ARTICLE Scarring
is a specific feature of human wound healing. It is both aesthetically disfiguring
and can cause functional problems as well. In genetically predisposed individuals
excessive, hypertrophic scarring and the formation of keloids arise as a
result of abnormal wound healing. They are at least in part due to an overproduction
of collagen types I and III [1].
Tamoxifen, which has been in use as chemotherapy of breast cancer for
two decades, has an additional inhibitory effect on the proliferation
of fibroblasts, and has been applied recently in the treatment of retroperitoneal
fibrosis and desmoid tumors [2-7].
Hu et al. [8] have shown a marked effect of the nonsteroidal
anti-estrogen tamoxifen on human fibroblasts involved in wound healing.
Mainly using cell biological methods such as cell proliferation or collagen
lattice contraction, they noted a reversible inhibitory effect on normal
skin fibroblasts as well as an influence on fibroblast morphology.
Similarly, Bruzzese et al. [9] investigated the suspected anti-proliferative
effect of lysine acetylsalicylate (LAS), which among other nonsteroidal
anti-inflammatory drugs has been shown to interfere with cell growth.
Earlier, Bernhardt et al. [10] described an inhibitory effect of
LAS at high concentrations on vascular smooth muscle cell proliferation,
but no effect on immune cells has been noticed in vitro [11]. Also,
Red'kin et al. [12] observed mitostatic effects on hematopoetic
stem cells after administering LAS.
Methods
Patients
Punch biopsies from the keloid or scarred area were obtained from 6
patients after informed consent according to the guidelines of the ethics
committee of the University of Leipzig. All biopsies were taken from the
upper chest, back or upper arm area. Skin fibroblasts from 3 age-matched
donors were used as normal dermal fibroblast controls from corresponding
sites.
Cell culture
Monolayer cultures of fibroblasts were established by outgrowth from
punch biopsies or dissected tissue obtained from the surgery unit of the
University Dermatology Department and maintained under standard conditions
(37° C, 5% (v/v) CO2 in air, 95% (v/v) relative humidity)
with Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum,
L-glutamine (292 mug/ml), penicillin (200 IE/ml), streptomycin (200 mug/ml)
and ascorbic acid (50 mug/ml) [13, 14]. In all experiments, fibroblasts
between passages 4 and 7 were used under identical experimental conditions.
Drug
Lysine acetylsalicylate (LAS; Bayer AG, Leverkusen Germany, Aspisol)
was dissolved in sterile water (180 mg/ml) and diluted to final concentrations
of 20, 200, 1,000, or 2,000 mug/ml in DMEM. After 24 h growth in DMEM
(initial density 300,000 cells/75 cm2 cell culture flask),
the medium was replaced with fresh medium in the LAS concentrations indicated
above.
Cell proliferation assay
To determine a possible cytotoxic effect of LAS, we assayed cell proliferation
with a commercial test kit (CellTiter 96 AQ, Promega, Mannheim) according
to the manufacturer's directions. Briefly, 2,000 cells per standard well
of flat-bottom 96-well plates were seeded and incubated in DMEM for 16
h at 37° C and 5% CO2. Then, LAS was added in the final
concentrations as indicated above. Proliferation was measured with fibroblasts
from three donors in triplicate (Dynatech MR 5,000 plate reader) after
24 h, 4 or 7 days, respectively, and compared to controls incubated only
with DMEM.
RNA extraction and Northern blot analysis
Northern blot analysis was carried out as described [15]. Briefly, mRNA
was isolated from 70 to 80 % confluent human cell cultures, quiescent
dermal fibroblasts as control and fibroblasts from the keloid patients
using the QuickPrep Micro mRNA purification kit (Amersham-Pharmacia-Biotech,
Freiburg). Cells gave comparable results between passages four to eight.
Approximately 500 ng mRNA was fractionated by electrophoresis on a 1.2%
agarose gel containing 1% formaldehyde followed by transfer to nylon membranes
(Porablot NY plus, Macherey & Nagel, Germany). Filters were baked
for 2 h at 80° C and hybridized with 100 ng/ml digoxigenin-UTP-labeled
RNA (riboprobe) for 16 h. The hybrids were made visible by an enzyme-linked
immunoassay and a subsequent enzyme-catalyzed chemiluminescent reaction
with CSPD (Tropix, Roche Diagnostica, Mannheim, Germany). The riboprobe
for human glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was transcribed
in vitro from a cloned 389-bp PCR product into the SmaI site of
cloning vector pBluescript SK+ (Stratagene, Heidelberg). The
probes for human MMP 1 and human TIMP 1 were obtained from cloned RT-PCR
products. The human procollagen alpha1(I) (Hf677) and procollagen alpha1(III)
(Hf934) clones were a gift from Prof. Thomas Krieg, Dermatology, University
of Cologne.
Optical densities of bands corresponding to the genes of interest were
measured using a computer-assisted imaging system (Kodak Image Station
440 CF). The values obtained were normalized to those of GAPDH in the
same experiment, and will be referred to as the relative optical density.
Statistical analysis
The linearity of the dose-response curve in the cell growth experiments
was verified by an F test. The significance of growth differences between
treated and non-treated fibroblasts was tested by an unpaired two-tailed
Student's t-test.
Results
Influence of LAS on the proliferation of fibroblasts
from healthy donors and keloid lesions
The proliferation of fibroblasts derived from normal skin (average of
populations from 3 different donors) was not substantially affected by
20 µg/ml LAS, after neither 1, 3 or 7 days (Fig.
1).
Treatment with 200 µg/ml LAS caused a marked reduction of proliferation
at the later time points (day 3 and 7) compared to the untreated controls.
The maximal growth inhibition could be observed at all time points after
exposure to 1,000 and 2,000 mug/ml LAS with no significant difference
between these concentrations at days 3 and 7. The inhibition reached between
83% and 86%, when the control was set to 100% proliferation.
Keloid fibroblasts basically behaved in a similar manner, with slight
differences, however. The keloid fibroblast populations (average from
6 different donors) showed an increased rate of proliferation after 3
and 7 days incubation in the non-exposed control. LAS concentrations of
20 mug/ml did not influence this increased proliferation rate. However,
high concentrations of LAS caused a relatively stronger growth reduction
in keloid fibroblasts compared to normal skin fibroblasts. The differences
between the two tested keloid fibroblast populations were greater than
differences between normal and keloid fibroblasts, and generally the differences
between normal and keloid fibroblasts were statistically not significant,
unless specifically marked.
Interestingly, in either type of cell, after 3 days of culture a decrease
in extinction corresponding to reduced proliferation was noticed. After
7 days, the untreated and 20 mug/ml LAS-treated fibroblasts proliferated
again, compared to day 3 with 200 µg/ml LAS, untreated fibroblasts
regained some proliferative capacity (absorbance 0.951 at day 7 vs. 0.647
at day 3), whereas keloid fibroblasts stayed at the same reduced level
as on day 3 (absorbance around 0.800).
Influence of LAS on normal skin and keloid fibroblasts
at the level of mRNA expression
In Figure 2 we present
typical Northern data of mRNA prepared from normal skin fibroblasts and
keloid fibroblasts after hybridization with riboprobes for procollagen
alpha1(I), procollagen alpha1(III), MMP 1, TIMP 1, and GAPDH. In Figure
3, the diagram with calculated densitometric values depicts the course
of expression in different fibroblast populations after exposure to increasing
amounts of LAS. After 6 days of incubation with 0, 20 or 200 mug/ml LAS,
respectively, normal skin fibroblasts show markedly increased expression
of procollagen alpha1(I): 2.6 times at 20 mug/ml and 3.3 times at 200
mug/ml, compared to untreated fibroblasts. In contrast, keloid fibroblasts,
which show the expected elevated baseline of about double the expression
level of normal skin fibroblasts, had only about 1.6 times increased levels
of procollagen alpha1(I) at a dose of 20 mug/ml and returned to the original
level at 200 mug/ml LAS. For procollagen alpha1(III), we noticed a similar
increase in synthesis after treatment with 200 mug/ml LAS in normal fibroblasts,
whereas at 20 mug/ml no increase was observed. Interestingly, the increase
in procollagen alpha1(III) in keloid fibroblasts was much more pronounced
at 20 mug/ml LAS (2.4 times) and 200 µg/ml (2.0 times) than for procollagen
I.
Among other MMPs, MMP 1 or interstitial collagenase 1, plays a key role
in the turnover of extracellular matrix proteins. To determine whether
MMP 1 is affected by LAS, we measured the expression of MMP 1 at the mRNA
level and also the expression of its natural inhibitor, TIMP 1. However
the densitometric data show that the expression of both genes was not
influenced by different doses of LAS.
Discussion
We investigated the influence of LAS on the proliferation and gene expression
of normal skin fibroblasts and human keloid fibroblasts in vitro.
Our experiments showed similar results for fibroblasts derived from skin
as other authors obtained for cells from nasal polyps [9].
Cell proliferation
We tested in vitro the expected anti-proliferative activity at
concentrations in a range of 20-2,000 mg/ml in culture medium containing
10% FCS. According to findings by Bayer and Beaven [16], it could be expected
that some of the inhibitory effect of LAS would be antagonized by the
serum. However, we used medium containing 10% FCS, because at 2% serum
the proliferation of fibroblasts usually does not proceed completely normally.
We demonstrated that LAS has a dose-dependent anti-proliferative effect
on both normal and keloid fibroblasts. After exposure to 20 mug/ml LAS,
there was no effect on proliferation of the cells during the observation
period. An effect on the proliferation became clear only at a dose of
200-2,000 mug/ml. After 1 day at 200 µg/ml, the effect was not pronounced,
but was clearly visible after 3 days, and even more after 7 days. Interestingly,
there was no significant difference between normal skin and keloid fibroblasts,
whereas the differences between different populations of keloid fibroblasts
were more pronounced. At doses of 1,000 or 2,000 mug/ml LAS, we observed
a significant inhibition of proliferation in all cells regardless of the
type. The cells were still viable at 2,000 mug/ml LAS, which suggests
a cytostatic effect of LAS on fibroblasts similar to the effect of indomethacin
[16].
Since the concentration of LAS that most effectively inhibits fibroblast
proliferation is markedly higher (> 200 mug/ml) than that currently
used in low-dose treatment of patients, further clinical trials should
address the potential benefit of high-dose LAS treatment of keloids. These
trials most likely will focus on topical application, since high systemic
levels of LAS (> 200 mg/l) might be toxic, as stated by the manufacturer
in the directions for professionals "Fachinformation Aspisol".
Gene expression
Metabolism of matrix proteins such as collagens I and III is a major
event during connective tissue turnover and wound healing. If this process
is not tightly controlled, it apparently can cause the formation of keloids.
Recently, an elegant study by Nirodi et al. [17] showed differences
between normal and keloid fibroblasts at the level of chemokine regulation,
whereas the present study is focused on matrix protein expression. A recent
study investigated an influence of aspirin and related drugs on wound
healing [18] and found a retardation of the process in the presence of
aspirin. After we confirmed the anti-proliferative effect of LAS on fibroblasts
in our experiments, we also looked at the molecular level for an influence.
The earliest effect on matrix gene expression can be seen at the mRNA
level, where we measured procollagen I and III expression as well as the
key collagen turnover enzyme MMP 1 and its inhibitor TIMP 1.
Clearly, the data show that in normal and keloid fibroblasts the expression
level of collagen I and, somewhat more pronounced at 200 mug/ml, collagen
III, is dose-dependently increased under the influence of LAS. Whether
this is caused by enhanced transcription or by altered mRNA stability,
cannot be distinguished from our results. Surprisingly, procollagen I
(and to some extent procollagen III) expression is even more increased
in normal skin fibroblasts compared to keloid fibroblasts. For the other
two genes, we did not observe LAS-dependent differences in gene expression.
The result could be explained with the influence of LAS on proliferation:
under high doses of LAS the fibroblasts do not proliferate, which means
more energy is available for the production and accumulation of cell matrix.
The observation that keloid fibroblasts produce less procollagen mRNA
than normal fibroblasts under the influence of LAS could support the proposed
suppression of excess matrix protein synthesis.
Further experiments should investigate how LAS inhibits the proliferation
of normal and keloid fibroblasts. It is well known that acetyl salicylates
interfere with the prostaglandin pathway by inhibiting cyclooxygenase
I. Thus, a possible action of LAS in the prostaglandin pathway has to
be examined.
The initial experiments reported here have been carried out in monolayer
cultures which are well established for studies of proliferation rates
and mRNA levels [1]. However, to obtain more detailed answers on the mode
of action under the influence of LAS and, possibly, the influence of growth
factors or hormones, three-dimensional experimental conditions such as
collagen gels will be better suited. Several reports on differences between
expression in monolayer cultures and three-dimensional culture systems
have been published [19, 20].
CONCLUSION
Acknowledgements
The authors would like to thank Mrs. H. Gedicke for her skilful technical
assistance.
Article accepted on 10/12/01
REFERENCES
1. Chau D, Mancoll JS, Lee S, Zhao J, Phillips LG, Gittes GK, Longaker
MT. Tamoxifen downregulates TGF-beta production in keloid fibroblasts.
Ann Plast Surg 1998; 40: 490-3.
2. Jung-Testas I, Baulieu EE. Effects of steroid hormones and antihormones
in cultured cells. Exp Clin Endocrinol 1985; 86: 151-64.
3. El Yazidi I, Renaud F, Laurent M, Courtois Y, Boilly-Marer Y. Production
and oestrogen regulation of FGF1 in normal and cancer breast cells. Biochim
Biophys Acta 1998; 1403: 127-40.
4. Pellicciari C, Mangiarotti R, Bottone MG, Danova M, Wang E. Identification
of resting cells by dual-parameter flow cytometry of statin expression
and DNA content. Cytometry 1995; 21: 329-37.
5. Sertoli MR, Bruzzi P, Pronzato P, Queirolo P, Amoroso D, Del Mastro
L, Venturini. M, Vigani A, Bertelli G, Campora E. Randomized cooperative
study of perioperative chemotherapy in breast cancer. J Clin Oncol 1995;
13: 2712-21.
6. al-Musawi D, Mitchenere P, al-Akraa M. Idiopathic retroperitoneal
fibrosis treated with tamoxifen only. Br J Urol 1998; 82: 442-3.
7. Hashimoto M, Inoue S, Ogawa S, Conrad C, Muramatsu M, Shackelford
D, Masliah E. Rapid fragmentation of vimentin in human skin fibroblasts
exposed to tamoxifen: a possible involvement of caspase-3. Biochem
Biophys Res Commun 1998; 247: 401-6
8. Hu D, Hughes MA, Cherry GW. Topical tamoxifen-a potential therapeutic
regime in treating excessive dermal scarring? Br J Plast Surg 1998;
51: 462-9.
9. Bruzzese N, Sica G, Iacopino F, Paludetti G, Schiavino D, Nucera
E, Scarano E, Patriarca G. Growth inhibition of fibroblasts from nasal
polyps and normal skin by lysine acetylsalicylate. Allergy 1998;
53: 431-4.
10. Bernhardt J, Rogalla K, Luscher TF, Buhler FR, Resink TJ. Acetylsalicylic
acid, at high concentrations, inhibits vascular smooth muscle cell proliferation.
J Cardiovasc Pharmacol 1993; 21: 973-6.
11. Uhlenbruck G, Lotzerich H, Bernhardt J, Rogalla K. Acetylsalicylic
acid has no effects on various isolated immune cells in vitro.
Eur J Appl Physiol 1993; 66: 473-6.
12. Red'kin IuV, Stepina NI, Lisnikova VE. The mitostatic activity of
benzylpenicillin and aspisol in an experiment [Article in Russian]. Eksp
Klin Farmakol 1994; 57: 44-8.
13. Gebauer S, Saalbach A, Mittag M, Neubauer H, Adelmann-Grill B, Petri
JB, Herrmann K. Expression of adhesion molecules during wound healing
- characterization of granulation fibroblasts. J. für Anästhesie
und Intensivbehandlung - Current Aspects in Wound Healing J A I 1996;
3: 32-42.
14. Petri JB, Saalbach A, Pierer M, Haupt B, Haustein UF, Herrmann K.
In vitro analysis of adhesion molecule expression and gel contraction
of human granulation fibroblasts. Wound Rep Regen 1997; 5: 69-76.
15. Petri JB, König S, Haupt B, Haustein UF, Herrmann K. Molecular
analysis of different phases in human wound healing. Experimental Dermatology
1997; 6: 133-9.
16. Bayer BM, Beaven MA. Evidence that indomethacin reversibly inhibits
growth in the G1 phase of the cell cycle. Biochem Pharmacol 1979; 28:
441-3.
17. Nirodi CS, Devalaraja R, Nanney LB, Arrindell S, Russell S, Trupin
J, Richmond A. Chemokine and chemokine receptor expression in keloid and
normal fibroblasts. Wound Repair and Regeneration 2000; 8: 371-82.
18. Yoshizawa T, Watanabe S, Hirose M, Yamamoto J, Osada T, Sato K,
Oide H, Kitamura T, Takei Y, Ogihara T, Miwa H, Miyazaki A, Sato N. Effects
of growth factors on aspirin-induced inhibition of wound repair in a rabbit
gastric epithelial cell model. Aliment Pharmacol Ther 2000; (suppl.
1): 176-82.
19. Sato M, Ishikawa O, Miyachi Y. Distinct pattern of collagen gene
expression are seen in normal and keloid fibroblasts grown in three-dimensional
culture. Br J Dermatol 1998; 138: 938-43.
20. Mauch C, Hatamochi A, Scharffetter K, Krieg T. Regulation of collagen
synthesis in fibroblasts within a three-dimensional collagen gel. Exp
Cell Res 1988; 178: 493-503.
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