Anti-Fibrotic and Anti-Inflammatory Role of NO-Sensitive Guanylyl Cyclase in Murine Lung.

Pulmonary fibrosis is a chronic and progressive disease with limited therapeutic options. Nitric oxide (NO) is suggested to reduce the progression of pulmonary fibrosis via NO-sensitive guanylyl cyclase (NO-GC). The exact effects of NO-GC during pulmonary fibrosis are still elusive. Here, we used a NO-GC knockout mouse (GCKO) and examined fibrosis and inflammation after bleomycin treatment. Compared to wildtype (WT), GCKO mice showed an increased fibrotic reaction, as myofibroblast occurrence (p = 0.0007), collagen content (p = 0.0006), and mortality (p = 0.0009) were significantly increased. After fibrosis induction, lymphocyte accumulations were observed in the lungs of GCKO but not in WT littermates. In addition, the total number of immune cells, specifically lymphocytes (p = <0.0001) and neutrophils (p = 0.0047), were significantly higher in the bronchoalveolar lavage fluid (BALF) of GCKO animals compared to WT, indicating an increased inflammatory response in the absence of NO-GC. The pronounced fibrotic response in GCKO mice was paralleled by significantly increased levels of transforming growth factor ß (TGFß) in BALF (p = 0.0207), which correlated with the total number of immune cells. Taken together, our data show the effect of NO-GC deletion in the pathology of lung fibrosis and the effect on immune cells in BALF. In summary, our results show that NO-GC has anti-inflammatory and anti-fibrotic properties in the murine lung, very likely by attenuating TGFß-mediated effects.

NO-sensitive guanylyl cyclase discriminates pericyte-derived interstitial from intra-alveolar myofibroblasts in murine pulmonary fibrosis.

BACKGROUND: The origin of αSMA-positive myofibroblasts, key players within organ fibrosis, is still not fully elucidated. Pericytes have been discussed as myofibroblast progenitors in several organs including the lung. METHODS: Using tamoxifen-inducible PDGFRß-tdTomato mice (PDGFRß-CreERT2; R26tdTomato) lineage of lung pericytes was traced. To induce lung fibrosis, a single orotracheal dose of bleomycin was given. Lung tissue was investigated by immunofluorescence analyses, hydroxyproline collagen assay and RT-qPCR. RESULTS: Lineage tracing combined with immunofluorescence for nitric oxide-sensitive guanylyl cyclase (NO-GC) as marker for PDGFRß-positive pericytes allows differentiating two types of αSMA-expressing myofibroblasts in murine pulmonary fibrosis: (1) interstitial myofibroblasts that localize in the alveolar wall, derive from PDGFRß+ pericytes, express NO-GC and produce collagen 1. (2) intra-alveolar myofibroblasts which do not derive from pericytes (but express PDGFRß de novo after injury), are negative for NO-GC, have a large multipolar shape and appear to spread over several alveoli within the injured areas. Moreover, NO-GC expression is reduced during fibrosis, i.e., after pericyte-to-myofibroblast transition. CONCLUSION: In summary, αSMA/PDGFRß-positive myofibroblasts should not be addressed as a homogeneous target cell type within pulmonary fibrosis.

A modular systems biological modelling framework studies cyclic nucleotide signaling in platelets.

BACKGROUND: The cyclic nucleotides cAMP and cGMP inhibit platelet activation. Different platelet signaling modules work together. We develop here a modelling framework to integrate different signaling modules and apply it to platelets. RESULTS: We introduce a novel standardized bilinear coupling mechanism allowing sub model debugging and standardization of coupling with optimal data driven modelling by methods from optimization. Besides cAMP signaling our model considers specific cGMP effects including external stimuli by drugs. Moreover, the output of the cGMP module serves as input for a modular model of VASP phosphorylation and for the activity of cAMP and cGMP pathways in platelets. Experimental data driven modeling allows us to design models with quantitative output. We use the condensed information about involved regulation and system responses for modeling drug effects and obtaining optimal experimental settings. Stepwise further validation of our model is given by direct experimental data. CONCLUSIONS: We present a general framework for model integration using modules and their stimulus responses. We demonstrate it by a multi-modular model for platelet signaling focusing on cGMP and VASP phosphorylation. Moreover, this allows to estimate drug action on any of the inhibitory cyclic nucleotide pathways (cGMP, cAMP) and is supported by experimental data.

NO-sensitive guanylyl cyclase in the lung.

In the late 1960s, several labatories identified guanylyl cyclase (GC) as the cGMP-producing enzyme. Subsequently, two different types of GC were described that differed in their cellular localization. Primarily found in the cytosol, nitric oxide (NO)-sensitive guanylyl cyclase (NO-GC) acts as receptor for the signalling molecule NO, in contrast the membrane-bound isoenzyme is activated by natriuretic peptides. The lung compared with other tissues exhibits the highest expression of NO-GC. The enzyme has been purified from lung for biochemical analysis. Although expressed in smooth muscle cells (SMCs) and in pericytes, the function of NO-GC in lung, especially in pericytes, is still not fully elucidated. However, pharmacological compounds that target NO-GC are available and have been implemented for the therapy of pulmonary arterial hypertension. In addition, NO-GC has been suggested as drug target for the therapy of asthma, acute respiratory distress syndrome and pulmonary fibrosis. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.