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PubMed1989

Skin fibroblasts from patients with the genetic disorder hereditary hemorrhagic telangiectasia compared with ataxia-telangiectasia fibroblasts in their response to the radiomimetic drug neocarzinostatin.

Skin fibroblasts from patients with hereditary hemorrhagic telangiectasia (HHT) were compared with fibroblasts from patients with the genetic disorder ataxia-telangiectasia (A-T), and with SV40-transformed A-T fibroblasts, regarding their sensitivity to the radiomimetic drug neocarzinostatin (NCS). Whereas A-T fibroblasts were found to be hypersensitive to NCS at low concentrations (as measured by the cellular survival test), as previously reported, the HHT fibroblasts were more resistant to NCS and behaved as normal fibroblasts. The SV40-transformed A-T cells also resembled normal fibroblasts in their response to NCS in the colony formation test. In the DNA synthesis test, HHT strains of fibroblasts did not fully resemble untransformed and SV40-transformed A-T cells that continued to synthesize DNA following NCS treatment, since NCS inhibited DNA synthesis in HHT fibroblasts by 5 to 15%; whereas the same treatment in normal fibroblasts reduced DNA synthesis by 40%.

Antibiotics, Antineoplastic↗

PubMed1997

Cellular retinol-binding protein-1 is transiently expressed in granulation tissue fibroblasts and differentially expressed in fibroblasts cultured from different organs.

We have reported that cellular retinol-binding protein-1 (CRBP-1) is transiently expressed by arterial smooth muscle cells during experimental intimal repair (P. Neuville, A. Geinoz, G. Benzonana, M. Redard, F. Gabbiani, P. Ropraz, G. Gabbiani: Am J Pathol 1997, 150:509-521). We have examined here the expression of CRBP-1 during wound healing after a full-thickness rat skin wound. CRBP-1 was transiently expressed by a significant proportion of fibroblastic cells including myofibroblasts. Expression started 4 days after wounding, reached a maximum at 12 days, and persisted up to 30 days when a scar was formed. After wound closure, most CRBP-1-containing fibroblastic cells underwent apoptosis. We have further investigated CRBP-1 expression in rat fibroblasts cultured from different organs. CRBP-1 was abundant in lung and heart fibroblasts and was detected in decreasing amounts in muscle, tendon, subcutaneous tissue, and granulation tissue fibroblasts. Dermis fibroblasts contained no detectable levels of CRBP-1. All-trans retinoic acid and transforming growth factor-beta1 inhibited cell proliferation and increased CRBP-1 expression in fibroblastic populations except dermis fibroblasts. We demonstrate that during granulation tissue formation a subpopulation of fibroblastic cells express CRBP-1 de novo. We also demonstrate that CRBP-1 expression by fibroblasts is regulated in vitro by retinoic acid and transforming growth factor-beta1. Our results suggest that CRBP-1 and possibly retinoic acid play a role in the evolution of granulation tissue.

Animals↗

PubMed1984

Reduced response of scleroderma fibroblasts to fibroblast growth factor.

Reactivity of scleroderma fibroblasts to lymphoid cell-derived fibroblast growth factor (FGF) was assessed in this study. The fibroblasts from the sclerotic lesion failed to respond to FGF, whereas those from scleroedematous lesions responded equally to normal fibroblast. Response of the fibroblast from sclerotic lesion was also lower than that of the fibroblast from mature scar. Fibroblasts obtained from three different layers of healthy skin, papillary dermis, reticular dermis, and reticular-subcutaneous layer, responded equally to FGF, whereas the fibroblast of reticular dermis from sclerotic lesion failed to respond to FGF. It is suggested that the fibroblast of reticular dermis in scleroderma is variously activated by some unknown factors, so that they do not have enough reserve to respond to further stimuli.

Animals↗

PubMedDate not supplied

Effects of basic fibroblast factor (bFGF) on MMP-2, TIMP-2, and type-I collagen levels in human lung carcinoma fibroblasts.

Matrix metalloproteinases (MMP's) and tissue inhibitors of metalloproteinases (TIMP's) possess a preponderant role in the metabolism of the major extracellular matrix protein, collagen, and are thought to be important in the mechanism of tumor invasion. Lung cancer occupies the first position in mortality and the second position in incidence, among all cancers. In the present investigation, we studied the effect of basic fibroblast growth factor (bFGF) on collagen, matrix metalloproteinase-2 (MMP-2), and tissue metalloproteinase inhibitor-2 (TIMP-2) levels in normal and carcinoma lung tissue fibroblast cultures. MMP-2 was selected because of its high specificity in the degradation of type IV collagen, major component of the basal membrane. The effect of bFGF on MMP-2, TIMP-2, total collagen, and type I collagen levels of normal and carcinoma lung fibroblast cultures was investigated at 0, 10, and 100 ng/ml. Statistical analysis was carried out using the Mann-Whitney-U test and possible correlations were searched using the Spearman correlation analysis method. MMP-2, TIMP-2, total collagen, and type-1 collagen levels based on cell counts (10(3) cells) showed no statistically significant differences between the carcinoma and normal fibroblast cultures. However, positive correlations were found between MMP-2 and TIMP-2 in normal (P = 0.016) and carcinoma (P = 0.001) tissue fibroblast cultures. Positive correlations were also found between total collagen and TIMP-2 levels in normal and carcinoma tissue fibroblast cultures (P = 0.002 and P = 0.032). Total collagen and TIMP-2 levels also showed positive and strong correlations in all cultures except in 100 ng/ml bFGF concentrations. In addition, type I collagen and MMP-2 levels showed positive significant correlations only in normal and carcinoma control cultures, while type I collagen and TIMP-2 levels showed positive correlations in all cultures except carcinoma fibroblasts at 100 ng/ml bFGF. It may be concluded that bFGF does not affect MMP-2, TIMP-2, total collagen, and type-1 collagen levels in fibroblast cultures grown from human carcinoma and normal lung tissues. However, bFGF was noted, in vitro, to disturb the equilibrium which normally exists between the four parameters, both in normal and carcinoma tissue fibroblasts.

Carcinoma↗

PubMed2001

Cell surface heparan sulfate proteoglycans control the response of renal interstitial fibroblasts to fibroblast growth factor-2.

BACKGROUND: While the progression of renal disease to end stage is strongly correlated with tubulointerstitial changes, the control of the fibrotic process within the interstitium is poorly understood. Basic fibroblast growth factor (FGF-2) has been implicated as a major growth factor involved in fibroblast activation and extracellular matrix synthesis. Furthermore, in many cells, the activity of FGF-2 is controlled by a low-affinity but high-capacity interaction with heparan sulfate (HS) proteoglycans (PGs), such as members of the syndecan family. These molecules are likely to be central to the control of interstitial fibrosis, but as yet, there has been no characterization of their synthesis by interstitial cells. METHODS: The expression of HSPG on the surface of NRK 49F fibroblasts was demonstrated by immunohistochemistry and by metabolic labeling with [(35)S]-sulfate. HSs were characterized by specific enzymatic digestion, size exclusion chromatography, and anion exchange chromatography. The mRNA for syndecan 1 through syndecan 4 in the fibroblasts was detected by semiquantitative reverse transcription-polymerase chain reaction. Fibroblast proliferation was measured by the MTT assay. RESULTS: Immunohistochemistry and [(35)S]-sulfate-labeling demonstrated that renal fibroblasts expressed HSPGs on their surface. Furthermore, enzymatic removal of these HS (but not chondroitin sulfate) glycosaminoglycan (GAG) chains, or inhibition of GAG sulfation, abolished the proliferative response of both NRK cells and primary human cortical fibroblasts to FGF-2 but not to platelet-derived growth factor. The addition of conditioned medium, containing HS-GAG fragments, restored the proliferative response to FGF-2, confirming the specificity of the interaction. Finally, the mRNA for all four syndecans was detected in the fibroblasts, and that for syndecan 1 in particular was up-regulated by FGF-2. CONCLUSIONS: The present study demonstrates that the expression of cell surface HSPG was essential for the proliferation of renal fibroblasts in response to FGF-2, and therefore may play a major role in the development and persistence of a proliferating phenotype during interstitial nephritis.

Animals↗

PubMed2004

[Effects of substance P on granulation tissue fibroblasts proliferation and expression of basic fibroblast growth factor mRNA].

OBJECTIVE: To explore the proliferation-promoting effect of sensory neuropeptide substance P (SP) on the cultured granulation tissue fibroblasts in vitro and its regulative effect on the gene expression of basic fibroblast growth factor (bFGF) mRNA. METHODS: The proliferation-promoting effect of cultured granulation tissue fibroblasts was observed by means of MTT; the regulative effect of SP on gene expression of fibroblast bFGF by RT-PCR. The time and dose-efficiency relations were also observed. RESULTS: There was a significant proliferation-promoting effect of SP on the cultured granulation tissue fibroblasts in vitro in a remarkable dose-dependent fashion. However, bFGF antibody only partly exerted its inhibitive effect. SP could induce the bFGF mRNA expression of the fibroblasts at the 3rd and 6th hour (P < 0.01). SP could promote the bFGF mRNA expression of the fibroblasts in the concentration of 10(-9) - 10(-5) mol/L and peaked in the concentration of 10(-7) mol/L. CONCLUSIONS: SP has a significant proliferation-promoting effect on the granulation tissue fibroblasts, which is correlated with SP inducing bFGF mRNA expression of fibroblasts.

Animals↗

PubMed2000

Basic fibroblast growth factor expression is increased in human renal fibrogenesis and may mediate autocrine fibroblast proliferation.

BACKGROUND: Interstitial fibroblasts play a critical role in renal fibrogenesis, and autocrine proliferation of these cells may account for continuous matrix synthesis. Basic fibroblast growth factor (FGF-2) is mitogenic for most cells and exerts intracrine, autocrine, and paracrine effects on epithelial and mesenchymal cells. The aims of the present studies were to localize and quantitate the expression of FGF-2 in normal and pathologic human kidneys and to study the in vitro effects of FGF-2 on proliferation, differentiation, and matrix production of isolated cortical kidney fibroblasts. METHODS: FGF-2 protein expression was localized by immunofluoresence double labelings in normal and fibrotic human kidneys. Subsequently, interstitial FGF-2 labeling was determined semiquantitatively in 8 normal kidneys and 39 kidneys with variable degrees of interstitial fibrosis and was correlated with the morphometrically determined interstitial cortical volume. In addition, FGF-2 expression was quantitated by immunoblot analysis in three normal and six fibrotic kidneys. FGF-2 mRNA was localized by in situ hybridizations. Seven primary cortical fibroblast lines were established, and expression of FGF-2 and FGF receptor-1 (FGFR-1) were examined. The effects of FGF-2 on cell proliferation were determined by bromodeoxyuridine incorporation and cell counts, those on differentiation into myofibroblasts by staining for alpha-smooth muscle actin, and those on matrix synthesis by enzyme-linked immunosorbent assay for collagen type I and fibronectin. Finally, proliferative activity in vivo was evaluated by expression of MIB-1 (Ki-67 antigen). RESULTS: In normal kidneys, FGF-2 expression was confined to glomerular, vascular, and a few tubular as well as interstitial fibroblast-like cells. The expression of FGF-2 protein was increased in human kidneys, with tubulointerstitial scarring correlating with the degree of interstitial fibrosis (r = 0.84, P < 0.01). Immunoblot analyses confirmed a significant increase in FGF-2 protein expression in kidneys with interstitial scarring. In situ hybridization studies demonstrated low-level detection of FGF-2 mRNA in normal kidneys. However, FGF-2 mRNA expression was robustly up-regulated in interstitial and tubular cells in end-stage kidneys, indicating that these cells are the source of excess FGF-2 protein. Primary cortical fibroblasts express FGF-2 and FGFR-1 in vitro. FGF-2 induced a robust growth response in these cells that could be blocked specifically by a neutralizing FGF-2 antibody. Interestingly, the addition of the neutralizing antibody alone did reduce basal proliferation up to 31.5%. In addition, FGF-2 induced expression of alpha-smooth muscle actin up to 1.6-fold, but no significant effect was observed on the synthesis of collagen type I and fibronectin. Finally, staining for MIB-1 revealed a good correlation of interstitial FGF-2 positivity with interstitial and tubular proliferative activity (r = 0.71, P < 0.01 for interstitial proliferation, N = 30). CONCLUSIONS: Interstitial FGF-2 protein and mRNA expression correlate with interstitial scarring. FGF-2 is a strong mitogen for cortical kidney fibroblasts and may promote autocrine fibroblast growth. Expression of FGF-2 correlates with interstitial and tubular proliferation in vivo.

Adult↗

PubMed1998

Inhibition of type I collagen production by dermal fibroblasts upon contact with activated T cells: different sensitivity to inhibition between systemic sclerosis and control fibroblasts.

OBJECTIVE: To assess the role of T lymphocyte-fibroblast contact in type I collagen production by cultured dermal fibroblasts from normal individuals and from patients with diffuse systemic sclerosis (SSc). METHODS: Cell membranes were prepared from activated CD4+ and CD8+ T cells, or type 1 T helper (Th1) clones, and added to confluent fibroblast monolayers. Type I collagen production was measured in culture supernatants, and messenger RNA (mRNA) levels of type I procollagen alpha1 (pro alpha1[I]) and matrix metalloproteinase 1 (MMP-1) were evaluated by Northern hybridization analysis. RESULTS: Dose-dependent inhibition of type I collagen production was observed with CD4+ and CD8+ T cells from both SSc patients and controls. Inhibition of type I collagen was significantly less pronounced in fibroblasts from SSc patients than in fibroblasts from controls (P < 0.02). Inhibition was not reversed by the addition of exogenous transforming growth factor beta, interleukin-4, interleukin-1 receptor antagonist, anti-tumor necrosis factor, anti-CD40, or indomethacin, whereas anti-interferon-gamma (IFNgamma) reversed Th1-mediated inhibition. This inhibitory activity was specific for type I collagen, since mRNA levels of pro alpha1(I) were decreased, whereas mRNA levels of MMP-1 were strongly increased. CONCLUSION: The production of type I collagen by skin fibroblasts is specifically down-regulated by membranes from activated T cells. The contact-dependent regulatory activity exerted by T cells on fibroblasts depends, at least in part, on the presence of membrane-associated IFNgamma. However, SSc fibroblasts are more resistant to inhibition than are fibroblasts from normal individuals.

Aged↗

PubMed1997

Characterization of adult human heart fibroblasts in culture: a comparative study of growth, proliferation and collagen production in human and rabbit cardiac fibroblasts and their response to transforming growth factor-beta1.

Fibroblasts constitute the majority of non-myocyte cells in the heart, but little is known about the biology of human cardiac fibroblasts. We have cultured and characterized fibroblasts from young adult human heart and compared their biological features with those of cells obtained from the ventricular tissue of sex- and developmental-stage-matched rabbits. Cell morphology and growth was determined by light microscopy and by measuring protein synthesis. The proliferative capacity of cells was determined by measuring the doubling time and the level of incorporation of 3H-thymidine into DNA under normal conditions of cell culture and in response to several growth factors. Production of collagen type I was determined at the mRNA level by assessing the steady state level of pro alpha1 20(l) collagen mRNA in cardiac fibroblasts under normal culture conditions and in response to transforming growth factor-beta (TGF-beta), and at the protein level by immuno-slot blot analysis. Ventricular tissue from adult human heart was used successfully for the preparation of passagable (up to nine passages) cultured cardiac fibroblasts. They had a significantly larger surface area per cell than rabbit cells. Under normal culture conditions, they showed a lower rate of DNA synthesis and longer doubling time than rabbit cardiac fibroblasts at the matching passage. The magnitude of growth-factor-induced changes in the incorporation of 3H-thymidine into DNA was lower in human cells than in rabbit cardiac fibroblasts, as was the level of mRNA for pro alpha1(l) collagen. The TGF-beta1-induced increase in pro alpha1(l) collagen mRNA was modest in human cardiac fibroblasts. Thus, significant differences exist between the biological properties of human and rabbit cardiac fibroblasts in culture.

Adult↗

PubMed2002

Differences in motility pattern between human buccal fibroblasts and periodontal and skin fibroblasts.

Migration of fibroblasts from surrounding normal tissue into the wound bed is an important requirement for successful wound healing. This study investigated the motility pattern of buccal, periodontal and skin fibroblasts to determine whether differences in the wound healing efficiency at these sites can be explained by differences in the motile behavior of their respective fibroblast populations. The migratory characteristics were studied in a two-dimensional culture system. The migration of single cells was time-lapse video recorded at intervals of 15 min for a period of 6 h using a computer-assisted microscope work-station. For evaluation of cell morphology, cell contours were recognized semiautomatically and used for determination of cell area, cell spreading and number and length of processes. We found that the cellular displacement of the buccal fibroblasts was only approximately 50% of the cellular displacement of periodontal and skin fibroblasts. The decreased cellular displacement of the buccal fibroblasts was found to be due to both lower cellular speed and less persistence in direction. The buccal fibroblasts also displayed smaller areas and longer processes. The differences in cellular morphology and motility pattern amongst the three fibroblast types could not be explained by differences in secretion of extracellular matrix components and are therefore believed to reflect phenotypic differences amongst fibroblast subpopulations.

Actins↗

PubMed1998

Fibroblasts from post-burn hypertrophic scar tissue synthesize less decorin than normal dermal fibroblasts.

1. Fibroblast cultures were established from biopsies of hypertrophic scar and normal dermis taken from nine patients recovering from second- and third-degree burns. The capacity of these fibroblasts to synthesize the small proteoglycan decorin was assessed by quantitative Western blot analysis of conditioned medium collected from confluent cultures. Levels of mRNA for decorin were assessed by quantitative Northern analysis. Since transforming growth factor-beta 1 is implicated in various fibrotic conditions, including post-burn hypertrophic scar, its effect on decorin synthesis by these paired fibroblast cell strains was assessed. 2. Production of decorin was lower in all cell strains of hypertrophic scar fibroblasts tested, compared with normal dermal fibroblasts cultured from the same patients (mean 49 +/- 23%; P < 0.001, n = 9). Levels of mRNA for decorin were also lower (mean 59 +/- 28%; P < 0.02, n = 7) but those for biglycan and versican were not significantly different. Four pairs of cell strains were examined at more than one passage and the differences in decorin protein were found to be phenotypically persistent. Treatment of confluent cultures with transforming growth factor-beta 1 for 3 days caused a reduction in both decorin protein and mRNA in all six strains of hypertrophic scar fibroblasts tested and in five of six strains of normal dermal fibroblasts. An increase in the length of the dermatan sulphate chain on decorin, a previously reported characteristic of this glycosaminoglycan in hypertrophic scar, was seen in all but two of the strains treated with transforming growth factor-beta 1. The depression of decorin synthesis by transforming growth factor-beta 1 was reversed on removal of the agent and passaging the fibroblasts. 3. The reduced capacity of fibroblasts in hypertrophic scar tissue to synthesize decorin may have implications for the development of the condition since this small proteoglycan is involved in tissue organization and may also play a role in modulating the activity in vivo of fibrogenic cytokines such as transforming growth factor-beta 1.

Adolescent↗

PubMed2000

Allogenicity of cryopreserved human fibroblasts: cryopreservation does not downregulate the allogenicity of fibroblasts making up the matrices of allografts.

BACKGROUND: Although cryopreserved tissue allografts are being widely used, long-term degeneration of implanted cryopreserved allografts has become a problem. Although immunologic rejection has been suggested to play a part in this degeneration, cryopreserved allografts are considered to be less immunogenic than fresh allografts. OBJECTIVE: We investigated the effect of cryopreservation on the allogenicity of the fibroblasts that make up the matrices of allografts. METHODS: Fibroblast cell strains obtained from surgically resected lung specimens were used. Allogenicity-related antigens expressed on the cell surface (human leukocyte antigen 1, human leukocyte antigen 2, and intercellular adhesion molecule 1), stimulation indices during 1-way mixed lymphocyte-fibroblast cell culture, and proliferation indices of freshly passaged fibroblasts and cryopreserved fibroblasts stored for 1, 4, and 24 weeks were examined. Flow cytometric analysis with monoclonal antibodies was used to test for cell surface antigens, and a colorimetric methyl-thiazol-diphenyl-tetrazolium assay was used to assess stimulation indices and fibroblast proliferation indices. The effect of exogenous interferon-gamma on the degree of expression of human leukocyte antigen 1, human leukocyte antigen 2, and intercellular adhesion molecule 1 was examined simultaneously. RESULTS: The proliferation indices of fibroblasts were well maintained by cryopreservation. Expression of human leukocyte antigen 1, human leukocyte antigen 2, and intercellular adhesion molecule 1 by fibroblasts was significantly upregulated by interferon-gamma, and cryopreservation did not downregulate this expression. CONCLUSION: Our study suggests that although the fibroblast cell component may be beneficial in restoring allograft function properties initially, it may render the implanted allograft more immunogenic, ultimately resulting in greater rejection and inflammatory responses by the host and, in turn, degeneration of the graft.

Analysis of Variance↗

PubMed1985

Cell culture of embryonic chick duodenal cells: preparation of epithelial-fibroblast bilayers and homotypic cultures of fibroblasts and epithelial cells.

A method for the primary cell culture of trypsin-dissociated embryonic chick duodenum is described. Both heterotypic (epithelial cells and fibroblasts together) and homotypic (highly enriched cultures of epithelial cells or fibroblasts alone) cell cultures were established. Dispersed duodenal epithelial cells and fibroblasts grown in 10% fetal bovine serum (FBS) spontaneously aggregated and proliferated as a bilayer of cells with the epithelial cells growing on top of the fibroblasts. Changing the serum supplement to 6% chicken serum (CS) and 4% FBS when the fibroblast monolayer reached confluence resulted in epithelial cell proliferation. Homotypic cultures of epithelial cells and fibroblasts were prepared and analyzed by scanning electron microscopy and transmission electron microscopy. Fibroblasts, isolated by differential adhesion and grown in 10% FBS, did not demonstrate measurable alkaline phosphatase activity. Homotypic epithelial cell cultures, isolated by floating them off the fibroblasts with collagenase, and maintained on collagen in 6% CS/4% FBS, demonstrated higher alkaline phosphatase-specific activity (16.1 +/- 2.3 U/mg protein) compared with epithelial-fibroblast bilayer cell cultures (12.1 +/- 1.3 U/mg protein).

Alkaline Phosphatase↗

PubMed1996

Venous ulcer fibroblasts compared with normal fibroblasts show differences in collagen but not fibronectin production under both normal and hypoxic conditions.

Previous immunocytochemical analysis showed that the base of venous ulcers was deficient in fibronectin compared with surrounding "normal" dermis. Here, we investigate whether impaired synthetic ability of ulcer fibroblasts could underlie this observation. Ulcer fibroblasts, established in culture from biopsies of the edge of chronic venous leg ulcers, were compared with normal fibroblasts grown from biopsies of site-and age-matched normal skin for their ability to synthesize matrix molecules. Collagen and fibronectin synthesis were measured following metabolic labeling, as collagenase susceptible counts and counts with gelatin affinity, respectively. More collagen was produced by normal fibroblasts than ulcer fibroblasts, both when the cells were cultured on plastic and in collagen gels. In fibronectin synthesis, however, there was no major difference between the two cell types on either substratum. The hypoxic environment to which ulcer fibroblasts are exposed may have caused the intrinsic differences in collagen synthesis by the two fibroblast types. When we tested the effect of culturing cells under hypoxic conditions, both cell types produced less collagen, especially normal fibroblasts grown in a collagen gel, but there was no effect of hypoxia on fibronectin synthesis. We conclude that venous ulcer edge-derived fibroblasts have an impaired ability to synthesize collagen in vitro, but synthesize fibronectin normally. Therefore, the low level of fibronectin found in venous ulcers is not likely to be due to the inability of ulcer cells to produce it or to the response to hypoxic conditions but may be due to the degradation of synthesized fibronectin by proteases present in these ulcers.

Cells, Cultured↗

PubMed2005

Conditioned medium from keloid keratinocyte/keloid fibroblast coculture induces contraction of fibroblast-populated collagen lattices.

BACKGROUND: Keloid scars represent a pathological response to cutaneous injury. Overproliferation of fibroblasts and overproduction of collagen characterize these abnormal scars. The pathology of these scars remains poorly understood. The role of epithelial-mesenchymal interactions in keloid pathogenesis and scar contracture has recently been explored. OBJECTIVES: To test our hypothesis that epithelial-mesenchymal interactions play a major role in modulating keloid scar contracture. METHODS: A coculture model was employed wherein keloid and normal keratinocytes were cocultured with keloid or normal fibroblasts, and the conditioned media from day 5 cocultures were collected to study the effect of the paracrine secretions on contraction of an in vitro fibroblast-populated collagen lattice (FPCL) model. RESULTS: Keloid keratinocyte/keloid fibroblast coculture conditioned media brought about increased contraction of the collagen lattice compared with non-cocultured conditioned media. When keloid fibroblasts populated the collagen lattice, significantly increased lattice contraction was induced compared with lattices populated by normal fibroblasts. The addition of antitransforming growth factor (TGF)-beta neutralizing antibody to the conditioned media produced an attenuation of the contraction of the FPCLs. When keloid and normal fibroblasts were cultured on chamber slides and treated with conditioned media from coculture and non-coculture series, immunohistochemical analysis demonstrated an increased expression of alpha-smooth muscle actin (a marker for fibroblast differentiation into myofibroblasts) in fibroblasts exposed to conditioned media from coculture. CONCLUSIONS: These data indicate that epithelial-mesenchymal interactions are likely to play a major role in scar contracture and scar pathogenesis, and underscore the role of TGF-beta1 as a key player in keloid pathogenesis.

Actins↗

PubMed1991

Constitutive expression of a 92-kD gelatinase (type V collagenase) by rheumatoid synovial fibroblasts and its induction in normal human fibroblasts by inflammatory cytokines.

Synovial fibroblasts freshly isolated from the rheumatoid joint are characterized by their marked connective tissue degradative ability. This phenotype includes the ability to secrete large amounts of the matrix-degrading metalloproteinases, collagenase, and stromelysin. We have found that another aspect of this phenotype is the constitutive expression at both protein and mRNA levels of a 92-kD gelatinolytic metalloproteinase, which is not secreted by normal dermal or lung fibroblasts and is immunologically cross-reactive with a type V collagenase expressed by activated macrophages and neutrophils. Expression of this 92-kD metalloproteinase confers upon the fibroblasts the capacity to degrade collagenase- and stromelysin-resistant interstitial elements, such as collagen types IV, V and XI. In contrast to the 92-kD metalloproteinase, a 68-kD gelatinase (type IV collagenase) was expressed by all fibroblast types studied, indicating that its regulation is distinct from that of the 92-kD gelatinase. To identify what cytokines may be important in the induction of the rheumatoid synovial phenotype, including expression of the 92-kD gelatinase, we exposed normal dermal fibroblasts to a number of cytokines including many known or considered likely to be present in rheumatoid synovial fluid and tissue. Although IL-1 beta, tumor necrosis factor-alpha, lymphotoxin, platelet-derived growth factor, and basic fibroblast growth factor were capable of stimulating fibroblasts to secrete collagenase, only tumor necrosis factor-alpha, lymphotoxin, and IL-1 beta were able to induce expression of the 92-kD gelatinase, demonstrating discordant regulation of the two metalloproteinases. Expression of the 68-kD gelatinase was independent of that of the 92-kD gelatinase, as demonstrated at the protein and mRNA levels. Late passage rheumatoid synovial fibroblasts, which no longer constitutively expressed the 92-kD gelatinase, displayed an accentuated response to IL-1 beta when compared to normal dermal fibroblasts. Thus, in addition to IL-1 beta, tumor necrosis factor-alpha or lymphotoxin may contribute to the expression of a specific rheumatoid synovial phenotype in vivo that is associated with progressive matrix destruction.

Arthritis, Rheumatoid↗

PubMed1992

Cytokine regulation of human lung fibroblast hyaluronan (hyaluronic acid) production. Evidence for cytokine-regulated hyaluronan (hyaluronic acid) degradation and human lung fibroblast-derived hyaluronidase.

We characterized the mechanisms by which recombinant (r) tumor necrosis factor (TNF), IFN-gamma, and IL-1, alone and in combination, regulate human lung fibroblast hyaluronic acid (HA) production. Each cytokine stimulated fibroblast HA production. The combination of rTNF and rIFN-gamma resulted in a synergistic increase in the production of high molecular weight HA. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous decrease in HA degradation. In contrast, when rTNF and rIL-1 were combined, an additive increase in low molecular weight HA was noted. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous increase in HA degradation. Human lung fibroblasts contained a hyaluronidase that, at pH 3.7, depolymerized high molecular weight HA to 10-40 kD end products of digestion. However, hyaluronidase activity did not correlate with fibroblast HA degradation. Instead, HA degradation correlated with fibroblast-HA binding, which was increased by rIL-1 plus rTNF and decreased by rIFN-gamma plus rTNF. Recombinant IL-1 and rTNF weakly stimulated and rIL-1 and rTNF in combination further augmented the levels of CD44 mRNA in lung fibroblasts. In contrast, rIFN-gamma did not significantly alter the levels of CD44 mRNA in unstimulated or rTNF stimulated cells. These studies demonstrate that rIL-1, rTNF, and rIFN-gamma have complex effects on biosynthesis and degradation which alter the quantity and molecular weight of the HA produced by lung fibroblasts. They also show that fibroblast HA degradation is mediated by a previously unrecognized lysosomal-type hyaluronidase whose function may be regulated by altering fibroblast-HA binding. Lastly, they suggest that the CD44 HA receptor may be involved in this process.

Adult↗

PubMed1995

Human orbital fibroblasts in culture express ganglioside profiles distinct from those in dermal fibroblasts.

Orbital fibroblasts appear phenotypically distinct from those derived from dermis and other extraorbital anatomical sites. In this study, we examined the profile of gangliosides expressed by orbital and dermal fibroblasts. Gangliosides have a wide range of functions including modulation of transmembrane signal transduction. The aim of this study was to examine the hypothesis that a differential expression of gangliosides by orbital and nonorbital fibroblasts could constitute an important determinant of the immunological properties peculiar to the orbit. Moreover, these differences could provide a molecular basis for the site-specific involvement of the orbit in Graves' ophthalmopathy. Total lipids were extracted from confluent cultures of six different orbital and six dermal fibroblast strains, and purified gangliosides were subjected to two-dimensional thin layer chromatographic analysis. Orbital and dermal fibroblasts contained qualitatively similar ganglioside contents, with two major peaks, one migrating in the mono- and the other in the disialoganglioside regions of the chromatogram. In orbital fibroblasts, the densities of these two peaks were nearly equal, whereas in dermal fibroblasts, the monosialoganglioside peak was 5- to 6-fold greater. Minor ganglioside peaks were resolved and were equally abundant in orbital and dermal fibroblasts. Ganglioside profiles were invariant with respect to treatment of fibroblasts with interferon-gamma. These differences in expression of the two major ganglioside species may be relevant to the peculiarities associated with normal and pathological events in orbital connective tissue.

Cells, Cultured↗