In idiopathic pulmonary fibrosis (IPF) structural properties of the extracellular matrix (ECM) are altered and influence cellular responses through cell-matrix interactions. Scaffolds (decellularized tissue) derived from subpleural healthy and IPF lungs were examined regarding biomechanical properties and ECM composition of proteins (the matrisome). Scaffolds were repopulated with healthy fibroblasts cultured under static stretch with heavy isotope amino acids (SILAC), to examine newly synthesized proteins over time. IPF scaffolds were characterized by increased tissue density, stiffness, ultimate force, and differential expressions of matrisome proteins compared to healthy scaffolds. Collagens, proteoglycans, and ECM glycoproteins were increased in IPF scaffolds, however while specific basement membrane (BM) proteins such as laminins and collagen IV were decreased, nidogen-2 was also increased. Findings were confirmed with histology, clearly showing a disorganized BM. Fibroblasts produced scaffold-specific proteins mimicking preexisting scaffold composition, where 11 out of 20 BM proteins were differentially expressed, along with increased periostin and proteoglycans production. We demonstrate how matrisome changes affect fibroblast activity using novel approaches to study temporal differences, where IPF scaffolds support a disorganized BM and upregulation of disease-associated proteins. These matrix-directed cellular responses emphasize the IPF matrisome and specifically the BM components as important factors for disease progression.

Pulmonary fibrosis is characterized by excessive accumulation of connective tissue, along with activated extracellular matrix (ECM)‐producing cells, myofibroblasts. The pathological mechanisms are not well known, however serotonin (5‐HT) and 5‐HT class 2 (5‐HT₂) receptors have been associated with fibrosis. The aim of the present study was to investigate the role of 5‐HT_2B receptors in fibrosis, using small molecular 5‐HT_2B receptor antagonists EXT5 and EXT9, with slightly different receptor affinity. Myofibroblast differentiation [production of alpha‐smooth muscle actin (α‐SMA)] and ECM synthesis were quantified in vitro, and the effects of the receptor antagonists were evaluated. Pulmonary fibrosis was also modeled in mice by subcutaneous bleomycin administrations (under light isoflurane anesthesia), and the effects of receptor antagonists on tissue density, collagen‐producing cells, myofibroblasts and decorin expression were investigated. In addition, cytokine expression was analyzed in serum. Lung fibroblasts displayed an increased α‐SMA (P < 0.05) and total proteoglycan production (P < 0.01) when cultured with TGF‐β1 together with 5‐HT, which were significantly reduced with both receptor antagonists. Following treatment with EXT5 or EXT9, tissue density, expression of decorin, number of collagen‐producing cells, and myofibroblasts were significantly decreased in vivo compared to bleomycin‐treated mice. Receptor antagonization also significantly reduced systemic levels of TNF‐α and IL‐1β, indicating a role in systemic inflammation. In conclusion, 5‐HT_2B receptor antagonists have potential to prevent myofibroblast differentiation, in vitro and in vivo, with subsequent effect on matrix deposition. The attenuating effects of 5‐HT_2B receptor antagonists on fibrotic tissue remodeling suggest these receptors as novel targets for the treatment of pulmonary fibrosis.

Silver nanoparticles (AgNPs) are commonly used in commercial and medical applications. However, AgNPs may induce toxicity, extracellular matrix (ECM) changes and inflammatory responses. Fibroblasts are key players in remodeling processes and major producers of the ECM. The aims of this study were to explore the effect of AgNPs on cell viability, both ex vivo in murine precision cut lung slices (PCLS) and in vitro in human lung fibroblasts (HFL-1), and immunomodulatory responses in fibroblasts. PCLS and HFL-1 were exposed to AgNPs with different sizes, 10 nm and 75 nm, at concentrations 2 µg/mL and 10 μg/mL. Changes in synthesis of ECM proteins, growth factors and cytokines were analyzed in HFL-1. Ag10 and Ag75 affected cell viability, with significantly reduced metabolic activities at 10 μg/mL in both PCLS and HFL-1 after 48 h. AgNPs significantly increased procollagen I synthesis and release of IL-8, prostaglandin E₂, RANTES and eotaxin, whereas reduced IL-6 release was observed in HFL-1 after 72 h. Our data indicate toxic effects of AgNP exposure on cell viability ex vivo and in vitro with altered procollagen and proinflammatory cytokine secretion in fibroblasts over time. Hence, careful characterizations of AgNPs are of importance, and future studies should include timepoints beyond 24 h.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with an unmet need of biomarkers that can aid in the diagnostic and prognostic assessment of the disease and response to treatment. In this two-part explorative proteomic study, we demonstrate how proteins associated with tissue remodeling, inflammation and chemotaxis such as MMP7, CXCL13 and CCL19 are released in response to aberrant extracellular matrix (ECM) in IPF lung. We used a novel ex vivo model where decellularized lung tissue from IPF patients and healthy donors were repopulated with healthy fibroblasts to monitor locally released mediators. Results were validated in longitudinally collected serum samples from 38 IPF patients and from 77 healthy controls. We demonstrate how proteins elevated in the ex vivo model (e.g., MMP7), and other serum proteins found elevated in IPF patients such as HGF, VEGFA, MCP-3, IL-6 and TNFRSF12A, are associated with disease severity and progression and their response to antifibrotic treatment. Our study supports the model’s applicability in studying mechanisms involved in IPF and provides additional evidence for both established and potentially new biomarkers in IPF.

Interstitial lung disease (ILD) encompasses a heterogeneous group of more than 200 conditions, of which primarily idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia, hypersensitivity pneumonitis, ILD associated with autoimmune diseases and sarcoidosis may present a progressive fibrosing (PF) phenotype. Despite different aetiology and histopathological patterns, the PF-ILDs have similarities regarding disease mechanisms with self-sustaining fibrosis, which suggests that the diseases may share common pathogenetic pathways. Previous studies show an enhanced activation of serotonergic signaling in pulmonary fibrosis, and the serotonin (5-HT)₂ receptors have been implicated to have important roles in observed profibrotic actions. Our research findings in support by others, demonstrate antifibrotic effects with 5-HT_2B receptor antagonists, alleviating several key events common for the fibrotic diseases such as myofibroblast differentiation and connective tissue deposition. In this review, we will address the potential role of 5-HT and in particular the 5-HT_2B receptors in three PF-ILDs: ILD associated with systemic sclerosis (SSc-ILD), ILD associated with rheumatoid arthritis (RA-ILD) and IPF. Highlighting the converging pathways in these diseases discloses the 5-HT_2B receptor as a potential disease target for PF-ILDs, which today have an urgent unmet need for therapeutic strategies.