Effects of Mitomycin-C on Normal Dermal Fibroblasts

To evaluate differences in fibroblast autocrine growth factor production by human fetal, keloid, and normal adult dermal fibroblasts. Serum-free cell lines of fetal, keloid, and normal adult dermal fibroblasts were established. Cell counts were performed and supernatants collected at 4, 24, and 72 hours. Cell-free supernatants were quantitatively assayed for transforming growth factor beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF). Population doubling times for fetal, keloid, and normal adult fibroblasts were 120.0, 88.1, and 128.4 hours, respectively. Differences in population doubling times did not reach statistical significance. Statistically significant differences between TGF-beta1 levels from fetal and normal adult fibroblasts were seen at 24 and 72 hours. Significant differences between TGF-beta1 levels from keloid and normal adult fibroblasts were also seen at 24 and 72 hours. Fetal fibroblasts demonstrated higher levels of bFGF than normal adult fibroblasts at each time point, but these differences were not statistically significant. No significant differences were observed between keloid and normal adult bFGF levels. Both fetal and keloid fibroblasts produce significantly more TGF-beta1 than normal adult fibroblasts. Our data and the data of others suggest that fetal fibroblasts produce more bFGF than adult fibroblasts. The serum-free model we describe can be used to quantitatively measure autocrine growth factor production by cells that underlie clinically different types of wound healing. This model provides information that may allow us to better treat and prevent undesirable scarring.

To evaluate the effects of tamoxifen on the growth and autocrine growth factor production of human dermal fibroblasts from the face. In vitro study of normal adult dermal fibroblast cells developed from surgical specimens in a serum-free model. Cell cultures were exposed to 5-, 8-, 12-, 16-, and 50-microg/mL concentrations of tamoxifen solution. Cell counts were performed, and the cell-free supernatants were collected at 0, 1, 3, 5, and 7 days after the initial exposure. Population doubling times were calculated, and supernatants were quantitatively assayed for basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF) beta1. Tamoxifen appears to delay cellular proliferation rates in a dose-dependent manner up to a concentration of 12 microg/mL. Higher concentrations, approaching 50 microg/mL, appear to have a toxic effect on cell growth. The analysis of growth factor production revealed decreased levels of bFGF and VEGF but no change in the levels of TGF-beta1. The in vitro findings of delayed cell proliferation and decreased production of VEGF and bFGF in cells exposed to tamoxifen are consistent with previous in vivo reports of delayed wound healing but improved scar formation. The in vitro findings of growth factor modulation by tamoxifen provide cellular and molecular evidence supporting the clinical use of tamoxifen to ultimately improve scar formation.

An in vitro model was used to determine the effect of superpulsed CO2 laser energy on normal dermal and keloid-producing fibroblast proliferation and release of growth factors. Growth factors assayed included basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1). bFGF is mitogenic, inhibits collagen production, and stabilizes cellular phenotype. TGF-beta1 stimulates growth and collagen secretion and is thought to be integral to keloid formation. Growth in a serum-free medium allowed measurement of these growth factors without confounding variables. Keloid and normal dermal fibroblasts cell lines were established from facial skin samples using standard explant techniques. Samples consisted of three separate keloid and three separate normal dermal fibroblast cell lines. Cells were used at passage 4 to seed 24-well trays at a concentration of 6 x 10(4) cells per milliliter in serum-free medium. At 48 hours, 18.8 percent of each cell well was exposed to a fluence of 2.4, 4.7, and 7.3 J/cm2 using the superpulsed CO2 laser. Cell viability and counts were established at four time points: 0 (time of superpulsed CO2 laser treatment), 24, 72, and 120 hours. Supernatants were collected and assessed for bFGF and TGF-beta1 using a sandwich enzyme immunoassay. All cell lines demonstrated logarithmic growth through 120 hours (conclusion of experiment), with a statistically significant shorter population doubling time for keloid fibroblasts (p < 0.05). Use of the superpulsed CO2 laser shortened population doubling times relative to that of controls; the differences were statistically significant in keloid dermal fibroblasts when fluences of 2.4 and 4.7 J/cm2 were used (p < 0.05 and 0.01, respectively). bFGF was present in greater levels in normal dermal fibroblasts than in keloid dermal fibroblasts. Application of superpulsed CO2 demonstrated a trend toward increased bFGF secretion in both fibroblast types; the increase was significant in the keloid group at 4.7J/cm2. A consistent trend in suppression of TGF-beta1 was seen in both groups exposed to superpulsed CO2, with the maximal effect occurring at 4.7 J/cm2. Serum-free culture sustains logarithmic cell growth and allows growth factor measurement without confounding variables from serum-containing media. Superpulsed CO2 enhances fibroblast replication and seems to stimulate bFGF secretion and to inhibit TGF-beta1 secretion. Given the function of these growth factors, the application of superpulsed CO2 may support normalized wound healing. These findings may explain the beneficial effects of laser resurfacing on a cellular level and support the use of superpulsed CO2 in the management of keloid scar tissue.

Increased Expression of Integrin αvβ5 Induces the Myofibroblastic Differentiation of Dermal Fibroblasts

A TGF-β1-Dependent Autocrine Loop Regulates the Structure of Focal Adhesions in Hypertrophic Scar Fibroblasts

Endothelin 1 (ET-1) has been implicated in the pathogenesis of fibrotic and inflammatory diseases, including scleroderma. In addition to modulating vascular tone and extracellular matrix turnover, ET-1 up-regulates cell surface adhesion molecules including intercellular adhesion molecule 1 (ICAM-1), which is key to cell-cell and cell-matrix adhesion and leukocyte infiltration. This study was undertaken to delineate the signal transduction pathways utilized by ET-1 and compare them with those adopted by proinflammatory cytokine interleukin-1beta (IL-1beta) in normal and scleroderma dermal fibroblasts. Protein expression induced by ET-1 and IL-1beta on normal dermal fibroblasts, with or without signaling inhibitors, was detected by enzyme-linked immunosorbent assay, while messenger RNA (mRNA) levels were analyzed by LightCycler polymerase chain reaction. Expression of protein kinase Cdelta (PKCdelta) and PKCepsilon protein in normal dermal fibroblasts and scleroderma dermal fibroblasts was determined by Western blotting, and PKCepsilon involvement in ET-1 signaling was confirmed through transfection of an ICAM-1 promoter construct into murine PKCepsilon-/- fibroblasts. NF-kappaB activation was confirmed via electrophoretic mobility supershift assay, and analysis of the ICAM-1 promoter region was achieved via transfection of deletion constructs into human dermal fibroblasts. In normal dermal fibroblasts, ET-1 induced ICAM-1 mRNA and surface protein expression in a dose- and time-dependent manner via both receptor subtypes, ET(A) and ET(B); antagonism of both abolished the ET-1 response. MEK was involved in the signaling cascade, but phosphatidylinositol 3-kinase and p38 MAPK were not. Key to the cascade was activation of NF-kappaB, achieved by ligation of either receptor subtype. PKCepsilon activation led to downstream activation of MEK and, in part, NF-kappaB. IL-1beta signaling required NF-kappaB and MEK activation, along with activation of PKCdelta. ET-1 and IL-1beta each utilized the same ICAM-1 promoter region and the same NF-kappaB site at -157 bp. Responses to ET-1 and IL-1beta differed in scleroderma dermal fibroblasts, with ET-1 sensitivity decreasing and IL-1beta responses remaining intact. Expression of PKCepsilon and PKCdelta in scleroderma dermal fibroblasts was also altered. The findings of this study indicate that differences in sensitivity to ET-1 and IL-1beta in scleroderma dermal fibroblasts may be explained by altered expression of the PKC isoforms and cytokine receptors.

Dermal fibroblasts play essential roles in wound healing. However, they lose their normal regenerative functions under certain pathologic conditions such as in chronic diabetic wounds. Here, we show that substance P (SP) rescues the malfunctions of dermal fibroblasts in diabetes. SP increased the proliferation of diabetic dermal fibroblasts dose-dependently, although the effect was lower compared to the SP-stimulated proliferation of normal dermal fibroblasts. In contrast to normal dermal fibroblasts, SP increased the expression level of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) in diabetic dermal fibroblast hence, rescuing their angiogenic potential. The cellular characteristics of diabetic dermal fibroblasts modulated by SP would be able to accelerate the wound healing process through faster wound contraction and improved angiogenesis in diabetic chronic wounds. Moreover, SP pretreatment into dermal fibroblasts isolated from diabetic patients would be a promising strategy to develop autologous cell therapy for treating diabetic chronic wounds.

Background: Scleroderma skin overexpresses the platelet-derived growth factor receptor β-subunit (PDGFR-β) in dermal vessels and PDGFR-β messenger RNA in cultured fibroblasts. Moreover, increased levels of PDGF and stimulatory autoantibodies to PDGFR have been identified in the serum of scleroderma patients. Objective: Imatinib being an inhibitor of tyrosine kinase receptors such as PDGFR, its effect on scleroderma fibroblasts was evaluated in vitro as a preclinical therapeutic step. Methods: The effect of imatinib on fibroblasts grown from normal or involved/uninvolved scleroderma skin was studied by Western blot and the methyltetrazolium test. The pattern of distribution of PDGFR-β in scleroderma versus normal skin was studied by immunohistochemistry. Results: In vitro, imatinib inhibited the proliferation of normal dermal and scleroderma fibroblasts at least partly via the inhibition of the phosphorylation of PDGFR. PDGFR-β was expressed in the epidermis and adnexae in 5 lesional scleroderma biopsies and not in controls. Conclusion: This study suggests that imatinib can serve as therapy to limit dermal fibroblast proliferation in scleroderma.

To evaluate the effects of mitomycin on the growth of human dermal fibroblast and immortalized human keratinocyte line (HaCat cell), particularly the effect of mitomycin on intracellular messenger RNA (mRNA) synthesis of collagen and growth factors of fibroblast. The normal dermal fibroblast and HaCat cell were cultured in vitro. Cell cultures were exposed to 0.4 and 0.04 mg/ml of mitomycin solution, and serum-free culture medium was used as control. The cellular morphology change, growth characteristics, cell proliferation, and apoptosis were observed at different intervals. For the fibroblasts, the mRNA expression changes of transforming growth factor (TGF)-β1, basic fibroblast growth factor (bFGF), procollagen I, and III were detected by reverse transcription polymerase chain reaction (RT-PCR). The cultured normal human skin fibroblast and HaCat cell grew exponentially. A 5-min exposure to mitomycin at either 0.4 or 0.04 mg/ml caused marked dose-dependent cell proliferation inhibition on both fibroblasts and HaCat cells. Cell morphology changed, cell density decreased, and the growth curves were without an exponential phase. The fibroblast proliferated on the 5th day after the 5-min exposure of mitomycin at 0.04 mg/ml. Meanwhile, 5-min application of mitomycin at either 0.04 or 0.4 mg/ml induced fibroblast apoptosis but not necrosis. The apoptosis rate of the fibroblast increased with a higher concentration of mytomycin (p<0.05). A 5-min exposure to mitomycin at 0.4 mg/ml resulted in a marked decrease in the mRNA production of TGF-β1, procollagen I and III, and a marked increase in the mRNA production of bFGF. Mitomycin can inhibit fibroblast proliferation, induce fibroblast apoptosis, and regulate intracellular protein expression on mRNA levels. In addition, mitomycin can inhibit HaCat cell proliferation, so epithelial cell needs more protecting to avoid mitomycin's side effect when it is applied clinically.

Effect of nitric oxide and peroxynitrite on type I collagen synthesis in normal and scleroderma dermal fibroblasts

Our objective was to study the regulation of intercellular adhesion molecule-1 (ICAM-1) expression by cytokines on cultured fibroblasts obtained from systemic sclerosis and normal skin. ICAM-1 expression on dermal fibroblasts obtained from diffuse systemic sclerosis patients with early disease (< or = 2 years) and normal dermal fibroblasts incubated with and without cytokines was measured by radioimmunoassay and flow cytometry. Systemic sclerosis dermal fibroblasts expressed lower basal levels of ICAM-1 than did normal dermal fibroblasts. Both the normal and systemic sclerosis dermal fibroblasts increased their cell surface expression of ICAM-1 in response to interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in a dose-dependent fashion. Systemic sclerosis dermal fibroblasts appeared to be hyperresponsive to IL-1 beta, TNF-alpha, and IFN-gamma. ICAM-1 expression in response to cytokine stimulation increased to a greater degree on systemic sclerosis compared to normal dermal fibroblasts. The enhanced ICAM-1 expression may play a role in the retention of leukocytes involved in systemic sclerosis skin lesions.

Effects of the immunosuppressant rapamycin on the expression of human α2(I) collagen and matrix metalloproteinase 1 genes in scleroderma dermal fibroblasts

Intravenously Injected Human Fibroblasts Home to Skin Wounds, Deliver Type VII Collagen, and Promote Wound Healing

Parathyroid hormone-related protein is a positive regulator of keratinocyte growth factor expression by normal dermal fibroblasts

Characteristics of interferon induced tryptophan metabolism in human cells in vitro