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PubMed1994

Collagen type I is not under autocrine control by transforming growth factor-beta 1 in normal and scleroderma fibroblasts.

BACKGROUND: The ability of transforming growth factor-beta (TGF-beta) to induce synthesis of extracellular matrix proteins stimulated this study in which we address the hypothesis that TGF-beta can induce, in normal fibroblasts, the sustained, elevated collagen synthesis characteristic of the scleroderma fibroblast. EXPERIMENTAL DESIGN: Fibroblasts were studied for synthesis of and responsiveness to TGF-beta. Secreted TGF-beta levels were determined in a bioassay and at the transcriptional level in a series of scleroderma (SSc) and normal fibroblasts. The ability of cells to interact functionally with a 3-dimensional collagen matrix after TGF-beta treatment was examined. The kinetics of TGF-beta-induced fibrosis in fibroblasts was studied. RESULTS: SSc fibroblasts were not characterized by elevated TGF-beta synthesis. There was no evidence of coordinate regulation of TGF-beta and collagen over passage number. Repeated pulses of 200 pM of TGF-beta did not significantly induce sustained procollagen alpha 1(I) mRNA synthesis in normal fibroblasts, and this treatment did not significantly alter the characteristics of normal fibroblasts in a collagen gel. mRNA for both collagen and TGF-beta type II receptor was induced by TGF-beta in both SSc and control cells. SSc fibroblasts were found to have an impaired ability to activate the small latent complex of TGF-beta. CONCLUSIONS: Our data give no support to the hypothesis that TGF-beta can maintain the SSc phenotype in vitro or that it is able to induce this phenotype. The inducibility of TGF-beta receptor mRNA in SSc fibroblasts after exposure to TGF-beta suggests that the lack of sustained elevation in collagen synthesis is not due to lack of responsiveness by the fibroblasts but is rather a reflection of the transient nature of TGF-beta-induced fibrosis.

Cell Division↗
PubMed1995

Potential genoprotective role for UDP-glucuronosyltransferases in chemical carcinogenesis: initiation of micronuclei by benzo(a)pyrene and benzo(e)pyrene in UDP-glucuronosyltransferase-deficient cultured rat skin fibroblasts.

UDP-glucuronosyltransferases (UGTs) are cytoprotective and may also be genoprotective. Since over 10% of the population have hereditary deficiencies in UGTs, this family of enzymes could constitute an important determinant of susceptibility to chemical carcinogenesis, teratogenesis, and neurodegeneration. Fibroblasts contain Phase I and II drug-metabolizing enzymes, including UGTs, and undergo mitosis, rendering them susceptible to xenobiotic genotoxicity associated with micronucleus formation, which is thought to reflect carcinogenic initiation. Accordingly, skin fibroblasts may provide an accessible model for elucidating genoprotective mechanisms in both animals and humans and for characterizing the potential role of UGTs as determinants of individual toxicological susceptibility. To test this hypothesis, the carcinogen/teratogen benzo(a)pyrene [B(a)P], or its noncarcinogenic B(e)P isomer, was incubated with cultured skin fibroblasts obtained from male RHA-J/J rats. These rats have a hereditary homozygous deficiency in bilirubin UGT and demonstrate reduced xenobiotic glucuronidation, enhanced cytochrome P-450-catalyzed bioactivation, covalent binding, and toxicity of acetaminophen and B(a)P. Control fibroblasts were cultured from UGT-normal congenic homozygous male RHA-(+/+) rats and male Wistar rats. The cells were incubated with 10 microM B(a)P or B(e)P either for assessment of micronucleus formation or for quantifying the bioactivation and covalent binding of B(a)P and the glucuronidation of its hydroxylated metabolites. Compared to control fibroblasts incubated only with buffer, micronucleus formation was not enhanced by either DMSO vehicle or B(e)P. In contrast, B(a)P significantly enhanced micronucleus formation in all cells, and UGT-deficient cells (RHA-J/J) had a > 2-fold higher B(a)P-initiated micronucleus formation compared to UGT-normal cells (RHA-(+/+)) (P < 0.05). Glucuronidation of total B(a)P metabolites was 10% lower in RHA-J/J UGT-deficient fibroblasts, and the covalent binding of B(a)P to protein, reflective of an electrophilic reactive intermediate and DNA-alkylating agent, was up to 3-fold higher in RHA-J/J UGT-deficient fibroblasts or fibroblast homogenates compared to UGT-normal controls (P < 0.05). In fibroblast homogenates, addition of the UGT cosubstrate UDP-glucuronic acid reduced B(a)P covalent binding, corroborating the cytoprotective importance of UGTs. There was a highly significant correlation between decreasing glucuronidation of B(a)P metabolites and increasing bioactivation and covalent binding of B(a)P (r = -0.889; P = 0.018) in fibroblasts from RHA-J/J and RHA-(+/+) rat strains, indicating an important genoprotective role for UGT. These results provide the first evidence that hereditary UGT deficiencies may enhance susceptibility to chemical carcinogenesis and suggest that skin fibroblasts may provide a useful and highly sensitive model for human risk assessment.

Animals↗
PubMed1993

Synthesis of collagen and fibronectin in fibroblasts derived from healthy and hyperplastic gingivae.

The purpose of the present study was to compare protein and matrix molecular (collagen and fibronectin) metabolism among fibroblasts cultured from two types of hyperplastic gingiva and normal tissues. Cell cultures were obtained from tissue explants of normal gingiva, the periodontal ligament (PDL), and phenytoin-induced and idiopathic fibromatotic gingiva. The synthetic capacity of matrix proteins of each isolated fibroblast type was studied by metabolic labeling (3H-proline), biochemical purification and characterization. Our results showed that fibroblasts derived from phenytoin-induced gingival hyperplasia synthesized a greater amount of total protein and collagen than the rest of the cell types, based on both the amount of total protein and protein/micrograms DNA. In contrast, fibroblasts obtained from idiopathic gingival fibromatosis exhibited a similar potential for both protein and collagen synthesis as normal gingival and PDL fibroblasts. Normal PDL fibroblasts displayed a significantly higher degree of collagen synthesis than normal gingival fibroblasts; however, no difference in protein production between them was noted. A few variations in fibronectin synthesis/microgram DNA among various fibroblasts from normal and hyperplastic tissues were noted. PDL fibroblasts seemed to produce a greater amount of fibronectin than all of the other cell types. Interestingly, fibroblasts obtained from idiopathic gingival fibromatosis showed a higher DNA content than other fibroblasts, although a decrease in synthesized fibronectin/microgram DNA was noted. In conclusion, the pathogenic mechanism of phenytoin-induced hyperplasia and idiopathic gingival fibromatosis may be different due to their distinct profile of cell proliferation and matrix protein synthesis.

Cells, Cultured↗
PubMed1996

Fibroblast radiosensitivity in vitro and lung fibrosis in vivo: comparison between a fibrosis-prone and fibrosis-resistant mouse strain.

Radiation-induced pneumonitis and fibrosis in the lung after treatment to the thoracic cavity for malignant disease currently limit the maximum tolerated dose to that region. It has been suggested that heterogeneity in susceptibility to radiation-induced fibrosis exists in the population, implying that the lung tolerance dose is defined by a sensitive subset of the patient population. Studies of radiotherapy patients have indicated that the survival at 2 Gy (SF2) of cultured skin fibroblasts correlates with the incidence and severity of postirradiation damage in a number of tissues, suggesting that this assay may be a useful predictor of late tissue effects. The goal of the studies presented here was to determine if the radiosensitivity of fibroblasts in vitro isolated from mouse lungs was correlated with the severity of radiation-induced fibrosis in the lungs of two inbred strains of mice previously shown to differ markedly in their susceptibility to radiation-induced lung fibrosis: the C3Hf/Kam strain, classified as fibrosis-resistant, and the C57BL/6J strain, classified as fibrosis-prone. Quantitative measurements of lung fibrosis after irradiation were compared to SF2 values for fibroblasts of skin and lung cultured from each strain. Lung fibrosis was quantified, using computerized image analysis, as the percentage of fibrosis on Masson's Trichrome-stained lung sections from both strains after single doses of radiation to the thorax. For the measurements of SF2, fibroblasts plated at the second passage and grown to confluence were given single doses of radiation ranging from 0 to 6 Gy. Survival curves were constructed and SF2 values obtained from a linear-quadratic fit to the data. The radiosensitivity of fibroblasts from the lung and skin of SCID mice was determined and served as a positive control. The percentage of radiation-induced lung fibrosis was significantly different between the two strains, 5.1% and 0.2% in the C57 strain and C3H strain, respectively. Follow-up of long-term survivors (two mice) from the C3H strain did not change this conclusion. However, the lung fibroblast SF2 for the C57BL/6J strain (fibrosis-prone), 0.50 +/- 0.03, was not statistically different from the C3Hf/Kam strain (fibrosis-resistant), 0.55 +/- 0.07. These data indicate that in vitro radiosensitivity of lung fibroblasts as assayed by survival at 2 Gy does not correlate with the development of lung fibrosis in this mouse model. The SF2 for lung fibroblasts from SCID mice was 0.10. Similar SF2 values were obtained for both the C3Hf/Kam mouse lung and skin fibroblasts, 0.55 and 0.56, respectively, and C57BL/6J mouse lung and skin fibroblasts, 0.50 and 0.52, respectively, indicating that the radiosensitivity of fibroblasts isolated from lung and skin within a strain is the same.

Animals↗
PubMed1996

Induction of fibroblast apoptosis by anti-CD44 antibody: implications for the treatment of fibroproliferative lung disease.

Fibroblast migration and proliferation within the alveolar wall and airspace after lung injury can lead to the development of fibrosis. Fibroblast cell surface CD44 is an adhesion receptor for provisional matrix proteins and mediates fibroblast invasion into fibrin matrices. Here we show that incubation of cultured fibroblasts with an anti-CD44 monoclonal antibody induces fibroblast detachment from the substratum and morphological changes compatible with apoptosis. In addition, we show that anti-CD44 monoclonal antibody rapidly induces fibroblast apoptosis within fibrin matrices. The effect of anti-CD44 antibody on induction of fibroblast apoptosis occurred within 8 hours and was dose dependent. Anti-CD44 antibody also induced fibroblast apoptosis in suspension. Furthermore, fibroblasts plated on anti-CD44-antibody-coated surfaces initially attached and spread on the antibody; however, after an 8-hour incubation time, many of the cells developed characteristic morphological features of apoptosis. Collectively, these data indicate that apoptosis did not result solely due to detachment from the substratum. Our results identify a new function for fibroblast cell surface CD44 related to the control of cell viability. We suggest this function may be important in fibroblast population control and could potentially be exploited in designing anti-fibrotic therapies.

Antibodies, Monoclonal↗
PubMed1997

Regulation of the functional activity of mast cells and fibroblasts by mononuclear cells in murine and human chronic graft-vs.-host disease.

Mast cell (MC)-fibroblast-immunocompetent cell interactions may play a role in the inflammatory and fibrotic processes present in chronic graft-vs.-host disease (cGVHD). Interactions between these cell types were examined in both murine and human cGVHD models. To this purpose, cell supernatants from mice or humans with cGVHD and from controls were incubated for 6 days with either rat peritoneal MCs cocultured with 3T3 fibroblasts or with 3T3 fibroblasts alone. Supernatants in the murine model were of splenocytes from either mice with cGVHD or syngeneic controls (B-->B). Supernatants in the human model were of peripheral blood mononuclear cells (PBMC) from cGVHD patients. Two groups of controls were used in the human model-patients who had undergone bone marrow transplantation (BMT) without developing cGVHD and patients with hematological malignancies who had not undergone bone marrow transplantation (pre-BMT). Histamine release was measured in MC/fibroblast cocultures incubated with the cell supernatants. Prostaglandin E2 (PGE2) and [3H]-thymidine incorporation were measured in both MC/fibroblast cocultures or 3T3 fibroblasts alone, incubated with the cell supernatants. In the murine model, the cGVHD supernatant caused significantly more histamine release from MCs than the syngeneic supernatant or medium alone. Moreover, cGVHD and syngeneic supernatants, compared with medium alone, inhibited 3T3 fibroblast proliferation. PGE2 production by 3T3 fibroblasts was higher after incubation with the cGVHD supernatant than with the syngeneic supernatant or in medium alone. Incubation of fibroblasts with supernatants and indomethacin decreased PGE2 production and increased [3H]-thymidine incorporation. In humans, PBMC supernatants from cGVHD patients, as well as from BMT and pre-BMT controls, also displayed histamine releasing activity when cocultured with rat MCs. As with the murine cGVHD splenocyte supernatant, the human cGVHD supernatant decreased fibroblast [3H]-thymidine uptake, but the presence of MCs in the culture abrogated this inhibitory effect. In addition, the human cGVHD supernatant was found to contain high levels of PGE2 and interleukin-1 beta (IL-1 beta). The addition of neutralizing anti-IL-1 beta antibodies to the cGVHD supernatant partially inhibited its histamine-releasing activity. Skin biopsies of involved areas in cGVHD patients revealed significantly reduced numbers of MCs and showed signs of MC degranulation compared with biopsies from pre-BMT controls. Immunocompetent cell supernatants from both mice and humans with cGVHD increased basal histamine release by MCs and reduced fibroblast proliferation. The murine cGVHD supernatant also enhanced PGE2 production by 3T3 fibroblasts. Our findings indicate that complex interactions between immunocompetent cells, MCs, and fibroblasts probably play a role in cGVHD pathogenesis.

3T3 Cells↗
PubMed1998

In vitro procollagen synthesis and proliferative phenotype of bronchial fibroblasts from normal and asthmatic subjects.

Asthma is characterized histologically by a bronchial subepithelial fibrosis. Cytokines and other mediators released in the asthmatic chronic inflammatory microenvironment can activate the repair process that leads to fibroblast proliferation and collagen synthesis. To our knowledge, there are no data regarding the effect of a chronic inflammatory microenvironment on the phenotype of human bronchial fibroblasts. In the present study, we address this issue by comparing bronchial fibroblasts isolated from normal and asthmatic subjects in terms of: (a) proliferation over cell passage; (b) in vitro lifespan; (c) proliferative response to transforming growth factor-beta 1, platelet-derived growth factor-BB, dexamethasone, and retinoic acid; and (d) base-line synthesis of procollagens I and III. Bronchial fibroblasts from asthmatic subjects demonstrated lower DNA synthesis with cell passage than bronchial fibroblasts from normals. The in vitro lifespan of asthmatic bronchial fibroblasts was lower than in those from normal subjects and was significantly correlated with airway responsiveness. Platelet-derived growth factor-BB and dexamethasone increased 3H-thymidine incorporation in asthmatic bronchial fibroblasts without having any significant effect on normal fibroblast proliferation. Transforming growth factor-beta 1 and retinoic acid had no significant effect on bronchial fibroblast proliferation. Base-line procollagens I and III synthesis measurements showed no differences between normal and asthmatic fibroblasts. Taken together, these results indicate that the chronic inflammatory microenvironment found in asthma can modulate some aspects of bronchial fibroblast phenotype.

Adult↗
PubMed1999

Discrepant expression of neprilysin on fibroblasts in diffuse systemic sclerosis.

OBJECTIVE: Neprilysin (NEP; EC3.4.24.11) is an ectopeptidase mainly produced by fibroblasts and cleaving a large number of neuropeptides. We previously found increased plasma circulating levels of NEP in patients with systemic sclerosis (SSc), but in SSc fibroblasts derived from the diffuse subset NEP was present in lower concentration. We evaluate in vitro fibroblasts of both subsets of the disease, diffuse and limited, the intracellular levels of NEP, and its expression as CD10 on the cellular surface. METHODS: Fibroblasts, derived from biopsies taken from affected skin of 8 patients with the limited subset and 5 with the diffuse subset, were grown in vitro and intracellular levels of NEP activity were measured with a fluorometric method, while CD10 surface expression was evaluated by FACS analysis. Cell proliferation was assessed by 3HThymidine incorporation. RESULTS: Intracellular NEP activity was significantly increased in diffuse (7.02+/-4.8 pg/ml/min 10(6) cells) compared to limited SSc (1.11+/-2.0) and control fibroblasts (1.41+/-0.9). CD10 expression was significantly impaired on diffuse SSc fibroblasts (47.3+/-15%) compared to controls (74.6+/-11%) and the limited subset (82.7+/-11%). Cell proliferation of diffuse SSc fibroblasts was strikingly higher than controls and limited SSc fibroblasts. CONCLUSION: These results confirm that NEP is produced by fibroblasts and indicate that in diffuse SSc fibroblasts NEP is produced in higher quantities, while the expression of the enzyme on the cell surface is significantly reduced. This condition may affect the proliferation rate of fibroblasts as well as the metabolism of various peptides.

Cell Division↗
PubMed2004

Site-matched papillary and reticular human dermal fibroblasts differ in their release of specific growth factors/cytokines and in their interaction with keratinocytes.

The interfollicular dermis of adult human skin is partitioned into histologically and physiologically distinct papillary and reticular zones. Each of these zones contains a unique population of fibroblasts that differ in respect to their proliferation kinetics, rates at which they contract type I collagen gels, and in their relative production of decorin and versican. Here, site-matched papillary and reticular dermal fibroblasts couples were compared to determine whether each population interacted with keratinocytes in an equivalent or different manner. Papillary and reticular fibroblasts grown in monolayer culture differed significantly from each other in their release of keratinocyte growth factor (KGF) and granulocyte-macrophage colony stimulating factor (GM-CSF) into culture medium. Some matched fibroblast couples also differed in their constitutive release of interleukin-6 (IL-6). Papillary fibroblasts produced a higher ratio of GM-CSF to KGF than did corresponding reticular fibroblasts. Interactions between site-matched papillary and reticular couples were also assayed in a three-dimensional culture system where fibroblasts and keratinocytes were randomly mixed, incorporated into type I collagen gels, and allowed to sort. Keratinocytes formed distinctive cellular masses in which the keratinocytes were organized such that the exterior most layer of cells exhibited characteristics of basal keratinocytes and the interior most cells exhibited characteristics of terminally differentiated keratinocytes. In the presence of papillary dermal fibroblasts, keratinocyte masses were highly symmetrical and cells expressed all levels of differentiation markers. In contrast, keratinocyte masses that formed in the presence of reticular fibroblasts tended to have irregular shapes, and terminal differentiation was suppressed. Furthermore, basement membrane formation was retarded in the presence of reticular cells. These studies indicate that site-matched papillary and reticular dermal fibroblasts qualitatively differ in their support of epidermal cells, with papillary cells interacting more effectively than corresponding reticular cells.

Adult↗
PubMed2005

Regulation of neuronal death and calcitonin gene-related peptide by fibroblast growth factor-2 and FGFR3 after peripheral nerve injury: evidence from mouse mutants.

The presence and regulation of basic fibroblast growth factor and its high-affinity tyrosine kinase receptor FGFR3 in sensory neurons during development and after peripheral nerve injury suggest a physiological role of the fibroblast growth factor-2 system for survival and maintenance of sensory neurons. Here we investigated L5 spinal ganglia of intact and lesioned fibroblast growth factor-2 knock-out and FGFR3 knock-out mice. Quantification of sensory neurons in intact L5 spinal ganglia revealed no differences between wild-types and mutant mice. After sciatic nerve axotomy, the normally occurring neuron loss in wild-type mice was significantly reduced in both knock-out strains suggesting that fibroblast growth factor-2 is involved in neuronal death mediated via FGFR3. In addition, the number of chromatolytic and eccentric cells was significantly increased in fibroblast growth factor-2 knock-out mice indicating a transient protection of injured spinal ganglia neurons in the absence of fibroblast growth factor-2. The expression of the neuropeptide calcitonin gene-related peptide in sensory neurons of intact fibroblast growth factor-2 knock-out and FGFR3 knock-out mice was not changed in comparison to adequate wild-types. Fibroblast growth factor-2 wild-type and FGFR3 wild-type mice showed a lesion-induced decrease of calcitonin gene-related peptide-positive neurons in ipsilateral L5 spinal ganglia whereas the loss of calcitonin gene-related peptide-immunoreactive sensory neurons is reduced in the absence of fibroblast growth factor-2 or FGFR3, respectively. In addition, FGFR3 wild-type and knock-out mice displayed a contralateral reduction of the neuropeptide after axotomy. These results suggest that endogenous fibroblast growth factor-2 and FGFR3 are crucially involved in the regulation of survival and calcitonin gene-related peptide expression of lumbar sensory neurons after lesion, but not during development.

Animals↗
PubMed1999

Visual input regulates the expression of basic fibroblast growth factor and its receptor.

Emerging evidence indicates that the expression of trophic factors in the brain is regulated in an activity-dependent manner, which suggests an involvement of trophic factors in events controlled by input activity. We have investigated the possibility that visual sensory input impacts the expression of basic fibroblast growth factor and its receptor in the brain. Rats were maintained for seven days in darkness and then re-exposed to normal illumination for 0, 1, 3 or 6 h. We assessed relative levels of basic fibroblast growth factor and fibroblast growth factor receptor messenger RNAs using nuclease protection assays, and examined possible changes in the phenotypic expression of basic fibroblast growth factor and its receptor using immunohistochemistry. There was a significant decrease in levels of basic fibroblast growth factor and fibroblast growth factor receptor messenger RNAs as a result of dark rearing, and levels of messenger RNAs increased progressively with light re-exposure. Changes in messenger RNAs were observed primarily in the cerebral cortex (caudal portion) and were accompanied by alterations in the staining intensity and density of cells exhibiting basic fibroblast growth factor and fibroblast growth factor receptor phenotypes. Regulation of the basic fibroblast growth factor system by sensory input suggests that basic fibroblast growth factor, and perhaps other trophic factors, are mediators of the effects of experience on the structure and function of the CNS.

Animals↗
PubMed1992

An acidic fibroblast growth factor protein generated by alternate splicing acts like an antagonist.

Polymerase chain reaction amplification of cDNA for acidic fibroblast growth factor in several lines of cultured human cells revealed two forms of mRNA. The novel smaller mRNA lacks the entire second coding exon of the acidic fibroblast growth factor gene, whereas the previously identified mRNA consists of three coding exons. The truncated variant of acidic fibroblast growth factor (aFGF') is only 60 amino acids long with an apparent molecular mass of 6.7 kD on sodium dodecyl sulfate gels in contrast to 18 kD for the full-length acidic fibroblast growth factor. aFGF' elicits only minimal fibroblast proliferation and antagonizes the effects of acidic fibroblast growth factor when added exogenously to or when coexpressed with aFGF in BALB/c/3T3 fibroblasts. Thus, the truncated variant of acidic fibroblast growth factor may provide fibroblasts with a unique mechanism for endogenous regulation of their responses to acidic fibroblast growth factor.

Amino Acid Sequence↗
PubMed2005

Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta.

Keratinocyte growth factor (KGF) is secreted by fibroblasts and protects from pulmonary fibrosis in animal models. Interleukin (IL)-1beta is the most potent inducer of KGF in fibroblasts, acting through the c-Jun pathway. We evaluated in vitro KGF production by human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF, n = 10) and from control subjects (n = 7) at baseline and after IL-1beta stimulation. Basal KGF secretion by IPF fibroblasts was similar to controls. In fibroblasts from control subjects, IL-1beta increased c-Jun expression, c-Jun activation, and KGF secretion. SP600125, a specific c-Jun N-terminal kinase (JNK) inhibitor, inhibited the effect of IL-1beta. By contrast, in IPF fibroblasts, IL-1beta did not increase c-Jun expression and c-Jun activation, and weakly increased KGF secretion, whereas SP600125 had no effect. IL-1beta similarly increased JunB expression in fibroblasts from patients with IPF and control subjects. Total JNK content was not different in either unstimulated or IL-1beta-stimulated IPF and control fibroblasts. IL-1beta increased phosphorylated JNK in control and IPF fibroblasts, but this increase was weaker and heterogeneous in IPF. Altogether, our results demonstrate a dysregulation of KGF secretion by IPF fibroblasts. The weak response to IL-1beta is associated with a defect of c-Jun expression and activation and a defect of JNK activation.

Adult↗
PubMed2002

Expression profiles of craniosynostosis-derived fibroblasts.

BACKGROUND: Craniosynostosis syndromes, a group of connective disorders characterized by abnormalities in vault osteogenesis and premature fusion of bone sutures, are associated with point mutations in FGF receptor family members. The cellular phenotype is characterized by abnormal extracellular matrix turnover. MATERIAL AND METHODS: We used primary cultures of periosteal fibroblasts derived from two different craniosynostosis syndromes, the Apert and Crouzon syndromes. The FGFR2 third immunoglobulin-like domain and its flanking linker regions were analyzed for mutation. DNA microarrays containing 19,200 cDNAs were used to study the gene expression profiles of Apert and Crouzon fibroblasts. The pathologic cells were compared to wild-type human periosteal fibroblasts. RESULTS: The P253R missense mutation and the G338R mutation were observed in Apert and Crouzon fibroblasts, respectively. The genetic profiles, as evaluated by DNA microarrays, yielded different clusters of expressed sequence tag (ESTs) expression within the experiment. Expression profiles from craniosynostosis-derived fibroblasts differ from those of wild-type fibroblasts (288 human ESTs, p< 0.01, pFDR = 0.12). Furthermore, two ESTs clusters discriminate the Crouzon from Apert fibroblasts. The differentially expressed genes cover a broad range of functional activities, including (1) bone differentiation, (2) cell-cycle regulation, (3) apoptotic stimulation, and (4) signaling transduction, cytoskeleton, and vesicular transport. CONCLUSIONS: The transcriptional program of craniosynostosis fibroblasts differs from that of wild-type fibroblasts. Expression profiles of Crouzon and Apert fibroblasts can also be distinguished by two EST expression clusters, thus hinting at a different genetic background.

Acrocephalosyndactylia↗
PubMed1997

Basic fibroblast growth factor induces apoptosis in myofibroblastic cells isolated from rat palatal mucosa.

The effect of basic fibroblast growth factor (bFGF) on apoptosis in normal rat palatal fibroblasts and rat palatal scar fibroblasts was examined by the TUNEL method in order to clarify the mechanism of apoptosis induction in myofibroblasts during the scar formation process. A percentage of scar fibroblasts undergoing apoptosis was significantly higher than that of palatal fibroblasts when they were treated with bFGF succeeding to serum starvation. Palatal fibroblasts, phenotypically modulated into myofibroblasts by the pretreatment with transforming growth factor-beta 1 (TGF-beta 1), similarly showed a higher level of apoptosis induction by bFGF-treatment. TGF-beta 1 elevated protein and mRNA level of FGF receptor (FGFR) in palatal fibroblasts. Tyrosine autophosphorylation of FGFR upon stimulation by bFGF was significantly higher in scar fibroblasts than in normal palatal fibroblasts. These findings suggested that bFGF may be a potential stimulator of apoptosis in myofibroblasts during palatal scar formation and that FGFR may be responsible for this process.

Animals↗
PubMedDate not supplied

Characteristics of growth, morphology, contractility, and protein expression of fibroblasts derived from keloid.

Phenotypic alterations of keloid-derived fibroblasts were characterized by comparison with the phenotypes of normal fibroblasts from the same patient. Explant cultures of keloids showed unique features. Keloid explants contracted considerably and reduced their size during culture, whereas the size of normal skin explants remained unchanged. Enlarged cells were found among fibroblasts which had grown out of all the explants and were morphologically distinct from fibroblasts; however, keloid explants produced many more of them than did the normal tissues. The growth rate of fibroblast colonies formed from normal explants was five times higher than keloid explants. Keloid fibroblasts which had been serially cultivated contracted lattices of collagen gels at a rate similar to normal fibroblasts. Proteins extracted from serially cultivated fibroblasts were mapped on polyacrylamide two-dimensional electrophoretic gels. No significant qualitative alterations in protein expression in keloid cells were found as compared with normal fibroblasts. However, some quantitative changes were found between the two. A computer-assisted image analyzer detected 151 polypeptide spots--50 spots (33%) of which increased their amounts in keloid cells, whereas 34 spots (22.5%) decreased in comparison with normal fibroblasts. Sixteen major polypeptides were identified as known proteins with the aid of time-of-flight mass spectrometry. The level of expression of these identified proteins was similar between normal and keloid cells, except stathmin whose expression was suppressed in keloid fibroblasts.

Journal Article↗
PubMedDate not supplied

Differential expression of platelet-derived growth factor receptors in porcine fibroblasts cultured from skin and granulation tissue.

This study investigated the biological response of fibroblasts cultured from uninjured skin and granulation tissue from different stages of healing wounds to the three isoforms of platelet-derived growth factor. Fibroblasts were derived by explant culture from the skin or the granulation tissue that formed within open mesh nylon Schilling-Hunt chambers (postoperative days 10, 20, 30, and 50) which had been implanted subcutaneously in the backs of domestic pigs. Cells were cultured under identical conditions in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Mitogenic activity was measured with (3)H-thymidine incorporation into DNA. Fibroblasts from normal skin responded equally well to all of the platelet-derived growth factor isoforms in the mitogenic assays. All of the wound fibroblasts exhibited a decreased response to platelet-derived growth factor compared with those from skin. Granulation tissue fibroblasts responded to platelet-derived growth factor BB, less to platelet-derived growth factor AB, and poorly to platelet-derived growth factor AA. These results correlated with a significantly decreased growth rate of fibroblasts in culture from both 30- and 50-day postsurgical wound tissue compared with normal skin. Western blot studies of cell membrane extracts showed that wound fibroblasts contained less than 20% as many platelet-derived growth factor-alpha receptors as found in fibroblasts cultured from normal skin. No significant difference in the amount of platelet-derived growth factor-beta receptor was detected. The decreases in platelet-derived growth factor-alpha receptors are sufficient to account for the diminished response of the wound fibroblasts to all platelet-derived growth factor isoforms and the differential loss of responsiveness to platelet-derived growth factor AA. These results show that fibroblasts derived from granulation tissue of pig skin wounds exhibit a decreased growth response to platelet-derived growth factor and a decreased growth rate in culture media as compared with fibroblasts derived from uninjured skin. How these differences may relate to the physiologic characteristics of normal and healing-impaired wounds is considered.

Journal Article↗
PubMed2001

Alpha2-Macroglobulin Modulates Interactions between Lymphocytes and Fibroblasts.

The aim of the present study was to evaluate the influence of apha2-macroglobulin (alpha(2)M) on lymphocyte adhesion to fibroblasts. Peripheral blood lymphocytes from healthy donors and two fibroblast lines (human diploid embryo fibroblasts M-19 and mouse transformed fibroblasts L929) were used in the experiments. alpha(2)M treatment of fibroblast monolayer appeared to result in the enhancement of lymphocyte adhesion to fibroblasts. The number of attached lymphocytes was increased by 2-2.5 times. It should be noted that the effect of alpha(2)M didn't depend on the conformational molecule changes, since either native or methylamine or plasmin transformed alpha(2)M approximately at the same fashion increased the lymphocyte adhesion to both allogeneic and xenogeneic fibroblasts. B lymphocytes were predominant cells that were attached to fibroblast monolayer without alpha(2)M treatment. However the percentage of adherent T lymphocytes was increased substantially after the fibroblast monolayer treatment by alpha(2)M. Subpopulation analysis has shown that fibroblast pretreatment by alpha(2)M didn't result in a selective adhesion of CD4(+) or CD8(+) T lymphocytes, but increased the adhesiveness for both T lymphocyte subpopulations. The data obtained demonstrate that besides its participation in the processes of fibroblast adhesion alpha(2)M is capable to modify the contact interaction of these cells with lymphocytes that may have an influence on the functional consequences of this process.

Journal Article↗