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PubMed1993

Production of fibroblast-pneumocyte-like factor by fetal rabbit lung fibroblasts: isolation and effects of it and related factors on fetal type II cells in vitro.

Fetal lung fibroblasts interact with type II epithelial cells, inducing their maturation. This interaction arises by secretion of factors which alter fetal type II cell function. To analyze these factors, conditioned medium (CM) was produced by exposing serum-free minimum essential medium, with [35S]methionine (5 microCi/ml), to confluent cultures of fetal rabbit lung fibroblasts. This medium was tested for ability to stimulate [3H]choline incorporation by fetal type II cells and subsequently fractionated on molecular weight filtration columns P60 (2.5cm x 90cm; NMW cutoff, 60kd; 1M acetic acid) and A1.5m (2.5cm x 90cm; NMW cutoff, 1,500kd; Tris-buffered saline) and a hydroxyapatite column (HT) (1.5cm x 30cm; NaCl and 0.01-0.3M phosphate). Crude medium stimulated choline incorporation into phosphatidylcholine. [35S]methionine was resolved in void volume material and in material of apparent molecular weight of 6000 daltons on the P60 filtration column. Filtration on the A1.5m column showed two major fractions with radiolabel incorporation. Each of these was resolved into two subfractions on HT chromatography. The high molecular mass fraction contained material which stimulated [3H]choline incorporation by fetal type II cells. The low molecular mass fraction tended to inhibit [3H]choline incorporation. The second subfractions of both the first and second primary fractions inhibited [3H]thymidine incorporation into DNA by fetal type II cells. SDS-PAGE electrophoresis and autoradiography showed that under reducing conditions, each peak contained several proteins. However few of these displayed radioactivity. These results indicate that protein factors produced by fetal lung fibroblasts may be involved in regulating both differentiation and replication of fetal type II cells.

Animals↗
PubMed1987

Alteration of the microtubule organization in aging WI-38 fibroblasts. A comparative study with embryonic hamster lung fibroblasts.

The microtubule organization in human WI-38 fibroblasts subcultivated in vitro has been investigated using nocodazole, a reversible inhibitor of the microtubules. Two phenotypes were observed. The typical fibroblast cells, called Type 1 cells, showed, after nocodazole treatment, a centripetal depolymerization wave of the microtubules and the giant Type 2 cells which have a more heterogeneous behaviour. Some of the cells clearly showed a centrifugal depolymerization of the microtubules, others a mixed behavior and less than 1% displayed the same behavior as the Type 1 cells. Confirming previous data obtained with Hamster fibroblasts (Raes et al., 1983, 1984), these results suggest a modification in the microtubule organization which could account for the aberrant division of some WI-38 cells in aged cultures. The relevance of this observation for the emergence of the morphologically different Type 2 cells and for cell division impairment in serially in vitro cultivated cells is discussed.

Aging↗
PubMed1994

Characterization of the immunophenotype and functional properties of fibroblast-like synoviocytes in comparison to skin fibroblasts and umbilical vein endothelial cells.

We characterized the immunophenotype as well as functional properties--phagocytosis, the uptake of acetylated LDL, and the expression of HLA class II antigens, adhesion molecules, and cytokine mRNA--of fibroblast-like synoviocytes from rheumatoid arthritis synovium. Skin fibroblasts (FB) and umbilical vein endothelial cells (HUVEC) were studied in parallel. Cytofluorometric immunophenotyping by use of 84 mAb and 2 lectins and immunofluorescence microscopy indicated a high degree of homology between the three cell types. Only staining with mAb to von Willebrand factor (vWF) and CD31 and the lectin UEA-I appeared specific to HUVEC, whereas the mAb 5B5 to prolyl 4-hydroxylase that has been reported to be specific to FB stained HUVEC as well as synoviocytes and FB. All of the cells phagocytosed fluorescent latex beads of 1.7 and 2.6 microns in size. The uptake of acetylated LDL could be shown by HUVEC and, surprisingly, by synoviocytes, but not by FB. The induction of HLA-DR, -DP, and -DQ by IFN-gamma on the three cell types showed a similar dose-dependence. The upregulation of ICAM-1 by IL-1 alpha, TNF-alpha, and IFN-gamma appeared similar, whereas the induction of VCAM-1 by IL-1 alpha, IL-4, TNF-alpha, and IFN-gamma showed differences between the three cell types. ELAM-1 was expressed only on HUVEC after treatment with IL-1 alpha and TNF-alpha. The capacity of the cells to produce cytokines was studied at the level of mRNA by reverse transcription and PCR. All three cell types expressed the mRNA of IL-1 alpha, IL-6, IL-8, GM-CSF, and TGF-beta 1 spontaneously or after LPS stimulation, but never TNF-alpha mRNA. Our results indicate a high degree of relationship between the three cell types. In contrast to HUVEC, none of the markers and functional properties investigated appear specific to FB. Therefore, the issue of the origin of fibroblast-like synoviocytes and the role of vascular endothelial cells in the inflamed synovium is discussed.

Antibodies, Monoclonal↗
PubMed1987

Normal rabbit aqueous humour, fibronectin, and fibroblast conditioned medium are chemoattractant to Tenon's capsule fibroblasts.

Some of the chemotactic and chemokinetic properties of rabbit Tenon's fibroblasts were examined in a 48-well micro-chemotaxis chamber. Normal rabbit aqueous humour, fibronectin, and fibroblast conditioned medium were used in the assay, and all were shown to be chemoattractant. In addition, aqueous humour was shown to be powerfully chemotactic. Since the failure of human trabeculectomies is associated with migration of fibroblasts to the operation site, the study of the chemoattractant influences acting on these cells may allow manipulation of their behaviour in order to influence favourably the outcome of surgery.

Animals↗
PubMed2006

The Ras-MAPK pathway downregulates Caveolin-1 in rodent fibroblast but not in human fibroblasts: implications in the resistance to oncogene-mediated transformation.

Normal human diploid fibroblasts (HDFs) are refractory to oncogene-mediated transformations in vitro, compared with rodent fibroblasts. As successful oncogene-mediated transformations of normal HDFs have been reported using the human telomerase catalytic subunit, it has been considered that telomerase activity contributes to the species-specific transformability. However, these transformed HDFs are much less malignant compared with those of rodent cells, suggesting the existence of undefined mechanisms that render HDFs resistant to malignant transformation. Here, cDNA microarray analysis identified caveolin-1 as one of the possible cellular factors involved in such mechanisms. The mitogen-activated protein kinases (MAPK) pathway downregulates Caveolin-1 in rodent fibroblasts, transformed by coexpression of the SV40 early region and activated H-Ras. In contrast, the coexpression of these two oncogenes in HDFs failed to reduce the expression level of Caveolin-1. These results strongly suggest the presence of critical differences in events following the phosphorylation of ERK during the activation process of the MAPK signaling pathway between human and rodent cells, as the ERK protein was similarly phosphorylated in both systems. Furthermore, the small interfering RNA-mediated suppression of Caveolin-1 facilitated the oncogene-mediated transformation of normal HDFs, clearly indicating that the differences in the transformability between human and rodent cells are due, at least in part, to the mechanism responsible for the resistance to Ras-induced Caveolin-1 downregulation in HDFs.

Animals↗
PubMed2005

Platelet-derived growth factor and reactive oxygen species (ROS) regulate Ras protein levels in primary human fibroblasts via ERK1/2. Amplification of ROS and Ras in systemic sclerosis fibroblasts.

The levels of Ras proteins in human primary fibroblasts are regulated by PDGF (platelet-derived growth factor). PDGF induced post-transcriptionally Ha-Ras by stimulating reactive oxygen species (ROS) and ERK1/2. Activation of ERK1/2 and high ROS levels stabilize Ha-Ras protein, by inhibiting proteasomal degradation. We found a remarkable example in vivo of amplification of this circuitry in fibroblasts derived from systemic sclerosis (scleroderma) lesions, producing vast excess of ROS and undergoing rapid senescence. High ROS, Ha-Ras, and active ERK1/2 stimulated collagen synthesis, DNA damage, and accelerated senescence. Conversely ROS or Ras inhibition interrupted the signaling cascade and restored the normal phenotype. We conclude that in primary fibroblasts stabilization of Ras protein by ROS and ERK1/2 amplifies the response of the cells to growth factors and in systemic sclerosis represents a critical factor in the onset and progression of the disease.

Apoptosis↗
PubMed1985

The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants.

Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus-transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus-transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and -independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator.

Animals↗
PubMed1990

c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts display more decondensed nucleosomal organization than normal fibroblasts.

We have compared the nucleosomal organization of c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts with that of normal fibroblasts by using micrococcal nuclease (MNase) as a probe for the chromatin structure. The bulk chromatin from asynchronously and exponentially growing ras-transformed cells was much more sensitive to MNase digestion than chromatin from the normal cells. Southern hybridization analyses of the MNase digests with probes specific for the ornithine decarboxylase (odc) and c-myc genes showed that the coding and/or 3' end regions of these growth-inducible genes carry a nucleosomal organization both in ras-transformed and normal cells. Studies with cells synchronized by serum starvation showed that in both cell lines the nucleosomal organization of chromatin is relatively condensed at the quiescent state, becomes highly decondensed during the late G1 phase of the cell cycle, and starts again to condense during the S phase. However, in ras-transformed cells the decondensation state stayed much longer than in normal cells. Moreover, irrespective of the phase of the cell cycle the bulk chromatin as well as that of the odc and c-myc genes was more sensitive to MNase digestion in the ras-transformed cell than in the normal fibroblast. Decondensation of the chromatin was also observed in the normal c-Ha-ras protooncogene-transfected cells, but to a lesser extent than in the mutant ras-transformed cells. Whether the increased degree of chromatin decondensation plays a regulatory role in the increased expression of many growth-related genes in the ras-transformed cells remains an interesting object of further study.

Animals↗
PubMed1979

Effects of leukocyte and fibroblast interferon on events in the fibroblast cell cycle.

Serum-depleted human foetal skin fibroblasts were stimulated by addition of 10% foetal calf serum to proliferate synchronously for at least one cell cycle. This proliferation was suppressed by leukocyte or fibroblast interferon (IF), which prolonged the G1 phase and diminished the rate of DNA synthesis during the S phase in a dose-dependent manner. When used in identical concentration, as judged in terms of units of antiviral activity, fibroblast IF had more pronounced effects on cell cycle events than leukocyte IF. Interferon exerted its effect in early G1, before the cells were irreversibly committed to DNA synthesis.

Cell Cycle↗
PubMed2005

Protein kinase-A-dependent osteoprotegerin production on interleukin-1 stimulation in human gingival fibroblasts is distinct from periodontal ligament fibroblasts.

Periodontitis, a chronic inflammatory disease, is characterized by increased expression of interleukin (IL)-1 and other inflammatory mediators resulting in extensive osteoclast formation and bone loss. Expression of receptor activator of nuclear factor kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), by osteoblasts is important to regulate osteoclast differentiation. The aim of the present study was to investigate the regulatory effects of IL-1 on RANKL and OPG production by mesenchymal fibroblasts in periodontal tissue. Human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) were stimulated with IL-1alpha with or without protein synthesis inhibitor cycloheximide (CHX), protein kinase A (PKA) inhibitors, protein kinase C (PKC) inhibitors and prostaglandin E(2) (PGE(2)) inhibitor. In some experiments, the cultured cells were directly stimulated with either PKA or PKC activators. In HGF, IL-1alpha-stimulated OPG mRNA expression was high and could be reduced by CHX. PKA inhibitor completely abrogated IL-1alpha-induced OPG mRNA expression and OPG production. Endogenous PGE(2) further enhanced IL-1alpha-induced OPG production in HGF. In PDL, RANKL mRNA expression was greatly augmented by IL-1alpha. IL-1alpha induced OPG mRNA expression and protein production. PKC inhibitor partially reduced IL-1alpha-induced OPG production and PKC activator enhanced OPG production in PDL. The IL-1alpha-stimulated OPG mRNA expression in HGF was greater than PDL. These results provide new evidence for the possible osteoclastogenesis-inhibitory function of HGF through PKA activity pathway. PDL utilized PKC for OPG production. Thus, we emphasize that HGF and PDL have different characteristics of host defence mechanism against inflammatory process.

Adult↗
PubMed1976

The role of adherent cells in the immune response. Fibroblasts and products released by fibroblasts and peritoneal cells can substitute for adherent cells.

The primary immune response to sheep erythrocytes in adherent cell-depleted cultures was restored by adding a critical number of peritoneal cells. Complete substitution was achieved also with supernatants from allogeneic and syngeneic peritoneal cells. Both living fibroblasts and supernatants from fibroblast cultures were found to be highly efficient substitutes for adherent cells in both syngeneic and allogeneic systems. Supernatants from non-antigen-reated peritoneal cells and fibroblasts caused increased DNA synthesis and induction of polyclonal antibody synthesis in normal spleen cells. Thus, adherent cells need not function in the immune response by presenting antigen to the B cells via 'IgT' or by releasing signal-2 activity, which acts on lymphocytes that have already received signal 1.

Animals↗
PubMed2006

Altered growth characteristics of skin fibroblasts from wild-derived mice, and genetic loci regulating fibroblast clone size.

Mouse fibroblast senescence in vitro is an important model for the study of aging at cellular level. However, common laboratory mouse strains may have lost some important allele variations related to aging processes. In this study, growth in vitro of tail skin fibroblasts (TSFs) derived from a wild-derived stock, Pohnpei (Pohn) mice, differed from growth of control C57BL/6 J (B6) TSFs. Pohn TSFs exhibited higher proliferative ability, fewer apoptotic cells, decreased expression of Cip1, smaller surface areas, fewer cells positive for senescence associated-beta-galactosidase (SA-beta-gal) and greater resistance to H(2)O(2)-induced SA-beta-gal staining and Cip1 expression. These data suggest that TSFs from Pohn mice resist cellular senescence-like changes. Using large clone ratio (LCR) as the phenotype, a quantitative trait locus (QTL) analysis in a Pohn/B6 backcross population found four QTLs for LCR: Fcs1 on Chr 3 at 55 CM; Fcs2 on Chr X at 50 CM; Fcs3 on Chr 4 at 51 CM and Fcs4 on Chr 10 at 25 CM. Together, these four QTLs explain 26.1% of the variations in LCRs in the N2 population. These are the first QTLs reported that regulate fibroblast growth. Glutathione S transferase mu (GST-mu) genes are overrepresented in the 95% confidence interval of Fcs1, and Pohn TSFs have higher H(2)O(2)-induced GST-mu 4, 5 and 7 mRNA levels than B6 TSFs. These enzymes may protect Pohn TSFs from oxidation.

Animals↗
PubMed2000

Fibroblast biology. Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis.

There is growing evidence that activated synovial fibroblasts, as part of a complex cellular network, play an important role in the pathogenesis of rheumatoid arthritis. In recent years, significant progress has been made in elucidating the specific features of these fibroblasts. It has been understood that although macrophage and lymphocyte secreted factors contribute to their activation, rheumatoid arthritis synovial fibroblasts (RA-SFs) do not merely respond to stimulation by pro-inflammatory cytokines, but show a complex pattern of molecular changes also maintained in the absence of external stimulation. This pattern of activation is characterized by alterations in the expression of regulatory genes and signaling cascades, as well as changes in pathways leading to apoptosis. These together result in the upregulation of adhesion molecules that mediate the attachment of RA-SFs to the extracellular matrix and in the overexpression of matrix degrading enzymes that mediate the progressive destruction of the joints. In addition, activated RA-SFs exert specific effects on other cell types such as macrophages and lymphocytes. While the initiating step in the activation of RA-SFs remains elusive, several key pathways of RA-SF activation have been identified. However, there is so far no single, specific marker for this phenotype of RA-SF. It appears that activated RA-SFs are characterized by a set of specific properties which together lead to their aggressive behavior.

Animals↗
PubMed2005

Relaxin modulates fibroblast function, collagen production, and matrix metalloproteinase-2 expression by cardiac fibroblasts.

Cardiac fibrosis is a hallmark of heart disease and involves recruitment, proliferation, and differentiation of extracellular matrix-producing fibroblasts, leading to overproduction of collagen within the myocardium. In this study, the effects of relaxin in inhibiting these processes were investigated. We used neonatal rat atrial and ventricular fibroblasts, which respond to pro-fibrotic stimuli (i.e., transforming growth factor-beta and angiotensin II) and naturally express the relaxin receptor LGR7. Relaxin significantly inhibited TGF-beta- and angiotensin II-mediated fibroblast function and collagen production over a 72-h period, while increasing MMP-2 expression and activity in the presence of both profibrotic factors (all P < .05). These studies demonstrate that relaxin may have therapeutic potential in diseased states characterized by cardiac fibrosis.

Angiotensin II↗
PubMed1976

Studies of human diploid fibroblast growth. I. Responses of normal and hypopituitary cells to fibroblast growth factor, insulin, and serum.

We have assayed the growth stimulating activity of bovine insulin, fibroblast growth factor (FGF), and fetal bovine serum (FBS) in diploid human fibroblasts from normal and idiopathic hypopituitary donors. All three factors stimulated DNA synthesis in cells arrested by serum starvation. FGF was active at concentrations as low as 5 ng/ml with maximum effect at 100 ng/ml. FGF stimualted DNA synthesis at lower concentrations than did insulin and also produced a greater maximum response. Only serum was capable of supporting cell division and growth, but FGF accellerated this growth rate when it was added to serum-containing medium. Hydrocortisone, actinomycin D, and cycloheximide inhibit FGF stimulation. There was no significant difference between fibroblasts from normal and hypopituitary donors.

Animals↗
PubMed1992

The dependency of collagen fibrillogenesis in vitro on fibroblast culture conditions. Fibroblasts in mono- and multi-layers.

The extracellular matrix produced by monolayer and tridimensional cultures of fibroblasts was investigated using histochemical and ultrastructural methods. In monolayer cultures, collagen and proteoglycans produced by fibroblasts could not be organized into morphologically recognizable structures. Tridimensional fibroblast cultures produced a well organized matrix with periodic, parallel ordered collagen fibrils of 50 nm diameter, criss-crossed by alcianophylic segments 6-10 nm thick in diameter and 100-300 nm in length, parallel to each other, perpendicular to the collagen fibrils and spaced 67 nm from each other. Some alcianophylic segments lay perpendicular to the above described ones, with maximum lengths of 65-70 nm. Alcianophylic segments are the ultrastructural evidence of structural proteoglycans. These observations suggest that the culture conditions influence the collagen and proteoglycans secretion, so that the final organization of the matrix results quite different.

Alcian Blue↗
PubMed2005

Identification of the difference in extracellular matrix and adhesion molecules of cultured human gingival fibroblasts versus juvenile hyaline fibromatosis gingival fibroblasts using cDNA microarray analysis.

BACKGROUND: A difference from the normal range in collagen profile and perivascular hyaline deposition in the dermis and gingiva has been demonstrated histopathologically in juvenile hyaline fibromatosis (JHF), which is an autosomal recessive disease. The aim of this study was to understand the mechanism of gingival overgrowth in JHF, and to observe differences in the expression of genes regulating extracellular matrix organization. METHODS: Human gingival fibroblasts (GF) were obtained from individuals who have clinically healthy gingival tissue. JHF-GF were obtained from a patient who underwent a gingivectomy. Cultured fibroblast cells were examined visually using a phase contrast microscope. Total RNA from both cell types was isolated, and after biotin-deoxyuridine triphosphate (dUTP) labeling of cDNA, hybridization was performed with a pathway-specific gene expression profiling array membrane. Extracellular matrix (ECM) and adhesion molecule (AM) mRNA expressions in GF and JHF-GF were analyzed, and microarray data on genes modulating ECM remodeling were confirmed with reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Cell morphology differences were observed between fibroblast types. Although type I collagen gene expression levels were almost the same, decreased type IV collagen expression was noted in JHF-GF versus GF. Decreased matrix metalloproteinase (MMP) and increased tissue inhibitor of matrix metalloproteinase (TIMP) transcripts were noted in JHF-GF versus GF. Increased fibronectin and decreased laminin mRNA expression were observed in JHF-GF when compared to GF. The present findings suggest that GF and JHF-GF differ not only morphologically but also in the expression level of ECM and AM genes involving connective tissue turnover and remodeling. CONCLUSIONS: Results from these analyses may be helpful to clarify the nature of overgrowth mechanisms, especially regarding enzymes and their inhibitors. This information is important in understanding the remodeling of ECM. The gingival overgrowth that is observed in JHF patients may be explained by a decreased level of MMPs and increased blockage of MMPs with TIMPs.

Adult↗
PubMed1988

Potentiation of fibroblast spreading by extracellular matrix from fibroblasts derived from phenytoin-induced gingival overgrowth.

Cell attachment and spreading appear when a cell, on contact with an appropriate substratum, adheres and changes its shape and accommodates to the substratum. The transition from a non-spreading to a spreading state is a prerequisite for growth. Cell-free extracellular matrix (ECM) was produced by fibroblast-like cells from normal gingiva (N-ECM) and phenytoin-induced gingival overgrowth (PHT-ECM). The effect of the ECM on cell attachment and spreading of human gingival fibroblasts was studied in the presence of 2% serum. Within 30 min after seeding 40% of the normal fibroblast cells showed an advanced flattening on PHT-ECM-prepared dishes, compared with 10% on normal ECM-prepared dishes and 5% on uncoated plastic dishes. The results indicate that cells derived from PHT-induced gingival overgrowth produce an ECM with special properties, which could regulate cell functions such as cell attachment and spreading.

Adolescent↗