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At least 91 records · Page 5Linked to original sources
PubMed2003

Humic acid induces oxidative DNA damage, growth retardation, and apoptosis in human primary fibroblasts.

Humic acid (HA) has been implicated as an etiological factor of Blackfoot disease endemic in the southwest coast of Taiwan. Dysfunction of endothelial cells and vasculopathy have been proposed to explain the onset of ulcerous changes at extremities. However, little is known about the effect of HA on activities of cells in these nonhealing wounds. In the present study, we demonstrate that HA adversely affects the growth properties of fibroblasts, one of the key players in wound repair. HA treatment caused growth arrest and apoptosis in human foreskin fibroblasts (HFF). This was accompanied by a significant increase in the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in cellular DNA. The increased fluorescence in dichlorofluorescin (H2DCF)-stained and HA-treated cells suggests the involvement of reactive oxygen species (ROS) in HA-induced biological effects. Conversely, vitamin E pretreatment, which significantly reduced the 8-OHdG formation in HA-treated cells, alleviated the growth-inhibitory and apoptosis-inducing effects of HA. These results indicate that HA initiates oxidative damages to fibroblasts, and leads to their dwindling growth potential and survival. The present study suggests that HA-induced growth retardation and apoptosis of fibroblasts may play a role in the pathogenesis of Blackfoot disease.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1993

Controlled induction of focal adhesion disassembly and migration in primary fibroblasts.

Fibroblast migration is an integral component of biological processes such as wound healing and embryogenesis. Previous experiments examining fibroblast locomotion from tissue explants have shown that migrating fibroblasts lack, or contain only transient, focal adhesions (focal contacts). Focal adhesions are specialized regions of tight cell-matrix interaction, assembled by a complex process of transmembrane signalling. Although the explant model has been used for studying several aspects of fibroblast locomotion, it is limited by the lack of control over migration, and only a small percentage of the cells actually locomoting. Therefore, we have developed an in vitro model for cultured fibroblast strains where the presence or absence of focal adhesions can be manipulated, and in the latter case 70% of these cells become locomotory. The stimulus used to decrease the percentage of cells containing focal adhesions, and hence enhance locomotion, was newborn rat heart-conditioned medium (HCM). Addition of HCM to rat embryo fibroblasts induced both chemokinesis and chemotaxis. Cells disassembled focal adhesions on a variety of extracellular matrix substrates after approximately 6 h of stimulation with HCM; conversely, removal of HCM promoted reformation of focal adhesions within 12-24 h. HCM-stimulated fibroblasts which lacked focal adhesions concomitantly lacked F-actin stress fibers and focal concentrations of vinculin and talin. Therefore, fibroblast migration can be readily controlled in an on-off manner through conditioned medium, which influences the absence or presence of focal adhesions.

3T3 Cells↗
PubMed1999

Human primary fibroblasts in vitro express a purinergic P2X7 receptor coupled to ion fluxes, microvesicle formation and IL-6 release.

We have investigated reponses to extracellular ATP in human fibroblasts obtained by skin biopsies. Our data show that these cells express a P2X7 purinergic receptor, as judged by (1) RT-PCR with specific primers, (2) reactivity with a specific anti-P2X7 antiserum, (3) activation by the selective P2X agonist benzoylbenzoylATP and (4) stimulation of transmembrane ion fluxes. Stimulation with benzoylbenzoylATP, and to a lesser extent with ATP, also caused striking morphological changes and increased formation of cytoplasmic microvesicles. These changes were fully reversible upon nucleotide removal. Two known blockers of P2X receptors, oxidised ATP and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, inhibited the morphological changes fully and the ion fluxes partially. The residual rise in intracellular Ca2+ levels and membrane depolarization observed in the presence of the inhibitors were dependent upon activation of a P2Y-type receptor exhibiting a peculiar pharmacological profile, in that CTP was the preferred agonist. ATP stimulation triggered release of the pro-inflammatory cytokine IL-6 in fibroblasts pre-treated with PMA and bacterial endotoxin. These observations reveal a novel pathway for fibroblast activation and for their recruitment in the inflammatory response.

Blotting, Western↗
PubMed2000

Peroxynitrite activates the phosphoinositide 3-kinase/Akt pathway in human skin primary fibroblasts.

Peroxynitrite is a potent oxidizing and nitrating species formed in a diffusion-limited reaction between nitrogen monoxide and superoxide. It induces apoptosis through unknown mechanisms and is believed to interfere with receptor tyrosine kinase signalling through nitration of tyrosine residues. One pathway emanating from receptor tyrosine kinases is that leading to activation of the anti-apoptotic kinase Akt. In the present study we provide evidence that peroxynitrite, administered to cells using two different delivery systems, results in the dose- and time-dependent activation of Akt. Akt activation is rapid and followed by phosphorylation of glycogen synthase kinase-3, an established substrate of Akt. Akt activation is inhibited in the presence of the phosphoinositide 3-kinase (PI-3K) inhibitors wortmannin and LY294002, and by treatment with the platelet-derived growth factor (PDGF) receptor (PDGFR) inhibitor AG1295, indicating a requirement for PDGFR and PI-3K in mediating peroxynitrite-induced Akt activation. Accordingly, the PDGFR-A and PDGFR-B isoforms were shown to undergo rapid tyrosine phosphorylation on treatment with peroxynitrite. Prior exposure of cells to peroxynitrite interferes with PDGF-induced Akt phosphorylation. Our findings suggest that Akt activation occurs as an acute response to peroxynitrite treatment and could play an important role in influencing cell survival and/or alter the cellular response to other growth regulatory signals.

Androstadienes↗
PubMed1990

[Primary fibroblastic tumor of the breast. Review of the literature apropos of a case of desmoid tumor of the breast posing nosologic problems with a low-grade fibrosarcoma].

The authors present a case of fibromatosis in the breast in a young nulliparous patient. These are rare tumours comparable to abdominal desmoid tumours. They are characterised by their ability to recur if spread locally, that is why they have to be widely removed. After having studied the anatomical and clinical characteristics as well as the biological ones of breast fibromatoses the authors discuss the problems of nosology which are often very difficult for this kind of a tumour and also of the therapy that has to be adapted.

Adolescent↗
PubMed1989

TGF beta inhibits Go/S-phase transition in primary fibroblasts. Loss of response to the antigrowth effect of TGF beta is observed after immortalization.

Transforming growth factor beta (TGF beta) may act as a negative growth regulator in secondary cultures of rodent fibroblasts by preventing quiescent cells from re-entering the cell cycle. Although TGF beta inhibits the serum induced transition from the Go to the S phase it does not inhibit induction of fos, myc, and JE. Actively growing cells are also inhibited in their proliferation by picomolar amounts of TGF beta. In contrast several established cell lines are not inhibited by TGF beta and in these it can cause quiescent cells to resume proliferation. This resistance to TGF beta can be traced back to the early divisions of immortal cells emerging from a senescence crisis, suggesting that alterations in the response of normal fibroblasts to TGF beta are concurrent with an unlimited proliferative potential. Induction of DNA synthesis by TGF beta is preceded in 5 established cell lines by induction of PDGF B chain mRNA as well as of other mRNAs associated with cell replication such as fos, myc, and JE. None of these genes is induced by TGF beta alone in presenescent cells. These results indicate that TGF beta can negatively regulate growth of normal presenescent fibroblasts. Alterations in the responsiveness to TGF beta can be detected immediately after immortalization which may contribute to the increased proliferative potential of these cells.

Animals↗
PubMed1994

Expression of the human glucocerebrosidase and arylsulfatase A genes in murine and patient primary fibroblasts transduced by an adeno-associated virus vector.

We have constructed two recombinant adeno-associated virus (AAV) vectors (pJJ-3GC and pJJ-3ASA) which contained either the human glucocerebrosidase (GC) or arylsulfatase A (ASA) cDNA under the control of an SV40 promoter. These plasmids were co-transfected to 293 cells with a helper plasmid containing trans-acting AAV genes required for packaging the vectors. The two recombinant vectors successfully infected murine and patient fibroblasts. The human glucocerebrosidase and arylsulfatase A genes were expressed at high levels in the cells as determined by Western blot analysis, enzyme assay and immunochemical staining. GC enzyme activity in Gaucher patient fibroblasts (GM-0877) infected by AAV-GC was 15-fold higher than in non-infected cells. ASA enzyme activity in MLD 557g cells infected by AAV-ASA was up to 500-fold higher than in the metachromatic leukodystrophy (MLD) control cells. Southern blotting results showed that the vector integrated 1-2 copies of pJJ-3GC and ASA in the targeted cell genome. These two vectors will be useful in studying AAV-mediated transfer of the GC and ASA genes in cells and animals.

Animals↗
PubMed1996

Double transduction with GTP cyclohydrolase I and tyrosine hydroxylase is necessary for spontaneous synthesis of L-DOPA by primary fibroblasts.

Gene transfer of tyrosine hydroxylase (TH) in animal models of Parkinson's disease (PD), using either genetically modified cells or recombinant virus vectors, has produced partial restoration of behavioral and biochemical deficits. The limited success of this approach may be related to the availability of the cofactor, tetrahydrobiopterin (BH4), because neither the dopamine-depleted striatum nor the cells used for gene transfer possess a sufficient amount of BH4 to support TH activity. To determine the role of BH4 in gene therapy, fibroblast cells transduced with the gene for TH were additionally modified with the gene for GTP cyclohydrolase l; an enzyme critical for BH4 synthesis. In contrast to cells transduced with only TH, doubly transduced fibroblasts spontaneously produced both BH4 and 3, 4-dihydroxy-L-phenylalanine. To examine further the importance of GTP cyclohydrolase I in gene therapy for PD, in vivo micro-dialysis was used to assess the biochemical changes in the dopamine-denervated striatum containing grafts of genetically modified fibroblasts. Only denervated striata grafted with fibro-blasts possessing both TH and GTP cyclohydrolase I genes displayed biochemical restoration. However, no significant differences from controls were observed in apomorphine-induced rotation. This is partly attributable to a limited duration of gene expression in vivo. These differences between fibroblasts transduced with TH alone and those additionally modified with the GTP cyclohydrolase I gene indicate that BH4 is critical for biochemical restoration in a rat model of PD and that GTP cyclohydrolase I is sufficient for production of BH4.

Animals↗
PubMed1998

Cell size, cell cycle, and alpha-smooth muscle actin expression by primary human lung fibroblasts.

Primary human lung fibroblasts were separated into small (group I), intermediate (group II), and large (group III) subpopulations by unit gravity sedimentation (1 G). The three subsets retained differences in cell size for up to 15 days of primary culture. Flow cytometric (fluorescence-activated cell sorter) measurements of forward-angle light scatter agreed well with fibroblast volume measured by image analysis and confirmed the utility of forward-angle light scatter for discriminating size subpopulations. Group II fibroblasts accumulated most rapidly by 8 days of culture and also contained the greatest proportion of S and G2/M phase cells as determined by fluorescence-activated cell sorter. Fibroblasts that were immunoreactive with antibodies to alpha-smooth muscle actin (alpha-SMA) were found only in group III. In situ end labeling of fragmented DNA detected apoptotic cells in both groups II and III, but double labeling for in situ end labeling and alpha-SMA revealed apoptotic cells in both the alpha-SMA-positive and -negative populations. These results demonstrate that primary human lung fibroblasts behave as predicted by classic models of cell cycle progression and differentiation. However, they do not support the hypothesis that the expression of alpha-actin is related to apoptosis. We also describe a simple and reproducible method for the high-yield isolation of human lung fibroblast subsets of differing proliferative potential and phenotype.

Actins↗
PubMed1969

Cholesterol requirement of primary diploid human fibroblasts.

Primary cultures of fibroblast-like cells were obtained from skin and articular cartilage of human donors in the age bracket of 1 to 15 years. For growth these cultures required 1 mg/liter of cholesterol added to Medium A2 plus acetyl choline, Na pyruvate, and D-galactosamine HCl (APG) containing 10% lipoprotein-free human serum. Established cell lines did not require cholesterol for growth. Eagle's medium could be used in place of Medium A2 plus APG with the same results. Desmosterol could replace cholesterol but lansterol or 7 dehydrocholesterol could not. Other cholesterol precursors were tested and found to be inactive. With the proviso that cholesterol precursors entered the cell and had to be converted to cholesterol to function, it was concluded that the particular primaries studied lacked a functional enzyme system to reduce the double bond at carbon 7.

Adolescent↗
PubMed1996

Identification of a human dermal macrophage population responsible for constitutive restraint of primary dermal fibroblast proliferation.

Primary human dermal cell suspensions prepared from the papillary dermis of keratomed skin strips were used to investigate the effect of indigenous dermal macrophages (HLA-DR+, CD11c+ CD11b+ CD1c- phagolysosome+) upon dermal fibroblast proliferation. Rapid dermal fibroblast expansion was induced upon immunomagnetic bead removal of CD11b+ or CD11c+ cells as well as by removal of more inclusive subsets contained within the DR+ population, but the removal of mast cells, endothelial cells, and CD1c+ dermal Langerhans cells from dermal cell suspensions failed to result in proliferation of the remaining cell subsets. Removal of 1B10+ fibroblasts from macrophage depleted (CD11b-) dermal cell suspensions essentially abrogated the unrestrained proliferation of the CD11b- dermal cells. Flow cytometric cell cycle analysis of cultured macrophage-depleted dermal cells confirmed that the unrestrained proliferating cells contain procollagen I+ as well as procollagen I- dermal fibroblasts. Inhibition of primary fibroblast expansion by adding a supernatant from unfractionated dermal cells suggested that a growth-inhibitory soluble activity of >30,000 kDa dominates the cytokine mixture released by unfractionated fresh dermal cells ex vivo. Inhibitory activity counterbalanced positive fibroblast growth- stimulatory cytokines released by dermal cells because neutralizing antibodies to insulin-like growth factor 1 and interleukin-1 beta resulted in decreased CD11b- dermal cell fibroblast proliferation. These data indicated an important role for dermal macrophages of the DR+ CD11b+ CD11c+ DC1c- phenotype in the normal homeostatic restraint of primary human dermal fibroblast proliferation.

Adult↗
PubMed2001

Biocompatibility of NDGA-polymerized collagen fibers. I. Evaluation of cytotoxicity with tendon fibroblasts in vitro.

The material properties of tendon type I collagen fibers polymerized with nordihydroguaiaretic acid (NDGA) are equivalent to native tendon, suggesting that NDGA crosslinking may provide a viable approach to stabilizing collagenous materials for repairing ruptured, lacerated, or surgically transected fibrous tissues, such as tendon and ligament (Koob & Hernandez, Biomaterials, in press). Using standard cytotoxicity tests, the present study evaluated the biocompatibility of these fibers with cultured bovine tendon fibroblasts. Primary fibroblasts obtained from calf digital extensor tendons were exposed to NDGA, reaction products generated from the polymerization protocol, and the crosslinked fibers. NDGA was cytotoxic to these cells at concentrations above 100 microM. NDGA oxidation products were similarly cytotoxic. At concentrations below 100 microM, fibroblast viability was not affected by NDGA or its oxidation products. At these lower concentrations, fibroblast proliferation was unaffected compared to controls not exposed to NDGA. Fibers crosslinked with NDGA contained no unreacted NDGA, but they did contain soluble reaction products that were cytotoxic to tendon fibroblasts in both the elution and the direct contact tests. Washing the fibers in 70% ethanol and phosphate-buffered saline eliminated cytotoxicity of the fibers. Ethanol simultaneously sterilized the fibers. Tensile tests established that the ethanol/phosphate buffer wash did not adversely affect the material properties of the fibers. The results of these experiments indicate that NDGA-crosslinked fibers can be rendered nontoxic to tendon fibroblasts and may provide a novel approach for producing biologically based, biocompatible, tendon bioprostheses.

Animals↗
PubMed2005

Global gene expression analysis of keloid fibroblasts in response to electron beam irradiation reveals the involvement of interleukin-6 pathway.

Keloid is a dermal fibroproliferative lesion of unknown etiology that commonly recurs after surgical excision. Post-operative adjuvant electron beam (EB) irradiation has been successfully used to reduce keloid recurrences. To provide new insights into the molecular mechanism behind the effect of EB irradiation, we used a cDNA microarray screening of more than 5000 genes to assess early changes in gene expression between EB-irradiated and non-irradiated keloid and non-lesional dermal fibroblasts. Primary fibroblast cultures from keloid and associated non-lesional dermis obtained from five patients were exposed to 15 Gy EB irradiation and analyzed after 15 min incubation. Early response to EB irradiation showed that 96 (1.8%) genes were modulated 2-fold or more in keloid fibroblasts. Upregulated genes accounted for 29.2% (28 genes), whereas downregulated genes comprised 70.8% (68 genes), indicating a silencing of many genes in keloid fibroblasts after EB irradiation. Many of the downregulated genes play roles in the enhancement of cell proliferation and extracellular matrix production, whereas several of the upregulated genes involves in the promotion of apoptosis and extracellular matrix (ECM) degradation. Using emerging bioinformatic tools and further corroboration, the interleukin 6 (IL-6) signaling pathway was found to be mainly involved in EB irradiation response. We also showed co-expression of IL-6 and its specific receptor (IL-6Ralpha) in keloid fibroblasts that points to the existence of an IL-6 autocrine loop in these cells. These results suggested that at the molecular level, EB irradiation might hinder keloid formation by regularizing disturbances in the homeostatic equilibrium between inducer and inhibitor activities in the matrix system most likely through the IL-6 pathway. Our study provides clues for the molecular mechanism(s) behind the beneficial effect of EB irradiation in reducing keloid recurrences and may help develop alternative strategies for the therapy and prophylaxis of this lesion.

Adult↗
PubMed2001

Loss of functional caveolae during senescence of human fibroblasts.

Primary human fibroblasts have a finite replicative lifespan in culture that culminates in a unique state of growth arrest, termed senescence that is accompanied by distinct morphological and biochemical alterations. Senescent cell responses to extracellular stimuli are believed to be altered at a point after receptors are bound by ligand, leading to improper integration of the signals which initiate DNA replication. In this study we demonstrate that one of the key organizing membrane microdomains for receptor signaling, caveolae, are absent in senescent cells. A comparison of young and senescent cells indicated that senescent cells contained a higher total amount of caveolins 1 and 2 but had significantly less of both proteins in the caveolar fraction. Additionally, caveolar fractions from senescent cells completely lacked the tyrosine-kinase activity associated with functional caveolae. Furthermore, old cells had little caveolar protein exposed to the outer plasma membrane as estimated by using an in vivo biotinylation assay and no detectable caveolin 1 on the cell surface when processed for immunofluoresence and confocal microscopy. Together, these data suggest that a fundamental loss of signal integration at the plasma membrane of senescent cells is due to the loss of signaling competent caveolae.

Caveolae↗
PubMed2001

Treatment of Parkinson's Disease by Genetically Modified Immortalized Fibroblasts.

Primary rat fibroblast cells were immortalized by genetic modification of SV40 large T antigen (LT(Ag)) gene and called RFLT. This cell line was non-tumorigenic after grafting into nud-mouse and rat. The LT(Ag)gene stopped expression when the cells were transplanted in rat striatum, but it resumed the expression ability after the transplanted cells were recovered from striatum and cultured in vitro.TH gene and GCH gene were transfected into RFLT, respectively, and two types of transfected cells, RFLT-TH and RFLT-GCH, were obtained. In mixed culture with these two cell lines, DA was detected with HPLC-ECD. Implanting mixture of those cells into the striatum of PD rats significantly decreased their rotational asymmetry for up to 12 weeks. The expression of TH gene was proved by TH immunohistochemical staining in the sections of rat brain. The establishment of the genetically modified immortalized cells may play role in the gene therapy of PD.

Journal Article↗
PubMed1989

Retroviral-mediated transfer and expression of hepatitis B e antigen in human primary skin fibroblasts and Epstein-Barr virus-transformed B lymphocytes.

Previously, an amphotropic retroviral expression system coding for the neomycin resistance gene was developed and used to synthesize hepatitis B e antigen (HBeAg) and hepatitis B core/e antigen (HBc/eAg) in transfected mouse NIH 3T3 fibroblasts (A. McLachlan et al., 1987, J. Virol. 61, 683-692). In the present study, these transfected cell lines were infected with a helper amphotropic murine leukemia virus resulting in the production of infectious recombinant retrovirus. The recombinant retrovirus was examined for its capacity to transmit resistance to the antibiotic, G418, and to express hepatitis B virus antigens in mouse NIH 3T3 fibroblasts, human primary skin fibroblasts, and Epstein-Barr virus (EBV)-transformed B lymphocytes. A mouse NIH 3T3 fibroblast clone was generated which produced recombinant retrovirus with the capacity to transmit HBeAg expression to these murine and human cell lines. In contrast, it was not possible to transmit HBc/eAg synthesis efficiently to these cell lines by recombinant retroviral infection. The difference between the efficiencies of transmission of HBeAg and HBc/eAg expression by recombinant retroviral-mediated infection was not predicted as the expression vector coding for HBc/eAg synthesis differs only by the deletion of approximately 90 nucleotides of HBV DNA sequence from the vector coding for HBeAg synthesis.

Adult↗