NH2 in the tibialis anterior muscle of a murine model. Needle tracts were clearly discernible with MRI at 4 days postinjection. This work establishes Gd(III) macrocycle-conjugated peptide amphiphiles as effective tracking agents for peptide amphiphile materials in vivo over the timescale of days.

Electron paramagnetic resonance oxygen imaging of a rabbit tumor using localized spin probe delivery.

PURPOSE Application ofin vivo electron paramagnetic resonance (EPR) oxygen imaging (EPROI) to tumors larger than those of mice requires development of both instrumental and medical aspects of imaging. METHODS 250 MHz EPR oxygen imaging was performed using a loop-gap resonator with a volume exceeding 100cm3. The paramagnetic spin probe was injected directly into the femoral artery feeding the rabbit leg/tumor. RESULTS The authors present continuous wave and electron spin echo EPR oxygen images of a large size (4 cm) VX-2 tumor located on the leg of a New Zealand white rabbit. CONCLUSIONS This study demonstrates the feasibility of continuous wave and electron spin echo oxygen imaging modalities for investigation of volumes of tumor and normal tissue relevant to large animals. The injection of the spin probe directly into the artery feeding a rabbit leg will allow one to reduce, by over one order of magnitude, the amount of spin probe used as compared to whole animal IV injection.

Sensitivity to tumor microvasculature without contrast agents in high spectral and spatial resolution MR images

Contrast‐enhanced (CE)‐MRI is sensitive to cancers but can produce adverse reactions and suffers from insufficient specificity and morphological detail. This research investigated whether high spectral and spatial resolution (HiSS) MRI detects tumor vasculature without contrast agents, based on the sensitivity of the water resonance line shape to tumor blood vessels. HiSS data from AT6.1 tumors inoculated in the hind legs of rats (N = 8) were collected pre‐ and post–blood pool contrast agent (iron‐oxide particles) injection. The waterline in small voxels was significantly more asymmetric at the tumor rim compared to the tumor center and normal muscle (P < 0.003). Composite images were synthesized, with the intensity in each voxel determined by the Fourier component (FC) of the water resonance having the greatest relative image contrast at that position. We tested whether regions with high contrast in FC images (FCIs) contain vasculature by comparing FCIs with CE‐MRI as the “gold standard” of vascular density. The FCIs had 75% ± 13% sensitivity, 74% ± 10% specificity, and 91% ± 4% positive predictive value (PPV) for vasculature detection at the tumor rim. These results suggest that tumor microvasculature can be detected using HiSS imaging without the use of contrast agents. Magn Reson Med 61:291–298, 2009.

Use of a reference tissue and blood vessel to measure the arterial input function in DCEMRI

Accurate measurement of the arterial input function is critical for quantitative evaluation of dynamic contrast enhanced magnetic resonance imaging data. Use of the reference tissue method to derive a local arterial input function avoided large errors associated with direct arterial measurements, but relied on literature values for Ktrans and ve. We demonstrate that accurate values of Ktrans and ve in a reference tissue can be measured by comparing contrast media concentration in a reference tissue to plasma concentrations measured directly in a local artery after the 1–2 passes of the contrast media bolus—when plasma concentration is low and can be measured accurately. The values of Ktrans and ve calculated for the reference tissue can then be used to derive a more complete arterial input function including the first pass of the contrast bolus. This new approach was demonstrated using dynamic contrast enhanced magnetic resonance imaging data from rodent hind limb. Values obtained for Ktrans and ve in muscle, and the shape and amplitude of the derived arterial input function are consistent with published results. Magn Reson Med, 2010.

Characterization of response to radiation mediated gene therapy by means of multimodality imaging.

Imaging techniques are under development to facilitate early analysis of spatial patterns of tumor response to combined radiation and antivascular gene therapy. A genetically modified, replication defective adenoviral vector (Ad.EGR‐TNFα), injected intratumorally, mediates infected cells to express tumor necrosis factor alpha (TNFα), which is increased after exposure to radiation. The goal of this study was to characterize an image based “signature” for response to this combined radiation and gene therapy in mice with human prostate xenografts. This study is part of an imaged guided therapy project where such a signature would be useful in guiding subsequent treatments. Changes in the tumor micro‐environment were assessed using MRI registered with electron paramagnetic resonance imaging which provides images of tissue oxygenation. Dynamic contrast‐enhanced MRI was used to assess tissue perfusion. When compared with null vector (control) treatment, the ratio of contrast agent (Gd‐DTPA‐BMA) washout rate to uptake rate was lower (P = 0.001) after treatment, suggesting a more balanced perfusion. Concomitantly, oxygenation significantly increased in the treated animals and decreased or did not change in the control animals (P < 0.025). This is the first report of minimally invasive, quantitative, absolute oxygen measurements correlated with tissue perfusion in vivo. Magn Reson Med, 2009.