©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Vascular Endothelial Growth Factor Expression in Cultured Keratinocytes
IMPLICATIONS FOR NORMAL AND IMPAIRED WOUND HEALING (*)

Stefan Frank (1)(§)(¶), Griseldis Hübner (1)(§)(**), Georg Breier (2), Michael T. Longaker (3), David G. Greenhalgh (4), Sabine Werner (1)(§§)

From the (1) Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany, the (2) Max-Planck-Institut für physiologische und klinische Forschung, Parkstrasse 1, 61231 Bad Nauheim, Germany, the (3) Institute of Reconstructive Plastic Surgery, New York University School of Medicine, New York, New York 10016, and the (4) Department of Surgery, Shriners Burns Institute and the University of Cincinnati, Cincinnati, Ohio 45229

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

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 , 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.


MATERIALS AND METHODS

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.

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, 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.

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.


RESULTS

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.




DISCUSSION

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-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.

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- (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.

In summary, we have identified several positive regulators of VEGF gene expression in keratinocytes, whereby EGF, TGF-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.

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.


FOOTNOTES

*
This work was supported by a grant from the Fritz-Thyssen-Stiftung (to S. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors have equally contributed to this work.

Supported by a fellowship from the Schering Forschungsgemeinschaft.

**
Recipient of a Boehringer Ingelheim fellowship.

§§
To whom correspondence should be addressed: Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany. Tel.: 49-89-8578-2269/2271; Fax: 49-89-8578-2814.

The abbreviations used are: EGF, epidermal growth factor; TGF-, 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.

G. Hübner, unpublished data.


ACKNOWLEDGEMENTS

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.


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