Biomedical subjects

Gunilla Westergren-Thorsson

Publications and source records attributed to Gunilla Westergren-Thorsson.

At least 19 recordsLinked to original sources

PubMed2006

Tissue fibrocytes in patients with mild asthma: a possible link to thickness of reticular basement membrane?

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. METHODS: Patients with mild asthma (n = 9) were recruited and divided into two categories based on whether or not fibroblast-like cells could be established from BALF. Non-asthmatic healthy subjects (n = 5) were used as controls. Colocalization of the fibrocyte markers CD34, CD45RO, procollagen I, and alpha-smooth muscle actin (alpha-SMA) were identified in bronchial biopsies from patients and controls by confocal microscopy. Kruskall-Wallis method was used to calculate statistical significance and Spearman coefficient of rank correlation was used to assess the degree of association. RESULTS: In patients with BALF fibroblasts, a 14-fold increase of tissue cells expressing CD34/CD45RO/alpha-SMA and a 16-fold increase of tissue cells expressing CD34/procollagen I was observed when compared to controls (p < 0.05). In contrast, patients without BALF fibroblasts displayed a 2-fold increase when compared to controls (p < 0.05). Fibrocytes were localized close to the basement membrane which was significantly thicker in patients with BALF fibroblasts when compared to the other two groups of subjects. Furthermore, basement membrane thickness could be correlated to the number of fibrocytes in tissue (r = 0.711). Fibroblasts-like cells were cultured from BALF where 17.6% of these cells expressed CD34, CD45RO and alpha-SMA. CONCLUSION: These findings indicate a correlation between recruited fibrocytes in tissue and thickness of basement membrane. Fibroblast progenitor cells may therefore be important in airway remodeling in steroid-naive patients with mild asthma.

Adult↗

PubMed2006

2DDB - a bioinformatics solution for analysis of quantitative proteomics data.

BACKGROUND: We present 2DDB, a bioinformatics solution for storage, integration and analysis of quantitative proteomics data. As the data complexity and the rate with which it is produced increases in the proteomics field, the need for flexible analysis software increases. RESULTS: 2DDB is based on a core data model describing fundamentals such as experiment description and identified proteins. The extended data models are built on top of the core data model to capture more specific aspects of the data. A number of public databases and bioinformatical tools have been integrated giving the user access to large amounts of relevant data. A statistical and graphical package, R, is used for statistical and graphical analysis. The current implementation handles quantitative data from 2D gel electrophoresis and multidimensional liquid chromatography/mass spectrometry experiments. CONCLUSION: The software has successfully been employed in a number of projects ranging from quantitative liquid-chromatography-mass spectrometry based analysis of transforming growth factor-beta stimulated fi-broblasts to 2D gel electrophoresis/mass spectrometry analysis of biopsies from human cervix. The software is available for download at SourceForge.

Computational Biology↗

PubMed2006

Functional and phenotypical comparison of myofibroblasts derived from biopsies and bronchoalveolar lavage in mild asthma and scleroderma.

BACKGROUND: Activated fibroblasts, which have previously been obtained from bronchoalveolar lavage fluid (BALF), are proposed to be important cells in the fibrotic processes of asthma and scleroderma (SSc). We have studied the motility for BALF derived fibroblasts in patients with SSc that may explain the presence of these cells in the airway lumen. Furthermore, we have compared phenotypic alterations in activated fibroblasts from BALF and bronchial biopsies from patients with mild asthma and SSc that may account for the distinct fibrotic responses. METHODS: Fibroblasts were cultured from BALF and bronchial biopsies from patients with mild asthma and SSc. The motility was studied using a cell migration assay. Western Blotting was used to study the expression of alpha-smooth muscle actin (alpha-SMA), ED-A fibronectin, and serine arginine splicing factor 20 (SRp20). The protein expression pattern was analyzed to reveal potential biomarkers using two-dimensional electrophoresis (2-DE) and sequencing dual matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-TOF). The Mann-Whitney method was used to calculate statistical significance. RESULTS: Increased migration and levels of ED-A fibronectin were observed in BALF fibroblasts from both groups of patients, supported by increased expression of RhoA, Rac1, and the splicing factor SRp20. However, these observations were exclusively accompanied by increased expression of alpha-SMA in patients with mild asthma. Compared to BALF fibroblasts in mild asthma, fibroblasts in SSc displayed a differential protein expression pattern of cytoskeletal- and scavenger proteins. These identified proteins facilitate cell migration, oxidative stress, and the excessive deposition of extracellular matrix observed in patients with SSc. CONCLUSION: This study demonstrates a possible origin for fibroblasts in the airway lumen in patients with SSc and important differences between fibroblast phenotypes in mild asthma and SSc. The findings may explain the distinct fibrotic processes and highlight the motile BALF fibroblast as a potential target cell in these disorders.

Adult↗

PubMed2006

Specific haptoglobin expression in bronchoalveolar lavage during differentiation of circulating fibroblast progenitor cells in mild asthma.

Haptoglobin is an acute-phase glycoprotein considered to be involved in tissue repair and is produced by fibroblasts and inflammatory cells. By using a gel-based proteomic approach, we show for the first time a possible role for haptoglobin in the differentiation of fibroblast progenitor cells, termed fibrocytes, in patients with mild asthma. Bronchoalveolar lavage fluid (BALF) was performed to sample circulating fibrocytes from patients with mild asthma and nonasthmatic control subjects. Fibrocytes from the airway lumen were characterized by triple staining of the markers CD34/CD45R0/alpha-smooth muscle actin, and subjected to confocal microscopy. The protein expression pattern was analyzed using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF). Elevated levels of haptoglobin expression in BALF was reported in a sub-group of patients with mild asthma (p < 0.05) when compared to the other subjects. In addition, this increase in haptoglobin was accompanied by differentiation of fibrocytes into fibroblast-like cells. When further analyzing the expression pattern of haptoglobin isoforms, a heterozygous expression was detected in the patients where fibrocyte differentiation could be observed. These data raise the possibility that an acute and specific inflammatory state facilitates the differentiation of fibroblast progenitor cells into activated fibroblasts. Furthermore, this study proposes a novel role for haptoglobin in airway remodeling in patients with asthma.

Asthma↗

PubMed2005

Lung fibroblast proteoglycan production induced by serum is inhibited by budesonide and formoterol.

Proteoglycans contribute to extracellular matrix remodeling in asthmatic airways. We investigated the effects of budesonide, a glucocorticoid, and formoterol, a long-acting beta2-adrenergic agonist, on serum-induced proteoglycan production by human lung fibroblasts. In 10% serum, total proteoglycan production was increased 1.5-fold (P < 0.01) compared with basal production in 0.4% serum. Budesonide (10(-8) M) reduced this increase by 44% (P < 0.01) and, whereas formoterol (10(-10)-10(-8) M) had no inhibitory effects, the drug combination abolished the increase (P < 0.01) without affecting fibroblast proliferation. This synergistic effect required functional glucocorticoid and beta-adrenergic receptors. The production of the proteoglycans decorin, biglycan, perlecan, and versican was increased 2.5- to 5-fold (P < 0.01) in 10% serum. Combination treatment with budesonide (10(-8) M) and formoterol (10(-10) M) abolished this increase to a significantly greater extent than either drug alone. In 10% serum, only versican mRNA was increased 1.4-fold (P < 0.05), whereas decorin mRNA was reduced to 39% (P < 0.01) of basal expression. These serum effects were counteracted by the drug combination, but there were no significant differences between the combination and either drug alone. Thus, the budesonide and formoterol combination seems to synergistically control serum-induced proteoglycan production, primarily at the post-transcriptional level. In conclusion, the proteoglycan upregulation characteristic of asthmatic airways may be limited by combination therapy with budesonide and formoterol.

Adrenergic beta-Agonists↗

PubMed2005

Regulation of the chondroitin/dermatan fine structure by transforming growth factor-beta1 through effects on polymer-modifying enzymes.

The chondroitin/dermatan sulfate proteoglycans (CS/DSPGs), biglycan, decorin, and versican play several important roles in extracellular matrix influencing matrix organization, cell proliferation, and recruitment. Moreover, they bind and regulate growth factors in the extracellular matrix. We have previously shown that cultured human lung fibroblasts treated with transforming growth factor-beta (TGF-beta) alone or in combination with epidermal growth factor and platelet-derived growth factor, increase the production of these PGs. In this report, we describe that the structure of their galactosaminoglycan side chains is altered, albeit there is no alteration of polysaccharide length. The findings showed that iduronic acid content is reduced by 50% in decorin and biglycan, whereas 4-O-sulfation is increased 2-fold in versican. To unravel the mechanism behind these changes, the activities of chondroitin C-5 epimerase and of O-sulfotransferases in cellular fractions prepared from fibroblasts were quantitated, and transcript levels of the relevant sulfotransferases were measured by real time polymerase chain reaction (RT-PCR). The C-5 epimerase activity was reduced by 25% in TGF-beta1 treated cells and 50% in fibroblasts treated with the growth factor combination. No change in activity in dermatan 4-O sulfotransferase was observed, and only a minor decrease in dermatan 4-O-sulfotransferase-1 (D4ST-1) mRNA was observed. On the other hand, chondroitin 4-O sulfotransferase activity increased 2-fold upon TGF-beta1 treatment and 3-fold upon treatment with the growth factor combination. This is in agreement with a 2-fold up-regulation of chondroitin-4-O-sulfotransferase 1 (C4ST-1) mRNA, and no changes in chondroitin-4-O-sulfotransferase 2 (C4ST-2) mRNA. Thus, cellular activity and transcript level correlated well with the changes in the structure of the dermatan/chondroitin sulfate chains.

Carbohydrate Epimerases↗

PubMed2005

Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate.

Certain structures of heparan sulfate (HS) inhibit cell proliferation of fibroblasts. Whether this inhibition is dependent on inhibition of mitogenic signaling pathways or nuclear translocation of HS is unknown. In this study we investigated possible mechanism(s) and structural requirements by which antiproliferative glycosaminoglycans exert their effects on mitogen-activated protein kinase (MAP kinase) phosphorylation, a key intermediate in cell signaling, followed by quantitative proteomic analysis of nuclear proteins by stable isotope coded affinity tags, multidimensional chromatography and tandem mass spectrometry. Serum starved human lung fibroblasts were stimulated with serum, platelet derived growth factor (PDGF-BB) or epidermal growth factor (EGF) in the presence of structurally different glycosaminoglycans. Antiproliferative heparan sulfate with a high content of 2-O-sulfated iduronic acid (IdoA-2SO4) and heavily sulfated glucosamine, and the structurally related glycosaminoglycan heparin inhibited significantly serum stimulated MAP kinase phosphorylation, by at least 80% when stimulated by serum and HS6. We hypothesized that the inhibition of the MAP kinase pathway will have effect in the nuclear proteome. Therefore an isotope coded affinity tag (ICAT) reagent labeling of nuclear proteins and tandem mass spectrometry was applied, resulting in the identification and quantification of 206 proteins. Several nuclear proteins were found to be induced or repressed due to HS stimulation, where the repression EBNA-2 co-activator and the induction of PML protein were of special interest. These results show that heparan sulfate with high content of (IdoA-2SO4) and heavily sulfated glucosamine specifically inhibits MAP kinase activation with a subsequent change in the nuclear proteome of the fibroblast.

Binding Sites↗

PubMed2004

Presence of activated mobile fibroblasts in bronchoalveolar lavage from patients with mild asthma.

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.

Adolescent↗

PubMed2004

Lung fibroblast clones from normal and fibrotic subjects differ in hyaluronan and decorin production and rate of proliferation.

Development of fibrosis involves an increase in the deposition of connective tissue components including collagens, fibronectin and proteoglycans. One hypothesis to account for matrix deposition in fibrosis is that fibroblast with differing matrix producing capacity are involved in the fibrotic process. To test this hypothesis, primary fibroblast cultures and clones derived from these primary lines were established from the lung tissue of control patients and patients with pulmonary fibrosis. The primary lines and derived clones were studied in relation to their capacity to proliferate and to produce proteoglycans and hyaluronan. Primary fibroblast cultures and clones from normal subjects and patients with lung fibrosis differed considerably, with up to 13-fold difference, in both hyaluronan and proteoglycan production. The major proteoglycan produced was decorin in both controls and cultures from fibrotic patients, while cultures from patients with lung fibrosis had a higher expression of mRNA for both collagen and decorin. Clones derived from a primary line from a fibrotic patient secreted 3-fold greater amounts of decorin than those from a control subject. Furthermore, a negative correlation between proliferation and synthesis of decorin was noted. We suggest that different fibroblast clones accumulate in the lung, and that specific cell populations of high decorin producing fibroblasts may exist which are crucial in the pathogenesis of fibrosis.

Cell Proliferation↗

PubMed2003

Biglycan and decorin induce morphological and cytoskeletal changes involving signalling by the small GTPases RhoA and Rac1 resulting in lung fibroblast migration.

Biglycan and decorin are small chondroitin/dermatan sulphate proteoglycans in the extracellular matrix of connective tissue that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins. We show for the first time that biglycan and decorin induce morphological and cytoskeletal changes in fibroblasts, resulting in an increase in migration. Biglycan changed the cell shape of fibroblasts with formation of long protruding filamentous processes. This was also seen for decorin but to a lesser extent. Using fluorescence staining of F-actin fibres it was possible to show that these long filamentous processes were supported by long thick bundles of actin, together with an induced formation of stress fibres after stimulation with biglycan and decorin. Moreover, a reorganisation of alpha-smooth muscle actin was clearly seen in these cultures. Decorin also stimulated alpha-smooth muscle actin expression in the cells. Using cDNA Atlas Arrays we were also able to show that the mRNA level of a number of the intracellular regulators and effectors involved in cell migration were increased. For example, the focal adhesion proteins paxillin and zyxin, and some of the small Rho GTPases such as RhoA, Rac1 and Cdc42 were upregulated. After treatment with biglycan or decorin, additional results showed an increased activation of RhoA (1.8- and 1.5-fold, respectively) and Rac1 (1.8- and 1.5-fold, respectively) after 15 minutes. These factors are known to be involved in fibroblast migration, and as expected a 1.3- to 1.6-fold increase in migration could be observed after stimulation with biglycan or decorin. This induced migration was caused by the core protein, as treatment with glycosaminoglycan chains alone did not have any effect. In summary, these data indicate that biglycan- and decorin-induced fibroblast cytoskeletal and signalling changes result in an increased cell migration, and demonstrate their potential role in the remodelling process.

Actins↗

PubMed2003

Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry: mapping the nuclear proteome of human fibroblasts.

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.

Cell Nucleus↗

PubMed2003

Activation of platelet-derived growth factor pathway in human asthmatic pulmonary-derived mesenchymal cells.

Cell cultures of mesenchymal type were obtained from biopsies taken after bronchoscopy from patients with asthma. It was possible to achieve outgrowth of fibroblast-like cells from these lung biopsies, which stained for alpha-smooth actin indicating that they were of myofibroblast type. Morphologically, two types of myofibroblasts could be observed: one intermediate form with more stretched cell shape and lamellipodia protrusions, and one more differentiated compact form of myofibroblast. The intermediate form was the most dominant type in these patients, indicating an active ongoing remodelling process. Further studies showed that platelet-derived growth factor (PDGF) might be the factor that stimulates the formation of the intermediate type of myofibroblasts, since it enhance migration of normal human lung fibroblasts 4-fold compared to control through an induced formation of stress fibers and lamellipodia protrusions. Additionally, intracellular signalling pathways involved in migration, such as RhoA and MAPkinase were stimulated 1.5-fold and 3.5-fold, respectively. By using two-dimensional (2-D) gel electrophoresis and protein identification by peptide mass finger printing matrix assisted laser desporption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS) it was possible to confirm that PDGF affected the synthesis of proteins involved in the remodelling process, such as collagen VI and post-translational forms thereof. PDGF also stimulated the production of FK506 binding protein of 65 kDa, a protein involved in smooth muscle differentiation, and proteins involved in the rearrangement of the cytoskeleton connected to migration such as the actin related protein ARP3, the T-complex protein and the heat shock protein 60. We demonstrate that PDGF has a potential pathological role in asthma and formation of subepithelial fibrosis by inducing changes in the proteome.

Asthma↗

PubMed2002

Tumour necrosis factor-alpha interacts with biglycan and decorin.

Several interactions of cytokines with extracellular matrix molecules are mediated by proteoglycans, such as biglycan and decorin. Using surface plasmon resonance, we show for the first time that tumour necrosis factor-alpha (TNF-alpha) binds to both biglycan and decorin with K(d)s of 0.81 microM and 1.23 microM respectively, a binding that was confirmed by Scatchard plots using a solid phase assay. Binding occurs preferentially via the core protein, shown by lower K(d)s, 0.26 microM and 0.81 microM for biglycan and decorin respectively. There was also binding to dermatan sulphate, with a K(d) of 10.53 microM. The function of this interaction between TNF-alpha and biglycan and decorin is not known, but we suggest that the differential localisation of the proteoglycans enables the cytokines to be immobilised in different environments.

Animals↗

PubMed2002

Proteoglycan and proteome profiling of central human pulmonary fibrotic tissue utilizing miniaturized sample preparation: a feasibility study.

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.

Actins↗

PubMed2002

Proteoglycan production in disomic and trisomy 7-carrying human synovial cells.

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.

Cells, Cultured↗

PubMed2002

Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects.

Asthma is characterized by an airway remodeling process involving altered extracellular matrix deposition such as collagen, fibronectin and proteoglycans. Proteoglycans determine tissue mechanical properties and are involved in many important biological aspects. Not surprisingly, it has been suggested that proteoglycan deposition may alter airway properties in asthma including airway hyperresponsiveness. In chronically inflamed airway tissues, fibroblasts likely represent an activated fibrotic phenotype that contributes to the excessive deposition of different extracellular matrix components. To investigate whether this was the case for proteoglycans, the production of hyaluronan, perlecan, versican, small heparan sulphate proteoglycans (HSPGs), decorin and biglycan was quantified in the culture medium of primary bronchial fibroblast cultures, established from four normal and six asthmatic subjects. Values were further correlated to the airway responsiveness (PC(20) methacholine) of donor subjects. Fibroblasts from subjects with the most hyperresponsive airways produced up to four times more total proteoglycans than cells from subjects with less hyperresponsive or normoresponsive airways. We observed a significant negative correlation between the PC(20) and perlecan, small HSPGs and biglycan, while such correlation was absent for decorin and close to significant for hyaluronan and versican. Altered proteoglycan metabolism by bronchial fibroblasts may contribute to the increased proteoglycan deposition in the bronchial mucosa and to airway hyperresponsiveness characterizing asthma.

Asthma↗

PubMed2002

Biglycan isoforms with differences in polysaccharide substitution and core protein in human lung fibroblasts.

Biglycan is widely distributed in the extracellular matrix and is a member of the small proteoglycan family characterized by a core protein with leucine-rich repeat motifs. We show in this paper for the first time that biglycan from human lung fibroblasts can be expressed as different isoforms. These isoforms can be separated from the predominant form of biglycan by hydrophobic interaction chromatography, where the more hydrophobic isoforms are retarded. The newly found isoforms of biglycan have a smaller core protein substituted with smaller glycosaminoglycan chains, migrating on SDS/PAGE at between 110 and 200 kDa. These molecules were identified as biglycan using MALDI-TOF MS. Identification of C-terminal peptides together with glycosylation of the N-terminal glycosaminoglycan sites excludes the possibility of terminal proteolytic cleavage. The biglycan isoforms are N-glycosylated, which demonstrates that a lack in N-glycosylation is not the reason for a smaller core. Two components revealed by RT-PCR indicate alternative splicing, which could be located in regions of the protein that have not been identified, with the exclusion of sites of glycosylations. Analyses of glycosaminoglycan chain length of the isoforms show that besides the normally occurring glycosaminoglycan chains, there is a mixture of shorter glycosaminoglycan chains. Structural analysis shows that these glycosaminoglycan chains contain a lower proportion of iduronic acid (61%) relative to glucuronic acid when compared to the glycosaminoglycan chain of the predominant form of biglycan (71%). We can anticipate that variation in structure of biglycan can cause changes in the connective tissue formation depending on its ability to bind matrix molecules, as well as cytokines.

Alternative Splicing↗

PubMed2002

Myofibroblast accumulation correlates with the formation of fibrotic tissue in a rat air pouch model.

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.

Animals↗