| Objective:This study aimed to explore the relationship between ADAM33 and inflammatory cytokines,pulmonary function,eosinophilic inflammation indicators and airway vascular remodeling.It is also sought to discuss the effect of ADAM33 gene silencing on the proliferation,apoptosis,migration,cell cycle of airway vascular smooth muscle cells(VSMCs)and explore the possible molecular mechanism.Furthermore,it aimed to investigate the effect of ADAM33 gene silencing in VSMCs on the proliferation and lumen formation of airway vascular endothelial cells(VECs)in a co-culture system and the possible regulatory mechanism.Methods:1)The research subjects were selected from the patients in outpatient and inpatient departments of the First Affiliated Hospital of Xinjiang Medical University during January 2018 to December 2019,including 103asthma cases and 60 physical examination volunteers.The clinical data,peripheral blood and serum samples were collected from all the research subjects,and 38 samples of lung tissue were obtained from the above observers.The expression of inflammatory cytokines IFN-γ,IL-4,IL-13,IL-17a,IL-10,1L-9 and IL-21 in serum of the research subjects were detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of ADAM33 m RNA in the peripheral blood were measured by quantitative Real-time PCR(q RT-PCR).The expression and distribution of ADAM33 in lung tissues were determined by immunohistochemistry(IHC).Additionally,the correlation between ADAM33 expression levels in asthma patients and the clinical and pathological parameters,including inflammatory cytokines,EOS count,peripheral blood T-Ig E level,pulmonary function,FENO value,airway wall thickness(WAt/Pbm),airway smooth muscle thickness(WAm/Pbm),airway smooth muscle nucleus number(N/Pbm)and airway vascular density(A/mm~2)were analyzed.2)Human aortic smooth muscle cells(HASMCs)were used as experimental cells.After the measurement of the background expression of ADAM33m RNA in HASMCs by q RT-PCR,the ADAM33 gene-silencing HASMC cell lines were constructed using lentiviral transfection technology.The changes in the proliferation,migration,apoptosis and cell cycle of HASMCs after ADAM33knockdown were detected by CCK-8 proliferation experiment,Transwell migration and flow cytometry(FCM)assay.The changes in the PI3K/Akt pathway and its downstream signaling molecules(PI3K、Akt、m TOR、ERK,Rho,ROCK,Bcl-2,Bax,FOXO1 and Cyclin D1)at m RNA and protein levels were determined by q RT-PCR and Western-blotting.3)The HASMCs and human pulmonary microvascular endothelial cells(HPMECs)were used to construct a cell co-culture system.A further CCK-8proliferation assay and a Transwell lumen formation experiment were used to observe the effect of ADAM33 gene silencing on the proliferation and lumen formation of HPMECs in a co-culture system.An ELISA assay and Western-blotting detected the effects of ADAM33 gene silencing on s ADAM33,VEGFA,VEGER,ang1 and ang2 in the co-culture system,along with the changes in the target molecules in Tie2/PI3K/Akt signaling pathway and the phosphorylation levels of AKT and m TOR.Results:1)The expression of IFN-γ,IL-4,IL-13,IL-17a,IL-9 and IL-21 was significantly increased in moderate and severe asthma groups,while the expression of IL-10 was decreased in the moderate and severe asthma groups.The expression of ADAM33m RNA in the peripheral blood of the asthma groups was higher than in that of the control group,and it indicated a difference:severe>Moderate>mild.The immunohistochemical staining of ADAM33 in lung tissue showed that ADAM33 was expressed mainly in epithelial cells,smooth muscle cells and interstitial inflammatory cells in the lung tissue of asthma patients,and also can be found in airway VSMCs.The airway vascular remodeling values WAt/Pbm and A/mm~2were higher in the mild asthma group and moderate-severe asthma group than in the control group,while WAm/Pbm and N/Pbm were significantly higher in the moderate-severe asthma group,but there was no difference between the mild asthma group and the control group.The expressions of IL-4,IL-13,IL-17a and IL-21 in asthma patients were positively correlated with the level of ADAM33m RNA,while the expression of IL-10 was negatively correlated with the level of ADAM33m RNA.The ADAM33 level in peripheral blood was negatively correlated with FEV1%and FVC%,positively correlated with FENO value,and had no significant correlation with FEV1/FVC,EOS count or T-Ig E.The level of ADAM33 in peripheral blood was positively correlated with airway vascular remodeling indexes At/Pbm,WAm/Pbm,N/Pbm and A/mm~2.2)Silencing the ADAM33 gene could significantly inhibited the proliferation and migration of HASMCs,promoted apoptosis and G0/G1 cell cycle arrest of HASMCs.Compared with the control group and the LV-NC group,the expression levels of PI3K,Rho,Bcl-2,FOXO1 and Cyclin D1m RNA in the LV-ADAM33 group were significantly decreased,while the expression levels of Bax m RNA was increased.The protein expression levels of PI3K,p-Akt,p-ERK,p-m TOR,ROCK,Bcl-2,p-FOXO1 and Cyclin D1 were also significantly lower than in the control group and the LV-NC group,while the protein expression of Bax was higher.3)ADAM33 gene silencing also affected the ability of HASMCs to secrete inflammatory cytokines,resulting in the decreased expression of TNF-αand the increased expression of IFN-γ.The content of s ADAM33 was very low when HPMECs were cultured alone,and was significantly increased when HASMCs and HPMECs were co-cultured.Silencing the ADAM33 gene expression of HASMCs in the co-culture system decreased the expression of s ADAM33 and inhibited the proliferation and lumen formation of HPMECs.After silencing the expression of ADAM33 gene in HASMCs in co-culture system,the decreased expression of pro-angiogenic factors(VEGFA,VEGFR2,ang1,ang2)and the decreased protein levels of Tie2,PI3K,p-Akt and P-m TOR in Tie2/PI3K/Akt signaling pathway were also observed.Conclusion:1)The expression of ADAM33 in asthma patients is related to the stimulation of inflammatory cytokines,which may be directly or indirectly regulated by the complex network formed by inflammatory cytokines.ADAM33 is correlated with pulmonary function parameters FEV1%、FVC%and airways eosinophilic inflammation index FENO,which can reflect the severity of airflow restriction and eosinophilic inflammation in asthma patients.ADAM33 is also closely related to the pathological indicators of airway vascular remodeling,and plays an important role in airway remodeling in asthma.2)Silencing the expression of the ADAM33 gene could regulate the proliferation,migration,apoptosis and cell cycle in airway VSMCs,which may be mediated by the PI3K/Akt signaling pathway and downstream signaling molecules.3)Silencing the expression of the ADAM33 gene could reduce the release of s ADAM33 from the membrane of the airway VSMCs,regulate the proliferation and lumen formation of airway VECs by reducing the expression of VEGF/VEGFR and inhibiting the activities of the Angs/Tie2 and PI3K/Akt/m TOR signaling pathways,participate in the airway vascular remodeling. |
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