The Relationship Between The Expression Level Of ADAM33Protein And Airway Smooth Muscle Cell Mechanical Properties

Asthma is a common chronic respiratory disease, caused by multiple genetic andenvironmental factors. It is a publicly known medicine puzzle, seriously jeopardinghuman health. What a pity is that until now the mechanism(s) of its pathogenesis is notfully understood. It is showed previously that the pathogenesis of asthma involvedairway tissue remodeling, airway inflammation and airway hyperresponsiveness (AHR).It is common recognized that the possible mechanism of asthma’s pathogenesis is thechange of the mechanical properties of airway smooth muscle cell (ASMC), which canreact strongly under some stimulation, causing AHR and leading to airway obstruction.So it is important to reveal the role of airway smooth muscle cells’ mechanicscharacteristics in the etiology and pathogenesis of asthma.A disintegrin and metalloproteinase33(ADAM33) has recently been identified asan asthma susceptibility gene through positional cloning and genomewide screening.Subsequently, the correlation between ADAM33gene and airway inflammation, airwaytissue remodeling have been illuminated. However, AHR which is caused by the changeof the mechanical properties of ASMC is equally, if not more important than airwaytissue remodeling and airway inflammation in asthma, and it is showed that theexpression of ADAM33mainly in the mesenchymal tissue of asthma patients, such asairway smooth muscle, fibroblast and basement membrane, is higher than that of normal.So it is intuitive to put question to that in asthma, whether the protein level of ADAM33is altered, which may correlate with mechanics of the ASMC, even modulate themechanics of ASMC, and then eventually work together in inducing the development ofasthma. In view of this, in this study we sensitized Sprague Dawley (SD) rats withovalbumin for up to12weeks as animal models of chronic asthma, isolated and culturedASMC in vitro, then measured protein expression of ADAM33, together with stiffnessand contractility, traction force generation, and cytoskeleton structure of the ASMC thatwere obtained from the animal models at different time points of sensitization. Then wemeasured the change of ASMC mechanics in response to regulating the expression ofADAM33protein, studying the relationship of ADAM33protein and ASMC mechanics.The specific and primary experiment and results are as follows:①The protein expression level of ADAM33in the ASMC from rats in responseto OVA sensitization for up to12weeks. First, we separated the SD rats into4groups in random. The rats were injected with1%OVA and10%Al(OH)3in NaCl solution on thefirst and seventh day, then from Day15the rats were sensitized by OVA three times perweek for4weeks,6weeks, and12weeks, respectively, to mimic chronic asthma atdifferent stage. Rats treated in the same protocol but with saline instead of OVA wereused as control. Primary ASMCs were isolated from the SD rats and cultured in vitro. Inthis study all the cells used were between2-5passages. First, when ASMCs grew to90%confluence, cells were lysed to extract total proteins, then the proteins were loadedin equal volume of solution into10%SDS-PAGE, western blot analysis was performedto measure the expression of ADAM33protein. Our results shows that the proteinexpression of ADAM33in ASMC from rats of OVA-sensitized increased significantlythan control (p<0.001), the increase was the greatest when the rats were sensitized for4weeks and became gradually less afterwards.②Measure the mechanical properties of ASMCs from rats of either with orwithout OVA-sensitization. From the view point of airway function, ASMC mechanicsis generally recognized as the common final pathway to airway hyperresponsivess(AHR) that characterize asthma. So in this study, we use Optical Magnetic TwistingCytometry (OMTC), Fourier Transform Traction Microscopy (FTTM), Transwell andImmunofluorescence staining measure the mechanical properties of ASMC,including cell stiffness, contractility, cell traction force, cell migration and theexpression of cytoskeleton structure protein. Our results show that, as compared tocontrol, cell stiffness (G0) in the OVA-sensitized rats was significant increased when therats were sensitized for4weeks (p<0.05) and became gradually less afterward, however,the contractility of ASMC in response to KCl is not significant change. Similarly, thetraction force generated by ASMCs was significantly greater with the rats that had beensensitized by OVA for4weeks (p<0.05), and slightly but not significantly enhanced forthe longer duration of OVA-sensitization. The migration of ASMCs was significantlygreater with the rats that had been sensitized by OVA for4weeks (p<0.001). It seemsthat ASMC from the OVA-sensitized rats exhibited slightly higher levels offluorescence intensity for both F-actin and vinculin as compared to those from thecontrol. In particular, ASMC from the rats sensitized by OVA for4weeks exhibited thegreatest level of vinculin expression (p<0.001).③Analyze the correlation between the expression level of ADAM33protein andmechanical properties of ASMC. Our results show that OVA-induced ADAM33proteinexpression and the altered mechanical properties of ASMC in response to OVA-sensitization is similar, which seemed to indicate that the protein expression ofADAM33being correlated with mechanics of ASMC. Further analysis of these resultswith respect of Pearson correlation coefficient indeed resulted in largely positivecoefficients between the tested results of ADAM33protein expression and the measuredmechanical properties including the cell stiffness, cell traction force, cell migration andthe levels of F-actin and vinculin (Pearson correlation coefficient was0.864,0.716,0.67,0.662,0.774, respectively, as shown in Table3.1).④Measure the change of mechanical properties of ASMC in response toregulating ADAM33protein expression. Transfect the ASMCs from the rats sensitizedby OVA for4weeks, which exhibited highest protein expression of ADAM33andmechanics behaviors, to down-regulate the expression of ADAM33by the use ofLentiviral vector showed as pLVT453, and GFP-vector transfected ASMCs as control(GFP). Our results show that, as compared to non-treated ASMCs (4weeks) andGFP-vector control, cell stiffness and cell traction force of ASMCs from pLVT453weresignificant decreased in response to down-regulating the expression of ADAM33protein (p<0.05).All these results together demonstrate that, ADAM33protein strongly positivelycorrelated with the mechanical properties of ASMCs, and directly regulated themechanics of ASMCs, further induced AHR which plays key role in asthma, suggestingthat there is directly link between ADAM33protein and mechanics of ASMCs.Research the effect of ADAM33protein in regulating mechanics of ASMC and thecorresponding mechanism, may help to further uncover the pathogenesis of asthma, andthus may help find ADAM33-based novel therapeutics to treat asthma.