The Mechanism Of Mechanical Stress Inducing Airway Inflammation And Remodeling By Regulating Lactate Metabolism Of Endothelial Cells Through Piezo1 In Asthma

Objective:Asthma is a common respiratory disease characterized by airway inflammation,variable airflow limitation and airway hyperresponsiveness.With the prolongation of the course of disease,the structure and composition of the airway wall can also be changed gradually,namely,airway remodeling.Airway inflammation was previously believed to be the main mechanism contributing to airway remodeling in asthma,but recent studies found that the mechanical stress was also involved in airway inflammation and remodeling.However,there is a lack of research on the target of mechanical force,and the mechanism of mechanical stress leading to airway remodeling in asthma is uncertain so far.Piezo1 is an important mechanically sensitive ion channel discovered in recent years,which is highly expressed in vascular endothelial cells.So far,no study which concentrated on the association between Piezo1 and asthma has been published.Endothelial cells are sensitive to mechanical force,and mechanical stress stimulation can regulate lactate metabolism of endothelial cells.Studies have shown that lactate can recruit immune cells and activate downstream inflammation pathways.Therefore,this study attempts to verify the following hypotheses:1.The pulmonary vascular endothelial cells of asthma patients are in an abnormal mechanical environment and the expression of mechanical receptor Piezo1 is up-regulated in asthma patients.2.Mechanical stress can affect lactate metabolism of endothelial cells through Piezo1,thus stimulating downstream immune cells to produce inflammatory factors,and ultimately leading to airway inflammation and remodeling in asthma.Materials and Methods:1.Clinical study:A total of 25 subjects underwent ventilation/perfusion single photon emission computed tomography（V/P SPECT）,including 20 patients with asthma and 5 healthy controls.Clinical data including gender,age,body mass index and lung function were collected for each subject.Lung tissue specimens from 13subjects who underwent video-assisted thoracoscopic surgery for lung nodules and post-operation biopsy confirmed benign lesions（inflammatory granuloma）were collected.7 subjects were diagnosed as asthma before operation（asthma group）and 6subjects had no disease history（heathy control group）.By single cell RNA sequencing（sc RNA-seq）of lung tissue,the expression of Piezo1 m RNA in each cell group of lung tissue and the differences in the expression of Piezo1 m RNA in endothelial cells between patients with asthma and control group were assayed.The distribution and expression of Piezo1 protein in the lung tissues of patients with asthma and healthy controls were also analyzed by immunohistochemical staining.2.Animal experiment:Chronic mouse asthma model was established by ovalbumin（OVA）/Al（OH）₃ adjuvant sensitization and challenge.The expression of Piezo1 m RNA in the endothelial cells of the mouse asthma model was analyzed by single-cell RNA sequencing,and the proportion of Piezo1-positive cells in the pulmonary vascular endothelial cells of the mouse asthma model was determined by flow cytometry.Piezo1 agonist Yoda1,Piezo1 inhibitors Gs MTx4 was given to the mouse asthma model,and endothelial cells Piezo1 gene specificity knockout mouse asthma model was established,HE,Masson and AB-PAS staining were used to observe the mice airway inflammatory cell infiltration,subepithelial collagen deposition and goblet cell hyperplasia degree,western blot was used to detectα-smooth muscle actin（α-SMA）and type I collagen protein expression level.Acetylcholine（Ach）or angiotensin II（AT II）were aerosolized to simulate the effect of mechanical force on pulmonary vascular endothelial cells in wild-type mice and endothelial cells Piezo1 gene specificity knockout mice.HE and Masson staining were used to observe the infiltration of airway inflammatory cells and the degree of subepithelial collagen deposition in mice.Quantitative real time polymerase chain reaction（RT-PCR）was used to detect the expression ofα-SMA m RNA in lung tissues of the mice.3.Targeted metabonomic and sc RNA-seq:Lung tissue samples were obtained from 6 subjects after pulmonary nodules operation,including 3 asthmatic patients and3 healthy control subjects.Endothelial cells were selected by antibody magnetic bead for targeted metabonomic detection to analyze the differences in endothelial cell metabolites,especially lactate levels,between patients with asthma and control group.Further analysis of the single cell RNA sequencing results of human lung tissue was performed to investigate the expression of pyruvate dehydrogenase kinase isozyme 4（PDK4）m RNA in patients with asthma and control group.4.Animal experiment and cells in vitro experiment:In the wild-type mouse asthma model and endothelial cells Piezo1 gene targeted knockout mouse asthma model,AT II was aerosolized to simulate the effect of mechanical stress on the endothelial cells.RT-PCR was used to detect the expression of PDK4 m RNA in the pulmonary vascular endothelial cells of mice.Human umbilical vein endothelial cells（HUVEC）were exogenous treated with different concentrations of Piezo1 agonist Yoda1,and the amount of lactate released by the endothelial cells was detected with a lactate content detection kit.The HUVEC Piezo1 gene was silenced with sh RNA plasmid by RNA interference technique.The expression of downstream PDK4 m RNA and monocarborxylate transporter 1（MCT1）m RNA were analyzed by RT-PCR,and the concentration of lactate released by endothelial cells was detected.HUVEC PDK4gene was silenced by si RNA plasmid,and the expression of downstream MCT1m RNA and lactate were detected by the same method.By analyzing the results of single cell RNA sequencing of human lung tissue,the cytokines highly expressed in macrophages of patients with asthma were screened out.The macrophages were stimulated exogenously with different concentrations of lactate,and the m RNA expression of the selected cytokines was detected by RT-PCR.In the endothelial-macrophage co-culture model,Piezo1/PDK4 genes were silenced with sh RNA/si RNA plasmids,respectively,and m RNA expression of cytokines produced by macrophages were detected by RT-PCR.Results:V/P SPECT images showed that,compared with healthy controls,regional ventilation obstruction was presented as well as disturbance of blood flow due to hypoxic vasoconstriction or other mechanisms was also existed in the patients with asthma.The degree of ventilation/perfusion defects was more severe in asthma patients than in healthy controls（P<0.001）.The degree of ventilation and perfusion impairment presented on V/P SPECT images was correlated with FEV₁%,and the higher the degree of ventilation/perfusion impairment,the lower FEV1%was（r=-0.74,P<0.001）.Lung tissue single cell RNA sequencing results of patients with asthma and control group indicated that Piezo1 was highly expressed in pulmonary vascular endothelial cells.Compared with the control group,the expression of Piezo1 m RNA in endothelial cells of asthma patients was up-regulated.The quantitative analysis of immunohistochemical staining positive area also showed that the expression of Piezo1was increased in the lung tissues of patients with asthma（P=0.0099）.Single cell RNA sequencing results of mouse lung tissue indicated that Piezo1was up-regulated in endothelial cells of chronic mouse model of asthma.Flow cytometry results also showed that the proportion of Piezo1 positive cells in pulmonary vascular endothelial cells of the mouse asthma model was increased（P=0.002）.HE,Masson and AB-PAS staining results showed that Piezo1 agonist Yoda1 caused increased inflammatory cell infiltration,increased subepithelial collagen deposition,and more obvious goblet cell proliferation in the mouse asthma model.Western blot indicated that Piezo1 agonist Yoda1 resulted in increased expression of type I collagen in lung tissue of the mouse asthma model.The Piezo1 inhibitor Gs MTx4 alleviated these changes in the mouse asthma model.These changes were also alleviated in the endothelial cell Piezo1 gene was specificity knockout mouse asthma model.The wild-type mice were treated with atomized Ach or AT II to simulate mechanical force,HE and Masson staining of wild-type mice showed inflammatory cell infiltration and subepithelial collagen deposition,and the m RNA expression ofα-SMA was increased（P<0.05）.The targeted knockout of Piezo1 gene in endothelial cells inhibited inflammatory cell infiltration,subepithelial collagen deposition,andα-SMA m RNA expression induced by Ach or AT II（P<0.05）.The results of targeted metabolomics indicated that the content of lactate in pulmonary vascular endothelial cells of asthma patients was increased（P=0.01）.Single cell RNA sequencing of lung tissue indicated that the expression of PDK4m RNA,a gene related to lactate metabolism,was up-regulated in endothelial cells of asthmatic patients（P<0.0001）.The expression of PDK4 m RNA in the endothelial cells was up-regulated in the mice who was treated by AT II（P=0.036）.The expression of PDK4 m RNA in the pulmonary vascular endothelial cells of mice,whose endothelial cells Piezo1 gene was targeted knockout,was down-regulated（P=0.015）.In vitro cell experiments showed that,after stimulated by exogenous 5μM and50μM Yoda1,the release of lactate of endothelial cells was increased（P<0.05）.After Piezo1 gene was silenced by sh RNA,the expression of PDK4 m RNA and MCT1m RNA in endothelial cells was down-regulated,and the release of lactate of endothelial cells was also decreased（P<0.05）.After PDK4 gene was silenced with si RNA,MCT1 m RNA expression level was down-regulated and release of lactate of endothelial cells was also decreased（P<0.05）.Single cell RNA sequencing results of human lung tissue indicated that the m RNA expression of CXCL2 and CCL8 chemokines in macrophages were up-regulated in patients with asthma（both P<0.001）.The m RNA expressions of CXCL2 and CCL8were upregulated in macrophages after treated by exogenous lactate stimulation with different concentrations.When lactate concentration was up to 20μM,compared with control group,the differences of chemokine m RNA expression were statistically significant（all P<0.05）.In endothelial cell-macrophage co-culture model,when Piezo1 or PDK4 genes were silenced with sh RNA/si RNA in endothelial cells,the m RNA expression of CXCL2 and CCL8 in macrophages were down-regulated（both P<0.05）Conclusion:1.V/P SPECT showed that airway obstruction due to bronchial constriction was presented in patients with asthma.And blood flow disturbance,which was induced by hypoxic vasoconstriction and other mechanisms was also existed in patients with asthma.Therefore,it provided us clues that there may be abnormal respiratory mechanics and hemodynamics in the lung tissue of patients with asthma,and the mechanical environment of pulmonary vascular endothelial cells may be changed and stimulated by abnormal mechanical stress.2.Mechanically sensitive ion channel Piezo1 was specifically highly expressed in pulmonary vascular endothelial cells,and the expression of Piezo1 was up-regulated in endothelial cells of asthma patients,compared to the control group.3.Piezo1 was upregulated in pulmonary vascular endothelial cells of the chronic mouse asthma model induced by OVA/Al（OH）₃.Through the Piezo1 in the endothelial cells,mechanical stress can promote airway inflammation and remodeling in the mouse asthma model.Activation of Piezo1 increased the degree of airway inflammation and remodeling in the mouse asthma model,while inhibition of Piezo1or targeted knockout of the Piezo1 gene in endothelial cells reduced the degree of airway inflammation and remodeling in the mouse asthma model.4.Atomization of Ach and AT II simulates mechanical stress effects on pulmonary vascular endothelial cells in mice,resulting in airway inflammation and remodeling in wild-type mice.Targeted knockout of endothelial cells Piezo1 gene in mouse alleviated the effects of simulated mechanical stress on airway inflammation and remodeling in mice.5.Through the Piezo1-PDK4-MCT1 pathway,mechanical stress up-regulates the metabolism of lactate in endothelial cells,and endothelial cells release lactate to promote the production of chemokines CXCL2 and CCL8 in macrophages,thereby promoting asthma airway inflammation and remodeling.6.Mechanical stress stimulates Piezo1 in endothelial cells and triggers the“endothelial cell original pathway”,leading to airway inflammation and remodeling in asthma.It is different from the classical“epithelial cell original pathway”in which allergens and other physical and chemical factors stimulate epithelial cells to trigger inflammatory cascades.