title: Effects of CB-VEGF-A injection in rat flap models for improved survival.
abstract: BACKGROUND
  Various carriers have been tested as drug delivery systems in an attempt to sustain the action of growth factors. Gene therapy has also been adopted to achieve lasting effects but without satisfactory results. Because the authors believe that the angiogenic effect of vascular endothelial growth factor (VEGF) can be enhanced by anchoring the fusion protein composed of the Clostridium-derived collagen-binding domain and recombinant VEGF-A164 (CB-VEGF-A) in the tissue, they examined the changes in blood flow of random pattern flaps following treatment of the dorsal region of the rat with the fusion proteins before skin flap elevation.
  
  
  METHODS
  The authors administered CB-VEGF-A subcutaneously into the dorsal region of Sprague-Dawley rats 7 days before creation of skin flaps, and compared the necrosis rate observed on the seventh day after flap elevation with that of vehicle controls. The authors also performed comparison with a group treated by subcutaneous administration of non-collagen-binding domain-binding VEGF. The skin flaps were also examined histologically.
  
  
  RESULTS
  The flap necrosis rate was lower in the CB-VEGF-A group (36.7 ± 7.4 percent) than in the control group (48.2 ± 5.4 percent). However, no improvement was observed in the non-collagen-binding domain-binding VEGF group. Moreover, histologic examination revealed an increase in the subcutaneous blood vessel counts.
  
  
  CONCLUSIONS
  CB-VEGF-A has an angiogenic effect on rat dorsal skin flaps and improves flap survival.
authors.affiliation: Kanagawa, Okayama, and Kyoto, Japan From the Department of Plastic and Aesthetic Surgery, Kitasato University School of Medicine; the Department of Bacteriology, Dentistry, and Pharmaceutical Sciences, Okayama University Graduate School of Medicine; and the Department of Immunology and Cell Biology and the Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University.
authors.forename: Minekatsu
authors.initials: M
authors.lastname: Akimoto
authors.forename: Akira
authors.initials: A
authors.lastname: Takeda
authors.forename: Osamu
authors.initials: O
authors.lastname: Matsushita
authors.forename: Joe
authors.initials: J
authors.lastname: Inoue
authors.forename: Keiko
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hattori
authors.forename: Natsuko
authors.initials: N
authors.lastname: Kounoike
authors.forename: Eiju
authors.initials: E
authors.lastname: Uchinuma
mesh_terms: D000818:Animals; D003013:Clostridium; D003094:Collagen; D005260:Female; D006085:Graft Survival / Q000187:drug effects*; D007279:Injections, Subcutaneous; D051379:Mice; D008807:Mice, Inbred BALB C; D023421:Models, Animal; D018919:Neovascularization, Physiologic / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D013524:Surgical Flaps / Q000098:blood supply*; D042461:Vascular Endothelial Growth Factor A / Q000008:administration & dosage* / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D003094:Collagen
references.citation: Plouët J, Schilling J, Gospodarowicz D. Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT-20 cells. EMBO J. 1989;8:3801–3806.
references.citation: Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989;246:1306–1309.
references.citation: Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983;219:983–985.
references.citation: Senger DR, Perruzzi CA, Feder J, Dvorak HF. A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res. 1986;46:5629–5632.
references.citation: Isner JM, Pieczek A, Schainfeld R, et al.. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet 1996;348:370–374.
references.citation: Kusumanto YH, van Weel V, Mulder NH, et al.. Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: A double-blind randomized trial. Hum Gene Ther. 2006;17:683–691.
references.citation: Rajagopalan S, Mohler ER III, Lederman RJ, et al.. Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: A phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation 2003;108:1933–1938.
references.citation: Ylä-Herttuala S, Alitalo K. Gene transfer as a tool to induce therapeutic vascular growth. Nat Med. 2003;9:694–701.
references.citation: McGregor IA, Morgan G. Axial and random pattern flaps. Br J Plast Surg. 1973;26:202–213.
references.citation: Koshima I, Soeda S. Inferior epigastric artery skin flaps without rectus abdominis muscle. Br J Plast Surg. 1989;42:645–648.
references.citation: Song YG, Chen GZ, Song YL. The free thigh flap: A new free flap concept based on the septocutaneous artery. Br J Plast Surg. 1984;37:149–159.
references.citation: Koshima I, Fukuda H, Utunomiya R, Soeda S. The anterolateral thigh flap; variations in its vascular pedicle. Br J Plast Surg. 1989;42:260–262.
references.citation: Angrigiani C, Grilli D, Siebert J. Latissimus dorsi musculocutaneous flap without muscle. Plast Reconstr Surg. 1995;96:1608–1614.
references.citation: Allen RJ, Tucker C Jr. Superior gluteal artery perforator free flap for breast reconstruction. Plast Reconstr Surg. 1995;95:1207–1212.
references.citation: Koshima I, Moriguchi T, Soeda S, Kawata S, Ohta S, Ikeda A. The gluteal perforator-based flap for repair of sacral pressure sores. Plast Reconstr Surg. 1993;91:678–683.
references.citation: Koshima I, Inagawa K, Yamamoto M, Moriguchi T. New microsurgical breast reconstruction using free paraumbilical perforator adiposal flaps. Plast Reconstr Surg. 2000;106:61–65.
references.citation: Kimura N. A microdissected thin tensor fasciae latae perforator flap. Plast Reconstr Surg. 2002;109:69–80.
references.citation: Kimura N, Saitoh M, Okamura T, Hirata Y, Itoh Y, Sumiya N. Concept and anatomical basis of microdissected tailoring method for free flap transfer. Plast Reconstr Surg. 2009;123:152–162.
references.citation: Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev. 1997;18:4–25.
references.citation: Matsushita O, Yoshihara K, Katayama S, Minami J, Okabe A. Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene. J Bacteriol. 1994;176:149–156.
references.citation: Matsushita O, Jung CM, Minami J, Katayama S, Nishi N, Okabe A. A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase. J Biol Chem. 1998;273:3643–3648.
references.citation: Matsushita O, Koide T, Kobayashi R, Nagata K, Okabe A. Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase. J Biol Chem. 2001;276:8761–8770.
references.citation: Nishi N, Matsushita O, Yuube K, Miyanaka H, Okabe A, Wada F. Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain. Proc Natl Acad Sci USA. 1998;95:7018–7023.
references.citation: Breier G, Albrecht U, Sterrer S, Risau W. Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development 1992;114:521–532.
references.citation: Urlaub G, Chasin LA. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci USA. 1980;77:4216–4220.
references.citation: Scahill SJ, Devos R, Van der Heyden J, Fiers W. Expression and characterization of the product of a human immune interferon cDNA gene in Chinese hamster ovary cells. Proc Natl Acad Sci USA. 1983;80:4654–4658.
references.citation: Gavard J, Gutkind JS. VEGF controls endothelial-cell permeability by promoting the beta-arrestin-dependent endocytosis of VE-cadherin. Nat Cell Biol. 2006;8:1223–1234.
references.citation: Chen L, Endler A, Uchida K, et al.. Int6/eIf3e silencing promotes functional blood vessel outgrowth and enhances wound healing by upregulating hypoxia-induced factor 2alpha expression. Circulation 2010;122:910–919.
references.citation: Taylor GI, Minabe T. The angiosomes of the mammals and other vertebrates. Plast Reconstr Surg. 1992;89:181–215.
references.citation: Bayramiçli M, Yilmaz B, Numanoğlu A. Contraction of experimental skin flaps. Ann Plast Surg. 1998;41:185–190.
references.citation: Akimoto M, Nishimaki T, Arai Y, Uchinuma E, Yamauchi H, Kameda Y. Hes1 regulates formations of the hypophyseal pars tuberalis and the hypothalamus. Cell Tissue Res. 2010;340:509–521.
references.citation: Risau W. Mechanisms of angiogenesis. Nature 1997;386:671–674.
references.citation: Risau W, Flamme I. Vasculogenesis. Annu Rev Cell Dev Biol. 1995;11:73–91.
references.citation: Mustonen T, Alitalo K. Endothelial receptor tyrosine kinases involved in angiogenesis. J Cell Biol. 1995;129:895–898.
references.citation: Ferrara N, Carver-Moore K, Chen H, et al.. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996;380:439–442.
references.citation: Ferrara N, Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors. Nat Med. 1999;5:1359–1364.
references.citation: Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev. 1992;13:18–32.
references.citation: Carmeliet P, Ng YS, Nuyens D, et al.. Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Nat Med. 1999;5:495–502.
references.citation: Carmeliet P, Ferreira V, Breier G, et al.. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996;380:435–439.
references.citation: Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem. 1992;267:26031–26037.
references.citation: Yoshihara K, Matsushita O, Minami J, Okabe A. Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase. J Bacteriol. 1994;176:6489–6496.
references.citation: Matsushita O, Jung CM, Katayama S, Minami J, Takahashi Y, Okabe A. Gene duplication and multiplicity of collagenases in Clostridium histolyticum. J Bacteriol. 1999;181:923–933.
references.citation: Philominathan ST, Matsushita O, Gensure R, Sakon J. Ca2+-induced linker transformation leads to a compact and rigid collagen-binding domain of Clostridium histolyticum collagenase. FEBS J. 2009;276:3589–3601.
references.citation: Shima DT, Deutsch U, D'Amore PA. Hypoxic induction of vascular endothelial growth factor (VEGF) in human epithelial cells is mediated by increases in mRNA stability. FEBS Lett. 1995;370:203–208.
references.citation: Bates DO. Vascular endothelial growth factors and vascular permeability. Cardiovasc Res. 2010;87:262–271.
references.citation: Tabata Y. Biomaterial technology for tissue engineering applications. J R Soc Interface 2009;6(Suppl 3):S311–S324.
references.citation: Takahashi T, Ogasawara T, Kishimoto J, et al.. Synergistic effects of FGF-2 with insulin or IGF-I on the proliferation of human auricular chondrocytes. Cell Transplant. 2005;14:683–693.
references.citation: Spanholtz TA, Theodorou P, Holzbach T, Wutzler S, Giunta RE, Machens HG. Vascular endothelial growth factor (VEGF165) plus basic fibroblast growth factor (bFGF) producing cells induce a mature and stable vascular network: A future therapy for ischemically challenged tissue. J Surg Res. 2011;171:329–338.
references.citation: Takeshita S, Zheng LP, Brogi E, et al.. Therapeutic angiogenesis: A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest. 1994;93:662–670.
references.citation: Baumgartner I, Pieczek A, Manor O, et al.. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 1998;97:1114–1123.
references.citation: Schratzberger P, Kirchmair R, Vale PR, Losordo DW. Therapeutic angiogenesis by gene transfer in critical limb and myocardial ischemia. Curr Pharm Des. 2003;9:1041–1047.
references.citation: Taub PJ, Marmur JD, Zhang WX, et al.. Locally administered vascular endothelial growth factor cDNA increases survival of ischemic experimental skin flaps. Plast Reconstr Surg. 1998;102:2033–2039.
references.citation: Zhang F, Yang F, Hu EC, Sones W, Lei M, Lineaweaver WC. Vascular endothelial growth factor gene therapy in improvement of skin paddle survival in a rat TRAM flap model. J Reconstr Microsurg. 2005;21:391–396.
references.citation: Gurunluoglu R, Meirer R, Shafighi M, Huemer GM, Yilmaz B, Piza-Katzer H. Gene therapy with adenovirus-mediated VEGF enhances skin flap prefabrication. Microsurgery 2005;25:433–441.
references.citation: de Freitas AL, Gomes HC, Lisboa BC, Arias V, Han SW, Ferreira LM. Effect of gene therapy with vascular endothelial growth factor after abdominoplasty on TRAM flap viability in a rat model. Plast Reconstr Surg. 2010;125:1343–1351.
references.citation: Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1:27–31.
references.citation: Larcher F, Murillas R, Bolontrade M, Conti CJ, Jorcano JL. VEGF/VPF overexpression in skin of transgenic mice induces angiogenesis, vascular hyperpermeability and accelerated tumor development. Oncogene 1998;17:303–311.
references.citation: Suri C, McClain J, Thurston G, et al.. Increased vascularization in mice overexpressing angiopoietin-1. Science 1998;282:468–471.