New avenues for Epac in inflammation and tissue remodeling in COPD

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease of the lung mainly caused by cigarette smoke. The main characteristic of COPD is a progressive loss of lung function due to inflammation and tissue remodeling, including airway fibrosis and emphysema. Little is known about the molecular mechanisms that cause the development and progression of COPD. This thesis aims to acquire more knowledge about these processes, to be able to make steps forward to improve current therapy for COPD. In this thesis the main focus is on the effector protein of the signaling molecule cyclic AMP, “exchange protein directly activated by cyclic AMP” (Epac), which has two isoforms, Epac1 and Epac2. In airway smooth muscle cells we show that Epac inhibits cigarette smoke extract (CSE)-induced inflammation. Interestingly, Epac1 protein expression is downregulated in lung tissue from COPD patients, which may result from increased expression of miRNA-7. In epithelial cells CSE reduces the expression of the adhesion molecule E-cadherin and the protein AKAP9, leading to reduction of the epithelial barrier that protects the airways against detrimental stimuli. AKAPs form complexes to localize proteins, including Epac, to specific cell compartments, thereby regulating cellular functions. In vivo studies demonstrated that Epac1 and Epac2 are indeed involved in inflammatory and remodeling processes induced by cigarette smoke that are also seen in COPD patients. The experiments described and discussed in this thesis reveal potential new drug targets that may lead to improved therapy of COPD

Multiple facets of cAMP signalling and physiological impact:cAMP compartmentalization in the lung

Therapies involving elevation of the endogenous suppressor cyclic AMP (cAMP) are currently used in the treatment of several chronic inflammatory disorders, including chronic obstructive pulmonary disease (COPD). Characteristics of COPD are airway obstruction, airway inflammation and airway remodelling, processes encompassed by increased airway smooth muscle mass, epithelial changes, goblet cell and submucosal gland hyperplasia. In addition to inflammatory cells, airway smooth muscle cells and (myo)fibroblasts, epithelial cells underpin a variety of key responses in the airways such as inflammatory cytokine release, airway remodelling, mucus hypersecretion and airway barrier function. Cigarette smoke, being next to environmental pollution the main cause of COPD, is believed to cause epithelial hyperpermeability by disrupting the barrier function. Here we will focus on the most recent progress on compartmentalized signalling by cAMP. In addition to G protein-coupled receptors, adenylyl cyclases, cAMP-specific phospho-diesterases (PDEs) maintain compartmentalized cAMP signalling. Intriguingly, spatially discrete cAMP-sensing signalling complexes seem also to involve distinct members of the A-kinase anchoring (AKAP) superfamily and IQ motif containing GTPase activating protein (IQGAPs). In this review, we will highlight the interaction between cAMP and the epithelial barrier to retain proper lung function and to alleviate COPD symptoms and focus on the possible molecular mechanisms involved in this process. Future studies should include the development of cAMP-sensing multiprotein complex specific disruptors and/or stabilizers to orchestrate cellular functions. Compartmentalized cAMP signalling regulates important cellular processes in the lung and may serve as a therapeutic target

PI3K inhibition reduces murine and human liver fibrogenesis in precisioncut liver slices

Background: Liver fibrosis results from continuous inflammation and injury. Despite its high prevalence worldwide, no approved antifibrotic therapies exist. Omipalisib is a selective inhibitor of the PI3K/mTOR pathway that controls nutrient metabolism, growth and proliferation. It has shown antifibrotic properties in vitro. While clinical trials for idiopathic pulmonary fibrosis have been initiated, an in-depth preclinical evaluation is lacking. We evaluated omipalisib's effects on fibrogenesis using the ex vivo model of murine and human precision-cut tissue slices (PCTS). Methods: Murine and human liver and jejunum PCTS were incubated with omipalisib up to 10 mu M for 48 h. PI3K pathway activation was assessed through protein kinase B (Akt) phosphorylation and antifibrotic efficacy was determined via a spectrum of fibrosis markers at the transcriptional and translational level. Results: During incubation of PCTS the PI3K pathway was activated and incubation with omipalisib prevented Akt phosphorylation (IC50 = 20 and 1.5 nM for mouse and human, respectively). Viability of mouse and human liver PCTS was compromised only at the high concentration of 10 and 1-5 mu M, respectively. However, viability of jejunum PCTS decreased with 0.1 (mouse) and 0.01 mu M (human). Spontaneously increased fibrosis related genes and proteins were significantly and similarly suppressed in mouse and in human liver PCTS. Conclusions: Omipalisib has antifibrotic properties in ex vivo mouse and human liver PCTS, but higher concentrations showed toxicity in jejunum PCTS. While the PI3K/mTOR pathway appears to be a promising target for the treatment of liver fibrosis, PCTS revealed likely side effects in the intestine at higher doses

Effects of (a Combination of) the Beta2-Adrenoceptor Agonist Indacaterol and the Muscarinic Receptor Antagonist Glycopyrrolate on Intrapulmonary Airway Constriction

Expression of bronchodilatory β2-adrenoceptors and bronchoconstrictive muscarinic M3-receptors alter with airway size. In COPD, (a combination of) β2-agonists and muscarinic M3-antagonists (anticholinergics) are used as bronchodilators. We studied whether differential receptor expression in large and small airways affects the response to β2-agonists and anticholinergics in COPD. Bronchoprotection by indacaterol (β2-agonist) and glycopyrrolate (anticholinergic) against methacholine- and EFS-induced constrictions of large and small airways was measured in guinea pig and human lung slices using video-assisted microscopy. In guinea pig lung slices, glycopyrrolate (1, 3 and 10 nM) concentration-dependently protected against methacholine- and EFS-induced constrictions, with no differences between large and small intrapulmonary airways. Indacaterol (0.01, 0.1, 1 and 10 μM) also provided concentration-dependent protection, which was greater in large airways against methacholine and in small airways against EFS. Indacaterol (10 μM) and glycopyrrolate (10 nM) normalized small airway hyperresponsiveness in COPD lung slices. Synergy of low indacaterol (10 nM) and glycopyrrolate (1 nM) concentrations was greater in LPS-challenged guinea pigs (COPD model) compared to saline-challenged controls. In conclusion, glycopyrrolate similarly protects large and small airways, whereas the protective effect of indacaterol in the small, but not the large, airways depends on the contractile stimulus used. Moreover, findings in a guinea pig model indicate that the synergistic bronchoprotective effect of indacaterol and glycopyrrolate is enhanced in COPD

Small airway hyperresponsiveness in COPD: relationship between structure and function in lung slices

The direct relationship between pulmonary structural changes and airway hyperresponsiveness (AHR) in chronic obstructive pulmonary disease (COPD) is unclear. We investigated AHR in relation to airway and parenchymal structural changes in a guinea pig model of COPD and in COPD patients. Precision-cut lung slices (PCLS) were prepared from guinea pigs challenged with lipopolysaccharide or saline two times weekly for 12 wk. Peripheral PCLS were obtained from patients with mild to moderate COPD and non-COPD controls. AHR to methacholine was measured in large and small airways using video-assisted microscopy. Airway smooth muscle mass and alveolar airspace size were determined in the same slices. A mathematical model was used to identify potential changes in biomechanical properties underlying AHR. In guinea pigs, lipopolysaccharide increased the sensitivity of large (>150 μm) airways toward methacholine by 4.4-fold and the maximal constriction of small airways

Transcriptomic characterization of culture-associated changes in murine and human precision-cut tissue slices

Our knowledge of complex pathological mechanisms underlying organ fibrosis is predominantly derived from animal studies. However, relevance of animal models for human disease is limited; therefore, an ex vivo model of human precision-cut tissue slices (PCTS) might become an indispensable tool in fibrosis research and drug development by bridging the animal-human translational gap. This study, presented as two parts, provides comprehensive characterization of the dynamic transcriptional changes in PCTS during culture by RNA sequencing. Part I investigates the differences in culture-induced responses in murine and human PCTS derived from healthy liver, kidney and gut. Part II delineates the molecular processes in cultured human PCTS generated from diseased liver, kidney and ileum. We demonstrated that culture was associated with extensive transcriptional changes and impacted PCTS in a universal way across the organs and two species by triggering an inflammatory response and fibrosis-related extracellular matrix (ECM) remodelling. All PCTS shared mRNA upregulation of IL-11 and ECM-degrading enzymes MMP3 and MMP10. Slice preparation and culturing activated numerous pathways across all PCTS, especially those involved in inflammation (IL-6, IL-8 and HMGB1 signalling) and tissue remodelling (osteoarthritis pathway and integrin signalling). Despite the converging effects of culture, PCTS display species-, organ- and pathology-specific differences in the regulation of genes and canonical pathways. The underlying pathology in human diseased PCTS endures and influences biological processes like cytokine release. Our study reinforces the use of PCTS as an ex vivo fibrosis model and supports future studies towards its validation as a preclinical tool for drug development

Anti-Inflammatory Role of the cAMP Effectors Epac and PKA: Implications in Chronic Obstructive Pulmonary Disease

Cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (IL-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (ASM) cells, may contribute to the development of chronic obstructive pulmonary disease (COPD). Despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic AMP (cAMP), little is known about its exact mechanism of action. We report here that next to the β2-agonist fenoterol, direct and specific activation of either exchange protein directly activated by cAMP (Epac) or protein kinase A (PKA) reduced cigarette smoke extract (CSE)-induced IL-8 mRNA expression and protein release by human ASM cells. CSE-induced IκBα-degradation and p65 nuclear translocation, processes that were primarily reversed by Epac activation. Further, CSE increased extracellular signal-regulated kinase (ERK) phosphorylation, which was selectively reduced by PKA activation. CSE decreased Epac1 expression, but did not affect Epac2 and PKA expression. Importantly, Epac1 expression was also reduced in lung tissue from COPD patients. In conclusion, Epac and PKA decrease CSE-induced IL-8 release by human ASM cells via inhibition of NF-κB and ERK, respectively, pointing at these cAMP effectors as potential targets for anti-inflammatory therapy in COPD. However, cigarette smoke exposure may reduce anti-inflammatory effects of cAMP elevating agents via down-regulation of Epac1

Performance of non-invasive tests and histology for the prediction of clinical outcomes in patients with non-alcoholic fatty liver disease: an individual participant data meta-analysis

BackgroundHistologically assessed liver fibrosis stage has prognostic significance in patients with non-alcoholic fatty liver disease (NAFLD) and is accepted as a surrogate endpoint in clinical trials for non-cirrhotic NAFLD. Our aim was to compare the prognostic performance of non-invasive tests with liver histology in patients with NAFLD.MethodsThis was an individual participant data meta-analysis of the prognostic performance of histologically assessed fibrosis stage (F0–4), liver stiffness measured by vibration-controlled transient elastography (LSM-VCTE), fibrosis-4 index (FIB-4), and NAFLD fibrosis score (NFS) in patients with NAFLD. The literature was searched for a previously published systematic review on the diagnostic accuracy of imaging and simple non-invasive tests and updated to Jan 12, 2022 for this study. Studies were identified through PubMed/MEDLINE, EMBASE, and CENTRAL, and authors were contacted for individual participant data, including outcome data, with a minimum of 12 months of follow-up. The primary outcome was a composite endpoint of all-cause mortality, hepatocellular carcinoma, liver transplantation, or cirrhosis complications (ie, ascites, variceal bleeding, hepatic encephalopathy, or progression to a MELD score ≥15). We calculated aggregated survival curves for trichotomised groups and compared them using stratified log-rank tests (histology: F0–2 vs F3 vs F4; LSM: 2·67; NFS: 0·676), calculated areas under the time-dependent receiver operating characteristic curves (tAUC), and performed Cox proportional-hazards regression to adjust for confounding. This study was registered with PROSPERO, CRD42022312226.FindingsOf 65 eligible studies, we included data on 2518 patients with biopsy-proven NAFLD from 25 studies (1126 [44·7%] were female, median age was 54 years [IQR 44–63), and 1161 [46·1%] had type 2 diabetes). After a median follow-up of 57 months [IQR 33–91], the composite endpoint was observed in 145 (5·8%) patients. Stratified log-rank tests showed significant differences between the trichotomised patient groups (p<0·0001 for all comparisons). The tAUC at 5 years were 0·72 (95% CI 0·62–0·81) for histology, 0·76 (0·70–0·83) for LSM-VCTE, 0·74 (0·64–0·82) for FIB-4, and 0·70 (0·63–0·80) for NFS. All index tests were significant predictors of the primary outcome after adjustment for confounders in the Cox regression.InterpretationSimple non-invasive tests performed as well as histologically assessed fibrosis in predicting clinical outcomes in patients with NAFLD and could be considered as alternatives to liver biopsy in some cases