Ching Hsuan Tung

Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells

The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications. We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles. The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10–30 pg of superparamagnetic iron per cell. Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells. Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples. In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells.

Factor XIII Deficiency Causes Cardiac Rupture, Impairs Wound Healing, and Aggravates Cardiac Remodeling in Mice With Myocardial Infarction

Background— Identification of key molecular players in myocardial healing could lead to improved therapies, reduction of scar formation, and heart failure after myocardial infarction (MI). We hypothesized that clotting factor XIII (FXIII), a transglutaminase involved in wound healing, may play an important role in MI given prior clinical and mouse model data. Methods and Results— To determine whether a truly causative relationship existed between FXIII activity and myocardial healing, we prospectively studied myocardial repair in FXIII-deficient mice. All FXIII−/− and FXIII−/+ (FXIII activity <5% and 70%) mice died within 5 days after MI from left ventricular rupture. In contradistinction, FXIII−/− mice that received 5 days of intravenous FXIII replacement therapy had normal survival rates; however, cardiac MRI demonstrated worse left ventricular remodeling in these reconstituted FXIII−/− mice. Using a FXIII-sensitive molecular imaging agent, we found significantly greater FXIII activity in wild-type mice and FXIII−/− mice receiving supplemental FXIII than in FXIII−/− mice (P<0.05). In FXIII−/− but not in reconstituted FXIII−/− mice, histology revealed diminished neutrophil migration into the MI. Reverse transcriptase–polymerase chain reaction studies suggested that the impaired inflammatory response in FXIII−/− mice was independent of intercellular adhesion molecule and lipopolysaccharide-induced CXC chemokine, both important for cell migration. After MI, expression of matrix metalloproteinase-9 was 650% higher and collagen-1 was 53% lower in FXIII−/− mice, establishing an imbalance in extracellular matrix turnover and providing a possible mechanism for the observed cardiac rupture in the FXIII−/− mice. Conclusions— These data suggest that FXIII has an important role in murine myocardial healing after infarction.

In Vivo Imaging of β-Galactosidase Activity Using Far Red Fluorescent Switch

β-Galactosidase (β-gal) has been widely used as a transgene reporter enzyme, and several substrates are available for its in vitro detection. The ability to image β-gal expression in living animals would further extend the use of this reporter. Here we show that DDAOG, a conjugate of β-galactoside and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO), is not only a chromogenic β-gal substrate but that the cleavage product has far-red fluorescence properties detectable by imaging. Importantly, the cleavage substrate shows a 50-nm red shift, enabling its specific detection in a background of intact probe, a highly desirable feature for in vivo imaging. Specifically, we show that β-gal-expressing 9L gliomas are readily detectable by red fluorescence imaging in comparison with the native 9L gliomas. We furthermore show that herpes simplex virus amplicon-mediated LacZ gene transfer into tumors can be transiently and thus serially visualized over time. The results indicated that in vivo real-time detection of β-gal activity is possible by fluorescence imaging technology.

Molecular Imaging of Factor XIIIa Activity in Thrombosis Using a Novel, Near-Infrared Fluorescent Contrast Agent That Covalently Links to Thrombi

Background—Activated factor XIII (FXIIIa) mediates fibrinolytic resistance and is a hallmark of newly formed thrombi. In vivo imaging of FXIIIa activity could further elucidate the role of this molecule in thrombosis and other biological processes and aid in the clinical detection of acute thrombi. Methods and Results—An FXIIIa-sensitive near-infrared fluorescence imaging agent (A15) was engineered by conjugating a near-infrared fluorochrome to a peptide ligand derived from the amino terminus of &agr;2-antiplasmin. To evaluate the molecular specificity of A15 for FXIIIa, a control agent (C15) was also synthesized by modifying a single key glutamine residue in A15. Fluorescence imaging experiments with A15 demonstrated stronger thrombosis enhancement in human plasma clots in vitro (P < 0.001 versus C15 clots and other controls). A15 was found to be highly specific for the active site of FXIIIa and was covalently bound to fibrin. In vivo murine experiments with A15 demonstrated significant signal enhancement in acute intravascular thrombi (P <0.05 versus C15 group). Minimal A15 enhancement was seen in older aged thrombi (>24 hours), consistent with an expected decline of FXIIIa activity over time. Imaging results were confirmed on correlative histopathology and fluorescence microscopy. Conclusions—A15 is a novel optical imaging agent that is specifically crosslinked to fibrin by FXIIIa, permitting detection of FXIIIa activity in experimental thrombi in vivo. This agent should permit assessment of FXIIIa activity in a broad range of biological processes and could aid in the clinical diagnosis of acute thrombi.

Adenovirus-mediated expression of antisense urokinase plasminogen activator receptor and antisense cathepsin B inhibits tumor growth, invasion, and angiogenesis in gliomas.

We have shown previously that urokinase plasminogen activator receptor (uPAR) and cathepsin B are overexpressed during glioma progression, particularly at the leading edge of the tumor. In the present study, we simultaneously down-regulated uPAR and cathepsin B in SNB19 glioma cell monolayer or SNB19 spheroids using an adenoviral vector carrying antisense uPAR and antisense cathepsin B and a combination of these genes as determined by Western blot analysis. The Ad-uPAR-Cath B-infected cells revealed a marked reduction in tumor growth and invasiveness as compared with the parental and vector controls. In vitro and in vivo angiogenic assays demonstrated inhibition of capillary-like structure formation and microvessel formation after Ad-uPAR-Cath B infection of SNB19 cells when compared with Ad-cytomegalovirus (CMV)-infected or mock-infected controls. Furthermore, using a near infrared fluorescence probe, in vivo imaging for cathepsin B indicated low/undetectable levels of fluorescence after injection of the Ad-uPAR-Cath B construct into pre-established s.c. tumors as compared with Ad-CMV-treated and untreated tumors. The effect with bicistronic construct (Ad-uPAR-Cath B) was much higher than with single (Ad-uPAR/Ad-Cath B) constructs. These results indicate that the down-regulation of cathepsin B and uPAR plays a significant role in inhibiting tumor growth, invasion, and angiogenesis. Hence, the targeting of these two proteases may be a potential therapy for brain tumors and other cancers.

Near-Infrared Fluorescent Imaging of Cerebral Thrombi and Blood–Brain Barrier Disruption in a Mouse Model of Cerebral Venous Sinus Thrombosis

An intravital microscopy imaging method was developed to visualize active cerebral thrombus and blood–brain barrier (BBB) disruption using Near Infrared Fluorescent (NIRF) probes. A circular craniotomy was made in CD-1 mice. Thrombi were formed by applying 10%-FeCl3 to the entire exposed superior sagittal sinus (SSS, 5 mm), or to the posterior 2.5 mm of the SSS for 5 mins. Control animals were pretreated with heparin (50 U/kg) before thrombus induction. Three hours after thrombus formation, a FXIIIa-targeted NIRF imaging probe (A15) was intravenously injected, and the SSS was imaged by intravital microscopy. This was followed by injection of indocyanine green (ICG) to assess BBB permeability. The A15 optical probe bound to thrombus, and the fluorescent signal emitted by the bound agent corresponded well with histologically confirmed thrombus. A15 initially remained intravascular, followed by excretion and subsequent decrease in all tissues except for thrombus, where it was retained. The subsequent ICG was also intravascular immediately after injection, but then began to leak into the cerebral parenchyma at 3 to 5 mins. The sites of leakage were adjacent to thrombosed areas. Heparin pretreatment prevented thrombus formation and reduced ICG leakage significantly. This demonstrates the feasibility of simultaneous in vivo monitoring of thrombus and BBB permeability in an animal model of cerebral venous thrombosis.

Novel Factor XIII Probes for Blood Coagulation Imaging

Thrombosis plays a central role in most cardiovascular diseases, which include myocardial infarction, pulmonary embolism, and deep venous thrombosis. A better ability to diagnose these potentially life-threatening conditions is necessary to minimize the associated morbidity and mortality. Thrombosis starts with the activation of thrombin, which converts soluble fibrinogen to fibrin. Thrombin initiates fibrin assembly and the polymerized fibrin is further crosslinked by activated blood coagulation factor XIII (FX13), a transglutaminase. 7] FX13 is a key player in the final stages of blood coagulation; it provides the clot with sufficient stiffness and the necessary resistance against thrombolytic enzymes by incorporating plasmin inhibitors into the crosslinked clot. In the study described herein, we designed new thrombosisspecific diagnostic probes based on the FX13 transglutaminase activity and previously identified peptide substrates. The specific peptide sequence, N13QEQVSPLTLLK24, extracted from 2-antiplasmin ( 2AP), the primary inhibitor of plasmin-mediated fibrinolysis, 9] was used as the primary template for imaging probe synthesis. It has been shown previously that this peptide crosslinks with fibrin in a clot in a specific manner. FX13 catalyzes the formation of a covalent bond between the glutamine residue at position 14 in one fibrin chain and the amino group of a lysine residue in a different chain. The glutamine residue at position 16 is significantly less reactive than glutamine 14. We hypothesized that an effective imaging probe could be covalently attached to blood clots. In particular, two types of imaging probes were synthesized for imaging of blood coagulation: one is a near-infrared (NIR) fluorescence probe and the other is a gadolinium (Gd)-chelating magnetic resonance (MR) probe. To prepare the NIR fluorescence probe (A15), an acetyl glycine group was added to the N terminus of the 2AP fragment selected for capping. In addition, a tryptophan residue and a cysteine residue were added to the C terminus for quantitation and conjugation purposes, respectively. The peptide was synthesized by solid-phase peptide synthesis, characterized by mass spectrometry (calcd: 1701.9; found: [M 1] 1701.6), and then reacted with a sulfhydryl reactive fluorochrome (Alexa Fluor 680C2 maleimide; Molecular Probes, Eugene, OR) through the C-terminal cysteine sidechain, in sodium acetate buffer, pH 6.5 (Scheme 1). The HPLC-purified final product, A15, had excitation and emission wavelengths of 679 and 702 nm, respectively. A control probe (CA15) only modified by replacing glutamine residue at position 14 with an alanine residue was also synthesized.

In-vivo imaging of tumor associated urokinase-type plasminogen activator activity

The ability to image tumor associated protease in vivo has biological and clinical implications. In the present study, we describe the development and validation of a urokinase-type plasminogen activator (uPA) sensitive fluorescence imaging probe. The activation of our probe is highly specific to uPA in both enzymatic and cellular-based assays. In two distinct in-vivo tumor models (human colon adenocarcinoma HT-29 and human fibrosarcoma HT-1080), the observed fluorescence changes correlate well with tumor associated uPA activity. The signal intensities of the tumors are about three-fold higher in animals with probe injections. Our results suggest a direct detection method for uPA activity in vivo and the approach can be used for monitoring tumor growth and development.

DNA binding chelates for nonviral gene delivery imaging.

Noninvasive in vivo monitoring of gene delivery would provide a critically important information regarding the spatial distribution, local concentration, kinetics of removal and/or biodegradation of the expression vector. We developed a novel approach to noninvasive gene delivery imaging using heterobifunctional peptide-based chelates (PBC) bearing double-stranded DNA-binding groups and a technetium-binding amino acid motif. One of such chelates: Gly-Cys(Acm)-Gly-Cys(Acm)-Gly-Lys4-Lys-(N-ε-[4-(psoralen-8-yloxy)]butyrate)-NH2 has been characterized and labeled with reducedu200999mtc pertechnetate (oxotechnetate). the psoralen moiety (a dna binding group of pbc) allowed linking to double-stranded dna upon short-term irradiation with the near uv range light (>320u2009nm). Approximately 30–40% of addedu200999mTc-labeled PBC was nonextractable and co-eluted with a model pCMV-GFP vector during the gel-permeation chromatography. Nuclear imaging of ‘naked’ DNA and DNA complexes with lipid-based transfection reagents (‘lipoplexes’) has been performed after systemic or local administration ofu200999mTc-PBC-labeled DNA in mice. Imaging results were corroborated with the biodistribution usingu200999mTc-PBC andu200932P-labeled DNA and lipoplexes. A markedly different biodistribution ofu200999mTc PBC-labeled DNA and lipoplexes was observed with the latter being rapidly trapped in the liver, spleen and lung.u200999mTc PBC-DNA was used as an imaging tracer during in vivo transfection of B16 melanoma by local injection of ‘naked’u200999mTc PBC-DNA and corresponding lipoplexes. As demonstrated by nuclear imaging,u200999mTc PBC-DNA lipoplexes showed a slower elimination from the site of injection thanu200999mTc PBC-DNA alone. This result correlated with a higher expression of marker mRNA and green fluorescent protein as determined using RT-PCR and immunohistochemistry, respectively.

Fortschritte in der optischen Bildgebung

ZusammenfassungEine Reihe optischer Bildgebungsmodalitäten hat in den letzten Jahren deutliche Fortschritte gemacht. Neben technischen Verbesserungen im Bereich der Bilderzeugung und -rekonstruktion sind in jüngster Zeit “intelligente” optische Fluorochrome entwickelt worden, die eine sensitive Detektion molekularer Ziele (z. B. endogener Enzyme) in vivo ermöglichen. Die Kombination neuer Bildgebungstechniken mit intelligenten Kontrastmitteln birgt ein großes diagnostisches Potenzial für zukünftige klinische und experimentelle Anwendungen. Im Folgenden sollen die Grundlagen der optischen Bildgebung erläutert und der aktuelle Stand der Entwicklung dargelegt werden.AbstractDifferent optical imaging technologies have significantly progressed over the last years. Besides advances in imaging techniques and image reconstruction, new “smart” optical contrast agents have been developed which can be used to detect molecular targets (such as endogenous enzymes) in vivo. The combination of novel imaging technologies coupled with smart agents bears great diagnostic potential both clinically and experimentally. This overview outlines the basic principles of optical imaging and summarizes the current state of the art.