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  • Angiogenic Effects Despite Limited Cell Survival of Bone Marrow-Derived Mesenchymal Stem Cells under Ischemia THORACIC AND CARDIOVASCULAR SURGEON Hoffmann, J., Glassford, A. J., Doyle, T. C., Robbins, R. C., Schrepfer, S., Pelletier, M. P. 2010; 58 (3): 136-142

    Abstract

    Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent and secrete angiogenic factors, which could help patients with occlusive arterial diseases. We hypothesize that MSCs, in comparison to fibroblasts, survive better under hypoxic conditions in vitro and in vivo. MSCs and fibroblasts from L2G mice expressing firefly luciferase and GFP were cultured in normoxic and hypoxic conditions for 24 hours. In vitro cell viability was tested by detecting apoptosis and necrosis. MSCs released higher amounts of VEGF (281.1 +/- 62.6 pg/ml) under hypoxic conditions compared to normoxia (154.9 +/- 52.3 pg/ml, p = NS), but were less tolerant to hypoxia (45 +/- 7.9%) than fibroblasts (28.1 +/- 3.6%, p = NS). A hindlimb ischemia model was created by ligating the femoral artery of 18 FVB mice. After one week, 1 x 106 cells (MSCs, fibroblasts or saline) were injected into the limb muscles of each animal (n = 6 per group). Bioluminescence measurement to assess the viability of luciferase positive cells showed significant proliferation of MSCs on day four compared to fibroblasts (p = 0.001). Three weeks after cell delivery, the capillary to muscle fiber ratio of ischemic areas was analyzed. In the MSC group, vessel density was significantly higher than in the fibroblast or control group (0.5 +/- 0.08 and 0.3 +/- 0.03). Under hypoxia, MSCs produced more VEGF compared to normal conditions and MSC transplantation into murine ischemic limbs led to an increase in vessel density, although MSC survival was limited. This study suggests that MSC transplantation may be an effective and clinically relevant tool in the therapy of occlusive arterial diseases.

    View details for DOI 10.1055/s-0029-1240758

    View details for Web of Science ID 000276420900001

    View details for PubMedID 20379963

  • Hepatocyte Growth Factor or Vascular Endothelial Growth Factor Gene Transfer Maximizes Mesenchymal Stem Cell-Based Myocardial Salvage After Acute Myocardial Infarction CIRCULATION Deuse, T., Peter, C., Fedak, P. W., Doyle, T., Reichenspurner, H., Zimmermann, W. H., Eschenhagen, T., Stein, W., Wu, J. C., Robbins, R. C., Schrepfer, S. 2009; 120 (11): S247-S254

    Abstract

    Mesenchymal stem cell (MSC)-based regenerative strategies were investigated to treat acute myocardial infarction and improve left ventricular function.Murine AMI was induced by coronary ligation with subsequent injection of MSCs, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), or MSCs +HGF/VEGF into the border zone. Left ventricular ejection fraction was calculated using micro-computed tomography imaging after 6 months. HGF and VEGF protein injection (with or without concomitant MSC injection) significantly and similarly improved the left ventricular ejection fraction and reduced scar size compared with the MSC group, suggesting that myocardial recovery was due to the cytokines rather than myocardial regeneration. To provide sustained paracrine effects, HGF or VEGF overexpressing MSCs were generated (MSC-HGF, MSC-VEGF). MSC-HGF and MSC-VEGF showed significantly increased in vitro proliferation and increased in vivo proliferation within the border zone. Cytokine production correlated with MSC survival. MSC-HGF- and MSC-VEGF-treated animals showed smaller scar sizes, increased peri-infarct vessel densities, and better preserved left ventricular function when compared with MSCs transfected with empty vector. Murine cardiomyocytes were exposed to hypoxic in vitro conditions. The LDH release was reduced, fewer cardiomyocytes were apoptotic, and Akt activity was increased if cardiomyocytes were maintained in conditioned medium obtained from MSC-HGF or MSC-VEGF cultures.This study showed that (1) elevating the tissue levels of HGF and VEGF after acute myocardial infarction seems to be a promising reparative therapeutic approach, (2) HGF and VEGF are cardioprotective by increasing the tolerance of cardiomyocytes to ischemia, reducing cardiomyocyte apoptosis and increasing prosurvival Akt activation, and (3) MSC-HGF and MSC-VEGF are a valuable source for increased cytokine production and maximize the beneficial effect of MSC-based repair strategies.

    View details for DOI 10.1161/CIRCULATIONAHA.108.843680

    View details for Web of Science ID 000269773000035

    View details for PubMedID 19752375

  • siRNA silencing of keratinocyte-specific GFP expression in a transgenic mouse skin model GENE THERAPY Gonzalez-Gonzalez, E., Ra, H., Hickerson, R. P., Wang, Q., Piyawattanametha, W., Mandella, M. J., Kino, G. S., Leake, D., Avilion, A. A., Solgaard, O., Doyle, T. C., Contag, C. H., Kaspar, R. L. 2009; 16 (8): 963-972

    Abstract

    Small interfering RNAs (siRNAs) can be designed to specifically and potently target and silence a mutant allele, with little or no effect on the corresponding wild-type allele expression, presenting an opportunity for therapeutic intervention. Although several siRNAs have entered clinical trials, the development of siRNA therapeutics as a new drug class will require the development of improved delivery technologies. In this study, a reporter mouse model (transgenic click beetle luciferase/humanized monster green fluorescent protein) was developed to enable the study of siRNA delivery to skin; in this transgenic mouse, green fluorescent protein reporter gene expression is confined to the epidermis. Intradermal injection of siRNAs targeting the reporter gene resulted in marked reduction of green fluorescent protein expression in the localized treatment areas as measured by histology, real-time quantitative polymerase chain reaction and intravital imaging using a dual-axes confocal fluorescence microscope. These results indicate that this transgenic mouse skin model, coupled with in vivo imaging, will be useful for development of efficient and 'patient-friendly' siRNA delivery techniques and should facilitate the translation of siRNA-based therapeutics to the clinic for treatment of skin disorders.

    View details for DOI 10.1038/gt.2009.62

    View details for Web of Science ID 000268916800004

    View details for PubMedID 19474811

  • Stem Cell-Mediated Accelerated Bone Healing Observed with in Vivo Molecular and Small Animal Imaging Technologies in a Model of Skeletal Injury JOURNAL OF ORTHOPAEDIC RESEARCH Lee, S., Padmanabhan, P., Ray, P., Gambhir, S. S., Doyle, T., Contag, C., Goodman, S. B., Biswal, S. 2009; 27 (3): 295-302

    Abstract

    Adult stem cells are promising therapeutic reagents for skeletal regeneration. We hope to validate by molecular imaging technologies the in vivo life cycle of adipose-derived multipotent cells (ADMCs) in an animal model of skeletal injury. Primary ADMCs were lentivirally transfected with a fusion reporter gene and injected intravenously into mice with bone injury or sham operation. Bioluminescence imaging (BLI), [(18)F]FHBG (9-(fluoro-hydroxy-methyl-butyl-guanine)-micro-PET, [(18)F]Fluoride ion micro-PET and micro-CT were performed to monitor stem cells and their effect. Bioluminescence microscopy and immunohistochemistry were done for histological confirmation. BLI showed ADMC's traffic from the lungs then to the injury site. BLI microscopy and immunohistochemistry confirmed the ADMCs in the bone defect. Micro-CT measurements showed increased bone healing in the cell-injected group compared to the noninjected group at postoperative day 7 (p < 0.05). Systemically administered ADMC's traffic to the site of skeletal injury and facilitate bone healing, as demonstrated by molecular and small animal imaging. Molecular imaging technologies can validate the usage of adult adipose tissue-derived multipotent cells to promote fracture healing. Imaging can in the future help establish therapeutic strategies including dosage and administration route.

    View details for DOI 10.1002/jor.20736

    View details for Web of Science ID 000263307200003

    View details for PubMedID 18752273

  • Regulation of maternal and fetal hemodynamics by heme oxygenase in mice BIOLOGY OF REPRODUCTION Zhao, H., Wong, R. J., Doyle, T. C., Nayak, N., Vreman, H. J., Contag, C. H., Stevenson, D. K. 2008; 78 (4): 744-751

    Abstract

    Heme oxygenase (HMOX) regulates vascular tone and blood pressure through the production of carbon monoxide (CO), a vasodilator derived from the heme degradation pathway. During pregnancy, the maternal circulation undergoes significant adaptations to accommodate the hemodynamic demands of the developing fetus. Our objective was to investigate the role of HMOX on maternal and fetal hemodynamics during pregnancy in a mouse model. We measured and compared maternal tissue and placental HMOX activity and endogenous CO production, represented by excreted CO and carboxyhemoglobin levels, during pregnancy (Embryonic Days 12.5-15.5) to nonpregnant controls. Micro-ultrasound was used to monitor maternal abdominal aorta diameters as well as blood flow velocities and diameters of fetal umbilical arteries. Tin mesoporphyrin, a potent HMOX inhibitor, was used to inhibit HMOX activity. Changes in maternal vascular tone were monitored by tail cuff blood pressure measurements. Effects of HMOX inhibition on placental structures were assessed by histology. We showed that maternal tissue and placental HMOX activity and CO production were significantly elevated during pregnancy. When HMOX in the placenta was inhibited, maternal and fetal hemodynamics underwent significant changes, with maternal blood pressures increasing. We concluded that increases in maternal tissue and placental HMOX activity contribute to the regulation of peripheral vascular resistance and therefore are important for the maintenance of normal maternal vascular tone and fetal hemodynamic functions during pregnancy.

    View details for DOI 10.1095/biolreprod.107.064899

    View details for Web of Science ID 000254217500020

    View details for PubMedID 18094356

  • Visualizing fungal infections in living mice using bioluminescent pathogenic Candida albicans strains transformed with the firefly luciferase gene MICROBIAL PATHOGENESIS Doyle, T. C., Nawotka, K. A., Kawahara, C. B., Francis, K. P., Contag, P. R. 2006; 40 (2): 82-90

    Abstract

    Animal studies with Candida albicans have provided models for understanding fungal virulence and antifungal drug development. To non-invasively monitor long-term Candida murine infections, clinical isolates were stably transformed with a codon-optimized luciferase gene to constitutively express luciferase. Chronic systemic infections were established in mice with engineered strains, and bioluminescent signals were apparent from kidneys by non-invasive imaging using charged-coupled device cameras. These infections were established in immune-competent mice, and bioluminescence was detectable in animals that showed no physiological consequence of infection, as well as those visually succumbing to the disease. Similarly, bioluminescence was measured from the vaginal tissue of mice infected vaginally. Fungal loads determined by plating vaginal lavages showed a similar pattern to the bioluminescent signals measured, and fungal infection could be detected in animals for over 30 days post infection by both modalities. The effect of the antifungal drug miconazole was tested in this model, and clearance in animals was apparent by both direct imaging and fungal load determination. The use of bioluminescence to monitor these and other models of Candida infections will greatly speed up the analysis of drug development studies, both in ease of visualizing infections and decreasing numbers of animals required to run such studies.

    View details for DOI 10.1016/j.micpath.2005.11.003

    View details for Web of Science ID 000235912600005

    View details for PubMedID 16426810

  • Expression of firefly luciferase in Candida albicans and its use in the selection of stable transformants MICROBIAL PATHOGENESIS Doyle, T. C., Nawotka, K. A., Purchio, A. F., Akin, A. R., Francis, K. P., Contag, P. R. 2006; 40 (2): 69-81

    Abstract

    The infectious yeast Candida albicans is a model organism for understanding the mechanisms of fungal pathogenicity. We describe the functional expression of the firefly luciferase gene, a reporter commonly used to tag genes in many other cellular systems. Due to a non-standard codon usage by this yeast, the CUG codons were first mutated to UUG to allow functional expression. When integrated into the chromosome of C. albicans with a strong constitutive promoter, cells bioluminesce when provided with luciferin substrate in their media. When fused to the inducible promoter from the HWP1 gene, expression and bioluminescence was only detected in cultures conditioning hyphal growth. We further used the luciferase gene as a selection to isolate transformed cell lines from clinical isolates of C. albicans, using a high-density screening strategy that purifies transformed colonies by virtue of light emission. This strategy requires no drug or auxotrophic selectable marker, and we were thus able to generate stable transformants of clinical isolates that are identical to the parental strain in all aspects tested, other than their bioluminescence. The firefly luciferase gene can, therefore, be used as a sensitive reporter to analyze gene function both in laboratory and clinical isolates of this medically important yeast.

    View details for DOI 10.1016/j.micpath.2005.11.002

    View details for Web of Science ID 000235912600004

    View details for PubMedID 16427765

  • Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation TRANSPLANTATION Cao, Y. A., Bachmann, M. H., Beilhack, A., Yang, Y., Tanaka, M., Swijnenburg, R. J., Reeves, R., Taylor-Edwards, C., Schulz, S., Doyle, T. C., Fathman, C. G., Robbins, R. C., Herzenberg, L. A., Negrin, R. S., Contag, C. H. 2005; 80 (1): 134-139

    Abstract

    Tissue regeneration and transplantation of solid organs involve complex processes that can only be studied in the context of the living organism, and methods of analyzing these processes in vivo are essential for development of effective transplantation and regeneration procedures. We utilized in vivo bioluminescence imaging (BLI) to noninvasively visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that constitutively expresses luciferase. Dynamic early events of hematopoietic reconstitution were accessible and engraftment from as few as 200 transplanted whole bone marrow (BM) cells resulted in bioluminescent foci in lethally irradiated, syngeneic recipients. The transplantation of autologous pancreatic Langerhans islets and of allogeneic heart revealed the tempo of transplant degeneration or immune rejection over time. This imaging approach is sensitive and reproducible, permits study of the dynamic range of the entire process of transplantation, and will greatly enhance studies across various disciplines involving transplantation.

    View details for DOI 10.1097/01.TP.0000164347.50559.A3

    View details for Web of Science ID 000230473800023

    View details for PubMedID 16003245

  • Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo JOURNAL OF BIOMEDICAL OPTICS Zhao, H., Doyle, T. C., Coquoz, O., Kalish, F., Rice, B. W., Contag, C. H. 2005; 10 (4)

    Abstract

    In vivo bioluminescence imaging depends on light emitted by luciferases in the body overcoming the effect of tissue attenuation. Understanding this relationship is essential for detection and quantification of signal. We have studied four codon optimized luciferases with different emission spectra, including enzymes from firefly (FLuc), click beetle (CBGr68, CBRed) and Renilla reniformins (hRLuc). At 25 degrees C, the in vitro lambda(max) of these reporters are 578, 543, 615, and 480 nm, respectively; at body temperature, 37 degrees C, the brightness increases and the firefly enzyme demonstrates a 34-nm spectral red shift. Spectral shifts and attenuation due to tissue effects were evaluated using a series of 20-nm bandpass filters and a cooled charge-coupled device (CCD) camera. Attenuation increased and the spectra of emitted light was red shifted for signals originating from deeper within the body relative to superficial origins. The tissue attenuation of signals from CBGr68 and hRLuc was greater than from those of Fluc and CBRed. To further probe tissue effects, broad spectral emitters were created through gene fusions between CBGr68 and CBRed. These resulted in enzymes with broader emission spectra, featuring two peaks whose intensities are differentially affected by temperature and tissue depth. These spectral measurement data allow for improved understanding of how these reporters can be used in vivo and what they can reveal about biological processes in living subjects.

    View details for DOI 10.1117/1.2032388

    View details for Web of Science ID 000232799200010

    View details for PubMedID 16178634

  • In vivo bioluminescence imaging for integrated studies of infection CELLULAR MICROBIOLOGY Doyle, T. C., Burns, S. M., Contag, C. H. 2004; 6 (4): 303-317

    Abstract

    Understanding biological processes in the context of intact organ systems with fine temporal resolution has required the development of imaging strategies that reveal cellular and molecular changes in the living body. Reporter genes that confer optical signatures on a given biological process have been used widely in cell biology and have been used more recently to interrogate biological processes in living animal models of human biology and disease. The use of internal biological sources of light, luciferases, to tag cells, pathogens, and genes has proved to be a versatile tool to provide in vivo indicators that can be detected externally. The application of this technology to the study of animal models of infectious disease has not only provided insights into disease processes, but has also revealed new mechanisms by which pathogens may avoid host defences during infection.

    View details for DOI 10.1111/j.1462-5822.2004.00378.x

    View details for Web of Science ID 000220006600001

    View details for PubMedID 15009023

  • Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals. Molecular imaging Zhao, H., Doyle, T. C., Wong, R. J., Cao, Y., Stevenson, D. K., Piwnica-Worms, D., Contag, C. H. 2004; 3 (1): 43-54

    Abstract

    In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which analog is most suitable for activity measurements in live cells and living animals, we characterized 10 CLZN analogs using Renilla luciferase (Rluc) as the reporter enzyme. For each analog, we monitored enzyme activity, auto-oxidation, and efficiency of cellular uptake. All CLZN analogs tested showed higher auto-oxidation signals in serum than was observed in phosphate buffer or medium, mainly as a result of auto-oxidation by binding to albumin. CLZN-f, -h, and -e analogs showed 4- to 8-fold greater Rluc activity, relative to CLZN-native, in cells expressing the enzyme from a stable integrant. In studies using living mice expressing Rluc in hepatocytes, administration of CLZN-e and -native produced the highest signal. Furthermore, distinct temporal differences in signal for each analog were revealed following intravenous or intraperitoneal delivery. We conclude that the CLZN analogs that are presently available vary with respect to hRluc utilization in culture and in vivo, and that the effective use of CLZN-utilizing enzymes in living animals depends on the selection of an appropriate substrate.

    View details for PubMedID 15142411

  • Movement of yeast cortical actin cytoskeleton visualized in vivo PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Doyle, T., Botstein, D. 1996; 93 (9): 3886-3891

    Abstract

    Fusion proteins between the green fluorescent protein (GFP) and the cytoskeleton proteins Act1p (actin), Sac6p (yeast fimbrin homolog), and Abp1p in budding yeast (Saccharomyces cerevisiae) localize to the cortical actin patches. The actin fusions could not function as the sole actin source in yeast, but fusions between the actin-binding proteins Abp1p and Sac6p complement fully the phenotypes associated with their gene deletions. Direct observation in vivo reveals that the actin cortical patches move. Movement of actin patches is constrained to the asymmetric distribution of the patches in growing cells, and this movement is greatly reduced when metabolic inhibitors such as sodium azide are added. Fusion protein-labeled patches are normally distributed during the yeast cell cycle and during mating. In vivo observation made possible the visualization of actin patches during sporulation as well.

    View details for Web of Science ID A1996UK55700028

    View details for PubMedID 8632984

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