Mark D. Bednarski

VEGF Gene Delivery to Muscle: Potential Role for Vasculogenesis in Adults

Constitutive expression of VEGF after implantation of genetically engineered myoblasts into non-ischemic muscle led to an increase in vascular structures. Previously, effects of VEGF delivery to adult muscle have only been reported in ischemic tissues. The resulting vascular structures were reminiscent of those formed during embryonic vasculogenesis, rather than angiogenesis, sprouting from preexisting vessels. Initially, VEGF caused an accumulation of endothelial cells and macrophages, followed by networks of vascular channels and hemangiomas with locally high serum VEGF levels. No effects were evident in adjacent tissue or contralateral legs, where low serum VEGF was detected. These data suggest that the induction by VEGF of angiogenesis or vasculogenesis may be dose-dependent. Furthermore, VEGF expression must be carefully modulated, as overexpression is deleterious.

Apparent Diffusion Coefficient: a quantitative parameter for in vivo tumor characterization

PURPOSEnThe purpose of the this study was to evaluate the potential of diffusion weighted imaging (DWI) to distinguish different tissue compartments in early, intermediate and advanced tumor stages.nnnMATERIALS AND METHODSnTwenty-two male mice were induced with squamous cell tumor (SCCVII) and scanned with a clinical 1.5 T scanner. T1-SE, T2-FSE, diffusion weighted Line-Scan-MRI and contrast enhanced T1-SE were obtained from mice with early (tumor volume 10-100 mm(3)), intermediate (200-600 mm(3)), advanced tumors (600-1000 mm(3)) and tumor necrosis (>1500 mm(3)). The apparent diffusion coefficient (ADC) of different tumor compartments was calculated offline with a pixel-by-pixel method. The animals were sacrificed immediately after scanning and histopathologic correlation was performed.nnnRESULTSnIn early stages of tumor development, tumors appeared homogeneous on diffusion weighted images with an ADC of 0.64+/-0.06 x 10(-3) mm(2)/s. With tumor progression the ADC in the rim areas of tumor increased significantly (intermediate stage: 0.70+/-0.11 x 10(-3) mm(2)/s; advanced stage: 0.88+/-0.11 x 10(-3) mm(2)/s; tumor necrosis 1.03+/-0.06 x 10(-3) mm(2)/s), whereas the ADC in viable tumor remained constant. Histologically the areas with an increased ADC correlated well with areas of necrosis (reduced cell density).nnnCONCLUSIONnThe ADC is a non-invasive technique to monitor changes in the biological structure of tumor tissue during tumor progression. Thus, DWI is a potential diagnostic tool for in-vivo tissue characterization.

Angiogenesis Is Required for Successful Bone Induction During Distraction Osteogenesis

The role of angiogenesis during mechanically induced bone formation is incompletely understood. The relationship between the mechanical environment, angiogenesis, and bone formation was determined in a rat distraction osteogenesis model. Disruption of either the mechanical environment or endothelial cell proliferation blocked angiogenesis and bone formation. This study further defines the role of the mechanical environment and angiogenesis during distraction osteogenesis.

ICAM-1 expression in autoimmune encephalitis visualized using magnetic resonance imaging

The expression of leukocyte adhesion molecules in the intact brains of mice with experimental autoimmune encephalitis (EAE) was visualized by Magnetic Resonance Imaging (MRI) through the use of a new, target-specific MR contrast agent. Antibody-conjugated paramagnetic liposomes (ACPLs) were designed to achieve in vivo targeting of molecules expressed on vascular endothelium, while providing sufficient signal enhancement at these sites for detection by MRI. ACPLs targeted to intercellular adhesion molecule-1 (ICAM-1), an endothelial leukocyte receptor upregulated on cerebral microvasculature during EAE, were administered to diseased mice. Fluorescence microscopy confirmed that fluorescently-tagged ACPLs were localized to central nervous system (CNS) microvasculature in a pattern consistent with ICAM-1 upregulation described immunohistochemically. High resolution MRI of mouse brains ex vivo demonstrated that ACPL binding conferred significant enhancement of signal intensity (SI) as compared to control images. These results suggest that ACPLs can be used as MRI contrast agents to visualize specific molecules expressed on vascular endothelium during disease.

Magnetic resonance image–guided proteomics of human glioblastoma multiforme

To investigate the correlation between gadolinium contrast‐enhancement patterns on T1‐weighted magnetic resonance (MR) images and spatial changes in protein expression profiles in human glioblastoma multiforme (GBM) and the use of imaging as a noninvasive technique to evaluate the heterogeneity of solid tumors prior to microarray analysis.

Dinitroazetidines Are a Novel Class of Anticancer Agents and Hypoxia-Activated Radiation Sensitizers Developed from Highly Energetic Materials

In an effort to develop cancer therapies that maximize cytotoxicity, while minimizing unwanted side effects, we studied a series of novel compounds based on the highly energetic heterocyclic scaffold, dinitroazetidine. In this study, we report the preclinical validation of 1-bromoacetyl-3,3-dinitroazetidine (ABDNAZ), a representative lead compound currently in a phase I clinical trial in patients with cancer. In tumor cell culture, ABDNAZ generated reactive free radicals in a concentration- and time-dependent manner, modulating intracellular redox status and triggering apoptosis. When administered to mice as a single agent, ABDNAZ exhibited greater cytotoxicity than cisplatin or tirapazamine under hypoxic conditions. However, compared with cisplatin, ABDNAZ was better tolerated at submaximal doses, yielding significant tumor growth inhibition in the absence of systemic toxicity. Similarly, when combined with radiation, ABDNAZ accentuated antitumor efficacy along with the therapeutic index. Toxicity studies indicated that ABDNAZ was not myelosuppressive and no dose-limiting toxicity was apparent following daily administration for 14 days. Taken together, our findings offer preclinical proof-of-concept for ABDNAZ as a promising new anticancer agent with a favorable toxicity profile, either as a chemotherapeutic agent or a radiosensitizer.

Vascular-targeted molecular imaging using functionalized polymerized vesicles.

In this review we will discuss the use of multivalent polymerized vesicles (PVs) combined with magnetic resonance imaging (MRI) and gamma scintigraphy to image expression of vascular molecular receptors in vivo. Specifically, we will present our data on the use of this technology to design imaging agents toward specific vascular receptors in both a mouse and rabbit tumor model and in the EAE mouse, a model for human multiple sclerosis (MS). Examples will be shown where the in vivo specificity of the targeted molecular imaging agents was validated in the animal models. Since the PVs are designed to carry either contrast or therapeutic agents or both, we can potentially use vascular‐targeted imaging for selecting patients and guiding vascular‐targeted therapies in these patients. Using this combined vascular‐targeted imaging and therapy approach, personalized treatment can potentially be delivered, maximizing efficacy and minimizing side effects. J. Magn. Reson. Imaging 2002;16:388–393. Published 2002 Wiley‐Liss, Inc.

Combined vascular targeted imaging and therapy: a paradigm for personalized treatment.

In order to be successful in personalizing treatment, methods for selecting patients as well as good surrogate biomarkers for monitoring the effects of treatment are required in addition to development of an efficacious targeted therapy. We have developed a polymerized nanoparticle platform technology that will allow us to put different targeting moieties on the surface of the particles in addition to loading the particles with different contrast and therapeutic agents. We have proven that these nanoparticles can be targeted to endothelial receptors and different payloads of contrast and therapeutic agents have been delivered to target cells with high target to background ratios. Using this combined vascular targeted imaging and therapy approach, we are optimistic that personalized treatment regimens can be developed for different disease processes such as cancer, inflammation, and ischemia. J. Cell. Biochem. Suppl. 39: 65–71, 2002.

Comparing genomic and histologic correlations to radiographic changes in tumors

RATIONALE AND OBJECTIVESnTo investigate the correlation between the temporal changes in T1- and T2-weighted contrast-enhanced magnetic resonance imaging (MRI), histologic evaluation, and genomic analysis using oligonucleotide microarrays in a murine squamous cell carcinoma tumor models.nnnMATERIALS AND METHODSnThe squamous cell carcinoma (SCC VII) cell line was used to initiate subcutaneous tumors in mice. This mouse model has been used as a model for human head and neck carcinomas. Animals were imaged using contrast enhanced MRI (CE-MRI). Different stages of tumor growth were defined based on changes in the T1- and T2-weighted MRI patterns. The contrast enhancing (CE) and nonenhancing (NE) regions of the tumors were marked and biopsied for oligonucleotide microarray and histologic analysis. Tumors with no differential contrast enhancement were used as controls.nnnRESULTSnDistinct temporal stages of tumor progression can be defined using both T1- and T2-weighted CE-MRI and microarray analysis. The early stage tumors show a homogeneous contrast enhancement pattern in the T1- and T2-weighted images with no significant differential gene expression from the center and periphery of the tumor. The more advanced tumors that show discrete regions of contrast enhancement in the post-contrast T1-weighted MRIs and tissues from the CE and NE regions show distinctly differential gene expression profiles. Histologic analysis (hematoxylin-eosin stain) showed that the samples obtained from the periphery and center of the early stage tumors and the CE and NE regions from these more advanced tumors were similar. The gene expression profiles of late-stage tumors that showed changes in T2-weighted MRI signal intensity were consistent with tissue degradation in the NE region, which also showed characteristic signs of tissue necrosis in histologic analysis.nnnCONCLUSIONnThese results show that temporal changes in T1- and T2-weighted CE-MRI are related to distinct gene expression profiles, and histologic analysis may not be sufficient to detect these detailed changes. As tumors progress, discrete regions of post-contrast T1 enhancement are identified; these regions have distinct gene expression patterns despite similar histologic features. In late-stage tumors, regions of T2 signal changes are observed which correspond with tissue necrosis.

Gene expression profiles, histologic analysis, and imaging of squamous cell carcinoma model treated with focused ultrasound beams.

OBJECTIVEnThe purpose of our study was to evaluate the effect of short-pulse high-intensity focused ultrasound (HIFU) on inducing cell death in a head and neck cancer model (SCCVII [squamous cell carcinoma]) compared with continuous HIFU to get a better understanding of the biologic changes caused by HIFU therapy.nnnMATERIALS AND METHODSnHIFU was applied to 12 SCCVII tumors in C3H/Km mice using a dual sonography system (imaging, 6 MHz; therapeutic, 1 MHz). A continuous HIFU mode (total time, 20 seconds; intensity, 6,730.6 W/cm2) and a short-pulse HIFU mode (frequency, 0.5 Hz; pulse duration, 50 milliseconds; total time, 16.5 minutes; intensity, 134.4 W/cm2) was applied. Three hours later, MR images were obtained on a 1.5-T scanner. After imaging, the treated and untreated control tumor tissue samples were taken out for histology and oligonucleotide microarray analysis.nnnRESULTSnProminent changes were observed in the MR images in the continuous HIFU mode, whereas the short-pulse HIFU mode showed no discernible changes. Histology (H and E, TUNEL [terminal deoxynucleotidyl transferase-mediated dUTP {deoxyuridine triphosphate} nick-end labeling], and immunohistochemistry) of the tumors treated with the continuous HIFU mode revealed areas of significant necrosis. In the short-pulse HIFU mode, the H and E staining showed multifocal areas of coagulation necrosis. TUNEL staining showed a high apoptotic index in both modes. Gene expression analysis revealed profound differences. In the continuous HIFU mode, 23 genes were up-regulated (> twofold change) and five genes were down-regulated (< twofold change), and in the short-pulse HIFU mode, 32 different genes were up-regulated and 16 genes were down-regulated.nnnCONCLUSIONnGenomic analysis might be included when investigating tissue changes after interventional therapy because it offers the potential to find molecular targets for imaging and therapeutic applications.