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At least 955 records · Page 53Linked to original sources
PubMed2003

Selective depletion of fibroblasts preserves morphology and the functional integrity of peritoneum in transgenic mice with peritoneal fibrosing syndrome.

BACKGROUND: A peritoneal fibrosing syndrome (PFS) can progressively reduce peritoneal ultrafiltration during chronic peritoneal dialysis in patients with renal failure. The pathogenesis of PFS is unclear and the role of peritoneal fibroblasts has not been evaluated experimentally. METHODS: We followed the fate of fibroblasts producing PFS in a mouse model using fibroblast-specific protein 1 (FSP1) as a marker. PFS was induced by daily peritoneal infusions of chlorhexidine gluconate (CHG) saline into transgenic mice expressing the thymidine kinase (Delta tk) gene under the control of the FSP1 promoter (FSP1.Delta tk mice). To demonstrate the role of fibroblasts in PFS, we treated these FSP1.Delta tk mice with a nucleoside analogue to induce DNA chain termination and fibroblast death. RESULTS: Mice receiving peritoneal infusions of CHG saline every other day for 2 weeks developed increasing numbers of FSP1+ fibroblasts in the subserosal layers of the visceral peritoneum. Mac-3+ monocytes (macrophages) subsequently accumulated over the next 2 weeks in association with increased deposition of type I collagen and increased endothelial vascularity (CD31+) in these subserosal tissues. Since these peritoneal fibroblasts expressed monocyte chemoattractant protein-1 (MCP-1), heat shock protein 47 (HSP47), and vascular endothelial growth factor (VEGF), we suspect they were partially responsible for macrophage recruitment, matrix production, and the neoangiogenesis in the subserosal tissue. Treatment of PFS in FSP1.Delta tk transgenic mice with a nucleoside analogue selectively reduced the numbers of peritoneal fibroblasts and attenuated the attendant changes in peritoneal histology. Rescuing the peritoneal membrane from chronic thickening and neoangiogenesis by reducing the number of fibroblasts also preserved ultrafiltration. CONCLUSION: Peritoneal fibroblasts play a pivotal role in PFS, and their deletion using a fibroblasts-specific transgene was effective in preventing peritoneal fibrogenesis.

Animals↗
PubMed2000

Influence of IgE-mediated activation of cultured human mast cells on proliferation and type I collagen production by human dermal fibroblasts.

BACKGROUND: Mast cells have been suggested to be involved in fibrotic conditions, but it still remains unknown whether IgE-mediated activation of human mast cells promotes fibrogenesis by human fibroblasts. OBJECTIVE: The purpose of this study was to determine whether IgE-mediated activation of cultured human mast cells can promote fibrogenesis by cultured human dermal fibroblasts. METHODS: Mast cells derived from human umbilical cord blood cells were incubated with IgE and then activated by anti-IgE, and histamine release was measured. IgE-sensitized mast cells were cocultured with fibroblasts from normal dermis and activated with anti-IgE to induce histamine release, after which proliferation and type I collagen synthesis by the fibroblasts were determined. RESULTS: Coculture of subconfluent human dermal fibroblasts with IgE-sensitized mast cells did not affect fibroblast proliferation. However, fibroblast proliferation was increased by activated mast cells, and a significant increase was observed in the presence of 10(5) or 3 x 10(5) mast cells/mL. The promotion of fibroblast proliferation by mast cells (3 x 10(5)/mL) was partly inhibited by ketotifen at a concentration that significantly reduced histamine release from mast cells. On the other hand, IgE-mediated activation of mast cells did not increase type I collagen production by confluent human dermal fibroblasts. CONCLUSION: IgE-mediated activation of cultured human mast cells could increase the proliferation of human dermal fibroblasts, but did not promote type I collagen production by the fibroblasts under the conditions tested.

Cell Division↗
PubMed1973

Roles of calcium, serum, plasma, and folic acid in the control of proliferation of normal and Rous sarcoma virus-infected chicken fibroblasts.

In a culture medium of pH 7.4 and a folic acid concentration of 100 mug/liter that contains 5% heat-inactivated chicken plasma rather than serum, the rate of proliferation of normal chicken fibroblasts is determined by the concentration of calcium. Proliferation, rapid when the calcium concentration is physiological, decreases when the calcium concentration is reduced. At a very low calcium concentration, in this culture medium, normal fibroblasts are maintained without proliferation, whereas those infected with Rous sarcoma virus proliferate rapidly. This proliferative inactivity of normal fibroblasts does not involve contact-inhibition, since the effect is observed at low, as well as higher, culture densities. When a physiological amount of calcium is added to cultures of normal fibroblasts that have been maintained in very low calcium-plasma medium for 3 days, labeled thymidine uptake and protein synthesis are strongly stimulated, and cell division follows. The use of heat-inactivated chicken serum, instead of plasma, in this medium appears to strongly sensitize normal fibroblasts to the mitogenic action of calcium. In a plasma-containing culture medium of physiological calcium concentration and a folate concentration of 5 mug/liter, neither normal nor Rous sarcoma virus-infected fibroblasts proliferate to an appreciable extent. The use of serum, however, instead of plasma results in rapid proliferation of both normal and infected cells, as does increase in the folate concentration of the plasma-containing medium to 100 mug/liter.The fact that while normal fibroblasts are maintained without proliferation in low calcium-plasma medium, Rous sarcoma virus-infected fibroblasts proliferate rapidly, indicates that the effect of calcium is regulatory rather than permissive. These results suggest that the proliferation of normal fibroblasts is initiated by a cellular function involving calcium, and that the autonomous proliferation of the neoplastic fibroblasts results either from increased calcium uptake or from an alteration or a hypass of that function. The results also suggest that serum contains a mitogenic factor(s) not present in plasma, possibly a "wound hormone" for fibroblasts.

Animals↗
PubMed1998

Correlation of polyamine and growth responses to N1,N11-diethylnorspermine in primary fetal fibroblasts derived from transgenic mice overexpressing spermidine/spermine N1-acetyltransferase.

A recently generated transgenic mouse line having activated polyamine catabolism due to systemic overexpression of spermidine/spermine N1-acetyltransferase (SSAT) was used to isolate primary fetal fibroblasts as a means to further elucidate the cellular consequences of activated polyamine catabolism. Basal levels of SSAT activity and steady-state mRNA in the transgenic fibroblasts were about approximately 20- and approximately 40-fold higher than in non-transgenic fibroblasts. Consistent with activated polyamine catabolism, there was an overaccumulation of putrescine and N1-acetylspermidine and a decrease in spermidine and spermine pools. Treatment with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) increased SSAT activity in the transgenic fibroblasts approximately 380-fold, whereas mRNA increased only approximately 3-fold, indicating post-mRNA regulation. SSAT activity in the nontransgenic fibroblasts increased approximately 200-fold. By Western blot, enzyme protein was found to increase approximately 46 times higher in the treated transgenic fibroblasts than non-transgenic fibroblasts: a value comparable to 36-fold differential in enzyme activity. With DENSPM treatment, spermidine pools were more rapidly depleted in the transgenic fibroblasts than in nontransgenic fibroblasts. Similarly, transgenic fibroblasts were much more sensitive to DENSPM-induced growth inhibition. This was not diminished by co-treatment with an inhibitor of polyamine oxidase, suggesting that growth inhibition was due to polyamine depletion per se as opposed to oxidative stress. Since the two fibroblasts were genetically identical except for the transgene, the various metabolic and growth response differences are directly attributable to overexpression of SSAT.

Acetyltransferases↗
PubMed2003

Disruption of basal JNK activity differentially affects key fibroblast functions important for wound healing.

We used both a gene knockout approach and pharmacologic modulation to study the implication of the JNK pathway in regulating fibroblast motility, capacity to contract mechanically unloaded collagen gels, and type I collagen gene expression in vitro. These parameters, which are important for tissue repair, are positively regulated by transforming growth factor (TGF)-beta, a cytokine viewed as playing a master role during wound healing. We demonstrate that basal JNK activity is critical for fibroblast motility because (a) mouse embryo jnk-/- fibroblasts exhibit significantly lower ability to close mechanically induced cell layer wounds than their wild-type (wt) counterparts, and (b) wound closure by human dermal fibroblasts is dramatically impaired by the specific JNK inhibitor SP600125. junAA fibroblasts, in which amino acids Ser63 and Ser73 of c-Jun are replaced by two Ala residues so that c-Jun cannot be phosphorylated by JNK, also exhibited impaired motility, suggesting that c-Jun phosphorylation by JNK is critical for fibroblast migration. In sharp contrast to their lesser motility on plastic, jnk-/- and junAA fibroblasts contracted free-floating, mechanically unloaded, collagen lattices markedly faster than wt fibroblasts. Furthermore, basal mRNA steady-state levels for types I and III collagen genes were similar in jnk-/- and wt fibroblasts. Likewise, overexpression of a dominant-negative mutant form of MKK4 in dermal fibroblasts did not affect collagen expression. We also demonstrate that basal JNK activity does not affect either TGF-beta-induced collagen gene expression or lattice contraction, whereas on the other hand, the blockage of motility initiated by JNK inhibition cannot be overcome by TGF-beta. Together these results demonstrate discrete, yet significant and highly specific, regulation of fibroblast functions important for wound healing by basal JNK activity.

Cell Movement↗
PubMed2006

Use of human fibroblasts in the development of a xenobiotic-free culture and delivery system for human keratinocytes.

Previous work has shown that keratinocytes can be cultured serum-free on an acid-functionalized, plasma-polymerized surface (for subsequent delivery to patients' wound beds) by inclusion of a fibroblast feeder layer. This study seeks to extend this work by substituting human for murine feeder cells in serum-free culture and examining the performance of keratinocytes expanded in this way to transfer to an in vitro human dermal wound bed model. We compared murine and human fibroblasts (both short-term dermal fibroblasts and a fetal lung fibroblast cell line MRC-5, which has a long history in human vaccine production), alternative methods for growth-arresting fibroblasts, establishing culture of cells serum-free, and the impact of culture with fibroblasts on the differentiation of the keratinocytes. Irradiated human and murine fibroblasts were equally effective in supporting initial keratinocyte expansion, both in the presence and absence of serum. Keratinocytes were significantly less differentiated, as assessed by measuring involucrin expression relative to DNA when grown serum-free with fibroblasts than when grown with serum. Initial cultures of fibroblasts and keratinocytes could be initiated serum-free but were much slower to establish than if serum were used. Transfer of keratinocytes from keratinocyte/fibroblast co-cultures cultured on a plasma polymer surface to a human dermal wound bed model was as successful as from monocultures in both serum and serum-free cultures. In summary, we have revisited a well-accepted methodology for expanding human keratinocytes for clinical use and avoided the use of bovine serum and a mouse fibroblast feeder layer by introducing an irradiated human fibroblast feeder layer.

Cell Differentiation↗
PubMed2003

Modulation of homologous gap junctional intercellular communication of human dermal fibroblasts via a paracrine factor(s) generated by squamous tumor cells.

Loss of gap junctional intercellular communication (GJIC) is a characteristic of cancer cells. Since a coordinated interaction of epithelial tumor cells with stromal cells is a prerequisite for tumor invasion and metastasis, the present study was designed to test the hypothesis that skin-derived tumor cells may modulate homologous and heterologous GJIC. While homologous GJIC of human dermal fibroblasts as well as epidermal keratinocytes was detected, no communication was measured between SCL-1 cells derived from squamous cell carcinoma of human skin. Interestingly, co-cultures of dermal fibroblasts and SCL-1 tumor cells in serum-containing medium resulted in a 52-70% lowering of the number of communicating fibroblasts. Furthermore, incubation of confluent fibroblast cultures with serum-free supernatant fractions (20-30 kDa) from tumor cells, termed the 20/30 fraction, lowered the homologous gap junction communication of fibroblasts by >90%. This novel aspect of down-regulated homologous GJIC of dermal fibroblasts, which is reversible, was neither mediated by alteration of the expression of connexin43, the major gap junctional protein of dermal fibroblasts, nor by aberrant localization of connexin43 in the plasma membrane. Furthermore, post-translational modifications of connexins, such as phosphorylation, was not measured by mobility shift studies. Tumor cell-mediated GJIC down-regulation between fibroblasts was suppressed using EGTA-containing serum-free tumor cell-derived supernatants suggesting that calcium ions (Ca2+) might mediate the transduction of this effect. The involvement of Ca2+ in down-regulation of homologous GJIC of fibroblasts was supported by an increase in fluorescence intensity of the intracellular calcium-sensitive indicator Fura-2 upon treatment of fibroblasts with the active 20/30 fraction. In conclusion, these data establish homologous GJIC of (stromal) fibroblasts as a parameter modulated by a paracrine acting factor(s) of epithelial tumor cells during tumor-stroma interaction of skin cells.

Base Sequence↗
PubMed1993

Endothelial cell induced modulation of cardiac fibroblast collagen metabolism.

OBJECTIVE: The aim was to examine the influence of vascular endothelial cells on rat cardiac fibroblast collagen synthesis and collagenase activity under co-culture conditions, and to determine whether angiotensin II or aldosterone influence endothelial cell induced modulation of fibroblast collagen metabolism. METHODS: Bovine aortic endothelial cells were grown to confluence on microporous membrane inserts and then co-cultured with cardiac fibroblasts obtained from adult rats, previously grown to confluence in 35 mm dishes supplemented with 0.4% fetal bovine serum. Collagen synthesis was measured by 3H-proline incorporation. Experiments were also conducted under similar conditions to quantitate collagenase activity by zymography in conditioned medium from fibroblasts co-cultured with endothelial cells. RESULTS: After 48 h co-culture, there was a 1.9-fold increase in fibroblast collagen synthesis when compared to fibroblasts alone (p < 0.001). Aldosterone (10(-8) M) or angiotensin II (10(-7) M) added to endothelial cells did not increase fibroblast collagen synthesis over co-cultures alone. Neither the aldosterone receptor antagonist spironolactone (10(-8) M) nor type I (DuP 753, 10(-8) M) or type II (PD 123319, 10(-8) M) angiotensin II receptor antagonists altered fibroblast collagen synthesis in co-cultures. A significant increment in collagenase activity was observed in co-cultured fibroblasts relative to collagenase activity of fibroblasts alone. CONCLUSIONS: Endothelial cells modulate both cardiac fibroblast collagen synthesis and degradation. The nature of the responsible signal(s) remains to be defined, but does not appear to be mediated or regulated by angiotensin II or aldosterone.

Aldosterone↗
PubMed1998

Endothelins: effect on matrix biosynthesis and proliferation in normal and scleroderma fibroblasts.

We investigated the effect of endothelin-1 (ET-1) in normal and systemic sclerosis (SSc) dermal fibroblasts. Collagen type I, collagen type III, and MMP-1 levels in culture supernatants were measured by competition ELISA and cellular mRNA expression was examined by Northern blotting. Mitogenic responses to ET-1 were assessed by [3H]TdR incorporation. ET receptor mRNA expression was examined by RT-PCR analysis of fibroblast RNA and with surface binding studies using radiolabeled ET receptor ligands and specific receptor antagonists. ET-1 enhanced release of collagen types I and III by control and SSc fibroblast strains, but the effects were significantly greater for control cells (p < 0.05). This effect appeared to involve both ETA and ETB receptor subtypes. SSc fibroblasts demonstrated lower constitutive MMP-1 production than control fibroblasts (p < 0.01), but ET-1 treatment decreased MMP-1 in normal fibroblasts to levels observed in SSc. Mitogenic response (percent control [3H]TdR incorporation) to ET-1 for SSc fibroblasts was 130 +/- 34, significantly less (p < 0.01) than that for normal fibroblasts strains (290 +/- 25). This response appeared to be predominantly mediated via the ETA receptor subtype. Surface binding studies suggested a significantly lower level of ETA binding sites in SSc compared with normal fibroblasts (p < 0.05). These data suggest that ET-1 induces a fibrogenic phenotype in normal dermal fibroblasts that resembles that seen in fibroblasts grown from lesional SSc skin. Moreover, SSc cells appear to be refractory to these effects, and this reduced responsiveness is associated with an altered ratio of ETA:ETB receptor expression, supporting a role for ET-1 in the fibrotic pathology of SSc.

Cell Division↗
PubMed2001

Abnormal balance between proliferation and apoptotic cell death in fibroblasts derived from keloid lesions.

A new culture model was developed to study the role of proliferation and apoptosis in the etiology of keloids. Fibroblasts were isolated from the superficial, central, and basal regions of six different keloid lesions by using Dulbecco's Modified Eagle Medium containing 10% fetal calf serum as a culture medium. The growth behavior of each fibroblast fraction was examined in short-term and long-term cultures, and the percentage of apoptotic cells was assessed by in situ end labeling of fragmented DNA. The fibroblasts obtained from the superficial and basal regions of keloid tissue showed population doubling times and saturation densities that were similar to those of age-matched normal fibroblasts. In contrast, the fibroblasts from the center of the keloid lesions showed significantly reduced doubling times (25.9 +/- 6.3 hours versus 43.5 +/- 6.3 hours for normal fibroblasts) and reached higher cell densities. In long-term culture, central keloid fibroblasts formed a stratified three-dimensional structure, contracted the self-produced extracellular matrix, and gave rise to nodular cell aggregates, mimicking the formation of keloid tissue. Apoptotic cells were detected in both normal and keloid-derived fibroblasts, but their numbers were twofold higher in normal cells compared with all keloid fibroblasts. To examine whether apoptosis mediates the therapeutic effect of ionizing radiation on keloids, the cells were exposed to gamma rays at a dose of 8 Gy. Under these conditions, a twofold increase in the population of apoptotic cells was detected. These results indicate that the balance between proliferation and apoptosis is impaired in keloid fibroblasts, which could be responsible for the formation of keloid tumors. The results also suggest that keloids contain at least two different fibroblast fractions that vary in growth behavior and extracellular matrix metabolism.

Adult↗
PubMed2002

Dupuytren's disease: physiologic changes in nodule and cord fibroblasts through aging in vitro.

The pathogenesis of the fibrotic disease Dupuytren's contracture remains unclear. The disease process includes two structurally distinct fibrotic elements, the nodule and the cord. It has been proposed that as the disease progresses, nodules develop into cords. To corroborate that hypothesis, the authors took advantage of cultured fibroblast differences found between gap junction intercellular communication and fibroblast-populated collagen lattice contraction. Paired fibroblast cell lines of nodules and cords derived from four patients with Dupuytren's disease were maintained in culture for at least eight passages. The presence of gap junction intercellular communication in nodule- and cord-derived fibroblasts was documented and reported as a coupling index. The contraction of free-floating nodule- or cord-derived collagen lattices was also documented and reported. Early passage (passage 4) cord-derived fibroblasts showed a significant increase in coupling index compared with passage 4 nodule-derived fibroblasts (4.0 +/- 0.4 versus 2.5 +/- 0.3, respectively), where p < or = 0.01. However, late passage (passage 8) nodule- and cord-derived fibroblasts were equivalent in their coupling index (4.1 +/- 0.4 versus 4.4 +/- 0.4, respectively). Early passage nodule-derived fibroblast-populated collagen lattices contracted by 64 percent, whereas late passage nodule-derived lattices showed less contraction, at only 40 percent. Early and late passage cord-derived lattices contracted 46 and 37 percent, respectively. All nodule- and cord-derived cell lines were statistically equivalent at lattice contraction by passage 8. These in vitro studies support the hypothesis that fibroblasts derived from Dupuytren's contracture nodules change their phenotype after undergoing repeated cell passage, acquiring a cord-like fibroblast phenotype. Dupuytren's nodules represent the early, active form of fibrosis in which cells are more proliferative, better at fibroblast-populated collagen lattice contraction, and display less gap junction intercellular communication. The speculation is that alterations in gap junction intercellular communication may be involved in the progression of Dupuytren's nodules to cords as the disease progresses.

Cell Communication↗
PubMed2002

Characterization of fibroblasts from rejecting tissue: the hyaluronan production is increased.

BACKGROUND: Interstitial edema of rejecting tissue can be partly attributed to the local accumulation of hyaluronan, which has strong water-binding capacity. The aim of the present study was to isolate fibroblasts from rejecting tissue and compare them, in terms of hyaluronan production and proliferation rate, with fibroblasts obtained from nontransplanted tissue. Furthermore, the fibroblast response to various cytokines involved in the rejection process was studied. METHODS: Fibroblasts were isolated from normal rat heart tissue and from cardiac allografts undergoing rejection. The various preparations were characterized with regard to hyaluronan production (radiometric assay) and cell proliferation ( H-thymidine incorporation). In addition, the effects of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-2 on these parameters were studied. RESULTS: Fibroblasts isolated from rejecting hearts displayed strongly up-regulated hyaluronan production and proliferation rate as compared with fibroblasts obtained from normal tissue. In the presence of TNF-alpha, the proliferation of nonconfluent cells was augmented, whereas in confluent cultures of fibroblasts from rejecting tissue, the proliferation was inhibited. IFN-gamma stimulated both hyaluronan secretion and proliferation in confluent fibroblasts from rejecting hearts but had no effect on fibroblasts from normal tissue. IL-2, finally, reduced the hyaluronan production of nonconfluent cells. CONCLUSIONS: The activation of fibroblasts is increased in rejecting tissue. As a result, the hyaluronan concentration is elevated which, in vivo, contributes to the formation of an interstitial edema and a subsequently increased tissue pressure. Several cytokines present at rejection are involved also in the regulation of fibroblast activity.

Animals↗
PubMed2001

Altered response of intestinal mucosal fibroblasts to profibrogenic cytokines in inflammatory bowel disease.

BACKGROUND AND AIMS: Fibrosis is a major complication of inflammatory bowel disease (IBD), which may be mediated by the intestinal fibroblast. Our aim was to isolate and characterize mucosal fibroblasts from histologically normal intestine (control), ulcerative colitis (UC), inflamed Crohn's disease (CD), and fibrosed CD intestine. METHODS: Fibroblasts were characterized by light and electron microscopy and immunohistochemistry. Fibroblast collagen secretion and proliferation were determined by 3H-proline and 3H-thymidine incorporation, and the effects of exposure to interleukin (IL)-1beta, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta1, insulin-like growth factor (IGF)-1, and macrophage colony stimulating factor (M-CSF) were determined. RESULTS: No difference in doubling time was observed between the fibroblast populations from UC and CD intestine. All proliferated faster than fibroblasts from control intestine. Collagen secretion from IBD fibroblasts, independent of type, was increased compared with control fibroblasts and PDGF, bFGF, and TGF-beta1-induced collagen secretion from IBD fibroblasts. CONCLUSIONS: These results suggest the presence of an activated subpopulation of fibroblasts in both UC and CD tissue irrespective of the presence of tissue fibrosis or disease type.

Actins↗
PubMed2002

Lysosomal cystine storage augments apoptosis in cultured human fibroblasts and renal tubular epithelial cells.

Nephropathic cystinosis is a lethal disorder of lysosomal cystine storage due to defective lysosomal cystine transport. How lysosomal cystine causes this multisystemic disorder culminating in end-stage renal disease is not known, because the cystine is isolated from cellular metabolism by the lysosomal membrane. It is here reported that in both normal and nephropathic cystinotic fibroblasts and cultured renal proximal tubule epithelial cells, increased lysosomal cystine causes an increased rate of apoptosis. In nephropathic cystinotic fibroblasts, the rate of apoptosis is 14.8% after exposure to TNF-alpha versus 7.8% in control normal fibroblasts. Anti-Fas antibodies and UV exposure induced apoptosis in 18.1% and 17.4% of nephropathic cystinotic fibroblasts, respectively, versus 5.2% and 7.1% in normal fibroblasts when analyzed by CaspACE (P < 0.05). Similar results were found when the cells were analyzed by TdT-mediated dUTP nick end labeling (TUNEL). When the cystine content of normal fibroblasts is increased by exposure to cystine dimethylester (CDME), the apoptotic rate is increased to the rate seen in nephropathic cystinotic cells. Decreasing the cystinotic cells' cystine content by use of cysteamine results in normalization of the apoptotic rate. Renal proximal tubule epithelial (RPTE) cells are much more sensitive to CDME than fibroblasts, reaching 43.8% apoptosis 6 h after exposure to CDME alone, compared with 38.2% when exposed to TNF-alpha alone. Serum withdrawal causes an apoptotic rate of 8.7% in nephropathic cystinotic fibroblasts, compared with 6.1% in normal fibroblasts. That rate increases to 37.3% in normal fibroblasts after CDME exposure. Fibroblasts from two cystinotic variants, benign ocular and intermediate cystinosis, do not display increased apoptosis with increased lysosomal cystine. It is concluded that enhanced apoptosis resulting from lysosomal cystine storage may lead to inappropriate cell death and decreased cell numbers in many tissues and hence contribute to the nephropathic cystinotic phenotype. The variant forms may represent co-segregation or linkage of rare alleles that confer resistance to apoptosis, moderating the cell loss and causing the milder disease expression.

Apoptosis↗
PubMed2004

The contractile properties and responses to tensional loading of Dupuytren's disease--derived fibroblasts are altered: a cause of the contracture?

Dupuytren's disease causes disability because of the development of finger flexion deformities, with distinct nodule and cord formation. This results in physical shortening of the diseased fascial tissue through a combination of cell-mediated contraction and matrix remodeling. It is this fixed tissue fabric shortening that prevents finger extension. In this experimental study, the relative contractile properties of Dupuytren nodule- and cord-derived fibroblasts were quantified in a culture force monitor model, in comparison with normal carpal ligament fibroblasts. Nine nodule, 10 cord, and four carpal ligament fibroblast cell lines were studied; each cell line was derived from a separate patient. The contractile forces generated by nodule and cord fibroblasts were significantly greater than the force generated by carpal ligament fibroblasts. There were also significant differences between nodule- and cord-derived fibroblasts, with the nodule cells demonstrating the greatest contractile force generation. The contraction profiles of both cord and nodule Dupuytren fibroblasts demonstrated delays in the attainment of tensional homeostasis, with an absence of a plateau phase by 20 hours. After the contraction phase, cell-seeded constructs were subjected to a series of four uniaxial mechanical overloads and cellular responses were monitored during each subsequent 30-minute period. Dupuytren nodule and cord fibroblast responses were significantly altered, compared with carpal ligament fibroblasts, exhibiting an increased and opposite response. Dupuytren fibroblasts, particularly nodule fibroblasts, exhibited increased force generation and a delay in reaching tensional homeostasis. The data suggest that these cells have an inherently higher basal tension and contractile ability. This results in increased shortening of the matrix, and the delay in reaching tensional homeostasis might exacerbate this response. These results represent a theoretical framework regarding the fundamental processes involved in the pathogenesis and progression of clinical flexion deformities in Dupuytren disease.

Biomechanical Phenomena↗
PubMed1995

Growth regulation in scleroderma fibroblasts: increased response to transforming growth factor-beta 1.

We investigated the responses of normal and scleroderma fibroblasts to various growth factors, especially transforming growth factor-beta 1 (TGF-beta 1). The effects of various growth factors on [3H]thymidine incorporation in normal and scleroderma fibroblasts were examined. [125I]-labeled platelet-derived growth factor (PDGF)-BB binding in scleroderma and normal fibroblasts was examined both in the presence and absence of TGF-beta 1 (1 ng/ml). Cytoplasmic protein was isolated and analyzed by Western blotting. Total RNA from fibroblasts was also isolated and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primer sets. Mitogenic responses to TGF-beta 1 (0.33-1 ng/ml) in seven scleroderma fibroblast strains were significantly greater than those in normal controls. [125I]-PDGF-BB binding to scleroderma fibroblasts was increased after TGF-beta 1 stimulation. The increased response to TGF-beta 1 was shown to be mediated through PDGF-like protein induction; TGF-beta 1-treated scleroderma fibroblasts produced greater amounts of 36-kD PDGF-like protein, which was reported previously as connective tissue growth factor (CTGF), than did TGF-beta 1-treated normal fibroblasts. TGF-beta 1 treatment also upregulated PDGF-alpha receptor expression in scleroderma fibroblasts but not in normal dermal fibroblasts. mRNA expression of CTGF and PDGF-alpha receptor was correlated with the above protein expression. These observations suggest that the increased growth response to TGF-beta 1 in scleroderma fibroblasts is mediated through the induction of CTGF and PDGF-alpha receptor.

Cell Division↗
PubMed1995

Mast cells induce T-cell adhesion to human fibroblasts by regulating intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression.

The capacity of mast cell products to mediate T-cell adhesion to fibroblasts was explored using heterotypic coculture systems or by exposing fibroblasts to mast-cell-conditioned media (MCCM), prepared by degranulating mast cells with calcium ionophore. Experimental results indicated that fibroblasts exposed to MCCM for 24 h bound fivefold more T cells than control fibroblasts. Binding was inhibited with intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1) neutralizing antibodies. Enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis revealed that fibroblasts exposed to MCCM markedly increased ICAM-1 and VCAM-1 surface expression by 4 h, with levels maximal at 16 h and returning toward baseline by 48 h. A dose-dependent response of ICAM-1 and VCAM-1 expression was noted using serial dilutions of MCCM or by altering the ratio of degranulated mast cells cocultured with fibroblasts. Similar results were obtained using human fibroblasts derived from the dermis, synovium, and lung, although lung fibroblasts were generally less responsive. Northern analysis confirmed that MCCM regulated ICAM-1 and VCAM-1 expression at the mRNA level. In summary, mast cell products stimulated fibroblast surface expression, steady-state mRNA levels, and functional expression of ICAM-1 and VCAM-1. Experimental data suggest that mast-cell-derived tumor necrosis factor-alpha may be in large part responsible for these observations, although further studies using human mast cells will be required. Using a skin-equivalent organotypic coculture model with fibroblasts admixed with mast cells, we observed increased ICAM-1 expression in both keratinocytes and fibroblasts after activation of the mast cells.

Adult↗
PubMed1996

Collagenase gene expression in cutis laxa fibroblasts is upregulated by transcriptional activation of the promoter gene through a 12-0-tetradecanoyl-phorbol-13-acetate (TPA)-responsive element.

Our previous work demonstrated that collagenase mRNA levels are increased in fibroblasts derived from patients with cutis laxa (CL). To pursue the mechanism of the upregulation of collagenase expression, we investigated transcriptional levels of the collagenase gene in CL fibroblasts. Fibroblasts cultured from the skin of three congenital CL patients were studied. Northern blot hybridization revealed 2.8- to 7.3-fold increases in collagenase mRNA levels in CL fibroblasts compared with normal cells. Nuclear run-off experiments demonstrated that the transcription rate of the collagenase gene in nuclei isolated from the same cells was 5.1- to 10.2-fold higher in the CL fibroblasts than in the controls. Transient transfection of a normal collagenase promoter-CAT construct into the cells further showed significantly enhanced transcriptional activity in CL but not in normal fibroblasts. Experiments of transient transfection of deleted or small substituted collagenase promoter-CAT constructs indicated that collagenase transcription in CL fibroblasts was activated the TPA-responsive element site of the collagenase promoter gene. Although the levels of Jun and Fos gene expression did not differ from those observed in normal fibroblasts, AP-1-binding activity, as measured by the ability to bind to an oligonucleotide containing a TPA-responsive element, was significantly elevated in CL fibroblasts as compared with normal fibroblasts. These data suggest that collagenase expression is upregulated at the transcriptional level by endogenous activation of DNA binding of AP-1 in CL fibroblasts [corrected].

Cells, Cultured↗