Alpha-1 Antitrypsin Inhibits Tumorigenesis and Progression of Colitis-Associated Colon Cancer through Suppression of Inflammatory Neutrophil-Activated Serine Proteases and IGFBP-3 Proteolysis.

Colitis-associated colon cancer (CAC) accompanies the massive infiltration of neutrophils during tumorigenesis and progression of CAC. Depletion of neutrophils in circulation results in significant inhibition of tumor incidence in CAC. However, the underlying mechanisms are largely unclear. In this study, we provide evidence for the crucial involvement of inflammatory neutrophil-activated serine proteases (NSPs) on the dysregulation of the anti-inflammatory and antitumor IGFBP-3/IGFBP-3R signaling axis in CAC using a chronic AOM/DSS mouse model. We also provide preclinical evidence for α1-antitrypsin (AAT) as a preventive and as a therapeutic for CAC. AAT administration not only prevented colitis-associated tumorigenesis but also inhibited established CAC. AOM/DSS treatment results in the significant activation of NSPs, leading to CAC through increased pro-inflammatory cytokines and decreased anti-inflammatory and antitumor IGFBP-3. Collectively, these data suggest that the NSPs proteolyze IGFBP-3, whereas AAT inhibits chronic colonic inflammation-induced NSP activity and subsequently suppresses IGFBP-3 proteolysis. Therefore, the anti-inflammatory and antitumor functions of the IGFBP-3/IGFBP-3R axis are restored. AAT mimicking small peptides also showed their inhibitory effects on NSP-induced IGFBP-3 proteolysis. These results suggest that targeting the NSP-IGFBP-3/IGFBP-3R axis using NSP inhibitors such as AAT and the AAT mimics and IGFBP-3R agonists could lead to novel approaches for the prevention and treatment of CAC.

Dendritic cell trafficking in tumor-bearing mice.

Prostate cancer is one of the leading causes of cancer deaths, with no curative treatments once it spreads. Alternative therapies, including immunotherapy, have shown limited efficacy. Dendritic cells (DC) have been widely used in the treatment of various malignancies. DC capture antigens and move to the lymphoid organs where they prime naive T cells. Interaction between DC and T cells are most active in lymph nodes and suppression of DC trafficking to lymph nodes impairs the immune response. In this work, we aimed to study trafficking of DC in vivo via various routes of delivery, to optimize the effectiveness of DC-based therapy. A DC labeling system was developed using 1,1'-dioctadecyltetramethyl indotricarbocyanine Iodine for in vivo fluorescent imaging. DC harvested from C57B/6 mice were matured, labeled, and injected intravenously, subcutaneously, or intratumorally, with or without antigen loading with whole tumor lysate, into C57B/6 mice inoculated with RM-1 murine prostate tumor cells. Signal intensity was measured in vivo and ex vivo. Signal intensity at the tumor site increased over time, suggesting trafficking of DC to the tumor with all modes of injection. Subcutaneous injection showed preferential trafficking to lymph nodes and tumor. Intravenous injection showed trafficking to lungs, intestines, and spleen. Subcutaneous injection of DC pulsed with whole tumor lysate resulted in the highest increase in signal intensity at the tumor site and lymph nodes, suggesting subcutaneous injection of primed DC leads to highest preferential trafficking of DC to the immunocompetent organs.

Sepiapterin Enhances Tumor Radio- and Chemosensitivities by Promoting Vascular Normalization.

Previously, we demonstrated that nitric oxide (NO) synthase (NOS) is uncoupled in a wide range of solid tumors and that restoring NOS coupling with the tetrahydrobiopterin precursor sepiapterin (SP) inhibits tumor progression. Endothelial dysfunction characterizes the poorly functional vasculature of solid tumors, and since NO is critical for regulation of endothelial function we asked whether SP, by recoupling NOS, improves tumor vasculature structure and function-enhancing chemotherapeutic delivery and response to radiotherapy. MMTV-neu mice with spontaneous breast tumors were treated with SP by oral gavage and evaluated by multispectral optoacoustic tomographic analysis of tumor HbO2 and by tissue staining for markers of hypoxia, blood perfusion, and markers of endothelial and smooth muscle proteins. Recoupling tumor NOS activity results in vascular normalization observed as reduced tumor hypoxia, improved tumor percentage of HbO2 and perfusion, as well as increased pericyte coverage of tumor blood vessels. The normalized vasculature and improved tumor oxygenation led to a greater than 2-fold increase in radiation-induced apoptosis compared with radiation or SP alone. High-performance liquid chromatography analysis of tumor doxorubicin levels showed a greater than 50% increase in doxorubicin uptake and a synergistic effect on tumor cell apoptosis. This study highlights for the first time the importance of NOS uncoupling and endothelial dysfunction in the development of tumor vasculature and presents a new approach for improving the tumoricidal efficacies of chemotherapy and radiotherapy.

Biodistribution and PET imaging of 89-zirconium labeled cerium oxide nanoparticles synthesized with several surface coatings.

Cerium oxide nanoparticles (CONPs) have unique surface chemistry allowing catalyst-like antioxidant properties, and are being investigated for several disease indications in medicine. Studies have utilized surface modified CONPs toward this application, but have been lacking in comprehensive biodistribution and pharmacokinetic data and a direct comparison to uncoated CONPs. We developed an enhanced single-pot synthesis of several coated CONPs and an efficient intrinsic core labeling of CONPs with the clinical PET isotope, zirconium-89, allowing detailed PET imaging and ex vivo biodistribution. All coated [89Zr]-CONPs showed benefit in terms of biodistribution compared to uncoated [89Zr]-CONPs, while retaining the intrinsic antioxidant properties. Among these, poly(acrylic acid) coated CONPs demonstrated excellent candidacy for clinical implementation due to their enhanced renal clearance and low reticuloendothelial system uptake. This work also demonstrates the value of intrinsic core labeling and PET imaging for evaluation of nanoparticle constructs to better inform future studies towards clinical use.

[18F]-Fluorinated Carboplatin and [111In]-Liposome for Image-Guided Drug Delivery.

Radiolabeled liposomes have been employed as diagnostic tools to monitor in vivo distribution of liposomes in real-time, which helps in optimizing the therapeutic efficacy of the liposomal drug delivery. This work utilizes the platform of [111In]-Liposome as a drug delivery vehicle, encapsulating a novel 18F-labeled carboplatin drug derivative ([18F]-FCP) as a dual-molecular imaging tool as both a radiolabeled drug and radiolabeled carrier. The approach has the potential for clinical translation in individual patients using a dual modal approach of clinically-relevant radionuclides of 18F positron emission tomography (PET) and 111In single photon emission computed tomography (SPECT). [111In]-Liposome was synthesized and evaluated in vivo by biodistribution and SPECT imaging. The [18F]-FCP encapsulated [111In]-Liposome nano-construct was investigated, in vivo, using an optimized dual-tracer PET and SPECT imaging in a nude mouse. The biodistribution data and SPECT imaging showed spleen and liver uptake of [111In]-Liposome and the subsequent clearance of activity with time. Dual-modality imaging of [18F]-FCP encapsulated [111In]-Liposome showed significant uptake in liver and spleen in both PET and SPECT images. Qualitative analysis of SPECT images and quantitative analysis of PET images showed the same pattern of activity during the imaging period and demonstrated the feasibility of dual-tracer imaging of a single dual-labeled nano-construct.

Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis.

Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity.

Sperm-associated antigen-17 gene is essential for motile cilia function and neonatal survival.

Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract infections, bronchiectasis, hydrocephaly, situs inversus, and male infertility. We generated knockout mice for the sperm-associated antigen-17 (Spag17) gene, which encodes a central pair (CP) protein present in the axonemes of cells with "9 + 2" motile cilia or flagella. The targeting of Spag17 resulted in a severe phenotype characterized by immotile nasal and tracheal cilia, reduced clearance of nasal mucus, profound respiratory distress associated with lung fluid accumulation and disruption of the alveolar epithelium, cerebral ventricular expansion consistent with emerging hydrocephalus, failure to suckle, and neonatal demise within 12 hours of birth. Ultrastructural analysis revealed the loss of one CP microtubule in approximately one quarter of tracheal cilia axonemes, an absence of a C1 microtubule projection, and other less frequent CP structural abnormalities. SPAG6 and SPAG16 (CP proteins that interact with SPAG17) were increased in tracheal tissue from SPAG17-deficient mice. We conclude that Spag17 plays a critical role in the function and structure of motile cilia, and that neonatal lethality is likely explained by impaired airway mucociliary clearance.

Microfluidic radiosynthesis and biodistribution of [18 F] 2-(5-fluoro-pentyl)-2-methyl malonic acid.

Microfluidics technology has emerged as a powerful tool for the radiosynthesis of positron emission tomography (PET) and single-photon emission computed tomography radiolabeled compounds. In this work, we have exploited a continuous flow microfluidic system (Advion, Inc., USA) for the [(18) F]-fluorine radiolabeling of the malonic acid derivative, [(18) F] 2-(5-fluoro-pentyl)-2-methyl malonic acid ([(18) F]-FPMA), also known as [(18) F]-ML-10, a radiotracer proposed as a potential apoptosis PET imaging agent. The radiosynthesis was developed using a new tosylated precursor. Radiofluorination was initially optimized by manual synthesis and served as a basis to optimize reaction parameters for the microfluidic radiosynthesis. Under optimized conditions, radio-thin-layer chromatography analysis showed 79% [(18) F]-fluorine incorporation prior to hydrolysis and purification. Following hydrolysis, the [(18) F]-FPMA was purified by C18 Sep-Pak, and the final product was analyzed by radio-HPLC (high-performance liquid chromatography). This resulted in a decay-corrected 60% radiochemical yield and ≥98% radiochemical purity. Biodistribution data demonstrated rapid blood clearance with less than 2% of intact [(18) F]-FPMA radioactivity remaining in the circulation 60 min post-injection. Most organs showed low accumulation of the radiotracer, and radioactivity was predominately cleared through kidneys (95% in 1 h). Radio-HPLC analysis of plasma and urine samples showed a stable radiotracer at least up to 60 min post-injection.

Mechanism and management of AKT inhibitor-induced hyperglycemia.

PURPOSE: Insulin-like growth factor-I receptor and phosphoinositide 3-kinase/AKT/mammalian target of rapamycin pathways are among the most active areas of drug discovery in cancer research. However, due to their integral roles in insulin signaling, inhibitors targeting these pathways often lead to hyperglycemia and hyperinsulinemia. We investigated the mechanism of hyperglycemia induced by GSK690693, a pan-AKT kinase inhibitor in clinical development, as well as methods to ameliorate these side effects. EXPERIMENTAL DESIGN: The effect of GSK690693 on blood glucose, insulin, and glucagon levels was characterized in mice. We then evaluated the effects of commonly prescribed antidiabetic agents on GSK690693-induced hyperglycemia. The mechanism of blood glucose increase was evaluated using fasting and tracer uptake studies and by measuring liver glycogen levels. Finally, approaches to manage AKT inhibitor-induced hyperglycemia were designed using fasting and low carbohydrate diet. RESULTS: We report that treatment with antidiabetic agents does not significantly affect GSK690693-induced hyperglycemia in rodents. However, administration of GSK690693 in mice significantly reduces liver glycogen (approximately 90%), suggesting that GSK690693 may inhibit glycogen synthesis and/or activate glycogenolysis. Consistent with this observation, fasting before drug administration reduces baseline liver glycogen levels and attenuates hyperglycemia. Further, GSK690693 also inhibits peripheral glucose uptake and introduction of a low-carbohydrate (7%) or 0% carbohydrate diet after GSK690693 administration effectively reduces diet-induced hyperglycemia in mice. CONCLUSIONS: The mechanism of GSK690693-induced hyperglycemia is related to peripheral insulin resistance, increased gluconeogenesis, and/or hepatic glycogenolysis. A combination of fasting and low carbohydrate diet can reduce the magnitude of hyperglycemia induced by an AKT inhibitor.

Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti-carcinoembryonic antigen single-chain Fv-Fc antibody fragments.

Antibody fragments are recognized as promising vehicles for delivery of imaging and therapeutic agents to tumor sites in vivo. The serum persistence of IgG1 and fragments with intact Fc region is controlled by the protective neonatal Fc receptor (FcRn) receptor. To modulate the half-life of engineered antibodies, we have mutated the Fc-FcRn binding site of chimeric anti-carcinoembryonic antigen (CEA) antibodies produced in a single-chain Fv-Fc format. The anti-CEA T84.66 single-chain Fv-Fc format wild-type and five mutants (I253A, H310A, H435Q, H435R, and H310A/H435Q, Kabat numbering system) expressed well in mammalian cell culture. After purification and characterization, effective in vitro antigen binding was shown by competition ELISA. Biodistribution studies in BALB/c mice using (125)I- and (131)I-labeled fragments revealed blood clearance rates from slowest to fastest as follows: wild-type > H435R > H435Q > I253A > H310A > H310A/H435Q. The terminal half-lives of the mutants ranged from 83.4 to 7.96 hours, whereas that of the wild-type was approximately 12 days. Additionally, (124)I-labeled wild-type, H435Q, I253A, H310A, and H310A/H435Q variants were evaluated in LS174T xenografted athymic mice by small animal positron emission tomography imaging, revealing localization to the CEA-positive xenografts. The slow clearing wild-type and H435Q constructs required longer to localize to the tumor and clear from the circulation. The I253A and H310A fragments showed intermediate behavior, whereas the H310A/H435Q variant quickly localized to the tumor site, rapidly cleared from the animal circulation and produced clear images. Thus, attenuating the Fc-FcRn interaction provides a way of controlling the antibody fragment serum half-life without compromising expression and tumor targeting.

Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging.

We have recently described the in vivo properties of an iodinated anti-p185HER2 engineered antibody fragment [minibody (scFv-C(H)3)2; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 +/- 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA), radiometal labeled, and evaluated in vivo. The tumor uptake of 111In-DOTA 10H8 minibody was 5.7 +/- 0.1% ID/g, similar to the radioiodinated 10H8 minibody. However, in addition to the expected liver clearance, the kidneys had unexpectedly high activity (34.0 +/- 4.0% ID/g). A minibody derived from a second anti-p185(HER2) antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using 64Cu-DOTA hu4D5v8 minibody, were 4.2 +/- 0.5% ID/g. Furthermore, in non-tumor-bearing mice, 111In-DOTA hu4D5v8 minibody exhibited similar elevated uptake in the kidneys (28.4 +/- 6.5% ID/g). Immunohistochemical staining of kidneys from non-tumor-bearing mice showed strong specific staining of the proximal tubules, and Western blot analysis of kidney lysate confirmed the presence of cross-reactive antigen. To further improve tumor uptake and normal tissue distribution, a larger hu4D5v8 fragment [(scFv-C(H)2-C(H)3)2; 105 kDa] was made, engineered to exhibit rapid clearance kinetics. This fragment, when evaluated by microPET, exhibited improved tumor targeting (12.2 +/- 2.4% ID/g) and reduced kidney uptake (13.1 +/- 1.5% ID/g). Thus, by manipulating the size and format of anti-p185(HER2) antibody fragments, the kidney activity was reduced and high or low expression of p185HER2 in xenografts could be distinguished by microPET imaging.

18F-Labeled Carboplatin Derivative for PET Imaging of Platinum Drug Distribution.

Increasing evidence indicates that reduced intracellular drug accumulation is the parameter most consistently associated with platinum drug resistance, emphasizing the need to directly measure the intratumor drug concentration. In the era of precision medicine and with the advent of powerful imaging and proteomics technologies, there is an opportunity to better understand drug resistance by exploiting these techniques to provide new knowledge on drug-target interactions. Here, we contribute to this endeavor by reporting on the development of an 18F-labeled carboplatin derivative (18F-FCP) that has the potential to image drug uptake and retention, including intratumoral distribution, by PET. Methods: Fluorinated carboplatin (19F-FCP) was synthesized using 19F-labeled 2-(5-fluoro-pentyl)-2-methyl malonic acid (19F-FPMA) as the labeling agent to coordinate with the cisplatin-aqua complex. It was then used to treat cell lines and compared with cisplatin and carboplatin at different concentrations. Manual radiosynthesis and characterization of 18F-FCP were performed using 18F-FPMA for coordination with the cisplatin-aqua complex. Automated radiosynthesis of 18F-FCP was optimized on the basis of manual synthesis procedures. The stability of 18F-FCP was verified using high-performance liquid chromatography. 18F-FCP was evaluated using ex vivo biodistribution and in vivo PET imaging in non-tumor-bearing animals as well as in KB-3-1 and COLO-205 tumor xenograft-bearing nude mice. Results: In vitro cytotoxicity studies demonstrated that 19F-FCP has an antitumor activity profile similar to that of the parent drug carboplatin. In vivo plasma and urine stability analysis showed intact 18F-FCP at 24 h after injection. PET imaging and biodistribution studies showed fast clearance from blood and major accumulation in the kidneys, indicating substantial renal clearance of 18F-FCP. Using 18F-FCP PET, we could image and identify the intratumor drug profile. Conclusion: Our results demonstrated that 19F-FCP, like carboplatin, retains antitumor activity in various cell lines. 18F-FCP could be a useful imaging tool for measuring the intratumor drug distribution. This strategy of using a new therapeutic carboplatin derivative to quantify and track platinum drugs in tumors using PET has the potential to translate into a clinically useful imaging tool for individual patients.

Development of a cell delivery system using alginate microbeads for tissue regeneration.

Alginate microbeads incorporating adipose-derived stem cells (ASCs) have potential for delivering viable cells capable of facilitating tissue regeneration. These microbeads are formed in calcium crosslinking solutions containing organic osmolytes to ensure physiological osmolality, but the comparative effects of these osmolytes on the microencapsulated cells are not known. In addition, delivery parameters needed to use microencapsulated cells for tissue regeneration remain unknown. We investigated the following parameters: (1) osmolyte effects on microbead diameter, cell viability and growth factor production; (2) the effect of the number of cells per microbead and the number of microbeads per unit volume on cell viability, growth factor production, and microbead degradation; (3) the ability of both degradable and non-degradable alginate microbeads to localize cells at the delivery site in vivo; and (4) whether alginate microbeads containing alginate-lyase elicit an inflammatory response after repeated exposure. Smallest microbead diameters were achieved using glucose as the osmolyte but cell viability and growth factor production did not depend on osmolyte type. As cell number per microbead or microbead number per well increased, growth factor production per cell decreased although percent cell viability was unchanged. The rate of cell release varied with the number of beads per well and with the number of cells per microbead. At the highest microbead density and at the lowest density of cells per microbead, cell release was delayed. Therefore fewer microbeads may be sufficient for clinical applications. Both degradable (0.22 U g-1) and non-degradable (0 U g-1) alginate microbeads localized cells at the delivery site. Degradable alginate microbeads delivered subcutaneously elicited a mild chronic inflammatory response on second exposure, but how this might impact repeated use of the technology remains to be determined.

Optical imaging of cardiac reporter gene expression in living rats.

BACKGROUND: Studies of cardiac gene transfer rely on postmortem analysis using histologic staining or enzyme assays. Noninvasive imaging of the temporal and spatial characteristics of cardiac gene expression in the same subject offers significant advantages. METHODS AND RESULTS: Rats underwent direct myocardial injection via left thoracotomy with adenovirus-expressing firefly luciferase (Ad-CMV-Fluc; n=30). The reporter substrate D-luciferin was injected intraperitoneally. Serial images were acquired by use of a cooled charged couple detector (CCD) camera. Results are expressed as relative light unit per minute (RLU/min). Rats transduced with 1x10(9) plaque-forming units show decremental cardiac luciferase activity over time: 152 070+/-21 170 (day 2), 195 806+/-62 630 (day 5), 7250+/-2941 (day 8), and 2040+/-971 RLU/min (day 14). To assess the detection sensitivity, serially diluted titers of Ad-CMV-Fluc were injected: 1x10(9) (195 393+/-14 896), 1x10(8) (33 777+/-18 179), 1x10(7) (417+/-91), 1x10(6) (185+/-64), 1x10(5) (53+/-1), and control (54+/-1) (P<0.05 for 1x10(9), 1x10(8), and 1x10(7) plaque-forming units versus control adenovirus-expressing mutant thymidine kinase [Ad-CMV-HSV1-sr39tk]; n=3). Finally, rats were euthanized, and in vitro luciferase activity correlated with in vivo CCD signals (r2=0.92). CONCLUSIONS: This study demonstrates for the first time the feasibility of imaging the location, magnitude, and time course of cardiac reporter gene expression in living rats. Cardiac gene therapy studies could be aided with wider application of this approach.

Positron emission tomography imaging of cardiac reporter gene expression in living rats.

BACKGROUND: Imaging reporter gene expression is useful for noninvasive monitoring of gene therapy. In this study, we imaged cardiac reporter gene expression in living rats using micro positron emission tomography (microPET). METHODS AND RESULTS: Rats (n=10) underwent intramyocardial injection with 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving herpes simplex virus type 1 mutant thymidine kinase (Ad-CMV-HSV1-sr39tk) as PET reporter gene. Control rats (n=4) received 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving firefly luciferase (Ad-CMV-Fluc). On days 2 to 4, microPET images were obtained after a tail vein injection of nitrogen-13 ammonia ([13N]-NH3) as myocardial perfusion tracer, followed by 9-(4-[18F]-fluoro-3 hydroxymethylbutyl) guanine ([18F]-FHBG) to assess HSV1-sr39tk expression. After imaging, hearts were removed for ex vivo [18F] gamma counting and thymidine kinase enzyme assay. Results show homogenous myocardial distribution of [13N]-NH3 on all microPET images. Rats injected with Ad-CMV-HSV1-sr39tk have significant [18F]-FHBG uptake in the anterolateral wall compared with background signal in controls. Gamma counting shows 20.0+/-4.4-fold increase of radioactivity, whereas enzyme assay shows 22.1+/-6.1-fold increase of thymidine kinase activity in Ad-CMV-HSV1-sr39tk injected rats (P<0.05). CONCLUSIONS: Successful imaging of cardiac HSV1-sr39tk expression was performed in living rats with microPET. The presence of [18F]-FHBG uptake is confirmed by gamma counting and the presence of HSV1-sr39TK protein by thymidine kinase enzyme assay. Cardiac reporter gene imaging by PET may eventually be applied toward human gene therapy studies.

Micro-positron emission tomography imaging of cardiac gene expression in rats using bicistronic adenoviral vector-mediated gene delivery.

BACKGROUND: We have previously validated the use of micro-positron emission tomography (microPET) for monitoring the expression of a single PET reporter gene in rat myocardium. We now report the use of a bicistronic adenoviral vector (Ad-CMV-D2R80a-IRES-HSV1-sr39tk) for linking the expression of 2 PET reporter genes, a mutant rat dopamine type 2 receptor (D2R80a) and a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk), with the aid of an internal ribosomal entry site (IRES). METHODS AND RESULTS: Rat H9c2 cardiomyoblasts transduced with increasing titers of Ad-CMV-D2R80a-IRES-HSV1-sr39tk (0 to 2.5x10(8) pfu) were assayed 48 hours later for reporter protein activities, which were found to correlate well with viral titer (r2=0.96, P<0.001 for D2R80A; r2=0.98, P<0.001 for HSV1-sr39TK) and each other (r2=0.97; P<0.001). Experimental (n=8) and control (n=6) athymic rats underwent intramyocardial injection of up to 2x10(9) pfu of Ad-CMV-D2R80a-IRES-HSV1-sr39tk and saline, respectively. Forty-eight hours later and weekly thereafter, rats were assessed for D2R80a-dependent myocardial accumulation of 3-(2-[18F]fluoroethyl)spiperone ([18F]-FESP) and HSV1-sr39tk-dependent sequestration of 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]-FHBG) using microPET. Longitudinal [18F]-FESP and [18F]-FHBG imaging of experimental rats revealed a good correlation between the cardiac expressions of the 2 PET reporter genes (r2=0.73; P<0.001). The location of adenovirus-mediated transgene expression, as inferred from microPET images, was confirmed by ex vivo gamma counting of explanted heart. CONCLUSIONS: The IRES-based bicistronic adenoviral vector can potentially be used in conjunction with PET for indirect imaging of therapeutic gene expression by replacing 1 of the 2 PET reporter genes with a therapeutic gene of choice.

Molecular imaging of cardiac cell transplantation in living animals using optical bioluminescence and positron emission tomography.

BACKGROUND: The current method of analyzing myocardial cell transplantation relies on postmortem histology. We sought to demonstrate the feasibility of monitoring transplanted cell survival in living animals using molecular imaging techniques. METHODS AND RESULTS: For optical bioluminescence charged-coupled device imaging, rats (n=20) underwent intramyocardial injection of embryonic rat H9c2 cardiomyoblasts (3x10(6) to 5x10(5)) expressing firefly luciferase (Fluc) reporter gene. Cardiac bioluminescence signals were present for more than 2 weeks with 3x10(6) cells: day 1 (627 000+/-15%), day 2 (346 100+/-21%), day 4 (112 800+/-20%), day 8 (78 860+/-24%), day 12 (67 780+/-12%), and day 16 (62 200+/-5% p x s(-1) x cm(2-1) x sr(-1)). For micro-positron emission tomography imaging, rats (n=20) received cardiomyoblasts (3x10(6)) expressing mutant herpes simplex type 1 thymidine kinase (HSV1-sr39tk) reporter gene. Detailed tomography of transplanted cells is shown by 9-(4-[18F]-fluoro-3hydroxymethylbutyl)guanine ([18F]-FHBG) reporter probe and nitrogen-13 ammonia ([13N]-NH3) perfusion images. Within the transplanted region, there was a 4.48+/-0.71-fold increase of in vivo [18F]-FHBG activity and a 4.01+/-0.51-fold increase of ex vivo gamma counting compared with control animals. Finally, the in vivo images of cell survival were confirmed by ex vivo autoradiography, histology, immunohistochemistry, and reporter protein assays. CONCLUSIONS: The location(s), magnitude, and survival duration of embryonic cardiomyoblasts were monitored noninvasively. With further development, molecular imaging studies should add critical insights into cardiac cell transplantation biology.

Positron-emission tomography reporter gene expression imaging in rat myocardium.

BACKGROUND: This study examines the quantitative accuracy, detection sensitivity, and time course of imaging the expression of a mutant herpes simplex type-1 virus thymidine kinase (HSV1-sr39tk) PET reporter gene in rat myocardium by using the PET reporter probe 9-(4-[18F]-Fluoro-3-Hydroxymethylbutyl)-Guanine ([18F]-FHBG) and a small-animal PET (microPET). METHODS AND RESULTS: In 40 rats, adenovirus expressing HSV1-sr39tk driven by a cytomegalovirus promoter (Ad-CMV-HSV1-sr39tk, 1x10(6) to 1x10(9) pfu) was injected through a thoracotomy directly into the left ventricular myocardium. After 3 days, myocardial perfusion was imaged with [13N]-ammonia for delineating the left ventricular myocardium, followed by imaging the expression of the reporter gene with intravenous [18F]-FHBG. The total myocardial [18F]-FHBG accumulation was quantified in percent of injected dose (%ID). Immunohistochemistry and autoradiography demonstrated HSV1-sr39tk enzyme (HSV1-sr39TK) and accumulation of [18F]-FHBG in the inoculated myocardium in 3 rats each. In 24 rats with various viral titers, the %ID was correlated with ex vivo well counting (r2=0.981, P<0.0001) and myocardial HSV1-sr39TK activity by tissue enzyme activity assay (r2=0.790, P<0.0001). Myocardial [18F]-FHBG accumulation was identified at viral titers down to 1x10(7) pfu. In 6 rats serially imaged up to day 17, myocardial [18F]-FHBG accumulation on microPET peaked on days 3 to 5 and was no longer identified on days 10 to 17. CONCLUSIONS: HSV1-sr39tk reporter gene expression can be monitored with [18F]-FHBG and microPET in rat myocardium quantitatively and serially with high detection sensitivity. Cardiac PET reporter gene imaging offers the potential of monitoring the expression of therapeutic genes in cardiac gene therapy.

The Role of Nitric Oxide Synthase Uncoupling in Tumor Progression.

UNLABELLED: Here, evidence suggests that nitric oxide synthases (NOS) of tumor cells, in contrast with normal tissues, synthesize predominantly superoxide and peroxynitrite. Based on high-performance liquid chromatography analysis, the underlying mechanism for this uncoupling is a reduced tetrahydrobiopterin:dihydrobiopterin ratio (BH4:BH2) found in breast, colorectal, epidermoid, and head and neck tumors compared with normal tissues. Increasing BH4:BH2 and reconstitution of coupled NOS activity in breast cancer cells with the BH4 salvage pathway precursor, sepiapterin, causes significant shifts in downstream signaling, including increased cGMP-dependent protein kinase (PKG) activity, decreased ß-catenin expression, and TCF4 promoter activity, and reduced NF-κB promoter activity. Sepiapterin inhibited breast tumor cell growth in vitro and in vivo as measured by a clonogenic assay, Ki67 staining, and 2[18F]fluoro-2-deoxy-D-glucose-deoxyglucose positron emission tomography (FDG-PET). In summary, using diverse tumor types, it is demonstrated that the BH4:BH2 ratio is lower in tumor tissues and, as a consequence, NOS activity generates more peroxynitrite and superoxide anion than nitric oxide, resulting in important tumor growth-promoting and antiapoptotic signaling properties. IMPLICATIONS: The synthetic BH4, Kuvan, is used to elevate BH4:BH2 in some phenylketonuria patients and to treat diseases associated with endothelial dysfunction, suggesting a novel, testable approach for correcting an abnormality of tumor metabolism to control tumor growth.

Spag17 deficiency results in skeletal malformations and bone abnormalities.

Height is the result of many growth and development processes. Most of the genes associated with height are known to play a role in skeletal development. Single-nucleotide polymorphisms in the SPAG17 gene have been associated with human height. However, it is not clear how this gene influences linear growth. Here we show that a targeted mutation in Spag17 leads to skeletal malformations. Hind limb length in mutants was significantly shorter than in wild-type mice. Studies revealed differences in maturation of femur and tibia suggesting alterations in limb patterning. Morphometric studies showed increased bone formation evidenced by increased trabecular bone area and the ratio of bone area to total area, leading to reductions in the ratio of marrow area/total area in the femur. Micro-CTs and von Kossa staining demonstrated increased mineral in the femur. Moreover, osteocalcin and osterix were more highly expressed in mutant mice than in wild-type mice femurs. These data suggest that femur bone shortening may be due to premature ossification. On the other hand, tibias appear to be shorter due to a delay in cartilage and bone development. Morphometric studies showed reduction in growth plate and bone formation. These defects did not affect bone mineralization, although the volume of primary bone and levels of osteocalcin and osterix were higher. Other skeletal malformations were observed including fused sternebrae, reduced mineralization in the skull, medial and metacarpal phalanges. Primary cilia from chondrocytes, osteoblasts, and embryonic fibroblasts (MEFs) isolated from knockout mice were shorter and fewer cells had primary cilia in comparison to cells from wild-type mice. In addition, Spag17 knockdown in wild-type MEFs by Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Our findings disclosed unexpected functions for Spag17 in the regulation of skeletal growth and mineralization, perhaps because of its role in primary cilia of chondrocytes and osteoblasts.