Bronchial asthma is a chronic inflammatory disease in which bronchial wall remodelling plays a significant role. This phenomenon is related to enhanced proliferation of airway smooth muscle cells, elevated extracellular matrix protein secretion and an increased number of myofibroblasts. Phenotypic fibroblast-to-myofibroblast transition represents one of the primary mechanisms by which myofibroblasts arise in fibrotic lung tissue. Fibroblast-to-myofibroblast transition requires a combination of several types of factors, the most important of which are divided into humoural and mechanical factors, as well as certain extracellular matrix proteins. Despite intensive research on the nature of this process, its underlying mechanisms during bronchial airway wall remodelling in asthma are not yet fully clarified. This review focuses on what is known about the nature of fibroblast-to-myofibroblast transition in asthma. We aim to consider possible mechanisms and conditions that may play an important role in fibroblast-to-myofibroblast transition but have not yet been discussed in this context. Recent studies have shown that some inherent and previously undescribed features of fibroblasts can also play a significant role in fibroblast-to-myofibroblast transition. Differences observed between asthmatic and non-asthmatic bronchial fibroblasts (e.g., response to transforming growth factor β, cell shape, elasticity, and protein expression profile) may have a crucial influence on this phenomenon. An accurate understanding and recognition of all factors affecting fibroblast-to-myofibroblast transition might provide an opportunity to discover efficient methods of counteracting this phenomenon.
Pathologic accumulation of myofibroblasts in asthmatic bronchi is regulated by extrinsic stimuli and by the intrinsic susceptibility of bronchial fibroblasts to transforming growth factor-β (TGF-β). The specific function of gap junctions and connexins in this process has remained unknown. Here, we investigated the role of connexin43 (Cx43) in TGF-β-induced myofibroblastic differentiation of fibroblasts derived from bronchoscopic biopsy specimens of patients with asthma and donors without asthma. Asthmatic fibroblasts expressed considerably higher levels of Cx43 and were more susceptible to TGF-β-induced myofibroblastic differentiation than were their nonasthmatic counterparts. TGF-β efficiently up-regulated Cx43 levels and activated the canonical Smad pathway in asthmatic cells. Ectopic Cx43 expression in nonasthmatic (Cx43) fibroblasts increased their predilection to TGF-β-induced Smad2 activation and fibroblast-myofibroblast transition. Transient Cx43 silencing in asthmatic (Cx43) fibroblasts by Cx43 small interfering RNA attenuated the TGF-β-triggered Smad2 activation and myofibroblast formation. Direct interactions of Smad2 and Cx43 with β-tubulin were demonstrated by co-immunoprecipitation assay, whereas the sensitivity of these interactions to TGF-β signaling was confirmed by Förster Resonance Energy Transfer analyses. Furthermore, inhibition of the TGF-β/Smad pathway attenuated TGF-β-triggered Cx43 up-regulation and myofibroblast differentiation of asthmatic fibroblasts. Chemical inhibition of gap junctional intercellular communication with 18 α-glycyrrhetinic acid did not affect the initiation of fibroblast-myofibroblast transition in asthmatic fibroblasts but interfered with the maintenance of their myofibroblastic phenotype. Collectively, our data identified Cx43 as a new player in the feedback mechanism regulating TGF-β/Smad-dependent differentiation of bronchial fibroblasts. Thus, our observations point to Cx43 as a novel profibrotic factor in asthma progression.
Investigating intestinal physiology in vitro remains challenging due to the lack of an effective primary enterocyte culture system. Recently developed protocols for growing organoids containing crypts and villus from adult mouse intestinal epithelium in Matrigel present an attractive alternative to the classical techniques. However, these approaches require the use of sophisticated and expensive serum-free medium supplemented with epithelial growth factor (EGF), Wnt agonist (R-spondin 1), and bone morphogenetic protein inhibitor (Noggin) in high concentrations. Here we demonstrate that is possible to use an isolated chicken embryonic intestinal epithelium to create such an organoid culture. Structures formed in Matrigel matrix in the first two days following isolation survive and enlarge during ensuing weeks. They have the appearance of empty spheres and comprise cells expressing cytokeratin (an epithelial cell marker), villin (a marker of enterocytes), and Sox-9 (a transcription factor characteristic of progenitors and stem cells of intestinal crypts). With chicken embryonic tissue as a source of organoids, prostaglandin E2 is as effective as R-spondin 1 and Noggin in promoting sustained growth and survival of epithelial spheroids.
The role of airway wall remodelling in bronchial asthma is well established. Myofibroblasts, the cells displaying features intermediate between fibroblasts and smooth muscle cells, are involved in this process but the mechanism of myofibroblasts activation in the onset of the disease remains obscure. Myofibroblasts can differentiate from various cell types, including resident fibroblasts, and the fibroblasts to myofibroblasts transition (FMT) can be reproduced in vitro. We aimed to investigate the process of FMT in human bronchial fibroblasts (HBF) derived from non-asthmatic (n = 7) and asthmatic (n = 7) subjects. We also tested whether cell-cell contacts affect FMT by using N-cadherin blocking antibody. HBF plated in low or high cell density were treated with TGF-β(1) up to one week to induce FMT. The percentage of myofibroblsts was counted and expression of α-smooth muscle actin was evaluated by cytoimmunofluorescence, flow cytometry and immunobloting. We demonstrated that the intensity of FMT induced by TGF-β(1)in vitro was strongly enhanced in asthmatic as compared to non-asthmatic HBF populations. This process was facilitated by low cell plating density in both groups of cultures. Furthermore, we proved that neither HBF-conditioned medium nor growth arrest in G(0)/G(1) phase of cell cycle could stop the TGF-β(1)-induced FMT in asthmatic cell populations. However, even in sparse asthmatic HBF, the blocking of N-cadherin resulted in the inhibition of FMT. Our findings show for the first time that the initial absence or an induced loss of cell-cell adhesions in asthmatic HBF populations is important for the completion of FMT.
Suppressive function of connexin(Cx)43 in carcinogenesis was recently contested by reports that showed a multifaceted function of Cx43 in cancer progression. These studies did not attempt to model the dynamics of intratumoral heterogeneity involved in the metastatic cascade. An unorthodox look at the phenotypic heterogeneity of prostate cancer cells in vitro enabled us to identify links between Cx43 functions and Snail-1-regulated functional speciation of invasive cells. Incomplete Snail-1-dependent phenotypic shifts accounted for the formation of phenotypically stable subclones of AT-2 cells. These subclones showed diverse predilection for invasive behavior. High Snail-1 and Cx43 levels accompanied high motility and nanomechanical elasticity of the fibroblastoid AT-2_Fi2 subclone, which determined its considerable invasiveness. Transforming growth factor-β and ectopic Snail-1 overexpression induced invasiveness and Cx43 expression in epithelioid AT-2 subclones and DU-145 cells. Functional links between Snail-1 function and Cx43 expression were confirmed by Cx43 downregulation and phenotypic shifts in AT-2_Fi2, DU-145 and MAT-LyLu cells upon Snail-1 silencing. Corresponding morphological changes and Snail-1 downregulation were seen upon Cx43 silencing in AT-2_Fi2 cells. This indicates that feedback loops between both proteins regulate cell invasive behavior. We demonstrate that Cx43 may differentially predispose prostate cancer cells for invasion in a coupling-dependent and coupling-independent manner. When extrapolated to in vivo conditions, these data show the complexity of Cx43 functions during the metastatic cascade of prostate cancer. They may explain how Cx43 confers a selective advantage during cooperative invasion of clonally evolving, invasive prostate cancer cell subpopulations.
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