From the
Recent in situ hybridization studies had demonstrated a
strong increase in vascular endothelial growth factor (VEGF) mRNA
expression in the hyperproliferative epithelium during wound healing.
To determine potential mediators of VEGF induction during this process,
we analyzed the regulation of VEGF expression in cultured human
keratinocytes. We found a large induction of VEGF expression upon
treatment of quiescent cells with serum, epidermal growth factor,
transforming growth factor-
During cutaneous wound repair, new tissue formation starts with
re-epithelialization and is followed by granulation tissue formation.
The latter process encompasses macrophage accumulation, fibroblast
ingrowth, matrix deposition, and angiogenesis
(1) . These events
are stimulated in part by a number of mitogens and chemotactic factors.
The soluble factors that stimulate angiogenesis in wound repair have
not been identified, however, several growth factors which are present
in a wound have been shown to be mitogenic for endothelial cells in
vitro and to be angiogenic in several in vivo assays.
These include different members of the fibroblast growth factor family
(for review, see Refs. 2-4), but also transforming growth factor
To analyze the
presence of VEGF proteins in the cell lysate, serum-treated and control
cultures from six 10-cm Petri dishes were scraped into 0.1 M
NaPO
For immunoprecipitation of
VEGF proteins, conditioned medium and control medium was prepared as
described above. VEGF-specific proteins were precipitated with a
polyclonal antibody directed against the amino terminus of VEGF
(Genzyme) and protein A-Sepharose beads. Beads were washed three times
with TNTG buffer (20 mM Tris, pH 7.2, 150 mM NaCl,
10% glycerol, 0.1% Triton X-100). Protein A-Sepharose-bound proteins
were eluted by a 5-min incubation at 95 °C in Laemmli sample buffer
and analyzed by Western blotting with the same VEGF antibody.
Recent in situ hybridization experiments had
demonstrated expression of VEGF and one of its receptors,
flt-1(19) , during wound healing. Highest levels of
VEGF mRNA were detected in keratinocytes at the wound edge and in
keratinocytes that migrated to cover the wound surface
(18) .
Besides a few mononuclear cells, VEGF expression was not found in other
cell types of the wound. This finding suggested an important role of
keratinocytes in wound angiogenesis. Since VEGF is highly specific for
endothelial cells it is likely to act in a paracrine manner on the
sprouting capillaries of the wound edge and the granulation tissue. The
exclusive detection of flt-1 in these cells
(20) supports this hypothesis.
Since keratinocytes are the
major source of VEGF in the wound, the identification of the factors
which regulate VEGF expression in these cells seems to be of particular
importance. We have therefore studied the regulation of VEGF expression
in vitro in cultured keratinocytes. Similar to the in vivo situation, quiescent keratinocytes in culture expressed only low
levels of VEGF mRNA. Due to the rapid induction of VEGF expression
in vivo(18) , we speculated that serum growth factors
which are released upon hemorrhage might be able to stimulate VEGF gene
expression. Our data demonstrate that VEGF mRNA and also protein
expression are indeed rapidly induced by serum in vitro. The
most potent activator of VEGF expression was EGF which is a major
mitogen for keratinocytes
(33, 34) . Furthermore, EGF
induces angiogenesis in vivo(5) . Thus, our finding
suggests that the angiogenic effect of this growth factor might be a
combination of a direct effect on endothelial cells and an indirect
effect which is based on the induction of VEGF expression in
keratinocytes. A similar mechanism has also been postulated for the
role of EGF-induced tumor angiogenesis in gliomas
(35) . In
addition to EGF, TGF-
Our hypothesis that serum-derived
factors, which are released upon local wound hemorrhage, could be
responsible for the induction of VEGF gene expression during wound
healing does explain the early onset of this process upon injury, but
it does not provide an explanation for the sustained expression of high
levels of VEGF mRNA during wound healing. Therefore, other sources of
VEGF-inducing factors could be present in the wound tissue. KGF is one
of the factors which might be responsible for the prolonged induction
of VEGF expression after injury. However, transgenic mice which express
a dominant-negative KGF receptor in basal keratinocytes of the
epidermis and therefore do not respond to KGF
(36) have normal
levels of VEGF mRNA in nonwounded and wounded skin (data not shown).
Thus, although KGF might normally contribute to the induction of VEGF
expression during wound healing, a lack of KGF expression or
responsiveness does not reduce VEGF expression, suggesting that other
factors can compensate.
Another possible source of VEGF-inducing
factors are mononuclear cells. Polymorphonuclear leukocytes represent a
first line of defense against bacteria and parasites and are the
predominant cell type of the early inflammatory response during repair.
Apart from phagocytosing particles and releasing toxic metabolites and
enzymes, they have also been shown to produce a series of
proinflammatory cytokines, including interleukin-1, interleukin-6, and
tumor necrosis factor-
In summary, we have
identified several positive regulators of VEGF gene expression in
keratinocytes, whereby EGF, TGF-
The up-regulation of
VEGF and its receptor during wound repair suggests an important role of
this growth factor in wound angiogenesis. To analyze the importance of
a correct regulation of VEGF expression for wound healing, we compared
the time course of VEGF expression during wound healing of healthy
control mice and genetically diabetic db/db mice. The latter are
characterized by a severe delay in wound healing and have been widely
used as a model for wound healing disorders
(21) . The reason for
the wound healing defect in these animals is still not completely
understood. It has been speculated that a reduced and delayed
infiltration of macrophages might contribute to the wound healing
abnormalities
(46) . Since macrophages are an important source of
growth factors and cytokines, expression of other mitogens in the wound
which are positively regulated by macrophage-derived cytokines might be
reduced. This has already been demonstrated for KGF expression which is
highly induced in cultured fibroblasts by macrophage-derived
proinflammatory cytokines
(47, 48) . Consistent with this
hypothesis, induction of KGF expression after injury has been shown to
be significantly reduced and delayed in db/db mice
(49) , and we
now demonstrate a similar effect for VEGF. In healthy control mice, a
significant induction of VEGF expression was observed within 24 h after
injury. Expression levels were high during the period when granulation
tissue formation normally occurs and only returned to the basal level
after completion of skin repair. In contrast, VEGF mRNA levels declined
in db/db mice within 3 days after injury, and only low levels of VEGF
transcripts were present during the period when wound angiogenesis
normally occurs. This finding demonstrates that the wound healing
defect in these animals is associated with reduced levels of VEGF
during skin repair, suggesting that up-regulation of VEGF expression is
essential for normal wound healing. Furthermore, our results suggest
that application of exogenous VEGF might have a beneficial effect on
wound healing in db/db mice, especially in combination with other
growth factors which stimulate different cell types in the wound.
We thank Irene Dick and Karin Angermeyer for excellent
technical assistance and Dr. P. H. Hofschneider, Dr. Werner Risau, and
Dr. Hans Smola for helpful discussions. The HaCaT keratinocyte cell
line was kindly provided by Dr. Norbert Fusenig and Dr. Petra Boukamp.
The human VEGF cDNA was a kind gift of Dr. H. Weich.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1, keratinocyte growth factor, or the
proinflammatory cytokine tumor necrosis factor
, respectively.
Since all these factors are present at the wound site during the early
phase of wound healing, they might also be responsible for VEGF
induction after cutaneous injury. To determine the importance of
increased VEGF production for wound repair, we compared the time course
of VEGF mRNA expression during wound healing of healthy control mice
with the kinetics of VEGF expression during skin repair of genetically
diabetic db/db mice which are characterized by impaired wound healing.
In normal mice we found elevated VEGF mRNA levels during the period
when granulation tissue formation occurs. In contrast, VEGF mRNA levels
even declined during this period in db/db mice, suggesting that a
defect in VEGF regulation might be associated with wound healing
disorders.
, epidermal growth factor (EGF)
(
)(5) and platelet-derived growth factor BB
(6) .
Furthermore, transforming growth factor-
(TGF-
) and tumor
necrosis factor-
(TNF-
) have been shown to be angiogenic
in vivo, although they inhibit endothelial cell growth in
vitro(7, 8, 9, 10, 11) .
The most recently discovered endothelial cell growth factor is vascular
endothelial growth factor (VEGF). VEGF is a dimeric glycoprotein with
structural homology to platelet-derived growth factor. Four different
human isoforms, VEGF
, VEGF
,
VEGF
, and VEGF
, have been isolated from
various sources and arise by alternative splicing of mRNA
(12) .
VEGF is a highly specific mitogen for endothelial cells in
vitro, and it has angiogenic properties in
vivo(13, 14, 15, 16) . Furthermore,
it enhances the permeability of local blood vessels. Since angiogenesis
and increased vascular permeability are characteristic features of
wound healing
(17) , VEGF may play an important role in tissue
repair. Recently, Brown et al.(18) demonstrated
expression of VEGF mRNA in proliferating keratinocytes of the newly
formed epithelium during wound healing. In contrast, expression of one
of the receptors for VEGF (flt-1)
(19) was found to be
up-regulated in the sprouting blood vessels at the wound edge and in
endothelial cells of the granulation tissue
(20) . These findings
suggest that keratinocyte-derived VEGF stimulates angiogenesis during
wound healing in a paracrine manner. In this study we have identified
potential mediators of VEGF mRNA induction after injury. Furthermore,
we provide evidence for a defect in VEGF regulation during wound
healing in genetically diabetic db/db mice which are characterized by a
severe delay in skin repair
(21) . The aberrant expression
pattern of VEGF mRNA during skin repair in db/db mice suggests that a
defect in VEGF regulation might be associated with the wound healing
abnormalities seen in these animals.
Animals
Balb/c mice were obtained from the
animal care facility of the Max-Planck-Institute of Biochemistry,
Martinsried. C57BL/KsJ-db/m mice were obtained from Jackson
Laboratories (Bar Harbor, ME). These mice were chosen because they
exhibit characteristics similar to those of human adult onset diabetes
as a result of a single autosomal recessive mutation on chromosome 4.
Only the homozygous animals develop diabetes
(21) . The animals
were 8-12 weeks of age at the start of the experiments. The
animal care facility was maintained by professionals who followed
federal guidelines, and all procedures were approved by the
Institutional Animal Care Utilization Committee or by the Local
Government of Bavaria (permission number 211-2531-16/93).
Wounding and Preparation of Wound Tissues
Two
independent wound healing experiments were performed with control mice
and diabetic mice, respectively. For every experiment 23 animals were
anesthetized under methoxyflurane (Metofane , Priman-Moore,
Inc). The animals' backs were shaved and wiped with 30% isopropyl
alcohol, and six full-thickness wounds (6 mm diameter, 3-4 mm
apart) were made on 20 mice/experiment by excising the skin and
panniculus carnosus. The wounds were allowed to dry in order
to form a scab. Animals were sacrificed with pentobarbital overdose,
and wounds from four animals were harvested at 1, 3, 5, 7, or 13 days
after wounding. An area of 7 mm in diameter which includes the complete
epithelial margins was excised at each time point. A similar amount of
skin from the backs of three non-wounded animals was used as a control.
Wound tissue was immediately frozen in liquid nitrogen and stored at
-70 °C until used for RNA isolation.
RNA Isolation and RNase Protection
Analysis
Isolation of total cellular RNA and RNase protection
analysis were performed as recently described
(22) . The increase
in VEGF mRNA levels was quantitated by laser scanning densitometry of
the autoradiograms. All experiments were repeated with a different set
of RNAs from an independent experiment. A 316-bp fragment corresponding
to nucleotides 139-455 of the murine VEGF cDNA
(23) and a 159-bp fragment corresponding to nucleotides
339-498 of human VEGF
cDNA
(24) were used as
templates.
Cell Culture
The human keratinocyte cell line
HaCaT
(25) was used for all tissue culture experiments. Cells
were cultured in Dulbecco`s modified Eagles medium (DMEM) with
10% fetal calf serum (FCS). For VEGF induction experiments, they were
grown to confluence without changing the medium and rendered quiescent
by a 16-h incubation in serum-free DMEM. Cells were then incubated for
varying periods in fresh DMEM containing serum, purified growth
factors, or cytokines. Aliquots of cells were harvested before and at
different time points after treatment with these reagents and used for
RNA isolation. FCS and DMEM were purchased from Life Technologies,
Inc., growth factors and cytokines were from Boehringer Mannheim
Biochemicals, and cycloheximide was from Sigma.
Western Blot Analysis of VEGF Proteins
5 ml of
DMEM/10-cm Petri dish were conditioned by quiescent HaCaT cells in the
presence or absence of 10% FCS. After 8 h the conditioned medium from
six Petri dishes was centrifuged for 10 min at 3000 revolutions/min to
remove cell debris. Heparin-binding proteins were precipitated from the
supernatant with 120 µl of heparin-Sepharose (1:1 slurry) overnight
at 4 °C. Heparin-Sepharose beads were precipitated by
centrifugation and washed three times in 20 mM Tris-HCl, pH
7.4, 0.3 M NaCl. Heparin-Sepharose-bound proteins were
extracted by a 5-min incubation in Laemmli sample buffer at 95 °C
and separated by SDS-gel electrophoresis. After transfer to
nitrocellulose membranes, VEGF proteins were detected using a
polyclonal antiserum directed against VEGF (Santa Cruz Biotechnology)
and an alkaline phosphatase detection system (Promega). Non-conditioned
medium containing 10% FCS was used as a control.
, pH 7.2, 1% Triton X-100, 2 M NaCl, 1%
aprotinin, and 2 mM phenylmethylsulfonyl fluoride. Insoluble
material was precipitated, and extracts were diluted to a final
concentration of 0.3 M NaCl. VEGF proteins were bound to
heparin-Sepharose (120 µl) overnight at 4 °C. Beads were washed
with 0.5 M NaPO
, pH 7.5, 0.1% Triton X-100, 0.3
M NaCl. Sepharose-bound proteins were eluted and analyzed by
Western blot analysis as described above.
Induction of VEGF mRNA and Protein Expression by
Serum
Recently Brown et al.(18) demonstrated
expression of VEGF mRNA in proliferating keratinocytes during wound
healing by in situ hybridization. To identify potential
mediators of VEGF induction in keratinocytes during wound healing, we
have studied the regulation of VEGF induction in vitro in
cultured human keratinocytes. Since local hemorrhage is one of the
initial events after skin injury, we have first tested the potency of
serum to stimulate VEGF expression. As shown in Fig. 1A,
low levels of VEGF mRNA were detected in quiescent keratinocytes. Upon
addition of 10% FCS, a large induction of VEGF mRNA expression was
observed. Within 90 min after serum stimulation, VEGF mRNA levels were
20-fold higher compared to the basal level. The effect of serum on VEGF
mRNA expression was long lasting, and elevated VEGF mRNA levels were
still observed 8 h after serum stimulation (Fig. 1A). 24
h after serum addition VEGF mRNA levels finally returned to the basal
level (data not shown).
Figure 1:
Induction of VEGF mRNA and protein
expression by serum in cultured keratinocytes. A, RNase
protection assay demonstrating the induction of VEGF mRNA expression by
FCS. Cells were rendered quiescent by serum starvation and stimulated
with 10% FCS for different time periods as indicated. Samples of 30
µg of total cellular RNA from these cells were analyzed for VEGF
mRNA expression. 1000 counts/min of the hybridization probe were used
as a size marker. B, three different forms of human VEGF mRNA
are shown schematically. A 159-nucleotide hybridization probe
(indicated with an arrow) which is complementary to the 3`-end
of human VEGF mRNA was used to detect VEGF transcripts. A 159-bp
protected fragment corresponding to the complete hybridization probe is
expected for transcripts encoding VEGF. Two shorter
protected fragments of 86 and 73 bp are generated by mRNA encoding
longer forms of VEGF. The fragments which are protected by the
different forms of VEGF mRNA are indicated with black bars below the RNAs. Serum-stimulated expression of VEGF protein is
shown in C. 30 ml of medium was conditioned for 8 h by
quiescent HaCaT keratinocytes in the presence or absence of 10% FCS.
Heparin-binding proteins were precipitated from the conditioned medium
using heparin-Sepharose beads and analyzed by immunoblotting for the
presence of VEGF protein. Non-conditioned medium containing 10% FCS was
used as a control. Two VEGF proteins of 22 and 24 kDa which were
detected in conditioned medium from serum-stimulated cells (lane
2) are indicated with an arrow. These proteins were not
present in conditioned medium from serum-starved cells (lane
1) or in non-conditioned medium containing 10% FCS (lane
3).
Four different VEGF proteins have recently
been identified in vitro which arise from differential
splicing in the 3`-end of VEGF mRNA
(12) . The hybridization
probe which we used for our protection assay experiments corresponds to
the 3`-end of the shortest form of VEGF (VEGF), and thus
enabled us to distinguish between this form of VEGF mRNA and longer
forms (Fig. 1B). A protected fragment corresponding to
the complete coding sequence of the hybridization probe is generated by
mRNA encoding VEGF
(upper band in
Fig. 1A), whereas two shorter protected fragments are
generated by mRNAs encoding longer variants of VEGF (lower bands in Fig. 1A). mRNAs encoding VEGF
and
other splice variants were induced to a similar extent
(Fig. 1A). To analyze whether induction of VEGF mRNA
expression correlates with the induction of immunoreactive VEGF
protein, conditioned medium was prepared from serum-treated and
non-treated keratinocytes and analyzed for the presence of VEGF
proteins. Because of the kinetics of VEGF induction at the mRNA level,
the conditioned medium was harvested after 8 h. Heparin-binding
proteins were enriched by their capacity to bind to heparin-Sepharose
and subsequently analyzed for the presence of VEGF protein by
immunoblot. As shown in Fig. 1C, two major forms of VEGF
with estimated molecular masses of 22 and 24 kDa were detected in
conditioned medium from serum-stimulated cells. The size of these
proteins correlates with the known molecular sizes of the VEGF gene
products. VEGF variants of identical size were also detected by
immunoprecipitation and subsequent immunoblotting with a different VEGF
antibody (data not shown). In contrast, the 34-kDa protein
(Fig. 1C) was not detected after immunoprecipitation,
suggesting that it is not a VEGF-specific protein. To exclude the
possibility that the VEGF-specific proteins in conditioned medium from
serum-stimulated cells result from serum-derived VEGF, non-conditioned
medium containing 10% fetal calf serum was used as a control.
VEGF-specific proteins of 22 and 24 kDa were also detected in the
lysate of serum-treated cells but not in control cells (data not
shown), suggesting that these proteins are partially retained in the
cytoplasm. Taken together these data demonstrate that induction of VEGF
mRNA by serum also correlates with induction of VEGF protein.
Induction of VEGF Expression by Serum Growth
Factors
To identify the serum components which are responsible
for the stimulatory effect on VEGF expression, we analyzed the effect
of purified serum growth factors on VEGF expression. As shown in
Fig. 2, A and C, epidermal growth factor was a
potent inducer of VEGF mRNA expression and increased VEGF mRNA levels
20-fold within 90 min, 50-fold within 5 h, and 60-fold within 8 h.
EGF-induced VEGF expression was long lasting, and high levels of VEGF
mRNA were still observed 24 h after addition of EGF. In contrast, the
major mitogen in platelets, platelet-derived growth factor BB, had no
effect on VEGF mRNA levels (data not shown), although other genes are
regulated in these cells by platelet-derived growth factor,(
)
demonstrating their general responsiveness to
platelet-derived growth factor. In addition to the keratinocyte mitogen
EGF, TGF-
1, which is a potent inhibitor of keratinocyte
proliferation
(26, 27, 28) also increased VEGF
mRNA levels 16-fold within 2 h (Fig. 2, A and
C). The kinetics of TGF-
1-induced VEGF expression was
different from the kinetics observed with EGF. Whereas EGF-induced VEGF
expression was long lasting, TGF
increased VEGF mRNA levels only
transiently, and 5 h after addition of the growth factor, VEGF
expression levels had significantly declined.
Figure 2:
Induction of VEGF mRNA expression by
purified serum growth factors. A, keratinocytes were rendered
quiescent by serum starvation. They were stimulated with 20 ng/ml EGF
or 1 ng/ml TGF-1 for 90 min, 2, 5, 8, or 24 h as indicated. 30
µg of total cellular RNA from these cells were analyzed by RNase
protection assay for VEGF mRNA expression. 50 µg of tRNA were used
as a negative control. 1000 counts/min of the hybridization probe were
used as a size marker. An ethidium bromide stain of 1 µg of the
same batch of RNA is shown in B. The EGF and
TGF-
1-induced increase in VEGF mRNA as assessed by laser scanning
densitometry of the autoradiogram is shown schematically in
C.
Keratinocyte Growth Factor Stimulates VEGF mRNA
Expression
Recently, we demonstrated a large induction of
keratinocyte growth factor (KGF) expression in the dermis during wound
healing
(22) . Since KGF is a potent mitogen for
keratinocytes
(29) , this finding suggested that dermally derived
KGF stimulates wound reepithelialization in a paracrine manner. To
assess whether KGF-induced keratinocyte proliferation is accompanied by
induction of VEGF mRNA in keratinocytes, we analyzed the effect of
purified KGF on VEGF expression. VEGF mRNA expression was rapidly
induced by KGF and slightly elevated VEGF mRNA levels were detected as
early as 15 min after addition of KGF (data not shown). Within 30 min
after KGF stimulation, VEGF mRNA levels were already 5-fold higher
compared to the basal level and a 8-10-fold induction of VEGF
mRNA expression was found within 2 h after exposure to KGF
(Fig. 3, A and B). Similar to EGF-induced VEGF
expression, high levels of VEGF mRNA were observed for at least 8 h
after addition of KGF (Fig. 4). Induction of VEGF expression by
KGF occurred in a dose-dependent manner. A maximal response was
obtained with 10 ng/ml KGF, but even at a very low concentration of KGF
(0.4 ng/ml) induction of VEGF could be detected (Fig. 3, A and C).
Figure 3:
Induction of VEGF mRNA expression by
keratinocyte growth factor. A, serum-starved keratinocytes
were treated for different time periods with 10 ng/ml KGF or for 2 h
with different concentrations of KGF as indicated. 30 µg of total
cellular RNA from these cells were analyzed for VEGF mRNA expression by
RNase protection analysis. 1000 counts/min of the hybridization probe
were used as a size marker. The time- and dose-dependent increase in
VEGF mRNA after KGF stimulation of keratinocytes as assessed by laser
scanning densitometry of the autoradiograms is shown schematically in
B.
Figure 4:
VEGF induction by KGF is independent of
de novo protein synthesis. Serum-starved keratinocytes were
stimulated for different time periods with 10 ng/ml KGF in the presence
or absence of 10 µg/ml of the protein synthesis inhibitor
cycloheximide as indicated. Since cycloheximide was dissolved in 5
µl of dimethyl sulfoxide (DMSO), medium containing 5
µl of the solvent was used as a control. 30 µg of total
cellular RNA from these cells were analyzed by RNase protection
analysis for the expression of VEGF mRNA. VEGF expression in cells
treated with 20% FCS is shown for comparison. 50 µg of tRNA were
used as a negative control. 1000 counts/min of the hybridization probe
were used as a size marker (A). The degree of VEGF mRNA
induction after 8 h as assessed by laser scanning densitometry of the
autoradiogram is shown schematically in
B.
Induction of VEGF Gene Expression Is Independent of de
Novo Protein Synthesis
The rapid induction of VEGF expression by
KGF suggested that VEGF induction could occur in the absence of de
novo protein synthesis. To address this question, the effect of
the protein synthesis inhibitor cycloheximide was analyzed. As shown in
Fig. 4
, A and B, the addition of 10 µg/ml
cycloheximide resulted in a significant superinduction of VEGF mRNA
expression. In contrast, serum-induced expression of VEGF protein was
completely abolished, demonstrating that protein synthesis was indeed
inhibited (data not shown). The induction of VEGF gene expression in
the presence of cycloheximide shows that VEGF gene activation is
independent of de novo protein synthesis. Therefore, the gene
encoding VEGF is a member of the growing family of primary response
genes.
VEGF Gene Expression Is Stimulated by Proinflammatory
Cytokines
Since the infiltration of polymorphonuclear leukocytes
followed by macrophages is another early event in wound repair, we
tested the ability of cytokines produced by these cells to induce VEGF
mRNA synthesis. As shown in Fig. 5, TNF-, which inhibits
keratinocyte growth in vitro(30) , is a very potent
activator of VEGF expression and a 50-fold stimulation of VEGF mRNA
expression was observed within 2 h after addition of 300 units/ml
TNF-
to keratinocytes. In contrast, IL-1
, which is mitogenic
for keratinocytes
(31) , was much less efficient and increased
VEGF mRNA levels less than 5-fold. Similar to TGF-
1-induced VEGF
expression, VEGF mRNA induction by these cytokines was only transient,
and 5 h after cytokine stimulation, VEGF expression levels had
significantly declined. Interleukin-6, which like IL-1 is also
mitogenic for keratinocytes
(32) , had no effect on VEGF
expression in these cells (data not shown).
Figure 5:
Induction of VEGF mRNA expression by
proinflammatory cytokines. Serum-starved keratinocytes were stimulated
for 2, 5, and 8 h with 300 units/ml TNF- or 100 units/ml
IL-1
. 30 µg of total cellular RNA from these cells were
analyzed by RNase protection analysis for the expression of VEGF mRNA.
The degree of VEGF mRNA induction as assessed by laser scanning
densitometry of the autoradiogram is shown schematically. The cytokine
used for VEGF induction is indicated in the
figure.
Defects in VEGF Expression during Wound Healing in db/db
Mice
To further determine the role of VEGF induction for normal
wound repair, we compared the time course of VEGF expression during
wound healing of db/db mice and healthy control mice. For this purpose
we isolated RNA from excisional full thickness wounds at different
intervals after wounding and performed RNase protection assays. 24
wounds from the backs of four mice were excised for each time point and
used for RNA isolation. Non-wounded back skin from the same area was
used as a control. As shown in Fig. 6A, two different
protected fragments were obtained with RNA from normal and wounded
skin, corresponding to differerent forms of VEGF mRNA
(Fig. 6C). The longer protected fragment which
corresponds to the complete coding sequence of the probe is generated
by mRNA encoding the shortest form of murine VEGF (VEGF),
whereas the shorter protected fragment is generated by mRNA encoding
longer forms of VEGF. As shown in Fig. 6A, a strong
induction of VEGF mRNA expression was observed within 24 h after injury
and highest VEGF mRNA levels were found between day 3 and 7 after
injury. Since this is the period of granulation tissue formation, our
data suggest that induction of VEGF might play a role in wound
angiogenesis. In non-wounded skin of db/db mice, VEGF mRNA levels were
significantly higher compared to skin of control mice
(Fig. 6B) However, after an initial increase in VEGF
mRNA levels within 24 h after injury, expression levels dramatically
declined during the time when granulation tissue formation normally
occurs and at day 5 after injury, VEGF mRNA expression was hardly
detectable (Fig. 6B). This finding demonstrates that the
wound healing defect seen in these animals is associated with reduced
VEGF expression during the time when wound angiogenesis normally
occurs.
Figure 6:
Regulation of VEGF expression during wound
healing in normal and db/db mice. Total cellular RNA (50 µg) from
non-wounded and wounded back skin of normal and db/db mice was analyzed
by RNase protection assay with an RNA hybridization probe complementary
to the 3`-end of the murine VEGF mRNA. The regulation of
VEGF mRNA expression in normal and db/db mice is shown in A and B, respectively. The gels were exposed for 24 h. The
time after injury is indicated on top of each lane. Control skin refers to non-wounded skin of normal mice; db/db skin refers to non-wounded skin of db/db mice. 50 µg of tRNA were
used as a negative control. 1000 counts/min of the hybridization probe
were added to the lane labeled probe. Three different forms of
murine VEGF mRNA (Breier et al., 1992) and the hybridization
probe (indicated with an arrow) are shown schematically in
C. A 316-bp protected fragment corresponding to the complete
hybridization probe is expected for VEGF transcripts encoding the
shortest form of murine VEGF (VEGF
). Two shorter
protected fragments are generated for longer forms of VEGF mRNA. The
fragments which are protected by the different forms of VEGF mRNA are
indicated with black bars below the RNAs. The smallest
protected fragment cannot be detected with the gel system which was
used in this experiment.
1 was also able to induce VEGF gene
expression, although this factor has been shown to inhibit keratinocyte
proliferation. Thus, induction of VEGF gene expression in keratinocytes
is not necessarily associated with stimulation of mitogenesis. Since
large amounts of TGF-
1 are released from platelets upon injury,
this factor might significantly contribute to the early induction of
VEGF expression after wounding.
(37, 38, 39) . These
cytokines as well as several growth factors are also produced by
activated macrophages
(11, 41, 42, 43, 44) which infiltrate the wound area following the
polymorphonuclear cells at 48-72 h after injury. Activated
macrophages subsequently initiate granulation tissue formation and
remain present in the wound tissue even beyond the initial inflammatory
phase
(1, 45) . The activation of the VEGF gene in
keratinocytes by IL-1
and particularly by TNF-
suggests that
these cytokines, together with macrophage-derived growth factors, might
significantly contribute to the long lasting high expression levels of
VEGF during skin repair. The large induction of VEGF mRNA expression by
TNF-
is of major biological importance since it could provide an
explanation for the potent angiogenic effect of this cytokine in
vivo(11) , a phenomenon which has not been completely
understood so far. Thus, it seems possible that TNF-
-stimulated
induction of VEGF in other surrounding cell types might indirectly
stimulate endothelial cell proliferation.
1, KGF, and TNF-
were the
most potent inducers. Since these growth factors and cytokines are
present in the wound during the early phase of skin repair, a
combination of these factors might also be responsible for VEGF
induction in vivo. Thus, we hypothesize that the initial
stimulation is caused by serum growth factors which are released upon
wound hemorrhage. The expression of some of these factors and also of
TNF-
by polymorphonuclear leukocytes and wound macrophages, as
well as high levels of KGF present in the wound, might further enhance
and sustain the high expression levels of VEGF.
,
transforming growth factor-
; TNF-
, tumor necrosis
factor-
; VEGF, vascular endothelial growth factor; bp, base
pair(s); DMEM, Dulbecco's modified Eagle's medium; FCS,
fetal calf serum; KGF, keratinocyte growth factor; IL, interleukin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.