In Vitro Angiogenic Properties of Plasmid DNA Encoding SDF-1α and VEGF165 Genes

  • Valeriya V. Solovyeva
  • Daria S. Chulpanova
  • Leysan G. Tazetdinova
  • Ilnur I. Salafutdinov
  • Ilia Y. Bozo
  • Artur A. Isaev
  • Roman V. Deev
  • Albert A. RizvanovEmail author


The stromal-derived factor-1 alpha (SDF-1α) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and exert a significant trophic function. SDF-1α is a chemoattractant for endothelial progenitor cells derived from bone marrow and promotes new blood vessel formation. VEGF regulates all types of vascular growth, stimulates angiogenesis, and is involved in the induction of lymphangiogenesis. The possibility of using these growth factors for regenerative medicine is currently under investigation. The angiogenic potential of a pBud-SDF-1α-VEGF165 bicistronic plasmid construct which simultaneously encodes VEGF165 and SDF-1α genes cDNA was evaluated in this study. The conditioned medium collected from HEK293T cells transfected with the pBud-SDF-1α-VEGF165 plasmid was shown to stimulate the formation of capillary-like structures by human umbilical vein-derived endothelial cells (HUVEC) on Matrigel and to increase the proliferative activity of these cells in vitro. Thus, the pBud-SDF-1α-VEGF165 plasmid exhibits angiogenic properties in cell cultures in vitro. As interest in the development of non-viral techniques for regenerative medicine increases, this plasmid which simultaneously expresses VEGF165 and SDF-1α may provide a platform for advanced methods of stimulating therapeutic angiogenesis.


Angiogenesis Vascular endothelial growth factor Stromal-derived factor Genetic modification Mesenchymal stem cells Endothelial cells 


Conflict of Interest

The authors declare that they have no conflict of interest.

Funding Information

The work was funded by the Human Stem Cells Institute and performed according to the Russian Government Program of Competitive Growth of the Kazan Federal University. AR was supported by the state assignment 20.5175.2017/6.7 of the Ministry of Education and Science of the Russian Federation and the President of the Russian Federation grant НШ-3076.2018.4. IS and VS were supported by the Russian Foundation for Basic Research grant 16-04-01567.

Compliance with Ethical Standards

The protocol was approved by the Biomedicine Ethics Expert Committee of the Kazan Federal University (No. 3, 23.03.2017). Written informed consent was obtained from the donors.


  1. 1.
    Ouma, G. O., Jonas, R. A., Usman, M. H., & Mohler, E. R., 3rd. (2012). Targets and delivery methods for therapeutic angiogenesis in peripheral artery disease. Vascular Medicine, 17(3), 174–192.CrossRefGoogle Scholar
  2. 2.
    Yla-Herttuala, S., Rissanen, T. T., Vajanto, I., & Hartikainen, J. (2007). Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine. Journal of the American College of Cardiology, 49(10), 1015–1026.CrossRefGoogle Scholar
  3. 3.
    Bulgin, D. (2015). Therapeutic Angiogenesis in Ischemic Tissues by Growth Factors and Bone Marrow Mononuclear Cells Administration: Biological Foundation and Clinical Prospects. Current Stem Cell Research & Therapy, 10(6), 509–522.CrossRefGoogle Scholar
  4. 4.
    Takeshita, S., Zheng, L. P., Brogi, E., Kearney, M., Pu, L. Q., Bunting, S., Ferrara, N., Symes, J. F., & Isner, J. M. (1994). Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. The Journal of Clinical Investigation, 93(2), 662–670.CrossRefGoogle Scholar
  5. 5.
    Bauters, C., Asahara, T., Zheng, L. P., Takeshita, S., Bunting, S., Ferrara, N., Symes, J. F., & Isner, J. M. (1995). Site-specific therapeutic angiogenesis after systemic administration of vascular endothelial growth factor. Journal of Vascular Surgery, 21(2), 314–324 discussion 324-315.CrossRefGoogle Scholar
  6. 6.
    Zisa, D., Shabbir, A., Mastri, M., Suzuki, G., & Lee, T. (2009). Intramuscular VEGF repairs the failing heart: role of host-derived growth factors and mobilization of progenitor cells. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 297(5), R1503–R1515.CrossRefGoogle Scholar
  7. 7.
    Sato, K., Laham, R. J., Pearlman, J. D., Novicki, D., Sellke, F. W., Simons, M., & Post, M. J. (2000). Efficacy of intracoronary versus intravenous FGF-2 in a pig model of chronic myocardial ischemia. The Annals of Thoracic Surgery, 70(6), 2113–2118.CrossRefGoogle Scholar
  8. 8.
    Efthimiadou, A., Asimakopoulos, B., Nikolettos, N., Giatromanolaki, A., Sivridis, E., Papachristou, D. N., & Kontoleon, E. (2006). Angiogenic effect of intramuscular administration of basic and acidic fibroblast growth factor on skeletal muscles and influence of exercise on muscle angiogenesis. British Journal of Sports Medicine, 40(1), 35–39 discussion 35-39.CrossRefGoogle Scholar
  9. 9.
    Morishita, R., Nakamura, S., Hayashi, S., Taniyama, Y., Moriguchi, A., Nagano, T., Taiji, M., Noguchi, H., Takeshita, S., Matsumoto, K., Nakamura, T., Higaki, J., & Ogihara, T. (1999). Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy. Hypertension, 33(6), 1379–1384.CrossRefGoogle Scholar
  10. 10.
    Li, W. W., Talcott, K. E., Zhai, A. W., Kruger, E. A., & Li, V. W. (2005). The role of therapeutic angiogenesis in tissue repair and regeneration. Advances in Skin & Wound Care, 18(9), 491–500 quiz 501-492.CrossRefGoogle Scholar
  11. 11.
    Takeshita, S., Pu, L. Q., Stein, L. A., Sniderman, A. D., Bunting, S., Ferrara, N., Isner, J. M., & Symes, J. F. (1994). Intramuscular administration of vascular endothelial growth factor induces dose-dependent collateral artery augmentation in a rabbit model of chronic limb ischemia. Circulation, 90(5 Pt 2), II228–II234.Google Scholar
  12. 12.
    Becit, N., Ceviz, M., Kocak, H., Yekeler, I., Unlu, Y., Celenk, C., & Akin, Y. (2001). The effect of vascular endothelial growth factor on angiogenesis: an experimental study. European Journal of Vascular and Endovascular Surgery : The Official Journal of the European Society for Vascular Surgery, 22(4), 310–316.CrossRefGoogle Scholar
  13. 13.
    Pearlman, J. D., Hibberd, M. G., Chuang, M. L., Harada, K., Lopez, J. J., Gladstone, S. R., Friedman, M., Sellke, F. W., & Simons, M. (1995). Magnetic resonance mapping demonstrates benefits of VEGF-induced myocardial angiogenesis. Nature Medicine, 1(10), 1085–1089.CrossRefGoogle Scholar
  14. 14.
    Kottakis, F., Polytarchou, C., Foltopoulou, P., Sanidas, I., Kampranis, S. C., & Tsichlis, P. N. (2011). FGF-2 regulates cell proliferation, migration, and angiogenesis through an NDY1/KDM2B-miR-101-EZH2 pathway. Molecular Cell, 43(2), 285–298.CrossRefGoogle Scholar
  15. 15.
    Laham, R. J., Rezaee, M., Post, M., Sellke, F. W., Braeckman, R. A., Hung, D., & Simons, M. (1999). Intracoronary and intravenous administration of basic fibroblast growth factor: myocardial and tissue distribution. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 27(7), 821–826.Google Scholar
  16. 16.
    Lazarous, D. F., Shou, M., Stiber, J. A., Dadhania, D. M., Thirumurti, V., Hodge, E., & Unger, E. F. (1997). Pharmacodynamics of basic fibroblast growth factor: route of administration determines myocardial and systemic distribution. Cardiovascular Research, 36(1), 78–85.CrossRefGoogle Scholar
  17. 17.
    Deepa, K., Rodionov, R. N., Weiss, N., & Parani, M. (2013). Transgenic expression and functional characterization of human platelet derived growth factor BB (hPDGF-BB) in tobacco (Nicotiana tabacum L.). Applied Biochemistry and Biotechnology, 171(6), 1390–1404.CrossRefGoogle Scholar
  18. 18.
    Choi, J. H., Kim, S., Sapkota, K., Park, S. E., & Kim, S. J. (2011). Expression and production of therapeutic recombinant human platelet-derived growth factor-BB in Pleurotus eryngii. Applied Biochemistry and Biotechnology, 165(2), 611–623.CrossRefGoogle Scholar
  19. 19.
    Yang, F., Xue, F., Guan, J., Zhang, Z., Yin, J., & Kang, Q. (2018). Stromal-Cell-Derived Factor (SDF) 1-Alpha Overexpression Promotes Bone Regeneration by Osteogenesis and Angiogenesis in Osteonecrosis of the Femoral Head. Cellular Physiology and Biochemistry, 46(6), 2561–2575.CrossRefGoogle Scholar
  20. 20.
    Deshane, J., Chen, S., Caballero, S., Grochot-Przeczek, A., Was, H., Li Calzi, S., Lach, R., Hock, T. D., Chen, B., Hill-Kapturczak, N., Siegal, G. P., Dulak, J., Jozkowicz, A., Grant, M. B., & Agarwal, A. (2007). Stromal cell-derived factor 1 promotes angiogenesis via a heme oxygenase 1-dependent mechanism. The Journal of Experimental Medicine, 204(3), 605–618.CrossRefGoogle Scholar
  21. 21.
    Yu, J. X., Huang, X. F., Lv, W. M., Ye, C. S., Peng, X. Z., Zhang, H., Xiao, L. B., & Wang, S. M. (2009). Combination of stromal-derived factor- 1alpha and vascular endothelial growth factor gene-modified endothelial progenitor cells is more effective for ischemic neovascularization. Journal of Vascular Surgery, 50(3), 608–616.CrossRefGoogle Scholar
  22. 22.
    Ho, T. K., Tsui, J., Xu, S., Leoni, P., Abraham, D. J., & Baker, D. M. (2010). Angiogenic effects of stromal cell-derived factor-1 (SDF-1/CXCL12) variants in vitro and the in vivo expressions of CXCL12 variants and CXCR4 in human critical leg ischemia. Journal of Vascular Surgery, 51(3), 689–699.CrossRefGoogle Scholar
  23. 23.
    Gorenoi, V., Brehm, M. U., Koch, A., & Hagen, A. (2017). Growth factors for angiogenesis in peripheral arterial disease. Cochrane Database of Systematic Reviews, (6), CD011741.Google Scholar
  24. 24.
    Inampudi, C., Akintoye, E., Ando, T., & Briasoulis, A. (2018). Angiogenesis in peripheral arterial disease. Current Opinion in Pharmacology, 39, 60–67.CrossRefGoogle Scholar
  25. 25.
    King, A., Balaji, S., Keswani, S. G., & Crombleholme, T. M. (2014). The Role of Stem Cells in Wound Angiogenesis. Advances in Wound Care, 3(10), 614–625.CrossRefGoogle Scholar
  26. 26.
    Hou, L., Kim, J. J., Woo, Y. J., & Huang, N. F. (2016). Stem cell-based therapies to promote angiogenesis in ischemic cardiovascular disease. American Journal of Physiology. Heart and Circulatory Physiology, 310(4), H455–H465.CrossRefGoogle Scholar
  27. 27.
    Solovyeva, V. V., Blatt, N. L., Shafigullina, A. K., & Rizvanov, A. A. (2012). Endogenous secretion of vascular endothelial growth factor by multipotent mesenchymal stromal cells derived from human third molar dental follicles. Cellular Transplantation and Tissue Engineering, 7(3), 155–158.Google Scholar
  28. 28.
    Fierro, F. A., Magner, N., Beegle, J., Dahlenburg, H., Logan White, J., Zhou, P., Pepper, K., Fury, B., Coleal-Bergum, D. P., Bauer, G., Gruenloh, W., Annett, G., Pifer, C., & Nolta, J. A. (2018). Mesenchymal stem/stromal cells genetically engineered to produce vascular endothelial growth factor for revascularization in wound healing and ischemic conditions. Transfusion. Google Scholar
  29. 29.
    Bortolotti, F., Ukovich, L., Razban, V., Martinelli, V., Ruozi, G., Pelos, B., Dore, F., Giacca, M., & Zacchigna, S. (2015). In vivo therapeutic potential of mesenchymal stromal cells depends on the source and the isolation procedure. Stem Cell Reports, 4(3), 332–339.CrossRefGoogle Scholar
  30. 30.
    Raval, Z., & Losordo, D. W. (2013). Cell therapy of peripheral arterial disease: from experimental findings to clinical trials. Circulation Research, 112(9), 1288–1302.CrossRefGoogle Scholar
  31. 31.
    Sanada, F., Taniyama, Y., Kanbara, Y., Otsu, R., Ikeda-Iwabu, Y., Carracedo, M., Rakugi, H., & Morishita, R. (2015). Gene therapy in peripheral artery disease. Expert Opinion on Biological Therapy, 15(3), 381–390.CrossRefGoogle Scholar
  32. 32.
    Yla-Herttuala, S., & Baker, A. H. (2017). Cardiovascular Gene Therapy: Past, Present, and Future. Molecular Therapy, 25(5), 1095–1106.CrossRefGoogle Scholar
  33. 33.
    Walder, C. E., Errett, C. J., Bunting, S., Lindquist, P., Ogez, J. R., Heinsohn, H. G., Ferrara, N., & Thomas, G. R. (1996). Vascular endothelial growth factor augments muscle blood flow and function in a rabbit model of chronic hindlimb ischemia. Journal of Cardiovascular Pharmacology, 27(1), 91–98.CrossRefGoogle Scholar
  34. 34.
    Chervyakov, Y. V., Staroverov, I. N., Vlasenko, O. N., Bozo, I. Y., Isaev, A. A., & Deev, R. V. (2016). Five-year results of treating patients with chronic lower limb ischaemia by means of gene engineering. Angiol Sosud Khir, 22(4), 38–44.Google Scholar
  35. 35.
    Kalinin, R. E., Suchkov, I. A., Deev, R. V., Mzhavanadze, N. D., & Krylov, A. A. (2018). Gene-mediated induction of angiogenesis in inoperable patients with atherosclerosis and diabetes mellitus. Angiol Sosud Khir, 24(2), 33–40.Google Scholar
  36. 36.
    Luo, R., Lu, Y., Liu, J., Cheng, J., & Chen, Y. (2019). Enhancement of the efficacy of mesenchymal stem cells in the treatment of ischemic diseases. Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie, 109, 2022–2034.CrossRefGoogle Scholar
  37. 37.
    Stewart, D. J., Hilton, J. D., Arnold, J. M., Gregoire, J., Rivard, A., Archer, S. L., Charbonneau, F., Cohen, E., Curtis, M., Buller, C. E., Mendelsohn, F. O., Dib, N., Page, P., Ducas, J., Plante, S., Sullivan, J., Macko, J., Rasmussen, C., Kessler, P. D., & Rasmussen, H. S. (2006). Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF(121) (AdVEGF121) versus maximum medical treatment. Gene Therapy, 13(21), 1503–1511.CrossRefGoogle Scholar
  38. 38.
    Niebuhr, A., Henry, T., Goldman, J., Baumgartner, I., van Belle, E., Gerss, J., Hirsch, A. T., & Nikol, S. (2012). Long-term safety of intramuscular gene transfer of non-viral FGF1 for peripheral artery disease. Gene Therapy, 19(3), 264–270.CrossRefGoogle Scholar
  39. 39.
    Shigematsu, H., Yasuda, K., Sasajima, T., Takano, T., Miyata, T., Ohta, T., Tanemoto, K., Obitsu, Y., Iwai, T., Ozaki, S., Ogihara, T., Morishita, R., & Group, H. G. F. S. (2011). Transfection of human HGF plasmid DNA improves limb salvage in Buerger's disease patients with critical limb ischemia. International Angiology : a Journal of the International Union of Angiology, 30(2), 140–149.Google Scholar
  40. 40.
    Lebas, B., Galley, J., Renaud-Gabardos, E., Pujol, F., Lenfant, F., Garmy-Susini, B., Chaufour, X., & Prats, A. C. (2017). Therapeutic Benefits and Adverse Effects of Combined Proangiogenic Gene Therapy in Mouse Critical Leg Ischemia. Annals of Vascular Surgery, 40, 252–261.CrossRefGoogle Scholar
  41. 41.
    Garanina, E. E., Mukhamedshina, Y. O., Salafutdinov, I. I., Kiyasov, A. P., Lima, L. M., Reis, H. J., Palotas, A., Islamov, R. R., & Rizvanov, A. A. (2016). Construction of recombinant adenovirus containing picorna-viral 2A-peptide sequence for the co-expression of neuro-protective growth factors in human umbilical cord blood cells. Spinal Cord, 54(6), 423–430.CrossRefGoogle Scholar
  42. 42.
    Zhuravleva, M. N., Khaliullin, M. R., Masgutov, R. F., Deev, R. V., & Rizvanov, A. A. (2017). Recombinant Plasmid DNA Construct Encoding Combination of vegf165 and bmp2 cDNAs Stimulates Osteogenesis and Angiogenesis In Vitro. Bionanoscience, 7(2), 288–293.CrossRefGoogle Scholar
  43. 43.
    Solovyeva, V. V., Salafutdinov, I. I., Tazetdinova, L. G., Masgutov, R. F., Khaiboullina, S. F., & Rizvanov, A. A. (2014). Genetic Modification of Adipose Derived Stem Cells with Recombinant Plasmid DNA pBud-VEGF-FGF2 Results in Increased of IL-8 and MCP-1 Secretion. Journal of Pure and Applied Microbiology, 8(Spl. Edn. 2), 523–528.Google Scholar
  44. 44.
    Khaiboullina, S. F., Rizvanov, A. A., Deyde, V. M., & St Jeor, S. C. (2005). Andes virus stimulates interferon-inducible MxA protein expression in endothelial cells. Journal of Medical Virology, 75(2), 267–275.CrossRefGoogle Scholar
  45. 45.
    Solovyeva, V. V., Salafutdinov, I. I., Martynova, E. V., Khaiboullina, S. F., & Rizvanov, A. A. (2013). Human Adipose Derived Stem Cells Do Not Alter Cytokine Secretion in Response To The Genetic Modification With pEGFP-N2 Plasmid DNA. World Applied Sciences Journal, 26(7), 968–972.Google Scholar
  46. 46.
    Carpentier, G. (2012). Contribution: angiogenesis analyzer. ImageJ News, 5(Available:
  47. 47.
    Apte, R. S., Chen, D. S., & Ferrara, N. (2019). VEGF in Signaling and Disease: Beyond Discovery and Development. Cell, 176(6), 1248–1264.CrossRefGoogle Scholar
  48. 48.
    Yue, X., & Tomanek, R. J. (2001). Effects of VEGF(165) and VEGF(121) on vasculogenesis and angiogenesis in cultured embryonic quail hearts. American Journal of Physiology. Heart and Circulatory Physiology, 280(5), H2240–H2247.CrossRefGoogle Scholar
  49. 49.
    Keyt, B. A., Berleau, L. T., Nguyen, H. V., Chen, H., Heinsohn, H., Vandlen, R., & Ferrara, N. (1996). The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. The Journal of Biological Chemistry, 271(13), 7788–7795.CrossRefGoogle Scholar
  50. 50.
    Neuhaus, T., Stier, S., Totzke, G., Gruenewald, E., Fronhoffs, S., Sachinidis, A., Vetter, H., & Ko, Y. D. (2003). Stromal cell-derived factor 1alpha (SDF-1alpha) induces gene-expression of early growth response-1 (Egr-1) and VEGF in human arterial endothelial cells and enhances VEGF induced cell proliferation. Cell Proliferation, 36(2), 75–86.CrossRefGoogle Scholar
  51. 51.
    Zheng, H., Fu, G., Dai, T., & Huang, H. (2007). Migration of endothelial progenitor cells mediated by stromal cell-derived factor-1alpha/CXCR4 via PI3K/Akt/eNOS signal transduction pathway. Journal of Cardiovascular Pharmacology, 50(3), 274–280.CrossRefGoogle Scholar
  52. 52.
    Inouye, S., Sahara-Miura, Y., Sato, J., & Suzuki, T. (2015). Codon optimization of genes for efficient protein expression in mammalian cells by selection of only preferred human codons. Protein Expression and Purification, 109, 47–54.CrossRefGoogle Scholar
  53. 53.
    Solovyeva, V. V., Kiyasov, A. P. and Rizvanov, A. A. (2016). Genetically Engineered Dental Stem Cells for Regenerative Medicine. Stem Cells Biol Reg, 93–107.Google Scholar
  54. 54.
    Serra, J., Alves, C. P. A., Brito, L., Monteiro, G. A., Cabral, J. M. S., Prazeres, D. M. F., & da Silva, C. L. (2019). Engineering of Human Mesenchymal Stem/Stromal Cells with Vascular Endothelial Growth Factor-Encoding Minicircles for Angiogenic Ex Vivo Gene Therapy. Human Gene Therapy, 30(3), 316–329.CrossRefGoogle Scholar
  55. 55.
    Hojati, Z., & Dehghanian, F. (2015). Enhanced expression of bioactive recombinant VEGF-111 with insertion of intronic sequence in mammalian cell lines. Applied Biochemistry and Biotechnology, 175(8), 3737–3749.CrossRefGoogle Scholar
  56. 56.
    Tang, J., Wang, J., Yang, J., Kong, X., Zheng, F., Guo, L., Zhang, L., & Huang, Y. (2009). Mesenchymal stem cells over-expressing SDF-1 promote angiogenesis and improve heart function in experimental myocardial infarction in rats. European Journal of Cardio-Thoracic Surgery : Official Journal of the European Association for Cardio-Thoracic Surgery, 36(4), 644–650.CrossRefGoogle Scholar
  57. 57.
    Zhang, L., Zhou, Y., Sun, X., Zhou, J., & Yang, P. (2017). CXCL12 overexpression promotes the angiogenesis potential of periodontal ligament stem cells. Scientific Reports, 7(1), 10286.CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Valeriya V. Solovyeva
    • 1
  • Daria S. Chulpanova
    • 1
  • Leysan G. Tazetdinova
    • 1
  • Ilnur I. Salafutdinov
    • 1
  • Ilia Y. Bozo
    • 3
    • 4
  • Artur A. Isaev
    • 2
  • Roman V. Deev
    • 5
  • Albert A. Rizvanov
    • 1
    Email author
  1. 1.Kazan Federal UniversityKazanRussia
  2. 2.Human Stem Cells InstituteMoscowRussia
  3. 3.Histograft, LLCMoscowRussia
  4. 4.Federal Medical Biophysical CenterFMBA of RussiaMoscowRussia
  5. 5.Ryazan State Medical UniversityRyazanRussia

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