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PubMed1983

Identification and characterization of specific growth hormone receptors in cultured human fibroblasts.

A significant impediment to studies of growth disorders in man has been the lack of an easily accessible target tissue for studies of GH action. As human fibroblasts have been shown to produce a somatomedin-C-like peptide in culture, the aim of this study was to investigate the interaction of GH with cultured fibroblasts. GH binding to human skin fibroblasts and to lung fibroblasts was examined. Specific binding of [125I]iodo human GH [( 125I]iodo hGH) was rapid, reversible, and time and temperature dependent. Maximal binding was achieved within 2 h at 30 C and specifically bound [125I]iodo-hGH was rapidly dissociable at this temperature. A linear relationship between specific binding of [125I]iodo-hGH and cell number was found and negligible degradation of labeled hormone occurred during the binding studies. Half-maximal inhibition of specific binding occurred at 30 ng/ml hGH. Lactogenic and nonprimate somatogenic hormones did not displace [125I]iodo-hGH at the concentrations tested. Scatchard analysis of [125I]iodo-hGH binding to human skin fibroblasts demonstrated a single class of binding sites with an affinity constant of 1.07 +/- 0.07 (SEM, n = 5) X 10(9) M-1 and a capacity of 8355 +/- 1880 site per cell. Similar GH-receptor characteristics were found on each of the fibroblast cell lines examined irrespective of the site of origin (skin or lung) or age of donor. These findings demonstrate for the first time specific GH receptors in cultured human fibroblast cell lines. The demonstration of GH receptors in human fibroblasts should encourage the search for similar receptors in tissues not previously considered to be target tissues for GH action. On the basis of these studies we suggest that cultured skin fibroblasts may be a suitable tissue for the study of GH-receptor status in patients with disorders of growth.

Adult↗

PubMed1995

Immunoglobulin A class fibroblast antibodies in patients with Graves' disease and pretibial myxedema.

The involvement of autoantibodies in the extrathyroidal manifestations of Graves' disease has been the subject of extensive investigation, with fairly inconclusive results to date. We investigated the presence of immunoglobulin A (IgA) and IgG antibodies in patients with Graves' disease and pretibial myxedema (PTM; n = 21) as well as those with Graves' disease with thyroid-associated ophthalmopathy (TAO; n = 10), Graves' disease with no clinical evidence of extrathyroidal manifestations (n = 11), Hashimoto's thyroiditis (n = 9), type 1 diabetes mellitus (n = 10), systemic lupus erythematosus (n = 9) and normal individuals (n = 17). We looked for antibodies to both retroocular muscle and dermal fibroblasts as well as to thyroid peroxidase, thyroid microsomal antigen, thyroglobulin, and human eye muscle membranes. IgA class antibodies to microsomal antigen (30-50% of patients), thyroid peroxidase (5-20%), and human eye muscle membrane (0-26%) antigens were found in the various groups of patients with Graves' disease. With each of these antigens, serum from patients with PTM showed the greatest binding. Highly significant IgA binding was shown by PTM serum to both dermal (P < 0.001) and retroocular muscle (P < 0.001) fibroblasts from 12 different donors. Serum from Graves' patients with and without TAO and that from Hashimoto's thyroiditis patients reacted significantly with 4 of the 12 fibroblasts lines. In contrast, IgG binding was only found for 3 of the 12 fibroblast lines using patient serum. The IgA binding to fibroblasts shown by PTM patients was predominantly of the IgA2 subclass. The activity was absorbed out by both fibroblasts and thyroid cells. In immunoblotting studies, PTM patient serum reacted with a 54-kilodalton dermal fibroblast antigen and a 66-kilodalton retroocular fibroblast antigen. No binding to these antigens was seen with serum from normal controls or patients without PTM. Further elucidation of the nature of this fibroblast antigen will help to determine the role of IgA autoantibodies in the extrathyroidal manifestations of Graves' disease.

Adult↗

PubMed1989

Characterization of pregnancy-specific beta 1-glycoprotein synthesized by human placental fibroblasts.

We have previously demonstrated that human placental fibroblasts produce a pregnancy-specific beta 1-glycoprotein (PS beta G) immunologically indistinguishable from placental PS beta G. This was confirmed by the immunocytochemical localization of PS beta G in these fibroblasts. In addition, placental fibroblasts contain all three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases which hybridize with the three PS beta G cDNAs (PSG16, PSG93, and PSG95) identified, although at 1.4-2.5% of the levels in human term placenta. The major PS beta G species synthesized by placental fibroblasts is a 62K glycopolypeptide formed from a 58K intracellular precursor polypeptide. However, the PS beta G species found in human placenta are one major glycoprotein of 72K and two minor ones of 64K and 54K. Poly(A)+ RNA from placental fibroblasts directed the synthesis of two polypeptides of 48K and 46K (major), whereas, poly(A)+ RNA from human placenta directed the synthesis of higher levels of four polypeptides of 50 K, 48 K (major), 46 K, and 36 K. Thus, the major PS beta G species found in fibroblasts and human placenta differ. The carbohydrate side-chains are essential for the stability of fibroblast PS beta G, because PS beta G synthesis in these fibroblasts could not be detected in the presence of tunicamycin, a protein glycosylation inhibitor which did not affect PS beta G mRNA expression. Our finding that a variant PS beta G species is produced in placental fibroblasts raises the possibility that the authentic placental PS beta G species may have different functions.

Alkaloids↗

PubMed2001

Apoptosis of granulosa cells and female infertility in achondroplastic mice expressing mutant fibroblast growth factor receptor 3G374R.

Fibroblast growth factors play an important role in the control of ovarian folliculogenesis, but the complete repertoire of ovarian receptors which can transduce the fibroblast growth factor signals and their precise localization in the ovary have not yet been characterized. The most common form of inherited human dwarfism results from a point mutation in the transmembrane region of fibroblast growth factor receptor 3. A mouse model for achondroplasia was generated by introducing the human mutation (glycine 380-arginine) into the mouse fibroblast growth factor receptor 3 (G374R) by a "knock-in" approach using gene targeting leading to a constitutively active receptor. This resulted in the development of dwarf mice that share many features with human achondroplasia. Here we report that female (fibroblast growth factor receptor 3 G374R) dwarf mice become infertile. While no significant changes were observed in the anatomical and histological appearance of ovaries of 3-wk-old dwarf mice, a dramatic difference was observed in ovaries of 3-month-old mice. The normal ovary consists mainly of healthy corpora lutea and follicles at different stages of development, whereas the ovaries of the dwarf mice remain small and contain mainly follicles with a progressive apoptosis in the granulosa cells, and no corpora lutea could be observed. The levels of LH, FSH, and progesterone were lower by 72.3%, 38.0%, and 40.0%, respectively, in the blood of the dwarf mice compared with normal mice, and the total bioactivity of pituitary FSH and LH was lower by 65.6% and 79.6%, respectively, in the dwarf mice compared with normal mice. However treatment with PMSG and human CG of the dwarf mice led to rapid follicular development and formation of corpora lutea. Interestingly, the expression of the tumor suppressor gene p53 was increased dramatically in ovaries of the dwarf mice. The presence of the fibroblast growth factor receptor 3 cellular receptors in both normal and dwarf animals was demonstrated by Western blot and immunostaining. However, the distribution of the fibroblast growth factor receptors in the two strains shows significant differences. In the normal ovaries fibroblast growth factor receptor 3 was homogeneously distributed on the cell membrane of the granulosa cells and was absent in theca as well as corpora lutea cells, whereas in dwarf mice ovaries it was highly clustered on granulosa cells and very often appears in endocytic vesicles. Aged oocytes were more frequently observed in preantral follicles of ovaries of the dwarf mice. Nevertheless, oocytes isolated from antral follicles resume their meiotic division at a high percentage, similar to oocytes obtained from normal ovaries. The results imply fibroblast growth factor receptor 3 involvement in the control of follicular development through regulation of granulosa cell growth and differentiation, and that unovulation in the dwarf mice could be overcome in part by administration of exogenous gonadotropins. Moreover, it is suggested that the infertile phenotype is partially due to defects in the pituitary-gonadal axis.

Achondroplasia↗

PubMed1994

The expression of proliferation-dependent antigens during the lifespan of normal and progeroid human fibroblasts in culture.

Normal human fibroblasts display a limited lifespan in culture, which is due to a steadily decreasing fraction of cells that are able to proliferate. Using antibodies that react with antigens present in proliferating cells only, in an indirect immunofluorescence assay, we have estimated the fraction of proliferating cells in cultures of normal human fibroblasts. Furthermore, we have estimated the rate of decline in the fraction of proliferating cells during the process of cellular ageing by application of the assay to normal human fibroblasts throughout their lifespan in culture. Werner's Syndrome is an autosomal recessive disease in which individuals display symptoms of ageing prematurely. Werner's Syndrome fibroblasts display a reduced lifespan in culture compared with normal human fibroblasts. Like normal human fibroblasts, the growth of Werner's Syndrome fibroblasts is characterised by a decreasing fraction of cells reacting with the proliferation-associated antibodies throughout their lifespan in culture. However, the rate of loss of proliferating cells in Werner's Syndrome fibroblasts during the process of cellular ageing is accelerated 5- to 6-fold compared with the rate determined for normal human fibroblasts.

Antigens↗

PubMed1998

Deficient epithelial-fibroblast heterocellular gap junction communication can be overcome by co-culture with an intermediate cell type but not by E-cadherin transgene expression.

Epithelial, fibroblast and intermediate cell lines were employed to examine the mechanism(s) essential for heterocellular gap junction intercellular communication in vitro. These cell lines were characterized extensively for cell type based on morphology, intermediate cytoskeletal proteins, cell adhesion molecules and their associated proteins, tight junction proteins as well as functional differentiation. All cell types expressed connexin43 and were dye-coupled in homocellular culture. Epithelial and intermediate cells or fibroblasts and intermediate cells readily assembled heterocellular connexin43-positive gap junction plaques when co-cultured, while gap junction plaques in mixed cultures of epithelial cells and fibroblasts were rare. Dye microinjection studies were used to show that there was little gap junction intercellular communication between epithelial cells and fibroblasts. However, intermediate cells were able to communicate with epithelial cells and, to a lesser extent, fibroblasts and could transfer dye to both epithelial cells and fibroblasts when all three cell types were cultured together. Fibroblasts that were stably transfected with a cDNA encoding E-cadherin had a greater tendency to aggregate and exhibited a more epithelial-like phenotype but heterocellular gap junction intercellular communication with epithelial cells, which endogenously express E-cadherin, was not enhanced. These results suggest that mutual expression of E-cadherin is insufficient to stimulate gap junction formation between epithelial cells and fibroblasts. Moreover, our results also demonstrate that communication gaps between epithelial cells and fibroblasts can be bridged by intermediate cells, a process that may be important in mammary gland development, growth, differentiation and cancer.

Animals↗

PubMed1988

Foetal and cancer patient fibroblasts produce an autocrine migration-stimulating factor not made by normal adult cells.

We have previously reported that (1) the migration of foetal and adult fibroblasts into three-dimensional collagen matrices is differentially affected by cell density, and (2) skin fibroblasts from cancer patients commonly display a foetal-like mode of migratory behaviour. Data presented here indicate that differences in the migration of these cell types are particularly apparent in cultures plated at high density (i.e. at cell confluence); under these conditions, foetal fibroblasts and the foetal-like fibroblasts of cancer patients migrate into the three-dimensional collagen matrix to a significantly greater extent than do normal adult cells. In this initial study concerned with the biochemical basis of these observations, we report that medium conditioned by either foetal or cancer patient fibroblasts stimulates the migration of confluent adult cells. This stimulation of migration is specific to confluent cells, as the migration of subconfluent adult fibroblasts is unaffected by these conditioned media. Gel filtration chromatography of foetal fibroblast-conditioned medium indicates that migration-stimulating activity is recovered in a single peak with an apparent molecular mass in the range of 50-60 (X 10(3]. The active migration stimulating factor (MSF) in both foetal and cancer patient fibroblast-conditioned media appears to be a protein stable at acid pH, but inactivated by heat, alkaline pH and reductive alkylation. MSF produced by foetal and cancer patient fibroblasts is presumably responsible for the characteristically elevated levels of migration displayed by these cells in confluent culture, thereby suggesting an autocrine mode of action for this factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Movement↗

PubMed1989

Differences between adult and foetal fibroblasts in the regulation of hyaluronate synthesis: correlation with migratory activity.

We have previously reported that confluent foetal fibroblasts migrate into three-dimensional collagen gel matrices to a significantly greater extent than do adult cells. Hyaluronic acid (HA) is a major constituent of the extracellular matrix deposited by fibroblasts and has been demonstrated to stimulate the migration of a number of different cell types. Previous studies have indicated that the synthesis of HA by normal adult skin fibroblasts declines significantly when the cells achieve confluence. Data presented in this paper indicate that foetal fibroblasts differ from adult cells in this respect, in that they do not show an inverse relationship between cell density and HA synthesis, i.e. confluent foetal fibroblasts continue to produce approximately the same amount of HA as do subconfluent cells. These data suggest that the synthesis of relatively high levels of HA by foetal fibroblasts at confluence may be causally related to the elevated migration displayed by these cells. In this context, a close correlation was observed between the level of HA synthesized by confluent foetal and adult fibroblasts and the differential migratory activity displayed by these cells. Such differences in HA synthesis and migratory behaviour were only apparent at cell confluence, with subconfluent foetal and adult fibroblasts being indistinguishable in terms of these two criteria. Our data further reveal that: (1) cell density affects the size class of HA synthesized by both foetal and adult cells; and that (2) there is a considerable degree of heterogeneity amongst the nine different fibroblast lines examined in this study in terms of the size class of HA that they produce.

Adult↗

PubMed1992

The mechanism involved in the inhibitory action of tranilast on collagen biosynthesis of keloid fibroblasts.

Tranilast, an anti-allergic drug inhibiting the release of substances such as histamine and prostaglandins from mast cells, was previously reported to suppress collagen synthesis of fibroblasts derived from keloid tissues. However, the inhibitory mechanism on collagen synthesis is unknown. We studied its inhibitory mechanism on collagen synthesis by culturing fibroblasts from keloid and hypertrophic scar tissues of humans. Collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue is greater than that from healthy human skin. Tranilast (3-100 microM) did not inhibit prolyl hydroxylase (the rate-limiting enzyme in collagen synthesis) activity. Tranilast (3-300 microM) suppressed the collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue but not healthy skin fibroblasts. Tranilast (30-300 microM) inhibited the release of transforming growth factor (TGF)-beta 1 from keloid fibroblasts, which enhances the collagen synthesis of keloid fibroblasts. Anti-TGF-beta 1 antibody (50 microliter/ml) inhibited the collagen synthesis, although diphenhydramine (10 microM) and indomethacin (10 microM) did not show any inhibition. These results suggest that tranilast inhibits collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue through suppressing the release of TGF-beta 1 from the fibroblasts themselves.

Cicatrix↗

PubMed1993

Epithelial-fibroblast interactions in bleomycin-induced lung injury and repair.

Intercellular communication between epithelial cells and fibroblasts of the alveolar wall contributes to regulatory control of each cell type. We examined whether lung injury and subsequent fibrosis are associated with disturbance of this mutual control system. Rats received bleomycin intratracheally, and after 10 days, when acute epithelial injury occurs, and at 6 weeks, when repair with fibrosis is found, pure populations of type 2 epithelial cells and lung fibroblasts were prepared to study interactions with respect to growth control. Epithelial cells were cultured alone, on a permeable filter over fibroblasts, and in co-culture with fibroblasts. The results showed that the low growth rate of normal epithelial cells increased when cells were exposed to fibroblast supernatants. This effect was also seen using cells from the 10-day bleomycin group, but it was diminished in the group treated for 6 weeks. However, epithelial cells from exposed or control rats did not show increased DNA synthesis when grown in contact with fibroblasts in co-culture. In contrast, fibroblast growth was inhibited when exposed to epithelial cell secretions in control cultures and when using cells from the 10-day bleomycin group. No inhibition of fibroblast growth by epithelial cells was found using cells from the fibrotic lungs. These results suggest that after lung injury by bleomycin, a fibroblast-secreted factor promotes epithelial growth; however, during repair, regenerating epithelial cells lose the ability to inhibit fibro-blast proliferation. These local changes in cellular control at the alveolar wall may be sufficient to produce pulmonary fibrosis.

Animals↗

PubMedDate not supplied

Influence of serum on adult and fetal dermal fibroblast migration, adhesion, and collagen expression.

The wound healing response to injury can be affected by many factors such as cell migration and extracellular matrix elaboration. The objective of this study was to examine the serum- and age-dependent effects on cell migration, adhesion, and collagen expression by skin fibroblasts. Dermal fibroblasts were isolated and plated with and without serum for up to 7 d. Cell migration was determined by quantitative image analysis, adhesion was quantified using a centrifugation assay, and collagen expression was assessed by PCR and immunohistochemical staining. Both adult and fetal fibroblasts migrated significantly faster in serum-containing medium compared to serum-free medium. There was no significant difference in migration between the two cell types in either serum-containing or serum-free medium. There was no significant difference in adhesion in the presence of serum, although there was a greater fraction of adherent fetal skin fibroblasts than adult fibroblasts in serum-free medium. Moreover, the adherent fraction of fetal fibroblasts in serum-free medium was not significantly different from that in serum-containing medium, suggesting that fetal skin fibroblasts possess serum-independent adhesion properties. Collagen mRNA expression was significantly up-regulated in serum-free compared to serum-containing medium for both cell types. With respect to collagen immunohistochemistry, both dermal fibroblast populations exhibited greater type I collagen compared to type III collagen staining. Quantitative assessment of collagen staining indicated significantly enhanced type I collagen secretion in the presence of serum by fetal skin fibroblasts. These findings suggest that intrinsic cellular characteristics may govern the observed differences in adult and fetal wound healing.

Age Factors↗

PubMed2001

Persistence of TGF-beta1 induction of increased fibroblast contractility.

Fibroblast contraction of collagen gels is regarded as a model of wound contraction. Transforming growth factor (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since fibroblasts isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in fibroblasts' contractility. To evaluate this question, confluent human fetal lung fibroblasts were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. Fibroblasts were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After 20 min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated fibroblasts caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated fibroblasts, respectively) than control fibroblasts (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-200 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by fibroblasts from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.

Adult↗

PubMed2002

Hypoxic activation of adventitial fibroblasts: role in vascular remodeling.

Substantial experimental evidence supports the idea that the fibroblast may play a significant role in the vascular response to injury, especially under hypoxic conditions. Fibroblasts have the ability to rapidly respond to hypoxic stress and to modulate their function to adapt rapidly to local vascular needs. Fibroblasts appear to be uniquely equipped to proliferate, transdifferentiate, and migrate under hypoxic conditions. Proliferative responses to hypoxia depend on the activation of Galpha(i) and Gq kinase family members, and on the subsequent stimulation of protein kinase C and mitogen-activated protein kinase family members. Extracellular nucleotides (eg, adenosine triphosphate [ATP]) are likely to be increased in the hypoxic adventitial compartment and can act as autocrine/paracrine modifiers of the hypoxia-induced proliferative response. The proliferative effects of ATP appear to be mediated largely through G-protein-coupled P2Y receptors in fetal and neonatal fibroblasts. Hypoxia, acting through Galpha(iota)-coupled pathways, also can directly up-regulate alpha-smooth muscle actin expression in fibroblast subpopulations, suggesting that hypoxia may play a direct role in mediating the "transdifferentiation" of fibroblasts into myofibroblasts in the vessel wall. In addition, chronic hypoxia causes stable (at least in vitro) phenotypic changes in fibroblasts that appear to be associated with changes in the signaling pathways used to elicit proliferation. However, it is also becoming clear that, similar to the heterogeneity described for vascular smooth muscle cells, numerous fibroblast subtypes exist in the vessel wall, and that each may respond in unique ways to hypoxia and other stimuli and thus serve special functions in response to injury. In fact, adventitia may be considered to be compartments in which cells with "stem-cell-like" characteristics reside. Future work is needed to determine more precisely the role of the fibroblast in the wide variety of vascular complications observed in many humans diseases, and in the genes and gene products that confer unique properties to this important vascular cell.

Adult↗

PubMed1993

Pentoxifylline inhibits the proliferation and glycosaminoglycan synthesis of cultured fibroblasts derived from patients with Graves' ophthalmopathy and pretibial myxoedema.

Excessive amounts of glycosaminoglycans accumulate in the extraocular muscles of patients with Graves' ophthalmopathy and in the affected skin of patients with pretibial myxoedema. It is widely accepted that fibroblasts are the sources of glycosaminoglycan synthesis. Pentoxifylline, an analogue of the methylxanthine theobromine, inhibits the proliferation and certain biosynthetic activities of fibroblasts derived from normal human skin and from skin of patients with some fibrotic disorders. Our objective was to determine whether pentoxifylline has similar effects on fibroblasts derived from patients with Graves' ophthalmopathy and pretibial myxoedema and could serve as a candidate for the treatment of these manifestations. Fibroblasts from the extraocular muscles of two patients with Graves' ophthalmopathy and normal extraocular muscles of two subjects with strabismus, as well as the affected skin of two patients with pretibial myxoedema were cultured in vitro in the presence and absence of pentoxifylline to assay its effect on the proliferation of fibroblasts and their production of glycosaminoglycans. In subconfluent fibroblast cultures, pentoxifylline treatment caused a dose-dependent inhibition of serum-driven fibroblast proliferation. In confluent fibroblast cultures both in the presence and absence of serum, exposure to pentoxifylline similarly resulted in a dose-dependent inhibition of glycosaminoglycan synthesis for all these different kinds of fibroblasts. These findings may form the rationale for a clinical trial using pentoxifylline for the treatment of Graves' ophthalmopathy and pretibial myxoedema.

Adult↗

PubMed1999

Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts.

The developmental potential of bovine fetal fibroblasts was evaluated using nuclear transfer. Fibroblasts from a 37-day-old fetus were fused to enucleated oocytes before activation. Nuclei of starved (cultured for 8 days in medium containing 0.5% serum) fibroblasts supported the development of reconstructed embryos to the blastocyst stage significantly better than those of non-starved fibroblasts (39% versus 20%; P < 0.05). When nuclear transfer morulae derived from starved or non-starved fibroblasts were used for re-cloning, the proportion of blastocysts (52 and 55%, respectively) obtained with these embryonic nuclei was significantly higher than it was with fibroblast nuclei used in the first round of nuclear transfer (P < 0.05 and P < 0.001, respectively). After transfer of blastocysts derived from non-starved and starved fibroblasts, respectively, 33% (1/3) and 78% (7/9) of recipients were pregnant on day 30 as assessed by ultrasonography. On day 90, the corresponding pregnancy rates were 33% (1/3) and 63% (5/8). Two live male twin calves, derived from non-starved fibroblasts, were delivered by Caesarean section at day 281 of gestation. This study demonstrates a positive effect of serum starvation on the efficiency of nuclear transfer using bovine fetal fibroblasts. The efficiency of nuclear transfer could be further increased by recloning.

Animals↗

PubMed2001

Extreme dark cytotoxicity of Nile Blue A in normal human fibroblasts.

Early reports using mouse models indicated that Nile Blue A (NBA) is taken up more efficiently by tumor cells than normal tissue and retards tumor growth. NBA also shows both dark toxicity and phototoxicity of human tumor cells in vitro. However, studies on the dark toxicity of NBA and the effects of NBA-mediated photodynamic treatment in normal human cells are lacking. In the current study we have examined the cytotoxicity of NBA in normal human fibroblasts, spontaneously immortalized Li-Fraumeni Syndrome (LFS) cells and three different human tumor cell lines. The normal human fibroblasts showed extreme sensitivity to NBA compared with LFS cells and the human tumor cell lines. Treatment with 0.1 microgram/mL of NBA for 1 h reduced the colony formation of normal human fibroblasts by greater than 95%, but had no significant effect on the colony formation of LFS cells. No significant numbers of apoptotic cells were detected in either normal human fibroblasts or LFS cells following this drug concentration. Thus, unlike photodynamic therapy with some other photosensitizers, the dark toxicity of NBA was not caused by apoptosis. Although the drug uptake was higher in normal human fibroblasts compared with LFS cells, the difference in sensitivity between normal human fibroblasts and LFS cells could not be accounted for by the difference in drug uptake alone. In addition, we could not detect any significant photocytotoxic effect of NBA in either normal human fibroblasts or LFS cells for a drug concentration of 0.05 microgram/mL at light exposures of up to 6.7 J/cm2. These data indicate an extreme sensitivity of normal human fibroblasts to NBA and an inability to produce a significant photocytotoxic effect on human cells using NBA concentrations that have relatively low toxicity for normal human fibroblasts.

Animals↗

PubMed1998

Autocrine transforming growth factor beta stimulation of extracellular matrix production by fibroblasts from fibrotic human gingiva.

Hereditary gingival fibromatosis (HGF) is a fibrotic enlargement of the gingiva. HGF gingiva contains large amounts of collagen and other extracellular matrix (ECM) molecules. In vitro, HGF fibroblasts produce greater amounts of the ECM components fibronectin (FN) and type 1 collagen than normal human gingival (GN) fibroblasts. Transforming growth factor beta (TGF beta) is a cytokine important in regulating tissue repair and regeneration after injury, and stimulating fibroblast proliferation and the production of FN and collagens. The objective of this study was to determine whether HGF fibroblasts produce TGF beta and, with the use of neutralizing antibodies to TGF beta isoforms, if their increased expression of FN and type 1 collagen is under autocrine TGF beta control. The HGF strains produced greater amounts of TGF beta1 and TGF beta2 (P < or = 0.003) as well as FN (P < or = 0.04) and type 1 collagen (P < or = 0.03) (measured by specific ELISA) than the GN strains. Treatment of HGF fibroblasts with anti-TGF beta1, beta2, or beta3, as well as a combination of all 3 antibodies, decreased their FN production by up to 60% (P < or = 0.04), and was able to decrease FN production by HGF fibroblasts to the levels of the GN fibroblasts. When used alone, the neutralizing antibodies decreased type 1 collagen production by the HGF fibroblasts by up to 40% (P = 0.014), and treatment with all 3 antibodies caused decreases of up to 55% (P = 0.0005). The results suggest that autocrine stimulation by the increased amounts of TGF beta isoforms made by HGF fibroblasts contributes to their increased production of FN and type 1 collagen.

Adult↗

PubMed2003

The putative collagen-binding peptide P-15 promotes fibroblast attachment to root shavings but not hydroxyapatite.

BACKGROUND: Regenerative periodontal treatment aims to restore the attachment of the periodontal ligament and gingival collagen fibers to both the cementum of the root surface and alveolar bone. Fibroblasts are the predominant cells of the periodontal ligament and gingiva and have important roles in the function and regeneration of the tooth-supporting apparatus. This study investigated whether a putative collagen-based cell-binding peptide (P-15) increases gingival fibroblast attachment to root shavings and bone replacement graft (BRG) materials. METHODS: Gingival and dermal fibroblast attachment to root shavings and BRG materials, and cell proliferation on root shavings and sections were measured fluorometrically. Root shavings and root sections obtained from periodontally healthy teeth were treated with P-15 at 2 concentrations (200 ng/g or 400 ng/g). Citric acid (CA)-treated root materials were also compared to untreated root shavings and root sections that served as negative control groups. RESULTS: Attachment of all cells to bone fragments (whether freeze-dried or demineralized) was significantly greater than to hydroxyapatite (HA)-based BRG materials. The addition of P-15 to HA did not significantly increase gingival or dermal fibroblast attachment. At a concentration of 400 ng/g, P-15 significantly increased gingival and dermal fibroblast attachment to root shavings as compared to untreated shavings. Bone fragments, HA-based BRG materials, and untreated root shavings inhibited gingival fibroblast proliferation. Treatment of root sections with P-15 did not have any effect on gingival fibroblast proliferation. CONCLUSIONS: P-15 is a potential alternative to CA for promoting fibroblast attachment to root surfaces. However, P-15 did not enhance fibroblast proliferation on root sections.

Adolescent↗