Mechanism Of Canonical Wnt And BMP Signaling Homeostasis Disruption Leading To Pulmonary Emphysema In Mice

Chronic Obstructive Pulmonary Disease（COPD）is a common,preventable and treatable disease that is defined as persistent respiratory symptoms and airflow limitation that is due to airways and/or alveolar abnormalities usually caused by significant exposure to noxious particles or gases.According to the World Health Organization（WHO）,COPD has become the third leading cause of death in the world,with more than300 million people unfortunately suffering from it.As one of the major manifestations of COPD,pulmonary emphysema is characterized by abnormal airspace enlargement of terminal bronchiole（including respiratory bronchioles,alveolar ducts,alveolar sacs and alveoli）and destruction of the alveolar wall leading to loss of surface area required for gas exchange.Emphysema causes loss of workforce and reduced quality of life,resulting in long-term suffering and even premature death.There are currently no drugs or treatments that can completely cure emphysema,and it has become an increasingly serious global public health problem that imposes a heavy economic burden of disease on individuals,families and society.The pathogenesis of emphysema is not fully understood,especially the molecular mechanisms involved in the pathological process.Based on the available clinical data and researches from animal models,it is clear that the occurrence and development of emphysema mainly involves the following aspects:increased levels of oxidative stress,recurrent chronic inflammatory responses,protease and anti-protease imbalance,and abnormal cell apoptosis.These factors are closely related and interact with each other to participate in the intricate process of emphysema.Although these hypotheses may partially explain some of the pathological features of emphysema,they remain controversial since there is no substantial progress has been made in the development of targeted drugs and therapies based on them.Therefore,it is particularly important to elucidate the gene regulatory networks that maintain homeostasis in lung tissues and to effectively unravel the upstream pathogenic mechanisms that contribute to the occurrence of emphysema.Recently,there is growing evidence that signaling pathways that regulate developmental processes in the lungs also play an important role in maintaining adult tissue homeostasis.Several of these pathways have been targeted pharmacologically in various in vivo and ex vivo models and show good promise.This project focuses on the role of canonical Wnt and BMP signaling pathways in the occurrence and progression of emphysema.Through in-depth studies of established emphysema mouse model,we have revealed the molecular mechanism of the disruption of homeostasis in canonical Wnt and BMP signaling pathways leading to emphysema in mice.First,by examining clinical tissue samples,we found that canonical Wnt and BMP signaling pathway activity was significantly downregulated in human emphysema tissues.And then,using the Tet-on and Cre-lox P site-specific recombination systems,we constructed SPC-Cre;p Nog transgenic mice overexpressing the Noggin gene in adult mouse alveolar type 2 epithelial（AT2）cells.Noggin overexpression has been shown to effectively inhibit both canonical Wnt and BMP signaling pathway activity in lung tissues.After induction of Noggin overexpression for different periods（15 days to 6 months）,we observed that the overall lung tissues of SPC-Cre;p Nog transgenic mice was abnormally swollen,the lung parenchyma lost its original compactness and elasticity,and many vacuolar-like structures of different sizes appeared at the edges of the lungs.Morphological observation of lung tissue sections showed significant alveolar airspace enlargement,disruption of alveolar septa,and a significant increase in mean linear intercept（MLI）,which were very similar to the histological features of human emphysema.To further confirm this phenotype,we examined the pathological and physiological indicators of emphysema.The results showed that the lung tissue of SPC-Cre;p Nog transgenic mice had increased levels of oxidative stress;excessive apoptosis;massive secretion of pro-inflammatory cytokines;increased hydrolytic protease activity;severely damaged extracellular matrix;and abnormal lung function indices consistent with emphysemtous features.These fingdings are in accordance with data reported in clinical or animal models of emphysema,and effectively demonstrate SPC-Cre;p Nog transgenic mice can be employed as a kind of animal model to simulate the characteristics of emphysema well.Next,using the SPC-Cre;p Nog emphysema model mice,we delved into the molecular mechanisms underlying the downregulation of canonical Wnt and BMP signaling pathways leading to emphysema.We first confirmed that the time point at which overexpression of Noggin triggered the emphysema phenotype occurred two days after administration of doxycycline（Dox）induction in vitro.Subsequent sc RNA-seq analysis of SPC-Cre;p Nog emphysema model mice harvested at this time point was performed with their control littermates,and we found anomalous numbers and ratios of multiple inflammatory cells in the lung tissue of SPC-Cre;p Nog emphysema model mice;GO functional enrichment of differentially expressed genes in AT2 cells was mainly located in immune response,cell proliferation and apoptosis,and cell differentiation and so on;The pseudotime analysis of AT2 cells suggested that the differentiation trajectory of some AT2 cells from SPC-Cre;p Nog emphysema model mice had been affected and were stuck in the original undifferentiated state.To further validate the results obtained by sc RNA-seq,we quantified the number of inflammatory cells of all types infiltrating the bronchoalveolar lavage fluid of the SPC-Cre;p Nog emphysema model mice using flow cytometry and confirmed that the proportion of macrophages was indeed significantly increased,which was also supported more visually by immunofluorescence staining for the macrophage biomarker Mac-3,indicating that macrophages play a dominant role in mediating the inflammatory response in the initiation phase of emphysema.Moreover,we verified that downregulation of BMP/Smad signaling pathway activity suppressed the expression of its target gene NFATc3,leading to a significant reduction in the expression levels of surfactant proteins SP-A and SP-D,which are regulated by NFATc3 as a transcription factor.Since SP-A and SP-D are deeply involved in the lung innate immune response and can influence macrophage activity,it can be inferred that downregulation of canonical BMP signaling pathway located upstream leaded to massive macrophage infiltration and mediated a series of inflammatory responses in the lungs.In addition,Noggin overexpression caused abnormal apoptosis of AT2 cells and inhibited their ability to self-renew and differentiate into AT1 cells.In summary,the disturbance of homeostasis within the canonical Wnt and BMP signaling pathway causes a violent inflammatory response on the one hand,resulting in inflammatory damage to lung tissue;on the other hand,it inhibits the ability of AT2 cells to proliferate and differentiate as stem cells in the alveolar region,making them unable to perform timely and effective repair after lung tissue damage.The combined effect of these two aspects rapidly triggers the occurance of emphysema.Subsequently,we designed genetic rescue experiments using p Mes-Ca Bmp RIa and Axin2^{Lac Z} transgenic mice to enhance the activity of canonical Wnt and BMP signaling pathways in SPC-Cre;p Nog emphysema model mice,respectively or simultaneously.After histological observation,pathological examination and lung function evaluation,it can be concluded that genetic enhancement of Wnt and BMP signaling pathway activity can effectively rescue the pathological structural changes in SPC-Cre;p Nog emphysema model mice.Specifically,the overall lung tissues of the emphysema model mice regained firmness and elasticity,with a significant reduction in alveolar airspace enlargement and a significant decrease in MLI;pathological features including oxidative stress,abnormal apoptosis,active immune response and damaged extracellular matrix were also significantly alleviated;and lung function parameters were also significantly improved.The success of this genetic rescue experiment reaffirms that disturbance of homeostasis within the canonical Wnt and BMP signaling pathway is one of the major causes of emphysema in mice.Finally,we combined the use of lithium chloride（Li Cl）and isoliquiritigenin（ISL）,activators of canonical Wnt/β-catenin and BMP/Smad signaling pathways,to perform in vitro rescue experiments on SPC-Cre;p Nog emphysema model mice.The results showed that both the preventive strategy of intraperitoneal injection of Li Cl and ISL at the beginning of Noggin overexpression induction（i.e.,without emphysema phenotype）and the curative strategy of Li Cl and ISL treatment after one month of Noggin overexpression（i.e.,with severe emphysema phenotype）significantly alleviated the histopathology of the emphysema model mice.Also,most of the pathological features and lung function indices were improved to a large extent.These results highlight the feasibility and effectiveness of small molecule drugs implemented to target the canonical Wnt and BMP signaling pathways in the prevention and treatment of emphysema.In conclusion,this project provides insight into the relationship between the canonical Wnt and BMP signaling pathways and the development of emphysema,and reveals the molecular mechanism of the disruption of homeostasis within the canonical Wnt and BMP signaling pathways leading to the occurrence of emphysema in mice.These findings may provide part of the theoretical basis for clinical formulation of ideas and strategies related to the prevention and treatment of emphysema,which have important theoretical value and practical significance.