Abstract
Objective: Topical pretreatment
with all-transretinoic acid (atRA) is known to
improve healing of cutaneous
wounds. We tested the effect of atRA on wound healing of
genetically diabetic db/db mice. It is known
that cutaneous wounds of db/db mice show delayed wound healing
due to impaired wound contraction, delayed
granulation tissue formation and underexpression of keratinocyte
growth factor (KGF). Methods: 0.1%
atRA in 100 mg aqueous gel was applied to the back skin of
db/db mice as well as to their normal heterozygous
littermates, db/+ mice, for five consecutive days, and 2
days after completion of the atRA treatment, two
round excisional wounds were created down the panniculus
carnosus with a 6-mm punch biopsy on the back
skin of each mouse. Results: After 5 days treatment with
0.1% atRA, significant hypertrophy of the epidermis
and dermis, neovascularization, and inflammatory cell invasion
were seen in the skin of the db/db mice, but
these effects were seen only weakly in db/+ mice. Wounds
in atRA-treated db/db mice closed more rapidly
than those in vehicle-treated db/db mice. KGF mRNA expression,
which is usually significantly lower
in db/db mice than in normal mice, in wounds of atRAtreated
db/db mice on day 1 of treatment was as strong
as in db/+ mice. Conclusion: Pretreatment with atRA reversed
the impaired wound healing in db/db mice.
Introduction
Retinoic acid (RA) improves the
clinical symptoms of acne vulgaris, psoriasis
and skin photoaging [1]. Histologically,
topical treatment with RA induces hypertrophy in the epidermis
and dermis via an increase in dermal vasculature
and invasion of inflammatory cells. RA has several contradictory
effects on wound healing. For example, systemic
application of RA has deleterious effect on healing of fullthickness
skin wounds and corneal wounds of diabetic animals
[2, 3]. On the other hand, systemic RA reverses delayed wound
healing in steroid-treated animals [4-7]. RA
is also known to improve wound healing of abraded skin by
acceleration of epidermal turnover when it is applied topically
prior to wounding [8]. Topical pretreatment with RA also
enhances healing of full-thickness excisional wounds
of photoaged and normal skin [9, 10]. These results suggest
that wound healing may be affected by the route or
schedule of retinoid administration, and that, at least in
some skin conditions, topical pretreatment may be the most
effective method of administration of all-trans-RA (atRA).
We hypothesized that impaired wound healing of genetically
diabetic db/db mice may be reversed by topical pretreatment
with atRA. Db/db mice show clinical symptoms
similar to insulin-resistant diabetes mellitus patients,
such as severe obesity, hyperglycemia, hyperinsulinemia,
and
insulin resistance [11-14]. These symptoms were derived from
genomic mutation in the leptin receptor. Db/db mice
are also known to show impaired cutaneous wound healing,
as is universally observed in diabetic patients [11]. Wound
healing of db/db mice is characterized by weak contraction,
slow inflammatory cell invasion and delayed granulation
formation. The characteristic anatomy of the skin of db/db
mice, including thick subdermal fat and an extremely thin
dermis, may also contribute to impairment of wound healing.
Many growth factors and their receptors, such as FGF,
FGF receptors, KGF, IGF and VEGF, have been found to be aberrantly
expressed in wounds of db/db mice [11, 12].
Of these, the factor that is considered to be the most important
for delayed wound healing in db/db mice is KGF
[15]. In normal mice, the mRNA levels of KGF are acutely
upregulated early in wound healing [16, 17]. However,
KGF mRNA levels in db/db mice are unchanged throughout the
healing process [15]. Since upregulation of KGF
mRNA is one of the earliest events of wound healing, KGF
is considered to be an important factor that might regulate
downstream events during wound healing.
Materials and methods
Animals
Female C57BLK/ksj db/db mice at 8-12 weeks of age (Saitama
Experimental Animals Supply Company, Saitama, Japan) and
their
normal littermates (db/+ mice) were used throughout this
study. Animal experiments were performed according to the
Laboratory
Animal Guidelines of Tokyo University. All animal procedures
were performed under general anesthesia with diethyl ether.
Three independent
experiments were performed, and 12 to 15 mice were used in
each experiment. The back skin of each mouse was shaved and
topically treated for five consecutive days with 100 mg 0.1%
atRA (Sigma Aldrich, Tokyo, Japan) in aqueous gel vehicle
or 100 mg
aqueous gel without atRA. Scaling was graded by three investigators
according to the visual scoring method of Effendy et al.
[18]:
0, no scaling; 1, weak scaling; 2, moderate scaling; 3, large
flakes, intense peeling.
On the 2nd day after completion of atRA treatment, two 6-mm
round excisional wounds, down the panniculus carnosus and
its associated
fascia, were created in each mouse. The wound area was calculated
as the product of the long and short diameters of each
wound. Mice were killed on days 1, 3, 7 and 14 by cervical
dislocation under general anesthesia with diethyl ether.
Wound tissue
was processed for histology and RT-PCR.
Histology
The mice were injected subcutaneously with 30 μg/g 5-bromo-2-deoxyuridine
(BrdU labeling reagent; Amersham Pharmacia, Shinjuku,
Tokyo) 3 and 24 h before they were killed. Skin samples were
fixed in 10% formalin, dehydrated and embedded in paraffin.
Paraffin sections were cut at a thickness of 5 μm. Sections
were rehydrated and stained with hematoxylin and eosin, and
with Gomori's
trichrome stain. Immunohistochemistry to determine the level
of BrdU incorporation into cells was performed as follows.
Sections
were incubated in 4 N HCl for 30 min at 37 °C followed by
0.1 M borax buffer, pH 9.0, for 10 min at room temperature.
The specimens
were then treated with anti-BrdU monoclonal antibody (Amersham
Pharmacia) followed by biotinylated anti-mouse IgG.
The specimens were then incubated in ABC reagent (Vector,
Burlingame, Calif.), and developed with 3,3′-diaminobenzidine.
RT-PCR
mRNA was extracted from a 2-mm wound margin using ISOGEN
LS (Nippon Gene, Shinjuku, Tokyo). The mRNA (1 μg in a total
volume of 20 μl) was transcribed to cDNA by AMV reverse transcriptase
(Promega Japan, Higashinihonnbashi, Tokyo) at 37°C
for 1 h. For semiquantitative detection of KGF and β-actin
cDNA, 1 μl cDNA in a total volume of 50 μl (50 mM KCl, 15
mM Tris-
HCl, pH 8.0, 4 mM MgCl2) was amplified with Ampli-Taq Gold
(PE Biosystems, Roppongi, Tokyo) in a PC-960G thermal cycler
(Cosmo Bio, Toyo, Tokyo). The sequences of the oligonucleotide
primers used for amplification of KGF were TTGCAATGAACAAGGAAGGA
and GAATTCTATCTTGCAATGAA, and those for β-actin were TGAGGAGCACCCTATGCTGC
and TAGCCCTCGTAGATGGG.
Amplification was carried out as follows: pre-PCR step of
10 min at 94 °C; 32 PCR cycles of 30 s at 94 °C, 30 s at
58 °C and 1 min at 72 °C; and a final step of 10 min at 72°C.
We had already confirmed in a preliminary study that the
PCR products
would be within the linear log phase under these conditions.
Results
The effects of topical application
of atRA
Scaling was more intense in db/db mice than in db/+ mice
following treatment with 0.1% atRA (Fig. 1). Although
0.1% atRA did not induce significant scaling in db/+ mice,
higher concentrations of atRA did induce significant scaling
in these mice, so the effect of atRA appears to be dose-dependent.
Histologically, the skin of db/+ mouse showed only
slight dermal and epidermal hypertrophy after treatment with
0.1% atRA. On the other hand, the skin of atRAtreated
db/db mice showed significant hypertrophy of epidermis and
dermis, scaling of stratum corneum, an increase
in cellularity and proliferation of subepidermal vessels
(Fig. 2). Elongation of the rete ridges and atrophy
of the sebaceous glands were also characteristic of atRAtreated
db/db mice. In vehicle-treated db/db mice, BrdU incorporation
was observed throughout the basal layer of the epidermis.
However, in atRA-treated db/db mice, BrdU
incorporation was greater in the follicular epidermis, but
not in the interfollicular epidermis (Fig. 3).
Wound healing of atRA- and vehicle-treated db/db mice
Day 0 after wounding
The wounds in vehicle-treated db/db mice were 20-30% larger
than the original wounds, but the wounds in atRAtreated
db/db mice did not show enlargement just after wounding (Fig.
4).
Day 1 after wounding
Vehicle-treated db/db mice on day 1 after wounding showed
only a small number of inflammatory cells in the
wound cleft. Only a small amount of extracellular matrix
had formed at this time. However, atRA-treated db/db mice
on day 1 showed massive invasion of inflammatory cells in
the wound cleft. In addition, a large amount of extracellular
matrix staining bluish with trichrome reagent had formed
within the granulation tissue, which connected the
granulation tissue with the wound margin (Fig. 5a, b).
Day 14 after wounding
Macroscopically, 78% of wounds in vehicle-treated db/db mice
had not closed on day 14. Histologically there was a
large amount of granulation tissue within the wide wound
cleft. Epidermal regeneration had started, but due to the
distance between the wound margins, there still was an uncovered
area within the cleft. On the other hand, 89% of
the wounds in atRA-treated db/db mice had already closed
by this time. Histologically the wound margins had come
close to each other, and reepithelialization was almost complete
(Fig. 5c, d).
Expression of KGF mRNA
mRNA expression of KGF at the time of wounding did not show
significant differences between atRA- and vehicletreated
db/db mice (data not shown). However, KGF mRNA in the wounds
of atRA-treated db/db mice on day 1
after wounding was significantly higher than in vehicleor
sodium lauryl sulfate-treated db/db mice (Fig. 6).
Discussion
In the study reported here we demonstrated
that (1) topical treatment with atRA had stronger
effects in db/db mice
than in db/+ mice, and that (2) atRA pretreatment reversed
delayed wound healing in db/db mice.
It is noteworthy that atRA induced cutaneous changes in db/db
mice much more strongly than in db/+ mice. This
does not mean that db/+ mice are totally refractory to the
effects of atRA, but that db/db mice are more sensitive to
atRA than db/+ mice, because the latter showed effects similar
to those seen in db/db mice when they were treated with
higher concentrations of atRA. It is not clear whether this
increased sensitivity was due to downstream events resulting
from the leptin receptor deficiency, or due to loss of barrier
function. Similar model-specific sensitivity has
been observed in other experiments. For example, atRA increases
the synthesis of collagen and hyaluronic acid in
UVB-irradiated mice, but not in non-irradiated hairless mice
[19, 20]. It has also been reported that atRA effectively
reverses steroid-retarded repair, which may be a result of
a mutual interaction between glucocorticoids and
retinoids in the process of inflammation, immunity and connective
tissue production [4-7]. It is interesting that
the sebaceous glands of db/db mice showed extremely strong
changes. Sebaceous glands almost completely disappeared
in db/db mice after treatment with 0.1% atRA, but those in
db/+ mice did not show significant changes
after the same treatment. Even with 0.4% atRA, about 30%
of sebaceous glands remained intact in db/+ mice, although
significant dermal hypertrophy was observed with this concentration
of atRA.
Pretreatment with 0.1% atRA had little effect on the wound
healing in normal mice, but it significantly accelerated
wound healing in db/db mice. This may reflect the biological
effects of atRA pretreatment in these animals. Delayed
wound healing in db/db mice seemed to be enhanced by treatment
with atRA. First, full-thickness wounds in
db/db mice had enlarged 20-40% just after wounding due to
their extremely thin dermis with a small amount of extracellular
matrix, which was the primary cause of delayed wound healing
in db/db mice during the early stages of
wound healing. Treatment with atRA made the dermis thick
and stiff preventing enlargement of excisional wounds in
db/db mice. Second, granulation tissue and extracellular
matrix formation induced by atRA may also accelerate
wound healing in db/db mice. This granulation tissue contains
huge numbers of inflammatory cells that release cytokines
and growth factors that enhance wound healing. In addition,
smooth muscle actin fiber involved in wound
granulation may exert a contractile force. The mechanism
of action of atRA in wound healing is
complicated. A study using a dominant-negative retinoic acid
receptor mutant strongly indicates that epidermal proliferation
and regeneration is initiated by heparin-binding epidermal
growth factor (HB-EGF) induced by atRA [21,
22]. Since atRA induction of HB-EGF occurs in suprabasal
cells prior to the onset of basal cell hyperproliferation,
this
mechanism may well explain the effectiveness of atRA pretreatment.
On the other hand, atRA may also enhance
macrophage function, and thus finally may enhance macrophage-derived
growth factors such as FGF, TGF and IGF-I
[6]. This is also plausible since macrophage dysfunction
is seen in db/db mice [23]. A more sensitive real-time RT-PCR
could detect quantitative differences in these macrophagederived
growth factors.
Although suppression of KGF is characteristic of wound healing
in db/db mice, KGF mRNA levels in unwounded
skin are almost the same between db/db and db/+ mice [20],
which was also confirmed by our RT-PCR (data not shown).
In addition, atRA-induced epidermal proliferation in db/db
mouse was not associated with KGF upregulation before
wounding. Taken together, atRA does not directly induce KGF
in unwounded skin but it causes the skin of db/db
mice to induce KGF. Previous studies of atRA application
to already existing
wounds have revealed conflicting results. Some investigators
have shown enhanced healing [7, 14, 24], but others
have shown retardation of healing [10, 25, 26]. On the other
hand, topical pretreatment with atRA has consistently
shown improvement in wound healing. In addition, our experimental
and clinical experience has revealed that atRA
worsens wound healing of full-thickness open wounds (unpublished
data). For these reasons we do not recommend
atRA application to diabetic ulcers. In practice, atRA may
enhance wound healing when it is applied before surgery
in diabetic patients. In the present study, we only evaluated
the effects of atRA pretreatment in diabetic wounds,
but it may be possible to apply this strategy to other healing-impaired
wounds, such as those in the skin of the elderly
or malnourished, or ischemic skin. Pretreatment with atRA
may prevent delayed healing, infection and dehiscence
in these potentially healing-impaired wounds.
Fig. 1 Scaling score of db/db mice
treated with 0.1% atRA, and of db/+ mice treated
with 0.1% or 0.4% atRA. Three
mice in each group were evaluated. The values are means ±
SD of duplicate measurements
from three independent experiments.
Fig. 2a-d H&E staining of the
back skin of db/+ mice (a vehicletreated mouse,
b 0.1% atRA-treated mouse) and db/db mice (c
vehicle-
treated mouse, d 0.1% atRA-treated mouse). Arrows indicate
sebaceous glands.
Fig. 3a-d BrdU staining of back
skin of db/db mice treated with either of vehicle
(a) or 0.1% atRA (b) for 5 days. c, d High-power
magnification of a and b (a, b ×100; c, d ×200)
Fig. 4 Wound area of db/+ and db/db mice. Round wounds of
diameter 6 mm were made on the back skin of mice pretreated
with either 0.1% atRA or vehicle. Wounds of db/+ mice had
almost closed by day 7 whether they had been treated
with 0.1% atRA or vehicle. On the other hand, vehicle-treated
db/db mice showed enlargement
of the wound area on day 1, which was followed by delayed
wound contraction. Pretreatment with atRA prevented
the wounds of db/db mice from expanding, which may have contributed
to the earlier wound closure. Data represent
means ± SD of duplicate measurements from three independent
experiments.
Fig. 5a-c Wound sections of db/db
mice pretreated with vehicle (a, c) or atRA (b,
d) were stained with Gomori's trichrome. Arrows
indicate the wound margin (a, b 1 day after wounding; c,
d 14 days after wounding). a, b ×200; c, d ×50
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