| 1. |
- Bjärdahlen, Anette, et al.
(författare)
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Myofibroblast accumulation correlates with the formation of fibrotic tissue in a rat air pouch model.
- 2002
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Ingår i: Journal of Rheumatology. - 0315-162X. ; 29:8, s. 1698-1707
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: The pathogenesis of arthritic joints involves cartilage degradation and pannus formation. It is well known that pannus influences the cartilage; however, the mechanism behind how the degrading cartilage interacts with pannus is not well known. To investigate this interplay, the expression of extracellular matrix (ECM) components in pannus and the degrading cartilage was analyzed. METHODS: Studies were performed using a rat air pouch model where cotton with viable or killed cartilage was implanted into 7-day-old pouches for 1-28 days. The remodeling of cartilage and the formation of tissue in the cotton was characterized histologically by quantitation of infiltrated cells. The amounts of collagen, hyaluronan, and proteoglycan were estimated. RESULTS: Implantation of homologous femoral head cartilage in cotton resulted in extensive remodeling of cartilage and formation of ECM in the cotton. In cotton without cartilage, fibroblasts and myofibroblasts were the predominant cells in the early stage of analyses. The ECM formed in cotton was of a fibrotic type, with mainly collagen and smaller amounts of proteoglycans correlating to the presence of myofibroblasts. In the cotton with cartilage, however, inflammatory cells such as neutrophils, macrophages, and lymphocytes dominated. Delayed accumulation of collagen and increased synthesis of proteoglycans occurred early in cotton with viable as well as non-viable cartilage. In later stages, the cell pattern changed and the myofibroblasts emerged together with an increasing collagen formation. CONCLUSION: The interaction between cartilage and the newly formed granulation tissue results in a faster degradation of cartilage molecules, which in turn leak into the surrounding ECM and affect the recruitment of myofibroblasts. This indicates the importance of the micromatrix.
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| 2. |
- Cheng, Fang, et al.
(författare)
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Differences in the uptake and nuclear localization of anti-proliferative heparan sulfate between human lung fibroblasts and human lung carcinoma cells
- 2001
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 83:4, s. 597-606
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate inhibits the proliferation of normal human lung fibroblasts (HFL-1) but not of a human lung carcinoma cell-line (A549). in this study we investigated possible mechanisms and structural requirements by which anti proliferative heparan sulfates exerts its effects on binding, uptake and subcellular localisation. Both HFL-1 and A549 cells were incubated with I-125- or rhodamine-labeled L-iduronate-rich antiproliferative heparan sulfate species as well as L-iduronate-poor inactive ones. The anti proliferative heparan sulfate was bound to the cell surface on both HFL-1 and A549 cells, but to a lesser extent and with less affinity to A549 cells. Both cell types bound the anti proliferative heparan sulfate with one high- and with one low affinity site. The L-iduronate-poor heparan sulfate bound to a lesser extent and with less affinity to both cell types compared to the anti proliferative heparan sulfate. The antiproliferative heparan sulfate accumulated in the cytoplasm of HFL-1 cells after 24 h incubation, but after 72 h it was found evenly distributed in the nucleus. The time-scale for anti proliferative activity correlated with nuclear localization. In contrast, in A549 cells it was only found near the nuclear membrane. The inactive heparan sulfate was taken up in considerably smaller amounts compared to the antiproliferative heparan sulfate and could not be detected in the nucleus of either HFL-1 or A549 cells. Our data suggest that the anti proliferative activity of L-iduronate-rich heparan sulfate on normal fibroblasts may be due to direct effects on nuclear processes, such as gene transcription.
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| 3. |
- Eklund, Erik, et al.
(författare)
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Proteoglycan production in disomic and trisomy 7-carrying human synovial cells.
- 2002
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Ingår i: Matrix Biology. - 1569-1802. ; 21:4, s. 325-335
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Tidskriftsartikel (refereegranskat)abstract
- To gain further insight into the synthesis and structure of the synovial matrix of joints, we have established cell cultures from synovial specimens and elaborated their production of hyaluronan and proteoglycans. The cultures secreted mainly the small proteoglycan decorin, but also considerable amounts of the related biglycan and the large proteoglycan versican. Only minor amounts of heparan sulfate proteoglycans were found. All cultures also had a high production of hyaluronan, which highlights the important role for normal joint function of these cells. In joint diseases, a common feature is the presence of an extra chromosome 7 (trisomy 7) in the synovial cells. To study the possible consequences of trisomy 7 on the synovial cell function, we extended our study to cultures that had been sub-cloned to contain high amounts of trisomy 7-carrying cells. These cell cultures had approximately four times more versican than their disomic counterparts in the cell culture medium, indicating that versican may be a mediator in the processes of joint destructive disorders. To find an explanation for this increase in versican, we investigated the expression/secretion of PDGF-AA and IL-6, cytokines with their genes located to chromosome 7. Indeed, both these cytokines were increased in the cultures with high frequencies of trisomy 7. We then added the two cytokines to cell cultures of disomic synovial cells, but only cells treated with IL-6 displayed an increased amount of versican. Thus, we suggest that the increased amount of versican in cultures of trisomy 7-carrying cells relates to an autocrine loop involving an increased IL-6 production.
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| 4. |
- Hesselstrand, Roger, et al.
(författare)
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The association between changes in skin echogenicity and the fibroblast production of biglycan and versican in systemic sclerosis
- 2002
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Ingår i: Clinical and Experimental Rheumatology. - 1593-098X. ; 20:3, s. 301-308
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Tidskriftsartikel (refereegranskat)abstract
- Objective To investigate a possible association between the longitudinal changes in skin involvement and the fibroblast production of proteoglycans in vitro, among patients with early and untreated systemic sclerosis (SSc). Methods In 11 patients, 6 with diffuse cutaneous systemic sclerosis (dSSc) and 5 with limited cutaneous systemic sclerosis (lSSc), and in 6 controls skin thickness and skin echogenicity, of the forearm was measured by high frequency (20 MHz) ultrasound, A skin biopsy was taken from the area of the ultrasound measurements, and from cultivated fibroblasts the production of the proteoglycans versican, perlecan, biglycan and decorin were measured. To investigate longitudinal changes in skin involvement, the ultrasound examination was repeated after 1-3 years. Results Compared to controls, SSc Patients had increased skin thickness at the first evaluation. Patients with dSSc had lower skin echogenicity than both patients with ISSc and the controls. Patients with greater changes in skin thickness and skin echogenicity produced more versican, whereas the production of biglycan and decorin was higher only, in patients with greater changes in skin echogenicity. There was a negative correlation between fibroblast production of biglycan and disease duration. Conclusion High fibroblast synthesis of the proteoglycans versican and biglycan is associated with changes in skin echogenicity and may predict more progressive skin sclerosis in SSc.
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| 5. |
- Larsen, Kristoffer, et al.
(författare)
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Presence of activated mobile fibroblasts in bronchoalveolar lavage from patients with mild asthma
- 2004
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Ingår i: American Journal of Respiratory and Critical Care Medicine. - 1535-4970. ; 170:10, s. 1049-1056
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Malmstrom, L, et al.
(författare)
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Proteomic 2DE database for spot selection, automated annotation, and data analysis
- 2002
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 1:2, s. 135-138
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Tidskriftsartikel (refereegranskat)abstract
- We present a software solution that enables faster and more accurate data analysis of 2DE/MALDI TOF MS data. The software supports data analysis through a number of automated data selection functions and advanced graphical tools. Once protein identities are determined using MALDI TOF MS, automated data retrieval from online databases provides biological information. The software, called 2DDB, reduces analysis time to a fraction without losing any quality compared to more manual data analysis. The database contains over 100 000 data entries, and selected parts can be reached at http://2ddb.org.
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| 7. |
- Malmström, Johan, et al.
(författare)
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A proteomic approach to mimic fibrosis disease evolvement by an in vitro cell line
- 2001
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Ingår i: Electrophoresis. - 0173-0835. ; 22:9, s. 1776-1784
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Tidskriftsartikel (refereegranskat)abstract
- Subepithelial fibrosis in asthma involves an increase in the thickening of the lamina reticularis and is due to increased deposition of collagen I, III and V, and fibronectin. The cause of the thickening of the reticular layer is not known in detail, however, it is proposed to be caused by bronchial myofibroblasts. The transformation of fibroblasts to myofibroblasts may be contributed by inflammatory cytokines. In this paper we have studied and compared in vivo tissue material with a human fibroblast target cell. A normal primary fetal fibroblast cell line and HFL-1 (human fibroblast lurg cells) were used as a comparison between fibroblasts from human central biopsies regarding morphology and cell proliferation. Both cell morphology and cell proliferation rate was similar between the different set of cell cultures. Furthermore, it could be concluded that fibroblasts cultures from patients with asthma were surrounded by more extracellular matrix molecules compared to the primary cell line HFL-1, which may mimic the in vivo situation during formation of fibrosis. We wanted to investigate if differential protein display by two-dimensional (2-D) gel electrophoresis and subsequent protein identification by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry could reveal proteins induced by cytokine stimulation that can be correlated to the transformation of normal human fetal lungs cells into a more myofibroblast like phenotype. After stimulation with transforming growth factor-beta (TGF-beta) several myofibroblast markers were found to be regulated. Especially cytoskeletal and cytoskeletal-associated proteins like actin isoforms and tropomyosin, proteins that are responsible for contraction as well as transportation of extra cellular matrix proteins, which are overproduced in the formation of fibrosis. These results indicate that TGF-beta, which is increased in a fibrotic process, participates in the transformation of fibroblasts to myofibroblasts.
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| 8. |
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| 9. |
- Malmström, Johan, et al.
(författare)
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Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry: Mapping the nuclear proteome of human fibroblasts.
- 2003
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 24:21, s. 3806-3814
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Malmström, Johan, et al.
(författare)
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Proteoglycan and proteome profiling of central human pulmonary fibrotic tissue utilizing miniaturized sample preparation : a feasibility study
- 2002
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Ingår i: Proteomics. - 1615-9861. ; 2:4, s. 394-404
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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