ADAM33 Modulation Alters Mechanics Of Airway Smooth Muscle Cells Through Rho/ROCK Pathway Mediated Phenotype Transition

Asthma is a common chronic respiratory inflammatory disease,but revealing the pathogenesis is still difficult.Asthma is characterized by airway remodeling and airway airway hyperresponsivness(AHR).In recent years,it has been found that asthma is also a genetic disease.ADAM33 gene was proved to be the susceptibility gene of asthma,and the expression of airway smooth muscle and basal membrane was significantly higher in the majority of asthma patients than in healthy controls.Many clinical and experimental evidence suggests that the overexpression of ADAM33 gene is associated with airway remodeling and airway hyper responsiveness,and this association is likely to be directly related to the mechanical behavior of airway smooth muscle.Up to now,there is no evidence to clarify the relationship between the biomechanical behavior of airway smooth muscle cells and the expression of ADAM33.Therefore,we hypothesized that ADAM33 overexpression could promote airway remodeling and the AHR by affecting the mechanical and biological behavior of airway smooth muscle.We found in our previous study that sADAM33 overexpression can significantly increase the stiffness and contractility of ASMCs,and the ability of lateral migration,reduce the migration in the Transwell.Confirmed that the overexpression of sADAM33 did affect the mechanical behavior of airway smooth muscle.However,no mechanism was revealed.Therefore,we make a further study for the effect of ADAM33 modulation on airway smooth muscle contraction,cyclic stretch,transient stretch,migration behavior and adhesion ability,and investigated its mechanism.This study may provide a useful reference to explain the pathogenesis and effective treatment of asthma.The main research contents and results are as follows:1)A preliminary study on the mechanism of the effect of ADAM33 expression on the ASMCs baseline stiffness and contractilityAfter surume deprived for 48 h,three contractile proteins was significantly upregulated by sADAM33 overexpression.The Rho-GTP activation was upregulated by sADAM33 overexpression too,about 2 fold higher compared with GFP group.However,all these regulation changed by sADAM33 overexpression can be effectively depressed by ROCK inhibitor H1152.All these evidence suggested that sADAM33 overexpression can induce the accumulation of contractile proteins and induced a hypercontractile phenotype of ASMCs in the end,and probably through Rho/ROCK pathway.We also collected the conditioned culture medium from sADAM33-overexpressed ASMCs,and confirmed the concentration of sADAM33 in it.Then use this conditioned culture medium to culture control ASMCs,found that the excessive sADAM33 in medium can upregulated contractile marker protein expression,and induced the phenotype transition.This result suggested sADAM33 can work as a soluble protien as paracrine.2)The effect of ADAM33 modulation on ASMCs responding to cyclic strain and transient stretch,and verify whether ADAM33 is a mechanical sensitive gene.After cyclic uniaxial stretch of 0.3Hz,5% for 48 h,sADAM33 overexpression can cooperate with uniaxial stretch effect and significantly upregulated contractile protein expression,and upregulated SM-MHC expression at a greater extent.It suggested that sADAM33 overexpression can upregulate SM-MHC expression much more and promote myosin filament assembling when ASMCs exposed to the cyclic strain,which mimic the tidal breath.This regulation may stabilize and form a longer myosin filament to generate a higher passive contraction.Therefore may lead to a hypercontractile phenotransition of ASMCs to contract much more much easier,to promote AHR.In addition,the ADAM33 expression was only upregulated in the earlier phase(24 h)during cyclic strain indicated that ADAM33 response mecahnical stimulation has time scale.After transient stretch,the traction force of ASMCs which sADAM33 overexpression was significantly higher than others,and keep higher traction force during the recovery.This suggested that sADAM33 overexpression may weaken the soften effect of ASMCs induced by deep inspiration compared with normal people,and accelerate re-stiffning of ASMCs.This founding was accordance with what happened in asthma airway.3)ADAM33 modulation regulated the migration behavious and adhesion ability of ASMCs.sADAM33 overexpression do not significantly alter the net displacement and migration speed of ASMCs in scratched line,but upregulated the directional efficiency and the intensity of direction angle.That's mean sADAM33 overexpression induced upregulation of migration ability may not through enhance the cells' migration speed,but through enhanced intercellular junctions which lead ASMCs migrated more likely with each other as collective migration,and induced a relatively intensity and stable migration direction.The single cell migration data reinforce this assumption,because sADAM33 overexpression reduced net displacement and migration speed.This may because sADAM33 overexpression induced ASMCs into a more contractile phenotype with the increased stiffness and reduced deformability,and thus reduced the ability of transmembrane migration.The above research results is important for further understand the role of ADAM33 in airway remodeling and AHR,and important for explain the contradictory result of our previous study.This study is not only the first time in the world to reveal the mechanism correlation between the ADAM33 overexpression and airway remodeling/AHR,but also provided a cell functional relation between ASMCs polymorphisms and asthma susceptibility,and provide important clues for the therapeutic strategy about control sADAM33 release.