Peptide-Functionalized Nanoinhibitor Restrains Brain Tumor Growth by Abrogating Mesenchymal-Epithelial Transition Factor (MET) Signaling.

Malignant gliomas are the most common primary brain tumors and are associated with aggressive growth, high morbidity, and mortality. Aberrant mesenchymal-epithelial transition factor (MET) activation occurs in approximately 30% of glioma patients and correlates with poor prognosis, elevated invasion, and increased drug resistance. Therefore, MET has emerged as an attractive target for glioma therapy. In this study, we developed a novel nanoinhibitor by conjugating MET-targeting cMBP peptides on the G4 dendrimer. Compared to the binding affinity of the free peptide ( KD = 3.96 × 10-7 M), the binding affinity of the nanoinhibitor to MET increased 3 orders of magnitude to 1.32 × 10-10 M. This nanoinhibitor efficiently reduced the proliferation and invasion of human glioblastoma U87MG cells in vitro by blocking MET signaling with remarkably attenuated levels of phosphorylated MET ( pMET) and its downstream signaling proteins, such as pAKT and pERK1/2. Although no obvious therapeutic effect was observed after treatment with free cBMP peptide, in vivo T2-weighted magnetic resonance imaging (MRI) showed a significant delay in tumor growth after intravenous injection of the nanoinhibitor. The medium survival in mouse models was extended by 59%, which is similar to the effects of PF-04217903, a small molecule MET inhibitor currently in clinical trials. Immunoblotting studies of tumor homogenate verified that the nanoinhibitor restrained glioma growth by blocking MET downstream signaling. pMET and its downstream proteins pAKT and pERK1/2, which are involved in the survival and invasion of cancer cells, decreased in the nanoinhibitor-treated group by 44.2%, 62.2%, and 32.3%, respectively, compared with those in the control group. In summary, we developed a peptide-functionalized MET nanoinhibitor that showed extremely high binding affinity to MET and effectively inhibited glioma growth by blocking MET downstream signaling. To the best of our knowledge, this is the first report of therapeutic inhibition of glioma growth by blocking MET signaling with a novel nanoinhibitor. Compared to antibodies and chemical inhibitors in clinical trials, the nanoinhibitor blocks MET signaling and provides a new approach for the treatment of glioma with the advantages of high efficiency, affordability, and, most importantly, potentially reduced drug resistance.

Manganese G8 dendrimers targeted to oxidation-specific epitopes: in vivo MR imaging of atherosclerosis.

PURPOSE: To determine if manganese (Mn) G8 dendrimers targeted to oxidation-specific epitopes (OSE) allow for in vivo detection of atherosclerotic lesions. MATERIALS AND METHODS: OSE have been identified as key factors in atherosclerotic plaque progression and destabilization. Mn offers a potentially clinically translatable alternative to gadolinium-based agents when bioretention and potential toxicity of gadolinium is anticipated. However, to be effective, high payloads of Mn must accumulate intracellularly in macrophages. It was hypothesized that G8 dendrimers targeted to OSE may allow delivery of high Mn payloads, thereby enabling in vivo detection of macrophage-rich plaques. G8 dendrimers were modified to allow conjugation with MnDTPA (758 Mn ion) and the antibody MDA2 that is targeted to malondialdehyde (MDA)-lysine epitopes. Both the untargeted and targeted G8 dendrimers were characterized and their in vivo efficacy evaluated in apoE(-/-) mice over a 96-hour time period after bolus administration of a 0.05 mmol Mn/kg dose using a clinical MR system (3T). RESULTS: Significant enhancement (normalized enhancement >60%, P = 0.0013) of atherosclerotic lesions was observed within a 72-hour time period following administration of the targeted dendrimers. The presence of Mn within atherosclerotic lesions was confirmed using spectroscopic methods (>8 µg Mn/g). Limited signal attenuation (<18%) and Mn deposition (<1 µg Mn/g) was observed in the arterial wall following injection of the untargeted material. CONCLUSION: This study demonstrates that manganese-labeled dendrimers, allowing a high Mn payload, targeted to OSE may allow in vivo image of atherosclerotic lesions.

Inflammatory bowel disease: MR- and SPECT/CT-based macrophage imaging for monitoring and evaluating disease activity in experimental mouse model--pilot study.

PURPOSE: To evaluate the feasibility of using magnetic resonance (MR) imaging and single photon emission computed tomography (SPECT)/computed tomography (CT) to visualize the in vivo recruitment of iron oxide-labeled macrophages and indium 111 ((111)In)-labeled macrophages in inflammatory bowel disease (IBD) and to monitor disease activity. MATERIALS AND METHODS: This study had institutional animal care and use committee approval. Twenty-seven C57/B6 mice with dextran sodium sulfate (DSS)-induced IBD and control mice were included. Peritoneal macrophages were harvested from seven thioglycollate-treated mice and were labeled with superparamagnetic iron oxide (SPIO) nanoparticles. Macrophage iron content was determined by using inductively coupled plasma mass spectrometry. SPIO nanoparticle-labeled macrophages (5 × 10(6)) were intravenously administered. Mice with DSS-induced IBD (n = 8) and control mice (n = 6) were imaged with a 9.4-T MR imaging unit at 0, 5, and 24 hours after macrophage administration. Percentage normalized enhancement (NE) was calculated for the intestinal wall and liver 24 hours after injection. Six mice with IBD coinjected with SPIO nanoparticles and (111)In oxine-labeled macrophages were imaged with MR imaging and SPECT/CT after 24 hours. The pharmacokinetics and biodistribution of the implanted macrophages were determined. Correlation between percentage NE and IBD scores was calculated. RESULTS: Ex vivo mass spectrometry revealed strong SPIO nanoparticle uptake (7.4 pg iron per cell). R2* correlated with cell number (r = 0.9813, P < .05). Percentage NE correlated with both clinical (r = 0.924) and pathologic (r = 0.795) IBD score. Cell circulation half-life in the first and second phases was 0.32 hour and 10.2 hours, respectively. SPECT/CT showed that approximately 3% of the injected dose was present in the intestines 24 hours after injection; this was confirmed at MR imaging and histologic examination. Indium 111-labeled cells were present in all tissue associated with the reticuloendothelial system or mononuclear phagocyte system at 24 hours. CONCLUSION: SPIO nanoparticles and (111)In-labeled macrophages could be observed in vivo at MR imaging and SPECT/CT in mice with IBD. Percentage NE at MR imaging correlates with disease activity.

The complex fate in plasma of gadolinium incorporated into high-density lipoproteins used for magnetic imaging of atherosclerotic plaques.

We have previously reported enhancing the imaging of atherosclerotic plaques in mice using reconstituted high density lipoproteins (HDL) as nanocarriers for the MRI contrast agent gadolinium (Gd). This study focuses on the underlying mechanisms of Gd delivery to atherosclerotic plaques. HDL, LDL, and VLDL particles containing Gd chelated to phosphatidyl ethanolamine (DTPA-DMPE) and a lipidic fluorophore were used to demonstrate the transfer of Gd-phospholipids among plasma lipoproteins in vitro and in vivo. To determine the basis of this transfer, the roles of phospholipid transfer protein (PLTP) and lipoprotein lipase (LpL) in mediating the migration of Gd-DTPA-DMPE among lipoproteins were investigated. The results indicated that neither was an important factor, suggesting that spontaneous transfer of Gd-DTPA-DMPE was the most probable mechanism. Finally, two independent mouse models were used to quantify the relative contributions of HDL and LDL reconstituted with Gd-DTPA-DMPE to plaque imaging enhancement by MR. Both sets of results suggested that Gd-DTPA-DMPE originally associated with LDL was about twice as effective as that injected in the form of Gd-HDL, and that some of Gd-HDL's effectiveness in vivo is indirect through transfer of the imaging agent to LDL. In conclusion, the fate of Gd-DTPA-DMPE associated with a particular type of lipoprotein is complex, and includes its transfer to other lipoprotein species that are then cleared from the plasma into tissues.

A versatile and tunable coating strategy allows control of nanocrystal delivery to cell types in the liver.

There are many liver diseases that could be treated with delivery of therapeutics such as DNA, proteins, or small molecules. Nanoparticles are often proposed as delivery vectors for such therapeutics; however, achieving nanoparticle accumulations in the therapeutically relevant hepatocytes is challenging. In order to address this issue, we have synthesized polymer coated, fluorescent iron oxide nanoparticles that bind and deliver DNA, as well as produce contrast for magnetic resonance imaging (MRI), fluorescence imaging, and transmission electron microscopy (TEM). The composition of the coating can be varied in a facile manner to increase the quantity of poly(ethylene glycol) (PEG) from 0% to 5%, 10%, or 25%, with the aim of reducing opsonization but maintaining DNA binding. We investigated the effect of the nanoparticle coating on DNA binding, cell uptake, cell transfection, and opsonization in vitro. Furthermore, we exploited MRI, fluorescence imaging, and TEM to investigate the distribution of the different formulations in the liver of mice. While MRI and fluorescence imaging showed that each formulation was heavily taken up in the liver at 24 h, the 10% PEG formulation was taken up by the therapeutically relevant hepatocytes more extensively than either the 0% PEG or the 5% PEG, indicating its potential for delivery of therapeutics to the liver.

The cardiomyocyte lineage is critical for optimization of stem cell therapy in a mouse model of myocardial infarction.

We recently described a murine embryonic stem cell (ESC) line engineered to express the activated Notch 4 receptor in a tetracycline (doxcycline; Dox) regulated fashion (tet-notch4 ESCs). Notch 4 induction in Flk1(+) hematopoietic and vascular progenitors from this line respecified them to a cardiovascular fate. We reasoned that these cells would be ideal for evaluating the contribution of the cardiomyocyte and vascular lineages to the functional improvement noted following stem cell transplantation in infarcted hearts. Flk-1(+) Tet-notch4 cells from d 3 embryoid bodies exposed to doxycycline (Dox(+)) were compared to uninduced (Dox(-)) Flk-1(+) cells. Mice underwent transplantation of 5 x 10(5) Dox(+) cells, Dox(-)cells, or an equal volume of serum-free medium after surgically induced myocardial infarction. The mean ejection fraction was 59 + or - 15, 46 + or - 17, and 39 + or - 13% in the Dox(+), Dox(-), and serum-free medium groups, respectively (P<0.05 for the differences among all 3 groups). Immunohistochemistry of hearts injected with Dox(+) grafts expressed myocardial and vascular markers, whereas grafts of Dox(-) cells expressed primarily vascular markers. We conclude that cardiovascular progenitors are more effective than vascular progenitors in improving function after myocardial infarction. The transplantation of appropriate cell types is critical for maximizing the benefit of cardiovascular cell therapy.-Adler, E. D., Chen, V. C., Bystrup, A., Kaplan, A. D., Giovannone, S., Briley-Saebo, K., Young, W., Kattman, S., Mani, V., Laflamme, M., Zhu, W.-Z., Fayad, Z., Keller, G. The cardiomyocyte lineage is critical for optimization of stem cell therapy in a mouse model of myocardial infarction.

Longitudinal tracking of human dendritic cells in murine models using magnetic resonance imaging.

Ex vivo generated dendritic cells are currently used to induce therapeutic immunity in solid tumors. Effective immune response requires dendritic cells to home and remain in lymphoid organs to allow for adequate interaction with T lymphocytes. The aim of the current study was to detect and track Feridex labeled human dendritic cells in murine models using magnetic resonance imaging. Human dendritic cells were incubated with Feridex and the effect of labeling on dendritic cells immune function was evaluated. Ex vivo dendritic cell phantoms were used to estimate sensitivity of the magnetic resonance methods and in vivo homing was evaluated after intravenous or subcutaneous injection. R2*-maps of liver, spleen, and draining lymph nodes were obtained and inductively coupled plasma mass spectrometry or relaxometry methods were used to quantify the Feridex tissue concentrations. Correlations between in vivo R2* values and iron content were then determined. Feridex labeling did not affect dendritic cell maturation or function. Phantom results indicated that it was possible to detect 125 dendritic cells within a given slice. Strong correlation between in vivo R2* values and iron deposition was observed. Importantly, Feridex-labeled dendritic cells were detected in the spleen for up to 2 weeks postintravenous injection. This study suggests that magnetic resonance imaging may be used to longitudinally track Feridex-labeled human dendritic cells for up to 2 weeks after injection.

Targeted molecular probes for imaging atherosclerotic lesions with magnetic resonance using antibodies that recognize oxidation-specific epitopes.

BACKGROUND: Oxidized low-density lipoprotein plays a key role in the initiation, progression, and destabilization of atherosclerotic plaques and is present in macrophages and the lipid pool. The aim of this study was to assess the feasibility of magnetic resonance imaging of atherosclerotic lesions in mice using micelles containing gadolinium and murine (MDA2 and E06) or human (IK17) antibodies that bind unique oxidation-specific epitopes. METHODS AND RESULTS: MDA2 micelles, E06 micelles, IK17 micelles, nonspecific IgG micelles, and untargeted micelles (no antibody) were prepared and characterized with respect to pharmacokinetics and biodistribution in wild-type and atherosclerotic apolipoprotein E-deficient (apoE(-/-)) mice. Magnetic resonance imaging was performed at 9.4 T over a 96-hour time interval after the administration of 0.075-mmol Gd/kg micelles. MDA2, E06, and IK17 micelles exhibited a longer plasma half-life than IgG or untargeted micelles in apoE(-/-) but not wild-type mice. In apoE(-/-) mice, MDA2 and IK17 micelles showed maximal arterial wall uptake at 72 hours and E06 micelles at 96 hours, manifested by 125% to 231% enhancement in magnetic resonance signal compared with adjacent muscle. Confocal microscopy revealed that MDA2, IK17, and E06 micelles accumulated within atherosclerotic lesions and specifically within macrophages. Intravenous injection of free MDA2 before imaging with MDA2 micelles resulted in significantly diminished magnetic resonance signal enhancement. IgG micelles and untargeted micelles showed minimal enhancement in apoE(-/-) mice. There was no significant signal enhancement with all micelles in wild-type mice. CONCLUSIONS: Magnetic resonance imaging with micelles containing gadolinium and oxidation-specific antibodies demonstrates specific targeting and excellent image quality of oxidation-rich atherosclerotic lesions.

Tyrosine polyethylene glycol (PEG)-micelle magnetic resonance contrast agent for the detection of lipid rich areas in atherosclerotic plaque.

Vulnerable or high-risk atherosclerotic plaques often exhibit large lipid cores and thin fibrous caps that can lead to deadly vascular events when they rupture. In this study, polyethylene glycol (PEG)-micelles that incorporate a gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) amphiphile were used as an MR contrast agent. In an approach inspired by lipoproteins, the micelles were functionalized with tyrosine residues, an aromatic, lipophilic amino acid, to reach the lipid-rich areas of atherosclerotic plaque in a highly efficient manner. These micelles were applied to apolipoprotein E(-/-) (ApoE(-/-)) mice as a model of atherosclerosis. The abdominal aortas of the animals were imaged using T(1)-weighted (T(1)W) high-resolution MRI at 9.4T before and up to 48 h after the administration of the micelles. PEG-micelles modified with 15% tyrosine residues yielded a significant enhancement of the abdominal aortic wall at 6 and 24 h postinjection (pi) as compared to unmodified micelles. Fluorescence microscopy on histological sections of the abdominal aorta showed a correlation between lipid-rich areas and the distribution of the functionalized contrast agent in plaque. Using a simple approach, we demonstrated that lipid-rich areas in atherosclerotic plaque of ApoE(-/-) mice can be detected by MRI using Gd-DTPA micelles.

Comparison of synthetic high density lipoprotein (HDL) contrast agents for MR imaging of atherosclerosis.

Determining arterial macrophage expression is an important goal in the molecular imaging of atherosclerosis. Here, we compare the efficacy of two synthetic, high density lipoprotein (HDL) based contrast agents for magnetic resonance imaging (MRI) of macrophage burden. Each form of HDL was labeled with gadolinium and rhodamine to allow MRI and fluorescence microscopy. Either the 37 or 18 amino acid peptide replaced the apolipoprotein A-I in these agents, which were termed 37pA-Gd or 18A-Gd. The diameters of 37pA-Gd and 18A-Gd are 7.6 and 8.0 nm, respectively, while the longitudinal relaxivities are 9.8 and 10.0 (mM s)(-1). 37pA has better lipid binding properties. In vitro tests with J774A.1 macrophages proved the particles possessed the functionality of HDL by eliciting cholesterol efflux and were taken up in a receptor-like fashion by the cells. Both agents produced enhancements in atherosclerotic plaques of apolipoprotein E knockout mice of approximately 90% (n = 7 per agent) and are macrophage specific as evidenced by confocal microscopy on aortic sections. The half-lives of 37pA-Gd and 18A-Gd are 2.6 and 2.1 h, respectively. Despite the more favorable lipid interactions of 37pA, both agents gave similar, excellent contrast for the detection of atherosclerotic macrophages using MRI.

High-relaxivity gadolinium-modified high-density lipoproteins as magnetic resonance imaging contrast agents.

There is an ongoing desire to produce high-relaxivity, Gd-based magnetic resonance imaging (MRI) contrast agents. These may allow for lower doses to be used, which is especially important in view of the current safety concerns surrounding Gd in patients. Here we report the synthesis of a high-relaxivity MRI contrast agent, by incorporating Gd-chelating lipids that coordinate two water molecules into high-density lipoprotein (q = 2 HDL). We compared the properties of q = 2 HDL with those of an analogous HDL particle labeled with Gd-chelating lipids that coordinate only one water molecule (q = 1 HDL). We found that the q = 2 HDL possessed an elevated r(1) of 41 mM(-1) s(-1) compared to 9 mM(-1) s(-1) for q = 1 HDL at 20 MHz, but the q = 2 HDL exhibited high R(2)* values at high fields, precluding imaging above 128 MHz. While carrying out this investigation we observed that enlarged, disrupted particles were formed when the synthesis was carried out above the lipid critical micelle concentration (cmc), indicating the importance of synthesis below the cmc when modifying lipoproteins in this manner. The high relaxivity of q = 2 HDL means it will be an efficacious contrast agent for future MR imaging studies.

Detection of neovessels in atherosclerotic plaques of rabbits using dynamic contrast enhanced MRI and 18F-FDG PET.

OBJECTIVE: The association of inflammatory cells and neovessels in atherosclerosis is considered a histological hallmark of high-risk active lesions. Therefore, the development and validation of noninvasive imaging techniques that allow for the detection of inflammation and neoangiogenesis in atherosclerosis would be of major clinical interest. Our aim was to test 2 techniques, black blood dynamic contrast enhanced MRI (DCE-MRI) and 18-fluorine-fluorodeoxyglucose (18F-FDG) PET, to quantify inflammation expressed as plaque neovessels content in a rabbit model of atherosclerosis. METHODS AND RESULTS: Atherosclerotic plaques were induced in the aorta of 10 rabbits by a combination of 2 endothelial abrasions and 4 months hyperlipidemic diet. Six rabbits underwent MRI during the injection of Gd-DTPA, whereas 4 rabbits were imaged after injection of 18F-FDG with PET. We found a positive correlation between neovessels count in atherosclerotic plaques and (1) Gd-DTPA uptake parameters evaluated by DCE-MRI (r=0.89, P=0.016) and (2) 18F-FDG uptake evaluated by PET (r=0.5, P=0.103 after clustered robust, Huber-White, standard errors analysis). CONCLUSIONS: DCE-MRI and 18F-FDG PET may allow for the evaluation of inflammation in atherosclerotic plaques of rabbits. These noninvasive imaging modalities could be proposed as clinical tools in the evaluation of lesion prognosis and monitoring of anti-angiogenic therapies.

An ApoA-I mimetic peptide high-density-lipoprotein-based MRI contrast agent for atherosclerotic plaque composition detection.

Cardiovascular disease is one of the prime causes of mortality throughout the world and there is a need for targeted and effective contrast agents to allow noninvasive imaging of the cholesterol-rich atherosclerotic plaques in arteries. A new, fully synthetic, high-density lipoprotein (HDL)-mimicking MRI contrast agent is developed, which enhances macrophage-rich areas of plaque in a mouse model of atherosclerosis by 94%. Confirmation of the targeting of this nanoparticulate agent is achieved using confocal microscopy by tracking a fluorescent lipid incorporated into the nanoparticle.

Clearance of iron oxide particles in rat liver: effect of hydrated particle size and coating material on liver metabolism.

OBJECTIVES: We sought to evaluate the effect of the particle size and coating material of various iron oxide preparations on the rate of rat liver clearance. MATERIALS AND METHODS: The following iron oxide formulations were used in this study: dextran-coated ferumoxide (size = 97 nm) and ferumoxtran-10 (size = 21 nm), carboxydextran-coated SHU555A (size = 69 nm) and fractionated SHU555A (size = 12 nm), and oxidized-starch coated materials either unformulated NC100150 (size = 15 nm) or formulated NC100150 injection (size = 12 nm). All formulations were administered to 165 rats at 2 dose levels. Quantitative liver R2* values were obtained during a 63-day time period. The concentration of iron oxide particles in the liver was determined by relaxometry, and these values were used to calculate the particle half-lives in the liver. RESULTS: After the administration of a high dose of iron oxide, the half-life of iron oxide particles in rat liver was 8 days for dextran-coated materials, 10 days for carboxydextran materials, 14 days for unformulated oxidized-starch, and 29 days for formulated oxidized-starch. CONCLUSIONS: The results of the study indicate that materials with similar coating but different sizes exhibited similar rates of liver clearance. It was, therefore, concluded that the coating material significantly influences the rate of iron oxide clearance in rat liver.

Intravascular contrast agent-enhanced MRI measuring contrast clearance and tumor blood volume and the effects of vascular modifiers in an experimental tumor.

PURPOSE: To examine the feasibility of using the MRI blood pool agent NC100150 for evaluation of tumor blood volume (TBV) estimates by both dynamic contrast-enhanced MRI (DCE-MRI) and susceptibility contrast MRI assays in an experimental tumor. Contrast agent clearance (K(trans); depends on perfusion and permeability) from the DCE-MRI time curves was estimated, and changes in TBV and K(trans) were measured after administration of two drugs that reduce perfusion by different mechanisms. METHODS AND MATERIALS: The DCE-MRI experiments were simulated with expected physiologic values for the C3H mouse mammary carcinoma. The C3H tumor was examined by DCE-MRI and susceptibility contrast MRI with NC100150 (NC100150 Injection, Clariscan; Amersham Health, Oslo, Norway) after treatment with either hydralazine or combretastatin (Oxigene, Boston, MA). RESULTS: Simulations showed that reliable estimates of changes in TBV and K(trans) could be performed with DCE-MRI. Hydralazine was shown to reduce TBV as measured by both assays and to reduce K(trans). Dynamic contrast-enhanced MRI also suggested that TBV and K(trans) were reduced in combretastatin-treated tumors, and the TBV reduction was confirmed by susceptibility contrast MRI. Data suggested the drug to affect mainly the total TBV, whereas microvessels as such seemed less altered. CONCLUSION: The study supports the use of the combined DCE-MRI and susceptibility contrast MRI assay with a blood pool agent in characterizing tumors and their response to treatment.

Imaging of oxidation-specific epitopes with targeted nanoparticles to detect high-risk atherosclerotic lesions: progress and future directions.

Oxidation-specific epitopes (OSE) within developing atherosclerotic lesions are key antigens that drive innate and adaptive immune responses in atherosclerosis, leading to chronic inflammation. Oxidized phospholipids and malondialdehyde-lysine epitopes are well-characterized OSE present in human atherosclerotic lesions, particularly in pathologically defined vulnerable plaques. Using murine and human OSE-specific antibodies as targeting agents, we have developed radionuclide and magnetic resonance based nanoparticles, containing gadolinium, manganese or lipid-coated ultrasmall superparamagnetic iron oxide, to non-invasively image OSE within experimental atherosclerotic lesions. These methods quantitate plaque burden, allow detection of lesion progression and regression, plaque stabilization, and accumulation of OSE within macrophage-rich areas of the artery wall, suggesting they detect the most active lesions. Future studies will focus on using "natural" antibodies, lipopeptides, and mimotopes for imaging applications. These approaches should enhance the clinical translation of this technique to image, monitor, evaluate efficacy of novel therapeutic agents, and guide optimal therapy of high-risk atherosclerotic lesions.

Myeloid-derived suppressor cells as a vehicle for tumor-specific oncolytic viral therapy.

One of the several impediments to effective oncolytic virus therapy of cancer remains a lack of tumor-specific targeting. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells induced by tumor factors in tumor-bearing hosts. The biodistribution kinetics of MDSC and other immune cell types in a murine hepatic colon cancer model was investigated through the use of tracking markers and MRI. MDSCs were superior to other immune cell types in preferential migration to tumors in comparison with other tissues. On the basis of this observation, we engineered a strain of vesicular stomatitis virus (VSV), an oncolytic rhabdovirus that bound MDSCs and used them as a delivery vehicle. Improving VSV-binding efficiency to MDSCs extended the long-term survival of mice bearing metastatic colon tumors compared with systemic administration of wild-type VSV alone. Survival was further extended by multiple injections of the engineered virus without significant toxicity. Notably, direct tumor killing was accentuated by promoting MDSC differentiation towards the classically activated M1-like phenotype. Our results offer a preclinical proof-of-concept for using MDSCs to facilitate and enhance the tumor-killing activity of tumor-targeted oncolytic therapeutics.

Increased expression of oxidation-specific epitopes and apoptosis are associated with haptoglobin genotype: possible implications for plaque progression in human atherosclerosis.

OBJECTIVES: The purpose of this study was to test the hypothesis that increased oxidative stress is associated with apoptosis in human plaques with the haptoglobin (Hp) 2-2 genotype. BACKGROUND: Intraplaque hemorrhage releases free hemoglobin (Hb). Impaired Hb clearance induces oxidative stress leading to plaque progression. The binding of Hp to Hb attenuates iron-induced oxidative reactions. METHODS: Twenty-six human aortic plaques were Hp genotyped. Hp2-2 plaques (n = 13) were compared with control (Hp1-1/2-1) (n = 13). The iron grade was measured by Perl's staining. Immunostaining was used to detect oxidation-specific epitopes (OSEs) reflecting oxidized phospholipids and malondialdehyde-like epitopes. The percentages of apoptotic cells and apoptotic morphological features were quantified. DNA fragmentation and active caspase-3 were measured by in situ end-labeling and immunohistochemistry, respectively. RESULTS: In Hp2-2 plaques, iron content was increased (1.22 ± 0.15 vs. 0.54 ± 0.08; p < 0.0001) along with expression of oxidized phospholipid- (78.9 ± 5.8 vs. 38.8 ± 3.8; p < 0.0001), and malondialdehyde-like OSEs (93.9 ± 7.9 vs. 54.7 ± 3.9; p < 0.0001). The total percentages of apoptotic cells (11.9 ± 0.44 vs. 3.5 ± 0.28; p < 0.0001), nuclear fragmentation (11.8 ± 0.50 vs. 3.3 ± 0.26; p < 0.0001), nuclear condensation (10.9 ± 0.58 vs. 3.4 ± 0.20; p < 0.0001), chromatin margination (14.2 ± 0.57 vs. 6.5 ± 0.37; p < 0.0001), cytoplasmic blebs (1.6 ± 0.28 vs. 0.8 ± 0.14; p < 0.002), and eosinophilia (10.8 ± 0.74 vs. 4.2 ± 0.27; p < 0.0001) were increased in Hp2-2 plaques. Furthermore, DNA fragmentation (119.9 ± 1.40 vs. 57.5 ± 0.80; p < 0.001), and active caspase-3 density (84.7 ± 7.62 vs. 50.6 ± 7.49; p < 0.004) were increased in Hp2-2 plaques. Logistic regression analysis identified correlation between the percentage of apoptotic cells and the density of OSEs (r = 0.56; p < 0.003). CONCLUSIONS: These findings provide insights into genetic predisposition to oxidative stress and the relationship between OSEs and macrophage apoptosis that may explain advanced atherosclerosis in human Hp2-2 plaques.

In vivo detection of oxidation-specific epitopes in atherosclerotic lesions using biocompatible manganese molecular magnetic imaging probes.

OBJECTIVES: This study sought to evaluate the in vivo magnetic resonance imaging (MRI) efficacy of manganese [Mn(II)] molecular imaging probes targeted to oxidation-specific epitopes (OSE). BACKGROUND: OSE are critical in the initiation, progression, and destabilization of atherosclerotic plaques. Gadolinium [Gd(III)]-based MRI agents can be associated with systemic toxicity. Mn is an endogenous, biocompatible, paramagnetic metal ion that has poor MR efficacy when chelated, but strong efficacy when released within cells. METHODS: Multimodal Mn micelles were generated to contain rhodamine for confocal microscopy and conjugated with either the murine monoclonal IgG antibody MDA2 targeted to malondialdehyde (MDA)-lysine epitopes or the human single-chain Fv antibody fragment IK17 targeted to MDA-like epitopes ("targeted micelles"). Micelle formulations were characterized in vitro and in vivo, and their MR efficacy (9.4-T) evaluated in apolipoprotein-deficient (apoE(-/-)) and low-density lipoprotein receptor negative (LDLR(-/-)) mice (0.05 mmol Mn/kg dose) (total of 120 mice for all experiments). In vivo competitive inhibition studies were performed to evaluate target specificity. Untargeted, MDA2-Gd, and IK17-Gd micelles (0.075 mmol Gd/kg) were included as controls. RESULTS: In vitro studies demonstrated that targeted Mn micelles accumulate in macrophages when pre-exposed to MDA-LDL with ∼10× increase in longitudinal relativity. Following intravenous injection, strong MR signal enhancement was observed 48 to 72 h after administration of targeted Mn micelles, with colocalization within intraplaque macrophages. Co-injection of free MDA2 with the MDA2-Mn micelles resulted in full suppression of MR signal in the arterial wall, confirming target specificity. Similar MR efficacy was noted in apoE(-/-) and LDLR(-/-) mice with aortic atherosclerosis. No significant differences in MR efficacy were noted between targeted Mn and Gd micelles. CONCLUSIONS: This study demonstrates that biocompatible multimodal Mn-based molecular imaging probes detect OSE within atherosclerotic plaques and may facilitate clinical translation of noninvasive imaging of human atherosclerosis.

Imaging of Oxidation-Specific Epitopes in Atherosclerosis and Macrophage-Rich Vulnerable Plaques.

Oxidative stress, and in particular oxidation of lipoproteins, is a hallmark of atherosclerosis. Upon entry of lipoproteins into the vessel wall, a cascade of pro-atherogenic pathways is initiated whereby the reaction of reactive oxygen species with substrates amenable to oxidation, such as polyunsaturated fatty acids, generates a variety of oxidation-specific epitopes on lipoproteins, proteins in the vessel wall, and apoptotic macrophages. Several of these oxidation-specific epitopes have been well characterized and specific murine and fully human antibodies have been generated in our laboratory to detect them in the vessel wall. We have developed radionuclide, gadolinium and iron oxide based MRI techniques to noninvasively image oxidation-specific epitopes in atherosclerotic lesions. These approaches quantitate plaque burden and also allow detection of atherosclerosis regression and plaque stabilization. In particular, gadolinium micelles or lipid-coated ultrasmall superparamagnetic iron oxide particles containing oxidation-specific antibodies accumulate within macrophages in the artery wall, suggesting they may image the most unstable plaques. Translation of these approaches to humans may allow a sensitive technique to image and monitor high-risk atherosclerotic lesions and may guide optimal therapeutic interventions.