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VEGF-A:
A TARGET OF THE HOSTILE MICROENVIRONMENT
OF THE CHRONIC WOUND
Sabine
A. Eming, Department of Dermatology,
University of Cologne, 50931 Cologne, Germany
THERE is increasing
evidence that the microenvironment of the chronic non-healing wound is
a hostile environment characterized by increased proinflammatory cytokines,
unbalanced proteolytic activity, and bacterial contaminants. However,
the consequences of these activities for cell function and repair mechanisms
are poorly characterized. We are interested to unravel molecular mechanisms
that contribute to a hostile wound environment and the pathology of wound
healing. Our recent studies on the vascular endothelial growth factor
(VEGF-A) highlight the importance of VEGF-A as pivotal mediator in tissue
repair. We identified novel mechanisms that impair VEGF-A-mediated repair
signals at the chronic wound site and which ultimately might compromise
the healing response.
Plasmin as modulator of VEGF-A activity
during wound repair
Vascular endothelial growth factor (VEGF) is an endothelial cell-specific
multifunctional cytokine that is a key regulator in physiologic and pathologic
processes of angiogenic remodelling (Ferrara 2004). By differential mRNA
splicing, the single human VEGF gene gives rise to at least six protein
isoforms VEGF121, 145, 165, 183, 189, and 206 (Tischer et al, 1991). Among
them, the 165-amino-acid isoform is the major gene product found in human
tissues. The splice variants differ primarily in the presence or the absence
of the heparin-binding domains encoded by exons 6 and 7, giving rise to
forms that differ in their heparin/heparansulfate binding ability, as
well as their affinities to VEGF receptors flt-1, flk-1/KDR and neuropilin-1
(Shibuya et al, 2001, Parker et al, 1993, Soker et al, 1998). Whereas
VEGF121 does not bind heparan-sulfate and is freely diffusible, VEGF189
binds heparin and is primarily associated with the cell surface and extracellular
matrix, and VEGF165 has intermediate properties (Parker et al, 1993, Houck
et al, 1992, Ortega et al, 1998). Further, native VEGF189 binds to flt-1
but not flk-1/KDR (Plouet et al, 1997). Native VEGF189 requires maturation
by urokinase (uPA) within the exon 6-encoded sequence to bind to flk-
1/KDR and to exert a mitogenic effect on endothelial cells (Plouet et
al, 1997). In contrast, plasmin digestion of VEGF165 decreases its mitogenic
activity for endothelial cells (Keyt et al, 1996b). Plasmin digestion
of VEGF165 yields two fragments: an amino-terminal homodimer (VEGF1-110)
containing the flt-1 and the flk-1/KDR receptor binding determinants encoded
by exons 3 and 4, respectively, and a carboxyl-terminal polypeptide comprising
the neuropilin-1 binding site encoded by exon 7 (VEGF111-165) (Keyt et
al, 1996b). Interestingly, the reduced mitogenic activity of the amino-terminal
homodimer VEGF1-110 is similar to that observed for VEGF121 (Plouet et
al, 1997, Keyt et al, 1996b). These findings suggest that differential
protease susceptibility, extracellular localization and/or receptor binding
may result in distinct functions
for different VEGF isoforms.
Recently, we demonstrated that the proteolysis of VEGF165 by plasmin is
increased in wound fluid collected from chronic non-healing wounds versus
healing wounds (Lauer et al, 2002). VEGF plays a critical role during
the angiogenic response in tissue repair (Brown et al, 1992, Detmar et
al, 1995), suggesting that VEGF degradation and loss of its biological
activity may contribute to an impaired wound healing response. These results
prompted us to introduce amino acid alterations at the plasmin sensitive
cleavage site of VEGF165 (Arg110/Ala111), in order to stabilize the VEGF165
molecule. Substitutions at either site result in VEGF165 products that,
while maintaining growth-promoting properties, are either fully or partially
plasmin-resistant. This type of modification would be expected to increase
the period that topically applied VEGF protein is active in the wound
environment, implying a potential clinical application. In order to further
characterize the biological relevance of the protease sensitivity of VEGF165
in vivo, in particular during cutaneous repair, in a recent study we investigated
the stability and activity of locally applied VEGF165- wild type (VEGF165-Wt)
or a VEGF165 mutant resistant to plasmin proteolysis (VEGF165A111P) in
a genetic mouse model of impaired healing (db/db mouse) (Roth et al, 2006).
These experiments provided the first in vivo data indicating that plasmin-catalyzed
cleavage is critical to regulate VEGF165 mediated angiogenesis. We chose
the particle-mediated gene transfer technology, a physical means of gene
delivery, to overexpress VEGF165 variants at the wound site. Histological
and functional data indicated that the improved healing response following
VEGF165-Wt application was based on the induction of a highly vascularised
granulation tissue as well as an accelerated reepithelialization process.
Wounds transfected with the cDNA coding for VEGF165A111P provoked an early
granulation tissue formation, which in regard to the vessel density and
cellular composition was similar to that induced by the wild type molecule.
However, vessel size was significantly increased in mutant versus wild
type treated wounds 8 and 12 days following wounding. Nevertheless, differences
in vessel size did not affect wound closure rate, which was similar in
VEGF165-Wt and VEGF165A111P transfected wounds. However, we found significant
differences regarding vessel regression in VEGF165-Wt and mutant transfected
wounds during later stages in the repair process. Whereas in wild type
transfected wounds capillary density resolved rapidly upon completion
of wound reepithelialization, VEGF165A111P transfected wounds were characterized
by a significant delayed involution of capillary density following wound
closure. This finding was consistent with a delayed and decreased number
of apoptotic endothelial cells in VEGF165A111P transfected wounds when
compared to VEGF165-Wt transfected wounds, and it suggested an increased
stability of vascular structures in VEGF165-mutant versus VEGF165-Wt treated
wounds.
VEGF-A is a critical survival factor for vascular endothelium, in particular
for immature vessels (Dor et al, 2001; Alon et al, 1995; Holash et al,
1999). The decreased endothelial cell apoptosis observed in mutant treated
wounds may therefore have resulted from an increased stability and prolonged
local activity of the VEGF165 mutant. This assumption was supported by
Western-blot analysis and CD31/TUNEL analysis, which demonstrated increased
stability and activity of the VEGF165 mutant within a highly proteolytic
wound environment. In addition, our data indicated that capillaries induced
by the VEGF mutant were characterized by an increased coating of perivascular
cells. VEGF165 is chemotactic for pericytes and in various models of angiogenic
remodeling ectopic application of VEGF-A has been shown to accelerate
pericyte coverage of newly formed blood vessels, which ultimately increased
vessel maturation (Alon et al, 1995; Grosskreutz et al, 1999; Benjamin
et al, 1998). Therefore, the prolonged vessel persistence in VEGF165A111P
transfected wounds might result from a combination of prolonged local
VEGF165 mutant activity and increased
pericyte coating. Different structural-functional properties of the VEGF165
mutant and the wild type protein could account
for the prolonged and enhanced activity of the mutant. We and others have
demonstrated that plasmin-catalyzed
cleavage of VEGF165 results in loss of its heparin-binding domain (Keyt
et al, 1996b; Lauer et al, 2000; Lauer et al, 2002), which is crucially
involved in diverse biochemical and functional properties (Park et al,
1993; Ortega et al, 1998; Carmeliet et al, 1999; Hutchings et al, 2003;
Soker et al, 1998; Dor et al, 2002; Ruhrberg et al, 2002). Inactivation
of the plasmin cleavage site should lead to the preservation/ integrity
of the heparin-binding domain in the VEGF165 molecule which enhances VEGF-receptor-affinity
and/or extracellular matrix interactions. The VEGF-A heparin-binding domain
has been identified as the epitope for neuropilin-1 (Nrp-1) binding (Soker
et al, 1998). Although, endothelial VEGF-A signaling described to date
is largely mediated via VEGFR-1 and/or -2 its mitogenic, migratory and
survival signaling can be significantly facilitated by signaling through
the membrane bound coreceptor Nrp-1 (Whitaker et al, 2002). In addition,
the
heparin-binding domain of VEGF-A isoforms is crucial for determining its
binding to extracellular matrix molecules (Park et al, 1993; Ortega et
al, 1998). Recent data have demonstrated that the angiogenic potential
of matrix associated isoforms is superior to soluble isoforms (Zisch et
al, 2003). Overall, effects mediated by the heparin binding domain may
act separately and/or in concert to enhance and prolong the activity of
the plasmin-resistant VEGF165 molecule in the db/db wound environment.
Our present data provided experimental evidence that a plasmin- resistant
VEGF165 variant exerts increased stability in the highly proteolytic environment
of an impaired healing wound with significant consequences to blood vessel
persistence.
Soluble VEGFR-1 as mediator in tissue repair
Beside analyzing VEGF-A processing, we were interested to identify potential
inhibitors of VEGF-A mediated actions in the microenvironment of the non-healing
wound. So far, soluble VEGFR-1 (sVEGFR-1), a splice variant of the membrane-bound
VEGFR-1, is considered the only naturally occurring specific inibitor
for VEGF-A. In vitro analysis demonstrated that sVEGFR-1 is a strong and
specific inhibitor of VEGF-A mediated actions and in vivo studies proved
that the recombinant secreted form of the extracellular region of VEGFR-1
is a potent inhibitor of angiogenesis (Kendall et al, 1993; Aiello et
al, 1995; Miotla et al, 2000; Mori et al, 2000). Potentially, sVEGFR-1
functions as an inert decoy receptor by binding VEGF-A and thereby regulating
the availability of VEGF related ligands for activation of VEGFR-2 (Flk-1/KDR),
the VEGF receptor principally involved in VEGF signalling (Hiratsuka et
al, 1998; Barleon et al, 1997; Kendall et al, 1993). Beside VEGF-A, VEGFR-1
binds the VEGF related proteins PIGF and VEGF-B, however, with much lower
affinity (Hornig et al, 2000; Kendall et al, 1993; Shibuya 2001). Although,
the precise function of PIGF and VEGF-B during cutaneous wound repair
is presently unknown, recent data indicate that membrane-bound VEGFR-1
might be a
critical signalling receptor for PIGF during cutaneous tissue repair (Failla
et al. 2000; Carmeliet et al. 2001). Expression
of sVEGFR-1 has been described in a variety of primary human endothelial
cells, in various cancer tissues and different biological fluids (Barleon
et al, 1997; Barleon et al, 2001; Banks et al, 1998; Hornig et al, 1999;
Hornig et al, 2000; Kendall et al, 1993; Tor et al, 2002; Vuerola et al,
2000). The significance of naturally occurring sVEGFR-
1 is unclear at this time. In a recent study we investigated the hypothesis
whether sVEGFR-1 plays a role during cutaneous wound repair and we evaluated
the expression of sVEGFR-1 in normal healing and chronic non-healing cutaneous
wounds (Eming et al, 2004). ELISA and Western blot analysis revealed that
sVEGFR-1 concentration in wound fluid obtained from chronic non-healing
wounds was significantly increased over levels in wound fluid obtained
from healing wounds.
To assess the heterogeneity of sVEGFR-1 concentrations among different
wound fluid samples, particular among chronic wound fluid samples, we
investigated the kinetics of sVEGFR-1 release at different stages during
the healing process. Progression in wound healing was evaluated by assessing
granulation tissue formation and re-epithelialization
by wound tracings at indicated time points. sVEGFR-1 levels quantified
in wound fluid collected from normal healing wounds were low at initial
postoperative days, similar to serum levels, increased during granulation
tissue formation up to a maximum and decreased with wound closure. During
a two-months follow up in our clinic some of the chronic wounds transformed
from a nonhealing in a healing state, characterized by granulation tissue
formation and finally wound closure. In these patients induction of granulation
tissue formation and wound closure was associated with a decrease in
sVEGFR-1 concentrations. The positive correlation between
healing progression and sVEGFR-1 decline was statistically significant
(r = 0.92, p < 0.0005). In contrast, sVEGFR-1 levels in chronic wounds
which did not develop granulation tissue and did not diminish in wound
size over a period of two months remained high. Interestingly, the kinetics
of sVEGFR-1 secretion in normal healing wounds resembled those described
for VEGF-A/PIGF expression during normal wound repair, indicating a temporal
correlation of sVEGFR-1 and VEGF ligand expression during wound angiogenesis
(Nissen et al, 1998; Failla et al, 2000; Carmeliet et al, 2001). This
observation supported the idea that during physiological angiogenesis
sVEGFR-1 may control a local overshooting response of the increasing VEGF
related ligands.
In contrast, induction of sVEGFR-1 expression to nonphysiological levels,
as measured in chronic non-healing wounds, indicate a disturbance of the
VEGF ligand/ sVEGFR-1 balance; potentially, this dysregulation may attenuate
vessel growth during granulation tissue formation and hence impair wound
closure.
In summary, our report revealed the expression of sVEGFR-1 during different
stages of healing suggesting a function of sVEGFR-1 during tissue repair.
Whether increased sVEGFR-1 levels in non-healing wounds interfere with
the activities of VEGF related ligands and potentially reduce angiogenesis
remains to be investigated in further studies. However, our results lead
to the intriguing hypothesis as to whether the sVEGFR-1 level detected
in wound fluid can be of prognostic value for differentiating an effective
or impaired wound healing response. An indicato for healing would be of
great value to assess disease severity and progression of the chronic
wound, and might serve as predictive indicator for the efficacy of a certain
therapy regime.
Conclusion
In recent studies, we and others provided evidence that proteolytic processing
of VEGF-A might be an important
event controlling VEGF-A activity in tissue repair, inflammation and cancer.
Indeed, our data indicates that increased proteolysis of VEGF-A in the
highly proteolytic microenvironment of the chronic wound leads to VEGF-A
inactivation and reduced VEGF165 availability at the wound site which
might contribute to an impaired healing response. Beside VEGF-A proteolysis
our studies on sVEGFR-1 suggest an additional mechanism which compromises
VEGF-A mediated angiogenesis in chronic nonhealing wounds. These observations
might have clinical impact. In the highly proteolytic environment of the
nonhealing human wound, a protease-resistant VEGF165 mutant might be more
effective in stimulating wound angiogenesis and to improve wound closure.
Furthermore, topical applied VEGF-A protein may shift the increased and
anti-angiogenic sVEGFR-1/VEGF-A balance of the non-healing wound to a
pro-angiogenic response which favours the healing response.
CORRESPONDING AUTHOR:
PD DrMed Sabine A. Eming
Department of Dermatology, University of Cologne
Joseph-Stelzmann Str. 9, D – 50931 Köln, Germany
Tel: +49 221 4784500 Fax: +49 221 4200988
E-mail: Sabine.Eming@uni-koeln.de
REFERENCES
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