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PubMed2003

Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell-surface components through the CD14/Toll-like receptor system.

We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin-8 (IL-8) responses of the cells to Salmonella lipopolysaccharide, a water-soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll-like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR-related molecules, i.e. TLR2, TLR4, MD-2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL-8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL-8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2- and TLR4-independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti-CD14 MAb.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2002

Application of interleukin-2-secreting syngeneic/allogeneic fibroblasts in the treatment of primary and metastatic brain tumors.

We found previously that mice injected intracerebrally (i.c.) with a mixture of malignant cells and allogeneic fibroblasts genetically engineered to secrete interleukin-2 (IL-2) survived longer than mice in various control groups. The primary goal of this study was to determine if an established i.c. glioma (Gl261) or breast carcinoma (SB-5b) could be treated by injection of IL-2-secreting allogeneic fibroblasts into the tumor region. As an additional objective, these results were compared with the effectiveness of injecting IL-2-secreting allogeneic fibroblasts prior to the introduction of the tumor cells as a means of preventing the development of an i.c. glioma or breast carcinoma. The results demonstrated that treatment of mice bearing an established i.c. glioma or breast carcinoma with IL-2-secreting allogeneic fibroblasts resulted in a prolonged survival. Furthermore, the results demonstrate a significant delay (P<.005) in the development of glioma in the animals treated with either allogeneic nonsecreting or IL-2-secreting fibroblasts prior to introduction of tumor cells. In addition, 50% of the animals pretreated with IL-2-secreting allogeneic fibroblasts injected subsequently with Gl261 glioma cells did not develop a tumor, whereas all of the animals injected with glioma cells alone and 92% of those treated with nonsecreting fibroblasts eventually died. Evidence also exists that long-term immunity was established in the treated animals because there was a significant prolongation of survival in comparison to naïve controls (P<.01) for those animals without evidence of glioma that previously had been immunized with treatment cells when challenged again with tumor cells. In a parallel experiment, 62% of the animals pretreated with nonsecreting allogeneic fibroblasts and 75% of the animals pretreated with allogeneic IL-2-secreting fibroblasts subsequently injected with SB-5b breast carcinoma cells did not develop tumors. The results indicate that IL-2-secreting allogeneic fibroblasts can be effective in the treatment of an established brain tumor. These data also suggest that i.c. injection of allogeneic IL-2-secreting fibroblasts is effective in prevention of the development of a brain tumor when the fibroblasts are introduced into the same site where the tumor is subsequently injected.

Animals↗
PubMed1990

The relationship of excess copper accumulation by fibroblasts from the brindled mouse model of Menkes disease to the primary defect.

Fibroblasts from the brindled mouse model of Menkes disease are known to accumulate excess copper. Most of the copper in the cytosol of these fibroblasts is bound to metallothionein (MT), which is elevated in Menkes or brindled mouse fibroblasts. Copper accumulation by normal fibroblasts containing excess MT was examined to determine if the excess copper accumulation phenotype was secondary to excess MT or associated with the primary defect in fibroblasts from the brindled mice. MT was induced in normal fibroblasts by copper, zinc or dexamethasone to levels comparable with those in brindled mice fibroblasts, as determined by radioimmunoassays. Normal fibroblasts containing excess MT accumulate copper normally, i.e. they do not exhibit the excess copper accumulation phenotype. Consistent with this result, copper efflux from normal fibroblasts containing excess MT was also normal. The data suggest that one function of the protein associated with the primary defect is to help determine how much copper is taken up and retained by fibroblasts and other cell types exhibiting the excess copper phenotype in Menkes disease. The capacity of this protein is apparently exceeded in normal fibroblasts if serum or albumin is not present extracellularly to limit total copper uptake. Consistent with a defect in an intracellular protein, the kinetics of copper transport by brindled mice fibroblasts were found to be normal.

Animals↗
PubMed2000

Defective extracellular matrix reorganization by chronic wound fibroblasts is associated with alterations in TIMP-1, TIMP-2, and MMP-2 activity.

Chronic leg wounds are characterized by defective remodeling of the extracellular matrix, failure of reepithelialization, and prolonged inflammation. The hypothesis that this defective extracellular matrix remodeling is associated with phenotypic differences in the activity of the matrix metalloproteinases and tissue inhibitors of metalloproteinases was studied in chronic wound and patient-matched normal fibroblasts in three-dimensional collagen lattice systems. Chronic wound fibroblasts exhibited no differences in morphology or proliferation (p > 0.1) compared with patient-matched uninvolved dermal fibroblasts. The ability of chronic wound fibroblasts to reorganize extracellular matrix was significantly impaired, however, in comparison to the uninvolved dermal fibroblasts (p < 0.01). This difference in extracellular matrix reorganization was not related to differences in proliferation within the collagen lattices (p > 0.05) or attachment to type I collagen (p > 0.1). Marked differences were evident in matrix metalloproteinase-2 activity between chronic wound and patient-matched normal fibroblasts. Whereas levels of pro-matrix metalloproteinase-2 were similar between the two fibroblast populations (p > 0.1), the chronic wound fibroblasts exhibited significantly decreased levels of the 62 kDa active form of matrix metalloproteinase-2 (p < 0.01). Reverse zymography and enzyme-linked immunosorbent assay demonstrated that the decreased matrix metalloproteinase-2 activity was associated with increased production of tissue inhibitors of metalloproteinase-1 and -2 by the chronic wound fibroblasts (p < 0.05). Increased production of tissue inhibitors of metalloproteinases in chronic wound fibroblasts was also reflected in decreased levels of matrix metalloproteinase-1 (p < 0.005). These data suggest that the impaired ability of chronic wound fibroblasts to reorganize extracellular matrix in vitro is related to decreased levels of active matrix metalloproteinase-2 and matrix metalloproteinase-1 resulting from increased production of tissue inhibitors of metalloproteinase-1 and -2 by chronic wound fibroblasts. These findings provide a mechanism to explain the impaired cellular responses and extracellular matrix reorganization observed in chronic leg wounds in vivo.

Cell Adhesion↗
PubMed2001

Dermal fibroblasts sustain proliferation of activated T cells via membrane-bound interleukin-15 upon long-term stimulation with tumor necrosis factor-alpha.

In chronic inflammatory conditions, mononuclear cells infiltrate the connective tissue attracted by fibroblast-secreted chemokines. The role of fibroblasts in sustaining the lymphocyte immune response upon cellular infiltration is so far unresolved. We here report that, upon prolonged stimulation with tumor necrosis factor-alpha, dermal fibroblasts enhance proliferation of activated T cells whereas unstimulated fibroblasts do not. T cell growth stimulation requires cell contact of tumor necrosis factor-alpha stimulated fibroblasts to T cells and is not due to soluble factors. Growth stimulation is substantially blocked by neutralizing antibodies to interleukin-15. Fluorescence-activated cell sorter analyses revealed that tumor necrosis factor alpha stimulated fibroblasts expose interleukin-15 in a membrane-bound form on the cell surface whereas nonstimulated fibroblasts and interferon-gamma treated fibroblasts do not. The amount of membrane interleukin-15 increases with the duration of tumor necrosis factor-alpha stimulation for at least 3 d. Unstimulated fibroblasts, however, accumulate interleukin-15 in the cytoplasm. No interleukin-15 could be detected in the culture supernatant. Immunohistochemical analyses confirmed membrane interleukin-15 on dermal fibroblasts in discoid lupus erythematosus skin lesions whereas no membrane interleukin-15 was found on the surface of fibroblasts in healthy skin. We conclude that dermal fibroblasts upon long-term tumor necrosis factor-alpha stimulation during chronic inflammation are involved via membrane-bound interleukin-15 in stimulating proliferation of accumulated, activated T cells.

Antibodies↗
PubMed2002

Fibroblast growth factor 2 regulation of mitral valve interstitial cell repair in vitro.

OBJECTIVE: Because elongated mitral valve interstitial cells have features of myofibroblasts, it is likely that these cells are essential for the repair of injured valve leaflets. We characterized the cellular morphology and pattern of repair of these interstitial cells in wounds produced in vitro and tested the hypothesis that fibroblast growth factor 2 enhances interstitial cell repair. METHODS: Mitral valve interstitial cells were plated onto glass coverslips, reached confluence after 1 week, and were wounded by passage of a spatula along the center of a monolayer, which created a linear wound with two edges. The wounds were observed from 0 to 96 hours by phase-contrast microscopy. Wounds were also fixed at 0, 2, and 24 hours and stained for fibroblast growth factor 2 and fibroblast growth factor receptor 1 by means of immunofluorescence and laser confocal microscopy. RESULTS: Cells in confluent monolayers and in the monolayer behind the wound edge formed a multilayered orthogonal pattern of elongated cells similar to fibroblasts. Cells along the wound edge migrated into the wound after 4 hours, and at 24 hours single cells with prominent lamellipodia and tails were present within the wound. There was overlapping of cells as well, similar to smooth muscle cells. Fibroblast growth factor 2 and fibroblast growth factor receptor 1 were present in the cells of the undisturbed confluent monolayer. They were upwardly regulated relative to the unwounded monolayer in the cells along the wound edge at 2 hours and in the monolayer behind the wound edge at 24 hours. In single cells that migrated into the wound, fibroblast growth factor 2 and fibroblast growth factor receptor 1 were prominent. Fibroblast growth factor 2 showed a 6-fold increase in concentration relative to unwounded cultures in conditioned medium from wounded cultures at 2 hours after wounding. Addition of a neutralizing antibody to fibroblast growth factor 2 significantly delayed wound closure at 24 to 96 hours. Addition of exogenous fibroblast growth factor 2 to cultures at the time of wounding did not enhance wound repair. CONCLUSION: Mitral valve interstitial cells have the ability to repair wounds, and fibroblast growth factor 2 is a modulator of these repair processes.

Animals↗
PubMed2001

Thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the proteolytically activated receptor-1 and a protein kinase C-dependent pathway.

Myofibroblasts are ultrastructurally and metabolically distinctive fibroblasts that express smooth muscle (SM)-alpha actin and are associated with various fibrotic lesions. The present study was undertaken to investigate the myofibroblast phenotype that appears after activation of normal lung fibroblasts by thrombin. We demonstrate that thrombin induces smooth muscle-alpha actin expression and rapid collagen gel contraction by normal lung fibroblasts via the proteolytically activated receptor-1 and independent of transforming growth factor-beta pathway. Using antisense oligonucleotides we demonstrate that a decreased level of PKCepsilon abolishes SM-alpha actin expression and collagen gel contraction induced by thrombin in normal lung fibroblasts. Inhibition of PKCepsilon translocation also abolishes thrombin-induced collagen gel contraction, SM-alpha actin increase, and its organization by normal lung fibroblasts, suggesting that activation of PKCepsilon is required for these effects. In normal lung fibroblasts PKCepsilon binds to SM-alpha actin after thrombin treatment, but in activated fibroblasts derived from scleroderma lung they associate even in untreated cells. This suggests that SM-alpha actin may serve as a substrate for PKCepsilon in lung fibroblasts when activated by thrombin. We propose that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via a PKC-dependent pathway. Thrombin-induced differentiation of normal lung fibroblasts to a myofibroblast phenotype resembles the phenotype observed in scleroderma lung fibroblasts. Therefore, we conclude that chronic exposure to thrombin after microvascular injury leads to activation of normal lung fibroblasts and to the appearance of a myofibroblast phenotype in vivo. Our study provides novel, compelling evidence that thrombin is an important mediator of the interstitial lung fibrosis associated with scleroderma.

Actins↗
PubMed1995

Identification and characterization of a fibroblast marker: FSP1.

We performed subtractive and differential hybridization for transcript comparison between murine fibroblasts and isogenic epithelium, and observed only a few novel intracellular genes which were relatively specific for fibroblasts. One such gene encodes a filament-associated, calcium-binding protein, fibroblast-specific protein 1 (FSP1). The promoter/enhancer region driving this gene is active in fibroblasts but not in epithelium, mesangial cells or embryonic endoderm. During development, FSP1 is first detected by in situ hybridization after day 8.5 as a postgastrulation event, and is associated with cells of mesenchymal origin or of fibroblastic phenotype. Polyclonal antiserum raised to recombinant FSP1 protein stained the cytoplasm of fibroblasts, but not epithelium. Only occasional cells stain with specific anti-FSP1 antibodies in normal parenchymal tissue. However, in kidneys fibrosing from persistent inflammation, many fibroblasts could be identified in interstitial sites of collagen deposition and also in tubular epithelium adjacent to the inflammatory process. This pattern of anti-FSP1 staining during tissue fibrosis suggests, as a hypothesis, that fibroblasts in some cases arise, as needed, from the local conversion of epithelium. Consistent with this notion that FSP1 may be involved in the transition from epithelium to fibroblasts are experiments in which the in vitro overexpression of FSP1 cDNA in tubular epithelium is accompanied by conversion to a mesenchymal phenotype, as characterized by a more stellate and elongated fibroblast-like appearance, a reduction in cytokeratin, and new expression of vimentin. Similarly, tubular epithelium submerged in type I collagen gels exhibited the conversion to a fibroblast phenotype which includes de novo expression of FSP1 and vimentin. Use of the FSP1 marker, therefore, should further facilitate both the in vivo studies of fibrogenesis and the mapping of cell fate among fibroblasts.

3T3 Cells↗
PubMed2000

Transforming growth factor-beta mRNA and protein in hypertrophic scar tissues and fibroblasts: antagonism by IFN-alpha and IFN-gamma in vitro and in vivo.

Hypertrophic scarring (HSc) following burn injury is a common, disfiguring, and functionally limiting form of dermal fibrosis, compromising recovery. Previously, elevated levels of transforming growth factor-beta1 (TGF-beta1), a fibrogenic cytokine, were found in wounds and serum of severely injured patients, antagonized in part by treatment with systemic interferon-alpha2b (IFN-alpha2b) both in vitro and in vivo. It is hypothesized that in wound healing after injury, platelets are an initial source of TGF-beta, but wound fibroblasts may be capable, after activation, of autoamplification of the initial response to injury by increasing TGF-beta mRNA and protein that may subsequently be responsive to IFN therapy with IFN-alpha or IFN-gamma or both. Using three pairs of site-matched HSc and normal fibroblasts from the same individuals, nonconfluent and near confluent fibroblasts were treated with TGF-beta, and cell proliferation and collagen production were assayed using cell counting and 18O2 isotopic uptake into hydroxyproline before analysis by gas chromatography-mass spectrometry (GC-MS). HSc and normal fibroblasts were assayed for the production of TGF-beta protein secretion using ELISA for TGF-beta1, TGF-beta2, and TGF-beta3 after acidification of medium samples from 96-h cultures. HSc and normal fibroblasts were treated with IFN-alpha2b or IFN-gamma or both for 96 h. Quantitative RT-PCR and Northern analysis were performed using newly synthesized internal standards for human TGF-beta1. TGF-beta stimulates both HSc and normal fibroblast proliferation. Collagen synthesis is greater in HSc than in normal fibroblasts and is maximally stimulated at 75 pM TGF-beta. TGF-beta stimulated collagen metabolism is antagonized by IFN-alpha or IFN-gamma or both in an additive fashion. HSc and normal fibroblasts not only possess the mRNA for TGF-beta1 but also secrete mature TGF-beta protein. Treatment of HSc and normal fibroblasts with IFN-alpha2b or IFN-gamma antagonizes TGF-beta protein production, and additive effects occur. RT-PCR demonstrates that after IFN treatment, downregulation of TGF-beta1 mRNA accounts in part for the reduction in protein secretion in HSc fibroblasts. Elevations of systemic TGF-beta may be due to wound fibroblasts. TGF-beta synthesis and antagonism of fibroblast TGF-beta protein secretion occurs with either IFN-alpha or IFN-gamma, in part by downregulation of TGF-beta1 mRNA levels.

Cicatrix, Hypertrophic↗
PubMed2005

Spontaneously immortalized mouse embryo fibroblasts: growth behavior of wild-type and vimentin-deficient cells in relation to mitochondrial structure and activity.

Dependent on the presence or absence of vimentin, primary mouse embryo fibroblasts exhibit different growth characteristics in vitro. While most Vim(+/+) fibroblasts stop dividing and die via apoptosis, a substantial fraction of cells immortalize and proliferate almost normally. Vim(-/-) fibroblasts cease to divide earlier, immortalize in vanishingly small numbers and thereafter proliferate extremely slowly. Early after immortalization, Vim(+/+) (imm) fibroblasts appear structurally almost normal, whereas Vim(-/-) (imm) fibroblasts equal postmitotic "crisis" cells, which are characterized by increased cell size, altered cell ultrastructure, nuclear enlargement, genome destabilization, structural degeneration of mitochondria, and diminution of mitochondrial respiratory activity. The differences between immortalized Vim(+/+) (imm) and Vim(-/-) (imm) fibroblasts persist during early cell cloning but disappear during serial subcultivation. At high cell passage, cloned, immortalized vim(-) fibroblasts grow nearly as fast as their cloned vim(+) counterparts, and also resemble them in size, ultrastructure, nuclear volume, and mitochondrial complement; they very likely employ redundancy to cope with the loss of vimentin function when adjusting structure and behavior to that of immortalized vim(+) fibroblasts. Reduction in nuclear size occurs via release of large amounts of filamentous chromatin into extracellular space; because it is complexed with extracellular matrix proteins, it tends to form clusters and to tightly stick to the surface of other cells, thus providing a potential for horizontal gene transfer. On the other hand, cloned vim(+) and vim(-) fibroblasts are equal in showing contact inhibition at young age and becoming anchorage-independent during serial subcultivation, as indicated by the formation of multilayered and -faceted cell sheets and huge spheroids on top of or in soft agar. With this, immortalized vim(-) fibroblasts reduce their adhesiveness to the substratum which, in their precrisis state and early after cloning, is much higher than that of their vim(+) counterparts. In addition, the coupling between the mitochondrial respiratory chain and oxidative phosphorylation is stronger in vim(+) than vim(-) fibroblasts. It appears from these data that after explantation of fibroblasts from the mouse embryo the primary cause of cell and mitochondrial degeneration, including genomic instability, is the mitochondrial production of reactive oxygen species in a vicious circle, and that vimentin provides partial protection from oxidative damage. As a matrix protein with specific in vitro and in vivo affinities for nuclear and mitochondrial, recombinogenic DNA, it may exert this effect preferentially at the genome level via its influence on recombination and repair processes, and in this way also assist the cells in immortalizing. Additional protection of mitochondria by vimentin may occur at the level of mitochondrial fatty acid metabolism.

Animals↗
PubMed2005

Stability and association with the cytomatrix of mitochondrial DNA in spontaneously immortalized mouse embryo fibroblasts containing or lacking the intermediate filament protein vimentin.

To extend previous observations demonstrating differences in number, morphology, and activity of mitochondria in spontaneously immortalized vim(+) and vim(-) fibroblasts derived from wild-type and vimentin knockout mice, some structural and functional aspects of mitochondrial genome performance and integrity in both types of cells were investigated. Primary Vim(+/+) and Vim(-/-) fibroblasts, which escaped terminal differentiation by immortalization were characterized by an almost twofold lower mtDNA content in comparison to that of their primary precursor cells, whereby the average mtDNA copy number in two clones of vim(+) cells was lower by a factor of 0.6 than that in four clones of vim(-) cells. However, during serial subcultivation up to high passage numbers, the vim(+) and vim() fibroblasts increased their mtDNA copy number 1.5- and 2.5-fold, respectively. While early-passage cells of the vim(+) and vim(-) fibroblast clones differed only slightly in the ratio between mtDNA content and mitochondrial mass represented by mtHSP70 protein, after ca. 300 population doublings the average mtDNA/mtmass ratio in the vim(+) and vim() cells was increased by a factor of 2 and 4.5, respectively. During subcultivation, both types of cells acquired the fully transformed phenotype. These findings suggest that cytoskeletal vimentin filaments exert a strong influence on the mechanisms controlling mtDNA copy number during serial subcultivation of immortalized mouse embryo fibroblasts, and that vimentin deficiency causes a disproportionately enhanced mtDNA content in high-passage vim(-) fibroblasts. Such a role of vimentin filaments was supported by the stronger retention potential for mtDNA and mtDNA polymerase (gamma) detected in vim(+) fibroblasts by Triton X-100 extraction of mitochondria and agaroseembedded cells. Moreover, although the vim(+) and vim(-) fibroblasts were equally active in generating free radicals, the vim(-) cells exhibited higher levels of immunologically detectable 8-oxoG and mismatch repair proteins MSH2 and MLH1 in their mitochondria. Because in vim(-) fibroblasts only one point mutation was detected in the mtDNA D-loop control region, these cells are apparently able to efficiently remove oxidatively damaged nucleobases. On the other hand, a number of large-scale mtDNA deletions were found in high-passage vim(-) fibroblasts, but not in low-passage vim(-) cells and vim(+) cells of both low and high passage. Large mtDNA deletions were also induced in young vim(-) fibroblasts by treatment with the DNA intercalator ethidium bromide, whereas no such deletions were found after treatment of vim(+) cells. These results indicate that in immortalized vim(-) fibroblasts the mitochondrial genome is prone to large-scale rearrangements, probably due to insufficient control of mtDNA repair and recombination processes in the absence of vimentin.

Adaptor Proteins, Signal Transducing↗
PubMed1981

Comparisons of the effects of chemical carcinogens in mixed cultures of rat hepatocytes and human fibroblasts.

Unscheduled DNA synthesis (UDS) was measured simultaneously in rat hepatocytes and human fibroblasts when combined cultures of the 2 cell types were exposed to procarcinogens. Human fibroblasts were preincubated with 5-bromo-2'-deoxyuridine (BRdU) to substitute for thymidine in the DNA. Hepatocyte DNA was separated from the heavier BRdU-substituted fibroblast DNA by isopycnic centrifugations in neutral cesium chloride and the specific activities of the DNA's were determined. In the presence of hepatocytes, benzo - [a]pyrene (BP) induced more UDS in the fibroblasts than in the hepatocytes. BP induced no UDS in the fibroblasts in the absence of hepatocytes. Diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) stimulated a significant amount of UDS only in the hepatocytes. Thus, the co-cultures of hepatocytes and fibroblasts responded with UDS to these chemical carcinogens in a manner that parallels the tissue specificity of the carcinogenicity of these chemicals in vivo. That is, the known hepatocarcinogens DEN and AAF, only stimulated significant UDS in the hepatocytes, whereas the non-hepatocarcinogen, BP, though activated in these cultures mainly by the hepatocytes, stimulated more UDS in the fibroblasts than in the hepatocytes. The amount of [3H]BP bound to DNA was investigated in the co-cultures and in cultures of fibroblasts alone. When co-cultures were exposed to [3H]BP the fibroblast DNA had approximately 3 times more BP bound to it (per microgram DNA) than did the hepatocyte DNA. The amount of [3H]BP bound to the DNA of cultures of fibroblasts alone was 29% of the amount bound to the DNA of the fibroblasts from the co-cultures. Thus, although the hepatocytes were mainly responsible for the activation of BP, more [3H]BP was bound to the fibroblast DNA. It is suggested that the intercellular distribution of carcinogenic metabolites may be a significant determinant of the carcinogenic effect.

2-Acetylaminofluorene↗
PubMed2001

Characterization of fibroblastic cell plasticity in the lamina propria of the rat uterine cervix at term.

Different organs contain fibroblasts with specific features and functions, indicating the complexity of fibroblast biology. In the rat cervical stroma, fibroblasts are preferentially located in the fibrous ring that surrounds the mucous layer. The purpose of this study was to investigate the morphological features and immunophenotype of fibroblastic cells of the uterine cervix in cycling, pregnant, and postpartum rats. Expression of the cytoskeletal proteins desmin, vimentin, and alpha-smooth muscle actin (alpha-SMA) were studied by immunohistochemistry. The optical density of immunohistochemical staining was quantified by image analysis. The ultrastructural features of fibroblastic cells were observed under transmission electron microscopy. Cervical fibroblastic cells always expressed vimentin and desmin but never alpha-SMA. During the first half of pregnancy (Day 5 [D5] to D14), desmin intensity values were similar to those of cycling and postpartum fibroblasts. In contrast, a strong expression of desmin was found from D15 to D22, with maximal expression at term (D23). Immunohistochemical expression for vimentin was constant throughout pregnancy and showed no differences with cycling and postpartum uterine cervices. Stromal cells from cycling and early pregnant rats displayed ultrastructural features characteristic of typical fibroblasts. In contrast, at the end of pregnancy, fibroblasts differentiated and showed increased secretory characteristics, reaching the ultrastructural features of a myofibroblast. Based on the differential expression of desmin and the electron microscopic observations, the foregoing results showed a modulation of the fibroblastic phenotype in the uterine cervix during pregnancy. To our knowledge, this is the first report that addresses the presence of myofibroblasts derived from resident fibroblasts in the fibrous ring of the rat uterine cervix. Fibroblastic-myofibroblastic cell plasticity may have implications in the physiological changes displayed in the uterine cervix during pregnancy, parturition, and postpartum involution.

Actins↗
PubMed2000

Stricture formation in Crohn's disease: the role of intestinal fibroblasts.

OBJECTIVE: To determine whether intestinal fibroblasts in patients with Crohn's disease (CD) have an enhanced capacity to reorganize collagen and thus cause stricture formation. SUMMARY BACKGROUND DATA: Stricture formation is a characteristic feature of CD that may distinguish it from other forms of inflammatory bowel disease. Methods Fibroblasts were obtained at surgery from the colon and ileum of patients with CD and ulcerative colitis (UC) and control patients. Primary fibroblast cultures were obtained by explant technique. Fibroblast contractile activity was measured using fibroblast-populated collagen lattices (FPCLs), in which the cultured fibroblasts were seeded in free-floating collagen gel matrices that they reorganize and contract. Fibroblast contractile activity was measured as the reduction of surface area (mm2) of collagen gel matrix at 24-hour intervals for 1 week. RESULTS: Fibroblasts from patients with CD displayed enhanced capacity to contract FPCL when compared to UC and control fibroblasts. This activity was maximal in fibroblasts recovered from strictured regions in CD. Fibroblasts from patients with UC had a contractile capacity similar to that of controls. Hydrocortisone inhibited this in vitro contractile activity in a dose-dependent manner. CONCLUSIONS: Intestinal fibroblasts in CD possess enhanced capacity for collagen reorganization and contractile activity in vitro. This activity may be responsible for stricture formation in CD.

Cell Line↗
PubMed1999

Differences between scar and dermal cultured fibroblasts derived from a patient with recurrent abdominal incision wound herniation.

Fibroblasts were derived from dermis and scar of a 47-year-old white man with a recurrent incisional hernia as a result of fractured ribs. The scar was thin and stretched, suggesting a defect in the maturation of granulation tissue. After surgical repair, biopsy specimens of discarded scar and skin were used to generate fibroblast cell lines. Fibroblasts maintained in medium containing 10% fetal bovine serum and antibiotic were studied between their third and eighth passage. By phase contrast microscopy, no structural differences were obvious, but it was noted that to pass scar fibroblasts, a more aggressive trypsin regimen was required. Immunohistologic and Western blot analysis of patient scar fibroblasts showed (1) more a smooth muscle actin within stress fibers, (2) increased expression of the vitronectin integrin receptor alpha(v) (CD 51), and (3) reduced expression of the collagen integrin receptor alpha2 (CD 49b). The expression of vinculin from focal adhesions or a tubulin from microtubules was the same among cell lines. Contractions of scar and dermal fibroblast-populated collagen lattice were compared. At 24 hours, contractions were 69 percent with newborn fibroblasts (normal); 68 percent for patient dermal fibroblasts; and only 48 percent for patient scar fibroblasts. The retarded contraction of scar fibroblast-populated collagen lattice was significant (p > or = 0.002). Myosin ATPase activity, critical for lattice contraction, and cell migration were equivalent among all cell lines. A plausible mechanism for the retardation of scar lattice contraction is disruption of fibroblasts and collagen interactions, for which the attachment of cells to collagen is altered. It is proposed that either the decrease in the expression of collagen integrin receptor alpha2 (CD 49b), an increase in the expression of the vitronectin receptor alpha(v) (CD 51), or a combination of both is responsible for disruption of collagen fibroblast interactions.

Actins↗
PubMed2005

Transforming growth factor-beta promotes pro-fibrotic behavior by serosal fibroblasts via PKC and ERK1/2 mitogen activated protein kinase cell signaling.

OBJECTIVE: To assess the role of fibroblasts, transforming growth factor (TGF)-beta, and cell signal pathways in promoting fibrosis in Crohn's disease (CD). SUMMARY BACKGROUND DATA: Intestinal strictures are a major source of morbidity in CD. Fibroblasts found at sites of stricture promote fibrogenesis. The mechanisms underlying this pro-fibrotic behavior remain elusive. METHODS: Fibroblasts were isolated from strictured and macroscopically normal serosa in patients with CD and from normal serosa in patients with colorectal cancer. Whole cell connective tissue growth factor (CTGF) and fibronectin expression were determined by Western blot analysis. Fibroblast type I collagen expression was evaluated by real-time PCR, while fibroblast contractile activity was measured using fibroblast populated collagen lattices. Cells were stimulated with TGF-beta1 and inhibitors of the protein kinase C (PKC) and ERK 1/2 mitogen activated protein (MAP) kinase cell signaling pathways. RESULTS: Stricture fibroblasts displayed enhanced constitutive expression of fibronectin. TGF-beta promoted fibroblast CTGF, fibronectin, and type I collagen expression and enhanced fibroblast contractile activity. Inhibition of PKC reduced basal collagen expression and contractile activity in Crohn's fibroblasts and attenuated the effect of TGF-beta on fibroblast CTGF, fibronectin, and collagen I expression as well as fibroblast contractility. ERK 1/2 inhibition had a similar effect on TGF-beta-induced CTGF and fibronectin expression. CONCLUSIONS: TGF-beta is a critical pro-fibrotic growth factor in CD, and its effects are mediated via PKC and ERK 1/2 MAP kinase cell signaling. These pathways may represent novel therapeutic targets for patients with CD characterized by recurrent intestinal stricture formation.

Adult↗
PubMed2004

Effects of commonly used immunosuppressants on graft-derived fibroblasts.

In acute rejection of transplanted organs intragraft fibroblasts increase their production of hyaluronan. Hyaluronan has strong water binding capacity and an increased tissue content of hyaluronan thus contributes to the development of interstitial oedema. The present study examined the effects of commonly used immunosuppressants (prednisolone, cyclosporin, tacrolimus, mycophenolic acid and sirolimus) on fibroblast proliferation, hyaluronan production and cell surface receptor expression. Fibroblasts isolated from rejecting tissue and from normal, non-transplanted tissue were studied in parallel. All substances investigated, except tacrolimus, were found to affect fibroblasts in one way or another. The most striking effect was the almost total inhibition of fibroblast proliferation in the presence of mycophenolic acid. Cyclosporin reduced the proliferation by about 50% and prednisolone had an inhibiting effect on hyaluronan production (50% reduction). These effects were observed on fibroblasts isolated from rat cardiac allografts undergoing rejection as well as on fibroblasts obtained from normal heart tissue. In contrast, sirolimus was found to stimulate the proliferation of fibroblasts from rejecting tissue (100% increase), but not that of normal fibroblasts. The majority of the fibroblasts expressed the hyaluronan receptor CD44, with a more intense expression in cultures of fibroblasts derived at rejection. None of the immunosuppressants affected the staining pattern (number of positive cells or intensity). The inhibitory effects of prednisolone, cyclosporin and mycophenolic acid on fibroblasts may contribute to the overall beneficial effects of these drugs when used for prevention or treatment of rejection.

Animals↗
PubMed2005

Fibroblast-specific protein 1 is a specific prognostic marker for renal survival in patients with IgAN.

BACKGROUND: There is little direct evidence that fibroblasts are involved in the progression of the renal interstitial fibrosis in human glomerulonephritis. With the availability of a new specific marker for fibroblasts, we determined the presence of fibroblasts in kidneys with IgA nephropathy (IgAN) and correlated their numbers with various clinical parameters. In particular, we also prospectively asked if the number of fibroblasts in the renal interstitium correlates with prognosis. METHODS: Cells positive for fibroblast-specific protein 1 (FSP1) were localized in renal biopsy specimens using immunohistochemistry with anti-FSP1 antibody. Clinical features were analyzed by one-way analysis of variance (ANOVA) with the Bonferroni correction. To assess the prognostic impact of the number of FSP1+ fibroblasts on renal survival in 142 patients with normal serum creatinine, the relationship between covariates to renal survival were evaluated univariately using the log-rank test and multivariately using Cox proportional hazards. RESULTS: Fibroblasts identified by their expression of FSP1 accumulate in areas showing severe interstitial fibrosis. Some tubular epithelial cells undergoing epithelial-mesenchymal transition (EMT) in fibrotic areas also express FSP1. Numbers of FSP1+ fibroblasts directly correlate with serum creatinine (r = 0.74, P < 0.0001) and inversely correlate with estimated creatinine clearance (r = -0.54, P < 0.0001), and by multivariate analysis, the clinical factors influencing renal survival are urinary protein excretion [> or = 1.0 g/day, relative risk (RR) = 4.20, P= 0.032], hypertension (RR 5.85, P = 0.0027), and > or = 20 FSP1+ fibroblasts per high power field (HPF) (RR 7.39, P = 0.0015). Staining for FSP1+ fibroblasts is largely nonoverlapping with alpha-smooth muscle actin+ (alpha-SMA) cells in the interstitium. CONCLUSION: The target protein FSP1 identifies human fibroblasts and tubular epithelium undergoing EMT, and distinguishes them from the diaspora of alpha-SMA+ vascular smooth muscle cells. FSP1+ fibroblasts are critically related to the progression of IgAN; consequently, staining FSP1 in renal biopsy specimens provides a valuable histologic index of progression.

Adolescent↗