Modulation Of ADAM33Gene Expression Alters Biomechanical Behavior Of Airway Smooth Muscle Cells

Asthma is a common chronic respiratory inflammatory disease, because bronchialhyperresponsiveness, allergens or other factors will cause a wide range of airwaynarrow, resulting in chest tightness, coughing, dyspnea. As one of the main parts ofairway, airway smooth muscle cell (ASMC) plays an important physiological role inairway contraction. Airway hyper-responsiveness (AHR) occurs when airway isstimulated by allergens causing clinical symptoms such as airway excessive constrictionand paroxysmal breathing disorder, and in turn making asthma a high-risk disease.Although contractile property of ASMCs has been identified for more than a century,the mechanism of pathological changes remains elusive. Airway smooth muscle is theeffector to control tracheal diameter, and its pathological contraction is the final factorof airway obstruction and breathing difficulties. It has played a key role in theeffectiveness of asthma treatment. A disintegrin and metalloprotease33(ADAM33) hasbeen identified as a susceptibility gene for asthma, being particularly associated withbronchial hyper-responsiveness. However, there is still no direct evidence to prove therelationship between the biomechanical behavior of ASMCs and ADAM33. Here, wegenerated transgenic ASMCs over-expressing or slincing ADAM33gene by lentiviraltransfection, and monitored the changes of biomechanical behaviors of the transgenicASMCs. The main experiments and results are as follows:①The expression level of ADAM33increased along with the severity of asthma.Firstly, we established an asthma model with SD (Sprague Dawley) rat using OVAsensitization with Aluminum hydroxide gel as an adjuvant. We treated the rats withOVA atomization for30minute every other day for12weeks. Lung tissues weresampled every two weeks from the treated and non-treated rats. Penh was assayed andairway tissue sections were observed with H&E staining. The results showed that wesuccessfully established an asthma model with SD rats. The transcriptional and proteinlevels of ADAM33gene in ASMCs were assessed by quantitative RT-PCR and Westernblot, respectively. As expected, the expression level of ADAM33increased during OVAatomization, positively correlated with asthma severity. This is consistent with Lee’sfindings in asthma patients.②Obtained ADAM33-over-expressing/silencing ASMCs.We firstly constructedADAM33over-expressing or silencing recombinant vectors, followed by lentiviral packaging, concentration, titer determination and lentiviral transfection. Non-transfected and GFP-vector transfected ASMCs were set as controls.24h after infection,GFP expression gradually increased and reached the peak at72h. Infection efficiencyreached95%when MOI=100. Quantitative RT-PCR demonstrated that thetranscriptional level of ADAM33was induced by8folds in overexpressor than incontrol (P﹤0.01), while silencing efficiency reached to75%(P﹤0.01). At protein level,similar results were obtained by Western blot analysis. No significant difference wasobserved between non-transfected control and GFP-vector control. It is indicated thatADAM33-over-expressing/silencing ASMCs were successfully engineered by lentiviraltransfection.③Change of ADAM33expression level affects cell migration and cellproliferation. MTT assay were implemented to investigate cells’ proliferation ability.ADAM33-over-expressing ASMCs had the same cell proliferation ability comparedwith controls, but the ability was lower in ADAM33silencing cells than controls (P﹤0.01). It should be noted that GFP-controls showed decreased cell proliferation compareto non-transfected controls, suggesting that lentiviral infection can suppress cellproliferation. Wound healing assay showed a dramatic increase of cell migration abilityin ADAM33overexpressor (P﹤0.01), indicating a role of ADAM33in lateral transferof ASMCs.④ADAM33can regulate the stiffness, contraction ability and traction force ofASMCs.Biomechanical characters such as traction force, contraction ability andstiffness were assessed by Fourier transform traction force microscopy (FTTM) andoptical magnetic twisting cytometry (OMTC). The results showed that the aboveinvestigated biomechanical characters were higher in ADAM33-over-expressingASMCs, and lower in ADAM33-silencing cells compared with controls. Laser scanningconfocal microscopy (LSCM) revealed the colocalization of ADAM33and vinculin,and distribution of some ADAM33along stress fiber. Furthermore, two cellbiomechanics-related genes, Calponin and Integrin-β1, were positively regulated byADAM33in ASMCs.In conclusion, we demonstrated that ADAM33regulated the cellular andbiomechanical behavior of ASMCs, including cell migration, cell proliferation, tractionforce, contraction ability and stiffness. It is suggested that ADAM33gene expressionpositively correlates with the biomechanical characters of ASMCs, and it plays acritical role in airway remodeling and AHR. These results will better our understandings on the mechanisms of AHR in asthma, and facilitate to explore effective therapies anddrugs.