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At least 685 records · Page 38Linked to original sources
PubMed1994

DNA interstrand crosslinking agents and human ocular fibroblasts: differential sensitivity to mitomycin-C and cis-diaminedichloroplatinum(II).

Although clinical application of mitomycin-C (MMC) as an adjunct to glaucoma filtration and pterygium ablation surgeries is based on its inhibitory effect on the local fibroblast proliferation, the extent to which fibroblasts of Tenon's capsule and adjacent tissues (ocular fibroblasts) are susceptible to MMC still remains to be investigated. In this context, we substantially compared the sensitivity of ocular fibroblasts to MMC with normal fibroblasts of other tissue origins and with Fanconi's anemia (FA) fibroblasts known to be supersensitive to MMC due to presumable genetic susceptibility. Since MMC kills cells mainly by forming DNA interstrand crosslinks, cis-diaminedichloroplatinum (CDDP), another crosslinking agent, was also used to evaluate the cytotoxic effect of crosslinks on ocular and other fibroblasts. Following the treatment with various concentrations of MMC or CDDP for 1 hr, fibroblasts were incubated for 14 days until colonies developed. Based on the colony number reflecting the cell number which survived, the regression lines of survival rates were plotted in a semilogarithmic fashion. D0 and D10, doses which reduced survival rate to 37% and 10%, respectively, were interpolated and compared among the three cell groups. Ocular fibroblasts were about 2.5 times more sensitive than other normal fibroblasts, but 4-5 times more resistant than FA fibroblasts, to MCC, since the mean D0 of ocular, other normal, and FA fibroblasts was 0.063, 0.171 and 0.015 micrograms ml-1, respectively, and the mean D10 of these was 0.154, 0.401 and 0.029 micrograms ml-1, respectively. On the other hand, ocular fibroblasts exhibited an isosensitivity to CDDP compared with other normal fibroblasts. FA fibroblasts showed a supersensitivity to CDDP as well. DNA synthesis assay analysed by immunocytochemistry using bromodeoxyuridine (BrdU) and anti-BrdU antibody showed a similar hypersensitivity to MMC but a normosensitivity to CDDP of ocular fibroblasts. These results may indicate that intracellular activation of MMC to the reactive reduced form progresses more effectively in ocular fibroblasts than in fibroblasts originating in other tissues. The hypersensitivity to MMC of ocular fibroblasts is not attributed to genetic susceptibility because of the normosensitivity to CDDP. CDDP may be a promising agent as an alternative to MMC in the ophthalmic fields.

Cell Division↗
PubMedDate not supplied

Differentiation of primary and secondary fibroblasts in cell culture systems.

As a function of the advancing development of Valo chicken, C3H mice, BN rats, and man in the embryonic, juvenile, adolescent, and senescent phases, stem cells and fibroblasts in the connective tissues of skin and lung differentiate along an 11-stage differentiation sequence in five compartments of the fibroblast stem cell system, when studied in primary ex vivo-in vitro systems. In the fibroblast stem cell system, three stem cells develop in the stem cell compartment along the cell lineage S1-S2-S3, three mitotic fibroblasts (MF) differentiate along the sequence MF I-MF II-MF III in the fibroblast progenitor compartment, three postmitotic fibroblasts (PMF) proceed in the fibroblast maturing compartment along the row PMF IV-PMF V-PMF VI. PMF VI is the terminally differentiated end cell of the fibroblast stem cell system. After a species- and tissue-specific period of high metabolic activity, PMF VI either dies as PMF VIIa in the fibroblast apoptosis compartment or transforms as PMF VIIb in the fibroblast transforming compartment. The reiterated appearance of the 11 cell types in primary stem cell and fibroblast populations and the reiterated age-related changes in the cell type composition of the primary stem cell and fibroblast populations make it very likely that stem cell, mitotic and postmitotic fibroblast equivalents exist in vivo and that age-related changes of the frequencies of the stem cell and fibroblast equivalents result from the progressing differentiation of stem cell, mitotic, and postmitotic fibroblast equivalents along the 11 stage differentiation sequence in the fibroblast equivalent stem cell system in vivo. Secondary fibroblast populations derived from connective tissue of prenatal and postnatal skin of Valo chicken, C3H mice, BN rats, and man, including the normal embryonic human lung fibroblast cell line WI38, were also found to develop along a terminal stem cell sequence. Thus, secondary fibroblast populations in vitro constitute a representative material for studies of general and special issues of cell biology, such as terminal differentiation, aging, apoptosis, and transformation, as long as stem cell system-specific concepts and methods are employed in such investigations.

Animals↗
PubMed2002

Expression of basic fibroblast growth factor is associated with poor outcome in non-Hodgkin's lymphoma.

It is now clear that angiogenesis and angiogenesis factors are important in the pathogenesis of haematological malignancies. High pretreatment levels of serum basic fibroblast growth factor have been shown to be associated with poor prognosis in patients with non-Hodgkin's lymphoma. The aim of this study was to evaluate whether non-Hodgkin's lymphoma cells express basic fibroblast growth factor and/or its receptor (fibroblast growth factor receptor-1) and whether basic fibroblast growth factor expression correlates with basic fibroblast growth factor serum levels, intratumoral microvessel density, and patient outcome. We measured basic fibroblast growth factor by enzyme-linked immunosorbent assay in sera taken from 58 patients with non-Hodgkin's lymphoma before treatment and in 19 of them also after treatment. Pathological specimens at diagnosis were evaluated by immunohistochemistry staining using polyoclonal antibody against factor-VIII-related antigen, basic fibroblast growth factor and fibroblast growth factor receptor-1 to determine the expression of the microvessel count and basic fibroblast growth factor and fibroblast growth factor receptor-1. The lymphoma specimens demonstrated positive staining for basic fibroblast growth factor (in 23%) and fibroblast growth factor receptor-1 (in 58.5%). The patients who expressed basic fibroblast growth factor had a significantly worse progression-free and overall survival than those who did not (P=0.003 and P=0.03 respectively), while patients expressing fibroblast growth factor receptor-1 were less likely to achieve complete remission than those lacking the receptor (33% vs 65%, P=0.047). There was no correlation of basic fibroblast growth factor staining with either serum basic fibroblast growth factor levels or microvessel count. Basic fibroblast growth factor serum levels did not change significantly after treatment These results suggest that non-Hodgkin's lymphoma specimens express basic fibroblast growth factor and its receptor (fibroblast growth factor receptor-1) and this expression is associated with poor patient outcome.

Adult↗
PubMed1994

Seizure-associated induction of basic fibroblast growth factor and its receptor in the rat brain.

In order to assess the role of neuronal activity in the regulation of the fibroblast growth factor system, we examined changes in levels of basic fibroblast growth factor and the expression of its receptor-1 following seizures. Epileptiform activity was induced by kainate injection and the rats displaying seizures were killed 3, 6, 12 and 24 h after injection. To identify basic fibroblast growth factor and fibroblast growth factor receptor-1 immunoreactivity, we used a monoclonal antibody that binds to the biological active form of basic fibroblast growth factor and a monoclonal antibody that recognizes fibroblast growth factor receptor-1. In normal brain tissue, fibroblast growth factor staining was widely distributed throughout the brain and appeared to be localized within the nucleus of astrocytes. Starting 6 h after seizures, there was a progressive increase in basic fibroblast growth factor immunoreactivity. The seizure-induced effect on basic fibroblast growth factor immunoreactivity was expressed in astrocytes as an enlargement of the nucleus and a spreading of the staining to the processes. This phenomenon was particularly strong in the cerebral cortex and hippocampus. The fibroblast growth factor receptor-1 immunoreactivity was virtually absent in control brain tissue. By 3 h post-seizure induction, there was an increase in fibroblast growth factor receptor-1 immunoreactivity in the molecular layer of the dentate gyrus. After 6 h, fibroblast growth factor receptor-1-positive cells appeared in the stratum oriens along the CA1, CA2 and CA3 hippocampal subfields. This effect gradually expanded to other brain regions and by 24 h fibroblast growth factor receptor-1 immunoreactivity was distributed throughout the hippocampus and cerebral cortex. Fluorescent double labelling indicated that the fibroblast growth factor receptor-1 immunoreactivity was expressed in astrocytes. At 24 h, some fibroblast growth factor receptor-1 immunoreactivity was also observed in neuron-like cells located throughout the cerebral cortex and hippocampus. Since our results indicate that seizure activity modulates the expression of basic fibroblast growth factor and fibroblast growth factor receptor-1 levels, it is also possible that physiological stimulation might have similar effects. In addition, our results suggest that the fibroblast growth factor system may have a role in plasticity events triggered by physiological activity.

Animals↗
PubMed1997

Effect of growth factors on the proliferation of ligament fibroblasts from skeletally mature rabbits.

Growth factors have been shown to stimulate fibroblast proliferation during wound and ligament healing. In this study, we analyzed individual effects of eight growth factors in vitro on the proliferation of fibroblasts from the medial collateral (MCL) and anterior cruciate (ACL) ligaments of skeletally mature rabbits. We compared the proliferative response of growth factor-treated and nontreated fibroblasts of both ligaments. The growth-factor treated fibroblasts of the MCL and ACL were also compared. We found that the fibroblasts exposed to epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-BB proliferated significantly more than untreated fibroblasts. Acidic fibroblast growth factor at a dose of 1.0 ng/ml caused significant increases in fibroblast proliferation only in the MCL. Transforming growth factor-beta 1, insulin-like growth factor-1, platelet-derived growth factor-AA, and interleukin-1 alpha did not significantly stimulate fibroblast proliferation. MCL fibroblasts generally did not proliferate significantly more than ACL fibroblasts with the exception of MCL fibroblasts exposed to the highest doses of basic fibroblast growth factor, acidic fibroblast growth factor and platelet-derived growth factor-BB. The data were also compared with those obtained earlier using fibroblasts from skeletally immature rabbits (Schmidt et al., JOR 1995). The proliferative response of both the MCL and the ACL fibroblasts was found to decrease with skeletal maturation. Thus, our findings suggest that animal age and fibroblast origin are important factors in determining the proliferative response to growth factors.

Animals↗
PubMed2005

Upregulation of transforming growth factor-beta1 and vascular endothelial growth factor in cultured keloid fibroblasts: relevance to angiogenic activity.

Keloids are tumor-like lesions that result from excessive scar formation during healing of wounds. Histologically, keloids show an increased blood vessel density compared with normal dermis or normal scars. However, the angiogenic activity of keloid fibroblasts remains unknown. In this study, we investigated angiogenic activity of keloid fibroblasts. Transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor (VEGF) were investigated as elements of the angiogenic factors. Expressions of TGF-beta1 and VEGF in conditioned medium were measured with enzyme-linked immunosorbent assay (EIA) and Northern blot analysis. Participation of TGF-beta1 in the production of VEGF was also investigated with addition of TGF-beta1 and a neutralizing anti-TGF-beta1 antibody. A modified Boyden chamber assay was performed to assess the chemotactic activity of vascular endothelial cells. Angiogenic activity in vivo was evaluated by neovascularization of nodules formed by implantation of fibroblasts into severe combined immunodeficiency (SCID) mice. EIA showed that the concentrations of TGF-beta1 and VEGF in conditioned medium were increased 2.5- and 6-fold, respectively, after the culture of keloid fibroblasts compared with normal fibroblasts. Northern blot analysis revealed that the expression of TGF-beta1 and VEGF mRNA was upregulated 3.6- and 6-fold, respectively, in keloid fibroblasts compared with normal fibroblasts. Addition of TGF-beta1 to keloid fibroblast cultures increased VEGF production by 3.5-fold, while there was a 6-fold in culture of normal fibroblasts. A neutralizing anti-TGF-beta1 antibody reduced VEGF secretion to control levels, suggesting that TGF-beta1 mediated the upregulation of VEGF expression. A modified Boyden chamber assay demonstrated that the chemotactic activity of vascular endothelial cells was more strongly (sevenfold) induced by keloid fibroblast-conditioned medium than by normal fibroblast-conditioned medium. Anti-VEGF antibody inhibited chemotaxis to basal levels. When SCID mice underwent implantation of fibroblasts into the back, the nodules formed by keloid fibroblasts were three times larger than those formed by normal fibroblasts. Although abundant neovascularization was observed in keloid fibroblast nodules, neovascularization was scarce in normal fibroblast nodules. Both in vitro and in vivo studies confirmed the significantly higher angiogenic activity of keloid fibroblasts compared with normal fibroblasts, and TGF-beta1 and VEGF were clearly shown to be involved. These results suggest that angiogenesis in keloids is promoted by endogenous TGF-beta1 and VEGF.

Adolescent↗
PubMed2003

Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia.

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O(2)), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 +/- 1.3 pg/ml versus 34.8 +/- 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients.

Angiogenesis Inducing Agents↗
PubMedDate not supplied

Cultured pressure ulcer fibroblasts show replicative senescence with elevated production of plasmin, plasminogen activator inhibitor-1, and transforming growth factor-beta1.

In a 16-patient study, cultured fibroblast populations from normal skin were able to replicate an average of 14.8 +/- 2.2 times before becoming senescent, while fibroblast populations from the ulcer bed reached the end of their replicative life span after 7.2 +/- 1.9 population doublings (p= 0.001). Fibroblast populations from 10 of 16 pressure ulcers became senescent after fewer than five population doublings, whereas when populations of fibroblasts from adjacent normal skin were studied, only 2 of 16 became senescent within this same time period. In addition, only an occasional fibroblast from normal skin stained positively for senescence-associated beta-galactosidase compared to approximately 50% of equally aged ulcer bed fibroblasts (p = 0.0060). Senescent ulcer bed fibroblasts secreted significantly more plasmin than early passage ulcer bed fibroblasts (p= 0.0237), nearly six times as much plasmin as early passage normal skin fibroblasts (p < 0.0001), three and a half times the level of normal skin fibroblasts of the same age (11.52 +/- 4.58 microg/mg protein; p= 0.0003), and more than one and a half times the level of senescent normal skin fibroblasts (p= 0.0525). Senescent pressure ulcer fibroblasts generated significantly more plasminogen activator inhibitor-1 (1179.27 +/- 25.37 ng/mg protein) than normal skin fibroblasts of the same age (132.16 +/- 16.20 ng/mg protein; p = 0.0357). Also, senescent ulcer bed fibroblasts produced higher levels of transforming growth factor-beta1, but these were not significantly different from senescent normal skin fibroblasts. Although senescent ulcer fibroblasts produce elevated levels of plasminogen activator inhibitor-1 and transforming growth factor-beta1, the ratio of these factors to plasmin levels suggests that this may have little influence on extracellular matrix synthesis or maintenance in the chronic wound. These data show that cultured fibroblasts from most patient pressure ulcers profile a wound environment that is associated with an increasing population of senescent fibroblasts; however, factors within the chronic wound environment that promote cellular senescence remain unclear. We have proposed that a prolonged inflammatory response may be a contributing factor to the chronic wound condition.

Cells, Cultured↗
PubMed1992

Differences in cell division and thymidine incorporation with rat and primate fibroblasts in collagen lattices.

Human and gorilla dermal fibroblasts, primate cells, suspended in a collagen lattice, do not divide for the first 3 days. In contrast, rat fibroblasts divide within 24 hr. In this study, the proliferation of rat fibroblasts were compared to primate fibroblasts. Rat fibroblasts in monolayer culture increase from 100,000 to 355,000 in 2 days, and human cells increase from 100,000 to 436,000 in the same period. An initial seeding of 100,000 rat fibroblasts suspended in collagen increased to 163,000 cells in 2 days. An initial 100,000 human fibroblasts seeded in collagen decreased to 80,000 cells in 2 days. Retarded proliferation of human and gorilla fibroblasts in collagen is unrelated to a defect in DNA synthesis. By autoradiography human fibroblasts suspended in collagen incorporate labelled thymidine. By flow cytometry analysis, the DNA concentrations of human fibroblasts suspended in collagen exhibited 41% in a 4N chromosome state, compared to 14% in monolayer culture. Nuclei of gorilla fibroblasts from collagen displayed 42% in a 4N state, compared to 19% in monolayer culture. With nuclei of rat fibroblasts from collagen, 14% were in a 4N state, compared to 9% in monolayer culture. Primate fibroblasts show a three-fold increase in the number of nuclei in a 4N state compared to rat fibroblasts suspended in collagen. After replating fibroblasts released from collagen in monolayer culture in the presence of 1 mM hydroxyurea (an inhibitor of DNA synthesis) primate fibroblasts doubled in 24 hr. Under identical conditions, rat fibroblasts showed no cell division.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Distribution of basic fibroblast growth factor in the developing rat brain.

Previous studies in vitro indicate that basic fibroblast growth factor participates in the survival, proliferation and differentiation of immature neural cells, predicting that it may have the same types of roles in vivo. In order to evaluate a possible role of basic fibroblast growth factor in neural development, we have examined its localization in the rodent brain at critical stages of development. We characterized basic fibroblast growth factor immunoreactivity at embryonic days 13 and 18, and postnatal days 1, 4, 6, 10, 20 and 90. Our results showed that basic fibroblast growth factor was transiently expressed by different cellular phenotypes throughout development. At embryonic day 13, basic fibroblast growth factor immunoreactivity was sparsely distributed in various cell phenotypes. At embryonic day 18, the primitive cerebral cortex showed basic fibroblast growth factor immunoreactivity within its emerging laminar structure, including the cortical plate and subplate regions. At postnatal day 1, basic fibroblast growth factor immunoreactivity was mostly concentrated in the hippocampal subfields cornu Ammon 1, cornu Ammon 2 and cornu Ammon 3, and neurons of the medical septum and the vertical limb of the diagonal band nuclei. At postnatal days 4-6, astrocyte-like cells showed basic fibroblast growth factor immunoreactivity for the first time during development. At this stage, basic fibroblast growth factor in the hippocampus was mostly shown within subfields cornu Ammon 2 and cornu Ammon 1. In the medical septum, just a few neuronal profiles were weakly stained, and basic fibroblast growth factor positive astrocytes appeared to accumulate around these basic fibroblast growth factor-stained neurons. At postnatal day 20, the adult pattern of basic fibroblast growth factor immunoreactivity was fully established. Astrocytes throughout the brain expressed basic fibroblast growth factor, and neuronal basic fibroblast growth factor was restricted to particular populations such as cingulate cortex and hippocampus. The cornu Ammon 2 subfield was the main neuronal location for basic fibroblast growth factor in the mature hippocampus. Our results showed that the cellular location of basic fibroblast growth factor changes during development, suggesting that basic fibroblast growth factor has multiple and evolving roles during histogenesis and differentiation of the CNS.

Animals↗
PubMed1992

Herpes simplex virus-infected human fibroblasts are resistant to and inhibit cytotoxic T-lymphocyte activity.

We examined the ability of human anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL) to lyse autologous human fibroblasts infected with HSV. In contrast to HSV-infected human Epstein-Barr virus-transformed B cells (LCL), which were lysed by HLA-restricted anti-HSV CTL, autologous fibroblasts infected with HSV were resistant to lysis. This resistance was not due to a lack of infectivity or production of HSV proteins since greater than 90% of the cells were infected and expressed abundant levels of viral proteins. HSV-infected human fibroblasts were also tested for susceptibility to lysis by alloantigen-specific CTL. Although allogeneic LCL and uninfected allogeneic fibroblasts were killed, human fibroblasts infected with HSV demonstrated a time-dependent resistance to lysis by alloantigen-specific CTL. HSV-infected human fibroblasts were not resistant to all forms of cell-mediated cytotoxicity since they were sensitive to antibody-dependent cellular cytotoxicity. Although one may suspect that the resistance of HSV-infected human fibroblasts to anti-HSV CTL and alloantigen-specific CTL-mediated lysis was due to a lack of major histocompatibility complex expression, Confer et al. (Proc. Natl. Acad. Sci. USA 87:3609-3613, 1990) previously demonstrated that incubation of human natural killer and lymphokine-activated killer cells with monolayers of human fibroblasts infected with HSV "disarmed" the killers in that they were unable to lyse sensitive target cells. We extend their results and show that incubation of anti-HSV CTL or alloantigen-specific CTL with uninfected fibroblasts did not affect their lytic activity, whereas CTL incubated with HSV-infected fibroblasts for 2 to 6 h rendered the CTL incapable of lysing their normally sensitive target cells. Indeed, human fibroblasts infected for merely 2 h with HSV were able to profoundly inhibit the cytotoxic activity of alloantigen-specific CTL. Thus, HSV-infected human fibroblasts are not inherently resistant to lysis by anti-HSV CTL or alloantigen-specific CTL, but rather contact of CTL with HSV-infected fibroblasts resulted in inactivation of the CTL. The inactivation of CTL appears to be HSV specific since incubation of alloantigen-specific CTL in sandwich assays with fibroblasts infected with HSV type 1 (HSV-1) or HSV-2 resulted in inactivation, whereas incubation of CTL with fibroblasts infected with adenovirus or vaccinia virus had no effect. Further, although incubation of alloantigen-specific CTL in sandwich assays with HSV-infected fibroblasts resulted in inhibition of CTL activity, exposure of CTL in Transwell cultures to cell-free supernatant from HSV-infected fibroblasts did not mediate this inhibitory effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Death↗
PubMed1999

Kinesin-like protein CENP-E is upregulated in rheumatoid synovial fibroblasts.

UNLABELLED: Articular destruction by invading synovial fibroblasts is a typical feature in rheumatoid arthritis (RA),. Recent data support the hypothesis that key players in this scenario are transformed-appearing synovial fibroblasts at the site of invasion into articular cartilage and bone. They maintain their aggressive phenotype toward cartilage, even when first cultured and thereafter coimplanted together with normal human cartilage into severe combined immunodeficient mice for and extended period of time. However, little is known about the upregulation of genes that leads to this aggressive fibroblast phenotype. To inhibit this progressive growth without interfering with pathways of physiological matrix remodelling, identification of pathways that operate specifically in RA synovial fibroblasts is required. In order to achieve this goal, identification of genes showing upregulation restricted to RA synovial fibroblasts is essential. AIMS: To identify specifically expressed genes using RNA arbitrarily primed (RAP)-polymerase chain reaction (PCR) for differential display in patients with RA. METHODS: RNA was extracted from cultured synovial fibroblasts from 10 patients with RA, four patients with osteoarthritis (OA), and one patient with psoriatic arthritis. RAP-PCR was performed using different arbitrary primers for first-strand and second-strand synthesis. First-strand and second-strand synthesis were performed using arbitrary primers: US6 (5'-GTGGTGACAG-3') for first strand, and Nuclear 1+ (5'ACGAAGAAGAG-3'), OPN28 (5'GCACCAGGGGG-3'), Kinase A2+(5'-GGTGCCTTTGG-3') and OPN24 (5'AGGGGCACCA-3') for second strand synthesis. PCR reactions were loaded onto 8 mol/l urea/6% polyacrylamide-sequencing gels and electrophoressed. Gel slices carrying the target fragment were then excised with a razor blade, eluated and reamplified. After verifying their correct size and purity on 4% agarose gels, the reamplified products derived from the single-strand confirmation polymorphism gel were cloned, and five clones per transcript were sequenced. Thereafter, a genbank analysis was performed. Quantitative reverse transcription PCRj of the segments was performed using the PCR MIMIC technique. In-situ expression of centromere kinesin-like protein-E (CENP-E) messenger (m)RNA in RA synovium was assessed using digoxigenin-labelled riboprobes, and CENP-E protein expression in fibroblasts and synovium was performed by immunogold-silver immunohistochemistry and cytochemistry. Functional analysis of CENP-E was done using different approaches (eg glucocorticoid stimulation, serum starvation and growth rate analysis of synovial fibroblasts that expressed CENP-E). RESULTS: In RA, amplification of a distinct PCR product suitable for sequencing could be observed. The indicated complementary DNA fragment of 434 base pairs from RA mRNA corresponded to nucleotides 6615-7048 in the human centromere kinesin-like protein CENP-E mRNA (GenBank accession No. emb/Z15005). The isolated sequence shared greater than 99% nucleic acid (P=2.9e(-169)) identify with the human centromere kinesin-like protein CENP-E. Two base changes at positions 6624 (A to C) and 6739 (A to G) did not result in alteration in the amino acid sequence, and therefore 100% amino acid identity could be confirmed. The amplification of 10 clones of the cloned RAP product revealed the presence of CENP-E mRNA in every fibroblast culture examined, showing from 50% (271.000 +/- 54.000 phosphor imager arbitrary units) up to fivefold (961.000 +/- 145.000 phosphor image arbitrary units) upregulation when compared with OA fibroblasts. Neither therapy with disease-modifying antirheumatic drugs such as methotrexate, gold, resochine or cyclosporine A, nor therapy with oral steroids influenced CENP-E expression in the RA fibroblasts. Of the eight RA fibroblast populations from RA patients who were receiving disease-modifying antirheumatic drugs, five showed CENP-E upregulation; and of the eight fibroblast populations from RA patients receiving steroids, four showed CENP-E upregulation. Numerous synovial cells of the patients with RA showed a positive in situ signal for the isolated CENP-E gene segment confirming CENP-E mRNA production in rheumatoid synovium, whereas in OA synovial tissue CENP-E mRNA could not be detected. In addition, CENP-E expression was independent from medication. This was further confirmed by analysis of the effect of prednisolone on CENP-E expression, which revealed no alteration in CENP-E mRNA after exposure to different (physiological) concentrations of prednisolone. Serum starvation also could not suppress CENP-E mRNA completely. DISCUSSION: Since its introduction in 1992, numerous variants of the differential display method and continuous improvements including RAP-PCR have proved to have both efficiency and reliability in examination of differentially regulated genes. The results of the present study reveal that RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts. The mRNA, which has been found to be upregulated in rheumatoid synovial fibroblasts, codes for a kinesin-like motor protein named CENP-E, which was first characterized in 1991. It is a member of a family of centromere-associated proteins, of which six (CENP-A to CENP-F) are currently known. CENP-E itself is a kinetochore motor, which accumulates transiently at kinetochores in the G2 phase of the cell cycle before mitosis takes place, appears to modulate chromosome movement and spindle elongation,and is degraded at the end of mitosis. The presence or upregulation of CENP-E has never been associated with RA.The three-dimensional structure of CENP-E includes a coiled-coil domain. This has important functions and shows links to known pathways in RA pathophysiology. Coiled-coil domains can also be found in jun and fos oncogene products, which are frequently upregulated in RA synovial fibroblasts. They are also involved in DNA binding and transactivation processes resembling the situation in AP-1 (Jun/Fos)-dependent DNA-binding in rheumatoid synovium. Most interestingly, these coiled-coil motifs are crucial for the assembly of viral proteins, and the upregulation of CENP-E might reflect the influence of infectious agents in RA synovium. We also performed experiments showing that serum starvation decreased, but did not completely inhibit CENP-E mRNA expression. This shows that CENP-E is related to, but does not completely depend on proliferation of these cells. In addition, we determined the growth rate of CENP-E high and low expressors, showing that it was independent from the amount of CENP-E expression. supporting the statement that upregulation of CENP-E reflects an activated RA fibroblast phenotype. In summary, the results of the present study support the hypothesis that CENP-E, presumably independently from medication, may not only be upregulated, but may also be involved in RA pathophysiology.

Adult↗
PubMedDate not supplied

Pachydermoperiostosis (primary hypertrophic osteoarthropathy): in vitro evidence for abnormal fibroblast proliferation.

Pachydermoperiostosis (PDP) is a disease characterized by the presence of pachydermia, periostosis and finger clubbing. Evidence that the skin and soft tissues are involved in the disease prompted the in vitro investigation of the behaviour of fibroblasts obtained from cutaneous biopsies of involved and apparently uninvolved PDP skin. PDP fibroblasts from affected skin demonstrated an abnormal proliferation, very rapid and tumultuous when compared to the growth of fibroblasts derived from apparently uninvolved skin and fibroblasts from the skin of healthy subjects. This characteristic was confirmed by the rate of thymidine incorporation, which was increased in PDP-affected fibroblasts (1152 dpm) compared to apparently non-PDP involved fibroblasts (273 dpm) and controls (262 dpm). Ultracentrifuged and non-centrifuged conditioned medium (CM) of fibroblasts affected or apparently not affected with PDP were used to evaluate the effect on the proliferation of healthy skin fibroblasts, compared to the effect of CM derived from healthy fibroblasts and from healthy fibroblasts incubated with 10% and 1% foetal calf serum. The CM of non-centrifuged PDP fibroblasts resulted in a statistically significant stimulation of fibroblast growth when compared to that expressed by ultracentrifuged PDP CM, healthy fibroblast CM and 10% stimulated CM. These data show that PDP fibroblasts maintain in vitro the capacity to proliferate at a higher rate than healthy fibroblasts and that in the CM residual cells and/or their debris may be present, inducing the abnormal growth of healthy fibroblasts. This evidence suggests that fibroblasts in PDP may play a role in the development of the disease.

Cell Division↗
PubMed1997

Basic fibroblast growth factor, its high- and low-affinity receptors, and their relationship to form-deprivation myopia in the chick.

Form deprivation myopia in chickens is a widely accepted model to study visually-regulated postnatal ocular growth. Recently we showed that basic fibroblast growth factor-2 provides a "stop" signal for the growing eye. To understand further its action, we have localized basic fibroblast growth factor-2 and its low- and high-affinity receptors in the chicken eye, and determined the localization of basic fibroblast growth factor receptors in the inner plexiform layer with respect to that of neurotransmitter systems known to play a role in form-deprivation myopia. By immunocytochemistry and in situ hybridization, two complementary methods, we found that nearly all cells in the retina, and scleral chondrocytes, contain basic fibroblast growth factor-2 protein and messenger RNA as well as high-affinity basic fibroblast growth factor receptor protein and messenger RNA. Immunocytochemical localization of basic fibroblast growth factor-2 binding sites (a high resolution alternative to autoradiography), combined with N-glycanase and heparitinase treatment or heparin competition, revealed additional binding sites in specific synaptic layers of the inner plexiform layer and low-affinity binding sites in the choroid and optic fibre layer. Some binding sites in the synaptic layers were found to co-stratify with neurites of dopamine-, vasoactive intestinal polypeptide- or enkephalin-containing amacrine cells, suggesting that basic fibroblast growth factor-2 could modulate synaptic transmission to or from these cells. Form deprivation did not affect the levels of basic fibroblast growth factor receptor-1 messenger RNA in retina/retinal pigment epithelium/choroid (Northern blotting), but it abolished the decrease in amount of extractable basic fibroblast growth factor normally observed in the dark (Western blotting). The results are discussed with respect to previous findings on basic fibroblast growth factor-2 and basic fibroblast growth factor receptor-1 localization in the avian and other vertebrate eyes, and their relevance to form-deprivation myopia. The widespread distribution of basic fibroblast growth factor-2 and its receptor makes it impossible to predict which cells might mediate the action of basic fibroblast growth factor-2 in form-deprivation myopia. However, the alteration in amounts of extractable retinal basic fibroblast growth factor-2 in form-deprived, dark-adapted retinas, in which basic fibroblast growth factor-2 probably serves as a "stop" signal for ocular growth, is consistent with a role for basic fibroblast growth factor-2 in the regulation of ocular growth.

Animals↗
PubMed2003

Effects of Aloe vera on gap junctional intercellular communication and proliferation of human diabetic and nondiabetic skin fibroblasts.

OBJECTIVE: To evaluate the effects of Aloe vera on gap junctional intercellular communication (GJIC) and proliferation of human skin fibroblasts in the presence or absence of basic fibroblast growth factor (FGF-2). DESIGN: In vitro study using human type II diabetic and nondiabetic skin fibroblast cell lines. SETTING AND SUBJECTS: Diabetic (n = 4) and nondiabetic (n = 4) human skin fibroblast cell lines were purchased from Coriell Institute for Medical Research (Camden, NJ). The cells were cultured with or without Aloe vera extract in increasing concentrations (0%, 0.625%, 1.25%, 2.5%, 5%, 10%, and 20%; v/v) in culture medium and with or without FGF-2 (30 ng/mL). MEASUREMENTS: GJIC was evaluated after 48-hour incubation with treatments by laser cytometry. Cells were counted after 72-hour incubation with treatments by using a Coulter counter. RESULTS: The rate of GJIC was greater (p < 0.01) for diabetic than for nondiabetic fibroblasts (3.5 +/- 0.1 versus 3.0 +/- 0.1% per minute during the first 4 minutes after photobleaching). GJIC was increased ( p < 0.05) for diabetic fibroblasts in the presence of 2.5% and 5% of Aloe vera extract (4.2 +/- 0.1 and 4.0 +/- 0.2 versus 3.5 +/- 0.1% per minute for control, respectively). FGF-2 stimulated (p < 0.01) GJIC for diabetic (4.0 +/- 0.1 versus 3.5 +/- 0.1% per minute for control) and nondiabetic (3.5 +/- 0.1 versus 3.0 +/- 0.1% per minute for control) fibroblasts. Aloe vera extract did not affect GJIC of nondiabetic fibroblast cultured without FGF-2. However, Aloe vera extract decreased (p < 0.05) FGF-2 stimulatory effects on GJIC of diabetic and nondiabetic fibroblasts. Proliferation of diabetic fibroblasts was increased (p < 0.05) by 1.25% and 2.5% Aloe vera extract in medium. Proliferation of nondiabetic fibroblasts was not affected by Aloe vera extract. FGF-2 increased (p < 0.05) proliferation of nondiabetic fibroblasts and FGF-2 did not affect proliferation of diabetic fibroblasts. Aloe vera extract decreased (p < 0.05) FGF-2 stimulatory effects on proliferation of nondiabetic fibroblasts. CONCLUSIONS: These data demonstrate that Aloe vera has the ability to stimulate GJIC and proliferation of human skin fibroblasts in diabetes mellitus. Furthermore, these results indicate that Aloe vera contains a compound(s) that neutralizes, binds with FGF-2 receptor, or otherwise alters signaling pathways for FGF-2. By affecting both GJIC and proliferation of diabetic fibroblasts, Aloe vera may improve wound healing in diabetes mellitus.

Aloe↗
PubMed2001

Differential effects of hexoses and sucrose, and platelet-derived growth factor isoforms on cyclooxygenase-1 and -2 mRNA expression in keloid, hypertrophic scar and granulation tissue fibroblasts.

Cyclooxygenase (COX) is the key enzyme in the formation of prostaglandins in inflammation. In the present study the effects of biomedically relevant hexose sugars (glucose, fructose, galactose, mannose) and sucrose disaccharide on the expression of COX-1 and COX-2 genes were evaluated in granulation tissue fibroblasts, hypertrophic scar fibroblasts and keloid fibroblasts. The effects of three isoforms (AA, AB and BB) of PDGF on COX gene expression in granulation tissue fibroblasts were also examined. All cell lines expressed COX-1 mRNA, whilst fibroblasts derived from abnormal scars did not express COX-2 mRNA. COX-1 mRNA expression was decreased by sugars in granulation tissue fibroblasts and increased in hypertrophic scar fibroblasts. No major changes were seen in keloid fibroblasts. On the other hand, COX-2 mRNA expression in granulation tissue fibroblasts was decreased dramatically in the presence of fructose, mannose and sucrose and moderately in the presence of galactose. All isoforms of PDGF increased COX-1 and COX-2 mRNA expression in granulation tissue fibroblasts, the most marked increases being elicited by PDGF-BB. All fibroblast cell lines studied expressed the COX-1 gene while the COX-2 gene was not expressed by abnormal scar-derived fibroblasts. Further, granulation tissue fibroblasts seemed to behave differently under the influence of sugars compared to hypertrophic scar fibroblasts whilst keloid fibroblasts seemed to be relatively unaffected by sugars. In addition, the PDGF-BB isoform is a potent inducer of COX-2 gene expression in wound fibroblasts. These findings may be relevant to the development of abnormal scars and indicate the need for further studies.

Cells, Cultured↗
PubMed1995

CD40 expression by human fibroblasts.

The purpose of this study was to determine whether human fibroblasts express CD40, a 50-kDa member of the tumor necrosis factor-alpha-receptor superfamily. CD40 is an important mitogenic receptor on B lymphocytes which regulates B lymphocyte proliferation and differentiation. Interestingly, CD40 mRNA was detected in human lung, gingival, synovial, dermal (foreskin), and spleen fibroblasts using the reverse-transcriptase polymerase chain reaction. Moreover, the CD40 protein was detected on cultured human fibroblasts using anti-CD40 mAbs (G28-5, EA-5) and flow cytometry and on fibroblasts in dermal tissue sections via in situ staining. In contrast to B lymphocytes, where CD40 expression is unregulated both by interleukin-4 and interferon (IFN-gamma), CD40 expression on cultured human fibroblasts could only be upregulated by IFN-gamma. IFN-gamma induced a 10-fold increase in CD40 mRNA and protein levels. Furthermore, via a two-color staining technique for CD40 expression and DNA content, IFN-gamma not only upregulated CD40 expression on cultured human fibroblasts, but also shifted fibroblasts into the G0/G1 phase of the cell cycle. This observation suggested that nonproliferating fibroblasts might display elevated levels of CD40. To test this hypothesis, CD40 expression was analyzed on fibroblasts in log phase growth vs fibroblasts which had reached confluency and were nonproliferating. Interestingly, confluent fibroblasts expressed higher levels of CD40 than fibroblasts in log phase growth. These data suggest that CD40 expression by human fibroblasts is related to cell growth. In summary, this report is the first to demonstrate that human fibroblasts from a variety of tissues display CD40. While the function of CD40 on fibroblasts is not yet known, it may facilitate fibroblast proliferation, an event important for tissue repair, and may facilitate inflammation via interaction with T lymphocytes and mast cells, which display the CD40 ligand.

Adjuvants, Immunologic↗
PubMed2006

Overproduction of collagen and diminished SOCS1 expression are causally linked in fibroblasts from idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and often fatal pulmonary disorder, and its pathology is characterized by parenchymal fibrosis. To investigate the characteristics of fibroblasts in IPF, we obtained eight fibroblast cell lines from lungs with IPF and eight lines from normal lungs. We found that the fibroblasts from IPF spontaneously produced higher amounts of type I collagen and had lower expression levels of SOCS1 than fibroblasts from normal lung. By using mouse fibroblasts, we demonstrated the causal relationship between them: the deficiency of SOCS1 in fibroblasts resulted in increased collagen production, whereas overexpression of SOCS1 suppressed collagen production. IFN-gamma suppressed spontaneous collagen production even in SOCS1-deficient fibroblasts, indicating that IFN-gamma inhibition is SOCS1-independent. In contrast, IFN-gamma suppressed the increase of collagen production induced by IL-4 in wild type fibroblasts but not SOCS1-deficient fibroblasts, suggesting IFN-gamma acted exclusively via SOCS1 in this case. Following IFN-gamma stimulation, the amount of SOCS1 mRNA expressed by IPF fibroblasts was comparable to that of normal fibroblasts. Thus, the extent of SOCS1 increase after stimulation by IFN-gamma was significantly higher in IPF fibroblasts. The extent to which IFN-gamma inhibited collagen production was also larger in IPF fibroblasts than in normal fibroblasts. These results suggest that the exaggerated production of collagen observed in fibroblasts from IPF is causally related to the diminished expression of SOCS1, and IPF fibroblasts are more susceptible to IFN-gamma because of decreased expression of SOCS1.

Animals↗