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Angiopoietin-1, but not Platelet-Derived Growth Factor-AB, Is a Cooperative Stimulator of Vascular Endothelial Growth Factor A-Accelerated Endothelial Cell Scratch Closure
Angiopoietin-1, but not Platelet-Derived Growth Factor-AB, Is a Cooperative Stimulator of Vascular Endothelial Growth Factor A-Accelerated Endothelial Cell Scratch Closure Alexander Alter,1 Dorothee Schmiedeck,2 Markus R. Fussnegger,1 Axel R. Pries,2 Wolfgang B. Freesmeyer,1 and Andreas Zakrzewicz,2 Berlin, Germany
Wound healing and the grow-in of free tissue grafts critically depend on blood vessel growth, i.e., on the angiogenic invasion of endothelial cells, which is critically reduced in smokers, in patients suffering from microangiopathies (e.g., in diabetes), or in those who are treated with immunosuppressives. Although several angiogenic factors have been tested to accelerate wound healing in such critically patients, their combinations have not yet been systematically investigated. This study was done to reveal which combination of proangiogenic with promaturating factors is the most effective in an endothelial wound closure assay. Human umbilical vein endothelial cells were isolated, cultured to confluence, and subjected to a scratch wound assay with the addition of vascular endothelial growth factor (VEGF)-A165, platelet-derived growth factor (PDGF)-AB, angiopoietin-1 (ANG1), or ANG2 and all of their 16 possible combinations. VEGF-A165 plus ANG1 was most effective at accelerating endothelial scratch closure. Moreover, VEGF-A165 stimulated wound closure in all combinations tested, while it was attenuated by PDGF-AB. Thus, with respect to their effects on endothelial cells, a combination of VEGF-A with ANG1 is the most promising and is superior to combinations with PDGF-AB.
INTRODUCTION Wound closure critically depends on the sprouting of blood vessel capillaries, which requires proliferation and migration of endothelial cells.1,2 Similarly, the grow-in of grafts is greatly promoted by the accompanying angiogenesis.3,4 However, the angiogenic response is reduced in patients suffering from diabetes mellitus and in smokers, thus leading
1 Department of Prosthetic Dentistry, Charite e Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany. 2 Institute of Physiology, ChariteeUniversita¨tsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany.
Correspondence to: Andreas Zakrzewicz, MD, PhD, Institute of Physiology, ChariteeUniversita¨tsmedizin Berlin, Campus Benjamin Franklin, Arnimallee 22, 14195 Berlin, Germany, E-mail: andreas. [email protected] Ann Vasc Surg 2009; 23: 239-245 DOI: 10.1016/j.avsg.2008.07.010 Ó Annals of Vascular Surgery Inc. Published online: October 29, 2008
to prolonged time of or even defects in wound healing.5-8 Angiogenesis is regulated by angiogenic factors, of which vascular endothelial growth factor A (VEGF-A) is the most prominent one.9,10 Expression of VEGF-A is induced by a drop in oxygen pressure as a result of increased tissue demand, a decrease in capillary density, or an inflammatory reaction.9,10 Once secreted by nonendothelial tissue cells (i.e., fibroblasts or epithelial cells), VEGF-A acts on endothelial cells by specific receptors, VEGF-R2 mainly,10 in a paracrine fashion. It then activates many endothelial functions, which in turn cause capillary sprouting.9,10 In addition to VEGF-A and as a consequence of bleeding, there are always blood platelets from which platelet-derived growth factor (PDGF) is secreted.11 PDGF is also well known to be angiogenic12 and may thus increase VEGFinduced angiogenesis. In addition, endothelial tyrosine kinase receptor TIE2 contributes to the maturation of capillaries.13 TIE2 activity is increased by angiopoietin-1 (ANG1) and (at physiological 239
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concentrations) decreased by ANG2.14,15 However, blood vessels grown by predominance of VEGF-A remain leaky.16-20 Therefore, VEGF-A was tested in combinations with PDGF21,22 or ANG1.23-26 Both combinations were able to induce the growth of more functional capillaries with near normal permeability. The present study was done to more systematically reveal by directly comparing in the same assay system which of all the possible 16 combinations of VEGF-A, PDGF, ANG1, and ANG2 is most effective at inducing endothelial cell growth and migration as a prerequisite of capillary sprouting.
MATERIALS AND METHODS Isolation and Cultivation of Cells Human umbilical vein endothelial cells (HUVECs) were isolated and cultivated as previously described.27 Briefly, HUVECs were removed from the veins of umbilical cords with 0.2% collagenase type II from Clostridium histolyticum (Biochrom, Berlin, Germany) for 15 min at 37 C. After incubation, the respective umbilical vein was rinsed with Hanks´ balanced salt solution (HBSS). The cell suspension was centrifuged and the supernatant discarded, and the cells were resuspended and cultivated to confluence in Endothelial Cell Basal Medium MVÒ with Cell-Growth-Supplement-Pack MVÒ (both from PromoCell, Heidelberg, Germany) at 37 C in a humidified atmosphere with 5% CO2.
Treatment of Cells with Test Agents Cells were seeded onto a 24-well test plate (TPP, Trasadingen, Switzerland), and each of 16 wells (except for nontreated controls) was treated with another test agent or a combination (Fig. 1). Test agents used were VEGF-A165 (stock solution 2.4 mg/mL in phosphate-buffered saline [PBS] with 1% bovine serum albumin [BSA], final concentration 24 ng/ mL), ANG1 (stock solution 16 mg/mL in PBS with 1% BSA, final concentration 16 ng/mL), ANG2 (stock solution 16 mg/mL in PBS with 1% BSA, final concentration 16 ng/mL), and PDGF (mostly PDGFAB heterodimers, stock solution 2 mg/mL in PBS with 1% BSA, final concentration 20 ng/mL) (all purchased from R&D Systems, Minneapolis, MN). To achieve significant effects, all test reagents were used approximately four times above their median effective concentration (EC50) as provided by the manufacturer’s data sheet. All possible combinations of the four substances and complete cell culture medium with 1% BSA as a negative control
Fig. 1. Combinations of test agents shown with group numbers and their corresponding factors
were tested. The 16 combinations are shown in Fig. 1. Scratch Wound Assay HUVECs were grown in test plate wells to 100% confluence. The monolayer was then scratched along a ruler using a 200 mL pipette tip. The initial scratch was photographed using a Nikon (Melville, NY) Diaphot inversion microscope equipped with Hoffman Modulation Contrast (Modulation Optics, Greenvale, NY) and a corresponding objective (plan 4/0.13 DL, 160/-, Phl; Nikon, Tokyo, Japan). Photos were taken with a Canon (Lake Success, NY) EOS 10D. Cells were then incubated for 5 hr at 37 C in a humidified atmosphere with 5% CO2. Cell migration was measured by comparing the initial photo with a photo following 5 hr incubation. Scratch width was measured offline using ImageJ 1.34s program for histometry (NIH, Bethesda, MD). The measured cleft width from one wound edge to the other was compared between test samples and nontreated controls. Immunofluorescence Staining Cells were fixed with methanol at 20 C for 10 min, washed in PBS containing 1% BSA, incubated with anti-von Willebrand Factor (vWF) antibody (F-3520; Sigma, Deisenhofen, Germany) 1:100 in PBS/BSA for 30 min at room temperature in a wet chamber, washed twice in PBS/BSA, followed by incubation with fluorescein isothiocyanate (FITC)elabeled monoclonal anti-rabbit (F-4890, Sigma) 1:20 in PBS/BSA for another 30 min, and
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Fig. 2. Influence of different combinations of angiogenic factors on scratch closure. First-passage HUVEC monolayers were scratched with a pipette tip, and scratch closure was measured 5 hr later in a control group (cell culture medium alone) or in the presence of additional VEGF-A165 plus ANG1, PDGF plus ANG2, or VEGF-A165 plus PDGF. Data are given as mean ± SD for n ¼ 7, *p 0.05.
washed again two times with PBS. Negative controls were incubated with the secondary FITC-labeled antibody alone. Samples were directly analyzed using an OrtholuxÒ microscope with a water immersion objective (25/0.60W Fluoreszenz) and a filter set I 2/3 for epifluorescence (Leitz, Wetzlar, Germany) or using a transilluminating light path. Photos were taken with a Canon EOS 10D. Statistical Analysis Endothelial cells were independently isolated from seven different umbilical cords. Each combination of test agents running from group 1 through 16 (Fig. 1) was investigated with HUVECs from each of these umbilical cords. Thus, within each group seven independent measurements were performed. (Three of these groups are shown in comparison to the control group, see Figure 2.) To reveal the effects of VEGF-A, e.g., samples from all groups without VEGF-A (groups 16, 2, 4, 6, 8, 10, 11, 14) or with VEGF-A (groups 1, 3, 5, 7, 9, 12, 13, 15) were pooled. The samples were paired according to umbilical cords and the factors which were present during the incubation time. For instance, HUVECs from umbilical cord number 1 in group 16 were paired with those from umbilical cord number one in group 1, and so forth. Thus, only one variable differed between paired samples, the test agent of interest. Data are given as mean ± standard deviation (SD) and statistical significance was assumed at p 0.05 as tested by Student´s t-test for paired samples.
RESULTS The endothelial origin of the cells cultured to confluence was confirmed by positive immunological staining for vWF (Fig. 2A, B). A scratch wound in these monolayers closed spontaneously within 24 hr (Fig. 2C-H). Even with the addition of growth factors and the resultant increase in velocity of wound closure,
there remained a measurable gap for at least up to 5 hr. Thus, the measurements were done at 0 and 5 hr following scratching. Four endothelial growth factors and all of their combinations were tested, and their effects were evaluated quantifying the velocity of wound closure as a composite measure of cell migration and proliferation. All groups without a single factor were tested against all groups with this same factor. This analysis revealed that a statistically significant increase in wound closure velocity (Fig. 3) was achieved by VEGF-A165 and ANG1, while PDGF-AB caused a decrease in both, but this decrease did not reach statistical significance. Compared to control cultures with complete cell culture medium, the increase in wound closure velocity (Fig. 4) was strongest with the addition of VEGF-A165 plus ANG1. By contrast, with the addition of PDGF-AB plus ANG2, wound closure slackened (Fig. 4).
DISCUSSION This study showed that out of 16 possible combinations of four angiogenic factors the combination of VEGF-A165 plus ANG1 was most effective at accelerating endothelial migration and proliferation in a scratch wound assay using HUVECs. Moreover, VEGF-A165 stimulated wound closure in all combinations tested, while it was attenuated by PDGF-AB. Wound closure in a scratch wound assay measured by the decrease of the width of the scratch in an endothelial monolayer results from the combination of cell proliferation and migration. Since both cell functions are central in capillary sprouting, this assay covers two main aspects of angiogenesis. Although differences between the factors tested would have been more pronounced in a cell culture medium without serum or without growth factors, the tests presented here were done in the presence of normal cell growth medium, to more closely simulate in vivo conditions, in which therapeutic treatment with angiogenic factors would just add these factors on top of those which are present near
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Fig. 3. Staining for vWF (A-D) and scratch closure (E-J). First-passage HUVECs are shown by transillumination microscopy (A) and by fluorescence microscopy following immunological staining of vWF (B). A negative control (FITC-labeled secondary antibody alone) and its corresponding transillumination picture are shown in D and
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C, respectively. Transillumination microscopy using Hoffmann modulation contrast shows a typical freshly drawn scratch (E, H) and follow-up at 5 hr (F, I) and 16 hr (G, J) under control conditions (E-G) or in the presence of VEGF-A165 (H-J). Scale bar ¼ 500 mm.
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Fig. 4. Influence of different combinations of four angiogenic factors on scratch closure. First-passage HUVEC monolayers were scratched with a pipette tip, and scratch closure was measured 5 hr later with the addition or not
of VEGF-A165, ANG1, PDGF, and ANG2. All combinations without one of these factors were tested against all combinations in which this special factor was added. Data are given as mean ± SD of n ¼ 56, *p 0.05.
a wound site anyway. In addition, the four factors were tested in all of 16 possible combinations. Doing so, VEGF-A165 stimulated wound closure in all combinations tested. VEGF-A is the most intensively investigated angiogenic factor.9,28 It is well known to stimulate endothelial proliferation and migration29 as well as other endothelial cell functions which play a role in capillary sprouting or the maintenance of mature blood vessels.30 Thus, the data presented here are in agreement with the literature. However, VEGF-A applied to induce angiogenesis in vivo not only increases capillary density but unfortunately correlates with an increase in capillary permeability for which the newly formed capillary networks do not properly work.31 Therefore, there was an increasing interest in combining VEGF-A with other factors, which promote the recruitment of perivascular cells and the maturation of blood vessels, thus sealing the capillaries, e.g., PDGF and ANG1. PDGF-AB binds to different endothelial receptors.12 Lack of PDGF in knockout mice results in a defect in blood vessel maturation.32 Accordingly, a combination of VEGF with PDGF was able to induce more mature blood vessels.33 Alternatively, VEGF-A has been tested in combination with ANG1.23,24 ANG1 activates endothelial tyrosine kinase receptor TIE2 and the phosphatidylinositol 3-kinase pathway.34,35 Again, this combination was also able to induce more mature blood vessels.23,24
The data presented here allow direct comparison of combinations of VEGF-A and PDGF-AB with those of VEGF-A and ANG1. This comparison showed that effects of VEGF-A165 on scratch closure are enhanced in combination with ANG1. Thus, combining VEGF with ANG1 could have two desired effects, prevention of increased vascular permeability36 and an additional increase in endothelial cell proliferation and migration. Since ANG1 has been shown to be a chemoattractant for endothelial cells,37 the increase in endothelial scratch closure is in agreement with the literature. Effects of VEGF-A and ANG1 will be mainly restricted to endothelial cells because their receptors are selectively expressed on these cells. This may help to reduce side effects in an in vivo situation and could be an additional advantage in a clinical setting. However, VEGF-A expression has been described during inflammatory reactions which, e.g., led to the failure of dental implants.38 The failure of these implants was probably a consequence of the inflammatory reaction in general, not of VEGF alone. Moreover, in combination with ANG1, the VEGFA-induced increase in vascular permeability will be prevented.39 Thus, most likely in a noninflammatory situation and in combination with ANG1, VEGF will induce angiogenesis by an increase in endothelial cell proliferation and migration and accelerate wound closure, thus preventing the failure of implants and promoting the grow-in of free tissue transplants.
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Furthermore, VEGF-A stimulates the expression of ANG2,40 which in turn has been shown to promote tumor necrosis factor-aeinduced inflammation.41 By contrast, activation of TIE2 via ANG1 stimulates the phosphatidylinositol 3-kinase pathway,42 thus inactivating FoxO1, a known transcription factor of ANG2.43 Thus, ANG1 will probably prevent VEGF-A-dependent ANG2-mediated inflammatory reactions. Although known as an angiogenic factor, PDGFAB reduced scratch closure in our study. This may be in agreement with data showing decreased capillary density through PDGF when combined with VEGF33 or increased capillary density while PDGF effects were blocked.44 Thus, the main effect of PDGF in the angiogenic process is thought to be the maturation of newly formed blood vessels.33 ANG2, initially introduced as an endogenous TIE2 antagonist,15 has recently been reported to activate TIE2.42 Unexpectedly, the combination of PDGF-AB with ANG2 significantly reduced scratch closure, which may be difficult to understand. However, we could not find any other tests of combined effects of PDGF-AB and ANG2 in the literature. Our data do at least predict a superiority of VEGF plus ANG1 over PDGF-AB plus ANG2. A clinical application of proangiogenic factors to promote and accelerate wound healing is the treatment of patients with defects in wound closure and high risk for failing implants. A prerequisite for such treatment is to reveal optimal combinations of angiogenic factors to improve their effects. This study suggests that VEGF plus ANG1 is the most promising combination of the four factors and 16 combinations tested.
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The expert technical assistance of G. Beyer is greatly acknowledged. 20.
REFERENCES 21. 1. Velazquez OC. Angiogenesis and vasculogenesis: inducing the growth of new blood vessels and wound healing by stimulation of bone marrow-derived progenitor cell mobilization and homing. J Vasc Surg 2007;45(Suppl. A): A39-A47. 2. Eming SA, Brachvogel B, Odorisio T, et al. Regulation of angiogenesis: wound healing as a model. Prog. Histochem. Cytochem 2007;42:115-170. 3. Nakanishi Y, Izumi K, Yoshizawa M, et al. The expression and production of vascular endothelial growth factor in oral mucosa equivalents. Int J Oral Maxillofac. Surg 2007;36:928-933. 4. Nissen NN, Polverini PJ, Koch AE, et al. Vascular endothelial growth factor mediates angiogenic activity during the
22.
23.
24.
proliferative phase of wound healing. Am J Pathol 1998;152:1445-1452. Bell GW, Large DM, Barclay SC. Oral health care in diabetes mellitus. SADJ 2000;55:158-165. Lehr HA. Microcirculatory dysfunction induced by cigarette smoking. Microcirculation 2000;7:367-384. Martin A, Komada MR, Sane DC. Abnormal angiogenesis in diabetes mellitus. Med Res Rev 2003;23:117-145. Mellado-Valero A, Ferrer Garcia JC, Herrera Ballester A, et al. Effects of diabetes on the osseointegration of dental implants. Med Oral Patol Oral Cir Bucal 2007;12:E38-E43. Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 2004;25:581-611. Risau W. Mechanisms of angiogenesis. Nature 1997;386: 671-674. Anitua E, Andia I, Ardanza B, et al. Autologous platelets as a source of proteins for healing and tissue regeneration. Thromb. Haemost 2004;91:4-15. D’Amore PA, Smith SR. Growth factor effects on cells of the vascular wall: a survey. Growth Factors 1993;8:61-75. Hoffmann J, Feng Y, vom Hagen F, et al. Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity. FASEB J 2005;19:2035-2036. Asahara T, Chen D, Takahashi T, et al. Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization. Circ Res 1998;83:233-240. Maisonpierre PC, Suri C, Jones PF, et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 1997;277:55-60. Detmar M, Brown LF, Schon MP, et al. Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J Invest Dermatol 1998;111:1-6. Dvorak HF, Brown LF, Detmar M, et al. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 1995;146:1029-1039. Hobbs SK, Monsky WL, Yuan F, et al. Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment. Proc Natl Acad Sci USA 1998;95: 4607-4612. Monsky WL, Fukumura D, Gohongi T, et al. Augmentation of transvascular transport of macromolecules and nanoparticles in tumors using vascular endothelial growth factor. Cancer Res 1999;59:4129-4135. Roberts WG, Palade GE. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J Cell Sci 1995;108:2369-2379. Hao X, Mansson-Broberg A, Blomberg P, et al. Angiogenic and cardiac functional effects of dual gene transfer of VEGF-A165 and PDGF-BB after myocardial infarction. Biochem Biophys Res Commun 2004;322:292-296. Richardson TP, Peters MC, Ennett AB, et al. Polymeric system for dual growth factor delivery. Nat Biotechnol 2001;19:1029-1034. Benest AV, Salmon AH, Wang W, et al. VEGF and angiopoietin-1 stimulate different angiogenic phenotypes that combine to enhance functional neovascularization in adult tissue. Microcirculation 2006;13:423-437. Chae JK, Kim I, Lim ST, et al. Coadministration of angiopoietin-1 and vascular endothelial growth factor enhances collateral vascularization. Arterioscler Thromb Vasc Biol 2000;20:2573-2578.
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25. Chen F, Tan Z, Dong CY, et al. Combination of VEGF165/ angiopoietin-1 gene and endothelial progenitor cells for therapeutic neovascularization. Eur J Pharmacol 2007;568: 222-230. 26. Yamauchi A, Ito Y, Morikawa M, et al. Pre-administration of angiopoietin-1 followed by VEGF induces functional and mature vascular formation in a rabbit ischemic model. J Gene Med 2003;5:994-1004. 27. Bongrazio M, Da Silva-Azevedo L, Bergmann EC, et al. Shear stress modulates the expression of thrombospondin1 and CD36 in endothelial cells in vitro and during shear stress-induced angiogenesis in vivo. Int J Immunopathol Pharmacol 2006;19:35-48. 28. Breier G, Risau W. The role of vascular endothelial growth factor in blood vessel formation. Trends Cell Biol 1996;6: 454-456. 29. Walsh DA. Pathophysiological mechanisms of angiogenesis. Adv Clin Chem. 2007;44:187-221. 30. Lee S, Chen TT, Barber CL, et al. Autocrine VEGF signaling is required for vascular homeostasis. Cell 2007;130:691-703. 31. Zacchigna S, Tasciotti E, Kusmic C, et al. In vivo imaging shows abnormal function of vascular endothelial growth factor-induced vasculature. Hum Gene Ther 2007;18: 515-524. 32. Lindahl P, Johansson BR, Leveen P, et al. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 1997;277:242-245. 33. Chen RR, Silva EA, Yuen WW, et al. Spatio-temporal VEGF and PDGF delivery patterns blood vessel formation and maturation. Pharm Res 2007;24:258-264. 34. Jones N, Master Z, Jones J, et al. Identification of Tek/Tie2 binding partners. Binding to a multifunctional docking site
Cooperativity of VEGF and ANG1
35.
36.
37.
38.
39. 40.
41.
42.
43.
44.
245
mediates cell survival and migration. J Biol Chem 1999;274:30896-30905. DeBusk LM, Hallahan DE, Lin PC. Akt is a major angiogenic mediator downstream of the Ang1/Tie2 signaling pathway. Exp Cell Res 2004;298:167-177. Thurston G, Suri C, Smith K, et al. Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 1999;286:2511-2514. Witzenbichler B, Westermann D, Knueppel S, et al. Protective role of angiopoietin-1 in endotoxic shock. Circulation 2005;111:97-105. Cornelini R, Artese L, Rubini C, et al. Vascular endothelial growth factor and microvessel density around healthy and failing dental implants. Int J Oral MaxillofacImplants 2001;16:389-393. Jain RK, Munn LL. Leaky vessels? Call Ang1!. Nat. Med 2000;6:131-132. Mandriota SJ, Pepper MS. Regulation of angiopoietin-2 mRNA levels in bovine microvascular endothelial cells by cytokines and hypoxia. Circ Res 1998;83:852-859. Fiedler U, Reiss Y, Scharpfenecker M, et al. Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation. Nat Med 2006;12:235-239. Daly C, Pasnikowski E, Burova E, et al. Angiopoietin-2 functions as an autocrine protective factor in stressed endothelial cells. Proc Natl Acad Sci USA 2006;103:15491-15496. Chlench S, Mecha Disassa N, Hohberg M, et al. Regulation of Foxo-1 and the angiopoietin-2/Tie2 system by shear stress. FEBS Lett 2007;581:673-680. Zymek P, Bujak M, Chatila K, et al. The role of platelet-derived growth factor signaling in healing myocardial infarcts. J Am Coll Cardiol 2006;48:2315-2323.
Wound healing and the grow-in of free tissue grafts critically depend on blood vessel growth, i.e., on the angiogenic invasion of endothelial cells, which is critically reduced in smokers, in patients suffering from microangiopathies (e.g., in diabetes), or in those who are treated with immunosuppressives. Although several angiogenic factors have been tested to accelerate wound healing in such critically patients, their combinations have not yet been systematically investigated. This study was done to reveal which combination of proangiogenic with promaturating factors is the most effective in an endothelial wound closure assay. Human umbilical vein endothelial cells were isolated, cultured to confluence, and subjected to a scratch wound assay with the addition of vascular endothelial growth factor (VEGF)-A165, platelet-derived growth factor (PDGF)-AB, angiopoietin-1 (ANG1), or ANG2 and all of their 16 possible combinations. VEGF-A165 plus ANG1 was most effective at accelerating endothelial scratch closure. Moreover, VEGF-A165 stimulated wound closure in all combinations tested, while it was attenuated by PDGF-AB. Thus, with respect to their effects on endothelial cells, a combination of VEGF-A with ANG1 is the most promising and is superior to combinations with PDGF-AB.
INTRODUCTION Wound closure critically depends on the sprouting of blood vessel capillaries, which requires proliferation and migration of endothelial cells.1,2 Similarly, the grow-in of grafts is greatly promoted by the accompanying angiogenesis.3,4 However, the angiogenic response is reduced in patients suffering from diabetes mellitus and in smokers, thus leading
1 Department of Prosthetic Dentistry, Charite e Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany. 2 Institute of Physiology, ChariteeUniversita¨tsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany.
Correspondence to: Andreas Zakrzewicz, MD, PhD, Institute of Physiology, ChariteeUniversita¨tsmedizin Berlin, Campus Benjamin Franklin, Arnimallee 22, 14195 Berlin, Germany, E-mail: andreas. [email protected] Ann Vasc Surg 2009; 23: 239-245 DOI: 10.1016/j.avsg.2008.07.010 Ó Annals of Vascular Surgery Inc. Published online: October 29, 2008
to prolonged time of or even defects in wound healing.5-8 Angiogenesis is regulated by angiogenic factors, of which vascular endothelial growth factor A (VEGF-A) is the most prominent one.9,10 Expression of VEGF-A is induced by a drop in oxygen pressure as a result of increased tissue demand, a decrease in capillary density, or an inflammatory reaction.9,10 Once secreted by nonendothelial tissue cells (i.e., fibroblasts or epithelial cells), VEGF-A acts on endothelial cells by specific receptors, VEGF-R2 mainly,10 in a paracrine fashion. It then activates many endothelial functions, which in turn cause capillary sprouting.9,10 In addition to VEGF-A and as a consequence of bleeding, there are always blood platelets from which platelet-derived growth factor (PDGF) is secreted.11 PDGF is also well known to be angiogenic12 and may thus increase VEGFinduced angiogenesis. In addition, endothelial tyrosine kinase receptor TIE2 contributes to the maturation of capillaries.13 TIE2 activity is increased by angiopoietin-1 (ANG1) and (at physiological 239
240 Alter et al.
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concentrations) decreased by ANG2.14,15 However, blood vessels grown by predominance of VEGF-A remain leaky.16-20 Therefore, VEGF-A was tested in combinations with PDGF21,22 or ANG1.23-26 Both combinations were able to induce the growth of more functional capillaries with near normal permeability. The present study was done to more systematically reveal by directly comparing in the same assay system which of all the possible 16 combinations of VEGF-A, PDGF, ANG1, and ANG2 is most effective at inducing endothelial cell growth and migration as a prerequisite of capillary sprouting.
MATERIALS AND METHODS Isolation and Cultivation of Cells Human umbilical vein endothelial cells (HUVECs) were isolated and cultivated as previously described.27 Briefly, HUVECs were removed from the veins of umbilical cords with 0.2% collagenase type II from Clostridium histolyticum (Biochrom, Berlin, Germany) for 15 min at 37 C. After incubation, the respective umbilical vein was rinsed with Hanks´ balanced salt solution (HBSS). The cell suspension was centrifuged and the supernatant discarded, and the cells were resuspended and cultivated to confluence in Endothelial Cell Basal Medium MVÒ with Cell-Growth-Supplement-Pack MVÒ (both from PromoCell, Heidelberg, Germany) at 37 C in a humidified atmosphere with 5% CO2.
Treatment of Cells with Test Agents Cells were seeded onto a 24-well test plate (TPP, Trasadingen, Switzerland), and each of 16 wells (except for nontreated controls) was treated with another test agent or a combination (Fig. 1). Test agents used were VEGF-A165 (stock solution 2.4 mg/mL in phosphate-buffered saline [PBS] with 1% bovine serum albumin [BSA], final concentration 24 ng/ mL), ANG1 (stock solution 16 mg/mL in PBS with 1% BSA, final concentration 16 ng/mL), ANG2 (stock solution 16 mg/mL in PBS with 1% BSA, final concentration 16 ng/mL), and PDGF (mostly PDGFAB heterodimers, stock solution 2 mg/mL in PBS with 1% BSA, final concentration 20 ng/mL) (all purchased from R&D Systems, Minneapolis, MN). To achieve significant effects, all test reagents were used approximately four times above their median effective concentration (EC50) as provided by the manufacturer’s data sheet. All possible combinations of the four substances and complete cell culture medium with 1% BSA as a negative control
Fig. 1. Combinations of test agents shown with group numbers and their corresponding factors
were tested. The 16 combinations are shown in Fig. 1. Scratch Wound Assay HUVECs were grown in test plate wells to 100% confluence. The monolayer was then scratched along a ruler using a 200 mL pipette tip. The initial scratch was photographed using a Nikon (Melville, NY) Diaphot inversion microscope equipped with Hoffman Modulation Contrast (Modulation Optics, Greenvale, NY) and a corresponding objective (plan 4/0.13 DL, 160/-, Phl; Nikon, Tokyo, Japan). Photos were taken with a Canon (Lake Success, NY) EOS 10D. Cells were then incubated for 5 hr at 37 C in a humidified atmosphere with 5% CO2. Cell migration was measured by comparing the initial photo with a photo following 5 hr incubation. Scratch width was measured offline using ImageJ 1.34s program for histometry (NIH, Bethesda, MD). The measured cleft width from one wound edge to the other was compared between test samples and nontreated controls. Immunofluorescence Staining Cells were fixed with methanol at 20 C for 10 min, washed in PBS containing 1% BSA, incubated with anti-von Willebrand Factor (vWF) antibody (F-3520; Sigma, Deisenhofen, Germany) 1:100 in PBS/BSA for 30 min at room temperature in a wet chamber, washed twice in PBS/BSA, followed by incubation with fluorescein isothiocyanate (FITC)elabeled monoclonal anti-rabbit (F-4890, Sigma) 1:20 in PBS/BSA for another 30 min, and
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Fig. 2. Influence of different combinations of angiogenic factors on scratch closure. First-passage HUVEC monolayers were scratched with a pipette tip, and scratch closure was measured 5 hr later in a control group (cell culture medium alone) or in the presence of additional VEGF-A165 plus ANG1, PDGF plus ANG2, or VEGF-A165 plus PDGF. Data are given as mean ± SD for n ¼ 7, *p 0.05.
washed again two times with PBS. Negative controls were incubated with the secondary FITC-labeled antibody alone. Samples were directly analyzed using an OrtholuxÒ microscope with a water immersion objective (25/0.60W Fluoreszenz) and a filter set I 2/3 for epifluorescence (Leitz, Wetzlar, Germany) or using a transilluminating light path. Photos were taken with a Canon EOS 10D. Statistical Analysis Endothelial cells were independently isolated from seven different umbilical cords. Each combination of test agents running from group 1 through 16 (Fig. 1) was investigated with HUVECs from each of these umbilical cords. Thus, within each group seven independent measurements were performed. (Three of these groups are shown in comparison to the control group, see Figure 2.) To reveal the effects of VEGF-A, e.g., samples from all groups without VEGF-A (groups 16, 2, 4, 6, 8, 10, 11, 14) or with VEGF-A (groups 1, 3, 5, 7, 9, 12, 13, 15) were pooled. The samples were paired according to umbilical cords and the factors which were present during the incubation time. For instance, HUVECs from umbilical cord number 1 in group 16 were paired with those from umbilical cord number one in group 1, and so forth. Thus, only one variable differed between paired samples, the test agent of interest. Data are given as mean ± standard deviation (SD) and statistical significance was assumed at p 0.05 as tested by Student´s t-test for paired samples.
RESULTS The endothelial origin of the cells cultured to confluence was confirmed by positive immunological staining for vWF (Fig. 2A, B). A scratch wound in these monolayers closed spontaneously within 24 hr (Fig. 2C-H). Even with the addition of growth factors and the resultant increase in velocity of wound closure,
there remained a measurable gap for at least up to 5 hr. Thus, the measurements were done at 0 and 5 hr following scratching. Four endothelial growth factors and all of their combinations were tested, and their effects were evaluated quantifying the velocity of wound closure as a composite measure of cell migration and proliferation. All groups without a single factor were tested against all groups with this same factor. This analysis revealed that a statistically significant increase in wound closure velocity (Fig. 3) was achieved by VEGF-A165 and ANG1, while PDGF-AB caused a decrease in both, but this decrease did not reach statistical significance. Compared to control cultures with complete cell culture medium, the increase in wound closure velocity (Fig. 4) was strongest with the addition of VEGF-A165 plus ANG1. By contrast, with the addition of PDGF-AB plus ANG2, wound closure slackened (Fig. 4).
DISCUSSION This study showed that out of 16 possible combinations of four angiogenic factors the combination of VEGF-A165 plus ANG1 was most effective at accelerating endothelial migration and proliferation in a scratch wound assay using HUVECs. Moreover, VEGF-A165 stimulated wound closure in all combinations tested, while it was attenuated by PDGF-AB. Wound closure in a scratch wound assay measured by the decrease of the width of the scratch in an endothelial monolayer results from the combination of cell proliferation and migration. Since both cell functions are central in capillary sprouting, this assay covers two main aspects of angiogenesis. Although differences between the factors tested would have been more pronounced in a cell culture medium without serum or without growth factors, the tests presented here were done in the presence of normal cell growth medium, to more closely simulate in vivo conditions, in which therapeutic treatment with angiogenic factors would just add these factors on top of those which are present near
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Fig. 3. Staining for vWF (A-D) and scratch closure (E-J). First-passage HUVECs are shown by transillumination microscopy (A) and by fluorescence microscopy following immunological staining of vWF (B). A negative control (FITC-labeled secondary antibody alone) and its corresponding transillumination picture are shown in D and
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C, respectively. Transillumination microscopy using Hoffmann modulation contrast shows a typical freshly drawn scratch (E, H) and follow-up at 5 hr (F, I) and 16 hr (G, J) under control conditions (E-G) or in the presence of VEGF-A165 (H-J). Scale bar ¼ 500 mm.
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Fig. 4. Influence of different combinations of four angiogenic factors on scratch closure. First-passage HUVEC monolayers were scratched with a pipette tip, and scratch closure was measured 5 hr later with the addition or not
of VEGF-A165, ANG1, PDGF, and ANG2. All combinations without one of these factors were tested against all combinations in which this special factor was added. Data are given as mean ± SD of n ¼ 56, *p 0.05.
a wound site anyway. In addition, the four factors were tested in all of 16 possible combinations. Doing so, VEGF-A165 stimulated wound closure in all combinations tested. VEGF-A is the most intensively investigated angiogenic factor.9,28 It is well known to stimulate endothelial proliferation and migration29 as well as other endothelial cell functions which play a role in capillary sprouting or the maintenance of mature blood vessels.30 Thus, the data presented here are in agreement with the literature. However, VEGF-A applied to induce angiogenesis in vivo not only increases capillary density but unfortunately correlates with an increase in capillary permeability for which the newly formed capillary networks do not properly work.31 Therefore, there was an increasing interest in combining VEGF-A with other factors, which promote the recruitment of perivascular cells and the maturation of blood vessels, thus sealing the capillaries, e.g., PDGF and ANG1. PDGF-AB binds to different endothelial receptors.12 Lack of PDGF in knockout mice results in a defect in blood vessel maturation.32 Accordingly, a combination of VEGF with PDGF was able to induce more mature blood vessels.33 Alternatively, VEGF-A has been tested in combination with ANG1.23,24 ANG1 activates endothelial tyrosine kinase receptor TIE2 and the phosphatidylinositol 3-kinase pathway.34,35 Again, this combination was also able to induce more mature blood vessels.23,24
The data presented here allow direct comparison of combinations of VEGF-A and PDGF-AB with those of VEGF-A and ANG1. This comparison showed that effects of VEGF-A165 on scratch closure are enhanced in combination with ANG1. Thus, combining VEGF with ANG1 could have two desired effects, prevention of increased vascular permeability36 and an additional increase in endothelial cell proliferation and migration. Since ANG1 has been shown to be a chemoattractant for endothelial cells,37 the increase in endothelial scratch closure is in agreement with the literature. Effects of VEGF-A and ANG1 will be mainly restricted to endothelial cells because their receptors are selectively expressed on these cells. This may help to reduce side effects in an in vivo situation and could be an additional advantage in a clinical setting. However, VEGF-A expression has been described during inflammatory reactions which, e.g., led to the failure of dental implants.38 The failure of these implants was probably a consequence of the inflammatory reaction in general, not of VEGF alone. Moreover, in combination with ANG1, the VEGFA-induced increase in vascular permeability will be prevented.39 Thus, most likely in a noninflammatory situation and in combination with ANG1, VEGF will induce angiogenesis by an increase in endothelial cell proliferation and migration and accelerate wound closure, thus preventing the failure of implants and promoting the grow-in of free tissue transplants.
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Furthermore, VEGF-A stimulates the expression of ANG2,40 which in turn has been shown to promote tumor necrosis factor-aeinduced inflammation.41 By contrast, activation of TIE2 via ANG1 stimulates the phosphatidylinositol 3-kinase pathway,42 thus inactivating FoxO1, a known transcription factor of ANG2.43 Thus, ANG1 will probably prevent VEGF-A-dependent ANG2-mediated inflammatory reactions. Although known as an angiogenic factor, PDGFAB reduced scratch closure in our study. This may be in agreement with data showing decreased capillary density through PDGF when combined with VEGF33 or increased capillary density while PDGF effects were blocked.44 Thus, the main effect of PDGF in the angiogenic process is thought to be the maturation of newly formed blood vessels.33 ANG2, initially introduced as an endogenous TIE2 antagonist,15 has recently been reported to activate TIE2.42 Unexpectedly, the combination of PDGF-AB with ANG2 significantly reduced scratch closure, which may be difficult to understand. However, we could not find any other tests of combined effects of PDGF-AB and ANG2 in the literature. Our data do at least predict a superiority of VEGF plus ANG1 over PDGF-AB plus ANG2. A clinical application of proangiogenic factors to promote and accelerate wound healing is the treatment of patients with defects in wound closure and high risk for failing implants. A prerequisite for such treatment is to reveal optimal combinations of angiogenic factors to improve their effects. This study suggests that VEGF plus ANG1 is the most promising combination of the four factors and 16 combinations tested.
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5. 6. 7. 8.
9. 10. 11.
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The expert technical assistance of G. Beyer is greatly acknowledged. 20.
REFERENCES 21. 1. Velazquez OC. Angiogenesis and vasculogenesis: inducing the growth of new blood vessels and wound healing by stimulation of bone marrow-derived progenitor cell mobilization and homing. J Vasc Surg 2007;45(Suppl. A): A39-A47. 2. Eming SA, Brachvogel B, Odorisio T, et al. Regulation of angiogenesis: wound healing as a model. Prog. Histochem. Cytochem 2007;42:115-170. 3. Nakanishi Y, Izumi K, Yoshizawa M, et al. The expression and production of vascular endothelial growth factor in oral mucosa equivalents. Int J Oral Maxillofac. Surg 2007;36:928-933. 4. Nissen NN, Polverini PJ, Koch AE, et al. Vascular endothelial growth factor mediates angiogenic activity during the
22.
23.
24.
proliferative phase of wound healing. Am J Pathol 1998;152:1445-1452. Bell GW, Large DM, Barclay SC. Oral health care in diabetes mellitus. SADJ 2000;55:158-165. Lehr HA. Microcirculatory dysfunction induced by cigarette smoking. Microcirculation 2000;7:367-384. Martin A, Komada MR, Sane DC. Abnormal angiogenesis in diabetes mellitus. Med Res Rev 2003;23:117-145. Mellado-Valero A, Ferrer Garcia JC, Herrera Ballester A, et al. Effects of diabetes on the osseointegration of dental implants. Med Oral Patol Oral Cir Bucal 2007;12:E38-E43. Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 2004;25:581-611. Risau W. Mechanisms of angiogenesis. Nature 1997;386: 671-674. Anitua E, Andia I, Ardanza B, et al. Autologous platelets as a source of proteins for healing and tissue regeneration. Thromb. Haemost 2004;91:4-15. D’Amore PA, Smith SR. Growth factor effects on cells of the vascular wall: a survey. Growth Factors 1993;8:61-75. Hoffmann J, Feng Y, vom Hagen F, et al. Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity. FASEB J 2005;19:2035-2036. Asahara T, Chen D, Takahashi T, et al. Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization. Circ Res 1998;83:233-240. Maisonpierre PC, Suri C, Jones PF, et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 1997;277:55-60. Detmar M, Brown LF, Schon MP, et al. Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J Invest Dermatol 1998;111:1-6. Dvorak HF, Brown LF, Detmar M, et al. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 1995;146:1029-1039. Hobbs SK, Monsky WL, Yuan F, et al. Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment. Proc Natl Acad Sci USA 1998;95: 4607-4612. Monsky WL, Fukumura D, Gohongi T, et al. Augmentation of transvascular transport of macromolecules and nanoparticles in tumors using vascular endothelial growth factor. Cancer Res 1999;59:4129-4135. Roberts WG, Palade GE. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J Cell Sci 1995;108:2369-2379. Hao X, Mansson-Broberg A, Blomberg P, et al. Angiogenic and cardiac functional effects of dual gene transfer of VEGF-A165 and PDGF-BB after myocardial infarction. Biochem Biophys Res Commun 2004;322:292-296. Richardson TP, Peters MC, Ennett AB, et al. Polymeric system for dual growth factor delivery. Nat Biotechnol 2001;19:1029-1034. Benest AV, Salmon AH, Wang W, et al. VEGF and angiopoietin-1 stimulate different angiogenic phenotypes that combine to enhance functional neovascularization in adult tissue. Microcirculation 2006;13:423-437. Chae JK, Kim I, Lim ST, et al. Coadministration of angiopoietin-1 and vascular endothelial growth factor enhances collateral vascularization. Arterioscler Thromb Vasc Biol 2000;20:2573-2578.
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25. Chen F, Tan Z, Dong CY, et al. Combination of VEGF165/ angiopoietin-1 gene and endothelial progenitor cells for therapeutic neovascularization. Eur J Pharmacol 2007;568: 222-230. 26. Yamauchi A, Ito Y, Morikawa M, et al. Pre-administration of angiopoietin-1 followed by VEGF induces functional and mature vascular formation in a rabbit ischemic model. J Gene Med 2003;5:994-1004. 27. Bongrazio M, Da Silva-Azevedo L, Bergmann EC, et al. Shear stress modulates the expression of thrombospondin1 and CD36 in endothelial cells in vitro and during shear stress-induced angiogenesis in vivo. Int J Immunopathol Pharmacol 2006;19:35-48. 28. Breier G, Risau W. The role of vascular endothelial growth factor in blood vessel formation. Trends Cell Biol 1996;6: 454-456. 29. Walsh DA. Pathophysiological mechanisms of angiogenesis. Adv Clin Chem. 2007;44:187-221. 30. Lee S, Chen TT, Barber CL, et al. Autocrine VEGF signaling is required for vascular homeostasis. Cell 2007;130:691-703. 31. Zacchigna S, Tasciotti E, Kusmic C, et al. In vivo imaging shows abnormal function of vascular endothelial growth factor-induced vasculature. Hum Gene Ther 2007;18: 515-524. 32. Lindahl P, Johansson BR, Leveen P, et al. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 1997;277:242-245. 33. Chen RR, Silva EA, Yuen WW, et al. Spatio-temporal VEGF and PDGF delivery patterns blood vessel formation and maturation. Pharm Res 2007;24:258-264. 34. Jones N, Master Z, Jones J, et al. Identification of Tek/Tie2 binding partners. Binding to a multifunctional docking site
Cooperativity of VEGF and ANG1
35.
36.
37.
38.
39. 40.
41.
42.
43.
44.
245
mediates cell survival and migration. J Biol Chem 1999;274:30896-30905. DeBusk LM, Hallahan DE, Lin PC. Akt is a major angiogenic mediator downstream of the Ang1/Tie2 signaling pathway. Exp Cell Res 2004;298:167-177. Thurston G, Suri C, Smith K, et al. Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 1999;286:2511-2514. Witzenbichler B, Westermann D, Knueppel S, et al. Protective role of angiopoietin-1 in endotoxic shock. Circulation 2005;111:97-105. Cornelini R, Artese L, Rubini C, et al. Vascular endothelial growth factor and microvessel density around healthy and failing dental implants. Int J Oral MaxillofacImplants 2001;16:389-393. Jain RK, Munn LL. Leaky vessels? Call Ang1!. Nat. Med 2000;6:131-132. Mandriota SJ, Pepper MS. Regulation of angiopoietin-2 mRNA levels in bovine microvascular endothelial cells by cytokines and hypoxia. Circ Res 1998;83:852-859. Fiedler U, Reiss Y, Scharpfenecker M, et al. Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation. Nat Med 2006;12:235-239. Daly C, Pasnikowski E, Burova E, et al. Angiopoietin-2 functions as an autocrine protective factor in stressed endothelial cells. Proc Natl Acad Sci USA 2006;103:15491-15496. Chlench S, Mecha Disassa N, Hohberg M, et al. Regulation of Foxo-1 and the angiopoietin-2/Tie2 system by shear stress. FEBS Lett 2007;581:673-680. Zymek P, Bujak M, Chatila K, et al. The role of platelet-derived growth factor signaling in healing myocardial infarcts. J Am Coll Cardiol 2006;48:2315-2323.