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PubMed1996

Developmentally regulated expression of IGF binding protein-3 (IGFBP-3) in human placental fibroblasts: effect of exogenous IGFBP-3 on IGF-1 action.

Preterm, human, placental fibroblasts exhibit growth rates, in vitro, that vary with gestational age. The observed increase in proliferation rate is associated with enhanced mitogenic responsiveness to IGF-I. IGFBPs can either potentiate or inhibit IGF action at the cellular level. The production of IGFBPs by placental fibroblasts was studied as potential modulators of their responsiveness to IGFs. Human placental fibroblasts were obtained at various gestational ages and maintained in culture. IGFBP-3 protein and mRNA expression were assessed by Northern and ligand blot analyses. First, media conditioned by fibroblasts, in culture, were subjected to ligand blot analysis. Multiple species of IGFBPs were present in each cell line tested. IGFBP-3, migrating as a doublet at approx. 38/42 kDa, was the predominant IGFBP species present. Other IGFBPs of 22-35 kDa were also present. The secretion of IGFBP-3 exhibited a marked decrease at 10-15 weeks gestation relative to 8-9 week fibroblasts but began to increase again by 19 weeks. We next studied the expression of IGFBP-3 mRNA. Total cellular RNA was obtained from rapidly growing cells and subjected to Northern analysis. Placental fibroblasts exhibited decreased steady state levels of IGFBP-3 mRNA at 10-15 weeks gestation consistent with its decreased protein expression. The ability of IGFBP-3 to influence IGF-1 stimulated DNA synthesis was studied in 10 week placental fibroblasts as measured by [3H]thymidine incorporation. IGFBP-3 inhibited IGF-1 (3.3 nM) stimulated DNA synthesis in a dose-dependent manner when added simultaneously with IGF-1 or preincubated with the cells for 48 h prior to the addition of IGF-1. By contrast, maximum effective concentrations of IGFBP-3 (52 nM) potentiated the effect of IGF-1 50-200% when preincubated with bovine fibroblasts for 48 h prior to the addition of IGF-1. These data suggest that IGFBP-3 production is developmentally regulated in human placental fibroblasts and inhibits their mitogenic response to IGF-1. The regulated expression of IGFBP-3 may contribute to the altered growth rate and IGF responsiveness exhibited by placental fibroblasts, in vitro.

Animals↗

PubMed1989

Basic fibroblast growth factor promotes in vitro survival and cholinergic development of rat septal neurons: comparison with the effects of nerve growth factor.

The effects of basic fibroblast growth factor and nerve growth factor on survival and transmitter expression of cultured rat embryonic (E18) septal neurons were studied. Two different culture media were used: (i) a horse serum-containing Leibowitz L-15 medium and (ii) a serum-free N1-supplemented Dulbecco's modified Eagle's medium. Addition of basic fibroblast growth factor to either culture medium enhanced neuronal survival in low density cultures after 4 days. The effects of basic fibroblast growth factor were dose-dependent and blocked by anti-basic fibroblast growth factor antibodies. In serum-containing L-15 medium nerve growth factor also promoted neuronal survival. Basic fibroblast growth factor and nerve growth factor supported neurons comprised both cholinergic and GABAergic subpopulations. The effects of basic fibroblast growth factor and nerve growth factor were not additive. In high density cultures using serum-containing L-15 medium survival of septal neurons was four times higher than in low density cultures after 4 days. Addition of basic fibroblast growth factor or nerve growth factor did not further augment neuronal survival in high density cultures. Maintenance of septal neurons at high density was not affected by antibodies to basic fibroblast growth factor and/or nerve growth factor. Addition of basic fibroblast growth factor or nerve growth factor to serum-containing L-15 medium at high cell density significantly enhanced choline acetyltransferase activity 3- and 7.5-fold, respectively, without affecting cell survival. In conclusion, next to nerve growth factor, basic fibroblast growth factor, which has been located in the hippocampal target area of septal neurons, appears to be another potent trophic factor for septal neurons.

Animals↗

PubMed1995

Protective effect of basic fibroblast growth factor-heparin and neurotoxic effect of platelet factor 4 on ischemic neuronal loss and learning disability in gerbils.

Platelet factor 4, which has a potent affinity for heparin, has been shown to inhibit the binding of basic fibroblast growth factor to the cell surface receptor and to counteract the biological activities of basic fibroblast growth factor in certain peripheral tissues. In the present in vitro [125I]basic fibroblast growth factor binding experiments, platelet factor 4 consistently inhibited the binding of iodinated basic fibroblast growth factor to cell membranes of the gerbil hippocampus. To investigate the in vivo function of endogenous basic fibroblast growth factor and/or basic fibroblast growth factor receptor possibly activated in the ischemic gerbil brain, we infused platelet factor 4 continuously into the left lateral ventricle with an osmotic minipump. When platelet factor 4 infusion was started within three days after a 3-min ischemic insult, it significantly enhanced ischemia-induced learning disability and ischemic neuronal loss in the CA1 region of the hippocampus, as demonstrated by the results of the step-down passive avoidance task and by subsequent histological examinations. Infusion of platelet factor 4 into the cerebral ventricle of intact gerbils did not affect learning ability or CA1 neuron number. Basic fibroblast growth factor-neutralizing antibody, when infused continuously in the cerebral ventricle, also exhibited a neurotoxic effect in ischemic but not intact gerbils. Basic fibroblast growth factor co-infused with heparin, but not basic fibroblast growth factor alone, rescued a significant number of ischemic neurons which were destined to degenerate without the infusion of heparinized basic fibroblast growth factor, and it prevented ischemia-induced learning disability.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Alternative adrenal chromaffin cell fates induced by basic fibroblast growth factor or cyclic AMP in vitro depend on a collaboration with the growth substrate.

Basic fibroblast growth factor stimulates cultured adrenal chromaffin cells to divide and to transform into sympathetic neurons, but the efficacies reported for these actions of basic fibroblast growth factor have varied widely. We have examined the effect of various growth substrates on basic fibroblast growth factor responses and here we show that the ability of basic fibroblast growth factor to transform neonatal rat chromaffin cells into sympathetic neurons depends on laminin as the culture substrate. On collagen, less than 5% of the cells were transformed into neurons by basic fibroblast growth factor, even when the culture was supplemented with heparin or heparan sulphate, but 65% of cells entered the S phase in the presence of basic fibroblast growth factor compared to 15% in its absence, showing that the basic fibroblast growth factor receptor is still active. On laminin, by contrast, over 60% of the cells transformed into neurons in response to the same concentrations of basic fibroblast growth factor, suggesting that an overlapping pool of cells change their phenotype depending on growth substrate. The cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP apparently mimicked the actions of basic fibroblast growth factor, promoting neuronal differentiation on laminin, but mitogenic stimulation on collagen. These data support the notion that collagen and laminin promote different instructions in chromaffin cells that can collaborate with the signals induced by basic fibroblast growth factor and 8-(4-chlorophenylthio)cyclic AMP to determine chromaffin or neuronal cell fates.

Adrenal Glands↗

PubMed1994

Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons.

The present study investigates the temporal and spatial changes of the cellular expression of basic fibroblast growth factor messenger RNA and immunoreactivity after a 6-hydroxydopamine-induced lesion in the nigrostriatal dopamine system. In situ hybridization revealed a sustained (from 4 h to two weeks) and strong (300-400% of control, at the peak intervals) increase of basic fibroblast growth factor messenger RNA in the pars compacta of the substantia nigra and the ventral tegmental area ipsilateral to the lesion. A short-lasting increase of basic fibroblast growth factor messenger RNA was observed in he ipsilateral pars reticulata of the substantia nigra (from 4-24 h, 300% of control) and neostriatum (24 h, 180% of control) as well as in the ipsilateral and contralateral hippocampus and neocortex (by 4 h, 200% of control). Brightfield microscopy showed an increased number of putative glial cells expressing the basic fibroblast growth factor messenger RNA signal. Basic fibroblast growth factor immunohistochemistry revealed on control brains the protein in the nuclei of glial cells throughout the forebrain and the midbrain and in the nuclei of neurons of the layer II of the retrosplenial granular cortex, the CA2 region of the hippocampus and the fasciola cinereum as well as in the nuclei of ependymal cells. The injection of 6-hydroxydopamine increased basic fibroblast growth factor immunoreactivity in the nuclei of astrocytes only within the ipsilateral substantia nigra and ventral tegmental area. By 2 h after the drug injection, the density of glial basic fibroblast growth factor-immunoreactive profiles was increased in the pars compacta of the substantia nigra and the ventral tegmental area. The density, size and intensity of the astroglial basic fibroblast growth factor immunoreactive nuclei were increased in the entire substantia nigra and the ventral tegmental area at 72 h, and peaked one week after the 6-hydroxydopamine injection. The saline injection promoted a time-dependent increase in the density of the glial basic fibroblast growth factor immunoreactivity but only in the ipsilateral pars compacta of the substantia nigra. In conclusion, the dopamine cell degeneration may give rise to extracellular signals activating the surrounding astroglia, leading to a sustained increased synthesis of astroglial basic fibroblast growth factor, which may exert neuroprotective action and increase repair on the nigrostriatal dopamine system.

Animals↗

PubMed1996

Modulation of clonogenicity, growth, and radiosensitivity of three human epidermoid tumor cell lines by a fibroblastic environment.

PURPOSE: To develop a model vitro system to examine the influence of fibroblasts on the growth and survival of human tumor cells after exposure to ionizing radiation. METHODS AND MATERIALS: The cell system of three epidermoid carcinoma cell lines derived from head and neck tumors having differing growth potentials and intrinsic radiosensitivities, as well as a low passage skin fibroblast strain from a normal human donor. The tumor cells were seeded for five days prior to exposure to radiation: (a) in the presence of different numbers of fibroblasts, (b) in conditioned medium from stationary fibroblast cultures, and (c) on an extracted fibroblastic matrix. RESULTS: When grown with fibroblasts, all three tumor cell lines showed increased clonogenicity and increased radioresistance. The radioprotective effect was maximal at a density of approximately 10(5) fibroblasts/100 mm Petri dish, and was greatest in the intrinsically radiosensitive tumor cell line. On the other hand, the effects of incubation with conditioned medium or on a fibroblastic matrix varied among the tumor cell lines. Thus, the protective effect afforded by coculture with fibroblasts must involve several cellular factors related to the fibroblast itself. CONCLUSIONS: These observations emphasize the importance of cultural conditions on the apparent radiosensitivity of human tumor cell lines, and suggest that the fibroblastic connective tissue enveloping the malignant cells should be considered when the aim is to establish a radiopredictive assay from surgical tumor fragments.

Carcinoma, Squamous Cell↗

PubMed1992

Mechanism of enhanced cyclic AMP stimulation by isoproterenol in aged human fibroblasts.

Human diploid lung fibroblasts (IMR-90) were used to investigate the reported increase in beta-adrenergic-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels in fibroblasts aged in culture. Under basal conditions cellular cAMP was 34.2 +/- 5.6 and 38.4 +/- 9.1 pmol/mg protein in early (PDL 22-24) and late (PDL 47-52) passage fibroblasts, respectively. Net release of cAMP from fibroblasts was 67.8 +/- 8.6 and 18.5 +/- 7.0 pmol/30 min/mg protein in early and late passage cultures, respectively. In confluent, early passage fibroblasts, cellular cAMP and net release of cAMP increased by 2.7-fold and 3.8-fold, respectively, after a 30 min incubation in 2 microM isoproterenol. In confluent late passage fibroblasts, isoproterenol incubation increased cellular cAMP and net release of cAMP by 7.8-fold and 26.1-fold, respectively. Adenosine failed to inhibit isoproterenol-induced stimulation of cAMP in early or late passage fibroblasts. There was no passage-related difference in basal, isoproterenol, or forskolin-stimulated adenylyl cyclase activity in crude fibroblast membrane preparations. The activity of cAMP-phosphodiesterase in sonicates of early and late passage IMR-90 was 9.61 +/- 1.15 and 5.81 +/- 1.11 pmol/min/mg protein respectively. Measurements of cAMP in subconfluent early passage fibroblasts indicated that mechanisms related to the reduced cell density in confluent late passage IMR-90 may, in part, account for the enhanced isoproterenol-induced cAMP levels observed in these cultures. The results suggest that the remainder of the enhanced cAMP response to isoproterenol of in vitro aged fibroblasts may be due to a lower cAMP phosphodiesterase activity in these cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1995

Decreased integrin alpha 2, but normal response to TGF-beta in scleroderma fibroblasts.

The distribution and amount of integrin alpha2 were studied in cultured fibroblasts from normal subjects and scleroderma patients by immunofluorescence and immunoblotting using a monoclonal antibody against the human integrin alpha2 subunit. Integrin alpha2 was concentrated at the perinuclear regions in a dot-like pattern in normal fibroblasts on the glass coverslips until the 14th day after planting, and the staining pattern of integrin alpha2 was gradually changed to a dispersed dot-like pattern by the 19th day as examined by immunofluorescence microscopy by using the anti-integrin alpha2 antibody. No difference was observed in the distribution patterns between normal and scleroderma fibroblasts. By immunoblotting study, the amount of integrin alpha2 in scleroderma fibroblasts (n = 10) was less than that of normal fibroblasts (n = 10) (P < 0.01) in both cytosol and cytoskeleton-associated fractions. Furthermore, transforming growth factor beta (TGF-beta) increased the amount of integrin alpha2 in both normal fibroblasts and scleroderma cells by 33%. The total amount of integrin alpha2 in TGF-beta-stimulated scleroderma fibroblasts was less than that in TGF-beta-stimulated normal fibroblasts. These findings suggest that the amount of integrin alpha2, a collagen receptor, is reduced in scleroderma fibroblasts, but the integrin alpha2 production by TGF-beta stimulation is not impaired in scleroderma fibroblasts.

Adult↗

PubMed1991

Regulation of glucocorticoid receptor expression in cultured fibroblasts from a patient with familial glucocorticoid resistance.

The thermolabile glucocorticoid receptor (GR) in fibroblasts from a patient with familial glucocorticoid resistance (FGR) was characterized by solution hybridization, Northern blot analysis and Western immunoblotting using an hGR and cRNA probe and a GR specific monoclonal antibody. Specific DNA binding was measured by binding of cytosolic GR to mouse mammary tumour virus (MMTV) DNA. Northern blot analysis of total cellular RNA isolated from the fibroblasts showed hybridization of the hGR probe to 7.0 and 6.1 kb RNA species. Basal expression of hGR mRNA was 1.8 times higher in fibroblasts derived from the patient compared to control fibroblasts as assayed by solution hybridization. Even though nonsignificant, dexamethasone treatment maximally caused at 60% down-regulation of GR mRNA in normal fibroblasts after 12 h but only a 40% down-regulation in fibroblasts from the patient. In both cases, the initial mRNA values were restored after 72 h. No difference in GR mRNA stability was observed between fibroblasts from the patient and from controls. The induction of the glucocorticoid-regulated gene metallothionein IIA (MTIIA) by dexamethasone and cadmium sulphate was studied at different temperatures using a cRNA probe for human MTIIA. At elevated temperatures, cadmium sulphate but not dexamethasone increased MTIIA mRNA levels approximately three-fold in fibroblasts from the patient, whereas in normal fibroblasts regardless of temperature both cadmium sulphate and dexamethasone increased MTIIA mRNA levels approximately three- and two-fold, respectively. Cytosolic GR from FGR-fibroblasts showed an increased specific binding to MMTV DNA at 4 degrees C. These data support our previous findings of a thermolabile GR, probably due to a defect intrinsic to the GR protein, in this patient with primary cortisol resistance and indicate a compensatory mechanism at the transcriptional level of GR expression. The data also indicate a receptor defect affecting specific DNA binding in vitro.

Binding Sites↗

PubMed2004

Critical involvement of stress-activated mitogen-activated protein kinases in the regulation of intracellular adhesion molecule-1 in serosal fibroblasts isolated from patients with Crohn's disease.

BACKGROUND: Stricture formation in Crohn's disease occurs as a result of persistent fibroblast activation. Chronic inflammation seen in patients with Crohn's disease leads to enhanced adhesion molecule expression in fibroblasts. Stress-activated mitogen-activated protein kinases are critical signaling pathways that control expression of intracellular adhesion molecule-1 (ICAM-1) in inflammation. The purpose of this study was to investigate the involvement of stress-activated mitogen-activated protein kinases in the regulation of ICAM-1 expression by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in serosal fibroblasts isolated from patients with Crohn's disease. STUDY DESIGN: Fibroblasts were isolated from serosal biopsies of strictures in patients with Crohn's disease and normal colon in patients with colorectal carcinoma. Cell surface and whole cell ICAM-1 expression were evaluated by flow cytometry and Western blot analysis, respectively. Cells were stimulated with TNF-alpha and IL-1beta. To determine the mitogen-activated protein kinase signaling pathway required for ICAM-1 induction, cells were pretreated with inhibitors to Jun N-terminal kinase, p38 kinase, and p42/44 kinase. RESULTS: Baseline ICAM-1 expression was higher (p < 0.001) in fibroblasts isolated from strictures in patients with Crohn's disease (3.2 +/- 0.3) as compared with nonstrictured Crohn's fibroblasts (2.1 +/- 0.3) and control fibroblasts (1.6 +/- 0.1). TNF-alpha and IL-1beta increased ICAM-1 expression in both control and Crohn's disease. Pretreatment of fibroblasts with the Jun N-terminal kinase inhibitor dimethylaminopurine abolished TNF-alpha- and IL-1beta-stimulated ICAM-1 expression. CONCLUSIONS: Serosal fibroblasts isolated from strictures of patients with Crohn's disease demonstrate enhanced expression of ICAM-1. TNF-alpha and IL-1beta upregulate ICAM-1 expression in serosal fibroblasts through a Jun N-terminal kinase signaling pathway. Specific inhibition of inflammatory signaling pathways could provide novel therapeutic targets for treatment of Crohn's disease.

Adult↗

PubMed2005

SB-431542 inhibits TGF-beta-induced contraction of collagen gel by normal and keloid fibroblasts.

BACKGROUND: Transforming growth factor (TGF)-beta induces fibroblast contraction, which is implicated in wound healing and keloid formation. SB-431542 is a novel specific inhibitor of TGF-beta type I receptor kinase activity. OBJECTIVE: We sought to determine whether SB-431542 inhibited TGF-beta-induced fibroblast contraction. METHODS: We used an in vitro type I collagen gel contraction assay with normal or keloid dermal fibroblasts incorporated. RESULTS: TGF-beta induced contraction of collagen gels with normal dermal fibroblasts incorporated, which was efficiently suppressed by SB-431542. Keloid fibroblasts showed higher basal contraction of collagen gels in the absence of TGF-beta than normal fibroblasts, which was enhanced by addition of TGF-beta. SB-431542 suppressed both the basal and TGF-beta-enhanced contraction of collagen gels by keloid fibroblasts. These inhibitory effects of SB-431542 were associated with suppression of TGF-beta-induced alpha-smooth muscle actin (alpha-SMA) expression and phosphorylation of Smad2 in normal and keloid fibroblasts. CONCLUSION: SB-431542 can suppress TGF-beta-induced contraction of collagen gel by normal and keloid dermal fibroblasts. Importantly, SB-431542 can inhibit basal contraction of collagen gel by keloid fibroblasts. These results suggest that an inhibitor of TGF-beta type I receptor kinase activity may have therapeutic potential for excessive skin contraction as observed in keloid.

Actins↗

PubMed2005

Altered fibroblast function following myocardial infarction.

Adequate wound healing and scar formation is an essential response to myocardial infarction (MI), and fibroblasts are primary cellular components regulating the process. How fibroblast functions are altered post-MI and to what extent these abnormalities persist in vitro is not well understood. Accordingly, we isolated myocardial fibroblasts from MI and non-MI (remote) regions at 7 days post-MI (n=35) and from the free wall and septum of unoperated control C57BL/6 mice (n=14). Proliferation was increased 182+/-28% in MI, but not in remote, fibroblasts compared with unoperated controls (P=0.01). Migration decreased 61+/-8%, adhesion to laminin decreased 79+/-8%, adhesion to collagen IV increased 196+/-27%, and collagen synthesis increased 169+/-24% in fibroblasts isolated from the MI region (all P<0.05). Migration, adhesion, and collagen synthesis changes were similar in remote fibroblasts, and the phenotypic differences were maintained through passage four. Transforming growth factor beta1 (TGFbeta1) is a bioactive molecule that has been shown to affect fibroblast function. Stimulation of unoperated control fibroblasts with 10 ng/ml TGFbeta(1) increased proliferation 137+/-7% (P=0.03 vs. unstimulated), increased adhesion to collagen IV 149+/-6% (P<0.01), and increased collagen I levels 187+/-10% (P=0.01). TGFbeta1 may, therefore, explain some of the changes in post-MI fibroblast phenotype. These data demonstrate for the first time region specific alterations in post-MI fibroblast biology that are maintained in vitro. Additionally, our model provides a novel in vitro template for examining the cellular mechanisms of wound healing and scar formation post-MI.

Angiotensin II↗

PubMed1986

Modulation of fibroblast proliferation and transformation by activated macrophages during postoperative peritoneal reepithelialization.

We studied the modulation of fibroblast proliferation and transformation by postoperative macrophages. One group of rabbits underwent resection and reanastomosis of the small bowel, after which macrophages were collected by peritoneal lavage. A second group of rabbits underwent peritoneal wall abrasion followed by collection of local fibroblast on postoperative days 4 and 8. Postoperative macrophages were added to five culture dishes containing fibroblasts. After 24 hours, tritiated thymidine was added to the culture dishes and incubated overnight. In two other dishes, which were incubated for up to 8 days, a 24-hour pulse of tritiated thymidine was added before culture termination. Postoperative day 4 fibroblasts demonstrated a greater increase in cell number during the culture interval compared to fibroblasts collected on postoperative day 8. By the second day of coculture with macrophages collected from different postoperative days, tritiated thymidine incorporation by day 4 fibroblasts was suppressed, especially by postoperative day 7 macrophages. Thereafter, a stimulation in tritiated thymidine uptake was found. In contrast, tritiated thymidine uptake by day 8 fibroblasts was accelerated by coculture with macrophages, especially those collected on postoperative day 7. Day 4 fibroblasts assumed a more spindly appearance when cocultured with macrophages than did day 8 fibroblasts. Taken together, these data suggest that macrophages activated in response to surgical injury may secrete substances that induce proliferation and transformation of fibroblasts.

Animals↗

PubMed2004

Human pulmonary fibroblasts exhibit altered interleukin-4 and interleukin-13 receptor subunit expression in idiopathic interstitial pneumonia.

Abnormal proliferation of pulmonary fibroblasts is a prominent feature of chronic pulmonary fibrotic diseases such as idiopathic interstitial pneumonia (IIP), but it is not presently clear how this proliferative response by lung fibroblasts can be therapeutically modulated. In the present study, we examined whether it was possible to selectively target primary human pulmonary fibroblasts grown out of surgical lung biopsies (SLBs) from IIP patients based on their expression of interleukin-4 receptor (IL-4R) and IL-13R subunits. Pulmonary fibroblast lines cultured from patients with the severest form of IIP, namely usual interstitial pneumonia, exhibited the greatest gene and protein expression of IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2 compared with primary pulmonary fibroblast lines grown from other IIP SLBs and normal SLBs. When exposed to increasing concentrations of a chimeric protein comprised of human IL-13 and a truncated version of Pseudomonas exotoxin (IL13-PE), the proliferation of primary usual interstitial pneumonia fibroblasts was inhibited to a much greater extent compared with fibroblast lines from nonspecific interstitial pneumonia and respiratory bronchiolitis/interstitial lung disease patient groups. Fibroblasts from normal patients exhibited minimal susceptibility to the cytotoxic effect of IL13-PE. IL13-PE-mediated targeting of IIP fibroblasts was dependent on their expression of IL-4Ralpha and IL-13Ralpha2. Thus, these data suggest that the abnormal proliferative properties of human lung fibroblasts from certain IIP patient groups can be modulated in a manner that is dependent on the IL-4 and IL-13 receptor subunit expression by these cells.

Base Sequence↗

PubMed1999

Overexpression of insulin-like growth factor-1 (IGF-I) receptor and the invasiveness of cultured keloid fibroblasts.

Keloid is a dermal fibroproliferative tissue of unknown etiology. Protein tyrosine kinases (PTKs) play an important role in the regulation of cell growth and differentiation. Activation of PTK cascades in keloid fibroblasts is thought to be closely linked to abnormal cell proliferation and migration. We determined the expression profile of PTK genes in normal skin and keloid fibroblasts using the homology cloning method with a degenerated primer. Eight PTK genes were expressed among a total of 46 receptor-type clones. The most abundant type of PTK receptors was the platelet-derived growth factor receptor in both fibroblasts. However, insulin-like growth factor-I receptor (IGF-IR) was overexpressed only in keloid-derived fibroblasts (9 of 24). Immunohistochemical analysis confirmed the high expression of IGF-IR in keloid fibroblasts, but not in normal fibroblasts. To examine the functional properties of the IGF-I/IGF-IR pathway, we investigated cell proliferation and invasion activities of both types of fibroblasts. The mitogenic effect of IGF-I on both fibroblasts was very weak compared with serum stimulation. In contrast, the invasive activity of keloid fibroblasts was markedly increased in the presence of IGF-I, and inhibited by a neutralizing antibody against IGF-IR. Our results indicate the involvement of activated IGF-I/IGF-IR in the pathogenesis of keloid by enhancing the invasive activity of fibroblasts.

Adolescent↗

PubMed1999

Effects of interferons on proliferation and collagen synthesis of rat palatal wound fibroblasts.

The purpose was to select drugs that specifically reduce collagen synthesis by palatal granulation fibroblasts without affecting their proliferation. Granulation fibroblasts were obtained from 8-day-old palatal mucoperiosteal wounds and normal fibroblasts from palatal tissue of unwounded rats. Cultured cells were treated with interferon-alpha2b, interferon-beta and interferon-gamma (0, 100, 1000, and 10000 U/ml). Cell proliferation was measured by [3H]thymidine incorporation. Collagen synthesis and non-collagenous protein synthesis were determined from the incorporation of [3H]proline. None of the interferons significantly inhibited the proliferation of either type of fibroblasts. Interferon-alpha2b had no effect on the variables studied at the dosages used. Interferon-beta reduced collagen synthesis of granulation fibroblasts without affecting their non-collagenous protein synthesis or protein synthesis by normal fibroblasts. Interferon-gamma reduced collagen synthesis of both types of fibroblast and the non-collagenous protein synthesis of granulation fibroblasts. These data show that interferon-beta specifically reduces collagen synthesis by oral granulation fibroblasts without affecting normal palatal fibroblasts.

Animals↗

PubMed1989

Selection of therapeutic agents for intraocular proliferative disease 3. Effects of fluoropyrimidines on cell-mediated contraction of human fibroblasts.

We evaluated the efficacy of 5-fluorouracil (5-FU) and 5-fluorouridine (5-FUR) as anticontractile agents in an in vitro model that measures the ability of human and rabbit fibroblasts to contract a collagen gel. Human fibroblasts were more contractile than rabbit fibroblasts, required higher concentrations of 5-FUR for inhibition of contraction, and were able to tolerate higher doses of the drug. 5-FU failed to inhibit contraction of human fibroblasts at concentrations up to 1.5 mM (200 micrograms ml-1), but significantly inhibited rabbit fibroblast contraction, although to a lesser degree than 5-FUR under similar conditions. 5-Fluorouridine (100 microM, 26 micrograms ml-1) inhibited contraction of human fibroblasts by more than 80%, whereas only 10 microM (2.6 micrograms ml-1) 5-FUR was required for 90% inhibition of rabbit fibroblast contraction. 5-FUR was cytotoxic to rabbit fibroblasts at concentrations of 200 microM (52 micrograms ml-1) or greater, but 800 microM was non-toxic to human fibroblasts after 96 hr exposure. At concentrations of 1.5 mM, 5-FU had no significant effects on the viability of either human or rabbit fibroblasts. These results suggest that 5-FUR may prove useful for the treatment of human ocular proliferative disorders in which contraction of fibrocellular elements is thought to play a part. They also suggest that fluoropyrimidine dosages found to be effective in a rabbit model of proliferative vitreoretinopathy may be less effective in humans and that 5-FUR levels shown to be toxic to rabbit-derived cells may be tolerated by humans cells.

Cells, Cultured↗

PubMed2001

Molecular characterization of fibroblasts isolated from human peritoneum and adhesions.

OBJECTIVE: To determine the response of adhesion and peritoneal fibroblasts to hypoxia. DESIGN: Prospective experimental study. SETTING: University medical center. PATIENT(S): Primary cultures of fibroblasts established from the peritoneal and adhesion tissues of the same patients (n = 2) to minimize genetic variations. INTERVENTION(S): Hypoxia treatment of the primary cultured fibroblast. MAIN OUTCOME MEASURE(S): Analyze the expression of extracellular matrix (ECM) components, metalloproteinases and their tissue inhibitors, growth factors, and cytokines in adhesion and peritoneal fibroblasts under normal and hypoxic conditions by reverse transcriptase/polymerase chain reaction analysis. RESULT(S): Compared to peritoneal fibroblasts, adhesion fibroblasts had a significant increase in the basal mRNA levels for collagen I, fibronectin, MMP-1, TIMP-1, TGF-beta 1, TGF-beta 2, and IL-10. Hypoxia resulted in a further increase in collagen 1, fibronectin, TIMP-1, TGF-beta 1, TGF-beta 2, IL-10, and IFN-gamma mRNA levels in both peritoneal and adhesion fibroblasts. The increase was more profound in adhesion fibroblasts. CONCLUSION(S): Hypoxia induces molecular changes in both peritoneal and adhesion fibroblasts, creating a milieu that favors adhesion development. The effect of hypoxia was more profound on adhesion fibroblasts.

Cell Hypoxia↗