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PubMed2000

Proliferative activity of intratumoral fibroblasts is closely correlated with lymph node and distant organ metastases of invasive ductal carcinoma of the breast.

Mitotic figures of fibroblasts are seen within invasive ductal carcinoma (IDC) of the breast. This suggests that the proliferative activity of fibroblasts may play an important role in IDC tumor progression. The purpose of this study was to examine whether the proliferative activity of fibroblasts can predict lymph node metastasis (LNM) or distant-organ metastasis (DOM) of IDCs. Two hundred four consecutive patients with IDC of the breast surgically treated at the National Cancer Center Hospital East constituted the basis of this study. Proliferative activity of fibroblasts was immunohistochemically evaluated by the mouse MIB-1 monoclonal antibody against Ki-67 antigen. The MIB-1 labeling index was the percentage of fibroblasts with positively stained nuclei, and fields for cell counting were selected in inner and outer areas within IDCs. In both areas, 300 fibroblasts were counted in each high-power field. The significance of proliferative activity of fibroblasts on LNM or DOM was compared with well-known prognostic parameters. Multivariate analyses demonstrated that a MIB-1 labeling index of more than 10% of fibroblasts in the inner area of IDCs significantly increased the relative risk of LNM and hazard rate of DOM (P < 0.001 and P = 0.007, respectively). The present study indicated that the metastatic ability of IDCs is closely dependent on proliferative activity of fibroblasts in the inner area.

Adult↗

PubMed1999

Dermal fibroblast proliferation is improved by beta-catenin overexpression and inhibited by E-cadherin expression.

Several recent studies have shown that proteins of the cadherin-catenin complex are not only involved in cell-cell adhesion but also in the proliferation and differentiation processes. For the first time, we investigated the effect of the quantity of cytoplasmic beta-catenin on dermal fibroblast proliferation by overexpressing human beta-catenin in human dermal fibroblasts. Our results show that dermal fibroblasts overexpressing normal beta-catenin or a stabilized beta-catenin mutant have a higher growth rate than control fibroblasts. Moreover, when confluence is reached, the number of fibroblasts is increased when the cells overexpress beta-catenin suggesting a role for beta-catenin in the regulation of contact growth arrest. Finally, by comparing proliferation in normal dermal fibroblasts and dermal fibroblasts expressing E-cadherin we observed a negative regulatory effect of E-cadherin expression on fibroblast proliferation. These data demonstrate the involvement of beta-catenin and cadherin in the dermal fibroblast proliferation process and in contact growth arrest.

Animals↗

PubMed1999

Regulation of ICAM-1-mediated fibroblast-T cell reciprocal interaction: implications for modulation of gut inflammation.

BACKGROUND & AIMS: Immune-nonimmune cell interactions modulate mucosal immunity. We investigated the expression of adhesion molecules by intestinal fibroblasts, the effect of immune cell-derived factor on fibroblast binding of T cells, and the consequences of interfering with adhesion molecule expression on fibroblast-T cell interaction. METHODS: Expression of fibroblast intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 surface and messenger RNA (mRNA) was measured before and after exposure to immune cell-derived supernatants. Fibroblasts were treated with antibodies to ICAM-1 or VCAM-1, or ICAM-1 antisense oligonucleotide Isis 2302, before a T-cell adhesion assay. RESULTS: Fibroblast activation by immune cell-derived cytokines enhanced ICAM-1 and VCAM-1 surface expression and mRNA as well as adhesiveness for T cells. Blockade with neutralizing antibodies showed that binding was almost exclusively dependent on ICAM-1. Isis 2302 specifically reduced fibroblast ICAM-1 mRNA and dose-dependently inhibited ICAM-1 surface expression and T-cell binding. CONCLUSIONS: ICAM-1 is essential for intestinal fibroblast binding of T cells, a phenomenon that is efficiently and specifically disrupted by ICAM-1 antisense oligonucleotides. These observations emphasize the crucial regulatory role of fibroblasts in mucosal immunity and their potential as targets for therapeutic intervention in intestinal inflammation.

Blotting, Northern↗

PubMed1997

Periodontal pathogen-related stimulation indicates unique phenotype of primary cultured human fibroblasts from gingiva and periodontal ligament: implications for oral health disease.

STATEMENT OF PROBLEM: The fibroblast is considered an important cellular component in periodontitis because it is the predominant cell type in periodontal connective tissue. PURPOSE: The purpose of this study was to test whether gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are heterogeneous in their production of inflammatory mediators associated with bone resorption in response to lipopolysaccharides from the gram-negative bacterium Porphyromonas gingivalis. To test this hypothesis, we (1) compared interleukin-6 production by cultured human PDLF and GF isolated from the same individual when exposed to P. gingivalis lipopolysaccharide; and (2) compared prostaglandin-E2 production by cultured human PDLF and GF isolated from the same individual when exposed to P. gingivalis lipopolysaccharide. MATERIAL AND METHODS: Human periodontal ligament and gingival fibroblasts were cultured from biopsies and exposed to P. gingivalis lipopolysaccharide. Levels of interleukin-6 and prostaglandin-E2 secreted into culture supernatant were measured by enzyme-linked immunosorbent assay and the data analyzed. Gingival fibroblasts secreted similar interleukin-6 and elevated prostaglandin-E2 levels compared with unstimulated cells. Periodontal ligament fibroblasts secreted elevated levels of both mediators compared with unstimulated cells. Secreted levels of interleukin-6 and prostaglandin-E2 by primary gingival and periodontal ligament fibroblast cultures appear to differ when stimulated with a periodontal pathogen-related virulence factor. CONCLUSION: The fibroblast of the periodontal ligament and the fibroblast of the gingiva may represent different phenotypes that play unique roles in tissue responses to implants.

Analysis of Variance↗

PubMed1995

In situ hybridization: a new technique to determine the origin of fibroblasts in cryopreserved aortic homograft valve explants.

Tissue degeneration reduces the durability of cryopreserved homografts. Earlier studies indicated that the presence of fibroblasts in homograft leaflets may contribute to increased valve longevity. These fibroblasts may be of recipient origin or represent surviving donor cells. We developed a method, based on in situ hybridization, to determine the origin of fibroblasts in homograft explants. In young pigs we performed aortic valve replacement with a cryopreserved porcine aortic homograft. A male homograft was implanted in a female pig, whereas two male recipients received a female homograft. After 3 to 4 months the homografts were explanted. Frozen sections were made and alternately examined with hematoxylin-eosin staining and in situ hybridization. With a biotinylated porcine Y chromosome-specific deoxyribonucleic acid probe, male fibroblasts could be clearly distinguished from female fibroblasts. In all leaflets we observed both donor and recipient fibroblasts. The distribution of these populations was marked in schematic drawings. Recipient fibroblasts mostly spread onto the leaflet surface but also penetrated the leaflet tissue. Remaining donor fibroblasts did not show morphologic signs of decreased viability on hematoxylin-eosin staining. In situ hybridization may become a useful technique in homograft research. In this porcine model, the fibroblasts in the aortic homograft explants were of both donor and recipient origin.

Animals↗

PubMed1997

Expression of acidic fibroblast growth factor in Barrett's esophagus and associated esophageal adenocarcinoma.

OBJECTIVE: Adenocarcinoma of the esophagus is generally attributed to the neoplastic transformation of intestinal metaplastic lesions (Barrett's esophagus). On the basis of our preliminary data that showed significant acidic fibroblast growth factor mRNA and protein expression in adenocarcinoma of the esophagus, we studied expression of fibroblast growth factor in esophageal adenocarcinoma and its precursor lesions, intestinal metaplasia, low-grade dysplasia, and high-grade dysplasia. Fibroblast growth factor belongs to a family of polypeptides that are involved in differentiation and cellular proliferation. METHODS: We examined 30 esophagectomy specimens that were resected for adenocarcinoma (n = 27) and high-grade dysplasia (n = 3). After confirmation of the diagnosis by routine light microscopy, the index lesions (invasive carcinomas) and adjoining Barrett's mucosa were evaluated with a monoclonal antibody against human acidic fibroblast growth factor. The results are expressed with the use of an immunoreactive score that allows distinction between weak, moderate, and strong immunoreactivity. RESULTS: Adenocarcinoma demonstrated a moderate-to-strong mean immunoreactive score of 8. In contrast, high-grade dysplasia demonstrated a weak-to-moderate mean score of 4.5, which was significantly different (p < 0.05). Intestinal metaplasia and low-grade dysplasia displayed even weaker expression of fibroblast growth factor, with a negligible immunoreactive score less than 1 (p < 0.005). Seventy-five percent of evaluable cases demonstrated an increasing degree of fibroblast growth factor expression in the spectrum of lesions ranging from metaplasia to dysplasia and carcinoma. CONCLUSIONS: These data indicate that in patients with adenocarcinoma arising in association with Barrett's esophagus, fibroblast growth factor is generally sequentially accumulated in the progression from metaplasia to neoplasia. This progression may affect future investigation into the role of fibroblast growth factors in tumorigenesis and, possibly, the application of fibroblast growth factor immunohistochemistry to diagnosis.

Adenocarcinoma↗

PubMed1992

Prostate and bone fibroblasts induce human prostate cancer growth in vivo: implications for bidirectional tumor-stromal cell interaction in prostate carcinoma growth and metastasis.

Prostate cancer selectively metastasizes to the axial skeleton to produce osteoblastic lesions, which suggests that bidirectional paracrine interactions exist between prostate cancer and bone cells. To evaluate the role of tumor-stromal cell interaction and stromal-specific growth factors in prostate cancer growth and dissemination, we coinoculated nontumorigenic human prostate cancer cells (LNCaP) and various tissue-specific fibroblasts subcutaneously in athymic mice. LNCaP tumors were induced most consistently by human bone fibroblasts (62%), followed by two prostate fibroblast cell lines (31% and 17%), but not by lung, kidney, or embryonic 3T3 fibroblasts. Carcinomas formed preferentially in male hosts, demonstrating in vivo androgen sensitivity. Immunohistochemical and biochemical techniques confirmed the human prostate component of these tumors and were paralleled by elevations in serum prostate specific antigen. In vitro mitogenic assays revealed a two-to three-fold bidirectional stimulation between LNCaP and bone or prostate fibroblast conditioned media, but not lung, kidney, or 3T3 fibroblast conditioned media. A novel method developed to deliver concentrated bone or prostate fibroblast conditioned media in vivo using a slowly absorbed matrix (gelfoam) also induced tumor formation, emphasizing the importance of fibroblast growth factors in LNCaP tumor formation. Northern analysis identified the stromal compartment as the primary source of extracellular matrix (collagen, fibronectin), while only LNCaP cells expressed transforming growth factor alpha. Although LNCaP and stromal cells express basic fibroblast growth factor (bFGF), the bidirectional paracrine-mediated mitogenic activity between these cells is not inhibited by anti-bFGF antibodies, suggesting that other undefined growth factors may be involved in stimulating LNCaP growth. These observations illustrate the importance of stromal-epithelial interaction in prostate tumor growth and suggest that extracellular matrix and paracrine-mediated growth factors play a role in prostate cancer growth and metastasis.

Androgens↗

PubMed1997

Promotion of mouse fibroblast proliferation by IgE-dependent activation of mouse mast cells: role for mast cell tumor necrosis factor-alpha and transforming growth factor-beta 1.

Pathologic fibroblast proliferation or tissue fibrosis develops in certain chronic allergic diseases and in a wide array of other inflammatory disorders in which mast cell activation is also a prominent feature. In this study we investigated a number of potential mechanisms by which IgE-dependent activation of mouse mast cells might influence the proliferation of mouse fibroblasts in vitro. We found that supernatants from in vitro-derived mast cells that had been activated by IgE and specific antigen (but not those from quiescent mast cells) promoted the proliferation of mouse embryonic skin or 3T3 fibroblasts, and we showed that this effect was detectable in the absence of fetal calf serum. We analyzed the kinetics with which the fibroblast-proliferative activity was secreted from bone marrow-derived cultured mast cells and found that it was released both rapidly (i.e., in 30 minutes or less) and for a more prolonged period (i.e., for more than 2 hours) after IgE-dependent mast cell activation. We then measured the levels at which the mast cells produce a number of cytokines that are known to affect fibroblasts (IL-1, IL-6, transforming growth factor-beta 1 [TGF-beta 1], and tumor necrosis factor-alpha [TNF-alpha]) and assessed their relative effects, as recombinant cytokines, on fibroblast proliferation. Our mast cells secreted high levels of TGF-beta 1 and TNF-alpha, intermediate amounts of IL-6, and low levels of IL-1. We titrated the fibroproliferative effects of each of these cytokines and determined that at a dose of 50 pg/ml their rank order of activity was TGF-beta 1 > TNF-alpha > IL-1 > IL-6, with all but IL-6 having significant effects. The ability of supernatants from activated bone marrow-derived cultured mast cells to promote fibroblast proliferation was partially diminished by absorption with neutralizing antibodies against either TNF-alpha or TGF-beta 1, and absorption of the supernatants with a combination of antibodies against TNF-alpha and TGF-beta 1 reduced their ability to induce fibroblast proliferation by approximately 50% (p < or = 0.001, n = 5). These findings show that IgE-dependent activation of mouse mast cells can result in the release of mediators that promote fibroblast proliferation in the absence of any other cell type and suggest that mast cell-derived TNF-alpha and TGF-beta 1 contribute substantially to this effect. They also suggest that these cytokines exert their effects through synergistic interactions with other mast cell mediators.

Animals↗

PubMed1994

Microvessel count and cerebrospinal fluid basic fibroblast growth factor in children with brain tumours.

Tumour growth is angiogenesis-dependent; brain tumours have more intense neovascularisation than other tumours and produce basic fibroblast growth factor, a potent angiogenic mediator. Because little is known about the release of basic fibroblast growth factor from brain tumours into extracellular fluids, we tested cerebrospinal fluid (CSF) from 26 children and young adults with brain tumours and 18 controls for basic fibroblast growth factor and for proliferative activity on cultured capillary endothelial cells. We also measured the density of microvessels in tumours by immunohistochemical staining. Basic fibroblast growth factor was detected in the CSF of 62% (16 of 26) patients with brain tumours but in none of the controls. Specimens with basic fibroblast growth factor stimulated DNA synthesis of capillary endothelial cells in vitro. Endothelial proliferative activity was blocked by neutralising antibodies to basic fibroblast growth factor. Basic fibroblast growth factor correlated with mitogenic activity in CSF in vitro (p < or = 0.0001), and with density of microvessels in histological sections (p < or = 0.005). A microvessel count of > or = 68 per 200 x field was associated with tumour recurrence (p = 0.005) and with mortality (p = 0.02). Basic fibroblast growth factor in brain tumours may mediate angiogenesis as measured by microvessel density in histological sections, so has potential as both a marker for neoplasia and a target for tumour treatments. Furthermore, evaluation of cerebrospinal fluid basic fibroblast growth factor, along with microvessel quantitation in biopsied tumours, may provide improved prognostic information for the management of patients with brain tumours.

Adolescent↗

PubMed1984

Human gingival fibroblast collagenase: purification and properties of precursor and active forms.

Human gingival fibroblast procollagenase has been purified to apparent homogeneity from serum-free and serum-supplemented fibroblast culture medium by a combination of ammonium sulfate precipitation, CM-cellulose chromatography, and gel-filtration on Bio-Gel P-150. Sodium dodecylsulfate polyacrylamide gel electrophoretic studies suggests that the purified fibroblast proenzyme is comprised of two closely related zymogens with the estimated Mr of 57,000 and 52,000. Upon densitometric scanning of the gels, the ratio of the two proenzyme forms was about 1 : 4 (57 : 52 kdal). Limited proteolysis of the fibroblast procollagenase with trypsin resulted in the conversion of both proenzyme forms into active enzyme forms of Mr 48,000 and 44,000, respectively. Amino acid analysis of the active enzymes and proenzyme forms revealed that the active enzymes contained fewer basic amino acids than do the proenzyme forms. The purified trypsin-activated fibroblast collagenase hydrolyzed type I collagen fibrils, cleaved tropocollagen in solution at 24 degrees C into TCA and TCB fragments, and cleaved the synthetic peptide substrate, DNP-peptide III, at the Gly-Ile bond. The gingival fibroblast collagenase exhibited a pH optimum of 7.5, was completely inhibited by EDTA or dithioerythritol but was not inhibited by N-ethylmaleimide or phenylmethylsulfonyl fluoride, and appeared to cleave human type III collagen approximately 10-fold faster than homologous type I collagen. In addition, comparison of the biochemical properties of the precursor and active forms of human gingival fibroblast collagenase with the precursor and active forms of human skin fibroblast collagenase, previously characterized by Stricklin and co-workers (Biochemistry 17: 2331-2337, 1978), revealed that they were similar in Mr, amino acid composition, and substrate specificity. Furthermore, the human gingival and skin fibroblast procollagenases were immunologically identical.

Amino Acids↗

PubMed1987

Comparison of virulent trypomastigotes with low-virulent ones derived from a single strain of Trypanosoma cruzi: infectivity to new fibroblasts from various organs of the mouse.

Previous experiments have shown that the infectivity of virulent trypomastigotes in L-cell culture was much more intensive not only for macrophages but also for newly cultured fibroblasts from the mouse heart as compared to low-virulent trypomastigotes in L-cell culture which had been derived from the same strain. On the other hand, the infectivity of virulent trypomastigotes for L-cells was less intensive. In the present work, new fibroblasts derived from muscles, skin, heart and kidney of newborn mice and established fibroblasts from mouse muscles were examined in terms of their susceptibility to trypomastigotes of different virulence. A slight difference of susceptibility to virulent trypomastigotes among new fibroblasts from different sources was found, i.e. fibroblasts derived from muscles and skin were more susceptible than those from heart and kidney. Growth of low-virulent trypomastigotes was suppressed in all new fibroblasts. This suppression was explained by the result that amastigote proliferation of a low-virulent strain was much slower in new fibroblasts from mouse muscles in spite of a similar penetrative ability of both kinds of trypomastigotes. Furthermore, a slight decrease in the susceptibility of new fibroblasts to virulent trypomastigotes was found depending on the length of time fibroblasts had been maintained.

Animals↗

PubMed2000

Changes in astrocytic basic fibroblast growth factor expression during and after prolonged exposure to escalating doses of amphetamine.

We have shown that brief exposure to amphetamine leads to sustained glutamate-dependent increases in expression of the neurotrophic, neuroprotective factor, basic fibroblast growth factor, in astrocytes in dopaminergic cell body regions and that blockade of basic fibroblast growth factor in this region prevents the development of behavioral sensitization to amphetamine. Here we examine the effects of prolonged exposure to an escalating-dose regimen of amphetamine known to induce long-lasting sensitization to amphetamine and leading to increases in neuronal dendritic length and spine density in nucleus accumbens and prefrontal cortex and to decreases in spine density in occipital cortex. Astrocytic basic fibroblast growth factor immunoreactivity was increased in both dopaminergic cell body and terminal regions one week after termination of a two-week amphetamine treatment (1-4mg/kg). These effects were not evident one week after a five-week treatment (1-9mg/kg) and, in fact, one month later basic fibroblast growth factor levels in cell body regions were decreased. In the occipital cortex, basic fibroblast growth factor immunoreactivity was decreased one week after the two-week amphetamine treatment, but was not different from that seen in saline-treated animals after the five-week treatment. Increased astrocytic basic fibroblast growth factor expression appears to be an early, but relatively prolonged, response to amphetamine exposure and seems to parallel structural changes induced by repeated drug exposure.These findings suggest that basic fibroblast growth factor may participate in the development of structural changes brought about by amphetamine. The fact that the basic fibroblast growth factor response is not maintained after prolonged intense exposure to amphetamine suggests that the factors that initially induce basic fibroblast growth factor expression are self-regulating.

Amphetamine↗

PubMed1997

A correlation between residual DNA double-strand breaks and clonogenic measurements of radiosensitivity in fibroblasts from preradiotherapy cervix cancer patients.

PURPOSE: To study the relationship between residual DNA damage and clonogenic measurements of radiosensitivity in fibroblasts from pretreatment cervix cancer patients. METHODS AND MATERIALS: Early passage vaginal fibroblasts from nine preradiotherapy cervix cancer patients and two radiosensitive skin fibroblast cell strains were studied. Cell survival was measured by clonogenic assay following both high and low dose rate irradiation. Residual DNA damage was measured using pulsed-field gel electrophoresis (PFGE) after irradiating radiolabeled, plateau-phase cells at 37 degrees C and allowing 24 h for repair. DNA damage was expressed both in terms of the residual damage slope (fitted to data from 60 to 150 Gy) and the fraction of activity released (FAR) following 150 Gy. RESULTS: The surviving fraction at 2 Gy (SF2) values after high dose rate irradiation for the vaginal fibroblasts ranged from 0.15 to 0.32 (a 2.2-fold difference). When the two radiosensitive cell strains were included, residual damage, expressed as the residual damage slope, correlated with alpha (r = 0.82, p = 0.002), D bar (r = -0.91, p < 0.001) and SF2 (p = -0.79, p = 0.004), and when the vaginal fibroblasts alone were studied, the residual damage slope again correlated with clonogenic survival, although less strongly [alpha (r = 0.66, p = 0.053), D bar (r = -0.83, p = 0.006), and SF2 (r = -0.63, p = 0.07)]. Within the group of vaginal fibroblasts there was a 4.0-fold difference in residual DNA damage slope. When residual damage was expressed as FAR at 150 Gy, then for all cell strains the correlations were alpha: r = 0.78, p = 0.004, D bar: r = -0.86, p = 0.001, and SF2: r = -0.78, p = 0.004, and for the vaginal fibroblast strains alone the correlations were alpha: r = 0.60, p = 0.088, D bar: r = -0.75, p = 0.02, and SF2: r = 0.62, p = 0.077. CONCLUSION: This study confirms previous findings that residual DNA damage correlates with clonogenic survival in fibroblasts. In addition, it demonstrates a correlation for fibroblasts from pretreatment cervix cancer patients demonstrating a relatively small range of SF2 values.

Cell Survival↗

PubMed1997

Possible monocytic origin of chondrosarcoma: in vitro transdifferentiation of HLA-DR blood monocyte-like cells from a patient with chondrosarcoma, into neo-fibroblasts and chondrocyte-like cells.

Nodules and multilayered areas composed of fibroblasts and chondrocyte-like cells embedded in an abundant extracellular matrix appeared spontaneously in in vitro culture of mononucleated blood cells taken from a patient with chondrosarcoma. Using specific antibodies it was demonstrated that the neo-fibroblasts which developed in the culture resulted from a direct transdifferentiation of monocytes expressing HLA-DR specificity. The experiment was carried out twice, once before surgery and then two years later. In both cases the spontaneous transdifferentiation of HLA-DR monocytes into neo-fibroblasts was observed. Previously it was shown that normal monocytes were also able to give rise in vitro to neo-fibroblasts. However, the latter are normally rapidly destroyed by cell-cell contact with T-cells. Normal T-cells adhere to normal neo-fibroblasts by which they are finally engulfed. As a result, the neo-fibroblasts lose their fibroblastic shape, no longer adhere to their support and die. Therefore the abnormal proliferation and persistence of neo-fibroblasts in pathological situations such as the present case may result either from an intrinsic defect in monocytes, T-cells or both. The question is whether or not this transdifferentiation process observed in vitro accounts for the development of chondrosarcoma in vivo. The present results suggest that in vivo chondrosarcoma may start in a necrotic zone (resulting for instance from trauma) and attract HLA-DR monocytes, where they accumulate and transdifferentiate into neo-fibroblasts and chondrocyte-like cells. The uncontrolled transdifferentiation of these HLA-DR monocytes resulting from a dysregulation of the immune system is probably linked to the malignant process which may have a retroviral origin. The question is raised regarding the embryologic origin of this special sub-population of blood monocytes in which pluripotential capabilities are retained; its origin may differ from that of the other circulating monocytes.

Cartilage↗

PubMed2001

Reactive oxygen and nitrogen metabolites modulate fibronectin-induced fibroblast migration in vitro.

Nitration of proteins by peroxynitrite may alter protein function. We hypothesized that reactive nitrogen species modulate fibronectin-induced fibroblast migration. To test this hypothesis, we evaluated fibroblast migration induced by fibronectin incubated with and without peroxynitrite. Peroxynitrite attenuated fibronectin-induced fibroblast migration in a dose-dependent manner but did not attenuate complement-activated serum-induced fibroblast migration. The reducing agents, deferoxamine and dithiothreitol (DTT), and L-tyrosine reversed the inhibition by peroxynitrite. PAPA-NONOate, a nitric oxide (NO) donor, and superoxide generated by the action of xanthine oxidase on lumazine or xanthine, also showed an inhibitory effect on fibroblast migration. The peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), caused a concentration-dependent inhibition of fibroblast migration. Peroxynitrite reduced fibronectin binding to fibroblasts and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of fibronectin binding to fibroblasts and suggest that peroxynitrite may play a role in regulation of fibroblast migration.

Blood↗

PubMed1997

Regulations of collagen synthesis by ascorbic acid, transforming growth factor-beta and interferon-gamma in human dermal fibroblasts cultured in three-dimensional collagen gel are photoaging- and aging-independent.

Decreased collagen synthesis and loss of responsiveness to growth factors are well known phenomena in in vivo or in vitro aged cells. Ascorbic acid and some cytokines such as transforming growth factor-beta and interferon-gamma are important regulators of collagen synthesis. To investigate the responsiveness of fibroblasts with regard to the photoaging and aging process, we examined the effect of ascorbic acid, TGF-beta, and IFN-gamma on collagen synthesis in dermal fibroblasts from three newborn foreskins (1 day old) and in both exposed and unexposed skin fibroblasts from 4 old individuals (60-76 years old) cultured in monolayer and in collagen gel. We demonstrated that basal levels of collagen synthesis decreased with increasing age. Photoaged fibroblasts in collagen gel showed greater basal collagen synthesis than aged fibroblasts in the same individuals, but similar basal collagen synthesis in monolayer cultures. Even though basal levels of collagen synthesis in collagen gel are downregulated in a photoaging- and aging-dependent manner, collagen synthesis by ascorbic acid in collagen gel, and by TGF-beta and IFN-gamma in both monolayer culture and collagen gel were regulated in a photoaging- and aging-independent manner. In monolayer culture, however, the responsiveness to ascorbic acid in newborn fibroblasts was greater than in photoaged and aged fibroblasts. Our results suggest that there are differences in collagen synthesis between photoaged and aged cells, depending on culture conditions. Responsiveness to ascorbic acid, TGF-beta and IFN-gamma related to collagen synthesis in photoaged and aged fibroblasts in collagen gel appears to be the same as in newborn fibroblasts, even though basal levels of collagen synthesis are downregulated in a photoaging- or aging-dependent manner.

Aged↗

PubMed2005

Microstructured cocultures of cardiac myocytes and fibroblasts: a two-dimensional in vitro model of cardiac tissue.

Cardiac myocytes and fibroblasts are essential elements of myocardial tissue structure and function. In vivo, myocytes constitute the majority of cardiac tissue volume, whereas fibroblasts dominate in numbers. In vitro, cardiac cell cultures are usually designed to exclude fibroblasts, which, because of their maintained proliferative potential, tend to overgrow the myocytes. Recent advances in microstructuring of cultures and cell growth on elastic membranes have greatly enhanced in vitro preservation of tissue properties and offer a novel platform technology for producing more in vivo-like models of myocardium. We used microfluidic techniques to grow two-dimensional structured cardiac tissue models, containing both myocytes and fibroblasts, and characterized cell morphology, distribution, and coupling using immunohistochemical techniques. In vitro findings were compared with in vivo ventricular cyto-architecture. Cardiac myocytes and fibroblasts, cultured on intersecting 30-microm-wide collagen tracks, acquire an in vivo-like phenotype. Their spatial arrangement closely resembles that observed in native tissue: Strands of highly aligned myocytes are surrounded by parallel threads of fibroblasts. In this in vitro system, fibroblasts form contacts with other fibroblasts and myocytes, which can support homogeneous and heterogeneous gap junctional coupling, as observed in vivo. We conclude that structured cocultures of cardiomyocytes and fibroblasts mimic in vivo ventricular tissue organization and provide a novel tool for in vitro research into cardiac electromechanical function.

Animals↗

PubMed2000

Cyclic stretch induces the release of growth promoting factors from cultured neonatal cardiomyocytes and cardiac fibroblasts.

Growth factors and hormones may play an autocrine/paracrine role in mechanical stress-induced cardiac hypertrophy. Using an in vitro model of mechanical stress, i.e. stretch of cardiomyocytes and cardiac fibroblasts, we tested the involvement of growth factors and hormones in this process. We found that conditioned medium (CM) derived from 4 h cyclicly (1 Hz) stretched cardiomyocytes increased the rate of protein synthesis in static cardiomyocytes by 8 +/- 3%. Moreover, CM derived from 2 h stretched fibroblasts increased the rate of protein synthesis in static fibroblasts as well as in static cardiomyocytes by 8 +/- 2 and 6 +/- 2%, respectively. Analysis of CM using size-exclusion HPLC showed that cardiomyocytes and fibroblasts released at least three factors with MW < or = 10 kD, their quantities being time-dependently increased by stretch. Subsequent analyses using immunoassays revealed that cardiomyocytes released atrial natriuretic peptide (ANP) and transforming growth factor-beta1 (TGFbeta1) being increased by 45 +/- 17 and 21 +/- 4% upon 4 h of stretch, respectively. Fibroblasts released TGFbeta1 and very low quantity of endothelin-1 (ET-1). The release of TGFbeta1 was significantly increased by 18 +/- 4% after 24 h of stretch in fibroblasts. Both cell types released no detectable amount of angiotensin II (Ang II). In conclusion, upon cyclic stretch cardiomyocytes and fibroblasts secrete growth factors and hormones which induce growth responses in cardiomyocytes and fibroblasts in an autocrine/paracrine way. TGFbeta secreted by cardiomyocytes and fibroblasts, and ANP secreted by cardiomyocytes are likely candidates. We found no evidence for the involvement of Ang II and ET-1 in autocrine/paracrine mechanisms between cardiac cell types.

Animals↗