RESUMO
The enzyme, nitric oxide-sensitive guanylyl cyclase (NO-GC), is activated by binding NO to its prosthetic heme group and catalyzes the formation of cGMP. The NO-GC is primarily known to mediate vascular smooth muscle relaxation in the lung, and inhaled NO has been successfully used as a selective pulmonary vasodilator. In comparison, NO-GC's impact on the regulation of airway tone is less acknowledged and, most importantly, little is known about the issue that NO-GC signaling is accomplished by two isoforms: NO-GC1 and NO-GC2, implying the existence of distinct "cGMP pools." Herein, we investigated the functional role of the NO-GC isoforms in respiration by measuring lung function parameters of isoform-specific knockout (KO) mice using noninvasive and invasive techniques. Our data revealed the participation and ongoing influence of NO-GC1-derived cGMP in the regulation of airway tone by showing that respiratory resistance was enhanced in NO-GC1-KOs and increased more pronouncedly after the challenge with the bronchoconstrictor methacholine. The tissue resistance and stiffness of NO-GC1-KOs were also higher because of narrowed airways that cause tissue distortion. Contrariwise, NO-GC2-KOs displayed reduced tissue elasticity, elastic recoil, and airway reactivity to methacholine, which did not even increase in an ovalbumin model of asthma that induced hyperresponsiveness in NO-GC1-KOs. In addition, conscious NO-GC2-KOs showed a higher breathing rate with a shorter duration of inspiration and expiration time, which remained faster even in the presence of bronchoconstrictors that slow down breathing. Thus, we provide evidence of two distinct NO/cGMP pathways in airways, accomplished by either NO-GC1 or NO-GC2, adjusting differentially the airway reactivity.
Assuntos
Broncoconstritores , Guanilato Ciclase , Animais , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Heme , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Ovalbumina , Isoformas de Proteínas/metabolismo , Guanilil Ciclase Solúvel/metabolismo , VasodilatadoresRESUMO
BACKGROUND: Asthma is an inflammatory disease of the respiratory system, and a major factor of increasing health care costs worldwide. The molecular actors leading to the development of chronic asthma are not fully understood and require further investigation. OBJECTIVE: The aim of this study was to monitor the proteome dynamics during asthma development from early inflammatory to late fibrotic stages. METHODS: A mouse asthma model was used to analyse the lung proteome at four time points during asthma development (0 weeks = control, 5, 8 and 12 weeks of treatment, n = 6 each). The model was analysed using lung function tests, immune cell counting and histology. Furthermore, a multi-fraction mass spectrometry-based proteome analysis was performed to achieve a comprehensive coverage and quantification of the lung proteome. RESULTS: At early stages, the mice showed predominant eosinophilic inflammation of the airways, which disappeared at later stages and was replaced by marked airway hyper-reactivity and fibrosis of the airways. 3325 proteins were quantified with 435 proteins found to be significantly differentially abundant between the experimental groups (ANOVA p-value ≤.05, maximum fold change ≥1.5). We applied hierarchical clustering to identify common protein abundance profiles along the asthma development and analysed these clusters using gene ontology annotation and enrichment analysis. We demonstrate the correlation of protein clusters with the course of asthma development, that is eosinophilic inflammation and fibrotic remodelling of the airways. CONCLUSIONS AND CLINICAL RELEVANCE: Proteome analysis revealed proteins that were previously described to be important during asthma chronification. Moreover, we identified additional proteins previously not described in the context of asthma. We provide a comprehensive data set of a long-term mouse model of asthma that may contribute to a better understanding and allow new insights into the progression and development of chronic asthma. Data are available via ProteomeXchange with identifier PXD011159.
Assuntos
Asma , Proteoma , Animais , Modelos Animais de Doenças , Ontologia Genética , Humanos , Pulmão , CamundongosRESUMO
Idiopathic pulmonary fibrosis (IPF) is a disease characterized by extensive fibrosis of the lung tissue. Wnt5a expression was observed to be upregulated in IPF and suggested to be involved in the progression of the disease. Interestingly, smooth muscle cells (SMC) are a major source of Wnt5a in IPF patients. However, no study has been conducted until now to investigate the precise role of smooth muscle-derived Wnt5a in IPF. Here, we used the bleomycin-induced lung fibrosis model in a conditional gene-deficient mouse, where the Wnt5a gene was excised from SMC. We show here that the excision of the Wnt5a gene in SMC led to significantly improved health conditions with minimized weight loss and improved lung function. This improvement was based on a significantly lower deposition of collagen in the lung with a reduced number of fibrotic foci in lung parenchyma. Furthermore, the bleomycin-induced cellular infiltration into the airways was not altered in the gene-deficient mice compared with wild-type mice. Thus, we demonstrate that the Wnt5a expression of SMC of the airways leads to aggravated fibrosis of the lung with poor clinical conditions. This aggravation was not an influence in the bleomycin-induced inflammatory processes but on the development of fibrotic foci in lung parenchyma and the deposition of collagen.
RESUMO
Allergic airway inflammation is accompanied by excessive generation of nitric oxide (NO). Beside its detrimental activity due to the generation of reactive nitrogen species, NO was found to modulate immune responses by activating the NO-sensitive Guanylyl Cyclases (NO-GCs) thereby mediating the formation of the second messenger cyclic GMP (cGMP). To investigate the contribution of the key-enzyme NO-GC on the development of Th2 immunity in vivo, we sensitized knock-out (KO) mice of the major isoform NO-GC1 to the model allergen ovalbumin (OVA). The loss of NO-GC1 attenuates the Th2 response leading to a reduction of airway inflammation and IgE production. Further, in vitro-generated OVA-presenting DCs of the KO induce only a weak Th2 response in the WT recipient mice upon re-exposure to OVA. In vitro, these NO-GC1 KO BMDCs develop a Th1-polarizing phenotype and display increased cyclic AMP (cAMP) formation, which is known to induce Th1-bias. According to our hypothesis of a NO-GC1/cGMP-dependent regulation of cAMP-levels we further demonstrate activity of the cGMP-activated cAMP-degrading phosphodiesterase 2 in DCs. Herewith, we show that activity of NO-GC1 in DCs is important for the magnitude and bias of the Th response in allergic airway disease most likely by counteracting intracellular cAMP.