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At least 739 records · Page 41Linked to original sources
PubMed2005

Effect of organ-specific fibroblasts on proliferation and differentiation of breast cancer cells.

Breast carcinomas contain both tumor cells and stromal cells, including fibroblasts, endothelial cells, and lymphocytes. Proliferation of breast cancer cells may be controlled partly by mesenchymal cells, especially fibroblasts. We studied effects of fibroblasts on tumorigenicity and histologic features of breast cancer cells vivo, and analyzed fibroblast-produced growth-promoting factors in vitro. Breast carcinoma cells from four lines, and fibroblasts from lines obtained from skin and breast tissue of four patients with breast cancer were used. A suitable number of breast tumor cells and fibroblasts were inoculated subcutaneously into nude mice; resulting tumors were examined. Then conditioned medium from fibroblasts was added to cultures of breast cancer cells to study growth effects, and growth-promoting factors from breast fibroblasts were analyzed. Co-inoculation of breast cancer cells with breast fibroblasts into mice significantly increased tumorigenicity and tumor size beyond those obtained with breast cancer cells alone. Histologically, tumors resulting from co-inoculation with breast fibroblasts showed a scirrhous pattern with extensive fibrosis, while those formed by breast cancer cells injected alone or co-inoculation with skin fibroblasts showed a solid pattern. Medium from breast fibroblasts significantly increased breast cancer cell growth in vitro, while the various skin fibroblasts did not all show this effect. Structural and functional interactions between organ-specific fibroblasts and breast cancer cells may importantly regulate breast cancer growth and progression.

Animals↗
PubMed2004

Role of nitric oxide in apoptosis of human peritoneal and adhesion fibroblasts after hypoxia.

OBJECTIVE: To study the modulation of the inducible nitric oxide synthase/nitric oxide (iNOS/NO) expression system in fibroblasts isolated from human peritoneum and adhesion tissues by hypoxia. DESIGN: Prospective experimental study. SETTING: University medical center. PATIENT(S): Cultures of fibroblasts from both peritoneum and adhesion tissues of five patients. INTERVENTION(S): Hypoxia treatment of the primary cultured fibroblasts. MAIN OUTCOME MEASURE(S): We used Western and Northern blots to determine whether iNOS mRNA and its protein were present in peritoneal and adhesion fibroblasts and whether this expression is modulated by hypoxia. Multiplex reverse transcription polymerase chain reaction (RT-PCR) technique was used to quantify type I collagen mRNA in response to NG-nitro-L-arginine methyl ester (L-NAME). A terminal deoxynucleotidyl transferase mediated dUTP nick-end-labeling (TUNEL) assay was used to quantify apoptosis in response to NO donor S-nitro-N-acetyl-penicillamine (SNAP) treatment. A Griess assay was used to measure NO levels. RESULT(S): Peritoneal fibroblasts have significantly higher NO levels than adhesion fibroblasts. Hypoxia decreased NO in peritoneal fibroblasts to levels observed for adhesion fibroblasts. In addition, hypoxia increased both mRNA and protein levels of the iNOS gene in peritoneal and adhesion fibroblasts. Augmentation of NO by SNAP treatment increased apoptosis in adhesion fibroblasts. In contrast, SNAP had no effect on apoptosis of peritoneal fibroblasts. Inhibition of NO by L-NAME treatment increased type I collagen mRNA levels in peritoneal fibroblasts. CONCLUSION(S): Our findings confirm that adhesion fibroblasts produce less NO than normal peritoneal fibroblasts; NO may be the mechanism responsible for the creation and persistence of the adhesion phenotype.

Apoptosis↗
PubMed2005

Viability and function of autologous and allogeneic fibroblasts seeded in dermal substitutes after implantation.

BACKGROUND: Fibroblast-seeded collagen sponges have been used for the treatment of skin defects and skin ulcers. However, the viability of the fibroblasts after implantation is still unknown. The objective of this study was to investigate the viability and distribution of autologous and allogeneic fibroblasts after implantation and to clarify which type is more effective for wound healing. MATERIALS AND METHODS: Skin samples of Hartley guinea pigs were retrieved and autologous fibroblasts were isolated and cultured. Fibroblasts isolated from the skin of a Strain2 guinea pig were used as allogeneic fibroblasts. Three full-thickness wounds were created on the backs of guinea pigs and an acellular collagen sponge, a collagen sponge seeded with autologous fibroblasts, and a collagen sponge seeded with allogeneic fibroblasts were transplanted. Before implantation, fibroblasts were labeled with PKH26. The guinea pigs were sacrificed 1, 2, and 3 weeks after implantation. The epithelization and contraction of the wounds were assessed, and the viability and distribution of the seeded fibroblasts were observed in cross sections. RESULTS: Three weeks after implantation, the PKH26-labeled autologous and allogeneic fibroblasts remained viable. In the wounds covered with the autologous fibroblast-seeded collagen sponge, the epithelization was fastest, and the percent wound contraction was smallest. In contrast, in the wounds covered with allogeneic fibroblasts, the epithelization was slowest and the percent contraction was largest. CONCLUSION: The allogeneic fibroblasts seeded in the collagen sponge survived and remained viable on the grafted area, but did not accelerate wound healing.

Animals↗
PubMed2003

Cyclooxygenase-2 is expressed in human fibroblasts isolated from intraperitoneal adhesions but not from normal peritoneal tissues.

OBJECTIVE: To determine whether the COX-2 gene is expressed in human fibroblasts isolated from normal peritoneal and adhesion tissues. DESIGN: Prospective experimental study. SETTING: University medical center. PATIENT(S): Five patients undergoing laparotomy for pelvic pain. Primary cultures of fibroblasts were taken from both peritoneum and adhesion tissues. INTERVENTION(S): Hypoxia treatment of the primary cultured fibroblasts. MAIN OUTCOME MEASURES: We used the multiplex reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry techniques to determine whether COX-2 mRNA and its protein were present in normal peritoneal and adhesion fibroblasts from the same patients. Total RNA was extracted from cultured fibroblasts and subjected to multiplex RT-PCR to detect the presence of COX-2 mRNA in these cells. Cultured fibroblasts from all tissues were also fixed on slides and stained with COX-2 monoclonal antibody labeled with immunofluorescence. RESULT(S): COX-2 mRNA and its protein were absent in normal peritoneal fibroblasts from all five subjects but were present in adhesion fibroblasts from the same patients, as indicated by the multiplex RT-PCR and immunohistochemistry techniques. Hypoxia treatment significantly induced the mRNA and COX-2 protein levels in normal peritoneal fibroblasts to levels seen in adhesion fibroblasts under normoxic conditions. However, hypoxia had no effects on COX-2 expression by adhesion fibroblasts. CONCLUSION(S): Adhesion fibroblasts develop a specific phenotype, an adhesion phenotype, which is in part characterized by the expression of COX-2. The expression of COX-2 mRNA in adhesion fibroblasts and the induction of COX-2 in peritoneal fibroblasts in response to hypoxia indicate a possible inflammatory response. Regulation of COX-2 may alter peritoneal healing and may provide the opportunity to reduce postoperative adhesion development.

Cyclooxygenase 2↗
PubMed2001

Impaired migration, integrin function, and actin cytoskeletal organization in dermal fibroblasts from a subset of aged human donors.

Deficits in the motility of fibroblasts contribute to age-related impairment of wound healing. We analyzed 'young' fibroblasts from four healthy donors 22-30 years old and 'aged' fibroblasts from six healthy donors 81-92 years old for migratory ability on type I collagen, secretion of matrix metalloproteases (MMPs), attachment to matrices and, expression and function of integrin alpha2beta1. Cells from each donor were analyzed separately in each experiment. Whereas migration of young fibroblasts was uniformly robust, three aged lines migrated well and three migrated poorly. Synthesis of MMP1 and TIMP1, but not MMP2 or MMP9, was increased in the aged fibroblasts relative to the young fibroblast lines irrespective of their motility. All lines of young and aged fibroblasts attached to plastic or collagen with similar efficiency. Although young and aged fibroblasts expressed comparable levels of the alpha2 integrin; the lines of aged fibroblasts that were poor migrators exhibited a significant reduction in alpha2beta1 function relative to fibroblasts with normal migratory capacities. Moreover, the lines of aged fibroblasts that exhibited poor migration demonstrated a disordered actin cytoskeleton and a reduced ability to contract collagen gels. In conclusion, aged fibroblasts, unlike young fibroblasts, displayed variable migratory capacities. Deficient migration by specific lines of aged fibroblasts was not related to the capacity to attach, express alpha2 integrin, or secrete MMPs and TIMP1, but was characterized by disorganized cytoskeletal actin and reduced alpha2beta1 function.

Actins↗
PubMed1998

A comparative analysis of the proteins between the fibroblasts from Werner's syndrome patients and age-matched normal individuals using two-dimensional gel electrophoresis.

Werner's syndrome (WS) is an autosomal recessive disorder causing symptoms of premature aging. The fibroblasts of WS patients have a shorter life-span than normal fibroblasts. We analyzed the fibroblast proteins from three WS patients and from three age-matched normal individuals using two-dimensional gel electrophoresis and image processing. The expressions of 12 proteins were shown to be augmented or suppressed in WS fibroblasts compared with normal fibroblasts: 11 of 12 spots on the electrophoresis gel of WS fibroblasts were denser than the corresponding spots of normal individual fibroblasts, while the remaining one spot was fainter in WS fibroblasts than in normal fibroblasts. The abundance of these proteins were compared to those of the corresponding proteins from normal fibroblasts at various cell passages in vitro reported in the TMIG-2DPAGE database. The result shows that the change in the protein patterns in in vitro aging did not necessarily correspond to the change in WS fibroblasts, except for three proteins abundant in WS fibroblasts, which increased their abundance during in vitro aging. These results suggest that the premature aging process of WS fibroblasts shares only part of the in vitro aging process of normal fibroblasts.

Adult↗
PubMed2003

Altered proliferative responses of dermal fibroblasts to TGF-beta1 may contribute to chronic venous stasis ulcer.

PURPOSE: Venous ulcer fibroblasts demonstrate decreased proliferative responses to growth factor stimulation, suggesting cellular senescence. However, the role of chronic venous insufficiency (CVI) disease progression and extracellular matrix (ECM) proteins in agonist-induced cellular proliferation is ill-defined. We hypothesize that CVI-induced fibroblast proliferative resistance to growth factors worsens with disease progression and is regulated by the composition of ECM. METHODS: Fibroblast explants were isolated from biopsy specimens from two patients without CVI and 16 patients with CVI of the lower calf (LC) and lower thigh (LT) and stratified according to CEAP disease severity: non-CVI (NC; n = 2), class 2-3 (n = 5), class 4 (n = 5), class 5 (n = 3), and class 6 (n = 3). Proliferation experiments were standardized with a neonatal foreskin fibroblast cell line (HS68). A 10-day course and dose response experiment with 0, 0.5, 1.0, 2.5, 5, 10, and 20 ng/mL of transforming growth factor-beta(1) (TGF-beta(1)) demonstrated maximal cell proliferation at 5 ng/mL of TGF-beta(1) on day 4. Under these conditions, CVI dermal fibroblasts were challenged with and without TGF-beta(1) and evaluated for proliferative responses on plates coated with polystyrene, collagen, and fibronectin. RESULTS: No differences in unstimulated proliferation were observed in LT and LC fibroblasts from patients with class 2-3 disease and LT fibroblasts from patients with class 4 and 5 disease, compared with NC and HS68 cells. LC fibroblasts from patients with class 4 disease (P <.05) and class 5 disease (P <.001), and LC (P <.001), and LT fibroblasts from patients with class 6 disease (P <.001) proliferated to a lesser degree than did NC and HS68 cells. The diminished proliferation observed in class 4 LC cells was reversible with TGF-beta(1) stimulation (P <.004); however, class 5 and class 6 LC and LT fibroblasts did not respond to stimulation with TGF-beta(1). Collagen increased proliferation of HS68 cells with (P <.05) and without (P <.01) TGF-beta(1), compared with cells grown on polystyrene, but did not increase proliferative responses in NC or CVI fibroblasts with and without TGF-beta(1). Similarly, fibronectin increased proliferation of HS68 cells (P <.05) compared with cells grown on polystyrene, but did not alter proliferation in CVI fibroblasts. Fibronectin did seem to inhibit TGF-beta(1)-induced proliferation observed in class 4 LC cells. CONCLUSION: These data indicate that clinical disease progression correlates with cellular dysfunction. Fibroblasts from patients with class 2-3 disease retain their unstimulated and agonist- induced proliferative capacity, compared with NC and HS68 cells. The onset of inflammatory skin changes (class 4 and class 5 disease) diminishes agonist-induced proliferation, and ulcer formation (class 6 disease) severely inhibits it. In addition, the composition of ECM does not affect TGF-beta(1)-induced proliferation of fibroblasts in CVI.

Cell Movement↗
PubMed1997

Stimulation of fibroblast proliferation and matrix contraction by wound fluid.

Fibroblast proliferation and fibroblast-mediated matrix contraction are critical to wound healing. Different cytokines have been shown to modulate fibroblast functions but little is known about the physiological role of these soluble factors during wound repair. In these experiments we characterized a fibroblast stimulating factor in wound fluid. Wound fluid was obtained from subcutaneously implanted polyvinyl alcohol sponges harvested 10 days post-wounding (pool of 100 Lewis rats). Normal dermal fibroblasts were obtained from Lewis rats by an explant technique, while wound fibroblasts were isolated from sponges harvested 10 days post-wounding. Proliferation in response to 0.5% and 10% fetal bovine serum was assessed by [3H]-thymidine incorporation. A fibroblast-populated collagen lattice was used for assaying contractile properties. Wound fibroblasts demonstrated markedly diminished proliferative and enhanced contractile properties compared to normal dermal fibroblasts. 10% wound fluid (v/v) stimulated proliferation of normal dermal fibroblasts (119%, p < 0.001) and wound fibroblasts (103%, p < 0.001). Wound fluid also stimulated collagen gel contraction by normal dermal fibroblasts (24% at 24 hr and 16% at 72 hr, p < 0.01), but not by wound fibroblasts. Separation by Sephadex G-100 gel filtration identified the active factor in wound fluid to have a molecular weight of about 100 kDa. Characterization of the soluble factor showed it to be a protein (ammonium sulfate precipitation), sensitive to trypsin digestion, heat resistant (56 degrees C, 30 min), and neuraminidase resistant. The isoelectric point appeared to be 7.0, as determined by ion exchange chromatography. Mitogenic proliferation of thymic lymphocytes was not affected by the active factor, suggesting cell target specificity. These data demonstrate that the wound environment contains high molecular weight protein(s) that promote fibroblast functions, essential for the healing process.

Animals↗
PubMedDate not supplied

Fibroblast senescence in pressure ulcers.

Pressure ulcers appear as localized chronic wounds in the middle of normal functioning skin. This study focuses on pressure ulcer fibroblasts cultured from the ulcer bed, ulcer margin, and normal, nonulcerated skin adjacent to the wound. From these areas we show that pressure ulcer fibroblasts become prematurely senescent. Verification of senescence was based on failure of cell populations to undergo a 0.5 population doubling after 1 week in culture, light microscopic appearance of late-passage senescent cells in culture, light microscopic verification of senescence in vivo and in vitro by anti-terminin staining, and ultrastructural identification of senescent fibroblasts by anti-terminin colloidal gold labeling. Although not all ulcer fibroblasts are senescent, there is a range of proliferative ability. The senescent phenotype of ulcer fibroblasts remains intact in vitro as these cells remain viable but are unable to complete DNA synthesis. Fibroblast senescence is not uniform in chronic wounds but varies within areas of the ulcer bed and from patient to patient. Although ulcer fibroblasts exhibit limited proliferative ability, fibroblasts from the ulcer margin and adjacent normal skin show a continued ability to divide. However, in all areas of the wound, the mean generation time of the fibroblast population is longer than commonly observed for cultured human diploid fibroblasts. At first passage, the mean generation time for fibroblasts from all patients is significantly different (p = .001, analysis of variance) among fibroblasts from the ulcer bed (4.2 2.2 days), ulcer margin (2.9 +/- 1.3 days), and adjacent normal skin (1.9 +/- 0.6 days). Analysis of the three cell groups by the post hoc Tukey test, using corrected p values shows that the difference between mean generation times among fibroblast populations from adjacent normal skin and ulcer bed are significantly different (p < 0.05), whereas the difference between fibroblasts from adjacent normal skin and ulcer margin and ulcer margin and ulcer bed are not significantly different (p > 0.05). These data may help to explain the poor response of certain pressure sores to aggressive medical treatment.

Analysis of Variance↗
PubMed2006

CCN2 is necessary for adhesive responses to transforming growth factor-beta1 in embryonic fibroblasts.

CCN2 is induced by transforming growth factor-beta (TGFbeta) in fibroblasts and is overexpressed in connective tissue disease. CCN2 has been proposed to be a downstream mediator of TGFbeta action in fibroblasts; however, the role of CCN2 in regulating this process unclear. By using embryonic fibroblasts isolated from ccn2-/- mice, we showed that CCN2 is required for a subset of responses to TGFbeta. Affymetrix genome-wide expression profiling revealed that 942 transcripts were induced by TGFbeta greater than 2-fold in ccn2+/+ fibroblasts, of which 345 were not induced in ccn2-/- fibroblasts, including pro-adhesive and matrix remodeling genes. Whereas TGFbeta properly induced a generic Smad3-responsive promoter in ccn2-/- fibroblasts, TGFbeta-induced activation of focal adhesion kinase (FAK) and Akt was reduced in ccn2-/- fibroblasts. Emphasizing the importance of FAK and Akt activation in CCN2-dependent transcriptional responses to TGFbeta in fibroblasts, CCN2-dependent transcripts were not induced by TGFbeta in fak-/- fibroblasts and were reduced by wortmannin in wild-type fibroblasts. Akt1 overexpression in ccn2-/- fibroblasts rescued the TGFbeta-induced transcription of CCN2-dependent mRNA. Finally, induction of TGFbeta-induced fibroblast adhesion to fibronectin and type I collagen was significantly diminished in ccn2-/- fibroblasts. Thus in embryonic fibroblasts, CCN2 is a necessary cofactor required for TGFbeta to activate the adhesive FAK/Akt/phosphatidylinositol 3-kinase cascade, FAK/Akt-dependent genes, and adhesion to matrix.

Androstadienes↗
PubMed1996

The acrodermatitis enteropathica mutation transiently affects zinc metabolism in human fibroblasts.

The acrodermatitis enteropathica (AE) mutation has been shown to affect zinc transport in human intestinal biopsies. However, whether the mutation is also expressed in human fibroblasts has not been determined. The activity of the zinc-dependent enzyme, 5' nucleotidase, and cell zinc content were measured in normal and AE fibroblasts 2 and 4 d after subculturing to determine the effect of the AE mutation on zinc metabolism. The activity of 5' nucleotidase in AE cells was 68% of normal at 2 d after subculturing. Although 5' nucleotidase activity had decreased significantly in both normal and AE fibroblasts at 4 d after subculturing, there was no significant difference between the two genotypes. The zinc content of AE fibroblasts was also significantly reduced. Acrodermatitis enteropathica fibroblasts contained 62% less zinc than normal fibroblasts at 2 d. By 4 d the normal fibroblast zinc content had decreased to that of the AE fibroblasts. The uptake and transport of 65Zn into AE fibroblasts at 2 d was measured because these cells exhibited reduced 5' nucleotidase activity and cell zinc content at this time. The uptake of zinc over a 90-min time period was the same in the two genotypes. However, AE fibroblasts incubated with 2-10 mumol Zn/L for 15 min had significantly slower zinc transport compared with normal fibroblasts. In both genotypes, Michaelis-Menten kinetics were observed. Normal and AE fibroblasts had similar affinities for zinc (Km), but AE fibroblasts exhibited a Vmax which was reduced by 38%. These results indicate that the phenotypic expression of the AE mutation occurs in a time-dependent manner, is not restricted to the intestine and is also transiently expressed in human fibroblasts, resulting in abnormal zinc metabolism in these cells.

5'-Nucleotidase↗
PubMed2005

Constitutively phosphorylated Smad3 interacts with Sp1 and p300 in scleroderma fibroblasts.

OBJECTIVE: To elucidate the role of transforming growth factor-beta (TGF-beta)/Smad signalling in the increased expression of the collagen gene in systemic sclerosis (SSc) fibroblasts. METHODS: Dermal fibroblasts from seven patients with diffuse SSc of recent onset and from seven healthy individuals were studied. The expression levels of Smad2, Smad3 and Smad4 proteins were determined by immunoblotting. Smad3 phosphorylation and the interaction of Smad3 with Sp1 or p300 were analysed using immunoprecipitation. The effects of overexpression of Smad proteins or Sp1 on the human alpha2(I) collagen gene transcription were investigated with chloramphenicol acetyltransferase (CAT) assays using the -772 COL1A2/CAT construct. RESULTS: Constitutive increased Smad3 phosphorylation was detected in SSc fibroblasts compared with normal fibroblasts. Increased interaction of Smad3 with Sp1 as well as p300 was also detected in SSc fibroblasts. The overexpression of Smad3 caused an increase of up to 5-fold in COL1A2 promoter activity in normal fibroblasts, while Smad3 caused a small increase in COL1A2 promoter activity in SSc fibroblasts. However, neither Smad2 nor Smad4 caused significant effects in COL1A2 promoter activity in normal fibroblasts or SSc fibroblasts. The overexpression of Sp1 caused further increase in COL1A2 promoter activity stimulated by TGF-beta in normal fibroblasts, but did not change COL1A2 promoter activity in the presence of TGF-beta in SSc fibroblasts. The combined overexpression of Smad3 and Sp1 significantly enhanced TGF-beta response in normal fibroblasts, but less markedly in SSc fibroblasts. CONCLUSIONS: These results suggested that SSc fibroblasts are less sensitive to exogenous TGF-beta stimulation because they are already activated by the autocrine TGF-beta loop.

Cells, Cultured↗
PubMed1994

Stimulation of extraocular muscle fibroblasts by cytokines and hypoxia: possible role in thyroid-associated ophthalmopathy.

OBJECTIVE: Smoking is a risk factor for the development of thyroid-associated ophthalmopathy, an inflammatory process primarily affecting the fibroblasts in extraocular muscles. We wished to determine whether the extraocular muscle fibroblasts are more sensitive than dermal fibroblasts to T-cell derived cytokines, as a reason for this anatomical localization, and whether hypoxia alters fibroblast function, as one explanation for the susceptibility conferred by smoking. DESIGN: Fibroblasts derived from the skin or extraocular muscles of healthy subjects were cultured with cytokines under normal (5% CO2:95% air) and hypoxic (5% CO2:95% N2) conditions. MEASUREMENTS: Glycosaminoglycan, protein and DNA synthesis were measured by assessing incorporation of D-6-3H-glucosamine, 3H-amino acids, and 3H-thymidine respectively. RESULTS: alpha-interferon and interleukin-6 had no effect on fibroblasts. gamma-interferon, tumour necrosis factor and interleukin-1 stimulated glycosaminoglycan synthesis; this effect was greater in orbital than in dermal fibroblasts with gamma-interferon and interleukin-1 (P < 0.05). The same cytokines stimulated total protein with a greater response in orbital fibroblasts with gamma-interferon. Interleukin-1 inhibited DNA synthesis in orbital fibroblasts but stimulated DNA synthesis in dermal fibroblasts (P < 0.01); tumour necrosis factor also displayed a differential effect (P < 0.01). Hypoxia caused a significant increase in glycosaminoglycan, protein and DNA synthesis in both types of fibroblasts, under both basal and cytokine-treated conditions (P < 0.05). CONCLUSIONS: Extraocular muscle fibroblasts respond differently from dermal fibroblasts following cytokine stimulation, which may explain in part the anatomical localization of ophthalmopathy. Hypoxia stimulates fibroblasts and this could contribute, as an enhancing factor, to the adverse effects of smoking on thyroid eye disease.

Cells, Cultured↗
PubMed1998

Intestinal fibroblasts regulate intestinal epithelial cell proliferation via hepatocyte growth factor.

Although the presence of subepithelial intestinal fibroblasts has been well recognized, the effects of fibroblasts on intestinal epithelial cell (IEC) growth are incompletely understood. In vitro studies were undertaken to evaluate the effects of fibroblasts on the proliferation of model IEC lines. IECs (Caco-2, T84, and IEC-6) were grown alone or in the presence of human intestinal (CCD-18), lung (CCD-37), or skin explant-derived fibroblasts. Cocultures were carried out directly on irradiated fibroblasts or by Transwell coculture technique with fibroblasts and epithelial cells separated by a porous filter. Cell proliferation was assessed by [3H]thymidine incorporation and cell counts. Hepatocyte growth factor (HGF) and c-met transcript expression in IECs and fibroblasts was examined by RT-PCR and Northern blotting; protein expression was evaluated by immunoblotting. Intestinal as well as lung and skin fibroblasts substantially stimulated proliferation of Caco-2, T84, and IEC-6 cells in both direct and Transwell cocultures. In addition, fibroblast-conditioned medium stimulated IEC proliferation, suggesting a paracrine mechanism. Anti-human HGF-neutralizing antibodies blocked the growth-promoting effects in both fibroblasts and fibroblast-conditioned medium. Recombinant human HGF dose dependently promoted IEC proliferation. HGF mRNA and protein expression was restricted to fibroblasts. High levels of c-met expression were found in Caco-2 and T84 cells; in contrast, expression in fibroblasts was weak. In summary, fibroblasts stimulate IEC proliferation through a paracrine mechanism mediated predominantly by HGF.

Cell Division↗
PubMed2000

Chronic hypoxia induces exaggerated growth responses in pulmonary artery adventitial fibroblasts: potential contribution of specific protein kinase c isozymes.

Enhanced proliferation of adventitial fibroblasts is a major contributor to the structural remodeling of the pulmonary artery (PA) that occurs during hypoxia-induced pulmonary hypertension. The mechanisms responsible for the exuberant growth of fibroblasts are unknown; however, protein kinase C (PKC) isozymes have previously been shown to be important in the enhanced growth properties of immature PA fibroblasts. We tested the hypotheses that PA adventitial fibroblasts from neonatal calves exposed chronically to hypoxia after birth would express augmented growth responses compared with fibroblasts from the control adventitia and that these properties would be associated with selective changes in expression of PKC isozymes. We studied the effects of serum, purified mitogens, and hypoxia on the growth of aggregate populations of fibroblasts isolated from the PA of neonatal control calves (Neo-C) and calves chronically exposed to hypoxia for 2 wk beginning on Day 1 of life (Neo-Hyp). Neo-Hyp fibroblasts demonstrated higher proliferative capabilities than did Neo-C cells in response to all the stimuli tested. Importantly, hypoxia was found to act synergistically with peptide mitogens (platelet-derived growth factor, basic fibroblast growth factor, insulin-like growth factor-I) to stimulate growth in Neo-Hyp but not in Neo-C cells. Using PKC-isozyme nonselective and selective inhibitors and immunoblot analysis, we found differences in utilization of PKC isozymes in Neo-Hyp and Neo-C fibroblasts and have identified PKC-betaI and -zeta as key contributors to the augmented growth of Neo-Hyp fibroblasts. Although the activity of PKC-betaI and -zeta isozymes was increased by hypoxia in serum-deprived Neo-C and Neo-Hyp fibroblasts, under normoxia, quiescent Neo-Hyp fibroblasts had higher PKC-zeta-specific activity than did Neo-C cells. These results suggest that neonatal PA adventitial fibroblasts acquire new growth properties in the setting of hypoxia- induced pulmonary hypertension and that the augmented proliferative characteristics of the Neo-Hyp fibroblasts might be associated with changes in specifc PKC isozyme expression and activation patterns.

Animals↗
PubMed2005

The implications of the upregulation of ICAM-1/VCAM-1 expression of corneal fibroblasts on the pathogenesis of allergic keratopathy.

OBJECTIVE: The present study investigated the expression of ICAM-1 and VCAM-1 on fibroblasts with interleukin (IL)-4 and/or tumor necrosis factor (TNF)-alpha stimulation and assessed the effect of eosinophil adhesion on fibroblast viability. METHODS: Primary cultured human corneal fibroblasts were incubated with IL-4, TNF-alpha, or their combination for 24 hours. Expression of ICAM-1 and VCAM-1 was examined by real-time quantitative PCR and flow cytometric analysis. Purified eosinophils were cocultured with activated fibroblasts, and the number of eosinophils adhered to fibroblasts and the number of damaged fibroblasts were counted using microscopy. In a separate trial, conjunctival and corneal impression cytology was performed on patients with atopic keratoconjunctivitis and corneal ulcers (eight eyes) to assess the status of the ocular surface epithelium and the presence of inflammatory cell infiltrates. RESULTS: Real-time quantitative PCR and flow cytometric analysis revealed that both mRNA and protein of VCAM-1 and ICAM-1 were upregulated by IL-4 and TNF-alpha. IL-5-primed eosinophils adhered to the corneal fibroblasts treated with IL-4 and TNF-alpha, and the fibroblasts were damaged by eosinophil adherence. Anti-ICAM-1 antibody and anti-VCAM-1 antibody inhibited the eosinophil adherence to fibroblasts and the fibroblast damage. Impression cytology revealed extensive infiltration of neutrophil and eosinophils among isolated ocular surface epithelial cells with advanced squamous metaplasia. CONCLUSIONS: Corneal fibroblasts expressed ICAM-1 and VCAM-1 when activated with IL-4 and TNF-alpha. Eosinophils can adhere to the activated fibroblasts and can induce subsequent fibroblast damage through these adhesion molecules. Eosinophil adhesion to fibroblasts may possibly contribute to the pathogenesis of severe persistent allergic corneal ulcers.

Cell Adhesion↗
PubMed2002

Adhesive defect in extracellular matrix tenascin-X-null fibroblasts: a possible mechanism of tumor invasion.

Extracellular matrix tenascin-X (TNX)-null mice, generated by disruption of the Tnx gene, display augmented invasion and metastasis of B16-BL6 melanoma tumor cells due to increased activities of matrix metalloproteinase (MMP)-2 and MMP-9. In this study, we investigated cell-matrix and cell-cell adhesions using TNX-null fibroblasts and wild-type fibroblasts. TNX-null fibroblasts exhibited a decreased attachment to fibronectin compared with that of wild-type fibroblasts. B16 melanoma cells were cocultured with wild-type or TNX-null fibroblasts, and the adhesion of B16 melanoma to the fibroblasts was assessed. B16 melanoma cells on wild-type fibroblasts proliferated and spread out in a horizontal direction, whereas those on TNX-null fibroblasts overlapped each other rather than migrating horizontally. These overlapping B16 melanoma cells on TNX-null fibroblasts peeled off faster than those on wild-type fibroblasts. To determine whether the decreased cell-matrix and cell-cell adhesions on TNX-null fibroblasts were due to increased MMP activity, the activities of MMPs in wild-type and TNX-null fibroblasts were compared by gelatinolytic assays. The analysis of MMPs from conditioned media demonstrated that almost the same levels of MMP activities were detected between wild-type and TNX-null fibroblasts. However, contrary to our expectations the activities of MMPs from conditioned media of B16 melanoma cells cocultured on TNX-null fibroblasts were rather reduced than those of B16 melanoma cells cocultured on wild-type. We concluded that the absence of TNX in the extracellular environment might play an important role in enhancement of the detachment of B16 melanoma cells.

Animals↗
PubMed2003

Increased binding and chemotactic capacities of PDGF-BB on fibroblasts in radiation pneumonitis.

Although pulmonary fibrosis is a frequent and serious consequence of radiotherapy for thoracic malignant diseases such as lung cancer, the pathogenesis of this radiation-induced lung disorder remains unclear. To clarify the mechanisms underlying radiation pneumonitis and pulmonary fibrosis, we investigated the expression of platelet-derived growth factor receptor (PDGFR) on fibroblasts obtained from irradiated rat lungs and on control fibroblasts. Whole lungs of male Wistar rats were irradiated with a single dose of 15 Gy, and lung fibroblasts were isolated at 4 weeks after the irradiation. The chemotactic response of irradiated lung fibroblasts to PDGF-BB was significantly higher than that of control lung fibroblasts, whereas there was no significant difference between irradiated lung fibroblasts and control lung fibroblasts in the response to PDGF-AA. Receptor binding assay showed more specific binding sites for PDGF-BB on irradiated lung fibroblasts than on control lung fibroblasts, and the displacement of (125)I-labeled PDGF binding to fibroblasts by unlabeled PDGF showed that (125)I-labeled PDGF-BB was displaced by PDGF-BB but not by PDGF-AA. These results suggest that the increased binding sites for PDGF-BB on irradiated lung fibroblasts correspond mainly to PDGFRB. Scatchard analysis of the saturation data demonstrated an approximately twofold increase both in the number of PDGF-BB binding sites and in the binding affinity in irradiated lung fibroblasts compared to that in control lung fibroblasts. Those results suggest that the increased chemotactic response of irradiated lung fibroblasts to PDGF-BB is related to the overexpression of PDGFRB, which may have an important role in the pathogenesis of radiation-induced pneumonitis and pulmonary fibrosis.

Animals↗