 |
|
Effect of Tamoxifen on Transforming Growth Factor ß1 Production by Keloid and Fetal Fibroblasts
Anthony A. Mikulec, MD;
Matthew M. Hanasono, MD;
Joanne Lum, BS;
James M. Kadleck;
Magdalena Kita;
R. James Koch, MD, MS
Arch Facial Plast Surg. 2001;3:111-114.
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) ß1. 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-ß1 expression.
Results Keloid fibroblasts show increased per-cell TGF-ß1 production
compared with fetal fibroblasts. Tamoxifen appeared to decrease per-cell TGF-ß1 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-ß1 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-ß1.
INTRODUCTION
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) ß1 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 ß is secreted by multiple cells, including
fibroblasts, and has 3 isoforms. Transforming growth factors ß1 and ß2 are overproduced by keloid fibroblasts compared
with normal fibroblasts.4 Exogenous TGF-ß2 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-ß1 on the rate of collagen synthesis in keloid fibroblasts,
fibroblasts from a hypertrophic scar, and normal skin fibroblasts. In response
to exogenous TGF-ß1, 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-ß1 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-ß 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-ß1 secretion in normal and keloid fibroblasts
in a fluence-dependent manner.15
This study sought to evaluate the effects of tamoxifen on TGF-ß1 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 x 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-ß 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 x 104
cells per milliliter.
GROWTH FACTOR ASSAYS
Expression of TGF-ß1 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-ß1 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-ß1 concentration
at each time point was divided by the number of viable cells to yield graphs
of TGF-ß1 concentration per cell at each time point (Figure 1 and Figure 2).
|
|
|
|
Figure 1. The average transforming growth
factor (TGF) ß1 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) ß1 concentration produced per cell in a serum-free
fetal fibroblast cell culture. Tamoxifen was added as tamoxifen citrate.
|
|
|
No TGF-ß1 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-ß1 concentration per
cell for that cell type. Tamoxifen, 8 and 12 µmol/L, yielded TGF-ß1 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-ß1. Tamoxifen,
8 and 16 µmol/L, again yielded similar but lower TGF-ß1
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-ß1 production to be significantly higher
for keloid fibroblasts on days 2 (P = .02) and 4
(P = .001). Unmodulated keloid fibroblasts exhibited
significantly more TGF-ß1 production on day 2 (P = .05) compared with keloid fibroblasts modulated with tamoxifen,
16 µmol/L. There was no statistically significant difference between
TGF-ß1 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-ß1 production by fetal
fibroblasts modulated with tamoxifen, 8 and 12 µmol/L. Unmodulated fetal
fibroblasts produced significantly more TGF-ß1 on day 4 (P = .004) than fibroblasts modulated with tamoxifen, 12
µmol/L.
COMMENT
The use of a serum-free protocol in this study allowed analysis of the
effect of tamoxifen on per-cell TGF-ß1 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-ß1 concentration per viable cell at each time point.
Transforming growth factor has 3 isoforms, of which TGF-ß1 is thought to stimulate greater collagen synthesis in keloids compared
with normal dermal fibroblasts.4 Transforming
growth factors ß1 and ß2 show increased expression
in keloid fibroblasts relative to normal fibroblasts.4
Fetal fibroblasts exhibit scar-free healing and have a lower level of TGF-ß1 than do normal fibroblasts,9 which
in turn have a lower level of TGF-ß1 and TGF-ß2 than do keloid fibroblasts.4 The present
study confirms these findings by showing that keloid fibroblasts have a higher
TGF-ß1 expression than do fetal fibroblasts.
Tamoxifen appears to decrease the per-cell level of TGF-ß1 in fetal fibroblasts in a concentration-dependent manner. The per-cell
levels of TGF-ß1 stayed fairly similar throughout the 4 days
of cell growth evaluated. While the absolute level of TGF-ß1
is likely partially cell passage number dependent, keloid fibroblasts in this
study exhibited a higher per-cell TGF-ß1 concentration than
did fetal cells.
Tamoxifen has been shown to decrease the overall level of TGF-ß
in keloid fibroblasts,13 but, to our knowledge,
its effect on the individual isomers of TGF-ß has not previously been
examined. This study demonstrates that tamoxifen decreases the per-cell concentration
of TGF-ß1 in keloid fibroblasts grown in serum-free media.
Higher concentrations of tamoxifen trended toward progressive decrements in
the TGF-ß1 level (in the present study) and in overall TGF-ß
levels.13 The inhibitory effect of tamoxifen
on TGF-ß1 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-ß1 production, bringing TGF-ß1 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-ß
isoforms. Transforming growth factors ß1 and ß2 appear to cause increased tissue fibrosis, while TGF-ß3
may serve to down-regulate its 2 cousins.4
Altering the level of at least one of these actors (TGF-ß1)
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-ß
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-ß1 production than do fetal fibroblasts.
2. The addition of tamoxifen results in decreased TGF-ß1
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-ß1 production
by tamoxifen-modulated fibroblasts without the confounding effects of exogenous
growth factors found in serum.
AUTHOR 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{at}stanford.edu).
From the Wound Healing and Tissue Engineering Laboratory, Division
of Otolaryngology/Head and Neck Surgery, Stanford University Medical Center,
Stanford, Calif.
REFERENCES
| |
1. Su CW, Alizadeh K, Boddie A, Lee RC. The problem scar. Clin Plast Surg. 1998;25:451-467.
ISI
| PUBMED
2. Di Cesare PE, Cheung DT, Perelman N, et al. Alteration of collagen composition and crosslinking in keloid tissues. Matrix. 1990;10:172-178.
ISI
| PUBMED
3. Younai S, Nichter LS, Wellisz T, et al. Modulation of collagen synthesis by transforming growth factor-ß
in keloid and hypertrophic scar fibroblasts. Ann Plast Surg. 1994;33:148-151.
FULL TEXT
|
ISI
| PUBMED
4. Lee TY, Chin GS, Kim WJH, Chau D, Gittes GK, Longaker MT. Expression of transforming growth factor-ß 1, 2, and 3 proteins
in keloids. Ann Plast Surg. 1999;43:179-184.
ISI
| PUBMED
5. Polo M, Smith PD, Kim YJ, Wang X, Ko F, Robson MC. Effect of TGF-ß2 on proliferative scar fibroblast cell
kinetics. Ann Plast Surg. 1999;43:185-190.
ISI
| PUBMED
6. Mackool RJ, Gittes GK, Longaker MT. Scarless healing: the fetal wound. Clin Plast Surg. 1998;25:357-365.
ISI
| PUBMED
7. Lorenz HP, Lin RY, Longaker MT, Whitby DJ, Adzick NS. The fetal fibroblast: the effector cell of scarless fetal skin repair. Plast Reconstr Surg. 1995;96:1251-1259.
ISI
| PUBMED
8. Ferguson MWJ, Howath GF. Marsupial models of scarless fetal wound healing. In: Adzick NS, Longaker MT, eds. Fetal Wound Healing. New York, NY: Elsevier Scientific Press; 1992:95-124.
9. Ihara S, Motobayashi Y. Wound closure in foetal rat skin. Development. 1992;114:573-582.
ABSTRACT
10. Martin P, Lewis J. Actin cables and epidermal movement in embryonic wound healing. Nature. 1992;360:179-182.
FULL TEXT
| PUBMED
11. Broker BJ, Chakrabarti R, Blynman T, Roesler J, Wang MB, Srivatsan ES. Comparison of growth factor expression in fetal and adult fibroblasts:
a preliminary report. Arch Otolaryngol Head Neck Surg. 1999;125:676-680.
FREE FULL TEXT
12. Mancoll JS, Macauley RL, Phillips LG. The inhibitory effect of tamoxifen on keloid fibroblasts. Surg Forum. 1996;47:718-720.
13. Chau D, Mancoll JS, Lee S, et al. Tamoxifen downregulates TGF-ß production in keloid fibroblasts. Ann Plast Surg. 1998;40:490-493.
FULL TEXT
|
ISI
| PUBMED
14. Koch RJ, Goode RL, Simpson GT. Serum-free keloid fibroblast cell culture: an in vitro model for the
study of aberrant wound healing. Plast Reconstr Surg. 1997;99:1094-1098.
ISI
| PUBMED
15. Nowak KC, McCormack M, Koch 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;105:2039-2048.
ISI
| PUBMED
16. Hong RH, Lum J, Koch RJ. Growth of keloid-producing fibroblasts in commercially available serum-free
media. Otolaryngol Head Neck Surg. 1999;121:469-473.
FULL TEXT
|
ISI
| PUBMED
THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES
Treatment of Keloids and Hypertrophic Scars: A Meta-analysis and Review of the Literature.
Leventhal et al.
Arch Facial Plast Surg 2006;8:362-368.
ABSTRACT
| FULL TEXT
Effects of tamoxifen on normal human dermal fibroblasts.
Ruffy et al.
Arch Facial Plast Surg 2006;8:329-332.
ABSTRACT
| FULL TEXT
Effect of Hyperbaric Oxygen on the Growth Factor Profile of Fibroblasts
Kang et al.
Arch Facial Plast Surg 2004;6:31-35.
ABSTRACT
| FULL TEXT
|
