The slides were stained with Sirius red to assess the collagen deposition. DNA damage and apoptosis in lung tissues The number of apoptotic cells detected by the TUNEL method has been considered to reflect the degree of lung injury.10,11 The TUNEL method was performed using the DeadEnd Colorimetric Apoptosis Detection system (Promega, Michigan, USA) as previously described.12 The number of positive cells for terminal transferase\mediated dUTP nick\end\labelling (TUNEL) was counted in the whole field of each section under a microscope with 200 magnification. the significance of TGF\1 on the early inflammatory phase (day 0 to day 7) or the fibrotic phase (day 7 to day 14) in this model. Results Transfection of sTGFRII gene at 3?days before or 4?days after bleomycin instillation significantly attenuated apoptosis, injury, and fibrosis at 7 or 14 days, respectively. This method does not require the use of viral vector or neutralising antibody, and it is therefore possible to avoid problems regarding the pathogenicity of the viral vector or immunocomplex. Conclusions This novel anti\TGF\1 strategy may Abarelix Acetate have clinical application in the treatment of lung injury and fibrosis. strong class=”kwd-title” Keywords: in vivo electroporation, pulmonary fibrosis, transforming growth factor\1, apoptosis, gene therapy Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic fibrosing interstitial pneumonia associated with the histopathological appearance of usual interstitial pneumonia on surgical lung biopsy. The median survival of patients with IPF is reported to be 3C4 years from the onset of respiratory symptoms.1 In spite of such poor prognosis, the aetiology of IPF is as yet unknown and no effective therapeutic strategy has been established. The effects of current immunosuppressive therapy with corticosteroids and cytotoxic agents are limited and the adverse effects cannot be ignored. Thus, establishment of an alternative therapeutic strategy is urgently needed. Transforming growth factor\1 (TGF\1) has multiple effects that may exacerbate fibrosis. There is a consistent increase in TGF\1 production in epithelial cells and macrophages in lung tissue from patients with IPF.2 Transient overexpression of active TGF\1 through the transfection of porcine TGF\1 cDNA to the rat lung, results in prolonged and severe interstitial and pleural fibrosis.3 In the bleomycin\induced pulmonary fibrosis model, TGF\1 is expressed in alveolar macrophages at the acute phase of inflammatory cell infiltration, and in epithelial cells at the later phase of pulmonary fibrosis.4 TGF\1 is also reported to be a critical mediator of pulmonary oedema in acute lung injury.5 We previously shown that TGF\1 could induce apoptosis of small airway epithelial cells.6 TGF\1 seems to be a primary factor which induces lung injury, which subsequently leads to pulmonary fibrosis. We previously shown that mutant MCP\1 gene transfection into muscle cells by in vivo electroporation prevents the development of bleomycin\induced pulmonary fibrosis in mice.7 Skeletal muscle cells infected with an expression plasmid can produce a secreted protein into the circulating blood.8 Abarelix Acetate Soluble TGFRII has been shown to inhibit bleomycin\induced pulmonary fibrosis in the hamster.9 To investigate the new anti\TGF\1 therapy in this model, we developed a transfection strategy using in vivo electroporation that comprises transfection of the sTGFRII gene into skeletal muscles as a biofactory for anti\TGF\1 therapy in the lungs. We hypothesised that muscle cells infected with the sTGFRII gene would secrete sTGFRII protein into the circulating blood, and that this protein would then capture TGF\1 in the lung tissue, thereby blocking its signalling. This novel strategy to inhibit Abarelix Acetate TGF\1 signalling should be considered in the treatment of lung injury and fibrosis. Methods Soluble TGFRII gene transfection into muscle cells by in vivo electroporation The entire extracellular domain of TGFRII fused to the FC portion of human IgG1 was cloned into the Xho1 and Xba1 sites of the Abarelix Acetate eukaryotic expression vector pCDM. Mice were anaesthetised by an intraperitoneal injection of pentobarbital sodium (Schering\Plough, San Diego, California, USA) and in vivo electroporation was performed as previously described.7 Briefly, the sTGFRII expression plasmid vector (50?g/50?l of saline) or empty vector pCDM was injected into the femoral muscle with a 27\gauge needle. Immediately after the plasmid injection, a pair of electrode needles (Tokiwa Science, Fukuoka, Japan) spaced 5?mm apart were inserted into the femoral muscle, one on each side of the injected site. Six 100\V square wave pulses (spaced 1?s apart) were applied with an electric pulse generator CUY201 (BTX Corp., San Diego, California, USA), and the wound was closed. Analysis of sTGFRII expression by measuring human IgG in serum At 1, 3, 5, 7, 10 and 14 days after gene transfection, three mice were killed at each time point and serum was obtained. Soluble TGFRII concentrations in serum were assayed with ELISA for human IgG1. Soluble TGFRII was detectable between 1 and 14 Rabbit Polyclonal to AQP12 days in the serum; it significantly increased between 3 and 10 days after gene transfer (fig 1?1).). Based on these findings, we injected the sTGFRII gene at 3?days before or 4?days after the bleomycin instillation in order to examine the significance of TGF\1 signalling on the early inflammatory phase (day 0 to day 7) or the fibrotic phase (day 7 to day 14) in this model, respectively. Open in a separate window Figure 1?Time course of human IgG concentration in the serum after intramuscular gene transfection. Data are shown as mean (SEM) from five mice. Significance was compared with mice of day 0 (*p 0.05). Model.