RESUMEN
Using a previously published model of human BPD this study examines whether preterm lung inflammatory cells produce transforming growth factor beta 1 (TGF-beta1), a cytokine pivotal in pathogenesis of bronchopulmonary dysplasia (BPD), and whether TGF-beta1 expression is regulated by inflammation. Lung inflammatory cells (neutrophils and macrophages) recovered in the broncho-alveolar (BAL) fluid of premature infants intubated for respiratory distress after birth expressed TGF-b1 mRNA and protein. Total and bioactive TGF-beta1 were abundantly found in the BAL fluid of the same infants. In cell culture stimulation by lipopolysaccharide (LPS) did not result in any further expression of total or bioactive TGF-beta1 by neonatal lung inflammatory cells over constitutive concentrations. In conclusion, lung inflammatory cells from premature infants are a source of TGF-beta1 but LPS does not regulate TGF-b1 production in these cells.
Asunto(s)
Displasia Broncopulmonar/metabolismo , Regulación de la Expresión Génica/fisiología , Pulmón/citología , Macrófagos/metabolismo , Neutrófilos/metabolismo , Nacimiento Prematuro , Factor de Crecimiento Transformador beta1/genética , Líquido del Lavado Bronquioalveolar , Células Cultivadas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Recién Nacido , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Embarazo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Regulation of transforming growth factor beta 1 (TGF-beta1) expression remains unclear. Inflammation has been inferred to play a major role in stimulating TGF-beta1 production since high concentrations of TGF-beta1 have been found in the lungs of patients with various diffuse inflammatory lung diseases. To establish an association between inflammation and TGF-beta1 expression, human alveolar epithelial (A549) cells were co-cultured with lipopolysaccharide (LPS), Tumor necrosis factor alpha (TNFalpha), Interleukin 1 beta (IL-1beta) and Interleukin 8 (IL-8) for 12 hours. Total and bioactive TGF-beta1 protein were then measured. A549 cells transiently transfected with a plasmid containing the TGF-beta1 promoter linked to a luciferase reported gene were then co-cultured with the same inflammatory peptides for 12 hours and TGF-beta1 promoter activity determined. Nuclear transcription factors AP-1 (c-jun) or NF-kappa (p65, p50 and p105) were over expressed in A549 cells transiently transfected with the TGF-beta1 promoter and TGF-beta1 promoter activity subsequently measured. Stimulation with inflammatory signals LPS, TNFalpha, IL-1beta, IL-8 resulted in no increase of total or bioactive TGF-beta1 activity above constitutive concentrations in vitro. TGF-beta1 promoter activity was also unchanged from baseline levels in response to the same inflammatory peptides. Expression of c-jun however led to significant increases of TGF-beta1 promoter activity over constitutive levels. In contrast p65 and p105 expression resulted in inhibition of TGF-beta1 promoter activity below baseline levels. We conclude that in a human alveolar epithelial cell line, inflammation does not regulate TGF-beta1 expression. These studies suggest that in lung pathologies such as asthma, lung fibrosis and CLD, TGF-beta1 production may involve pathways independent of inflammatory mediators LPS, TNFalpha, IL-1beta and IL-8.
Asunto(s)
Regulación de la Expresión Génica , Mediadores de Inflamación/fisiología , Alveolos Pulmonares/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Línea Celular , Combinación de Medicamentos , Humanos , Mediadores de Inflamación/farmacología , Interleucina-1/farmacología , Interleucina-8/farmacología , Lipopolisacáridos/farmacología , FN-kappa B/biosíntesis , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , Factor de Transcripción AP-1/biosíntesis , Transfección , Factor de Crecimiento Transformador alfa/farmacología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1RESUMEN
Pathologic lesions caused by catheter-based revascularization procedures for occlusive artery disease include disruption of the endothelium, exposure of extracellular matrix (ECM) proteins, and proliferation of vascular smooth muscle cells, which lead to neointima formation and restenosis. We have developed matrix-collagen-targeted retroviral vectors that are able to accumulate at sites of vascular injury (Hall et al., Hum. Gene Ther. 1997;8:2183-2192; Hall et al., Hum. Gene Ther. 2000;11:983-993). The primary tissue-targeting motif, adapted from the physiological surveillance sequence found in von Willebrand factor, served to localize and concentrate the vector within vascular lesions. In the present study, we evaluated the efficiency of this vector-targeting system in rats with nonligated balloon-injured carotid arteries. Both intraarterial (by retrograde femoral artery catheterization) and intravenous (via femoral vein) injection of a matrix-targeted vector enhanced transduction of neointimal cells ( approximately 20%) at severely denuded areas when compared with the nontargeted vector (<1%). Further, intraarterial instillation of a matrix-targeted, but not a nontargeted, vector bearing an antisense cyclin G1 construct inhibited neointima lesion formation in the injured carotid arteries. Taken together, these data indicate that strategic targeting of retroviral vectors to vascular lesions would have therapeutic potential in the management of vascular restenosis and many other disorders of uncontrolled proliferation where endothelial disruption, ECM remodeling, and collagen deposition form the nexus for preferential vector localization and concentration in vivo.
Asunto(s)
Traumatismos de las Arterias Carótidas/terapia , Técnicas de Transferencia de Gen , Vectores Genéticos , Células 3T3 , Angioplastia de Balón/efectos adversos , Animales , Western Blotting , Línea Celular , Colágeno/metabolismo , Humanos , Ratones , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Retroviridae/genética , Factores de Tiempo , Transducción Genética , beta-Galactosidasa/metabolismoRESUMEN
Vaso-occlusive sequelae following percutaneous transluminal coronary angioplasty (PTCA), including smooth muscle cell migration, proliferation, and attendant extracellular matrix production, often results in restenosis of the treated artery. To further understand the molecular mechanisms governing progressive intimal hyperplasia, we performed a molecular screen using differential display PCR on total RNA prepared from injured and normal carotid arterial segments to identify a subset of differentially expressed genes at t=7 days post-balloon catheter injury in a non-human primate. DNA sequence analysis of selected differentially expressed RNA by this procedure using 240 combinations of random primer pairs yielded 41 distinct cDNA sequences: 22 of which have significant sequence homology to previously identified meta-zoan genes, 15 GEMS (genes expressed in monkey neointima), and 4 GSMS (genes suppressed in monkey neointima) that have little homology to reported sequences. Among the up-regulated homologues include i) secreted growth regulatory factors, ii) membrane receptors, iii) transcription factors, iv) cell adhesion molecules, and v) extracellular matrix proteins; some of which have not been previously linked to vascular restenosis. In particular, Cyr61, a known angiogenesis inducer, was found to be highly expressed in the neointima lesion of the balloon-injured monkey artery. This finding provides the first links of Cyr61 to the pathogenesis of vascular restenosis, and identifies a novel locus for potential therapeutic intervention. These studies identified a number of known and unknown genes, whose up- or down-regulated expression during the proliferative phase of vascular restenosis makes them potential targets for therapeutic intervention.
Asunto(s)
Traumatismos de las Arterias Carótidas/genética , Cateterismo/efectos adversos , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Túnica Íntima/metabolismo , Animales , Arterias Carótidas/química , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/etiología , Traumatismos de las Arterias Carótidas/patología , Proteína 61 Rica en Cisteína , Genes/genética , Sustancias de Crecimiento/análisis , Sustancias de Crecimiento/genética , Proteínas Inmediatas-Precoces/análisis , Proteínas Inmediatas-Precoces/genética , Inmunohistoquímica , Macaca mulatta , ARN/genética , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Túnica Íntima/química , Túnica Íntima/patologíaRESUMEN
A major obstacle that limits the potential of human gene therapy is the inefficiency of gene delivery to appropriate sites in vivo. Previous studies demonstrated that the physiological surveillance function performed by von Willebrand factor (vWF) could be incorporated into retroviral vectors by molecular engineering of the MuLV ecotropic envelope (Env) protein. To advance the application of vWF targeting technology beyond laboratory animals, we prepared an extensive series of Env proteins bearing modified vWF-derived matrix-binding sequences and assembled these chimeric proteins into targeted vectors that are capable of transducing human cells. Initially, a dual envelope configuration was utilized, which required coexpression of a wild-type amphotropic Env. Subsequently, streamlined "escort" Env proteins were constructed wherein the inoperative receptor-binding domain of the targeting partner was replaced by the vWF-derived collagen-binding motif. Ultimately, an optimal construct was developed that exhibited properties of both extracellular matrix (ECM)-targeting and near wild-type amphotropic infectivity, and could be arrayed as a single envelope on a retroviral particle. On intraarterial instillation, enhanced focal transduction of neointimal cells (approximately 20%) was demonstrated in a rat model of balloon angioplasty. Moreover, transduction of tumor foci (approximately 1-3%) was detected after portal vein infusion of a matrix-targeted vector in a nude mouse model of liver metastasis. We conclude that the unique properties of these targeted injectable retroviral vectors would be suitable for improving therapeutic gene delivery in numerous clinical applications, including vascular restenosis, laser and other surgical procedures, orthopedic injuries, wound healing, ischemia, arthritis, inflammatory disease, and metastatic cancer.
Asunto(s)
Matriz Extracelular/metabolismo , Productos del Gen env/genética , Terapia Genética/métodos , Vectores Genéticos/farmacología , Factor de von Willebrand/genética , Factor de von Willebrand/farmacología , Secuencia de Aminoácidos , Animales , Traumatismos de las Arterias Carótidas/terapia , Línea Celular , Portadores de Fármacos , Vectores Genéticos/genética , Humanos , Virus de la Leucemia Murina/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Neoplasias Pancreáticas/secundario , Ratas , Ratas Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción Genética , Factor de von Willebrand/metabolismoRESUMEN
Fusion peptides are hydrophobic sequences located at the N terminus of the transmembrane (TM) envelope proteins of the orthomyxoviruses and paramyxoviruses and several retroviruses. The Moloney murine leukemia virus TM envelope protein, p15E, contains a hydrophobic stretch of amino acids at its N terminus followed by a region rich in glycine and threonine residues. A series of single amino acid substitutions were introduced into this region, and the resulting proteins were examined for their abilities to be properly processed and transported to the cell surface and to induce syncytia in cells expressing the ecotropic receptor. One substitution in the hydrophobic core and several substitutions in the glycine/threonine-rich region that prevented both cell-cell fusion and the transduction of NIH 3T3 cells when incorporated into retroviral vector particles were identified. In addition, one mutation that enhanced the fusogenicity of the resulting envelope protein was identified. The fusion-defective mutants trans dominantly interfered with the ability of the wild-type envelope protein to cause syncytium formation in a cell-cell fusion assay, although no trans-dominant inhibition of transduction was observed. Certain substitutions in the hydrophobic core that prevented envelope protein processing were also found. These data indicate that the N-terminal region of p15E is important both for viral fusion and for the correct processing and cell surface expression of the viral envelope protein.
Asunto(s)
Virus de la Leucemia Murina/genética , Proteínas Oncogénicas de Retroviridae/genética , Proteínas del Envoltorio Viral/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Análisis Mutacional de ADN , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Metastatic osteosarcoma is a potential target for gene therapy, because conventional therapies are only palliative and metastatic disease is invariably fatal. Overexpression of the cyclin G1 (CYCG1) gene is frequently observed in human osteosarcoma cells, and its continued expression is found to be essential for their survival. Previously, we reported that down-regulation of cyclin G1 protein expression induced cytostatic and cytocidal effects in human MG-63 osteosarcoma cells (Skotzko et al., Cancer Research, 1995). Here, we extend these findings in a tumorigenic MNNG/HOS cell line and report on the effective inhibition of tumor growth in vivo by an antisense cyclin G1 retroviral vector when delivered as concentrated high titer vector supernatants directly into rapidly growing subcutaneous tumors in athymic nude mice. Histologic sections from the antisense cyclin G1 vector-treated tumors showed decreased mitotic indices and increased stroma formation within the residual tumors. Furthermore, in situ analysis of the cell-cycle kinetics of residual tumor cells revealed a decrease in the number of cells in S and G2/M phases of the cell cycle concomittant with an accumulation of cells in the G1 phase. Taken together, these studies demonstrate in vivo efficacy of a high-titer antisense cyclin G1 retroviral vector in an animal model of osteosarcoma.
Asunto(s)
Ciclinas/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Osteosarcoma/terapia , Retroviridae/genética , Animales , Ciclo Celular , Ciclina G , Ciclina G1 , ADN sin Sentido/administración & dosificación , Modelos Animales de Enfermedad , Expresión Génica , Vectores Genéticos/genética , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Osteosarcoma/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: The contemporary treatment of coronary athero-occlusive disease by percutaneous transluminal coronary angioplasty is hampered by maladaptive wound healing, resulting in significant failure rates. Morbid sequelae include smooth muscle cell (SMC) hyperplasia and restenosis due to vascular neointima formation. METHODS AND RESULTS: In this study, we examined the inhibitory effects of a concentrated retroviral vector bearing an antisense cyclin G1 gene on aortic SMC proliferation in vitro and on neointima formation in vivo in a rat carotid injury model of restenosis. Retroviral vectors bearing an antisense cyclin G1 construct inhibited the proliferation of transduced aortic SMCs in 2- to 6-day cultures, concomitant with down-regulation of cyclin G1 protein expression and decreased [3H]thymidine incorporation into DNA. Morphological examination showed evidence of cytolysis, giant syncytia formation, and apoptotic changes evidenced by overt cell shrinkage, nuclear fragmentation, and specific immunostaining of nascent 3'-OH DNA ends generated by endonuclease-mediated DNA fragmentation. Pronounced "bystander effects" including neighboring cells were noted in aortic SMCs transduced with the antisense cyclin G1 vector, as determined by quantitative assays and fluorescent labeling of nontransduced cells. In an in vitro tissue injury model, the proliferation and migration of antisense cyclin G1 vector-transduced aortic SMCs were inhibited. Moreover, in vivo delivery of high-titer antisense cyclin G1 vector supernatant to the balloon-injured rat carotid artery in vivo resulted in a significant reduction in neointima formation. CONCLUSIONS: These findings represent the first demonstration of the inhibitory effects of an antisense cyclin G1 retroviral vector on nonneoplastic cell proliferation. Taken together, these data affirm the potential utility of antisense cyclin G1 constructs in the development of novel gene therapy approaches to vascular restenosis.
Asunto(s)
Arterias Carótidas/patología , Estenosis Carotídea/patología , Movimiento Celular/genética , Ciclinas/genética , Técnicas de Transferencia de Gen , Terapia Genética , Oligonucleótidos Antisentido/genética , Animales , Arterias Carótidas/metabolismo , Estenosis Carotídea/metabolismo , Estenosis Carotídea/terapia , División Celular/genética , Células Cultivadas , Ciclina G , Ciclina G1 , Ciclinas/biosíntesis , Regulación hacia Abajo , Vectores Genéticos , Oligonucleótidos Antisentido/administración & dosificación , Ratas , Ratas Wistar , Retroviridae , Túnica Íntima/metabolismo , Túnica Íntima/patologíaRESUMEN
Targeted gene delivery to vascular lesions is a major challenge in the development of gene therapy protocols for cardiovascular diseases. One approach would be to enable retroviral vectors to accumulate at sites of vascular injury and enhance local vector concentration. An early step in wound repair is the adhesion of platelets to collagen exposed from damaged vasculature. Hence, the Moloney murine leukemia virus (MoMLV) envelope (env) protein was engineered to incorporate a high-affinity collagen-binding domain derived from von Willebrand clotting factor, and expressed in Escherichia coli and in mammalian cells. The chimeric env protein bound tightly to collagen, and virions bearing this collagen-binding env protein exhibited viral titers approaching those of virions expressing wild-type (WT) env protein. The chimeric virions were concentrated on collagen matrices, and they retained their infectivity under conditions in which virions bearing WT env protein were washed away. Targeted delivery of the chimeric env protein to injured mouse aorta and selective binding of the collagen-targeted virions to injured rabbit artery were observed. In comparative studies, vascular smooth muscle cell transduction was demonstrated in catheter-injured carotid arteries following infusion of collagen-targeted virions but not of virions bearing WT env protein. Taken together, these observations demonstrate the ability of collagen-targeted virions to localize gene delivery to sites of vascular injury.
Asunto(s)
Arterias/metabolismo , Vectores Genéticos , Virus de la Leucemia Murina de Moloney , Proteínas Oncogénicas de Retroviridae/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Factor de von Willebrand/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Aorta/metabolismo , Arterias/lesiones , Línea Celular Transformada , Colágeno/metabolismo , Endotelio Vascular/metabolismo , Ingeniería Genética , Ratones , Datos de Secuencia Molecular , Músculo Liso Vascular/metabolismo , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Oncogénicas de Retroviridae/biosíntesis , Proteínas Oncogénicas de Retroviridae/genética , Vena Cava Inferior/metabolismo , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genética , Factor de von Willebrand/genéticaRESUMEN
Expression of the entire open reading frame of hepatitis A virus was achieved in a vaccinia virus/T7/EMCV hybrid system. The expressed antigens were characterized as particles with buoyant densities and sedimentation coefficients typical of empty capsids and pentamers of HAV. This was further confirmed by electron microscopy. All capsid proteins were determined as components of the particles which raised neutralizing antibodies in an immunized rabbit.