Background: Myofibroblasts, proposed as being derived from circulating fibrocytes, are considered to be important cells in thickening of the basement membrane in patients with asthma. We have studied the correlation of tissue fibrocyte levels to basement membrane thickness and the presence of fibrocytes in bronchoalveolar lavage fluid (BALF) in steroid-naive patients with mild asthma and controls.

Activated fibroblasts are suggested to be involved in the deposition of extracellular matrix in the formation of peribronchial fibrosis in asthma. We report the novel finding of activated elongated fibroblasts accompanied by elevated numbers of eosinophils in bronchoalveolar lavage fluid from 5 out of 12 patients with mild asthma (= 42%), whereas no fibroblasts were observed in the control subjects without asthma (n = 17). The elongated fibroblasts migrated twice as far when compared with fibroblasts from corresponding bronchial biopsies from the same patients, accompanied by an induced expression of RhoA and Rac1, indicating that the increased expression of these proteins are linked to increased migratory capabilities. Moreover, the elongated fibroblasts had an elevated production of the proteoglycans biglycan, versican, perlecan, and decorin, which correlated to an active cytoplasm in these cells. Differential expression patterns between the two fibroblast groups in motility-regulating proteins, such as cofilin, nuclear chloride ion channel protein, and heat-shock protein 20, were identified by two-dimensional electrophoresis and mass spectrometry. These findings indicate the presence of activated and mobile fibroblasts accompanied by an induced inflammatory response outside the airway epithelium in patients with mild asthma, results that may play a role in formation of airway fibrosis.

Introduction: Airway remodelling refers to a wide pattern of patophysiological mechanisms involving smooth muscle cell hyperplasia, increase of activated fibroblasts and myofibroblasts with deposition of extracellular matrix. In asthma, it includes alterations of the epithelial cell layer with goblet cell hyperplasia, thickening of basement membranes, peri-bronchial and peri-broncheolar fibrosis. Moreover, airway remodelling occurs not only in asthma but also in several pulmonary disorders such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and systemic sclerosis. Asthma treatment with inhaled corticosteroids does not fully prevent airway remodelling and thus have restricted influence on the natural course of the disease. Objectives: This review highlights the role of different fibroblast phenotypes and potential origins of these cells in airway remodelling. Results: During inflammatory conditions, such as asthma, fibroblasts can differentiate into an active, more contractile phenotype termed myofibroblast, with expression of stress fibres and alpha-smooth muscle actin. The origin of myofibroblasts has lately been debated, and three sources have been identified: recruitment and differentiation of resident tissue fibroblasts; fibrocytes -circulating progenitor cells; and epithelial-mesenchymal transition. Conclusion: It is clear that airway mesenchymal cells, including fibroblasts/ myofibroblasts, are more dynamic in terms of differentiation and origin than has previously been recognised. Considering that these cells are key players in the remodelling process, it is of utmost importance to characterise specific markers for the various fibroblast phenotypes and to explore factors that drive the differentiation to develop future diagnostic and therapeutic tools for asthma patients.

The objective of this study was to isolate fibrotic cells from human lung biopsies taken from different central pulmonary locations. A comparison was made of cell morphology, proteoglycan- and protein-expression in mesenchymal cell cultures obtained from human bronchial biopsies from patients with asthmatic-like disorders. We isolated viable cells from 10 out of the 12 biopsies. The fibroblast-like cells were positive for the biomarker a-smooth muscle actin, indicating that the cells were in an activated state. Two different types of fibroblast-like cells were observed from human pulmonary connective tissue; one of contractile type with lamellipodia that facilitate migration and a second cell type with an increased cell size, which most likely is of a synthetic phenotype. This is the first evidence of alterations in the proteoglycan expression pattern of versican, perlecan, biglycan and decorin which can be linked to the pathophysiological state of asthmatics proven by a combination of solid-phase extraction by reversed phase and by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Protein expression analysis using two-dimensional electrophoresis was interfaced to miniaturized sample preparation techniques using microcapillary extraction. Four protein groups were identified; cytoskeletal, adhesion, scavenger and metabolic proteins. These patient's proteomes showed a high degree of heterogeneity between patients but larger homogeneity within biopsies derived from different locations of the same patient.

Miniaturized liquid chromatography nanoseparation in combination with minigel fractionation of human primary cell nuclei is presented. We obtained high-sensitivity and high-throughput identification of expressed proteins by subcellular fractionation and nanocapillary liquid chromatography interfaced to both electrospray ionization (ESI)- and matrix-assisted laser desorption/ionisation (MALDI) tandem mass spectrometry. The reversed-phase nanocapillary eluents were applied directly onto the MALDI target plate as discrete crystal spots using in-line matrix infusion. When working with primary cells, only a limited amount of sample is available. To maximize the number of identified proteins from a restricted amount of sample, miniaturized sample preparation protocols and nanoflow separation is a necessity, especially when working with low-abundant proteins. From the same isolated nuclear sample, complementary separation of intact proteins by two-dimensional (2-D) gel electrophoresis was made. In total 594 gene products from the nuclear preparations were identified out of which 261 were unique. Several proteins involved in transcriptional events were identified such as TATA-binding protein, EBNA-co-activator, and interleukin enhancer binding proteins, indicating that sufficient proteomic depth is obtained to study transcriptional controlling events. Our results suggest that by sample prefractionation and downscaled nanoflow separation along with a combined mass spectrometry strategy, it is possible to identify a large number of nuclear proteins from human primary cells. These findings are of particular importance due to the disease link of these targets cells.