Published online before print December 28, 2006, doi: 10.1161/01.ATV.0000256459.06671.3c
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© 2007 American Heart Association, Inc.
Vascular Biology |
Chimeric VEGF-ENZ7/PlGF Specifically Binding to VEGFR-2 Accelerates Skin Wound Healing via Enhancement of Neovascularization
From the Division of Genetics (Y.Z., M.W., M.S.), Laboratory Animal Research Center and Amami Laboratory of Injurious Animals (T.K., S.H., C.K.), Institute of Medical Science, University of Tokyo, Japan.
Correspondence to Dr Masabumi Shibuya, Division of Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan. E-mail shibuya{at}ims.u-tokyo.ac.jp
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Abstract |
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Objective— VEGF-ENZ7/PlGF molecules composed of Orf virus-derived VEGF-ENZ7 and human PlGF1 were previously proven to be potent angiogenic factors stimulating angiogenesis without significant enhancement of vascular leakage and inflammation in vivo. For its future clinical application, there is a pressing need to better understand the beneficial effects of VEGF-ENZ7/PlGF during wound healing in adulthood.
Methods and Results— In this study, several angiogenic factors were administrated to skin punched wounds of both wild-type and diabetic mice. The treatment with VEGF-ENZ7/PlGF accelerated wound closure accompanied with enhanced angiogenesis, the process was occurring slightly faster than that in VEGF-A164 group. Moreover, the macrophage infiltration and lymphangiogenesis level in healed wounds were strikingly lower in VEGF-ENZ7/PlGF group than VEGF-A164 group, suggesting that the increased inflammation was the key issue preventing speedy wound healing of VEGF-A164–treated skin. Considering clinical safety, we further examined the antigenicity of chimeric VEGF-ENZ7/PlGF. Compared with the original VEGF-ENZ7, the immunogenicity of VEGF-ENZ7/PlGF molecules was markedly decreased in mice and squirrel monkeys with the increase of PlGF1 humanized ratio.
Conclusion— These results indicate that VEGF-ENZ7/PlGF molecules are superior to VEGF-A for the acceleration of either normal or delayed skin wound healing and might be regarded as potential drugs in therapeutic angiogenesis.
We report here that chimeric VEGF-ENZ7/PlGF molecules of low antigenicity accelerate skin wound healing with enhanced angiogenesis, less macrophage infiltration and lymphangiogenesis in both wild-type and diabetic mouse model. These findings clearly indicate that chimeric VEGF-ENZ7/PlGF molecules are superior to VEGF-A, and might be the potential drugs in therapeutic angiogenesis.
Key Words: VEGF-ENZ7/PlGF • wound healing • angiogenesis • lymphangiogenesis • inflammation
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Introduction |
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Skin, composed of keratinized stratified epidermis as well as collagen-rich dermis, primarily serves as a protective barrier against the environment. Severe skin injury or chronic ulcers may lead to disability or even death. Healing of skin wounds is a complex process which encompasses multiple sequential events such as fibrin clot formation, inflammation, angiogenesis, epthelialization, tissue formation and remodeling.1,2 Throughout these events, angiogenesis, the formation of new blood vessels from preexisting ones, is thought to be the crucial step under both physiological and pathological conditions.3,4 Therefore, angiogenic factors are proposed to be the potential molecules for promoting cutaneous wound closure.
Chronically incurable ulcers may occur if skin wounds do not proceed smoothly, particularly in diabetics because of the significant downregulation of vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF) around the wound.5–7 Tremendous efforts have been made to explore the use of growth factors for accelerating chronic or delayed skin wound closure and improving blood perfusion via the approaches of gene therapy, protein administration, or transplantation of bone marrow–derived cells. To date, the beneficial effects of VEGF, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), KGF, platelet-derived growth factor (PDGF), and angiopoietin (Ang) have been well investigated in different wound healing models, and some of them are even undergoing clinical trials.8–13 However, not all results have been satisfactory, for instance, VEGF-A caused severe vascular leakage and inflammation.14,15 On the other hand, the disadvantages of FGF, HGF, KGF, PDGF, and Ang1 are attributed to their short half-life time, non-endothelial cell specificity, and not as favorable effective as VEGF in terms of promoting angiogenesis. To address these issues, it is necessary to identify new endothelial cell–specific proangiogenic factors that induce neither vascular leakage nor severe inflammation.
Our group is now focusing on VEGF-ENZ7, a potent angiogenic factor encoded by Orf virus (strain NZ7).16,17 Recently, we constructed two humanized molecules composed of the VEGFR-2–specific ligand VEGF-ENZ7 and VEGFR-1–specific ligand human PlGF1.18 These humanized molecules, namely chimeric VEGF-ENZ7/PlGF, were proven to specifically bind VEGFR-2 and promote angiogenesis without significant enhancement of vascular leakage and inflammatory cell infiltration in a transgenic mouse model.19 Based on these promising findings, practically, it is worth examining the beneficial effects of VEGF-ENZ7/PlGF protein during wound healing. In the present study, by using both wild-type and genetically diabetic mice, we investigated the effects of VEGF-ENZ7/PlGF on punched skin wounds. Our results indicate that VEGF-ENZ7/PlGF accelerates wound closure with a histological well-reconstructed skin architecture, enhanced angiogenesis, as well as less macrophage infiltration and lymphangiogenesis, suggesting that humanized VEGF-ENZ7/PlGF may be better than VEGF-A as a potential growth factor for therapeutic angiogenesis.
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Methods |
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Immunogenicity Assay
Animal experiments were performed according to the guidelines of the Institute of Medical Science, the University of Tokyo. Groups of 8-week-old BALB/c mice received subcutaneous injection of VEGF-ENZ7 or 2 chimeric VEGF-ENZ7/PlGF molecules (chimera9 and chimera33) at a dose of 10 µg/mouse/week for 3 weeks. Mouse serum was collected and subjected to immunogenecity assays using the sandwich ELISA. A multi-well plate was coated with anti-His tag antibody (Abcam, Cambridge, United Kingdom). The antigens used were His-tagged VEGF-ENZ7, His-tagged chimera9, and His-tagged chimera33 expressed in Sf9 baculovirus system. Mouse serum was added as a capture antibody, and the detection antibody was horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Amersham Biosciences, Uppsala, Sweden). The plate was developed using TMB substrate (Sigma–Aldrich, St Louis, Mo), and optical density was recorded at 450 nm. The concentration of antibody in mouse serum against VEGF-ENZ7, chimera9, or chimera33 was calculated from a standard curve obtained from serial dilutions of VEGFR-2/Fc (R&D system, Minneapolis, Minn) as a positive capture antibody and HRP-conjugated anti-human IgG as a detection antibody (Amersham Biosciences, Uppsala, Sweden).
Groups of 4- to 17-year-old squirrel monkeys received subcutaneous injection of VEGF-ENZ7 (1 µg/monkey/week) or chimera33 (1 and 10 µg/monkey/week). Monkey serum was collected and subjected to immunogenicity assays using the sandwich ELISA. For VEGF-ENZ7, ELISA plate was coated with purified His-tagged VEGF-ENZ7. For chimera33, coating antibody was PlGF C-20 (Santa Cruz Biotechnology, Santa Cruz, Calif) and chimera33 was coated as antigen. Capture antibody was monkey serum and the detection antibody was HRP-conjugated protein A. The concentration of antibody in monkey serum against VEGF-ENZ7 or chimera33 was calculated from a standard curve obtained from serial dilutions of anti-His tag antibody as the positive capture antibody and HRP-conjugated protein A as the detection antibody.
Wound Healing Experiments
8-week-old male BALB/c or C57BLKS/Jm +/+ Leprdb (db/db) diabetic mice (CREA Japan Inc, Tokyo, Japan; 5 per group) were anesthetized, the dorsum was shaved and cleaned with 70% ethanol. Full-thickness skin wounds of 5-mm diameter were made aseptically using a biopsy punch (Kai Industries). Each wound was digitally photographed at the indicated time intervals. Changes in the wound were measured using a digital slide caliper and expressed as a percentage of the initial wound. Healed wounds and surrounding areas were collected for histological and immunohistochemical examination.
The growth factors used for wound healing therapy in this study were obtained as follows: subconfluent mouse embryonic fibroblast cells (MEF) were infected with adenovirus, which encoded ß-galactosidase, VEGF-A164, chimera9 or chimera33, respectively (provided by Dr Seppo Yla-Herttuala, University of Kuopio, Finland). Two days later, MEFs were cultured in serum-free Dulbecco’s Modified Eagle’s medium (DMEM) for another 3 days. Suspended cells and cell debris were separated from the serum-free conditional medium (CM) by centrifugation. Purified His-tagged VEGF-ENZ7 expressed in sf9 cells was mixed with the CM of Ad-LacZ-MEF. The concentrated CM was dialyzed against phosphate-buffered saline (PBS) and injected subcutaneously into mice with punched wounds in the dorsal skin at a dose of 2 µg/wound/10 days.
Histological Analysis and Immunohistochemistry
Tissue samples were fixed in 4% paraformaldehyde (PFA). For H&E staining, paraffin-embedded specimens were sectioned at 5-µm thickness and stained with hematoxylin and eosin using a standard protocol. Immunohistochemical staining for von Willebrand factor (vWF) was performed with anti-human vWF antibody (Dakopatts, Glostrup, Denmark) using a standard protocol.
Immunofluorescence Staining
The skin samples fixed in 4% PFA were frozen and sectioned at 10-µm thickness. After blocking, samples were incubated with a primary antibody and subsequently with the corresponding secondary antibody. Primary antibodies were rat anti-mouse F4/80 (Serotec, Oxford, United Kingdom) and rabbit anti-mouse LYVE-1 (Abcam, Cambridge, United Kingdom). The secondary antibodies were anti-rat IgG Alex 488 and anti-rabbit IgG Alex 488 (Molecular Probes, Eugene, Ore). Digital images were recorded by confocal microscopy.
Western Blotting
Proteins separated by SDS-PAGE were transferred to an Immobilon P membrane. The membrane was blocked with 5% bovine serum albumin (BSA) in PBS buffer containing 0.1% Tween20 (PBST) and subjected to primary antibody incubation. The primary antibodies were VEGF C-1 against mouse VEGF-A, PlGF C-20 against human PlGF1 (Santa Cruz Biotechnology, Santa Cruz, Calif), and VE3 against the loop3 region of VEGF-ENZ7 as well as chimeric VEGF-ENZ7/PlGF (generated in our laboratory). Corresponding HRP-conjugated secondary antibodies were used for detection of the primary antibody and developed with chemiluminescent regent.
Statistical Analysis
Data from at least 5 independent experiments are presented as mean±SD. Comparisons between different groups were analyzed with the Student unpaired t test. A value of P<0.05 was considered as statistically significant.
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Humanization at Nonessential Portions of VEGF-ENZ7 Molecule Results in Significantly Decreased Immunogenicity
Previously, we examined the angiogenic activity and side effects of 2 chimeric VEGF-ENZ7/PlGF molecules, which were humanized with PlGF1 at the non-essential portions of Orf virus derived VEGF-ENZ7, namely chimera9 and chimera33 in a transgenic mouse model.19 Although no obvious difference was found between them, chimera33 possessed much more portions from human PlGF1. The identities of chimera9 and chimera33 with human PlGF1 are 50.3% and 61.8% at the amino acid level, respectively, whereas the original VEGF-ENZ7 is only 27.3% identical with human PlGF1 (Figure 1A). Therefore, in terms of antigenicity, we speculated that VEGF-ENZ7/PlGF is better than VEGF-ENZ7 for therapeutic angiogenesis. To investigate the immunogenicity, His-tagged proteins including VEGF-ENZ7, chimera9, and chimera33 were subcutaneously injected into wild-type mice. Mouse serum was collected and subjected to immunogenicity assays by ELISA. We found that the immunogenicity of both chimera9 and chimera33 was significantly decreased compared with that of the original VEGF-ENZ7 (Figure 1B).
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In addition, using the same method, we tested the immunogenicity of chimera33 in squirrel monkey. VEGF-E chimera33 protein appears to be useful for middle and high-aged patients. When we used 16- to 17-year-old monkeys which correspond to about 50-year-old humans, we did not detect antigenicity of chimera33 against either regular (1 µg/monkey) or high-dose (10 µg/monkey) challenge (Figure 1C). When we used young monkeys of 4- to 5-year-old (corresponding to 12
15-year-old humans) for immunization, we detected a minor antigenicity (undetectable level to 84 ng/mL of antibody in serum). However, even in these cases with young animals, the amounts of antibody in serum were very low, which were less than 0.01% of regular immunization in rabbits (specific antibody generated in rabbits: usually higher than 1 mg/mL of serum). Neither mouse nor monkey used in these experiments showed any anaphylactic response or abnormal swelling at the local sites. These findings suggest that VEGF-ENZ7/PlGF molecules are much more useful than the original VEGF-ENZ7 for angiogenic therapy in clinic.
VEGF-ENZ7/PlGF Accelerates the Closure of Punched Skin Wounds as Effectively as VEGF-A164 in Both Wild-Type and Genetically Diabetic Mice
In K14-VEGF-A165, K14-VEGF-ENZ7, and K14-chimeric VEGF-ENZ7/PlGF transgenic mice, we have found that the healing speed of cutaneous wound was quicker than that of their wild-type littermates (data not shown). This result encouraged us to further investigate how the effect of directly introducing these growth factors into the wound site in wild-type mice is of the same BALB/c background with transgenic mice. Although deficient adenoviral vector is a good vehicle for drug delivery and has been wildly used in gene therapy, it was reported that adenovirus was easy to induce host immune responses resulting in inflammation of transduced tissues and efficient clearance of the administered vector.20,21 Therefore, the growth factors used for this wound healing assay were adenovirally expressed in MEF cells and baculovirally expressed in sf9 cells in vitro (Figure 2A). Measurements of wound size were performed every 2 days after skin damage, and the photographs taken at the indicated time points during wound healing revealed that all the growth factors including VEGF-A164, VEGF-ENZ7, chimera9, and chimera33 had the similar effect of accelerating wound closure in comparison to the control group treated with the CM of Ad-LacZ-infected MEF cells (Figure 2B). The quantitative analysis showed that early stage of VEGF-A164–treated wounds healed as quickly as wounds of the other 3 groups; however, wound closing gradually slowed in the late stage. All the wounds of the VEGF-A164 group had healed by day 21, compared with day 23 in the control group. It was noteworthy that the wounds of the VEGF-ENZ7, chimera9, and chimera33 groups closed at a very steady pace in both the early and late stages and had completely healed by day 19 (Figure 2C).
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Considering the application of VEGF-ENZ7/PlGF in chronic incurable or delayed wound healing which often happens in diabetics because of microangiopathy,22 we next examined the healing effect of VEGF-ENZ7/PlGF in the C57BLKS/Jm +/+ Leprdb (db/db) diabetic mouse model.23 In comparison with wild-type BALB/c mice treated with the CM of Ad-LacZ-MEF, 4 more days were needed for the complete healing of skin wounds in db/db mice treated with the same CM (Figure 3). Although, groups treated with the CM of Ad-VEGF-A164-MEF, Ad-chimera9-MEF, Ad-chimera33-MEF, or purified His-tagged VEGF-ENZ7 healed quicker than the control group, the healing process still took 2 days longer than in the nondiabetic mouse model. These results indicate that VEGF-ENZ7/PlGF accelerates the delayed wound healing in diabetic mice as well.
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VEGF-ENZ7/PlGF Induces Reepithelialization Accompanied with the Enhancement of Angiogenesis around the Wound Area
To examine the tissue-remodeling of wounds, skin samples were harvested from healed wounds, and a histological analysis was carried out. H&E staining showed that reepithelialization occurred at wound site in all experimental groups of both wild-type BALB/c and diabetic mice (Figure 4A). Immunohistochemical staining of the blood vessel endothelial specific marker vWF revealed that blood vessel numbers in the dermis of wounds were significantly increased in response to VEGF-A164, VEGF-ENZ7, chimera9, and chimera33 (Figure 4B). Quantification indicated that groups treated with the different angiogenic factors developed more than twice as many blood vessels in the healing wounds as the control group. However, among these angiogenic factor–treated groups there was no significant difference in blood vessel density around wound area, suggesting that chimeric VEGF-ENZ7/PlGF molecules are as effective as VEGF-A164 and VEGF-ENZ7 to promote angiogenesis in skin wound healing model (Figure 4C).
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Local Administration of VEGF-ENZ7/PlGF Does Not Induce the Elevation of Macrophage Infiltration to the Wound Site
Normally, inflammation is involved in the process of wound healing, and the recruited inflammatory cells could secrete numerous cytokines to accelerate wound repairing. However, severe inflammation in the late stage of wound healing will somehow prevent a speedy recovery. We hypothesized that the decreased healing speed in the VEGF-A164 group at the late stage was caused by severe inflammation. To verify this hypothesis, we proceeded to investigate macrophage infiltration in the healed skin wounds by immunofluorescence staining of the macrophage-specific marker F4/80. Numerous F4/80-positive macrophages were found in VEGF-A164–treated skin wounds of both wild-type and diabetic mice, numbers of which were markedly increased compared with those observed in other groups (Figure 5A). Quantitative analysis revealed that macrophage densities in the specimens from the VEGF-ENZ7, chimera9, and chimera33-treated groups were almost the same as those of the controls, whereas 1.6- and 2.1-fold increases in macrophage numbers were found in the samples of VEGF-A164–treated wild-type BALB/c and diabetic mice (Figure 5B). These results strongly suggest that severe inflammation in the late stage of the healing process is the main reason preventing wounds from quickly closing in VEGF-A164–treated mice.
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Administration of VEGF-A164 But Not Chimeric VEGF-ENZ7/PlGF Results in Enhancement of Lymphangiogenesis Around the Wound Area
It was reported that the infiltrated macrophages often secreted many cytokines and promoted lymphangiogenesis.24–26 Thus, we next examined lymphangiogenesis in healed skin wounds via immunofluorescence staining with a lymphatic specific marker, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). In the skin samples from the VEGF-A164–treated group, the LYVE-1–positive signal density was elevated, whereas in samples from the VEGF-ENZ7, chimera9, and chimera33-treated groups, the LYVE-1–positive signals were of a similar density to that in the control group (Figure 6A). Quantification indicated that in wild-type and genetically diabetic mice, lymphangiogenesis in the groups treated with VEGF-A164 was elevated 1.7- and 2.0-fold in comparison with that in the control group, respectively (Figure 6B). The enhancement of lymphangiogenesis in VEGF-A164–treated skin wounds is consistent with the result of elevated macrophage infiltration to the healed wound site, suggesting that the VEGF-C or VEGF-D secreted from macrophages might be the major cytokines promoting lymphangiogenesis via VEGFR-3 signaling. Interestingly, although much more macrophages were found in the wounds of diabetic mice, the lymphangiogenesis level in the same region of diabetic mice was lower than that in wild-type BALB/c mice. This result might be caused by the down-regulated expression level of a number of growth factors in diabetic mice.
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Discussion |
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During wound healing a number of cytokines such as KGF, EGF, PDGF, VEGF, FGF, and transforming growth factor (TGF) secreted by keratinocytes, neutrophils, macrophages, fibroblasts, and endothelial cells are involved in the healing process.27–29,4,30,31 However, not all of the cytokines are as necessary as VEGF for wound healing. More recently, it was reported that in KGF or even KGF/TGF-
double knockout mice, skin wounds healed at normal rates,32 and blocking FGF2 through vaccination did not result in an alteration to wound healing process.30 Here, we focused on the effect of 2 chimeric VEGF-ENZ7/PlGF molecules derived from VEGF-ENZ7 encoded by Orf virus stain NZ7 on wound healing. The VEGF-E gene in the genome of the Orf virus is not essential for viral replication, but promotes angiogenesis at local infectious site and increase in the efficiency of progenitor production. After humanization with PlGF1 at nonessential portions, we have recently shown that the chimeric VEGF-ENZ7/PlGF molecules are VEGFR-2–specific and potent angiogenic factors without the induction of severe tissue edema and inflammatory cell infiltration in a transgenic mouse model.19 In this study, we have further found a significant acceleration of the closure of skin punched wounds in both wild-type BALB/c and genetically diabetic mice via local administration of chimeric VEGF-ENZ7/PlGF proteins at the wound site without increased macrophage infiltration and lymphangiogenesis. Physiological inflammatory response is one of the crucial phases of the wound healing process, and inflammatory cells including neutrophil and macrophage are responsible for defense against bacterial invasion as well as degradation of debris within the wound site.33 A reduced macrophage infiltration at the wound site in a hemophilia B mouse model was reported to result in an increase in angiogenesis, suggesting that an abnormal increase in macrophage infiltration may induce edematous lesions and suppress angiogenesis as well as wound healing.34 This report is in accordance with our present findings: chimeric VEGF-ENZ7/PlGF, the growth factor with weak inflammatory activity attributable to no activation of VEGFR-1 compared with VEGF-A164, accelerated angiogenesis and skin wound healing with less macrophage recruitment.
In addition, our findings also revealed that the enhanced lymphangiogenesis level at the wound site of VEGF-A164–treated skin was correlated with the increased macrophage infiltration level. The role of macrophages in wound tissue is supplying proinflammatory chemoattractants to recruit as well as activate additional macrophages and growth factors such as VEGF-C responsible for cellular proliferation. Much evidence has proven that the major cytokine contributing to lymphangiogenesis secreted by macrophages is VEGF-C, which preferentially stimulates lymphangiogenesis but not angiogenesis in most cases. Recently, we have shown that VEGF-A and svVEGF derived from snake venom, which activate both VEGFR-1 and VEGFR-2, efficiently stimulate vascular permeability compared with VEGF-ENZ7.35 Thus, treatment of skin wounds with VEGF-A164 results in the enlargement of lymphatic vessels not only via VEGF-C from recruited macrophages but also via the high leakage of blood plasma fluid into tissues. Therefore, it seems reasonable that not only lymphangiogenesis but also the macrophage infiltration level in healed skin wounds treated with VEGF-ENZ7, chimera9, and chimera33 are in the same low range as those of the control group.
Although the identities of VEGF-ENZ7, chimera9, and chimera33 with murine PlGF1 at the amino acid level are not of great difference (28.1%, 35.8% and 41.5%, respectively), we have found the immunogenicity of chimera9 and chimera33 significantly decreased in comparison to that of Orf-virus derived VEGF-ENZ7 in mice (Figure 1A and 1B). Furthermore, aged monkeys even without diabetes did not show clear immune response to VEGF-E chimera33 protein (Figure 1C). A chronic wounding is often observed in aged and diabetic patients, and the immune system in diabetic patients is known to be lower than healthy people at the same age. Thus, the potential immunogenicity of the VEGF-E chimera proteins in diabetic patients may be very low, if any, because of the two factors, aging and basal disease (diabetis meritus). Based on these results, we suggest that the immunogenicity of chimera9 and chimera33 in human is much weaker than that of VEGF-ENZ7 or even undetectable.
Skin wound healing requires not only angiogenesis but also the proliferation of keratinocyte and other mesenchymal cells. Several different types of wound healing may exit, and in some types, particularly those in the aged people, chronic wounds might be mainly caused by a poor blood supply derived from atherosclerosis, resulting in a poor nutrition and poor proliferation of epithelial as well as mesenchymal cells. Therefore, we suggest that activation of VEGFRs with an appropriate growth factor such as VEGF-E chimera protein may be one of the efficient methods to promote the healing. FGF also could be useful for wound healing, but the molecular target is slightly different from VEGF-E: FGF does not activate directly the VEGFRs pathways but activates FGFRs expressed in a variety of cell types including endothelial cells. In addition, we should consider that angiogenic factors such as FGF family and HGF activate signaling pathways which are known to have transforming potential in various cells including gastric epithelial cells.36,37
It is well known that FGF-2 has been used in clinical skin wound healing with the combination of stabilizer. Early reports demonstrated that directly applied FGF-2 could not improve diabetic ulcer because of its short half life time and the local concentration of FGF-2 will decrease 50% within 4 hours.38,39 To control the release of growth factor, chitosan film as a stabilizer was used together with FGF-2 in wound healing assay. Even in such a condition, the diabetic mice treated with 2 µg FGF-2/chitosan film still had about 10% wound remaining by day 20.40 Our results indicated that in the same diabetic mouse model skin wounds almost closed via locally applied VEGF-ENZ7/PlGF at the dose of 2 µg/wound by day 20. Therefore, we suggest that chimeric VEGF-ENZ7/PlGF molecules have comparable wound healing effects to FGF-2 in the absence of stabilizer.
In conclusion, all the findings presented here clearly reveal that chimeric VEGF-ENZ7/PlGF of low immunogenicity has beneficial effects to accelerate both normal and delayed skin wound.
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Acknowledgments |
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We thank Dr. Seppo Yla-Herttuala at University of Kuopio, Finland for kindly providing Ad-VEGF-A164, Ad-chimera9, and Ad-chimera33 constructs.
Sources of Funding
This work was supported by Grant-in-Aid Special Project Research on Cancer-Bioscience 17014020 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, a grant from the program "Research for the Future" of the Japan Society for Promotion of Science, and the program "Promotion of Fundamental Research in Health Science" of the Organization for Pharmaceutical Safety and Research.
Disclosures
None.
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Footnotes |
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Original received August 23, 2006; final version accepted December 4, 2006.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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- Martin P. Wound healing–aiming for perfect skin regeneration. Science. 1997; 276: 75–81.
[Abstract/Free Full Text] - Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med. 1999; 341: 738–746.
[Free Full Text] - Tonnesen MG, Feng X, Clark RA. Angiogenesis in wound healing. J Investig Dermatol Symp Proc. 2000; 5: 40–46.[CrossRef][Medline] [Order article via Infotrieve]
- Bates DO, Jones RO. The role of vascular endothelial growth factor in wound healing. Int J Low Extrem Wounds. 2003; 2: 107–120.[Abstract]
- Walters DP, Gatling W, Mullee MA, Hill RD. The distribution and severity of diabetic foot disease: a community study with comparison to a non-diabetic group. Diabet Med. 1992; 9: 354–358.[Medline] [Order article via Infotrieve]
- Mason J, O’Keeffe C, McIntosh A, Hutchinson A, Booth A, Young RJ. A systematic review of foot ulcer in patients with Type 2 diabetes mellitus. I: prevention. I: prevention.Diabet Med. 1999; 16: 801–812.
- Werner S, Breeden M, Hubner G, Greenhalgh DG, Longaker MT. Induction of keratinocyte growth factor expression is reduced and delayed during wound healing in the genetically diabetic mouse. J Invest Dermatol. 1994; 103: 469–473.[CrossRef][Medline] [Order article via Infotrieve]
- Romano Di Peppe S, Mangoni A, Zambruno G, Spinetti G, Melillo G, Napolitano M, Capogrossi MC. Adenovirus-mediated VEGF(165) gene transfer enhances wound healing by promoting angiogenesis in CD1 diabetic mice. Gene Ther. 2002; 9: 1271–1277.[CrossRef][Medline] [Order article via Infotrieve]
- Obara K, Ishihara M, Ishizuka T, Fujita M, Ozeki Y, Maehara T, Saito Y, Yura H, Matsui T, Hattori H, Kikuchi M, Kurita A. Photocrosslinkable chitosan hydrogel containing fibroblast growth factor-2 stimulates wound healing in healing-impaired db/db mice. Biomaterials. 2003; 24: 3437–3444.[CrossRef][Medline] [Order article via Infotrieve]
- Kunugiza Y, Tomita N, Taniyama Y, Tomita T, Osako MK, Tamai K, Tanabe T, Kaneda Y, Yoshikawa H, Morishita R. Acceleration of wound healing by combined gene transfer of hepatocyte growth factor and prostacyclin synthase with Shima Jet. Gene Ther. 2006; 13: 1143–1152.[CrossRef][Medline] [Order article via Infotrieve]
- Geer DJ, Swartz DD, Andreadis ST. Biomimetic delivery of keratinocyte growth factor upon cellular demand for accelerated wound healing in vitro and in vivo. Am J Pathol. 2005; 167: 1575–1586.
[Abstract/Free Full Text] - Keswani SG, Katz AB, Lim FY, Zoltick P, Radu A, Alaee D, Herlyn M, Crombleholme TM. Adenoviral mediated gene transfer of PDGF-B enhances wound healing in type I and type II diabetic wounds. Wound Repair Regen. 2004; 12: 497–504.[CrossRef][Medline] [Order article via Infotrieve]
- Cho CH, Sung HK, Kim KT, Cheon HG, Oh GT, Hong HJ, Yoo OJ, Koh GY. COMP-angiopoietin-1 promotes wound healing through enhanced angiogenesis, lymphangiogenesis, and blood flow in a diabetic mouse model. Proc Natl Acad Sci U S A. 2006; 103: 4946–4951.
[Abstract/Free Full Text] - Vajanto I, Rissanen TT, Rutanen J, Hiltunen MO, Tuomisto TT, Arve K, Narvanen O, Manninen H, Rasanen H, Hippelainen M, Alhava E, Yla-Herttuala S. Evaluation of angiogenesis and side effects in ischemic rabbit hindlimbs after intramuscular injection of adenoviral vectors encoding VEGF and LacZ. J Gene Med. 2002; 4: 371–380.[CrossRef][Medline] [Order article via Infotrieve]
- Rissanen TT, Rutanen J, Yla-Herttuala S. Gene transfer for therapeutic vascular growth in myocardial and peripheral ischemia. Adv Genet. 2004; 52: 117–164.[CrossRef][Medline] [Order article via Infotrieve]
- Ogawa S, Oku A, Sawano A, Yamaguchi S, Yazaki Y, Shibuya M. A novel type of vascular endothelial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity without heparin-binding domain. J Biol Chem. 1998; 273: 31273–31282.
[Abstract/Free Full Text] - Shibuya M. Vascular endothelial growth factor receptor-2: its unique signaling and specific ligand, VEGF-E. Cancer Sci. 2003; 94: 751–756.[CrossRef][Medline] [Order article via Infotrieve]
- Kiba A, Yabana N, Shibuya M. A set of loop-1 and -3 structures in the novel vascular endothelial growth factor (VEGF) family member, VEGF-ENZ-7, is essential for the activation of VEGFR-2 signaling. J Biol Chem. 2003; 278: 13453–13461.
[Abstract/Free Full Text] - Zheng Y, Murakami M, Takahashi H, Yamauchi M, Kiba A, Yamaguchi S, Yabana N, Alitalo K, Shibuya M. Chimeric VEGF-ENZ7/PlGF Promotes Angiogenesis via VEGFR-2 without Significant Enhancement of Vascular Permeability and Inflammation. Arterioscler Thromb Vasc Biol. 2006; 26: 2019–2026.
[Abstract/Free Full Text] - Nazir SA, Metcalf JP. Innate immune response to adenovirus. J Investig Med. 2005; 53: 292–304.[Medline] [Order article via Infotrieve]
- Muruve DA. The innate immune response to adenovirus vectors. Hum Gene Ther. 2004; 15: 1157–1166.[CrossRef][Medline] [Order article via Infotrieve]
- Martin A, Komada MR, Sane DC. Abnormal angiogenesis in diabetes mellitus. Med Res Rev. 2003; 23: 117–145.[CrossRef][Medline] [Order article via Infotrieve]
- Coleman DL. Diabetes-obesity syndromes in mice. Diabetes. 1982; 31: 1–6.[Abstract]
- Cursiefen C, Chen L, Borges LP, Jackson D, Cao J, Radziejewski C, D’Amore PA, Dana MR, Wiegand SJ, Streilein JW. VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J Clin Invest. 2004; 113: 1040–1050.[CrossRef][Medline] [Order article via Infotrieve]
- Maruyama K, Ii M, Cursiefen C, Jackson DG, Keino H, Tomita M, Van Rooijen N, Takenaka H, D’Amore PA, Stein-Streilein J, Losordo DW, Streilein JW. Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages. J Clin Invest. 2005; 115: 2363–2372.[CrossRef][Medline] [Order article via Infotrieve]
- Kerjaschki D. The crucial role of macrophages in lymphangiogenesis. J Clin Invest. 2005; 115: 2316–2319.[CrossRef][Medline] [Order article via Infotrieve]
- Werner S. Keratinocyte growth factor: a unique player in epithelial repair processes. Cytokine Growth Factor Rev. 1998; 9: 153–165.[CrossRef][Medline] [Order article via Infotrieve]
- Shirakata Y, Kimura R, Nanba D, Iwamoto R, Tokumaru S, Morimoto C, Yokota K, Nakamura M, Sayama K, Mekada E, Higashiyama S, Hashimoto K. Heparin-binding EGF-like growth factor accelerates keratinocyte migration and skin wound healing. J Cell Sci. 2005; 118: 2363–2370.
[Abstract/Free Full Text] - Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev. 1999; 79: 1283–1316.
[Abstract/Free Full Text] - Plum SM, Vu HA, Mercer B, Fogler WE, Fortier AH. Generation of a specific immunological response to FGF-2 does not affect wound healing or reproduction. Immunopharmacol Immunotoxicol. 2004; 26: 29–41.[CrossRef][Medline] [Order article via Infotrieve]
- Rappolee DA, Mark D, Banda MJ, Werb Z. Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping. Science. 1988; 241: 708–712.
[Abstract/Free Full Text] - Guo L, Degenstein L, Fuchs E. Keratinocyte growth factor is required for hair development but not for wound healing. Genes Dev. 1996; 10: 165–175.
[Abstract/Free Full Text] - Martin P, D’Souza D, Martin J, Grose R, Cooper L, Maki R, McKercher SR. Wound healing in the PU. 1 null mouse–tissue repair is not dependent on inflammatory cells. Curr Biol. 2003; 13: 1122–1128.[CrossRef][Medline] [Order article via Infotrieve]
- Hoffman M, Harger A, Lenkowski A, Hedner U, Roberts HR, Monroe DM. Cutaneous wound healing is impaired in hemophilia B. Blood. 2006; 108: 3053–3060.
[Abstract/Free Full Text] - Takahashi H, Hattori S, Iwamatsu A, Takizawa H, Shibuya M. A novel snake venom vascular endothelial growth factor (VEGF) predominantly induces vascular permeability through preferential signaling via VEGF receptor-1. J Biol Chem. 2004; 279: 46304–46314.
[Abstract/Free Full Text] - Katoh M, Katoh M. FGF signaling network in the gastrointestinal tract. Int J Oncol. 2006; 29: 163–168.[Medline] [Order article via Infotrieve]
- Smolen GA, Sordella R, Muir B, Mohapatra G, Barmettler A, Archibald H, Kim WJ, Okimoto RA, Bell DW, Sgroi DC, Christensen JG, Settleman J, Haber DA. Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752. Proc Natl Acad Sci U S A. 2006; 103: 2316–2321.
[Abstract/Free Full Text] - Richard JL, Parer-Richard C, Daures JP, Clouet S, Vannereau D, Bringer J, Rodier M, Jacob C, Comte-Bardonnet M. Effect of topical basic fibroblast growth factor on the healing of chronic diabetic neuropathic ulcer of the foot. A pilot, randomized, double-blind, placebo-controlled study. Diabetes Care. 1995; 18: 64–69.[Abstract]
- Finetti G, Farina M. Recombinant human basic-fibroblastic growth factor: different medical dressings for clinical application in wound healing. Farmaco. 1992; 47: 967–978.[Medline] [Order article via Infotrieve]
- Mizuno K, Yamamura K, Yano K, Osada T, Saeki S, Takimoto N, Sakurai T, Nimura Y. Effect of chitosan film containing basic fibroblast growth factor on wound healing in genetically diabetic mice. J Biomed Mater Res. 2003; 64: 177–181.[CrossRef]
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