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Figure 1.
The average transforming growth
factor (TGF) β1 concentration produced per cell in a serum-free
keloid fibroblast cell culture. Tamoxifen was added as tamoxifen citrate.

The average transforming growth factor (TGF) β1 concentration produced per cell in a serum-free keloid fibroblast cell culture. Tamoxifen was added as tamoxifen citrate.

Figure 2.
The average transforming growth
factor (TGF) β1 concentration produced per cell in a serum-free
fetal fibroblast cell culture. Tamoxifen was added as tamoxifen citrate.

The average transforming growth factor (TGF) β1 concentration produced per cell in a serum-free fetal fibroblast cell culture. Tamoxifen was added as tamoxifen citrate.

1.
Su  CWAlizadeh  KBoddie  ALee  RC The problem scar. Clin Plast Surg. 1998;25451- 467
2.
Di Cesare  PECheung  DTPerelman  N  et al.  Alteration of collagen composition and crosslinking in keloid tissues. Matrix. 1990;10172- 178Article
3.
Younai  SNichter  LSWellisz  T  et al.  Modulation of collagen synthesis by transforming growth factor-β in keloid and hypertrophic scar fibroblasts. Ann Plast Surg. 1994;33148- 151Article
4.
Lee  TYChin  GSKim  WJHChau  DGittes  GKLongaker  MT Expression of transforming growth factor-β 1, 2, and 3 proteins in keloids. Ann Plast Surg. 1999;43179- 184
5.
Polo  MSmith  PDKim  YJWang  XKo  FRobson  MC Effect of TGF-β2 on proliferative scar fibroblast cell kinetics. Ann Plast Surg. 1999;43185- 190Article
6.
Mackool  RJGittes  GKLongaker  MT Scarless healing: the fetal wound. Clin Plast Surg. 1998;25357- 365
7.
Lorenz  HPLin  RYLongaker  MTWhitby  DJAdzick  NS The fetal fibroblast: the effector cell of scarless fetal skin repair. Plast Reconstr Surg. 1995;961251- 1259Article
8.
Ferguson  MWJHowath  GF Marsupial models of scarless fetal wound healing. Adzick  NSLongaker  MTeds.Fetal Wound Healing. New York, NY Elsevier Scientific Press1992;95- 124
9.
Ihara  SMotobayashi  Y Wound closure in foetal rat skin. Development. 1992;114573- 582
10.
Martin  PLewis  J Actin cables and epidermal movement in embryonic wound healing. Nature. 1992;360179- 182Article
11.
Broker  BJChakrabarti  RBlynman  TRoesler  JWang  MBSrivatsan  ES Comparison of growth factor expression in fetal and adult fibroblasts: a preliminary report. Arch Otolaryngol Head Neck Surg. 1999;125676- 680Article
12.
Mancoll  JSMacauley  RLPhillips  LG The inhibitory effect of tamoxifen on keloid fibroblasts. Surg Forum. 1996;47718- 720
13.
Chau  DMancoll  JSLee  S  et al.  Tamoxifen downregulates TGF-β production in keloid fibroblasts. Ann Plast Surg. 1998;40490- 493Article
14.
Koch  RJGoode  RLSimpson  GT Serum-free keloid fibroblast cell culture: an in vitro model for the study of aberrant wound healing. Plast Reconstr Surg. 1997;991094- 1098Article
15.
Nowak  KCMcCormack  MKoch  RJ The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors: a serum-free study. Plast Reconstr Surg. 2000;1052039- 2048Article
16.
Hong  RHLum  JKoch  RJ Growth of keloid-producing fibroblasts in commercially available serum-free media. Otolaryngol Head Neck Surg. 1999;121469473Article
Citations 0
Original Article
April 2001

Effect of Tamoxifen on Transforming Growth Factor β1 Production by Keloid and Fetal Fibroblasts

Author Affiliations

From the Wound Healing and Tissue Engineering Laboratory, Division of Otolaryngology/Head and Neck Surgery, Stanford University Medical Center, Stanford, Calif.

 

From the Wound Healing and Tissue Engineering Laboratory, Division of Otolaryngology/Head and Neck Surgery, Stanford University Medical Center, Stanford, Calif.

Arch Facial Plast Surg. 2001;3(2):111-114. doi:
Abstract

Background  Evidence suggests that keloid scar formation may be mediated, in part, by deranged growth factor activity, including that of transforming growth factor (TGF) β1. Tamoxifen citrate has shown promise in the treatment of keloids.

Objective  To evaluate the effect of tamoxifen on autocrine growth factor expression in keloid and fetal dermal fibroblasts, which exhibit scar-free healing.

Design  Serum-free cell lines of keloid and fetal dermal fibroblasts were established. Cell cultures were exposed to different concentrations of tamoxifen solution (8 and 12 or 16 µmol/L). Cell counts were performed and supernatants collected at 24, 48, and 96 hours. Cell-free supernatants were quantitatively assayed for TGF-β1 expression.

Results  Keloid fibroblasts show increased per-cell TGF-β1 production compared with fetal fibroblasts. Tamoxifen appeared to decrease per-cell TGF-β1 production at each of the time points evaluated.

Conclusions  Keloids likely arise due to locally insufficient or excessive concentrations of specific growth factors. The higher level of TGF-β1 produced by keloid cells compared with fetal fibroblasts could be related to the aberrant wound healing seen with keloids. The addition of tamoxifen may lead to improved wound healing in keloids by decreasing the expression of TGF-β1.

ABERRANT WOUND healing is a significant problem that affects millions of patients yearly. Keloids, for example, are characterized by the formation of exuberant scar tissue that does not flatten over time. They are associated with an abnormal proliferation of fibroblasts and an overproduction of extracellular matrix and collagen.1-2 Treatment for keloid scars is problematic, with no single modality producing uniformly satisfactory results.

Aberrant wound healing may be caused in part by deranged growth factor activity. Transforming growth factor (TGF) β1 is a key cytokine involved in the initiation and termination of tissue repair.3 Its sustained production likely underlies the development of tissue fibrosis. Transforming growth factor β is secreted by multiple cells, including fibroblasts, and has 3 isoforms. Transforming growth factors β1 and β2 are overproduced by keloid fibroblasts compared with normal fibroblasts.4 Exogenous TGF-β2 has been shown to increase the in vitro cell proliferation kinetics of keloid and burn hypertrophic scar fibroblasts.5 Younai et al3 investigated the in vitro effects of TGF-β1 on the rate of collagen synthesis in keloid fibroblasts, fibroblasts from a hypertrophic scar, and normal skin fibroblasts. In response to exogenous TGF-β1, keloid fibroblasts produced 12 times more collagen than did normal fibroblasts and 4 times more than did hypertrophic scar fibroblasts.

Fetal wounds heal without histologic evidence of scarring.6 Fibroblasts are the main effector of scarless healing in fetal tissue, and this healing can occur outside the fetal environment.7-10 Broker et al11 found an increase in messenger RNA expression of acidic and basic fibroblast growth factors and in TGF-β1 in adult fibroblasts compared with fetal fibroblasts. Their work suggests that differences in cytokine production may contribute to the suboptimal wound healing seen in adult wounds compared with the scarless healing of fetal wounds. Measurement of messenger RNA is indirect evidence of differences in growth factor production, and the logical next step is to directly assay for secreted growth factors.

Tamoxifen citrate is a synthetic nonsteroidal antiestrogen, used in the treatment of breast cancer. It has been shown to inhibit keloid fibroblast proliferation and decrease collagen production.12 Effects of tamoxifen include altering transcriptional synthesis, decreasing cellular proliferation, and modulating production of multiple polypeptide growth factors.12 Recently, Chau et al13 showed that tamoxifen decreased the total (all 3 isomers) TGF-β produced by keloid fibroblasts in cell culture in a dose-dependent manner. However, no comparison was made with other fibroblasts types, and fibroblasts were not grown in a strictly serum-free model.

Prior in vitro studies of fibroblast autocrine characteristics have been confounded by the presence of serum-containing tissue culture media, because serum contains growth factors. One of us (R.J.K.) helped develop a serum-free in vitro fibroblast model.14 Since the only growth factors present are products of the fibroblasts themselves, autocrine products may be assayed without exogenous contributions. This model has already been successfully used to test pulsed carbon dioxide laser energy as a potential wound healing modulator.15 Pulsed carbon dioxide laser energy stimulated basic fibroblast growth factor and inhibited TGF-β1 secretion in normal and keloid fibroblasts in a fluence-dependent manner.15

This study sought to evaluate the effects of tamoxifen on TGF-β1 production by fibroblasts from the 2 ends of the wound-healing spectrum: keloid fibroblasts, for exuberant, aberrant healing; and fetal fibroblasts, for scar-free healing.

MATERIALS AND METHODS
FIBROBLAST PRIMARY CULTURES

The keloid-producing fibroblast was established as a primary cell line from scar tissue obtained from the auricle of a white patient. Exemption to use operative specimens that would otherwise be discarded was obtained from the Human Subjects Committee of Stanford University, Stanford, Calif. Fetal fibroblasts derived from facial skin were obtained from a cell line repository (Coriell Laboratories, Camden, NJ).

Cell lines from each specimen were established and propagated in a serum-containing environment followed by a serum-free environment. Using a sterile technique under a laminar flow hood, the dermal specimen was minced into approximately 1-mm3 fragments on a Petri dish with a sterile scalpel blade. The specimens were washed in Dulbecco phosphate-buffered saline solution with a combination of 5% penicillin, streptomycin sulfate, and amphotericin B (GIBCO, Grand Island, NY). The specimens were then placed in scored 75-cm2 tissue culture flasks (T75; Falcon, Becton-Dickinson, Franklin Lakes, NJ) with 10 mL of culture medium (10% fetal calf serum in Dulbecco-modified Eagle medium with 1% levoglutamide and 1% penicillin–streptomycin sulfate–amphotericin B) (GIBCO). The specimens were then stored in a humidified incubator at 37°C with a 5% carbon dioxide atmosphere.

After 24 hours, the medium was changed with 5 mL of primary culture medium. The medium was then changed every 2 days until fibroblasts were visualized under light microscopy to be growing outward from the explanted tissue. At that time, the tissue was removed. With sufficient outgrowth of fibroblasts, cells were subcultured into 75-cm2 culture flasks. Primary culture medium was changed every third to fourth day. Successive cultures were passed at confluence.

CELL PLATING IN SERUM-FREE MEDIA

Experiments were performed with early passage cells (second through ninth passages). At the time of experimentation, confluent cells were released from the flask wall using 0.05% trypsin. The trypsin was inactivated using trypsin soybean inhibitor (GIBCO) in a 1:1 ratio. Cells were then suspended in a commercially available serum-free medium (UltraCULTURE; BioWhittaker, Walkersville, Md) that was previously shown to sustain fibroblast cell cultures for durations similar to those used in this study.16 Cells were counted in duplicate using phase-contrast microscopy and a hemacytometer. Viable cells were determined using trypan blue dye exclusion. Keloid and fetal fibroblasts were then seeded at a density of 6 × 104 cells per milliliter in each well of a 24-well plate (Falcon, Becton-Dickinson). Each cell line was cultured in triplicate.

TAMOXIFEN MODULATION

Tamoxifen was added to the appropriate wells in concentrations of 8 and 12 or 16 µmol/L after the fibroblasts were allowed 24 hours to attach to their wells. Tamoxifen, 8 µmol/L, has been shown to decrease the overall TGF-β level.13 Untreated cells from each cell line were used for controls. Keloid and fetal fibroblasts were incubated with and without tamoxifen in serum-free media for 1, 2, and 4 days. At each predetermined time point, cell-free supernatant was collected, in triplicate, from the testing wells. One-milliliter samples were stored at −75°C in microcentrifuge tubes for later growth factor assays.

Cell counts were performed using the cell proliferation reagent 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay (Boehringer Mannheim, Indianapolis, Ind) at 1, 2, and 4 days postinitiation for growth curve generation. The WST-1 assay is a colorimetric assay used in the quantification of cell proliferation and cell viability based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. It is a nonradioactive alternative to the tritium-thymidine incorporation assay. Assays were read using an automated plate reader (E1x800; Bio-Tek Instruments, Inc, Winooski, Vt). Optical densities were analyzed with software (KC4; Bio-Tek Instruments, Inc). Cell counts were determined by comparison with a standard curve derived from known cell quantities and corrected based on the initial seeding density of 6 × 104 cells per milliliter.

GROWTH FACTOR ASSAYS

Expression of TGF-β1 was evaluated for each of the triplicated postmodulation cell cultures by solid-phase enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn) at 3 representative time points: 1, 2, and 4 days. Unmodulated samples from each duplicated source were also evaluated by enzyme-linked immunosorbent assay at the 3 representative time points. Finally, cell-free samples of a commercially available serum-free media (UltraCULTURE) exposed and not exposed to tamoxifen were also assayed for TGF-β1 expression at each time point. Assays were read using an automated plate reader, and optical densities were analyzed with software (KC4).

RESULTS

Keloid and fetal fibroblasts exhibited growth in serum-free media. The configurations of the growth curves were similar regardless of the presence of tamoxifen but differed between fibroblast cell type. All 3 cell lines showed an initial decline in cell population after seeding, with a proliferative recovery after the third day. Growth curves were plotted for each cell line at each tamoxifen concentration (not shown). The TGF-β1 concentration at each time point was divided by the number of viable cells to yield graphs of TGF-β1 concentration per cell at each time point (Figure 1 and Figure 2).

No TGF-β1 was detected in the serum-free media with or without the addition of tamoxifen, as expected. Fetal cells without tamoxifen modulation exhibited the highest TGF-β1 concentration per cell for that cell type. Tamoxifen, 8 and 12 µmol/L, yielded TGF-β1 per-cell concentrations that were similar but lower than those for fetal fibroblasts without tamoxifen. Keloid cells incubated without tamoxifen showed the largest per-cell concentrations of TGF-β1. Tamoxifen, 8 and 16 µmol/L, again yielded similar but lower TGF-β1 concentrations per cell at each time point.

Statistical evaluation using the t test of the per-cell concentration for unmodulated (no tamoxifen) keloid and fetal fibroblasts showed TGF-β1 production to be significantly higher for keloid fibroblasts on days 2 (P = .02) and 4 (P = .001). Unmodulated keloid fibroblasts exhibited significantly more TGF-β1 production on day 2 (P = .05) compared with keloid fibroblasts modulated with tamoxifen, 16 µmol/L. There was no statistically significant difference between TGF-β1 production by keloid fibroblasts modulated with tamoxifen, 8 µmol/L, and those modulated with tamoxifen, 16 µmol/L, on any of the days evaluated.

There was no difference in TGF-β1 production by fetal fibroblasts modulated with tamoxifen, 8 and 12 µmol/L. Unmodulated fetal fibroblasts produced significantly more TGF-β1 on day 4 (P = .004) than fibroblasts modulated with tamoxifen, 12µ mol/L.

COMMENT

The use of a serum-free protocol in this study allowed analysis of the effect of tamoxifen on per-cell TGF-β1 production as a function of time. As the fibroblasts replicated, they were bathed in only their own autocrine growth factors instead of in serum that contains exogenous growth factors, as in previous studies.4, 13 The method used to evaluate cell growth at each time point (WST-1 assay) measured only viable cells. This allowed us to determine the average TGF-β1 concentration per viable cell at each time point.

Transforming growth factor has 3 isoforms, of which TGF-β1 is thought to stimulate greater collagen synthesis in keloids compared with normal dermal fibroblasts.4 Transforming growth factors β1 and β2 show increased expression in keloid fibroblasts relative to normal fibroblasts.4 Fetal fibroblasts exhibit scar-free healing and have a lower level of TGF-β1 than do normal fibroblasts,9 which in turn have a lower level of TGF-β1 and TGF-β2 than do keloid fibroblasts.4 The present study confirms these findings by showing that keloid fibroblasts have a higher TGF-β1 expression than do fetal fibroblasts.

Tamoxifen appears to decrease the per-cell level of TGF-β1 in fetal fibroblasts in a concentration-dependent manner. The per-cell levels of TGF-β1 stayed fairly similar throughout the 4 days of cell growth evaluated. While the absolute level of TGF-β1 is likely partially cell passage number dependent, keloid fibroblasts in this study exhibited a higher per-cell TGF-β1 concentration than did fetal cells.

Tamoxifen has been shown to decrease the overall level of TGF-β in keloid fibroblasts,13 but, to our knowledge, its effect on the individual isomers of TGF-β has not previously been examined. This study demonstrates that tamoxifen decreases the per-cell concentration of TGF-β1 in keloid fibroblasts grown in serum-free media. Higher concentrations of tamoxifen trended toward progressive decrements in the TGF-β1 level (in the present study) and in overall TGF-β levels.13 The inhibitory effect of tamoxifen on TGF-β1 production by keloid fibroblasts appeared to be consistent during the 4-day course of this experiment. Therefore, the addition of tamoxifen may lead to improved keloid wound healing by reducing the level of autocrine TGF-β1 production, bringing TGF-β1 production somewhat closer to that present in normal (and fetal) cells. This helps to explain the clinical usefulness of tamoxifen in keloid treatment.12

Wound healing results from a coordinated interplay of the 3 TGF-β isoforms. Transforming growth factors β1 and β2 appear to cause increased tissue fibrosis, while TGF-β3 may serve to down-regulate its 2 cousins.4 Altering the level of at least one of these actors (TGF-β1) may allow tamoxifen to modulate the aberrant wound healing seen in keloids. Further examination of the interplay of the comparative levels of the 3 TGF-β isoforms in fetal and keloid fibroblasts, which exhibit scar-free and aberrant healing, respectively, may lead to an improved understanding of the complex roles of this growth factor in wound healing.

The following are our conclusions:

1. Keloid fibroblasts exhibit greater propensity for scar formation and higher TGF-β1 production than do fetal fibroblasts.

2. The addition of tamoxifen results in decreased TGF-β1 production in keloid and fetal fibroblasts.

3. The serum-free protocol used in this study allows, for the first time to our knowledge, the evaluation of TGF-β1 production by tamoxifen-modulated fibroblasts without the confounding effects of exogenous growth factors found in serum.

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Article Information

Accepted for publication August 8, 2000.

Presented at the American Academy of Facial Plastic and Reconstructive Surgery Spring Meeting, Orlando, Fla, May 13, 2000.

Corresponding author and reprints: R. James Koch, MD, MS, Division of Otolaryngology/Head and Neck Surgery, Stanford University Medical Center, 300 Pasteur Dr, Stanford, CA 94305-5328 (e-mail: rjk@stanford.edu).

References
1.
Su  CWAlizadeh  KBoddie  ALee  RC The problem scar. Clin Plast Surg. 1998;25451- 467
2.
Di Cesare  PECheung  DTPerelman  N  et al.  Alteration of collagen composition and crosslinking in keloid tissues. Matrix. 1990;10172- 178Article
3.
Younai  SNichter  LSWellisz  T  et al.  Modulation of collagen synthesis by transforming growth factor-β in keloid and hypertrophic scar fibroblasts. Ann Plast Surg. 1994;33148- 151Article
4.
Lee  TYChin  GSKim  WJHChau  DGittes  GKLongaker  MT Expression of transforming growth factor-β 1, 2, and 3 proteins in keloids. Ann Plast Surg. 1999;43179- 184
5.
Polo  MSmith  PDKim  YJWang  XKo  FRobson  MC Effect of TGF-β2 on proliferative scar fibroblast cell kinetics. Ann Plast Surg. 1999;43185- 190Article
6.
Mackool  RJGittes  GKLongaker  MT Scarless healing: the fetal wound. Clin Plast Surg. 1998;25357- 365
7.
Lorenz  HPLin  RYLongaker  MTWhitby  DJAdzick  NS The fetal fibroblast: the effector cell of scarless fetal skin repair. Plast Reconstr Surg. 1995;961251- 1259Article
8.
Ferguson  MWJHowath  GF Marsupial models of scarless fetal wound healing. Adzick  NSLongaker  MTeds.Fetal Wound Healing. New York, NY Elsevier Scientific Press1992;95- 124
9.
Ihara  SMotobayashi  Y Wound closure in foetal rat skin. Development. 1992;114573- 582
10.
Martin  PLewis  J Actin cables and epidermal movement in embryonic wound healing. Nature. 1992;360179- 182Article
11.
Broker  BJChakrabarti  RBlynman  TRoesler  JWang  MBSrivatsan  ES Comparison of growth factor expression in fetal and adult fibroblasts: a preliminary report. Arch Otolaryngol Head Neck Surg. 1999;125676- 680Article
12.
Mancoll  JSMacauley  RLPhillips  LG The inhibitory effect of tamoxifen on keloid fibroblasts. Surg Forum. 1996;47718- 720
13.
Chau  DMancoll  JSLee  S  et al.  Tamoxifen downregulates TGF-β production in keloid fibroblasts. Ann Plast Surg. 1998;40490- 493Article
14.
Koch  RJGoode  RLSimpson  GT Serum-free keloid fibroblast cell culture: an in vitro model for the study of aberrant wound healing. Plast Reconstr Surg. 1997;991094- 1098Article
15.
Nowak  KCMcCormack  MKoch  RJ The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors: a serum-free study. Plast Reconstr Surg. 2000;1052039- 2048Article
16.
Hong  RHLum  JKoch  RJ Growth of keloid-producing fibroblasts in commercially available serum-free media. Otolaryngol Head Neck Surg. 1999;121469473Article
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