ARTICLE
Healing of wounds in fetal skin can occur without scar formation by a
process resembling regeneration rather than repair [1-4]. Wounded fetal
skin is able to heal rapidly with reduced inflammation [5, 6] and neovascularisation
[7]. The extracellular matrix deposited in fetal wounds contains essentially
the same structural components as that in the adult wound and the organisation
of collagen in the healed fetal wound is indistinguishable from the normal
surrounding tissue [8]. This non-scarring phenomenon is gestationally
regulated and there is a transition during the third trimester of gestation
from the "fetal-type" to the "adult-type" of healing. Mouse embryos and
fetuses up to embryonic day 16 (E16) heal by repairing wounds in a scar-free
manner, by contrast E18 fetuses heal in an "adult-type" fashion with subsequent
scar formation [8]. There is tissue specificity with regard to fetal non-scarring
healing. Scar formation is evident in fetal tissues such as diaphragmatic
skeletal muscle [9] and gastric tissue [10] whereas many studies have
shown that equivalent aged fetal skin does not scar [1, 2, 4, 8].
Growth factors play a major role in adult wound healing and their role
in fetal wound repair has been investigated [11]. One such set of growth
factors is the transforming growth factor beta (TGF-beta) family. They
are of particular importance in wound healing due to their ability to
modulate extracellular matrix (ECM) formation. They stimulate the synthesis
of multiple ECM components, including collagens, fibronectin, vitronectin,
tenascin, and proteoglycans [12, 13]. They suppress matrix degradation
by down regulating the expression of proteases, such as plasminogen activators
[14, 15] and stromelysin [16], and by inducing protease inhibitors, such
as plasminogen activator inhibitor-1 [15, 17] and tissue inhibitor of
metalloproteases-1 (TIMP-1) [14]. TGF-beta1 itself is bound to the extracellular
matrix, and can be released by proteases [18]. Furthermore the presence
of extracellular matrix has been found to down regulate the expression
of the TGF-beta1 gene [19]. Thus, TGF-beta may act as a feedback regulator
of extracellular matrix formation. TGF-beta is also involved in scar formation
as neutralization of TGF-beta1 and TGF-beta2 in adult wounds reduces scarring
in rat dermal wounds [20]. By contrast, exogenous addition of TGF-beta3
to dermal rat wounds results in reduced scar formation [21]. Therefore
it is clear that different isoforms of TGF-beta can have completely opposite
effects in wound repair although the mechanisms behind these differences
are still under investigation. The actions of the TGF-betas are mediated
through binding to specific cell membrane receptors. The type I and type
II receptors are high-affinity transmembrane serine-threonine kinases
that interact with one another and facilitate each other's signalling
[22]. The type I receptor requires the type II receptor to bind ligand,
while the type II receptor requires the type I receptor for signalling
[23, 24]. Signalling from the TGF-beta receptor complex involves various
second messengers including SMADs and transgenic analysis has highlighted
the importance of different SMAD signalling pathways for elucidating different
TGF-beta effects in wound healing [25].
Given the central role of the TGF-betas in the wound healing process
in adults, the influence of TGF-beta1 on fetal wound repair has been investigated.
The TGF-beta1 profile of fetal wounds is different from that of adult
wounds such that in the fetus there is rapid induction of TGF-beta1 within
1 hr of wounding with rapid clearance from the wound site so that by 18
hrs post-wounding levels of TGF-beta1 return to background levels [26].
Twelve hours after incising mouse fetal lips no upregulation of TGF-beta1
was detected within or at the wound margins although strong expression
of TGF-beta1 was observed at the wound margins in equivalent adult wounds
[11]. Additionally, in a model of human fetal wound repair, fetal skin
grafts on nude mice do not show any TGF-beta1 staining, but exogenous
addition of TGF-beta1 to these grafts results in abnormal fibrosis and
scar formation [27].
The expression of the TGF-beta receptors TGF-betaRI and TGF-betaRII
has been identified in adult excisional sheep wounds where both receptors
co-localise in both the wounded and unwounded skin and are present in
the same cell types as the TGF-beta ligands [28]. However no studies,
to date, have identified the expression of the TGF-beta receptors in fetal
dermal wound repair. Given the different roles that different isoforms
of TGF-beta have in wound repair we have investigated the expression of
TGF-beta1, TGF-beta2 and TGF-beta3 in fetal wound repair. Additionally,
we have characterised the expression of the TGF-beta receptors in fetal
and adult wounds to determine the importance of these ligands and receptors
in non-scarring fetal wound repair.
Materials and methods
Fetal murine surgical techniques
Time mated female MF1 mice were anaesthetised on day 16 of gestation.
A midline laparotomy was performed to access the pregnant uterus and a
purse string suture was placed through all layers of the uterine wall
on the anti-mesenteric surface. An incision was made to expose the left
flank of the fetus and a 2 mm linear full thickness incision was made
on the left flank of the fetus. The wound was left unsutured, the purse
string closed, and the maternal abdominal wound was repaired. The pregnancies
were continued until the fetuses were harvested or birth occurred on day
19-20 of gestation.
Adult murine surgical techniques
Male MF1 mice (16-20 weeks old) were anaesthetised and two standardised
1cm full thickness linear incisions were made using a scalpel blade on
the flanks of the animals extending 3.5-4.5 cm from the base of the skull,
1 cm either side of the spinal column. The wounds were left unsutured
and the animals allowed to recover from anaesthesia.
Wound harvest and sectioning
Fetal heads were decapitated, limbs and tails amputated and the remaining
body fixed in 4% buffered paraformaldehyde and embedded in paraffin wax.
Adult animals were killed by overdose of chloroform followed by cervical
dislocation. Fetal wounds were harvested at 0, 1, 3, 6, 12, 18, 24, 36,
48, 72 hrs and 28 days post-wounding. Adult wounds were harvested at 0,
0.5, 1, 3, 5, 7, 14 days post-wounding. These wounds were either fixed
in 4% buffered paraformaldehyde and embedded in paraffin wax, or fresh
frozen in optimum cutting temperature compound (OCT) for immunofluorescent
analysis. Wax sections were cut for in situ hybridisation analysis
using RNase treated solutions. Sections were cut at 3 µm on a microtome
and floated onto RNase treated glass slides, baked overnight at
37° C and stored at room temperature in RNase free containers. Cryosections
were cut at 7 µm, fixed in acetone for 20 min and stored at - 20°
C until required.
Immunofluorescent staining
Primary antibodies to TGF-beta1, TGF-betaRI and TGF-betaRII were obtained
from Santa Cruz Biotechnology, Inc. CA. Antibodies to TGF-beta2, and TGF-beta3
were obtained from R&D Systems, MN. Acetone fixed frozen sections
were used for all antibody staining. All primary antibodies were applied
at a 1:100 dilution and incubated at 4° C overnight in a humidified
chamber. They were rinsed three times in phosphate buffered saline (PBS)
and incubated for 1 hr at room temperature with anti-rabbit IgG biotinylated
secondary antibody (1:200) for TGF-beta1, TGF-betaRI and TGF-betaRII and
anti-goat IgG biotinylated secondary antibody (1:200) for TGF-beta2 and
TGF-beta3. This was rinsed with 2-3 washes of PBS before the final incubation
of FITC conjugated streptavidin (DAKO, CA) 1:40 dilution in PBS was applied
for 40 min at room temperature. The stained sections were finally mounted
in a non-fading water based medium (Gelvatol) and analysis of the immunofluorescence
was determined using a Leitz Aristoplan microscope. For verification of
staining negative controls included pre-adsorption of the antibodies with
an excess amount of the immunization peptide (TGF-beta1, TGF-betaRI and
TGF-betaRII Santa Cruz Biotechnology, Inc. CA) or excess ligand (TGF-beta2,
TGF-beta3 R&D Systems, MN) for 1 hr at room temperature and replacement
of the primary antibodies by either normal rabbit IgG or normal goat IgG
for TGF-beta1, TGF-betaRI, TGF-betaRII and TGF-beta2 and TGF-beta3 respectively.
On additional control sections, the primary and secondary antibodies were
left out to determine non-specific binding. All control sections had negligible
immunofluorescence.
In situ hybridisation
TGF-beta type I receptor [29], TGF-beta receptor type II both in pSV7d
vectors were generous gifts from Dr. Kohei Miyazono (Uppsala, Sweden).
As necessary, these vector containing probes were transformed into E.
coli cultures and stock cultures were grown using ampicillan as a
screen. Plasmid DNA was extracted and the cDNA probe excised using the
appropriate restriction enzymes. Probes were purified before labelling
with Digoxigenin (DIG)-dUTP (Boehringer-Mannheim, Mannheim, Germany).
In situ analysis was performed on wax sections using a standard
non-radioactive in situ protocol (Boehringer-Mannheim, Mannheim,
Germany). The wax sections were rehydrated, washed in diethyl pyrocarbonate-phosphate
buffered saline (DEPC-PBS) before proteinase K (10 µg/ml) treatment
for 15 min at room temperature. After further DEPC-PBS washes, sections
were fixed in 4% buffered paraformaldehyde and subsequently washed in
0.1 M triethanolamine containing 0.25% acetic anhydride. Hybridisation
of (DIG)-dUTP labeled probes occurred overnight at 60° C in a humidified
chamber. Washes were performed to remove unbound label including an RNase
A (25 µg/ml) wash at 37° C for 45 min. To prevent any non-specific
binding of the alkaline phosphatase-conjugated digoxigenin antibody (anti-DIG-AP
Fab) to the sections, they were treated with 10% sheep serum for a minimum
of 2-3 hrs before the overnight treatment of 1:5,000 dilution of the anti-DIG-AP
Fab (Boehringer-Mannheim, Mannheim, Germany) at
4° C. Further washes were performed to remove any unbound antibody.
Alkaline phosphatase was detected using 4-nitro blue tetrazolium chloride
(NBT), 5-bromo-4-chloro-3-indoyl-phosphate (BCIP) detection system (Boehringer-Mannheim,
Mannheim, Germany) such that a positive purple signal was produced. Sections
were counterstained using Mayer's carmalum to reveal cell morphology,
mounted in a non-fading water based medium (Gelvatol) and analysed using
a Leitz Aristoplan microscope.
The specificity of DNA binding to mRNA was determined. Before the addition
of the cDNA probe, control slides were incubated with DNase free-RNase
A (25 µg/ml) (Sigma, St. Louis, MI), and put through the normal in
situ hybridisation protocol. No binding of the DNA probe was observed
confirming binding specificity to the RNA target. To determine specificity
of the digoxigenin antibody (Boehringer-Mannheim, Mannheim, Germany) to
(DIG)-dUTP, DIG labeled cDNA probe was omitted form the protocol. No positive
signal was obtained indicating specificity of the antibody to the (DIG)-label
and not to the section.
Results
Expression of TGF-beta isoforms in fetal and adult wounds
TGF-beta1
In the unwounded fetal skin at E16, low level immunofluorescent staining
of TGF-beta1 was observed in the outer epithelial layers and around hair
follicles but no positively stained cells were observed in the dermis.
However, by 6 hrs post-wounding, distinct positively stained inflammatory
cells and fibroblasts were seen at both wound edges and in the wound space
(Fig. 1a). The number of these positively stained cells at the
wound site was observed to increase up until 24 hrs post-wounding (Fig.
1b) although the total staining was still low compared to adult wounds
at comparable stages of wound repair. An example of a 7 day adult wound
is shown in Figure 1c. By 48 hrs the staining had returned to the
normal basal expression throughout the skin.
TGF-beta2
In the fetus, basal staining for TGF-beta2 in unwounded tissue was higher
than for TGF-beta1. Strong immunofluorescence was observed in the epidermis
and hair follicles. Upon wounding increased numbers of TGF-beta2 positive
cells were observed randomly distributed throughout the dermis as can
be seen 3 hrs post wounding (Fig. 1d). The staining pattern of
these cells was very granular and their morphology resembled inflammatory
cells. By 18 hrs post wounding the number of TGF-beta2 positive cells
were still relatively high compared to TGF-beta1 but had returned to basal
levels (Fig. 1e). In the adult, epidermis, hair follicles and fibroblasts
throughout the dermis all stained positive for TGF-beta2. Considerably
greater numbers of positively stained cells were observed within the granulation
tissue of the adult wounds compared to fetal wounds. An example of a 3
day adult wound stained for TGF-beta2 is shown in Figure 1f.
TGF-beta3
Staining for TGF-beta3 in unwounded tissue in both the adult and the
fetus was similar to that of TGF-beta1 and TGF-beta2 in that epithelium
and hair follicles stained positive. Upon wounding increased immunofluorescence
was observed for TGF-beta3 in the epidermis of the fetal skin, and where
the epidermis at the wound edge formed a "ball" of cells around the exposed
dermis. This epidermal fluorescence of TGF-beta3 could be observed at
12 hrs post wounding (Fig. 1g). As the wound healed, the epidermis
continued to show strong positive staining for TGF-beta3 however, no upregulation
of TGF-beta3 staining was observed post-wounding although some faintly
stained fibroblasts in the dermis were staining positive for TGF-beta3
48 hrs post wounding (Fig. 1h). Intense epidermal staining was
also observed in adult wounds. Additionally, large numbers of fibroblasts
and inflammatory cells stained positively for TGF-beta3 in the granulation
tissue in the adult wounds. This was maximum between 5-7 days post-wounding
and an example is shown in Figure 1i. Once again, the staining
of fibroblasts and inflamatory cells was considerable greater in adult
wounds than in comparable fetal wounds.
In situ hybridisation analysis of TGF-beta receptors
in fetal wound healing
The expression of mRNA for the TGF-beta receptors was assessed by in
situ hybridisation using cDNA probes to TGF-betaRI and TGF-betaRII.
Various time points were assessed post-wounding which covered the whole
wound healing period of the fetal wound. We observed no positive mRNA
expression for TGF-betaRI or TGF-betaRII in the fetal skin (Fig. 2a,
c respectively). No expression was observed within the wound nor surrounding
the wound or in the normal dermis or in the fibroblasts nor the epidermis.
This observation held for all time points assessed, which ranged from
1 hr through to 28 days post injury. When other tissues within the fetus
were assessed, positive mRNA expression was found in the epithelium of
the villi and the crypts of the fetal alimentary tract for both the TGF-betaRI
(Fig. 3a) and the TGF-betaRII. These expression patterns were highly
reproducible and were observed at all the fetal time points examined.
TGF-beta receptor immunofluorescence in fetal wound healing
TGF-betaRI and TGF-betaRII protein expression was also assessed in fetal
wounds using immunofluorescent staining. Minimal TGF-betaRI or TGF-betaRII
receptor protein staining was observed in the fetal skin (Fig. 2b,
d respectively). Low level immunofluorescence for the TGF-betaRI could
be seen in the basal epidermal cells of the epidermis but following injury
no upregulation of either receptor was observed at any of the time points
assessed. By contrast, strong immunofluorescence was observed for both
TGF-betaRI (Fig. 3b) and TGF-betaRII within the fetal alimentary
tract at all time points studied on the same sections that had shown no
staining within the dermis and dermal wounds.
In situ hybridisation analysis of TGF-beta receptors in adult
wound healing
TGF-betaRI
The type I TGF-beta receptor mRNA was observed in adult skin and appeared
to be up-regulated post-wounding in the fibroblasts of the dermis particularly
at the wound margins. No expression was observed within the wound itself
or around hair follicles and blood vessels. Furthermore, no receptor mRNA
was observed in the epidermis. At 12 hrs post-wounding there was little
expression in the dermal fibroblasts and no expression within the wound
itself. However, increased expression in the dermal fibroblasts at the
wound margins and in the normal dermis was observed with time post-wounding.
This expression increased at
day 3, and day 5 and peaked at day 7 post-wounding (Fig. 4a-c).
At this point mRNA expression was present in the dermal fibroblasts and
around the hair follicles at the edges of the wound and in normal dermal
tissue. At 14 days post-wounding mRNA expression was still observed in
the dermis and around hair follicles at the wound margins but to a lesser
degree than that observed at earlier time points.
TGF-betaRII
The type II TGF-beta receptor mRNA was detected in the dermis either
side of the wound. Receptor mRNA was present in the fibroblasts especially
at the wound margins but again no expression was observed in the epidermis,
hair follicles or blood vessels. Similar to the type I receptor, TGF-betaRII
expression was not observed in the wound itself. By contrast to the type
I receptor, strong expression was observed at early time points post-wounding
and increased with time post-wounding, peaking between
1 and 3 days after injury (Fig. 4d-f). After 3 days post-wounding
receptor mRNA expression was reduced compared to the earlier times post-wounding
and expression was only observed in the dermis.
TGF-beta receptor I and receptor II immunofluorescence in adult wound
healing
Low level staining for TGF-betaRI was observed at the base of the wound
12 hrs post-wounding in the epithelial cells at the edges of the wound,
the dermal fibroblasts at the wound edge and in the mesenchyme of the
normal unwounded skin. By 3 days post-wounding, as the epidermis began
to migrate over the wound, strong epithelial staining at the leading edge
of the basal epidermal cells was observed. Inflammatory cells also stained
positively at the base of the wound. Maximum staining occurred between
5 and 7 days post-wounding in the basal epithelia and in fibroblasts within
the wound itself (Fig. 5a). By 14 days post-wounding, staining
had returned to normal levels. TGF-betaRII staining was observed in the
basal epithelial cells and dermal fibroblasts at the edges of the wound
12 hrs post-wounding. By 3 days post-wounding, intense epithelial staining
in the basal layers of the leading epidermal edge was observed. At the
base of the wound, inflammatory cells stained positive for TGF-betaRII.
At 5 and 7 days post-wounding, TGF-betaRII stained strongly within the
wound especially inflammatory cells and the epidermis (Fig. 5b).
However, by 14 days post-wounding, staining of the hair follicles, blood
vessels and dermal fibroblasts had returned to normal levels.
Discussion
We have examined the expression of two of the TGF-beta receptors, TGF-betaRI
and TGF-betaRII, and their ligands, TGF-beta1, TGF-beta2, TGF-beta3 during
adult and fetal murine dermal wound repair using in situ hybridisation
and immunofluorescent staining. Our studies have demonstrated that during
adult healing both the receptors and their ligands are expressed in response
to wounding and they are expressed by the same cell types and in a similar,
though not identical, temporal progression during the three phases of
wound repair. However, in the fetus, there is reduced expression of both
the TGF-beta receptors and their ligands that may influence the wound
healing response observed in early gestation.
In the adult, TGF-beta1, TGF-beta2 and TGF-beta3 were all rapidly expressed
by inflammatory cells migrating into the wound as early as 12 hrs after
injury and all three isoforms remained upregulated in response to injury
over the following 7 days. Strong epidermal staining was observed with
all ligands, which could reflect the known chemotactic properties of TGF-betas
on epithelial cells, promoting epithelial migration, differentiation,
but not proliferation [30, 31]. The source of TGF-beta1 and 2 within the
wound immediately post-injury is the degranulating platelets and the effects
of these TGF-betas in the wound could influence cell migration and infiltration
of inflammatory cells such as monocytes and macrophages within the first
phase of the wound healing process.
In the fetus, similar to studies by Whitby and Ferguson (1991) and Martin
et al. (1993) we found low level TGF-beta ligand expression in
both the wounded and unwounded skins. Studies by Martin et al.
(1993) revealed an increase in the expression of TGF-beta1 transcripts
within epithelial cells at the wound margin 1-3 hrs post-wounding. However,
no expression of TGF-beta3 was observed in the fetal wound and this study
was unable to detect TGF-beta2 due to endogenous background expression.
In our study in the much more developed E16 fetus we also saw rapid production
of TGF-beta1 particularly in the epidermis and also in inflammatory cells
and fibroblasts within the dermis, however the numbers of these cells
were low by comparison with the response observed in the adult. Additionally,
expression of both TGF-beta2 and TGF-beta3 were observed in the fetal
skin. Increased numbers of inflammatory cells adjacent to the wound site
were TGF-beta2 positive. Although TGF-beta3 expression was particularly
strong in the epithelium at the edge of the wound where the epithelium
had formed a "ball" of cells around the exposed wound margin, no clear
upregulation of this protein was observed in response to wounding. TGF-beta3
may be important in stimulating the rapid re-epithelialisation observed
in fetal wounds, due to its effects on promoting epithelial migration
[30, 31]. This may potentially contribute to the reduced scar formation
observed in fetal wound repair. Unlike in adult wound healing, the formation
of a fibrin clot does not occur in fetal wounds. This lack of platelet
degranulation and fibrinogenesis may also result in reduced levels of
TGF-beta1 and 2 at the wound site and additionally as TGF-beta1 has been
demonstrated to be autoinductive, binding at its own gene promoter to
increase its synthesis [32] this may account for the low level of TGF-beta1
in fetal wound repair.
TGF-beta receptor expression was upregulated in response to wounding
in adults. Messenger RNA expression for the TGF-beta receptors was observed
particularly in the margins of the adult wounds and in fibroblasts within
the adjacent dermis. This was in contrast to the staining pattern of its
protein observed in the epidermis and the granulation tissue of the wound.
The temporal expression of the type I and type II receptors did not appear
to be synchronised, with the TGF-betaRI not being expressed within the
wound until 3 days post-wounding, peaking at day 7 whereas the TGFbeta-RII
was expressed as early as 12 hrs post-wounding peaking at 3 days. As the
type II receptor binds the TGF-beta ligands before recruiting the TGF-betaRI
to form the signalling complex [33] it is possible that the binding of
the ligand to the type II receptor may cause upregulation of the TGF-beta
receptors. In support of this, in an ovine excisional adult wound healing
model, where the TGF-beta receptor protein expression was observed after
the ligand, it was suggested that the TGF-betas were able to induce their
receptor expression [28]. Altering the ratio of the TGF-betaRI and RII
at various stages during and after wound repair may be a mechanism for
altering the qualitative and quantitative cellular responses to TGF-beta
ligands.
In contrast to the clear upregulation of both the TGF-betaRI and the
TGF-betaRII in response to wounding with both mRNA and protein expression
in adult wounds, expression of the TGF-beta receptors in fetal skin was
very low for both the TGF-betaRI and the TGF-betaRII. Wounding did not
induce the expression of either of the receptors in E16 fetal skin. Although
no mRNA for either of the TGF-beta receptors was detected by in situ
hybridisation in the fetal skin they were clearly present in the alimentary
tract of the fetus. Several studies have previously shown that fetal abdominal
surgery causes fibrous intraperitoneal adhesions and scarring similar
to that which occurs postnatally [4, 10]. Therefore it is clear that not
all fetal tissue exhibits scar-less repair properties at the same gestational
age. Meuli et al. (1995) suggest that the motions present in the
fetal stomach and diaphragm may be involved in fetal scar formation and
that this movement may be reduced or absent in the non-scarring skin.
However an alternative explanation for fetal intestinal scarring post-surgery
could be the presence of high levels of TGF-beta type I and type II receptors
in the fetal gut compared to the fetal skin thereby allowing the effects
of TGF-beta ligands on collagen and extracellular matrix formation to
occur. During development, TGF-beta receptors may also appear earlier
in the gut as compared to the skin so that this tissue responds to the
low levels of TGF-beta released during fetal repair with an adult like
scarring response [11, 26]. The presence of low levels of TGF-betaRI and
TGF-betaRII in the fetal skin compared to the adult may be an additional
reason for the scar-free healing observed in this tissue. Not only do
fetal dermal wounds have little TGF-beta1 and TGFbeta2 compared to the
adult [11, 26] but the TGF-beta signalling receptors also appear to be
reduced, indicating a greatly reduced fetal TGF-beta signalling system
compared to adult wounds. The disparity between the minimal expression
of the TGF-beta receptors in fetal wounds and the obvious, albeit low
level, expression of their ligands is not easy to explain. However, it
may be possible that there is a fetal form of the TGF-beta receptor complex
which is not recognised by our antibodies or probes and/or that the TGF-beta
ligands bind to a different receptor.
Therefore the differences that we have observed in
TGF-beta receptor expression in adult and fetal wounds may contribute
to the different types of scar-forming and scar-free healing observed
following dermal wounding. Manipulation of the TGF-beta receptor profile
could be a potential therapeutic target in adult wound healing to prevent
scarring. *
Article accepted on 12/4/01Acknowledgements
This work was supported by a research grant from Smith and Nephew Group
Research, York, UK, for which we are extremely grateful.
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