ORIGINAL ARTICLE

Dexamethasone Induction of Keloid Regression

through Effective Suppression of VEGF Expression
and Keloid Fibroblast Proliferation
Wen-Sheng Wu1,2, Feng-Sheng Wang1, Kuender D. Yang1, Chao-Cheng Huang3 and Yur-Ren Kuo1,2

The biological mechanism underlying steroid therapy for treating keloids remains unclear. Analytical results
demonstrated that topical intra-lesional steroid injections suppress vascular endothelial growth factor (VEGF)
expression in keloid tissue and induce its regression in vivo. This study investigated whether glucocorticoid
(dexamethasone) downregulates VEGF expression and hinders keloid fibroblast (KF) proliferation in keloid
regression. Primary KF cultures were treated with various concentrations of dexamethasone, glucocorticoid
receptor (GR) antagonist (mifeprostone, RU-486), VEGF-A antibody, VEGF receptor-2 (VEGF-R2) antagonist
(SU-5416), and VEGF protein. Analytical results demonstrated that dexamethasone retarded KFs proliferation.
However, suppression of fibroblast proliferation by dexamethasone pre-treatment was reduced by adding
exogenous VEGF protein. Dexamethasone suppressed endogenous VEGF mRNA induction, protein expressed
by KFs, and angiogenesis activity detected by a tube-forming assay of human umbilical vein endothelial cells co-
cultured fibroblasts. These effects were reversed by pre-treatment with RU-486, and not by pre-treatment with
SU-5416. Thus, dexamethasone induces keloid regression via interaction with the GR and suppresses
endogenous VEGF expression and fibroblast proliferation. However, exogenous VEGF promotes fibroblast
proliferation through the GR-independent pathway. Modulation of VEGF production may comprise a valuable
treatment modality for keloids.
Journal of Investigative Dermatology (2006) 126, 1264–1271. doi:10.1038/sj.jid.5700274; published online 30 March 2006

INTRODUCTION The pathophysiological events resulting in keloid forma-

Keloid formation following wound healing frequently occurs in
African and Asian populations (Rockwell et al., 1989). Keloids,
which are pathological scars defined as benign cutaneous
tumors extending beyond wound margins, are distinguished by
substantial deposition of collagen in the dermis, resulting in an
imbalanced production and aggregation of extracellular matrix
(Diegelmann et al., 1979; Murray, 1994; Wu et al., 2004).
1
Department of Medical Research, Chang Gung Memorial Hospital at
Kaohsiung, Chang Gung University, Kaohsiung, Taiwan; 2Department of
Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital at
Kaohsiung, Chang Gung University, Kaohsiung, Taiwan and 3Department of
Pathology, Chang Gung Memorial Hospital at Kaohsiung, Chang Gung
University, Kaohsiung, Taiwan
Presented at the 50th Anniversary Meeting of the Plastic Surgical Research
Council, Toronto, Canada, May 18–21, 2005.
Correspondence: Dr Yur-Ren Kuo, Department of Plastic and Reconstructive
Surgery, Chang Gung Memorial Hospital at Kaohsiung, Chang Gung
University, Taiwan, 123 Ta-Pei Road, Niao-Sung Hsiang, Kaohsiung 83305,
Taiwan. E-mail: t1207816@ms22.hinet.net
Abbreviations: GR, glucocorticoid receptor; HRP, horseradish peroxidase;
HUVEC, human umbilical vein endothelial cell; IC, immunocytospin;
IGF, insulin growth factor; IHC, immunohistochemical staining; KF, keloid
fibroblast; PCNA, proliferating cell nuclear antigen; MTT, 3-[4,5-di-
methylthiazole]-2,5-diphenyletetrazolium bromide; TGF-b, transforming
growth factor-b; VEGF, vascular endothelial growth factor; VEGF-R, vascular
endothelial growth factor receptor
Received 11 March 2005; revised 27 January 2006; accepted 28 January
2006; published online 30 March 2006
tion remain unclear (Appleton et al., 1996). Research has
focused on investigating growth factors, such as vascular
endothelial growth factor (VEGF), transforming growth factor-
b (TGF-b), insulin growth factor (IGF), etc., that likely alter the
regeneration of the wound-healing cascade in regulating scar
development (Rockwell et al., 1989; Bennett and Schultz,
1993). Cytokines, such as IL-6, tumor necrosis factor-a, are
also associated with keloid scar formation (McCauley et al.,
1992; Xue et al., 2000). Excisional surgery, laser therapy,
radiotherapy, cryosurgery, 5-fluorouracil, and silicone sheet-
ing are frequently applied therapeutic modalities for mana-
ging keloids (Pollack and Goslen, 1982; Borgognoni, 2002;
Zouboulis et al., 2002; Kuo et al., 2004; Malaker et al., 2004;
Nanda and Reddy, 2004). These modalities, which amelio-
rate or eradicate keloid scar formation, have all obtained
controversial outcomes for regression and arresting of keloid
scar development (Shaffer et al., 2002). Intra-lesional
corticosteroid as a monotherapy or combined with surgery
for large keloids has proven efficient for flattening and
reducing keloids (Kiil, 1977; Boyadjiev et al., 1995; Patel and
Hall, 2000). However, the mechanistic role of steroids in
moderating keloid formation remains unclear.
Glucocorticoid dexamethasone has numerous effects
during wound-healing sequences. In vitro studies have
demonstrated that glucocorticoid dexamethasone suppresses
fibroblast proliferation of TGF-b (Slavin et al., 1994; Carroll

1264 Journal of Investigative Dermatology (2006), Volume 126 & 2006 The Society for Investigative Dermatology

et al., 2002). Recently, speculation based on experimental
data hypothesized that excess production of VEGF is crucial
to keloid tissue formation (Steinbrech et al., 1999; Gira
et al., 2004). However, few experimental studies have investi-
gated the relationship between VEGF expression and dexa-
methasone.
VEGF, a proangiogenic cytokine, has been implicated as
crucial to normal and pathological wound healing (Le et al.,
2004). As an angiogenic peptide composed a variety of
isoforms, VEGF is also a vascular permeability factor that
promotes neo-vascularization and cell growth (Sayah et al.,
1999; Carmeliet and Jain, 2000). The most abundant of
the four VEGF isoforms, VEGF-A, is typically utilized in
biological angiogenic studies (Ferrara et al., 1992; Thomas,
1996). Recently, Gira et al. (2004) indicated that VEGF
production is abundant in the underlying dermis of keloids. In
vitro studies have indicated that VEGF is expressed at higher
levels in keloid-derived fibroblasts than in normal skin
fibroblasts (Steinbrech et al., 1999; Gira et al., 2004).
Therefore, VEGF likely plays a significant role in keloid
formation by changing the extracellular matrix.
In the authors’ clinical experience, patients with keloids
demonstrated substantial regression following steroid (triam-
cinolone) injections. In this study, keloid tissues were
obtained before and after steroid injections and analyzed.
For in vitro study, human keloid fibroblasts (KFs) were
obtained as a cell line. Proliferating cell nuclear antigen
(PCNA) and VEGF expressions in keloid tissues were
determined utilizing immunohistochemical staining (IHC).
The mRNA expressions of growth factors in keloids, such as
VEGF, IGF-1, TGF-b, and VEGF-R2, were determined using in
situ hybridization, and quantitative real-time PCR. This study
investigated whether steroids (e.g. dexamethasone) can
effectively downregulate VEGF expression and suppress KF
proliferation under keloid regression. Cell proliferation of KFs
was assessed with a 3-[4,5-dimethylthiazole]-2,5-diphenyle-
tetrazolium bromide (MTT) assay and by quantifying PCNA
expression. Protein and mRNA expressions of VEGF were
demonstrated in KFs before and after experimental treatments.
RESULTS
VEGF involved in the steroid-induced keloid tissue regression
The IHC staining results indicated a significant reduction of
KF proliferation at 7 days after intra-lesional steroid injec-
tions, demonstrated by reduced PCNA expression in keloid
tissues after steroid injections. Simultaneously, the VEGF
expression in keloid tissue was significantly suppressed
following steroid injections (Figure 1). In situ hybridization
of mRNA expression of VEGF-A in keloid tissue demonstrated
marked suppression following intra-lesional steroid therapy
compared to keloid tissue before injections. The expression of
IGF-1 in keloid tissue was slightly suppressed compared with
that following intra-lesional steroid injection. No significant
differences before and after steroid injection were observed
for TGF-b1 and VEGF-R2 (Figure 2). These analytical results
suggest that VEGF is crucial to steroid-induced keloid
regression pathways.
W-S Wu et al.
Dexamethasone-Induced Keloid Regression
a b c
Pre-treatment Post-treatment Negative control
d e f
PCNA
g h i
VEGF
Figure 1. Histological changes in keloid tissues before and following steroid
treatment. (a–c) Patient with keloid formation produced by of ear piercing. In
histological hematoxylin and eosin staining, the untreated keloid tissues
indicated hyperkeratosis, thickness of the epidermis and dermis layers, and
overproduction of excess dense collagen fibers in the extracellular matrix.
Scale bar is (b) 10 mm and (c) 50 mm. (d–f) The IHC staining with an HRP-
diaminobenzidine staining kit indicated substantial PCNA expression (brown
color in cell nuclei; arrow) in keloid before (d) intra-lesional steroid
(triamcinolone) injection. Expression of PCNA in keloid biopsy tissue was
retarded after multiple steroid injections at 4-week intervals. (g–h) The IHC
staining results indicated VEGF expression (brown color; arrow) in keloid
tissue before (g) intra-lesional steroid treatment. Expression of VEGF in keloid
tissue was suppressed after steroid injections. Scale bar ¼ 50 mm.
Dexamethasone suppresses KF proliferation, but not when
exogenous VEGF is added
The effect of dexamethasone on cellular proliferation of KFs
in vitro was detected using the MTT assay. Proliferation
activities of KFs compared with that in the control group were
clearly suppressed by 10
7 M of dexamethasone treatment
after culturing for 96 hours (Figure 3a). Adding an optimal
dosage of exogenous VEGF protein alone without dexa-
methasone treatment significantly enhanced KF proliferation
compared with that in the control group (without treatment)
(Figure 3b). The VEGF monoclonal antibody treatment group
mimicked dexamethasone-suppressed cell proliferation. Add-
ing exogenous VEGF protein to the culture medium following
dexamethasone pre-treatment for 24 hours did not hinder KF
proliferation (Figure 3c). For immunocytospin (IC), positive
PCNA antibody staining determined KFs proliferation. The
PCNA expressions in KFs following dexamethasone (10
7 M)
treatment for 48 hours were identified in relatively smaller
amounts compared with that in the control group (without
treatment) (Figure 3d). No suppressive effect of PCNA
expression in KFs was observed by adding exogenous VEGF
protein after dexamethasone pre-treatment. This analytical
finding demonstrates that adding exogenous VEGF protein
can – via a pathway other than suppression by dexametha-
sone – independently induce fibroblast proliferation.
Dexamethasone suppressed VEGF-A expression in KFs
Treatment of KFs by dexamethasone (10
7 and 10
9 M)
significantly reduced mRNA expression of VEGF-A, as
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 W-S Wu et al.
Dexamethasone-Induced Keloid Regression
Pre-treatment Post-treatment Negative control
a b c KFs through the glucocorticoid receptor
VEGF-A
d e f and enhanced mRNA expression of VEGF-A (Figure 6a).
VEGFR2
g h i dexamethasone suppresses mRNA induction of VEGF in
IGF-1
j k l (Figure 6b). However, the suppressive effect observed in
TGF-
1
Figure 2. ISH for mRNA expression in keloid tissues before and at 7 days
following each intra-lesional steroid (triamcinolone) injection. (a–c) The ISH
staining identified marked VEGF-A expression (brown color; arrow) in keloid
tissue before intra-lesional steroid treatment compared with keloid tissue prior
to steroid injections. (d–f) Expression of VEGF-R2 in keloid tissue was not
significantly different before and following intra-lesional steroid therapy.
(g–i) Expression of IGF-1 in keloid tissue was slightly suppressed following
intra-lesional steroid injection, and was not significant different than that
in untreated keloid tissue. (j–l) No significant differences before and
after steroid injection were observed for TGF-b1. Scale bar ¼ 50 mm.
determined by quantitative real-time PCR (Figure 4a).
Expression of VEGF protein in culture supernatants deter-
mined by ELISA kits was substantially reduced in groups
treated with 10
7 M of dexamethasone for 96 hours (Figure
4b). The mRNA expression of VEGF in KFs was markedly
reduced by dexamethasone treatment for 24 hours. Expres-
sion of IGF-1 was also significantly reduced after 24 hours of
co-culturing. No significant differences were observed in
mRNA expressions of TGF-b1 in KFs treated with dexa-
methasone for 24 hours (Figure 4c). Experimental results were
similar to the expressions of VEGF, IGF-1 and TGF-b1 for KFs
after dexamethasone treatment for 48 hours. These analytical
results suggest that dexamethasone is a primary suppressor of
endogenous VEGF and IGF expressions, especially VEGF,
and not a factor in KF in vitro proliferation.
Dexamethasone reduced the angiogenic effect of KFs in HUVEC
co-culture
This study examined the effect of KFs on VEGF secretion
demonstrated by the amount of tube-like formations in the
co-culture of KFs with human umbilical vein endothelial
cells (HUVEC) (Figure 5). Tube-like formations were large
and extremely abundant in untreated KFs co-cultured
with HUVEC than in HUVEC culture alone. However, the
dexamethasone and VEGF mAb treatment groups down-
regulated tube formation compared with that in the KFs
without treatment group (Po0.05) (Figure 5).
1266 Journal of Investigative Dermatology (2006), Volume 126
Dexamethasone suppressed endogenous VEGF expression in
The mRNA expression of VEGF-A in KFs treated with dexa-
methasone (10
7 M) was substantially reduced. However,
pre-treatment with an glucocorticoid receptor (GR) antagonist,
mifepristone (RU-486), suppressed the dexamethasone effect
Pre-treatment with SU-5416, a VEGF-R2 antagonist, did
not hinder the suppressive effect of dexamethasone. Pre-
treatment with RU-486 and SU-5416 suppressed the effect
of dexamethasone. This analytical finding indicates that
KFs through GR and not the VEGF-R2 pathway. Cellular
proliferation detected by MTT assay was markedly reduced
by treatment with 10
7 M of dexamethasone for 48 hours
KFs proliferation following dexamethasone treatment was re-
versed in the RU-486 treatment group and not in the SU-5416
pre-treatment group. This finding indicates the likelihood that
dexamethasone-induced suppression of endogenous VEGF
synthesis in KFs is mediated via the GR pathway.
DISCUSSION
Despite clinical, histological, and in vitro observations, the
pathogenic mechanisms underlying keloid formation remain
unclear. Numerous approaches have been applied in treating
keloids (Pollack and Goslen, 1982; Rockwell et al., 1989;
Shaffer et al., 2002; Kuo et al., 2004). However, no single
treatment has proven entirely effective. Intra-lesional gluco-
corticoid treatment is typically utilized as local therapy in
keloid treatment (Kiil, 1977; Le et al., 2004). However, the
biosignal mechanisms of corticosteroid treatments in keloids
also remain unclear.
Based on the authors’ clinical observations, fibroblast
proliferation, as demonstrated by PCNA expression in keloid
tissues, was substantially suppressed following intra-lesional
steroid injection in vivo. This in vitro study demonstrated that
an optimal dosage of steroid (dexamethasone) significantly
hindered proliferation of cultured KFs, as detected by the
MTT assay. IC staining determined a reduction in the PCNA
expression of KFs in the dexamethasone-treated group
compared with that in the untreated control group. This
analytical finding suggests that intra-lesional steroid injec-
tions likely cause keloid regression by suppressing KF
proliferation.
The release and activation of growth factors and cytokines
are essential to keloid scar formation and proliferation
(Bennett and Schultz, 1993). VEGF, IGF, or TGF-b, etc.,
have been identified (Slavin et al., 1994; Bettinger et al.,
1996; Le et al., 2004). Previous in vitro experiments
suggested that corticosteroids can retard synthesis of
pro-collagen and TGF-b expressions in KFs in vitro (Slavin
et al., 1994; Carroll et al., 2002). However, in this study,
corticosteroid (dexamethasone) did not significantly reduce
mRNA expressions of TGF-b1 – by quantitative real-time PCR
in cultured-KFs – compared with those in untreated groups.
The mRNA expressions of TGF-b1, detected via in situ
hybridization in keloid tissues, were not substantially
 W-S Wu et al.
Dexamethasone-Induced Keloid Regression

a 1.0 b 1.0 Control m

Control VEGF protein
m

Absorbance at 550 nm
Dexan (10–9 M)

Absorbance at 550 nm
* 0.9 (100 ng/ml)
0.8 Dexan (10–7 M)
VEGF protein
# 0.8 (500 ng/ml)
0.6 &
# * 0.7 &
0.4
0.6
0.2 0.5

0.0 0.4
24 hours 48 hours 96 hours 48 hours 96 hours
c
Dexan – + + – – + + –
VEGF (Dexan pre-Tx) – – + – – – + – d e
VEGF mAb – – – + – – – +
1.0
Absorbance at 550 nm

0.9 *#

0.8 #
0.7 f g
0.6 *
0.5
0.4
48 hours 96 hours
Hours after treatment

Figure 3. Dexamethasone suppressed KF proliferation, but not when exogenous VEGF was added. (a) Proliferation activities of KFs (104 cells/well) compared
with that in the control group were hindered by dexamethasone treatment using a tetrazolium-based colorimetric MTT assay (#P ¼ 0.01 and *P ¼ 0.002 indicate
significant differences compared with the untreated control group). (b) Adding an optimal dosage of exogenous VEGF protein alone significantly promoted KF
proliferation compared with that in the control group (without treatment) (&P ¼ 0.01 and yP ¼ 0.02 indicate a significant difference compared with control group
(without treatment)). (c) The VEGF monoclonal antibody treatment group mimicked dexamethasone-suppressed cell proliferation, as indicated by MTT assay.
Adding exogenous VEGF protein (100 ng/ml) to culture medium after dexamethasone (10
7 M) pre-treatment for 24 hours did not suppress KF proliferation.
Experiments were repeated six times in each study (wP ¼ 0.001, #P ¼ 0.001 and *Po0.001 as compared with the untreated control group after 48 and 96 hours,
respectively). (d–g) IC staining of (d, brown deposition indicated by arrow) expressed PCNA suggested strong positive expressions of PCNA in untreated KFs.
(e) The PCNA expressions after dexamethasone (10
7 M) treatment for 48 hours were determined in relatively smaller amounts compared with those in the
control group. However, adding VEGF protein (100 ng/ml) (f) to the culture medium after dexamethasone pre-treatment did not suppress PCNA expression in
KFs. (g) The VEGF monoclonal antibody treatment mimicked dexamethasone-suppressed cell proliferation and suppressed PCNA expression. Scale bar ¼ 50 mm.

different before and following intra-lesion steroid injections. when exogenous VEGF protein was added following dexa-
These analytical findings indicate that keloid-formation inhi- methasone pre-treatment. Notably, adding exogenous VEGF
bition by dexamethasone may not be through the TGF-b1 protein alone significantly increased keloid fibroblasts pro-
pathway activated in the cultured strain of KFs. Factors other liferation. These findings indicate that an independent path-
than TGF-b1 likely downregulate KFs proliferation. way develops when exogenous VEGF protein in KFs is added
In situ hybridization and IHC staining results demonstrated to dexamethasone.
that endogenous VEGF expression in keloid tissues, and not Gadson et al. (1984) demonstrated that GRs are active in
other growth factors, was significantly suppressed following keloid-derived and normal human fibroblasts. Whether the
intra-lesional steroid injections. The mRNA expression of suppressive effect of dexamethasone on KFs is mediated
VEGF-A and IGF-1, especially VEGF-A, in cultured KFs was through GR requires elucidation. In this study, MTT assay
markedly suppressed in the dexamethasone treatment group. results indicated that pre-treatment with RU-486, a GR
Production of the VEGF protein, detected by ELISA kits in the antagonist, reversed the suppressive effect of dexamethasone
supernatant of cultured KFs, following dexamethasone and increased KF proliferation. Conversely, the effect of
treatment was significantly downregulated. In co-cultured dexamethasone, which inhibited VEGF-A mRNA expression
HUVEC cells and KFs, the amount of tube-like formations in cultured KFs, was also reversed by adding RU-486 before
was reduced in the dexamethasone treatment group com- dexamethasone treatment. However, pre-treatment with
pared with that in the untreated control group. These SU-5416, a VEGF-R2 antagonist, did not downregulate
analytical findings imply that VEGF contributes to fibroblastic dexamethasone suppression. Combined pretreatment with
activity in keloids and dexamethasone suppresses keloid SU-5416 and RU-486 hindered the dexamethasone-suppres-
formation by modulating endogenous VEGF expression in sive effect, indicating that dexamethasone through the GR
KFs. and not the VEGF-R2 pathway suppresses endogenous VEGF-
In this study, KF proliferation was reduced by dexametha- A mRNA expression in KFs. These analytical findings suggest
sone treatment; however, proliferation was not decreased that dexamethasone suppresses VEGF mRNA induction in

www.jidonline.org 1267
 W-S Wu et al.
Dexamethasone-Induced Keloid Regression

a Expression of VEGF-A mRNA

1.4 a b
(fold decrease over control)
Control
1.2 Dexan (10–9 M)
*#
1.0 Dexan (10–7 M)

0.8
0.6
c d
0.4 #
0.2 *
0.0

b 1000
VEGF protein (pg/106 cells)

*#
Control e
800 Dexan (10–9 M) 35 *#

Tube formation of HUVEC/units

Dexan (10–7 M)
30
600 #
25 #
400
20
*
200 15 *

0 10
5
c 1.4
(fold decrease over control)

# Control 0
1.2
Expression of mRNA

* Dexan
1.0
0.8
0.6
0.4 *
0.2
0.0
TGF-
1
VEGF-A
Figure 4. Dexamethasone suppressed VEGF expression in KFs. (a) Treatment
of KFs by dexamethasone (10
7 and 10
9 M) significantly reduced mRNA
expression of VEGF-A, as determined by quantitative real-time PCR,
compared with that in the untreated control group (#P ¼ 0.01 and *Po0.001
indicate significant differences compared with the untreated control group).
(b) Expression of VEGF protein in culture supernatants determined by ELISA
kits was significantly reduced in treatment with 10
7 M dexamethasone for
96 hours (#P ¼ 0.01 and *Po0.001 indicate significant differences compared
with the untreated control group). (c) The mRNA expression of VEGF in KFs
was markedly reduced by dexamethasone treatment for 24 hours (*P ¼ 0.001).
Expression of IGF-1 was significantly reduced following 24 hours of co-
culturing (#P ¼ 0.04). No significant difference was observed in mRNA
expression of TGF-b1 in KFs treated with dexamethasone for 24 hours.
A B C D
Figure 5. Co-culturing of KFs and HUVEC was utilized to determine
angiogenic responses. (a) Tube forming assay for HUVEC alone without KFs
# as negative control. (b) Tube formations were significantly larger and more
abundant in KFs without treatment and co-cultured with HUVEC than
cultured HUVEC alone. However, (c) VEGF monoclonal antibody and
(d) dexamethasone-treatment downregulated tube formation compared with
that in the untreated control group. Scale bar ¼ 50 mm. (e) Quantities of tube
formations from different treatments. Results were conducted using six
IGF-1
paired triplicate experiments. All values of Po0.05 are in comparison with
the control group.
by suppressing their transcription, and induces keloid
regression. Dexamethasone also has positive suppression
effect on IGF transcription and IGF-1 mRNA expression. No
direct interactions inhibit TGF-b expression after dexametha-
sone treatment in keloids. Taken together, these experimental
results indicate that dexamethasone suppresses KF prolifera-
tion and downregulates endogenous VEGF expression in KFs.
Modulation of VEGF production may be an effective strategy
for inducing keloid scar regression.

cultured KFs through the GR pathway. Adding exogenous
VEGF protein – through the VEGF-R on fibroblast membranes
induce KF proliferation and not by triggering endogenous
VEGF expression – may directly account for dexamethasone’s
inability to suppress exogenous VEGF signaling. However,
further investigation of biosignal mechanisms in GR modula-
tion of VEGF transcription and expression in fibroblast
proliferation is required.
In conclusion, a proposed model of such interactions
(Figure 7) shows that although many growth factors are
involved in keloid formation, steroid (dexamethasone)
through GR pathway inhibits endogenous VEGF expression
MATERIALS AND METHODS
Preparation of KFs and reagents
One-centimeter intra-lesional excisions or punch biopsy samples of
keloids were harvested from three untreated volunteer patients. All
procedures were approved by the Institutional Review Board of
Chang Gung Memorial Hospital (CGMH), Taiwan. The study was
conducted according to The Declaration of Helsinki Principles. The
dermis was isolated and sectioned into roughly 1-mm3 fragments
using a no. 11 blade. Collagenase II (Invitrogens, Carlsbad, CA)
750 U/ml was added to these fragments and incubated at 371C for
4 hours. This mixture was aspirated through 18, 21, and 23 G
needles four times for each needle. Samples were then centrifuged at

1268 Journal of Investigative Dermatology (2006), Volume 126


RU486 (pre-Tx) – – +
SU5416 (pre-Tx) – – –
Dexan – + + +
a Exogenous VEGF
Expression of VEGF-A mRNA
(fold decrease over control)
1.4
*#
1.2
1.0
0.8
0.6
0.4
0.2
* Keloid fibroblast
0.0
b 0.7
Absorbance at 550 nm
0.6 *#
*
0.5
0.4
Figure 6. Dexamethasone suppressed VEGF-A mRNA expression through
the GR. (a) The mRNA expression of VEGF-A in KFs treated with
dexamethasone (10
7 M) for 48 hours was markedly reduced. However,
pretreatment of GR antagonist, mifepristone (RU-486), suppressed the
dexamethasone effect and enhanced mRNA expression of VEGF-A. Pre-
treatment with SU-5416, a VEGF-R2 antagonist, did not hinder the
suppressive effect of dexamethasone. Pre-treatment combined with RU-486
and SU-5416 suppressed the suppressive effect of dexamethasone (*Po0.001
and #Po0.001 indicates a significant difference between the two groups).
(b) Cellular proliferation detected by MTT assay was significantly reduced by
treatment with 10
7 M dexamethasone for 48 hours. However, the suppressive
effect of fibroblast proliferation following dexamethasone treatment was
reversed with the RU-486 and not in the SU-5416 pre-treatment group.
*Po0.001 and #P ¼ 0.009 indicates a significant difference between the two
groups.
1,500 r.p.m. for 5 minutes. Supernatant was then decanted and cell
pellets were harvested. Cells were placed in scored 25-cm2 Falcon
T-25 flasks (Becton-Dickinson, Franklin Lakes, NJ) with 5 ml culture
medium in DMEM. The medium contained 10% fetal bovine serum
(Hyclones, Taurange, New Zealand) and antibiotics–antimycotics
(Gibcos, Carlsbad, CA). Cell cultures were stored at 371C in a
humidified incubator with 5% CO2. The medium was changed at
3-day intervals until fibroblast growth was confluent. With sufficient
fibroblast outgrowth, cells were then sub-cultured or passed into
75-cm2 Falcon T-75 flasks (Becton-Dickinson, Bedford, MA) using
0.05% trypsin (Gibcos) in phosphate-buffered saline. Sub-cultures
were then placed in serum-free DMEM without phenol red-contain-
ing 0.1% bovine serum albumin (Sigmas, St Louis, MO) and the
antibiotics. KFs utilized in these experiments were limited at
passages 3–8. All experiments were performed in duplicate and
repeated three times.
Cell survival and proliferation assay
The KFs (104 cell/well) were pre-treated with various concentrations
(10
7 and 10
9 M) of dexamethasone. Additionally, KFs were
removed by adding various doses of VEGF protein and antibody
W-S Wu et al.
Dexamethasone-Induced Keloid Regression
– + Steroid
+ + (e.g. dexamethasone)
+
↓
VEGFR
GR Cell proliferation ↑
VEGF ↓↓
Cell proliferation ↓
IGF-1 ↓
Transcription factors
#
Figure 7. Scheme for proposed mechanisms of steroids in keloid regression.
Steroid (dexamethasone) suppresses KF proliferation and downregulates
endogenous VEGF expression in KFs are proposed. Adding exogenous VEGF
stimulate cellular proliferation through VEGF-R. Steroid inhibition of KF
proliferation is by an interaction with VEGF and IGF-1 transcription and
expression, especially VEGF.
#
(Sigmas). The KFs were placed in a 96-well plate for detecting cell
survival and proliferation after culturing for 24, 48, and 96 hours.
Cells were evaluated with the tetrazolium-based colorimetric assay
(Roches, Mannheim, Germany). Tetrazolium salts (e.g. MTT) were
utilized for quantifying the viable cells, as these cells were cleaved
to form a blue Formazan dye only by dehydrogenous enzymes in
metabolically active cells. Acid-isopropanol (100 ml of 0.04 N HCl in
isopropanol) was then added and incubated for 12 hours. Colori-
metric changes were read with a microplate reader at a wavelength
of 550 nm (Molecular Devices Corp., Sunnyvale, CA).
Quantitative real-time PCR
The RNA samples isolated from confluent KFs were treated with
dexamethasone at different concentrations (10
7 and 10
9 M) for
48 hours using the TRI-zol reagent (Sigmas). The reagents contained
a monophasic solution of guanidine thiocyanate and phenol. Equal
amounts of RNA were reverse transcribed to cDNA using 0.5 mg/ml
oligo-dT primer (Promegas, Madison, WI), 10 mM dNTP and
Moloney murine leukemia virus reverse transcriptase and its buffer.
Amplification of VEGF-A product (forward primer, CCC ACT GAG
GAG TCC AAC AT; reverse primer, TTA TAC CGG GAT TTC TTG
CG; 197-bp expected), IGF-1 (forward primer, TCA ACA AGC CCA
CAG GGT AT; reverse primer, CCT GCA CTC CCT CTA CTT GC;
223-bp expected); TGF-b1 (forward primer, GGG ACT ATC CAC
CTG CAA GA; reverse primer, CCT CCT TGG CGT AGT AGT CG;
239-bp expected); VEGF-R2 (forward primer, GCC ACC ATG TTC
TCT AAT AGC AC, reverse primer, CAC TGC ATG CCT GGC AGG
TGT AG), and b-actin (Promegas) (forward primer, TCA TGA AGT
GTG ACG TTG ACA TCC GT; reverse primer, CTT AGA AGC ATT
TGC GGT GCA CGA TG) (285-bp expected) were carried out for
internal comparison. Real-time quantitation was based on the
iCycler assay, using a fluorogenic SYBR Green kit (Bio-Rad,
Hercules, CA) for PCR with the iCycler Instrument (Bio-Rad,
Hercules, CA). The pairs of primer for each target mRNA were the
same as described previously. The PCR reactions were carried out in
a total volume of 25 ml, which included SYBR Green kit with a Taq
DNA polymerase reaction buffer, 10 mM dNTP, 10 mM MgCl2, PCR-
grade water, 1 ml of template DNA, and primers. The reaction
www.jidonline.org 1269
 W-S Wu et al.
Dexamethasone-Induced Keloid Regression
conditions were as follows: pre-incubation at 951C for 5 minutes,
followed by 40 cycles (amplification) of 951C for 10 s and 551C for
30 s. All experiments were conducted at least three times.
Fluorescence emission spectra were monitored and analyzed. PCR
products were measured by the threshold cycles (CT), at which
specific fluorescence became detectable. The CT was used for
kinetic analysis and was proportional to the initial number of target
copies in the sample. A melting curve analysis was performed to
assess the specificity of the amplified PCR products.
In situ hybridization
In situ hybridization was performed utilizing RNA probes that target
mRNA expressions of VEGF-A, IGF-1, TGF-b1, and VEGF-R2 in
keloid tissue before and 1 week after each steroid treatment.
Amplification of specific cDNA was performed and individually
cloned into transcription vector pCRII-TOPO II vector (Invitrogens).
Amplicon sequences were identified by NCBI blast researching.
These cDNA clones were linearized at a suitable site. The RNA
probes were synthesized by in vitro transcription utilizing SP6 and
T7 RNA polymerase (Promegas). Antisense and sense RNA probes
were produced in the presence of digoxigenin-11-UTP using a
digoxigenin RNA labeling kit (Roches).
Tissue blocks were sectioned at 5-mm thick and mounted on
silane-coated slides and dried at 371C overnight. Tissue sections
were prefixed with 4% paraformaldehyde and washed for 5 minutes
in diethyl-pyrocarbonate (Calbiochems, Darmstadt, Germany)
–phosphate-buffered saline containing 0.2% Triton X-100 (Sigmas).
These tissue sections were then pretreated with 20mg/ml of
proteinases-K at 371C for 25 minutes. Sections were incubated with
0.25% acetic anhydride in 0.1 M triethanolamine for 10 minutes (pH
8.0). The RNA probes (3 ng/ml of digoxigenin labeled) were applied
to a buffer containing 50% formamide, 4  standard saline citrate,
10 mM dithiothreitol, 10% dextran sulfate, 5  Denhardt’s solution,
500 mg/ml yeast t-RNA, and 500 mg/ml salmon sperm DNA. The RNA
probes were denatured for 3 minutes at 1001C and applied to
sections for hybridization at 581C overnight in a humidified chamber
with 50% formamide and 5  standard saline citrate solutions.
Sections were washed in buffer I (100 mM Tris-HCl and 150 mM
NaCl, pH 8.0) for 10 minutes, blocked in 10% blocking reagent for
30 minutes, and incubated for 1 h with anti-rabbit digoxigenin
antibody conjugated horseradish peroxidase (HRP) (Roches). Excess
antibody was removed using Tris-buffered saline with Tween-20
(three times at 5 minutes). The HRP activity was developed for
15 minutes with biotinyl-tyramide and streptavidin–HRP. Finally,
tissue sections were washed with 1  Tris-buffered saline with
Tween-20 for 30 minutes. The HRP-diaminobenzidine staining
progressed until the brown precipitate (positive color) developed
under microscopic visualization. Tissue sections were counter-
stained with hematoxylin and mounted in aqueous mounting fluid
(R&D Inc., Minneapolis, MN).
ELISA for measurement of VEGF production
Cultured supernatants of KFs were harvested after treatment at
different concentrations of dexamethasone (10
7 and 10
9 M), and
stored at
801C until analysis. The concentration of VEGF was deter-
mined using a commercially available ELISA kit that detects soluble
isoforms of human VEGF (Quantikines, R&D Inc., Minneapolis,
1270 Journal of Investigative Dermatology (2006), Volume 126
MN). Colorimetric changes were determined by a microplate
reader at a wavelength of 550 nm (Molecular Devices Corp., CA).
The VEGF levels were normalized to the corresponding total cell
number that was determined using a hemacytometer after staining
with 0.4% trypan blue.
IHC staining
Primary antibodies of PCNA (Immunotech Corp., Marseille, France)
and VEGF (Santa Cruzs, Santa Cruz, CA) were determined by IHC
staining that detected VEGF and PCNA expressions from three pairs
of steroid-treated specimens. For IHC staining, the HRP-diamino-
benzidine system staining kit (R&D Inc., Minneapolis, MN). The IHC
stain was produced according the method described previously (Kuo
et al., 2005).
IC staining for PCNA expression
The KFs were harvested and cytospin (104 cells/slide) was assessed
following each treatment. These cells were fixed by air-drying the
cell sample with 100% methanol and frozen at
201C. IC staining of
PCNA in KFs was detected using HRP-diaminobenzidine tissue
staining kit (R&D Inc.) (Kuo et al., 2005). Detection of PCNA was
based on avidin–biotin complex formation with specific primary
antibodies against PCNA. Endogenous peroxidase activity was
blocked with 3% hydrogen peroxide for 10 minutes at room
temperature. Sample cells were then blocked with 3% bovine serum
albumin in phosphate-buffered saline to decrease nonspecific
antibody binding. Samples were then stained with anti-rat mono-
clonal PCNA antibody (Upstates, Charlottesville, VA) at a 1:200
dilution in phosphate-buffered saline. Reactive cells were then
incubated with biotinylated anti-mouse antibody as a secondary
antibody for 30 minutes. Visualization of specific binding sites for
PCNA localization employed an enzymatic conversion of the
chromogenic substrate diaminobenzidine into a brown precipitate
by HRP. Following counterstaining with hematoxylin, KFs were
mounted, cleared, and coverslipped. The KFs were analyzed under
microscopic visualization.
Measurement of angiogenic response in KFs
Angiogenic response of various KF treatments was examined using
an in vitro capillary/tube-forming assay (Yamakawa et al., 2003).
Differently treated KFs were cultured as a feed layer for 48 hours in
chamber slides coated with Matrigels (Becton-Dickinson, Bedford,
MA), seeded with HUVEC 104 cell/well as a co-culture, and
maintained in extracellular matrix medium containing 10% fetal
bovine serum (Cambrex BioScience, MD, USA). When KFs expres-
sing VEGF interact with HUVEC cells in culture, they form tube-like
structures. These tube-like formations were visualized and counted
using light microscopy at 4 hours after co-culturing.
Statistical analysis
Data sets were analyzed with a nonparametric analysis of variance
followed by the analysis of test to determine significance differences
among treated and untreated groups. A value of Po0.05 was
considered statistically significant.
CONFLICT OF INTEREST
The authors state no conflict of interest.

ACKNOWLEDGMENTS
We thank the National Science Council of the Taiwan, for financially
supporting this research under Contract No. NSC 91-2314-B-182A-116 and
NSC 92-2314-B-182A-061. Dr Justin M. Sacks in University of Pittsburgh, PA
is appreciated for his valuable discussion and English grammar correction.
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