Distinct Regulatory Effects of Myeloid Cell and Endothelial Cell NAPDH Oxidase 2 on Blood Pressure.

BACKGROUND: Hypertension caused by increased renin-angiotensin system activation is associated with elevated reactive oxygen species production. Previous studies implicate NADPH oxidase (Nox) proteins as important reactive oxygen species sources during renin-angiotensin system activation, with different Nox isoforms being potentially involved. Among these, Nox2 is expressed in multiple cell types, including endothelial cells, fibroblasts, immune cells, and microglia. Blood pressure (BP) is regulated at the central nervous system, renal, and vascular levels, but the cell-specific role of Nox2 in BP regulation is unknown. METHODS: We generated a novel mouse model with a floxed Nox2 gene and used Tie2-Cre, LysM Cre, or Cdh5-CreERT2 driver lines to develop cell-specific models of Nox2 perturbation to investigate its role in BP regulation. RESULTS: Unexpectedly, Nox2 deletion in myeloid but not endothelial cells resulted in a significant reduction in basal BP. Both Tie2-CreNox2 knockout (KO) mice (in which Nox2 was deficient in both endothelial cells and myeloid cells) and LysM CreNox2KO mice (in which Nox2 was deficient in myeloid cells) had significantly lower BP than littermate controls, whereas basal BP was unaltered in Cdh5-CreERT2 Nox2KO mice (in which Nox2 is deficient only in endothelial cells). The lower BP was attributable to an increased NO bioavailability that dynamically dilated resistance vessels in vivo under basal conditions without a change in renal function. Myeloid-specific Nox2 deletion had no effect on angiotensin II-induced hypertension, which, however, was blunted in Tie2-CreNox2KO mice, along with preservation of endothelium-dependent relaxation during angiotensin II stimulation. CONCLUSIONS: We identify a hitherto unrecognized modulation of basal BP by myeloid cell Nox2, whereas endothelial cell Nox2 regulates angiotensin II-induced hypertension. These results identify distinct cell-specific roles for Nox2 in BP regulation.

Dysfunction of the CNS-heart axis in mouse models of Huntington's disease.

Cardiac remodelling and contractile dysfunction occur during both acute and chronic disease processes including the accumulation of insoluble aggregates of misfolded amyloid proteins that are typical features of Alzheimer's, Parkinson's and Huntington's disease (HD). While HD has been described mainly as a neurological disease, multiple epidemiological studies have shown that HD patients exhibit a high incidence of cardiovascular events leading to heart failure, and that this is the second highest cause of death. Given that huntingtin is ubiquitously expressed, cardiomyocytes may be at risk of an HD-related dysfunction. In mice, the forced expression of an expanded polyQ repeat under the control of a cardiac specific promoter led to severe heart failure followed by reduced lifespan. However the mechanism leading to cardiac dysfunction in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that pre-symptomatic animals developed connexin-43 relocation and a significant deregulation of hypertrophic markers and Bdnf transcripts. In the symptomatic animals, pronounced functional changes were visualised by cardiac MRI revealing a contractile dysfunction, which might be a part of dilatated cardiomyopathy (DCM). This was accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis. To our surprise, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation, even at the end stage of disease. We postulate that the HD-related cardiomyopathy is caused by altered central autonomic pathways although the pathogenic effects of mutant HTT acting intrinsically in the heart may also be a contributing factor.

Magnetic Drug Targeting: Preclinical in Vivo Studies, Mathematical Modeling, and Extrapolation to Humans.

A sound theoretical rationale for the design of a magnetic nanocarrier capable of magnetic capture in vivo after intravenous administration could help elucidate the parameters necessary for in vivo magnetic tumor targeting. In this work, we utilized our long-circulating polymeric magnetic nanocarriers, encapsulating increasing amounts of superparamagnetic iron oxide nanoparticles (SPIONs) in a biocompatible oil carrier, to study the effects of SPION loading and of applied magnetic field strength on magnetic tumor targeting in CT26 tumor-bearing mice. Under controlled conditions, the in vivo magnetic targeting was quantified and found to be directly proportional to SPION loading and magnetic field strength. Highest SPION loading, however, resulted in a reduced blood circulation time and a plateauing of the magnetic targeting. Mathematical modeling was undertaken to compute the in vivo magnetic, viscoelastic, convective, and diffusive forces acting on the nanocapsules (NCs) in accordance with the Nacev-Shapiro construct, and this was then used to extrapolate to the expected behavior in humans. The model predicted that in the latter case, the NCs and magnetic forces applied here would have been sufficient to achieve successful targeting in humans. Lastly, an in vivo murine tumor growth delay study was performed using docetaxel (DTX)-encapsulated NCs. Magnetic targeting was found to offer enhanced therapeutic efficacy and improve mice survival compared to passive targeting at drug doses of ca. 5-8 mg of DTX/kg. This is, to our knowledge, the first study that truly bridges the gap between preclinical experiments and clinical translation in the field of magnetic drug targeting.

Cell-specific effects of Nox2 on the acute and chronic response to myocardial infarction.

BACKGROUND: Increased reactive oxygen species (ROS) production is involved in the process of adverse cardiac remodeling and development of heart failure after myocardial infarction (MI). NADPH oxidase-2 (Nox2) is a major ROS source within the heart and its activity increases after MI. Furthermore, genetic deletion of Nox2 is protective against post-MI cardiac remodeling. Nox2 levels may increase both in cardiomyocytes and endothelial cells and recent studies indicate cell-specific effects of Nox2, but it is not known which of these cell types is important in post-MI remodeling. METHODS AND RESULTS: We have generated transgenic mouse models in which Nox2 expression is targeted either to cardiomyocytes (cardio-Nox2TG) or endothelial cells (endo-Nox2TG). We here studied the response of cardio-Nox2TG mice, endo-Nox2TG mice and matched wild-type littermates (WT) to MI induced by permanent left coronary artery ligation up to 4weeks. Initial infarct size assessed by magnetic resonance imaging (MRI) and cardiac dysfunction were similar among groups. Cardiomyocyte hypertrophy and interstitial fibrosis were augmented in cardio-Nox2TG compared to WT after MI and post-MI survival tended to be worse whereas endo-Nox2TG mice showed no significant difference compared to WT. CONCLUSIONS: These results indicate that cardiomyocyte rather than endothelial cell Nox2 may have the more important role in post-MI remodeling.

Bone marrow transplantation modulates tissue macrophage phenotype and enhances cardiac recovery after subsequent acute myocardial infarction.

BACKGROUND: Bone marrow transplantation (BMT) is commonly used in experimental studies to investigate the contribution of BM-derived circulating cells to different disease processes. During studies investigating the cardiac response to acute myocardial infarction (MI) induced by permanent coronary ligation in mice that had previously undergone BMT, we found that BMT itself affects the remodelling response. METHODS AND RESULTS: Compared to matched naive mice, animals that had previously undergone BMT developed significantly less post-MI adverse remodelling, infarct thinning and contractile dysfunction as assessed by serial magnetic resonance imaging. Cardiac rupture in male mice was prevented. Histological analysis showed that the infarcts of mice that had undergone BMT had a significantly higher number of inflammatory cells, surviving cardiomyocytes and neovessels than control mice, as well as evidence of significant haemosiderin deposition. Flow cytometric and histological analyses demonstrated a higher number of alternatively activated (M2) macrophages in myocardium of the BMT group compared to control animals even before MI, and this increased further in the infarcts of the BMT mice after MI. CONCLUSIONS: The process of BMT itself substantially alters tissue macrophage phenotype and the subsequent response to acute MI. An increase in alternatively activated macrophages in this setting appears to enhance cardiac recovery after MI.

Toll-like receptor 9 prevents cardiac rupture after myocardial infarction in mice independently of inflammation.

We have reported that the Toll-like receptor 9 (TLR9) signaling pathway plays an important role in the development of pressure overload-induced inflammatory responses and heart failure. However, its role in cardiac remodeling after myocardial infarction has not been elucidated. TLR9-deficient and control C57Bl/6 wild-type mice were subjected to left coronary artery ligation. The survival rate 14 days postoperation was significantly lower in TLR9-deficient mice than that in wild-type mice with evidence of cardiac rupture in all dead mice. Cardiac magnetic resonance imaging showed no difference in infarct size and left ventricular wall thickness and function between TLR9-deficient and wild-type mice. There were no differences in the number of infiltrating inflammatory cells and the levels of inflammatory cytokine mRNA in infarct hearts between TLR9-deficient and wild-type mice. The number of α-smooth muscle actin (αSMA)-positive myofibroblasts and αSMA/Ki67-double-positive proliferative myofibroblasts was increased in the infarct and border areas in infarct hearts compared with those in sham-operated hearts in wild-type mice, but not in TLR9-deficient mice. The class B CpG oligonucleotide increased the phosphorylation level of NF-κB and the number of αSMA-positive and αSMA/Ki67-double-positive cells and these increases were attenuated by BAY1-7082, an NF-κB inhibitor, in cardiac fibroblasts isolated from wild-type hearts. The CpG oligonucleotide showed no effect on NF-κB activation or the number of αSMA-positive and αSMA/Ki67-double-positive cells in cardiac fibroblasts from TLR9-deficient hearts. Although the TLR9 signaling pathway is not involved in the acute inflammatory response in infarct hearts, it ameliorates cardiac rupture possibly by promoting proliferation and differentiation of cardiac fibroblasts.

The Shortening of MWNT-SPION Hybrids by Steam Treatment Improves Their Magnetic Resonance Imaging Properties In Vitro and In Vivo.

Carbon nanotubes (CNTs) have been advocated as promising nanocarriers in the biomedical field. Their high surface area and needle-like shape make these systems especially attractive for diagnostic and therapeutic applications. Biocompatibility, cell internalization, biodistribution, and pharmacokinetic profile have all been reported to be length dependent. In this study, further insights are gotten on the role that the length of CNTs plays when developing novel contrast agents for magnetic resonance imaging (MRI). Two samples of CNTs with different length distribution have been decorated with radio-labeled iron oxide nanoparticles. Despite characterization of the prepared hybrids reveals a similar degree of loading and size of the nanoparticles for both samples, the use of short CNTs is found to enhance the MRI properties of the developed contrast agents both in vitro and in vivo compared to their long counterparts.

Synthesis, Characterization, and Application of Core-Shell Co0.16Fe2.84O4@NaYF4(Yb, Er) and Fe3O4@NaYF4(Yb, Tm) Nanoparticle as Trimodal (MRI, PET/SPECT, and Optical) Imaging Agents.

Multimodal nanoparticulate materials are described, offering magnetic, radionuclide, and fluorescent imaging capabilities to exploit the complementary advantages of magnetic resonance imaging (MRI), positron emission tomography/single-photon emission commuted tomography (PET/SPECT), and optical imaging. They comprise Fe3O4@NaYF4 core/shell nanoparticles (NPs) with different cation dopants in the shell or core, including Co0.16Fe2.84O4@NaYF4(Yb, Er) and Fe3O4@NaYF4(Yb, Tm). These NPs are stabilized by bisphosphonate polyethylene glycol conjugates (BP-PEG), and then show a high transverse relaxivity (r2) up to 326 mM(-1) s(-1) at 3T, a high affinity to [(18)F]-fluoride or radiometal-bisphosphonate conjugates (e.g., (64)Cu and (99m)Tc), and fluorescent emissions from 500 to 800 nm under excitation at 980 nm. The biodistribution of intravenously administered particles determined by PET/MR imaging suggests that negatively charged Co0.16Fe2.84O4@NaYF4(Yb, Er)-BP-PEG (10K) NPs cleared from the blood pool more slowly than positively charged NPs Fe3O4@NaYF4(Yb, Tm)-BP-PEG (2K). Preliminary results in sentinel lymph node imaging in mice indicate the advantages of multimodal imaging.

High-frequency speckle tracking echocardiography in the assessment of left ventricular function and remodeling after murine myocardial infarction.

The objectives of this study were to assess the feasibility and accuracy of high-frequency speckle tracking echocardiography (STE) in a murine model of myocardial infarction (MI). STE is used clinically to quantify global and regional cardiac function, but its application in mice is challenging because of the small cardiac size and rapid heart rates. A high-frequency micro-ultrasound system with STE (Visualsonics Vevo 2100) was compared against magnetic resonance imaging (MRI) for the assessment of global left ventricular (LV) size and function after murine MI. Animals subjected to coronary ligation (n = 46) or sham ligation (n = 27) were studied 4 wk postoperatively. Regional and global deformation were also assessed. STE-derived LV ejection fraction (EF) and mass correlated well with MRI indexes (r = 0.93, 0.77, respectively; P < 0.001), as did STE-derived mass with postmortem values (r = 0.80, P < 0.001). Higher STE-derived volumes correlated positively with MRI-derived infarct size (P < 0.01). Global strain parameters were significantly reduced after MI (all P < 0.001) and strongly correlated with LV mass and MRI-derived infarct size as promising surrogates for the extent of remodeling and infarction, respectively (both P < 0.05). Regional strain analyses showed that radial strain and strain rate were relatively preserved in anterior basal segments after MI compared with more apical segments (P < 0.001); however, longitudinal strain and strain rate were significantly impaired both basally and distally (P < 0.001). Strain-derived parameters of dyssynchrony were significantly increased in the MI group (P < 0.01). Analysis time for STE was 210 ± 45 s with acceptable inter- and intraobserver variability. In conclusion, high-frequency STE enables quantitative assessment of regional and global function in the remodeling murine LV after MI.

Magnetically Decorated Multi-Walled Carbon Nanotubes as Dual MRI and SPECT Contrast Agents.

Carbon nanotubes (CNTs) have been proposed as one of the most promising nanomaterials to be used in biomedicine for their applications in drug/gene delivery as well as biomedical imaging. The present study developed radio-labeled iron oxide decorated multi-walled CNTs (MWNT) as dual magnetic resonance (MR) and single photon emission computed tomography (SPECT) imaging agents. Hybrids containing different amounts of iron oxide were synthesized by in situ generation. Physicochemical characterisations revealed the presence of superparamagnetic iron oxide nanoparticles (SPION) granted the magnetic properties of the hybrids. Further comprehensive examinations including high resolution transmission electron microscopy (HRTEM), fast Fourier transform simulations (FFT), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) assured the conformation of prepared SPION as γ-Fe2O3. High r2 relaxivities were obtained in both phantom and in vivo MRI compared to the clinically approved SPION Endorem®. The hybrids were successfully radio-labeled with technetium-99m through a functionalized bisphosphonate and enabled SPECT/CT imaging and γ-scintigraphy to quantitatively analyze the biodistribution in mice. No abnormality was found by histological examination and the presence of SPION and MWNT were identified by Perls stain and Neutral Red stain, respectively. TEM images of liver and spleen tissues showed the co-localization of SPION and MWNT within the same intracellular vesicles, indicating the in vivo stability of the hybrids after intravenous injection. The results demonstrated the capability of the present SPION-MWNT hybrids as dual MRI and SPECT contrast agents for in vivo use.

Assessment of inflammation with a very small iron-oxide particle in a murine model of reperfused myocardial infarction.

PURPOSE: To investigate a very small iron-oxide particle (VSOP) in a mouse model of acute ischemia-reperfusion to access the mechanism of such particles in areas of myocardial inflammation. MATERIALS AND METHODS: Animals were injected with VSOP at several time points, in a mouse model of acute myocardial infarction (MI), before and after MI. MRI was used to localize areas of VSOP enhancement, evaluate VSOP areas extension, and determine the related T2* values. Histology, electron microscopy, macrophage counting, and Evan's Blue staining were also performed. RESULTS: We found that areas of VSOP uptake decreased from 1 to 8 days post-MI while the related T2* values increased. T2* and VSOP areas, defined from MRI data, correlated well between 1 and 3 days post-MI but not at 7 days after injection. Histological analysis and electron microscopy showed colocalization of macrophages with areas of VSOP staining. However, there was no correlation between number of macrophages and the extension of the VSOP areas achieved by MR. We found that only areas of increased permeability (assessed by Evan's Blue staining) showed colocalization of macrophages and VSOP uptake. CONCLUSION: This study demonstrates that VSOP allows the assessment of myocardial inflammation associated with increased permeability during infarct healing in a mouse model of ischemia-reperfusion.

In vivo imaging using surface enhanced spatially offset raman spectroscopy (SESORS): balancing sampling frequency to improve overall image acquisition.

In the field of optical imaging, the ability to image tumors at depth with high selectivity and specificity remains a challenge. Surface enhanced resonance Raman scattering (SERRS) nanoparticles (NPs) can be employed as image contrast agents to specifically target cells in vivo; however, this technique typically requires time-intensive point-by-point acquisition of Raman spectra. Here, we combine the use of "spatially offset Raman spectroscopy" (SORS) with that of SERRS in a technique known as "surface enhanced spatially offset resonance Raman spectroscopy" (SESORRS) to image deep-seated tumors in vivo. Additionally, by accounting for the laser spot size, we report an experimental approach for detecting both the bulk tumor, subsequent delineation of tumor margins at high speed, and the identification of a deeper secondary region of interest with fewer measurements than are typically applied. To enhance light collection efficiency, four modifications were made to a previously described custom-built SORS system. Specifically, the following parameters were increased: (i) the numerical aperture (NA) of the lens, from 0.2 to 0.34; (ii) the working distance of the probe, from 9 mm to 40 mm; (iii) the NA of the fiber, from 0.2 to 0.34; and (iv) the fiber diameter, from 100 µm to 400 µm. To calculate the sampling frequency, which refers to the number of data point spectra obtained for each image, we considered the laser spot size of the elliptical beam (6 × 4 mm). Using SERRS contrast agents, we performed in vivo SESORRS imaging on a GL261-Luc mouse model of glioblastoma at four distinct sampling frequencies: par-sampling frequency (12 data points collected), and over-frequency sampling by factors of 2 (35 data points collected), 5 (176 data points collected), and 10 (651 data points collected). In comparison to the previously reported SORS system, the modified SORS instrument showed a 300% improvement in signal-to-noise ratios (SNR). The results demonstrate the ability to acquire distinct Raman spectra from deep-seated glioblastomas in mice through the skull using a low power density (6.5 mW/mm2) and 30-times shorter integration times than a previous report (0.5 s versus 15 s). The ability to map the whole head of the mouse and determine a specific region of interest using as few as 12 spectra (6 s total acquisition time) is achieved. Subsequent use of a higher sampling frequency demonstrates it is possible to delineate the tumor margins in the region of interest with greater certainty. In addition, SESORRS images indicate the emergence of a secondary tumor region deeper within the brain in agreement with MRI and H&E staining. In comparison to traditional Raman imaging approaches, this approach enables improvements in the detection of deep-seated tumors in vivo through depths of several millimeters due to improvements in SNR, spectral resolution, and depth acquisition. This approach offers an opportunity to navigate larger areas of tissues in shorter time frames than previously reported, identify regions of interest, and then image the same area with greater resolution using a higher sampling frequency. Moreover, using a SESORRS approach, we demonstrate that it is possible to detect secondary, deeper-seated lesions through the intact skull.

Noninvasive magnetic resonance imaging evaluation of endothelial permeability in murine atherosclerosis using an albumin-binding contrast agent.

BACKGROUND: Endothelial dysfunction promotes atherosclerosis and precedes acute cardiovascular events. We investigated whether in vivo magnetic resonance imaging with the use of an albumin-binding contrast agent, gadofosveset, could detect endothelial damage associated with atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. Furthermore, we tested whether magnetic resonance imaging could noninvasively assess endothelial function by measuring the endothelial-dependent vasodilation in response to acetylcholine. METHODS AND RESULTS: ApoE(-/-) mice were imaged at 4, 8, and 12 weeks after commencement of a high-fat diet. Statin-treated ApoE(-/-) mice were scanned after 12 weeks of a high-fat diet. Wild-type mice were imaged before and 48 hours after injection of Russell's viper venom, an endothelial toxin. Delayed enhancement magnetic resonance imaging and T1 mapping of the brachiocephalic artery, 30 minutes after injection of gadofosveset, showed increased vessel wall enhancement and relaxation rate (R(1)) with progression of atherosclerosis in ApoE(-/-)(R(1) [s(-1)]: R(4 weeks) 2.42±0.35, R(8 weeks) 3.45±0.54, R(12 weeks) 3.83±0.52) and Russell's viper venom-injected wild-type mice (R(1)=4.57±0.86). Conversely, wild-type (R(1)=2.15±0.34) and statin-treated ApoE(-/-) (R(1)=3.0±0.65) mice showed less enhancement. Uptake of gadofosveset correlated with Evans blue staining, morphological changes of endothelial cells, and widening of the cell-cell junctions, suggesting that uptake occurs in regions of increased vascular permeability. Endothelial-dependent vasomotor responses showed vasoconstriction of the arteries of the ApoE(-/-) (-22.22±7.95%) and Russell's viper venom-injected (-10.37±17.60%) mice compared with wild-type mice (32.45±12.35%). Statin treatment improved endothelium morphology and function (-8.12±8.22%). CONCLUSIONS: We demonstrate the noninvasive assessment of endothelial permeability and function with the use of an albumin-binding magnetic resonance contrast agent. Blood albumin leakage could be a surrogate marker for the in vivo evaluation of interventions that aim to restore the endothelium.

Flow-independent 3D whole-heart vessel wall imaging using an interleaved T2-preparation acquisition.

Current techniques to visualize the arterial vessel wall are limited in coverage because most of them are flow dependent. In this study, we present a novel technique for flow-independent vessel wall imaging that takes advantage of the differences in T2 relaxation time of arterial blood and surrounding tissues using the T2-preparation prepulse. The technique is based on the acquisition and subtraction of two data sets, one obtained with and one without T2-preparation prepulse. This approach allows for nulling the signal of arterial blood while maintaining signal from muscle and vessel wall. The result of the subtraction is a flow-independent black-blood vessel wall image. To minimize the motion sensitivity of the subtraction step, we developed an interleaved acquisition for the T2-preparation prepulse and non-T2-preparation prepulse images, which allows obtaining coronary vessel wall images from a whole-heart acquisition with minimal misregistration artefacts. In this article, we present the technique and preliminary results in healthy subjects.

Aurora A kinase inhibition induces accumulation of SCLC tumor cells in mitosis with restored interferon signaling to increase response to PD-L1.

Despite small cell lung cancers (SCLCs) having a high mutational burden, programmed death-ligand 1 (PD-L1) immunotherapy only modestly increases survival. A subset of SCLCs that lose their ASCL1 neuroendocrine phenotype and restore innate immune signaling (termed the "inflammatory" subtype) have durable responses to PD-L1. Some SCLCs are highly sensitive to Aurora kinase inhibitors, but early-phase trials show short-lived responses, suggesting effective therapeutic combinations are needed to increase their durability. Using immunocompetent SCLC genetically engineered mouse models (GEMMs) and syngeneic xenografts, we show durable efficacy with the combination of a highly specific Aurora A kinase inhibitor (LSN3321213) and PD-L1. LSN3321213 causes accumulation of tumor cells in mitosis with lower ASCL1 expression and higher expression of interferon target genes and antigen-presentation genes mimicking the inflammatory subtype in a cell-cycle-dependent manner. These data demonstrate that inflammatory gene expression is restored in mitosis in SCLC, which can be exploited by Aurora A kinase inhibition.

MRI-based prediction of adverse cardiac remodeling after murine myocardial infarction.

Myocardial infarction (MI) results in adverse cardiac remodeling leading to heart failure and increased mortality. Experimental mouse models of MI are extensively used to identify mechanisms underlying adverse remodeling, but the extent of remodeling that occurs may be highly variable and can limit the utility to discover new disease pathways. The ability to predict the development of significant late post-MI remodeling would be invaluable in conducting such studies by increasing throughput and efficiency. This study aimed to identify potential thresholds of cardiac magnetic resonance imaging (MRI) parameters measured early after murine MI that would predict the development of significant adverse remodeling at 4 wk. MI was achieved by permanent coronary ligation and animals (n = 84) were followed up for 4 wk subsequently. MRI was used to assess left ventricular (LV) volumes, mass and ejection fraction, as well as infarct size (IS). Late gadolinium enhancement cine-MRI was performed at 2 days with standard cine-MRI at 30 days post-MI. Utilizing multiple logistic regression, we found that IS >36%, at 2 days post-MI, was the overall best single predictor of adverse remodeling at 30 days (sensitivity 80.7%, specificity 88.9%; C-statistic of 0.939 from receiver-operating curve analysis). LV end-systolic volume (LVESV) >32 µl was also an excellent predictor comparable to IS. The combination of IS >36% and/or LVESV >32 µl provided the highest predictive values for late adverse remodeling among multiple predictors. This study demonstrates that MRI-based estimation of IS and ESV during the acute phase of murine MI are good predictors of subsequent adverse remodeling that may aid experimental design.

(99m)Tc-bisphosphonate-iron oxide nanoparticle conjugates for dual-modality biomedical imaging.

The combination of radionuclide-based imaging modalities such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) with magnetic resonance imaging (MRI) is likely to become the next generation of clinical scanners. Hence, there is a growing interest in the development of SPECT- and PET-MRI agents. To this end, we report a new class of dual-modality imaging agents based on the conjugation of radiolabeled bisphosphonates (BP) directly to the surface of superparamagnetic iron oxide (SPIO) nanoparticles. We demonstrate the high potential of BP-iron oxide conjugation using (99m)Tc-dipicolylamine(DPA)-alendronate, a BP-SPECT agent, and Endorem/Feridex, a liver MRI contrast agent based on SPIO. The labeling of SPIOs with (99m)Tc-DPA-alendronate can be performed in one step at room temperature if the SPIO is not coated with an organic polymer. Heating is needed if the nanoparticles are coated, as long as the coating is weakly bound as in the case of dextran in Endorem. The size of the radiolabeled Endorem (99m)Tc-DPA-ale-Endorem) was characterized by TEM (5 nm, Fe3O4 core) and DLS (106 ± 60 nm, Fe3O4 core + dextran). EDX, Dittmer-Lester, and radiolabeling studies demonstrate that the BP is bound to the nanoparticles and that it binds to the Fe3O4 cores of Endorem, and not its dextran coating. The bimodal imaging capabilities and excellent stability of these nanoparticles were confirmed using MRI and nanoSPECT-CT imaging, showing that (99m)Tc and Endorem co-localize in the liver and spleen In Vivo, as expected for particles of the composition and size of (99m)Tc-DPA-ale-Endorem. To the best of our knowledge, this is the first example of radiolabeling SPIOs with BP conjugates and the first example of radiolabeling SPIO nanoparticles directly onto the surface of the iron oxide core, and not its coating. This work lays down the basis for a new generation of SPECT/PET-MR imaging agents in which the BP group could be used to attach functionality to provide targeting, stealth/stability, and radionuclides to Fe3O4 nanoparticles using very simple methodology readily amenable to GMP.

Visualization of elastin using cardiac magnetic resonance imaging after myocardial infarction as inflammatory response.

The aim of this study was to investigate the merits of magnetic resonance imaging (MRI) using an elastin-binding contrast agent after myocardial infarction in mouse models with deletions of monocyte populations. Permanent ligation of the left anterior descending (LAD) artery was conducted in 10 wild-type mice and 10 each of three knockout models: CX3CR-/-, CCR2-/-, and MCP-1-/-. At 7 days and 30 days after permanent ligation, cardiac MRI was performed with a 7 T-Bruker horizontal scanner for in vivo detection of elastin with an elastin/tropoelastin-specific contrast agent (ESMA). Histology was performed with staining for elastin, collagen I and III, and F4/80. Real-time PCR was conducted to quantify the expression of genes for collagen I and III, F4/80, and tumor necrosis factor alpha (TNFα). Histological and ESMA-indicated elastin areas were strongly correlated (r = 0.8). 30 days after permanent ligation, CCR2-deficient mice demonstrated higher elastin levels in the scar relative to MCP-1-/- (p < 0.04) and wild-type mice (p < 0.02). The ejection fraction was lower in CCR2-deficient mice. In vivo MRI in mouse models of MI can detect elastin deposition after myocardial infarction, highlighting the pivotal role of elastin in myocardial remodeling in mouse models with deletions of monocyte populations.

In vivo hypoxia characterization using blood oxygen level dependent magnetic resonance imaging in a preclinical glioblastoma mouse model.

PURPOSE: Hypoxia measurements can provide crucial information regarding tumor aggressiveness, however current preclinical approaches are limited. Blood oxygen level dependent (BOLD) Magnetic Resonance Imaging (MRI) has the potential to continuously monitor tumor pathophysiology (including hypoxia). The aim of this preliminary work was to develop and evaluate BOLD MRI followed by post-image analysis to identify regions of hypoxia in a murine glioblastoma (GBM) model. METHODS: A murine orthotopic GBM model (GL261-luc2) was used and independent images were generated from multiple slices in four different mice. Image slices were randomized and split into training and validation cohorts. A 7 T MRI was used to acquire anatomical images using a fast-spin-echo (FSE) T2-weighted sequence. BOLD images were taken with a T2*-weighted gradient echo (GRE) and an oxygen challenge. Thirteen images were evaluated in a training cohort to develop the MRI sequence and optimize post-image analysis. An in-house MATLAB code was used to evaluate MR images and generate hypoxia maps for a range of thresholding and ΔT2* values, which were compared against respective pimonidazole sections to optimize image processing parameters. The remaining (n = 6) images were used as a validation group. Following imaging, mice were injected with pimonidazole and collected for immunohistochemistry (IHC). A test of correlation (Pearson's coefficient) and agreement (Bland-Altman plot) were conducted to evaluate the respective MRI slices and pimonidazole IHC sections. RESULTS: For the training cohort, the optimized parameters of "thresholding" (20 ≤ T2* ≤ 35 ms) and ΔT2* (±4 ms) yielded a Pearson's correlation of 0.697. These parameters were applied to the validation cohort confirming a strong Pearson's correlation (0.749) when comparing the respective analyzed MR and pimonidazole images. CONCLUSION: Our preliminary study supports the hypothesis that BOLD MRI is correlated with pimonidazole measurements of hypoxia in an orthotopic GBM mouse model. This technique has further potential to monitor hypoxia during tumor development and therapy.

Rotigaptide Infusion for the First 7 Days After Myocardial Infarction-Reperfusion Reduced Late Complexity of Myocardial Architecture of the Healing Border-Zone and Arrhythmia Inducibility.

Background Survivors of myocardial infarction are at increased risk of late ventricular arrhythmias, with infarct size and scar heterogeneity being key determinants of arrhythmic risk. Gap junctions facilitate the passage of small ions and morphogenic cell signaling between myocytes. We hypothesized that gap junctions enhancement during infarction-reperfusion modulates structural and electrophysiological remodeling and reduces late arrhythmogenesis. Methods and Results Infarction-reperfusion surgery was carried out in male Sprague-Dawley rats followed by 7 days of rotigaptide or saline administration. The in vivo and ex vivo arrhythmogenicity was characterized by programmed electrical stimulation 3 weeks later, followed by diffusion-weighted magnetic resonance imaging and Masson's trichrome histology. Three weeks after 7-day postinfarction administration of rotigaptide, ventricular tachycardia/ventricular fibrillation was induced on programmed electrical stimulation in 20% and 53% of rats, respectively (rotigaptide versus control), resulting in reduction of arrhythmia score (3.2 versus 1.4, P=0.018), associated with the reduced magnetic resonance imaging parameters fractional anisotropy (fractional anisotropy: -5% versus -15%; P=0.062) and mean diffusivity (mean diffusivity: 2% versus 6%, P=0.042), and remodeling of the 3-dimensional laminar structure of the infarct border zone with reduction of the mean (16° versus 19°, P=0.013) and the dispersion (9° versus 12°, P=0.015) of the myofiber transverse angle. There was no change in ECG features, spontaneous arrhythmias, or mortality. Conclusions Enhancement of gap junctions function by rotigaptide administered during the early healing phase in reperfused infarction reduces later complexity of infarct scar morphology and programmed electrical stimulation-induced arrhythmias, and merits further exploration as a feasible and practicable intervention in the acute myocardial infarction management to reduce late arrhythmic risk.