RESUMEN
BACKGROUND: Transforming growth factor beta inducible early gene-1 (TIEG-1), a member of the Krüppel-like factor, was identified as a primary response gene for TGF-ß. The role of TIEG-1 in skin repair has been mainly addressed in vivo on TIEG-1 null mice model and the mechanism remains unexplored. METHODS: We investigated the modulation of TIEG-1 expression in normal human skin fibroblasts by either down-expressing or overexpressing the gene. We evaluated reactive oxygen species production and the cell viability of treated cells. The effect of TIEG-1 overexpression was monitored by wound healing assay and immunofluorescence staining of actin fibers organization and alpha-smooth muscle actin (α-SMA). Western blots were carried out to identify the level of expression or phosphorylation of key proteins such as cofilin, Rho GTPases, and p38 mitogen-activated protein kinase (p38 MAPK). RESULTS: TIEG-1 down-regulation had a deleterious effect on the cell viability. It was significantly reduced (65±5%) and exposure to ultraviolet further increased this effect (47±3%). By contrast, cells overexpressing TIEG-1 had a reduced reactive oxygen species production (75%) compared to control and mock-transfected cells. This overexpression also resulted in formation of actin stress fibers and increased α-SMA expression and an enhanced wound healing feature. RhoB GTPase was upregulated and phosphorylation of cofilin and p38 MAPK was observed. CONCLUSION: TIEG-1 overexpression in normal human skin fibroblasts results in improved resistance to oxidative stress, myofibroblast-like conversion that involved RhoB signaling pathway with cofilin and p38 MAPK proteins activation. GENERAL SIGNIFICANCE: This study enlightens the role of TIEG-1 role in skin biology.
Asunto(s)
Citoesqueleto de Actina/química , Factores de Transcripción de la Respuesta de Crecimiento Precoz/fisiología , Fibroblastos/metabolismo , Factores de Transcripción de Tipo Kruppel/fisiología , Estrés Oxidativo , Factores Despolimerizantes de la Actina/metabolismo , Movimiento Celular , Células Cultivadas , Humanos , Fosforilación , Piel/citología , Cicatrización de Heridas , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Tendon injury is a major musculoskeletal disorder with a high public health impact. We propose a non-viral based strategy of gene therapy for the treatment of tendon injuries using histidylated vectors. Gene delivery of fibromodulin, a proteoglycan involved in collagen assembly was found to promote rat Achilles tendon repair in vivo and in vitro. In vivo liposome-based transfection of fibromodulin led to a better healing after surgical injury, biomechanical properties were better restored compared to untransfected control. These measures were confirmed by histological observations and scoring. To get better understandings of the mechanisms underlying fibromodulin transfection, an in vitro tendon healing model was developed. In vitro, polymer-based transfection of fibromodulin led to the best wound enclosure speed and a pronounced migration of tenocytes primary cultures was observed. These results suggest that fibromodulin non-viral gene therapy could be proposed as a new therapeutic strategy to accelerate tendon healing. FROM THE CLINICAL EDITOR: Tendon injury is relatively common and healing remains unsatisfactory. In this study, the effects of liposomal-based delivery of fibromodulin gene were investigated in a rat Achilles tendon injury model. The positive results observed would provide a new therapeutic strategy in clinical setting in the future.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Técnicas de Transferencia de Gen , Terapia Genética , Proteoglicanos/genética , Traumatismos de los Tendones/terapia , Tendón Calcáneo/patología , Adenoviridae/genética , Animales , Proteínas de la Matriz Extracelular/biosíntesis , Fibromodulina , Vectores Genéticos , Humanos , Liposomas/química , Masculino , Proteoglicanos/biosíntesis , Ratas , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/patología , Cicatrización de Heridas/genéticaRESUMEN
Microbubble oscillation at specific ultrasound settings leads to permeabilization of surrounding cells. This phenomenon, referred to as sonoporation, allows for the in vitro and in vivo delivery of extracellular molecules, including plasmid DNA. To date, the biological and physical mechanisms underlying this phenomenon are not fully understood. The aim of this study was to investigate the interactions between microbubbles and cells, as well as the intracellular routing of plasmid DNA and microbubbles, during and after sonoporation. High-speed imaging and fluorescence confocal microscopy of HeLa cells stably expressing enhanced green fluorescent protein fused with markers of cellular compartments were used for this investigation. Soft-shelled microbubbles were observed to enter cells during sonoporation using experimental parameters that led to optimal gene transfer. They interacted with the plasma membrane in a specific area stained with fluorescent cholera subunit B, a marker of lipid rafts. This process was not observed with hard-shelled microbubbles, which were not efficient in gene delivery under our conditions. The plasmid DNA was delivered to late endosomes after 3 h post-sonoporation, and a few were found in the nucleus after 6 h. Gene transfer efficacy was greatly inhibited when cells were treated with chlorpromazine, an inhibitor of the clathrin-dependent endocytosis pathway. In contrast, no significant alteration was observed when cells were treated with filipin III or genistein, both inhibitors of the caveolin-dependent pathway. This study emphasizes that microbubble-cell interactions do not occur randomly during sonoporation; microbubble penetration inside cells affects the efficacy of gene transfer at specific ultrasound settings; and plasmid DNA uptake is an active mechanism that involves the clathrin-dependent pathway.
Asunto(s)
Clatrina/metabolismo , Electroporación/métodos , Endocitosis/fisiología , Plásmidos/genética , Sonicación/métodos , Transfección/métodos , Permeabilidad de la Membrana Celular/fisiología , Permeabilidad de la Membrana Celular/efectos de la radiación , Endocitosis/efectos de la radiación , Células HeLa , Humanos , Microburbujas , Plásmidos/administración & dosificación , Plásmidos/química , Transducción de Señal/fisiología , Transducción de Señal/efectos de la radiación , Ondas UltrasónicasRESUMEN
Non-viral gene carriers for safe and efficient gene transfection have become of particular interest among researchers of different disciplines ranging from physical chemistry to biotechnology. Recently polymeric vectors have been extensively studied as potentially new gene transfer agents. Until now most of the research efforts were made to optimize the gene-to-polymer weight ratio of polyplexes for safe and efficient gene transfection. In this work, we report on the development of novel poly(allylamine) derivatives with different balance of the primary, secondary, tertiary, and quaternary amino groups. All derivatives were able to complex pDNA into polyplexes at low gene-to-polymer weight ratios i.e., 1:1 or 1:2. Moreover, the examined polyplexes were less cytotoxic and showed better transfection efficiency when compared to linear poly(ethyleneimine). These results indicate that the presence of quaternary ammonium groups is important in the formation of stable polyplexes. Polymers with all types of amino groups showed large potential for gene delivery. Furthermore, polyplexes with such derivatives were well internalized by cells and ended up into acidic late endosomes.
Asunto(s)
ADN/administración & dosificación , Técnicas de Transferencia de Gen , Poliaminas/administración & dosificación , Transporte Biológico , Supervivencia Celular/efectos de los fármacos , ADN/química , Endosomas/metabolismo , Células HeLa , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Plásmidos , Poliaminas/químicaRESUMEN
Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking.