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Vascular endothelial growth factor-A (VEGF-A) is a crucial mediator of vascular hyperpermeability, angiogenesis, and inflammation, processes intimately involved in tissue repair. Although much emphasis has been placed on understanding the synthesis and stability of VEGF-A mRNA, relatively little attention has been given to the study of the stability and processing of VEGF-A proteins themselves. In recent years, several studies indicated that VEGF-A protein activity is highly controlled through interaction with angiogenic or non-angiogenic mediators. We analyzed mechanisms that might control extracellular VEGF-A processing during wound repair. First, our studies provide evidence that VEGF-A protein is a target of proteases present in the microenvironment of human chronic non-healing wounds. Serine proteases, in particular plasmin cleave VEGF165 and digested VEGF fragments, showed decreased mitogenic activity. Inactivation of the plasmin cleavage site Arg110/Ala111 preserved the structural integrity and increased the angiogenic potency of VEGF165 when tested in an impaired healing mouse model. Secondly, we identified significantly increased levels of the potent VEGF-A inhibitor, the soluble form of the VEGF receptor VEGFR-1 (sVEGFR-1) in non-healing wounds when compared to healing wounds. Wound closure in healing and non-healing wounds correlated significantly with a decline in sVEGFR-1 levels. These observations support the concept that VEGF-A and VEGF-A receptors are important mediators in tissue repair. Further, our data provide mechanisms how VEGF-A-mediated interactions are disturbed during impaired healing.
vascular endothelial growth factor
Vascular Endothelial Growth Factor-a and Vascular Endothelial Growth Factor-a Receptors In Tissue Homeostasis And Repair
Vascular endothelial growth factor-A (VEGF-A), the most potent and specific vascular growth factor, is a key regulator in physiological and pathological angiogenesis (
). VEGF-A levels are regulated through transcriptional control and mRNA stability. Moreover, by differential mRNA splicing, the single human VEGF-A gene gives rise to at least eight isoforms (VEGF121, 145, 148, 162, 165, 183, 189, and 206) whose relative abundance varies among different tissues (
); the 165-amino-acid isoform is the major gene product found in human tissues (Figure 1). The domains encoded by exons 1–5 of the VEGF-A gene are present in all VEGF splice variants and contain information required for the recognition of the tyrosine kinase VEGF receptors 1 (VEGFR-1/fms-like tyrosine kinase 1) and 2 (VEGFR-2/KDR/Flk-1) (
). The isoforms are distinguished by the presence or absence of the peptides encoded by exons 6 and 7 of the VEGF-A gene that code for two independent heparin-binding domains. Substantial evidence indicates that differences in the expression of the heparin-binding domains are crucially involved in the diverse biochemical and functional properties of the VEGF-A splice forms, including binding to cell surfaces and extracellular matrix (
VEGF-A expression in normal skin is absent. However, mechanical injury in skin provokes a strong upregulation of VEGF-A expression, which correlates both temporally and spatially with the proliferation of new blood vessels (
) (Figure 2). In these initial studies, immunohistological and in situ hybridization analysis identified epidermal keratinocytes and infiltrating macrophages as the principal VEGF-A source during skin repair. Since then in vitro analysis revealed that also platelets, neutrophils, and mast cells are significant VEGF-A sources and might provide additional VEGF-A sources during wound angiogenesis. Wound healing studies in two different transgenic mouse VEGF-A-GFP reporter models gave contradictive results with regard to the sites of VEGF-A expression during repair (
). Whereas the model of Fukumura revealed VEGF-A expression predominantly in fibroblasts, Kishimoto showed primarily epidermal VEGF-A expression. These opposing results are most likely explained by the different promoter regions used. Furthermore, positional effects of the transgene might explain differences in transgene expression. Thirdly, in both studies, the human promoter was used which might be differentially regulated in murine models.
VEGF-A transcription is regulated by numerous external factors, including growth factors, proinflammatory cytokines, hormones, and cellular stress (
). One of the best-characterized factor of VEGF-A synthesis is hypoxia. In vitro analysis indicates that beside hypoxia, proinflammatory cytokines, and growth factors regulate VEGF-A expression during repair (
VEGF-A action during repair is thought to regulate different processes, including vascular permeability, the influx of inflammatory cells into the site of injury, migration and proliferation of pre-existing endothelial cells, and, as suggested recently, the recruitment of marrow-derived endothelial progenitor cells to the local wound site where they are able to accelerate repair (
. In this study, it was shown that in diabetic mice (db/db), which are significantly compromised in wound healing, VEGF-A mRNA and intracellular VEGF-A protein processing was dramatically decreased during the repair process. Wound angiogenesis is significantly impaired in these mice, thus providing a causative link between decreased VEGF-A activity, impaired wound angiogenesis, and delayed healing. Furthermore, the role of VEGF-A in tissue repair was revealed in a study where application of neutralizing antibodies caused a striking reduction in wound angiogenesis, fluid accumulation, and granulation tissue formation in a pig wound model (
). In addition, the role of VEGF-A in tissue repair has been demonstrated by the treatment of ischemic wounds with VEGF-A protein, or adenovirus-mediated VEGF165 gene transfer, which enhanced wound healing in streptozotocin-induced diabetic mice (
). Along these lines, recently, we and others provided evidence that the db/db delayed healing phenotype can be reversed by the local treatment of wounds with VEGF165 protein, most likely by promoting wound angiogenesis (
). Interestingly, transgenic mice overexpressing VEGF-A in the epidermis showed no difference in wound closure rate when compared to their wild-type littermates, illustrating the importance of using an impaired healing model for testing the effects of VEGF in tissue repair (
). Together, these data provide substantial evidence that VEGF-A activity is a crucial regulator of wound repair.
VEGF-A exerts its biological effects upon binding to two high-affinity receptor tyrosine kinases VEGFR-1 and VEGFR-2, and the recently described neuropilin-1 receptor, all of which are expressed predominantly but not exclusively on endothelial cells (Figure 3) (
). Non-endothelial cells that express VEGFRs and are known to be involved in wound repair include macrophages, neutrophils, pericytes, and stromal cells; whether VEGF signaling in these cells is functionally relevant in tissue repair is currently unknown. During skin repair, expression of VEGFR-1 and VEGFR-2 has been shown to be upregulated primarily in dermal capillaries and macrophages (
). In vivo studies support the hypothesis that epidermally derived VEGF can stimulate angiogenesis in a paracrine manner by demonstrating increased microvascular density in the skin of VEGF transgenic mice (
). Whereas most of endothelial VEGF-A signaling described to date is largely mediated via VEGFR-2, the function of VEGFR-1 is less clear. Strong experimental evidence indicates that VEGFR-1 on the vasculature may act primarily as a ligand-binding molecule during angiogenesis, rather than as a signaling tyrosine kinase; in VEGFR-1 null mutant mice, the abnormal overgrowth of endothelial cells has been shown to be lethal. In vitro studies revealed that VEGFR-1 on monocytes/macrophages promotes chemotaxis (
). Interestingly, recent data indicate that VEGFR-1-mediated function in wound repair is regulated in part by binding of placental growth factor (PlGF), another ligand of VEGFR-1. Healing of skin incisions in PlGF-deficient mice was significantly reduced when compared to control mice (
). This effect may be explained by pleiotrophic effects of PlGF on multiple cell types (endothelial cells, hematopoietic progenitors, macrophages, and neutrophils), involved in all stages of vascular growth in the adult (
). Nrp-1 was originally characterized as a semaphorin receptor that is important for guidance of developing nerves. Nrp-1 also plays a role in vasculogenesis as Nrp-1-null-mice are embryonic lethal and exhibit cardiovascular defects. Interestingly, beside VEGF-B, PlGF-2, and some VEGF-E variants, VEGF165 has been identified as the only VEGF-A isoform binding to the Nrp-1. The VEGF-A heparin-binding domain has been identified as the epitope for Nrp-1 binding (
suggest a role for Nrp-1 during wound angiogenesis. Using a murine model of dermal wound repair, this group showed abundant Nrp-1 expression on new vasculature in healing wounds. Mice treated with neutralizing anti-Nrp-1 antibodies exhibited a significant decrease in vascular density, indicating a functional role for Nrp-1 during wound angiogenesis.
Proteolytic Processing Of Vegf-a Isoforms In Wound Repair
In addition to mRNA-splicing, proteolytic mechanisms also generate VEGF-A variants with different biological activites (
). This observation suggests that the proteolytic microenvironment should be considered as a critical determinant controlling VEGF-A-mediated activities. Initial evidence supporting the hypothesis that VEGF-A proteolytic processing regulates its activity were derived from studies analyzing the activity of the VEGF189 isoform (
). Whereas native VEGF189 binds to VEGFR-1 but not VEGFR-2, maturation of native VEGF189 by urokinase (uPA) within the exon 6-encoded sequence resulted in its VEGFR-2 binding and exerted a mitogenic effect on endothelial cells. Whether VEGF189, in particular its uPA-mediated activation, plays a role in vivo has not been analyzed so far.
We and other investigators demonstrated the sensitivity of VEGF165 protein to serine proteases, in particular plasmin (
). Plasmin digestion of VEGF165 yields two fragments: an amino-terminal homodimer (VEGF1–110) containing VEGF receptor binding determinants and a carboxyl-terminal polypeptide comprising the heparin-binding domain (VEGF111–165) (
) (Figure 4). Whereas the heparin-binding affinity of the complete VEGF165 protein and the VEGF111–165 cleavage product were nearly equivalent, no heparin binding was observed for the VEGF1–110 cleavage product (
), indicating that the heparin-binding function of VEGF165 is completely mediated by the carboxyl-terminal domain. Loss of the carboxyl-terminal polypeptide through plasmin digestion significantly reduces VEGF165 mitogenic activity on HUVE cells (
), supporting the crucial significance of the heparin-binding domain for VEGF-A function. So far, little is known about the prevalence and biological significance of proteolytic digestion of VEGF-A, in particular in the in vivo situation.
Recently, we provided evidence for the biological significance of VEGF165 proteolytic processing in wound healing. Comprehensive morphological and functional data indicate that endothelial cell function and angiogenesis is significantly disturbed in human chronic non-healing wounds (
). We investigated the hypothesis whether there exists a causative link between impaired angiogenesis and decreased VEGF-A activity in chronic non-healing wounds. To test this hypothesis, we analyzed VEGF-A expression and protein integrity in chronic non-healing wounds. VEGF-A mRNA and protein expression was highly upregulated in non-healing wounds, and levels were comparable to normal healing wounds (
). Thus, these data prompted us to focus on the degradation pathway of VEGF165 protein. Our data demonstrated that in contrast to healing wounds, VEGF165 protein is a target of proteolytic processing in chronic non-healing wounds. SDS-PAGE analysis and protease inhibitor studies strongly indicated that serine proteases, in particular plasmin, are involved in VEGF165 processing in the context of the chronic wound environment. In contrast, inhibitors of matrix metalloproteinases (MMP) (EDTA, phenanthroline), of cystein proteinases (elastinal, leupeptin, E-64, TLCK, antipain), and of acidic proteases (pepstatin) had no effect on VEGF165 degradation in wound fluid collected from non-healing wounds.
To further investigate the biological consequence of plasmin-mediated VEGF165 cleavage, we identified the plasmin cleavage site in the VEGF165 molecule (Figure 4). Plasmin digestion of VEGF165 yielded two fragments: an amino-terminal homodimer (VEGF1–110), which was resistant to further cleavage, and a carboxyl-terminal polypeptide (VEGF111–165) (Keyt, 1996b;
). By the introduction of amino-acid alterations at the plasmin-sensitive cleavage site Arg110/Ala111, we generated plasmin-resistant VEGF165 mutants. Substitutions at either site resulted in VEGF165 products that, while maintaining growth-promoting properties, were fully plasmin-resistant. Interestingly, a plasmin-resistant VEGF165 mutant (Ala111 → Pro111) (VEGF165A111P) showed significantly increased stability when incubated in wound fluid derived from non-healing wounds (
). Taken together, our data demonstrated that in the highly proteolytic environment of human non-healing skin ulcers, VEGF165 degradation is increased as compared to normal-healing wounds and VEGF-A processing can be partially rescued by inactivation of the plasmin-sensitive cleavage site Arg110/Ala111. Furthermore, our data propose that reduced VEGF165 availability at the wound site may contribute to impaired healing.
described additional MMP cleavage sites in the VEGF-A protein. This group identified MMP-3, -7, -9, and -19, which efficiently cleaved the murine VEGF164 isoform; MMP-1 and -16 were less effectively. MMP cleavage appeared to occur in sequential steps at residues 135, 120, and finally at residue 113 (Figure 4). Similar to plasmin-mediated digestion, also for MMP cleavage the most N-terminal-proximal cleavage site resulted in dissociation of the VEGF165/164 heparin-binding motif.
Overall, these studies support the idea that proteolytic processing of VEGF-A is a crucial event in controlling VEGF-A activity. These findings reveal a novel aspect in the regulation of extracellular VEGF-A that holds significance for angiogenesis. Although MMPs are highly involved in tissue repair, the role of MMP-mediated processing of VEGF-A in wound healing is currently unclear.
reported that collagenase (MMP-1), an MMP intimately involved in wound repair, does not cleave human VEGF165. Data from our group demonstrated that VEGF165 protein stability in chronic wound fluid was not influenced by the classical MMP inhibitor ethylenediaminetetraacetic acid, proposing that in chronic wounds MMP-mediated cleavage of VEGF165 is not the primarily proteolytic pathway (
Plasmin Resistance Of Vegf165 Improves Wound Angiogenesis
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) (
). 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 vascularized granulation tissue as well as an accelerated re-epithelialization 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 (
). 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 (
). 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 (
). 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. As outlined above, the VEGF-A heparin-binding domain has been identified as the epitope for Nrp-1 binding (
). 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 (
). 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. Future studies will have to investigate whether extracellular processing of VEGF165 is a frequent regulatory event in other physiological or pathological situations of angiogenesis than tissue repair, such as inflammation and cancer.
analyzed the biological impact of MMP processing of VEGF-A in vitro and in vivo tumor models. Carcinoma cells were transfected with different VEGF-A variants and injected subcutaneously into a mouse model. Interestingly, tumors that expressed the MMP-cleaved VEGF fragment VEGF1–113 grew poorly and showed capillary dilation of existing vessels. In contrast, tumors expressing the MMP-resistant VEGF-A variant displayed faster growth kinetics, and the vasculature was characterized by increased capillary density with multiple and frequent branch points. Similar to the enhanced angiogenic response of the plasmin-resistant VEGF165 mutant during wound repair, altered structural–functional properties of MMP-resistant VEGF164 might account for the accelerated tumorangiogenesis and tumor growth observed.
Overall, these studies provide substantial evidence that VEGF-A proteolysis occurs in vivo and might add an additional control for VEGF-A-mediated processes. Different structural–functional properties might account for the altered VEGF activities of processed VEGF-A. Present studies suggest that in particular loss of the heparin-binding domain might result in VEGF-A fragments with altered functions. Rendering the VEGF-A molecule resistant to proteolytic cleavage might increase the portion of matrix-bound VEGF-A molecules, which are more efficient in supporting a more functional angiogenic response than soluble VEGF-A.
Soluble Vegfr-1: A Mediator In Tissue Repair
Besides analyzing VEGF-A processing, we were interested to identify potential inhibitors of VEGF-A-mediated actions in the microenvironment of non-healing wounds. So far, soluble VEGFR-1 (sVEGFR-1), a splice variant of the membrane-bound VEGFR-1, is considered the only naturally occurring specific inhibitor 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 (
). Potentially, sVEGFR-1 functions as an inert decoy receptor by binding VEGF-A and thereby regulating the availability of VEGF-related ligands for the activation of VEGFR-2 (Flk-1/KDR), the VEGF receptor principally involved in VEGF signaling (
). 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 signaling receptor for PIGF during cutaneous tissue repair (
). 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 (
). 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, particularly 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 2 month follow-up in our clinic, some of the chronic wounds transformed from a non-healing 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 2 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 (
). 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 non-physiological 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 indicator 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.
The authors' Institutional Review Board has approved human in vivo studies. All clinical investigations have been conducted according to the Declaration of Helsinki Principles, and written informed consent has been obtained from all patients. All animal studies have been approved by the institutional animal care and use committee.
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 cancerogenesis. Indeed, our data indicate 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. Besides VEGF-A proteolysis, our studies on sVEGFR-1 suggest an additional mechanism, which compromises VEGF-A-mediated angiogenesis in chronic non-healing wounds. These observations might have clinical impact. In the highly proteolytic environment of the non-healing human wound, a protease-resistant VEGF165 mutant might be more effective in stimulating wound angiogenesis and to improve wound closure. Further topical applied VEGF-A protein may shift the increased and antiangiogenic sVEGFR-1/VEGF-A balance of the non-healing wound to a proangiogenic response that favors repair. However, in tumor development, protease inhibition might increase the portion of matrix-bound VEGF-A, which could promote tumor growth. These observations are interesting and merit future studies, which analyze whether pathways of VEGF-A proteolysis might be different and potentially functional relevant in diverse disease states and organs.
Conflict Of Interest
The authors state no conflict of interest.
This work was supported by the Deutsche Forschungsgemeinschaft (SAE, FOR 265 and TK, SFB 589), and a grant of the European Community (SAE, LSHB-CT-2005-512102).
Suppression of retinal neovascularization in vivo by inhibition of VEGF using soluble VEGF-receptor chimeric proteins.