M.A. Boldireva - Ph.D. (Biol.), Research Scientist, Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex Ministry of Health of Russia, Moscow, Russia E-mail: mboldyreva@inbox.ru E.S. Zubkova - Junior Research Scientist, Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex Ministry of Health of Russia, Moscow, Russia E-mail: ver-mishel@mail.ru I.B. Beloglasova - Ph.D. (Biol.), Research Scientist, Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex Ministry of Health of Russia, Moscow, Russia E-mail: irene.beloglazova@gmail.com E.I. Ratner - Ph.D. (Biol.), Senior Research Scientist, Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex Ministry of Health of Russia, Moscow, Russia E-mail: eiratner@gmail.com O.Yu. Sukhareva - Ph.D. (Med.), Leading Research Scientist, Institute of Diabetes, Endocrinology Research Center Ministry of Health of Russia, Moscow, Russia E-mail: olgasukhareva@mail.ru Zh.A. Akopian - Ph.D. (Med.), Deputy Dean, Faculty of Fundamental Medicine, Lomonosov Moscow State University, Moscow, Russia E-mail: hanna@fbm.msu.ru M.V. Shestakova - Corresponding Member of RAS, Dr. Sc. (Med.), Director, Institute of Diabetes, Endocrinology Research Center, Ministry of Health of Russia, Moscow, Russia E-mail: shestakova.mv@gmail.com Ye.V. Parfyonova - Dr.Sc. (Med.), Professor, Director, Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex Ministry of Health of Russia, Moscow, Russia E-mail: yeparfyon@mail.ru

Cell therapy using of autologous progenitor cells is an intensively developing approach to promote tissue repair and rege-neration. We and others have demonstrated the potential impact of adipose tissue-derived stromal cells (ADSC) on angi-ogenic cell therapy for myocardial and limb ischemia. The main benefit of ADSC is that they can be easily harvested from patients by a simple, minimally invasive method and also easily cultured. However, the efficacy of cell therapy for ischemic diseases is restricted by pure variability of transplanted cells, which may be exposed to an inflamed hypoxic environment of damaged tissue, resulted in drastic reduction of their number and, thus, therapeutic effect. Since the initial success of cell therapy for ischemic diseases many attempts have been made to increase its efficacy. One of the promising approach is a combination of gene and cell therapy by transgenic expression in ischemic tissues of growth factors stimulating trans-planted cells survival and proliferation. The aim of this work is to study the feasibility of the increase therapeutic angiogenesis efficacy by combination of ADSC therapy with plasmid based PDGF-B gene therapy. PDGF-B is important regulator of ADSC function. It stimulates proliferation, migration and inhibits apoptosis of these cells, and increases their angiogenic potential. Using mouse hind limb ischemia model we assessed the efficacy of combination of ADSCs transplan-tation with plasmid based PDGF-B gene therapy for blood flow restoration and angiogenesis in ischemic limb. Intramuscular administration of plasmid encoding PDGF-B gene just after hind limb modeling were followed after 5 days by transcutaneous intramuscular injection of mouse ADSC isolated from C57/BL6 male syngeneic mice. Blood flow restoration was determined by laser Doppler perfusion measurements every 7 days during 21 days. At endpoint animals were sacrificed and skeletal muscle was evaluated for vessel density. Mice injected with PDGF-B plasmid followed by ADSCs transplantation showed significant increase in perfusion compared to single plasmid injection or ADSCs transplantation along. These findings were supported by significantly increased CD31+/SMA+ vessel density in animals that received combined gene and cell therapy compared to single gene or cell therapy. The results obtained provide the foundation for the development gene-cell therapy method for patient with critical limb ischemia which often results in limb amputation especially in diabetic patients.

 

1. Kannel W.B. Lipids, diabetes, and coronary heart disease: insights from the Framingham Study // Am. Heart J. 1985. V. 110. № 5. P. 1100-1107.
2. Rhee S.Y., Kim Y.S. Peripheral arterial disease in patients with type 2 diabetes mellitus. Diabetes Metab. J. 2015. V. 39. № 4. P. 283-290.
3. Thiruvoipati T., Kielhorn C.E., Armstrong E.J. Pe­ripheral artery disease in patients with diabetes: epidemiology, mechanisms, and outco­mes // World J. Diabetes. 2015. V. 6. № 7. P. 961-969.
4. Rümenapf G., Morbach S. What can I do with a patient with diabetes and critically impaired limb perfusion who cannot be revascularized - // Int. J. Low Extrem. Wounds. 2014. V. 13. № 4. P. 378-389.
5. Bondarenko O.N., Galstjan R. G., Dedov I.I. Osobennosti klinicheskogo techenija kriticheskojj ishemii nizhnikh konechnostejj i rol ehndovaskuljarnojj revaskuljarizacii u bolnykh sakharnym diabetom // Sakharnyjj diabet. 2015. T. 18. № 3. S. 57-69.
6. Nacionalnye rekomendacii po vedeniju pacientov s zabolevanijami arterijj nizhnikh konechnostejj (Rossijjskijj soglasitelnyjj dokument). M.: 2013. Dostupno po ssylke: http://www.chelsma.ru/files/misc/recommendations_lla.pdf.
7. Makarevich P.I., Rubina K.A., Dyjjkanov D.T., Tkachuk V.A., Parfenova E.V.Terapevticheskijj angiogenez s primeneniem faktorov rosta: sovremennoe sostojanie i perspektivy razvitija // Kardiologija. 2015. T. 55. № 9. S. 59-71.
8. Shevchenko E.K., Makarevich P.I., Tsokolaeva Z.I., Boldyreva M.A., Sysoeva V.Y., Tkachuk V.A., Parfyonova Ye.V. Transplantation of modified human adipose derived stromal cells expressing VEGF165 results in more efficient angiogenic response in ischemic skeletal muscle // J. Transl. Med. 2013. V. 11. 138 r.
9. Liang X., Ding Y., Zhang Y., Tse H..F, Lian Q. Paracrine mechanisms of mesenchymal stem cell-based therapy: current status and perspectives. Cell Transplant. 2014. V. 23. № 9. P. 1045-1059.
10. Kalinina N.I., Sysoeva V.JU., Rubina K.A., Parfenova E.V. Tkachuk V.A. Mezenkhimnye stvolovye kletki v processakh rosta i reparacii tkanejj // Acta Naturae. 2011. V. 3. № 4. P. 32-39.
11. Traktuev D.O., Parfenova E.V., Tkachuk V.A., March K.L. Stromalnye kletki zhirovojj tkani ? plasticheskijj tip kletok, obladajushhikh vysokim terapevticheskim potencialom // Citologija. 2006. T. 48. № 2. S. 83-94.
12. Lopatina T., Kalinina N., Karagyaur M., Stambolsky D., Rubina K., Revischin A., Pavlova G., Parfyonova Y., Tkachuk V. Adipose-derived stem cells stimulate regeneration of peripheral nerves: BDNF secreted by these cells promotes nerve healing and axon growth de novo // PLoS One. 2011. V. 6. № 3. e17899.
13. Hu X., Yu S.P., Fraser J.L., Lu Z., Ogle M.E., Wang J.A., Wei L. Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis // J. Thorac. Cardiovasc. Surg. 2008. V. 135. № 4. P. 799-808.
14. Efimenko A., Dzhoyashvili N., Kalinina N., Kochegura T. Akchurin R., Tkachuk V., Parfyonova Ye. Adipose-derived mesenchymal stromal cells from aged patients with coronary artery disease keep mesenchymal stromal cell properties but exhibit characteristics of aging and have impaired angiogenic potential // Stem Cells Transl. Med. 2014. V. 3. № 1. P. 32-41.
15. Dzhoyashvili N.A., Efimenko A.Y., Kochegura T.N., Kalinina N.I., Koptelova N.V., Sukhareva O.Y., Shestakova M.V., Akchurin R.S., Tkachuk V.A., Parfyonova Y.V. Disturbed angiogenic activity of adipose-derived stromal cells obtained from patients with coronary artery disease and diabetes mellitus type 2 // J. Transl. Med. 2014. V. 12. R. 337.
16. Efimenko A.Y., Kochegura T.N., Akopyan Z.A., Parfyonova Y.V. Autologous stem cell therapy: how aging and chronic diseases affect stem and progenitor cells // Biores. Open Access. 2015. V. 4. № 1. P. 26-38.
17. Kim W.S., Park H.S., Sung J.H. The pivotal role of PDGF and its receptor isoforms in adipose-derived stem cells // Histol. Histopathol. 2015. V. 30. № 7. P. 793-799.
18. Rubina K., Kalinina N., Efimenko A., Lopatina T., Melikhova V., Tsokolaeva Z., Sysoeva V., Tkachuk V., Parfyonova Y. Adipose stromal cells stimulate angiogenesis via promoting progenitor cell differentiation, secretion of angiogenic factors, and enhancing vessel maturation // Tissue Eng. Part A. 2009. V. 15. № 8. P. 2039-2050.
19. Zubkova E.S., Beloglazova I.B., Makarevich P.I., Boldyreva M.A., Sukhareva O.Y., Shestakova M.V., Dergilev K.V., Parfyonova Y.V., Menshikov M.Y. Regulation of adipose tissue stem cells angiogenic potential by tumor necrosis factor-alpha // J. Cell. Biochem. 2016. V. 117. № 1. P. 180-196.
20. Parfenova E.V., SHevchenko E.K., Makarevich P.I., CokolaevaZ.I., Boldyreva M.A., Tkachuk V.A. Gen PDGF-B opt trombocitarnogo faktora rosta cheloveka (optimizirovannyjj). Reshenie o vydache patenta ot 11.03.2016 po zajavke № 2014134753, ot 26.08.2014.
21. Traktuev D.O., Tsokolaeva Z.I., Shevelev A.A., Talitskiy K.A., Stepanova V.V., Johnstone B.H., Rahmat-Zade T.M., Kapustin A.N., Tkachuk V.A., March K.L., Parfyonova Y.V. Urokinase gene transfer augments angiogenesis in ischemic skeletal and myocardial muscle // Mol. Ther. 2007. V. 15. № 11. P. 1939-1946.
22. Takeshita S., Weir L., Chen D., Zheng L.P., Riessen R., Bauters C., Symes J.F., Ferrara N., Isner J.M. Therapeutic angiogenesis following arterial gene transfer of vascular endothelial growth factor in a rabbit model of hindlimb ischemia // Biochem. Biophys. Res. Commun. 1996. V. 227. № 2. P. 628-635.
23. Makarevich P., Tsokolaeva Z., Shevelev A., Rybalkin I., Shevchenko E., Beloglazova I., Vlasik T., Tkachuk V., Parfyonova Y. Combined transfer of human VEGF165 and HGF genes renders potent angiogenic effect in ischemic skeletal muscle // PLoS One. 2012. V. 7. № 6. e38776.
24. Makarevich P.I., Boldyreva M.A., Gluhanyuk E.V., Efimenko A.Y., Dergilev K.V., Shevchenko E.K., Sharonov G.V., Gallinger J.O., Rodina P.A., Sarkisyan S..S, Hu Y.C., Parfyonova Y.V. Enhanced angiogenesis in ischemic skeletal muscle after transplantation of cell sheets from baculovirus-transduced adipose-derived stromal cells expressing VEGF165 // Stem Cell Res. Ther. 2015. V. 6. R. 204.
25. Kholia S., Ranghino A., Garnieri P., Lopatina T., Deregibus M.C., Rispoli P., Brizzi M.F., Camussi G. Extracellular vesicles as new players in angiogenesis // Vascul. Pharmacol. 2016. V. 86. R. 64-70.
26. Gehmert S., Gehmert S., Hidayat M., Sultan M., Berner A., Klein S., Zellner J., Müller M., Prantl L. Angiogenesis: the role of PDGF-BB on adipose-tissue derived stem cells (ASCs) // Clin. Hemorheol. Microcirc. 2011. V. 48. № 1. R. 5-13.
27. Kim J.H., Park S.G., Song S.Y., Kim J.K., Sung J.H. Reactive oxygen species-responsive miR-210 regulates proliferation and migration of adipose-derived stem cells via PTPN2 // Cell Death Dis. 2013. V. 4. e588.
28. Lopatina T., Bruno S., Tetta C., Kalinina N., Porta M., Camussi G. Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential // Cell Commun. Signal. 2014. V. 12. R. 26.