The Role Of A Disintegrin And Metalloproteinase 33 And Interaction Of It And IFN-γ In Asthma

BackgroundAsthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. It is a problem worldwide, with an estimated 300 million affected individuals. In addition to the inflammatory response, there are characteristic structural changes, often described as airway remodeling, in the airways of asthma patients including children. It is seen even before the onset of symptoms.The pathologic features of airway remodeling include subepithelial fibrosis, airway smooth muscle hypertrophy and hyperplasia, Blood vessels in airway walls proliferate, Mucus hypersecretion results from increased numbers of goblet cells. The reason that caused the remodeling is not very clear. Now the aim at treatment of asthma is to control the onset and save lives. Up to now, not any drugs may reverse the airway remodeling of asthma patient.ADAM33, a recently discovered ADAM family member, has been identified as an asthma susceptibility gene on chromosome 20p13 encoding a metalloproteinase that contributes to airway remodeling. ADAM33 is a highly polymorphic gene containing more than 70 single nucleotide polymorphisms, some of which have been shown to be associated with asthma and AHR. ADAM33 is preferentially expressed by smooth muscle cells, myofibroblasts, and fibroblasts, but not by epithelial cells, T cells, or inflammatory cells. The selective expression of ADAM33 in cells of mesenchymal origin suggests that alterations in the activity of ADAM33 may cause abnormalities in the function of smooth muscle cells and fibroblasts, indicating a possible role for ADAM33 in airway remodeling. To date, there no related report about the role of ADAM33 in chronic asthma model has been seen.Recent understanding of asthma pathogenesis has shown that asthma is an allergic inflammatory disorder of the airways characterized as a imbalance in the expression of Th1/Th2 cytokines. IFN-γ, a Th1 cytokine, plays an important role in chronic inflammation of asthma. IFN-γcan strongly inhibit the activities of Th2 cytokines such as IL-5 and IL-13. The level of Th1 cytokin IFN-γhas been found to be dereased in the BALF from patients with asthma. IFN-γcan markly spressed the airway hyperreactivity in asthma model.IFN-γmay play a role in pathogenesis of remodeling in lung diease. IFN-γcan suppress bronchiolar inflammation and fibrosis, and abnormalities in pulmonary resistance and dynamic compliance in viral bronchiolitis. In the bleomycin-mouse model of lung fibrosis, IFN-γdownregulates TGF-B gene expression and suppresses both the proliferation of fibroblasts and collagen synthesis. In patients with idiopathic pulmonary fibrosis, treatment using IFN-γwith a low dose of prednisolone has been reported to reduce the levels of transcription of both TGF-β1 and connective tissue growth factor (CTGF) genes, and to improve lung function and blood gases.Studies in animal models of allergic bronchopulmonary inflammation, including investigations in genetargeted mice deficient in one or more Th2 cytokines, have suggested that the manifestations of asthma may be ameliorated by targeting these mediators. However, clinical trials involving anti-IL-5 or soluble IL-4 receptor have thus far proved to be disappointing. This discordance may be related to the failure of animal models involving short-term challenge with high levels of antigen to replicate many of the features of chronic human asthma or to the induction of various compensatory mechanisms in gene-targeted cytokine-deficient animals. Reliable assessment of potential treatments is more likely to be achieved using an experimental model of chronic allergic disease of the airways.In this study, we aimed at investigating the effects of AdCMV IFN-γon the expression levels of ADAM33 mRNA and protein in mouse fibroblasts and in the allergen-induced airway remodeling mice. To suppress the expression of a disintegrin and metalloproteinase 33(ADAM33) gene, we established ADAM33 pGCL-GFP shRNA lentivirus vectors, and successfully knocked down the expression of ADAM33 gene and protein. These provided an effective tool for the further studies on experimental animals of ADAM33 gene knockdowns and the specific mechanisms of ADAM33 gene in the pathogenesis of asthma.Chapter 1 The effect of murine IFN-γon a disintegrin and metalloproteinase-33 in allergen-induced mouse asthmamodelObjective To evaluate the effect of AdCMVmlFN-γand recombinant murine IFN-γ(rmIFN-γ) on the expression of ADAM33 and airway remodeling in ovalbumin (OVA)-induced mouse asthma models by preventive or therapeutic intervention.Methods Mice were sensitized on days 0 and 14 by intraperitoneal injections of OVA complexed with alum, and repeatedly challenged from days 24 to 41 by intranasal OVA to establish a mouse model of asthma characterized by airway inflammation and airway remodeling. Mice were treated with AdCMVmlFN-γor rmlFN-γon day 23 (1 day before OVA challenge) for preventive intervention or on day 42 for therapeutic intervention. The outcome measurements included ADAM33 mRNA and protein, airway inflammatory indices, eosinophil count, collagen deposition around the bronchus, and depth of airway smooth muscle by RT-PCR, western blot, HE, and Masson's staining.Results The OVA-sensitized/challenged mice developed extensive eosinophil inflammatory infiltration, airway smooth muscle hypertrophy, collagen deposition, and high-level expression of ADAM33. Preventive intervention with AdCMVmlFN-γsignificantly reduced the airway eosinophil infiltration, smooth muscle hyperplasia, sub-epithelial fibrosis, and ADAM33 expression in the OVA-sensitized/challenged mice. However, therapeutic intervention using AdCMVmlFN-γand rmlFN-γafter establishment of airway remodeling only partially reduced the expression of ADAM33 and airway remodeling.Conclusions ADAM33 may play a role in the process of airway smooth muscle hypertrophy and collagen deposition in asthma patients. Preventive intervention with AdCMVmlFN-γcan significantly inhibit the expression of ADAM33 and reduce airway remodeling, as compared with a normal, untreated control. However, therapeutic intervention with AdCMVmlFN-γwas only able to partially accomplish this response.Chapter 2 The effect of murine IFN-γon a disintegrin andmetalloproteinase-33 in murine fibroblast cellsObjective To evaluate the effect of AdCMVmlFN-γand recombinant murine IFN-γ(rmlFN-γ) on the expression of ADAM33 in mouse fibroblast (NIH/3T3). Methods The outcome measurements included mIFN-γ, ADAM33 mRNA and protein by ELISA, RT-PCR and western blot. Results In vitro, AdCMVmlFN-γand rmlFN-γreduced ADAM33 mRNA and protein expression by mouse fibroblast cells in a time- and dose-dependent manner. In the rmlFN-γgroup, ADAM33 mRNA and protein concentration began to slowly increase within 24-48 hours. The transgenic expression by AdCMVmlFN-γwas able to continuously inhibit ADAM33 mRNA and protein until 96h. Conclusions mIFN-γreduced ADAM33 mRNA and protein expression in a time- and dose-dependent manner. The effect of AdCMVmlFN-γis longer than rmlFN-γ.Chapter 3 Suppression of a disintegrin and metalloproteinase 33 in mouse fibroblast cells by Lentivirusshort hairpin RNAsObjective To suppress the expression of a disintegrin and metalloproteinase 33(ADAM33) gene, different short hairpin RNA(shRNA) eukaryotic expression vectors were constructed to find out the strongest antagonized function for ADAM33 expression. Methods Four different shRNA sequences specific to the pro-domain, metalloproteinase domain and transmembrane domain of ADAM33 gene were selected and their eukaryotic expression vector(pGCL-GFP shRNA1,2,3,4) were constructed respectively. The vectors with different ADAM33 shRNA were transfected into 3T3 cells. At 24, 48 and 72 hours after transfection, the transfection efficiency was identified by fluorescence microscope. The expression levels of ADAM33 mRNA and protein were validated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Results Recombinant plasmids were completely coincided with the designs by the sequences analysis. The recombinant ADAM33 eukaryotic expression vectors were constructed successfully. The expression level of ADAM33 mRNA and protein decreased to 49.3±2.52%和30.1±5.51% respectively (P<0.001) compared with the negative control, was significantly inhibited in 3T3 cells by pGCL-GFP shRNA3 at 48 hour after transfection. Conclusions The data suggested that different domains of ADAM33 have different effect on their expression, and transmembrane domain be the most important one. The expression of ADAM33 gene could be strongly suppressed by the specific shRNA sequences to the transmembrane domain.