bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cardiomyocyte hyperplasia after plasmid‐mediated vascular endothelial growth factor gene transfer in pigs with chronic myocardial ischemia
Authors: Laguens Rubén , Cabeza Meckert Patricia , Vera Janavel Gustavo , De Lorenzi Andrea , Lascano Elena , Negroni Jorge , del Valle Héctor , Cuniberti Luis , Martínez Verónica , Dulbecco Eduardo , Melo Carlos , Fernández Nahuel , Criscuolo Marcelo , Crottogini Alberto 
Published: February 2004

AbstractBackgroundFor over 40 years it has been proposed that cardiomyocyte hyperplasia may occur in hypertrophic human hearts. While this implies that heart myocytes can undergo cytokinesis, evidence of conventional cell division has been exceptionally reported. Recently, we found that gene transfer of vascular endothelial growth factor (VEGF) displays a mitogenic effect on adult cardiomyocytes. In the present study we searched for cardiomyocyte hyperplasia as evidence of VEGF‐induced cardiomyocyte cytokinesis.MethodsThree weeks after implanting an Ameroid constrictor at the origin of the left circumflex artery, 16 pigs were randomized to receive 10 direct intramyocardial injections of 3.8 mg of plasmid encoding for VEGF (pVEGF) or empty plasmid. Five weeks later, hearts were weighed, myocyte diameter was measured in tissue sections, and myocyte length and nuclei number were studied in isolated myocytes. A resting echocardiogram was performed immediately before reoperation and before sacrifice to evaluate global and regional left ventricular function. Investigators were blinded to the study groups and nature of the injectate until the end of data analysis.ResultsNo heart weight differences existed between groups. However, in the ischemic myocardium, pVEGF‐treated hearts had 22% more cardiomyocytes per unit volume and exhibited significantly more oligonucleated (1 or 2 nuclei) cardiomyocytes than hearts receiving empty plasmid.ConclusionsIn pigs with chronic myocardial ischemia, VEGF gene transfer induced cardiomyocyte cytokinesis, as revealed by cardiomyocyte hyperplasia. Our finding extends the previously reported mitogenic effect of VEGF on adult cardiomyocytes and supports the hypothesis that VEGF may have a therapeutic role in diseases characterized by myocardial cell loss. Copyright © 2004 John Wiley & Sons, Ltd.
Reducing mobilization of simian immunodeficiency virus based vectors by primer complementation
Authors: Grunwald Thomas , Pedersen Finn Skou , Wagner Ralf , Überla Klaus 
Published: February 2004

AbstractBackgroundHuman gene therapy vectors based on primate lentiviruses harbour in contrast to oncoretroviruses the risk of vector mobilization by human immunodeficiency viruses (HIV). Infection of cells transduced with a lentiviral vector by HIV could lead to packaging of the lentiviral vector RNA into HIV particles and transfer of the vector.MethodsA new approach based on primer complementation was developed to reduce the risk of vector mobilization. The primer binding site (PBS) of an SIV‐based vector was mutated abolishing tRNA primer binding and thus blocking reverse transcription. This block was efficiently by‐passed during vector production by providing an artificial tRNA matching the mutated PBS with titers reaching 106 infectious units/ml.ResultsPrimer‐complemented SIV vectors were mobilized from transduced cells by HIV‐1 >150‐fold less efficiently than vectors with wild‐type PBS. Mobilization of the primer‐complemented SIV vector by SIV was inhibited to a lesser extent indicating reduced efficacy of the primer complementation approach for preventing mobilization of lentiviral vectors by homologous virus. The analysis of the PBS of the vector DNA in target cells transduced with vectors containing mutated PBS in the absence of a matched tRNA suggests that formation of heterozygous particles followed by priming on the helper RNA and strand switch during reverse transcription can lead to mobilization of the primer‐complemented vector by SIV, but not HIV‐1. Although self‐inactivating vectors were more efficient in preventing vector mobilization by HIV‐1 than primer‐complemented vectors, mobilization remained undectable only if both approaches were combined.ConclusionsThe primer complementation approach should further reduce the risk of mobilization of self‐inactivating SIV‐based vectors by HIV‐1 and thus increase their safety. Copyright © 2004 John Wiley & Sons, Ltd.
Ex vivo gene transfer to mature skeletal muscle by using adenovirus helper cells
Authors: Kimura Shigemi , Ikezawa Makoto , Cao Baohong , Kanda Yasunari , Pruchnic Ryan , Cummins James , Huard Johnny , Miike Teruhisa , Suzuki Satoru 
Published: February 2004

AbstractBackgroundAdenoviral gene transfer to adult skeletal muscle is hindered by several major limitations, including host immune responses and maturation‐dependent loss of myofiber infectivity. Ex vivo gene delivery is more efficient than direct viral injection in surmounting maturation‐dependent adenoviral transduction. Here we investigated the use of helper cells to improve the efficiency of ex vivo gene transfer to adult mouse skeletal muscle.MethodsNew producer cells carrying the E1 gene of adenovirus type 5 (E32 cells) were developed using primary myoblasts from mdx mice. The E32 cells and 293 cells were infected with an E1‐deleted first‐generation adenovirus carrying the LacZ gene. These transduced helper cells were injected into the skeletal muscle of adult mdx and SCID mice.ResultsLacZ‐positive mature myofibers were detected in the skeletal muscle of adult mice sacrificed 5 days post‐injection. The gene transfer efficiency using 293 cells and E32 cells was 6.2 and 3.6 times higher than myoblast‐mediated gene transfer, respectively. Ex vivo gene transfer of these cell types led to a better outcome than did direct adenoviral injection.ConclusionsWe achieved more efficient adenoviral gene transduction by using 293 and E32 helper cells than by myoblast‐mediated gene transfer and direct viral injection. These helper cells also enabled adenoviral gene transfer to mature myofibers. The mechanisms by which this method facilitated adenoviral gene transfer to mature myofibers remains unclear; however, we hypothesize that the in vivo occurrence of cytopathic effects (CPE) in the transduced 293 and E32 helper cell populations facilitated the improved adenoviral transduction of myofibers. Copyright © 2004 John Wiley & Sons, Ltd.
In vitro and in vivo delivery of intact BAC DNA – comparison of different methods
Authors: Magin‐Lachmann C. , Kotzamanis G. , D'Aiuto L. , Cooke H. , Huxley C. , Wagner E. 
Published: February 2004

AbstractBackgroundThe ability to deliver large (>100 kb) fragments of DNA to mammalian cells in vitro and in vivo is becoming increasingly important with the availability of BAC and PAC constructs for gene expression. Here we investigate in vitro and in vivo delivery of BACs up to 157 kb.MethodsDifferent types of polyethylenimine (PEI) and Lipofectamine were used to deliver 150‐kb BAC (bacterial artificial chromosome) DNA to mouse and human cell lines in tissue culture and the level of EGFP expression compared. To assess the intactness of the DNA delivered, a BAC carrying oriP/EBNA‐1 was used to make stably transfected cell lines. Episomal DNA was then rescued into E. coli followed by analysis on a pulsed‐field gel. Three different methods of in vivo delivery were also assessed for delivery of BAC DNA; intravenous injection of DNA/PEI particles, intramuscular injection with electroporation and high‐volume injection into the tail vein.ResultsPEI22 (linear polymer form, 22 kDa) was found to be the most efficient method for delivery of 150‐kb BAC DNA to both cell lines in tissue culture. However, Lipofectamine 2000 was found to give a higher proportion of intact DNA than PEI22 in stably transformed colonies and almost all the DNA delivered by Lipofectamine 2000 was intact. Intravenous injection of DNA/PEI particles was found to be inefficient for delivery of BAC DNA. Intramuscular injection with electroporation of pure BAC DNA was very efficient and expression was maintained for 105 days. High‐volume injection of BAC DNA gave excellent expression in the liver and intact BAC DNA could be rescued 7 days after injection.ConclusionsThese results demonstrate efficient delivery of intact, large (up to 157 kb) DNA constructs for in vitro gene expression and in vivo gene therapy applications. Copyright © 2004 John Wiley & Sons, Ltd.
A synthetic peptide vector system for optimal gene delivery to corneal endothelium
Authors: Collins Louise , Fabre John W. 
Published: February 2004

AbstractBackgroundEfficient and non‐toxic gene delivery, preferably with non‐viral DNA vectors readily transferable to clinical practice, is generally regarded as a major limitation for gene therapy.MethodsA 31 amino acid, integrin‐targeted bifunctional synthetic peptide (polylysine‐molossin), and two (Lys)16‐containing control peptides, were assessed for ex vivo gene delivery to the rabbit cornea. Critical physical properties of polylysine‐molossin/DNA complexes were evaluated and both chloroquine and a 20 amino acid fusogenic peptide were used to promote endocytic exit.ResultsPolylysine‐molossin/DNA complexes and (Lys)16/DNA complexes at 10 µg/ml of DNA were much smaller and much more positively charged in non‐ionic isotonic medium (5% dextrose or 5% dextrose buffered to pH 7.4 in 10 mM Tris) when compared with culture medium or phosphate‐buffered saline (PBS). Addition of the fusogenic peptide (net charge −5) reversed the positive charge of complexes in PBS, and reduced the strong positive charge of polylysine‐molossin/DNA complexes in dextrose. Polylysine‐molossin/DNA complexes in 5% dextrose were much more effective for gene delivery to the cornea when compared with complexes in culture medium, and the fusogenic peptide was much more effective than chloroquine for promoting gene delivery. The optimal DNA/polylysine‐molossin/fusogenic peptide w/w ratio was 1 : 3 : 2 at 10 µg/ml of DNA. Essentially 100% of corneal endothelial cells were transfected under these optimal conditions, without any evidence of toxicity. Integrin‐targeting did not contribute significantly to gene delivery in this system.ConclusionsThis DNA vector system, consisting entirely of synthetic peptides, is ideally suited for clinical applications of gene therapy of the corneal endothelium. Copyright © 2004 John Wiley & Sons, Ltd.
Efficient transfection of non‐proliferating human airway epithelial cells with a synthetic vector system
Authors: Meng Qing‐Hai , Robinson Danielle , Jenkins R. Gisli , McAnulty Robin J. , Hart Stephen L. 
Published: February 2004

AbstractBackgroundThe poor transfection efficiency of most synthetic vectors in non‐dividing, airway epithelial cells is limiting the development of gene therapy for respiratory diseases such as cystic fibrosis.MethodsCultured airway epithelial cells, quiescent or replicating, were transfected with a lipid/peptide synthetic vector system and transfection efficiency assessed by luciferase assays or flow cytometry for green fluorescent protein expression. Tight junctions were disrupted by treatment with ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA). Cell division was inhibited by aphidicolin and assessed by 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation and assay by fluorescence microscopy or flow cytometry. In vivo transfection was performed by intratracheal instillation.ResultsInitial studies showed that this synthetic vector transfected 8% of non‐dividing epithelial cells in culture compared with 45% of subconfluent, dividing cells. However, formulation of the vector with EGTA, which disrupts cellular tight junctions, induced a four‐fold increase in the transfection efficiency of confluent, non‐mitotic cells. The EGTA enhancement of transfection also correlated with a four‐fold increase in vector binding to the cells, presumably via exposed basolateral receptors. Flow cytometry and double immunofluorescent staining for reporter gene expression and BrdU incorporation indicated efficient transfection of non‐proliferating cells in a confluent monolayer by this vector. Furthermore, in vivo transfection of murine lung by intratracheal administration showed a six‐fold enhancement of luciferase expression when lungs were pre‐treated with EGTA.ConclusionsThis study confirms the hypothesis that a lipid/peptide vector transfects non‐dividing cells with high efficiency which contributes to its previously reported efficiency in transfecting bronchial epithelium, making it a good choice for gene transfer studies in the lung. Copyright © 2004 John Wiley & Sons, Ltd.
Effective polyethylenimine‐mediated gene transfer into human endothelial cells
Authors: Zaric Violeta , Weltin Denis , Erbacher Patrick , Remy Jean‐Serge , Behr Jean‐Paul , Stephan Dominique 
Published: February 2004

AbstractBackgroundThe major advantage in choosing non‐viral vectors such as cationic polymers for in vitro and in vivo transfection is their higher biosafety than viral ones. Among the cationic polymers, polyethylenimines (PEIs) are promising molecules for gene delivery to a variety of cells. Efficient transfection of primary endothelial cells using PEIs could be regarded as an interesting strategy of treatment in some ischemic cardiovascular diseases.MethodsEfficacies of a 22‐kDa linear PEI (L‐PEI) and its glucose‐grafted derivative (L‐PEI‐Glc4) were compared for gene transfer into human umbilical vein endothelial cells (HUVEC) using the reporter gene luciferase. Cells were incubated for 2, 4 and 24 h with PEI/DNA complexes made in 150 mM sodium chloride (NaCl) or in 5% glucose solution. Luciferase activity was measured 24 h after the onset of transfection. The effects of low (2%) and high (30%) concentrations of serum on transfection efficacy were assessed as well. We then studied the intracellular fate of the PEI/DNA complexes labelled with the DNA intercalator YOYO‐1 using flow cytometry analysis (FACS) and confocal microscopy.ResultsPEI/DNA complexes formed in NaCl led to a higher transfection efficacy than those made in glucose. The optimal formulation, depending on the incubation time and the presence of serum in the medium, was obtained using DNA complexed to L‐PEI‐Glc4 and incubated for 4 h with the cells. This condition led to 50% fluorescent cells after GFP transfection. A high serum concentration diminished the L‐PEI associated toxicity but decreased L‐PEI‐Glc4 transfection efficiency. FACS analysis using both vectors showed that almost 90% of the cells had internalized the DNA complexes. Confocal microscopic observations showed a fast attachment of the complexes to the cell surface followed by inclusion into vesicles that migrated to the perinuclear region.ConclusionsIn this work, we defined the optimal conditions for gene delivery in HUVEC. These conditions were obtained when using derivatives L‐PEI and L‐PEI‐Glc4 complexed with DNA in 150 mM NaCl and added to cells for 2 and 4 h, respectively. Cellular trafficking of the complexes suggested that cell entry was not a limiting factor for gene delivery using PEI. This study underlined the interest in PEIs as efficient vectors for gene transfer into human endothelial cells. Copyright © 2004 John Wiley & Sons, Ltd.
Post‐secretion neutralization of transgene‐derived effect: soluble erythropoietin receptor/IgG1Fc expressed in liver neutralizes erythropoietin produced in muscle
Authors: Maruyama H. , Higuchi M. , Higuchi N. , Kameda S. , Saito M. , Sugawa M. , Matsuzaki J. , Neichi T. , Yokoyama S. , Miyazaki Y. , Miyazaki J. , Gejyo F. 
Published: February 2004

AbstractBackgroundThe regulation of transgene expression is a key issue for the development of safe gene therapy. Various strategies have been used to regulate protein production at the levels of transgene expression, transcription, translation, and secretion. Neutralization following secretion is another important backup system to prevent super‐therapeutic levels of a protein from being expressed by gene transfer.MethodsWe tested whether the soluble human erythropoietin receptor (EpoR)/IgG1Fc could neutralize the rat Epo at the post‐secretory level and suppress erythrocytosis.ResultsTo assess whether soluble human EpoR could bind rat Epo in vitro, we used the Epo‐dependent human leukemic cell line, AS‐E2. EpoR/IgG1Fc significantly inhibited the growth of AS‐E2 cells in Epo‐containing medium. To test this neutralization effect of EpoR/IgG1Fc in vivo, we first transferred pCAGGS‐Epo into rat muscle by in vivo electroporation, confirmed erythropoiesis for 3 weeks, and then delivered EpoR/IgG1Fc by liver‐targeted gene transfer via tail‐vein injection with hydrodynamics‐based transfection. Reticulocyte counts and hematocrit levels in rats that received pCAGGS‐EpoR/IgG1Fc injections were significantly lower than in rats that received pCAGGS‐EpoR, pCAGGS‐IgG1Fc, or no injection.ConclusionsThese results demonstrate that liver‐targeted pCAGGS‐EpoR/IgG1Fc transfer by tail‐vein injection with hydrodynamics‐based transfection is useful for neutralizing Epo delivered by in vivo electroporation. This backup strategy at the level of post‐secretion could facilitate the clinical application of gene therapy in the future. Copyright © 2004 John Wiley & Sons, Ltd.
Overexpression of human fibroblast growth factor 2 stimulates cell proliferation in an ex vivo model of articular chondrocyte transplantation
Authors: Madry Henning , Emkey Greg , Zurakowski David , Trippel Stephen B 
Published: February 2004

AbstractBackgroundGenetically engineered chondrocytes could be used to enhance cartilage repair. Fibroblast growth factor 2 (FGF‐2) is a mitogen for chondrocytes and may be a candidate for gene transfer approaches to stimulate chondrocyte proliferation. In the present study, we tested the hypothesis that human FGF‐2 (hFGF‐2) gene transfer into articular chondrocytes modulates cell proliferation in an ex vivo model of chondrocyte transplantation.MethodsTransfection of articular chondrocytes with an expression plasmid vector carrying the cDNA for hFGF‐2 under the control of the cytomegalovirus promoter/enhancer mediated transgene expression and synthesis of biologically relevant amounts of the recombinant hFGF‐2 protein. Articular chondrocytes transfected with the Escherichia coli β‐galactosidase (lacZ) gene or a hFGF‐2 cDNA were transplanted onto the surface of articular cartilage explants.ResultsThe tissue formed by the chondrocytes expressing hFGF‐2 was thicker and contained more cells than control cultures. Quantitative analysis of [3H]thymidine and [35S]sulfate incorporation in composite cultures revealed that hFGF‐2 transfection stimulated mitogenic activity in the new tissue but did not augment matrix glycosaminoglycan synthesis.ConclusionsThese data support the concept that chondrocytes overexpressing a hFGF‐2 cDNA selectively modulate cell proliferation in an ex vivo model of chondrocyte transplantation. These results suggest that therapeutic hFGF‐2 gene transfer may be applicable for the treatment of articular cartilage disorders, such as traumatic defects in which cellular repopulation is a therapeutic goal. Copyright © 2004 John Wiley & Sons, Ltd.
Protection and selection for gene therapy in the hematopoietic system
Authors: Milsom Michael D. , Fairbairn Leslie J. 
Published: February 2004

AbstractHematopoietic stem cell gene therapy is potentially curative for a number of inherited and acquired disorders. However, poor gene transfer and expression in repopulating hematopoietic stem cells attenuate this potential. Here we review potential means of conferring a selective advantage to hematopoietic stem cells and their progeny, and discuss the issues that surround the use of selective advantages in vivo. Copyright © 2004 John Wiley & Sons, Ltd.
Cycloheximide and actinomycin D delay death and affect bcl‐2, bax, and Ice gene expression in astrocytes under in vitro ischemia
Authors: Yu Albert Cheung Hoi , Yung Hon Wa , Hui Michael Hung Kit , Lau Lok Ting , Chen Xiao Qian , Collins Richard A. 
Published: February 2004

AbstractAn in vitro ischemia model was established and the effect of the metabolic inhibitors cycloheximide (CHX) and actinomycin D (ActD) on apoptosis in astrocytes under ischemia studied. CHX decreased by 75% the number of cells dying after 6 hr of ischemia compared with control cultures. TdT‐mediated dUTP nick end labelling (TUNEL) staining of comparable cultures was reduced by 40%. ActD decreased cell death by 60% compared with controls. The number of TUNEL‐positive cells was reduced by 38%. The nuclear shrinkage in TUNEL‐positive astrocytes in control cultures did not occur in ActD‐treated astrocytes, indicating that nuclear shrinkage and DNA fragmentation during apoptosis are two unrelated processes. Expression of bcl‐2 (α and β), bax, and Ice in astrocytes under similar ischemic conditions, as measured by quantitative reverse transcription‐polymerase chain reaction, indicated that ischemia down‐regulated bcl‐2 (α and β) and bax. Ice was initially down‐regulated from 0 to 4 hr, before returning to control levels after 8 hr of ischemia. ActD decreased the expression of these genes. CHX reduced the expression of bcl‐2 (α and β) but increased bax and Ice expression. It is hypothesized that the balance of proapoptotic (Bad, Bax) and antiapoptotic (Bcl‐2, Bcl‐Xl) proteins determines apoptosis. The data suggest that the ratio of Bcl‐2/Bad in astrocytes following ActD and CHX treatment does not decrease as much in untreated cells during ischemia. Our data indicate that it is the ratio of Bcl‐2 family members that plays a critical role in determining ischemia‐induced apoptosis. It is also important to note that ischemia‐induced apoptosis involves the regulation of RNA and protein synthesis. © 2003 Wiley‐Liss, Inc.
Widely expressed transcripts for chemokine receptor CXCR1 in identified glutamatergic, γ‐aminobutyric acidergic, and cholinergic neurons and astrocytes of the rat brain: A single‐cell reverse transcription‐multiplex polymerase chain reaction study
Authors: Danik M. , Puma C. , Quirion R. , Williams S. 
Published: February 2004

AbstractIncreasing evidence suggests that the chemokine interleukin (IL)‐8/CXCL8 plays important roles in CNS development, neuronal survival, modulation of excitability, and neuroimmune response. Recently, we have shown that CXCL8 can acutely modulate ion channel activity in septal neurons expressing receptors CXCR1 and/or CXCR2. This was a surprising finding, insofar as CXCR1 expression had not been described for the mammalian brain. Here we investigated whether CXCR1 transcripts are present in other brain regions, whether they are expressed at the single‐cell level in molecularly identified neurons and astrocytes, and how they are regulated during early postnatal development. In addition, possible cellular colocalization of CXCR1 and CXCR2 transcripts was examined. Semiquantitative reverse transcription‐polymerase chain reaction (RT‐PCR) revealed that CXCR1 mRNAs were expressed in the septum, striatum, hippocampus, cerebellum, and cortex (temporoparietal and entorhinal) at different levels and appeared to be regulated independently from CXCR2 during development. By using RT multiplex PCR on acutely dissociated cells from these brain regions, we show that CXCR1 transcripts were expressed in 83% of 84 sampled neurons displaying cholinergic (choline acetyltransferase mRNAs), γ‐aminobutyric acidergic (glutamic acid decarboxylases 65 and 67 mRNAs), or glutamatergic (vesicular glutamate transporters 1 and 2 mRNAs) phenotypes. CXCR1 and CXCR2 transcripts were colocalized in 45% of neurons sampled and also were present in some glial fibrillary acidic protein mRNA‐expressing astrocytes. This is the first study to demonstrate the widespread expression of CXCR1 transcripts in the brain and suggests that CXCR1 may have hitherto unsuspected roles in neuromodulation and inflammation. © 2003 Wiley‐Liss, Inc.