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PubMed1997

Keratinocytes contract human dermal extracellular matrix and reduce soluble fibronectin production by fibroblasts in a skin composite model.

Composites of human de-epidermised acellular dermis and normal adult human keratinocytes and fibroblasts were examined for the ability of cells to contract these composites. Image analysis of the outline of the composites showed that, in this model, keratinocytes alone or in the presence of fibroblasts caused highly significant contraction (of the order of 25% by day 12). There was no significant contraction of the dermis with fibroblasts alone or in the absence of cells. The presence or absence of basement membrane antigens did not influence the effect of keratinocytes on dermal contraction. Analysis of the conditioned media from these composites showed that the greatest fibronectin production was seen with fibroblasts alone in the presence of basement membrane. Keratinocytes alone produced little fibronectin irrespective of the presence of the basement membrane. If keratinocytes were present with fibroblasts, however, then fibronectin production was significantly reduced both in the presence and absence of the basement membrane, indicating that keratinocytes modify dermal fibroblast extracellular matrix production. This study shows that while keratinocytes and fibroblasts are clearly influencing each other's activity in this human skin composite model, under the circumstances we describe it is the keratinocyte and not the fibroblast which causes contraction of the human de-epidermised acellular dermis.

Adult↗

PubMed2000

Characterization of beta-galactosidase in leukocytes and fibroblasts of GM1 gangliosidosis heterozygotes compared to normal subjects.

OBJECTIVES: Characterization of beta-galactosidase in leukocytes and fibroblasts of heterozygotes for GM1 type I. DESIGN AND METHODS: Leukocyte and fibroblast beta-galactosidase activity was determined fluorimetrically using 4-methylumbelliferyl-beta-D-galactoside as an artificial substrate. Optimum pH, Km, Vmax and thermostability of the enzyme at 42 degrees C were determined. RESULTS: The leukocyte and fibroblast enzyme of heterozygotes have an optimum pH of 4.0 and 4.2, respectively. In normal subjects, the optimum pH was 4.2 in both cells, according to previous studies. The Km of the enzyme of heterozygotes was determined to be 0.65 mM in leukocytes and 0.59 mM in fibroblasts. The Vmax was determined in 167.21 nmol/h/mg of protein in heterozygotes leukocytes and 541.2 nmol/h/mg of protein in heterozygotes fibroblasts compared to 291.7 and 1768.1 nmol/h/mg of protein in controls leukocytes and fibroblasts, respectively. When leukocyte and fibroblast heterozygote beta-galactosidase was preincubated at 42 degrees C, after 80 min the residual activity was determined to be 25 to 30% of the initial activity. These results are similar to the control group. CONCLUSIONS: We have found significant differences between the two groups in some investigated parameters. Both fibroblasts and leukocytes showed a virtually similar level of reliability as source of enzyme for the detection of heterozygotes.

Cells, Cultured↗

PubMed2003

An analysis of replicative senescence in dermal fibroblasts derived from chronic leg wounds predicts that telomerase therapy would fail to reverse their disease-specific cellular and proteolytic phenotype.

The accumulation of senescent fibroblasts within tissues has been suggested to play an important role in mediating impaired dermal wound healing, which is a major clinical problem in the aged population. The concept that replicative senescence in wound fibroblasts results in reduced proliferation and the failure of refractory wounds to respond to treatment has therefore been proposed. However, in the chronic wounds of aged patients the precise relationship between the observed alteration in cellular responses with aging and replicative senescence remains to be determined. Using assays to assess cellular proliferation, senescence-associated staining beta-galactosidase, telomere length, and extracellular matrix reorganizational ability, chronic wound fibroblasts demonstrated no evidence of senescence. Furthermore, analysis of in vitro senesced fibroblasts demonstrated cellular responses that were distinct and, in many cases, diametrically opposed from those exhibited by chronic wound fibroblasts. Forced expression of telomerase within senescent fibroblasts reversed the senescent cellular phenotype, inhibiting extracellular matrix reorganizational ability, attachment, and matrix metalloproteinase production and thus produced cells with impaired key wound healing properties. It would appear therefore that the distinct phenotype of chronic wound fibroblasts is not simply due to the aging process, mediated through replicative senescence, but instead reflects disease-specific cellular alterations of the fibroblasts themselves.

Cell Adhesion↗

PubMed1998

Lack of insulin resistance in fibroblasts from subjects with polycystic ovary syndrome.

Insulin resistance in polycystic ovary syndrome (PCOS) is characterized by a novel defect in insulin signal transduction expressed in isolated human adipocytes as impaired insulin sensitivity for glucose transport and antilipolysis. To determine whether this is a generalized defect of a potentially genetic basis, or possibly a tissue-specific one, fibroblast cultures were established from age- and weight-matched obese normal cycling (NC; n = 5) and PCOS (n = 6) subjects. Adipocytes from the current PCOS subjects displayed impaired sensitivity for glucose transport stimulation (half-maximal effective concentration [EC50], 317 +/- 58 pmol/L in PCOS v 130 +/- 40 in NC; P < .025). Specific insulin binding was similar in fibroblasts from NC (0.57% +/- 0.10%/10(6) cells) and PCOS (0.45% +/- 0.10%) subjects. Fibroblasts from NC (4.9- +/- 0.5-fold stimulation) and PCOS (4.6- +/- 0.3-fold) subjects were equally responsive to insulin for stimulation of glucose incorporation into glycogen. Insulin sensitivity for glycogen synthesis in fibroblasts did not differ between NC (EC50, 9.6 +/- 0.9 nmol/L) and PCOS (9.1 +/- 0.9) cells. For thymidine incorporation into DNA, relative insulin responsiveness was similar in NC (2.3- +/- 0.3-fold stimulation) and PCOS (2.1- +/- 0.1-fold) fibroblasts. Insulin sensitivity for DNA synthesis was similar in NC (EC50, 12.9 +/- 2.4 nmol/L) and PCOS (7.6 +/- 1.3) cells. In summary, (1) insulin receptor binding is normal in PCOS fibroblasts; and (2) PCOS fibroblasts have normal insulin sensitivity and responsiveness for metabolic and mitogenic responses. Impaired insulin signal transduction, while present in adipocytes from a group of PCOS subjects, is not found in fibroblasts from the same subjects. This defect is not generalized to all cell types, but may be limited to specific tissues and responses.

Adipose Tissue↗

PubMed2003

Cell-matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan.

Normal human skin fibroblasts were grown in a three-dimensional collagen gel or in monolayer in the presence or absence of high molecular weight hyaluronan (HA) to assess the influence of extracellular HA on cell-matrix interactions. HA incorporated into the collagen gel or added to the culture medium did not modify lattice retraction with time. The effect was independent from HA molecular weight (from 7.5 x 10(5) to 2.7 x 10(6) Da) and concentration (from 0.1 up to 1 mg/ml). HA did not affect shape and distribution of fibroblasts within the gel, whereas it induced the actin filaments to organise into thicker cables running underneath the plasma membrane. The same phenomenon was observed in fibroblasts grown in monolayer. By contrast, vimentin cytoskeleton and cell-substrate focal adhesions were not modified by exogenous HA. The number of fibroblasts attached to HA-coated dishes was always significantly lower compared to plastic and to collagen type I-coated plates. By contrast, adhesion was not affected by soluble HA added to the medium nor by anti-CD44 and anti-RHAMM-IHABP polyclonals. After 24-h seeding on collagen type I or on plastic, cells were large and spread. Conversely, cells adherent to HA-coated surfaces were long, thin and aligned into rows; alcian blue showed that cells were attached to the plastic in between HA bundles. Therefore, normal human skin fibroblasts exhibit very scarce, if any, adhesion to matrix HA, either soluble or immobilised. Moreover, even at high concentration, HA molecules do not exert any visco-mechanical effect on lattice retraction and do not interfere with fibroblast-collagen interactions nor with focal adhesion contacts of fibroblasts with the substrate. This is probably relevant in organogenesis and wound repair. By contrast, HA greatly modifies the organisation of the actin cytoskeleton, suggesting that CD44-mediated signal transduction by HA may affect cell locomotion and orientation, as indicated by the fusiform shape of fibroblasts grown in the presence of immobilised HA. A role of HA in cell orientation could be relevant for the deposition of collagen fibrils in regeneration and tissue remodelling.

Actin Cytoskeleton↗

PubMed2001

Effects of silica on human lung fibroblast in culture.

Silica has been reported to directly stimulate cellular proliferation of human lung fibroblasts, and silica-treated macrophage supernatants induce fibroblast proliferation and some of their biosynthetic activities. Alveolar macrophages produce increased amount of tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta). Lung fibroblasts are producers of interleukin-6 (IL-6). We investigated the capacity of lung fibroblasts obtained from normal and silicosis subjects to elaborate IL-6 in response to TNF-alpha and to TGF-beta. Our data show that TNF-alpha and TGF-beta are able to stimulate the proliferation of human lung fibroblasts in culture, to increase the collagen production of the cells and are both able to increase IL-6 production by lung fibroblasts of patients with silicosis. We hypothesise that silica is able to stimulate lung fibroblast both directly, increasing the cell proliferation, and indirectly stimulating the release of factors (as TNF-alpha and TGF-beta) from activated alveolar macrophages, that are able to increase proliferative and biosynthetic activities of fibroblast.

Cell Division↗

PubMed2000

Nuclear transfer in cattle using colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells.

To assess the developmental potential of nuclear transfer embryos in cattle using mammary gland epithelial (MGE) cells derived from the colostrum, we compared the effectiveness of cloning using those cells and fibroblast cells derived from the ear. The fusion rate of the enucleated oocytes with fibroblast cells (75 +/- 4%) was significantly higher than that with MGE cells (56 +/- 7%, P<0.05). There were no significant differences in the cleavage rate (85 +/- 3% vs. 91+/- 2%) or in the developmental rate to the blastocyst stage (35 +/- 6% vs. 35 +/- 5%) using MGE cells vs. fibroblast cells as donor nuclei (P>0.05). After transfer of blastocysts derived from nuclear transfer embryos produced using MGE cells and fibroblast cells, 13% (4/31) and 16% (6/37) of recipient heifers were pregnant on Day 42 as assessed by ultrasonography, respectively. Two of the 4 and 4 of the 6 recipients of embryos with MGE cell- and fibroblast cell-derived nuclei, respectively, aborted within 150 days of pregnancy. Four live female calves were obtained from MGE cells or fibroblast cells. However, one died from internal hemorrhage of the arteria umbilicalis. The other three calves were normal and healthy. There were no differences in the pregnancy rate or calving rate when using MGE cells vs. fibroblast cells. Microsatellite DNA analyses confirmed that the cloned calves were genetically identical to the donor cows and different from the recipient heifers. We conclude that colostrum-derived MGE cells have the developmental potential to term by nuclear transfer, and the efficiency of development of those cloned embryos was the same as that of embryos obtained using fibroblast cells as donor nuclei, although there was a significant difference in the fusion rate. This method using MGE cells derived from colostrum, which is obtained easily and safely from live adult cows, is more advantageous for cloning with somatic cells.

Animals↗

PubMed1997

Effects of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro.

The effects of chitin and its derivatives on the proliferation of fibroblasts and on the production of cytokines were examined in vitro. Chitin and its derivatives showed almost no acceleratory effect on the proliferation of cultured fibroblasts. On the contrary, high-concentration 500 micrograms ml-1) D-glucosamine cultures supplemented with or without a 10% fetal calf serum (FCS) supplementation showed a significant (P < 0.05) reduction in the rate of proliferation of L929 fibroblast cells relative to control. High-concentration chitosan cultures supplemented with 10% FCS showed a significant (P < 0.05) reduction in the rate of L929 fibroblast proliferation. However, the inhibition of cell proliferation by high concentrations of chitosan did not show in cultures without FCS. Interleukin-8 (IL-8) was induced in the supernatants of rat primary cultured dermal fibroblasts stimulated with chitin and its derivatives. Chitin and its derivatives did not stimulate the production of IL-6 by mouse dermal primary cultured fibroblasts. IL-1 alpha, IL-1 beta and tumour necrosis factor-alpha were not detected in the fibroblast supernatants. These observations support the notion that cell proliferation is accelerated indirectly by chitin and its derivatives when these materials are used in vivo. In vivo findings of a angiogenesis and migration of neutrophils may be due to persistent release of IL-8 from fibroblasts.

Animals↗

PubMed1998

Antiproliferative and antifibrotic effects of mimosine on adult cardiac fibroblasts.

Prolyl 4-hydroxylase catalyzes the hydroxylation of collagen pro-alpha chains for the deposition of cardiac collagen. The effect of prolyl 4-hydroxylase on synthesis and degradation of collagen was studied in cultured adult cardiac fibroblasts using mimosine, a prolyl 4-hydroxylase inhibitor. Mimosine inhibited [3H]thymidine incorporation in cultured fibroblasts in a dose-dependent manner (100-600 microM). Immunofluorescence in fibroblasts and biochemical detection of mature type I collagen in culture serum revealed a strong inhibition of synthesis and secretion of mature collagens, respectively, in the presence of 200 microM mimosine. Western blot analysis for procollagen was carried out in cultured fibroblasts, and 200 microM mimosine treatment was associated with increased intracellular accumulation of procollagen from 4.14+/-0.27 to 10. 19+/-0.37 (arbitrary units). Immunofluorescence studies confirmed a marked increase of intracellular procollagens in fibroblasts treated with mimosine, which suggests a loss of coordinated monomeric procollagen synthesis and secretion of triple helical mature collagens. Modest inhibition of collagen type I mRNA abundance was observed in mimosine-treated fibroblasts, whereas no effect was noted for mRNAs of collagen type III, alpha-prolyl 4-hydroxylase or beta-prolyl 4-hydroxylase when compared to untreated control values. Treatment of fibroblasts with 200 microM mimosine was associated with elevation of matrix metalloproteinase (MMP)-9 activity. The cytotoxicity of mimosine treatment was found minimal at the concentrations indicated above. Thus the antifibrotic effects induced by mimosine on cultured adult cardiac fibroblasts was associated with inhibition of prolyl 4-hydroxylase and diminished extracellular secretion of procollagen, despite the reactive elevation of intracellular procollagen synthesis. We suggest that specific inhibition of prolyl 4-hydroxylase may provide a novel therapeutic approach for the modulation of cardiac fibrosis.

Animals↗

PubMed1989

Short-term keloid treatment in vivo with human interferon alfa-2b results in a selective and persistent normalization of keloidal fibroblast collagen, glycosaminoglycan, and collagenase production in vitro.

Two intralesional injections of interferon alfa-2b (1.5 million U per injection) into a progressively enlarging keloid resulted in a 41% reduction in its area. Fibroblasts cultured from the keloid before and 9 days after the initial injection were compared with fibroblasts cultured from the patient's normal skin with respect to proliferation and production of connective tissue matrix components and collagenase. There were no significant differences in the in vitro doubling times of keloidal fibroblasts before (p greater than 0.2) and after (p greater than 0.5) treatment with interferon alfa-2b, as well as of normal fibroblasts, in subconfluent cultures. Multiple passages of keloidal fibroblasts before interferon alfa-2b therapy assayed as confluent cultures produced more collagen (171%, 187%, and 204%), more glycosaminoglycans (153% and 141%), and less collagenase (26% and 31%) than the patient's own normal fibroblasts. In contrast, keloidal fibroblasts after interferon alfa-2b therapy persistently produced normal or subnormal amounts of collagen (107%, 73%, and 64%) and glycosaminoglycans (97% and 96%) and normalized levels of collagenase activity (96% and 86%). Normal amounts of fibronectin were produced by keloidal fibroblasts before and after treatment with interferon alfa-2b.

Cell Division↗

PubMed2001

Follistatin production by skin fibroblasts and its regulation by dexamethasone.

Activin and follistatin (FS) appear to play a role in the development of the skin and its appendages, in the inflammatory process, angiogenesis, and in wound healing. Although there is information on the expression of activin subunits and receptors in fibroblasts and keratinocytes, there are no reports on the regulation of FS expression in these cells. In the present study we analyzed the splicing variants of FS mRNAs in fibroblasts from genital and nongenital skin by RT-PCR and northern analysis, and examined the induction of FS mRNA and protein by hormones and growth factors in skin fibroblasts from human and nonhuman primates. FS mRNA was highly expressed in all fibroblast strains with similar expression regardless of donor species (human or monkey), donor age (neonate or adult), or the organ from which the fibroblast strains were established (skin or pituitary, genital or non-genital skin). Moreover, the band density corresponding to FS-288 was <5-10% of the value for FS-315 in skin fibroblasts as in all other tissues examined. Fibroblast FS mRNA and protein production were biphasically regulated by dexamethasone: low concentrations (0.01 and 0.1 nM) increased whereas higher concentrations (>1 nM) suppressed FS expression. On the other hand, androgens, activin and PACAP38 were without effect. These data establish cultured skin fibroblasts as a model to study FS gene expression in humans, and support a role for follistatin in the normal immune response and in the anti-inflammatory actions of glucocorticoids.

Adult↗

PubMed1997

Basic fibroblast growth factor-induced seizures in rats.

The neurotrophic effects of basic fibroblast growth factor on hippocampal neurons have been well documented. However, the acute effects of basic fibroblast growth factor on hippocampal electrical activities in vivo have not yet been studied. To assess the impact of basic fibroblast growth factor on hippocampal electroencephalographic (EEG) activity, two doses of basic fibroblast growth factor were injected into the right ventral hippocampus of freely moving animals. Unilateral injection of 50 ng of basic fibroblast growth factor into the dentate region of the hippocampus resulted in immediate EEG ictal discharges and behavioral seizures in 7/9 rats. These seizures lasted about 2 h following the injection. Injection of 25 ng of basic fibroblast growth factor into the same sites induced ictal discharges of approximately 60 min duration in 3/7 rats. Injection with heat-inactivated basic fibroblast growth factor into the same sites did not cause any EEG or behavioral modifications. These results suggest that in addition to growth-promoting effects, basic fibroblast growth factor is able to acutely alter the electrical activity of hippocampal neurons and cause seizures.

Animals↗

PubMed2000

The high molecular weight fibroblast growth factor-2 isoforms (21,000 mol. wt and 23,000 mol. wt) mediate neurotrophic activity on rat embryonic mesencephalic dopaminergic neurons in vitro.

Basic fibroblast growth factor is expressed in different isoforms which display tissue and species specificity and are differentially regulated during development and after experimental interventions. The differential regulation of the fibroblast growth factor-2 isoforms may indicate specific activities and functions of these molecules. The characterization of fibroblast growth factor-2 effects, however, is almost exclusively based on studies including the 18,000 mol. wt isoform. It is not yet known whether the high molecular weight fibroblast growth factor-2 isoforms (21,000 mol. wt, 23,000 mol. wt) exert similar or distinct activities in the nervous system. In the present study, we investigated the effects of the high molecular weight isoforms on dissociated rat mesencephalic dopaminergic neurons. For this purpose, recombinant fibroblast growth factor-2 isoforms, prepared in a histidine expression system, were administered on dopaminergic neurons in vitro, and Schwann cells over-expressing the high molecular weight isoforms were co-cultured with dopaminergic neurons. This is the first demonstration to show that the high molecular weight isoforms mediate a neurotrophic activity. Exogenous high molecular weight fibroblast growth factor-2 isoforms stimulated the survival of embryonic mesencephalic dopaminergic neurons and protected them from 6-hydroxydopamine neurotoxicity. In addition, co-culture of dopaminergic neurons with high molecular weight fibroblast growth factor-2 over-expressing Schwann cells revealed an increased survival and neurite formation of the mesencephalic dopaminergic neurons. These results suggest that the high molecular weight fibroblast growth factor-2 isoforms may serve as a new tool for the treatment of Parkinson's disease.

Animals↗

PubMed1999

Basic fibroblast growth factor in the primary olfactory pathway: mitogenic effect on ensheathing cells.

The mitogenic effect of basic fibroblast growth factor and nerve growth factor (2.5S) on olfactory ensheathing cell culture was examined by bromodeoxyuridine uptake. It was found that, at 10 ng/ml, basic fibroblast growth factor elicited about a three-fold increase in proliferation, while the stimulatory effect of nerve growth factor was considerably less. The increased proliferation resulting from basic fibroblast growth factor could be attributed to perlecan, which was shown to be expressed by ensheathing cell in culture. Perlecan is known to induce high-affinity binding of basic fibroblast growth factor to receptors on cell membranes. Immunohistochemical staining demonstrated that basic fibroblast growth factor was abundantly expressed in select regions of the lamina propria underlying the olfactory epithelium. In these regions, contiguous patches of olfactory epithelium also showed the presence of basic fibroblast growth factor. Although basic fibroblast growth factor was present on the periphery of nerve bundles in the olfactory nerve layer of the bulb, all other laminae did not demonstrate the presence of this factor. The immunohistochemistry and cell culture results show that regions of the lamina propria and small patches of the olfactory epithelium, by their presence of basic fibroblast growth factor, are potential sites of ensheathing cell proliferation in vivo.

Animals↗

PubMed1990

Alkaline phosphatase-positive and negative bone tumors. Ultrastructural and immunohistochemical studies of fibroblast-like tumor cells.

Bone tumors, which consist largely of fibroblast-like cells, were categorized into ALPase-positive (3 ossifying fibromas and 2 fibroblastic osteosarcomas) and negative (4 non-ossifying fibromas and 5 MFHs) groups. They were investigated as to their ultrastructure and immunophenotype using antibodies of fibroblast markers (collagen I, III, aminopeptidase M, dipeptidylpeptidase IV and factor XIIIa), classical macrophage markers (AACT and AAT) and vimentin. In the case of the ALPase-positive group, fibroblast-like cells showed short, branching rough endoplasmic reticulum with bundles of microfibrils in their cytoplasms. They were often intermingled with osteoblastic cells particularly in proximity to osteoid tissue. Furthermore, these cells expressed fibroblast markers of collagen I, aminopeptidase M, dipeptidylpeptidase IV and factor XIIIa. Fibroblast-like cells of the ALPase-negative group more or less revealed phagosomes in addition to fibroblastic features admixed with histiocyte-like cells. They expressed classical macrophage markers, but rarely fibroblast markers. The above findings indicated that derivation from different precursor cells should be proposed between the two groups and that the tumors in the ALPase-positive group might be intimately related to a certain population of the bone marrow stromal cells.

Alkaline Phosphatase↗

PubMed1996

Pharmacologic regulation of Dupuytren's fibroblast contraction in vitro.

Dupuytren's disease is associated with contraction of specialized fibroblasts present in the diseased palmar fascia. Pharmacologic agents were evaluated for their ability to promote or inhibit contraction of Dupuytren's fibroblasts in vitro using a collagen lattice contraction assay. In the first part of the study, lysophosphatidic acid (LPA), serotonin, angiotensin II, and prostaglandin F2 alpha were tested for their ability to promote Dupuytren's fibroblast contraction. Lysophosphatidic acid was found to significantly promote Dupuytren's fibroblast contraction as compared with controls. This response to LPA is dose dependent, with a half-maximal response of 0.07 microM. Angiotensin II, serotonin, and prostaglandin F2 alpha at 1 mM, induced a significant amount of contraction as compared to controls, but the amount of contraction was at least six times less than that observed for LPA. In the second part of the study, prostaglandins E1 and E2 or the calcium blockers nifedipine and verapamil were tested for their ability to inhibit LPA-promoted contraction. It was found that both types of inhibitors partially block LPA-promoted contraction of Dupuytren's fibroblasts. The effect of the various pharmacologic agents on normal palmar fibroblasts was not evaluated. The focus of this study was to examine the regulation of contraction of Dupuytren's fibroblasts. This study demonstrates that LPA is a potent agonist of Dupuytren's fibroblast contraction and that this contraction can be inhibited by specific pharmacologic agents. These findings provide a rational basis for investigating further the clinical use of the calcium channel blockers nifedipine or verapamil and prostaglandins E1 and E2 to control Dupuytren's disease and possibly other fibrotic conditions.

Alprostadil↗

PubMed2001

Differential vulnerability to oxidative stress in rat cardiac myocytes versus fibroblasts.

OBJECTIVES: This study was designed to test the hypothesis that cardiac myocytes have greater vulnerability to oxidative stress compared with cardiac fibroblasts. BACKGROUND: The function of cardiac myocytes differs from that of fibroblasts in the heart, but differences in their response to oxidative stress have not been extensively studied. METHODS: Cardiomyocytes and fibroblasts from F344 neonatal rat hearts were cultured and exposed to different concentrations of hydrogen peroxide (H(2)O(2)) and menadione (superoxide generator). The mitogen-activated protein kinase (MAPK) proteins were assayed after oxidative stress; cell death was determined by trypan blue staining and deoxyribonucleic acid (DNA) ladder electrophoresis. RESULTS: The cardiac myocytes were significantly more vulnerable than the fibroblasts to oxidative damage, showing substantial DNA fragmentation and consistently poor cell survival after exposure to H(2)O(2) (100 to 800 microM), while the cardiac fibroblasts demonstrated little or no DNA fragmentation, and superior cell survival rates both over time (from 1 to 72 h after 100 microM) and across increasing doses of H(2)O(2) (100 to 800 microM). The p42/44 extracellular signal-regulated kinases were phosphorylated in both cell types after exposure to H(2)O(2), but significantly more in cardiac fibroblasts. However, p38 MAPK and c-jun NH(2)-terminal kinase were phosphorylated more in the cardiac myocytes compared to cardiac fibroblasts. This was also the case after exposure to menadione. CONCLUSION: Taken together, these results suggest that oxidative stress causes greater injury and cell death in cardiac myocytes compared with cardiac fibroblasts. It is possible that the signaling differences via the MAPK family may partly mediate the observed differences in vulnerability and functional outcomes of the respective cell types.

Animals↗

PubMed2001

Fibroblast expression of C-C chemokines in response to orthopaedic biomaterial particle challenge in vitro.

C-C chemokines are soluble mediators that occur in a periprosthetic granuloma and influence recruitment, localization and activation of inflammatory cells. This study tested effects of titanium and polymethylmethacrylate (PMMA) particles on expression of selected C-C chemokines in cultured human fibroblasts. The C-C chemokines analyzed included monocyte chemoattractant protein-1. 2 (MCP-1. 2), monocyte inflammatory protein-1 alpha (MIP-1 alpha), and regulated on activation, normal T-cell expressed and secreted protein (RANTES). Interleukin-1 beta (IL-1 beta) served as a known stimulator of chemokine release while interleukin-6 (IL-6) expression served as a marker for fibroblast activation. Protein and mRNA signal levels were determined by ELISA and RT-PCR, respectively. The results demonstrated that exposure of fibroblasts to titanium and PMMA particles resulted in increased release of MCP-1 in a dose- and time-dependent manner. After 24 h, titanium particles maximally upregulated MCP-1 release 7-fold while PMMA particles increased MCP-1 levels 2-fold, when compared to unchallenged fibroblasts. MCP-2, MIP-1 alpha and RANTES levels remained unchanged following exposure of fibroblasts to titanium or PMMA particles at any concentration or time point tested. However, IL-1 beta stimulated release of MCP-1, MCP-2, and RANTES, but not MIP-1 alpha from the fibroblasts. IL-1 beta, not particles, exhibited the most prominent effect on MCP-1 mRNA levels. Increased release of MCP-1 from fibroblasts exposed to titanium and PMMA particles coincided with increased release of IL-6. This study suggests that release of chemoattractant factors from fibroblasts localized in periprosthetic membranes enhances the chronic inflammatory process leading to bone resorption and implant loosening.

Bone Cements↗