Enhanced angiogenesis in ischemic skeletal muscle after transplantation of cell sheets from baculovirus-transduced adipose-derived stromal cells expressing VEGF165.
BACKGROUND
Cell therapy using adipose-derived stromal cells (ADSC) is an intensively developing approach to promote angiogenesis and regeneration. Administration technique is crucial and among others minimal constructs - cell sheets (CS) have certain advantages. Delivery of CS allows transplantation of cells along with matrix proteins to facilitate engraftment. Cells' therapeutic potential can be also increased by expression of proangiogenic factors by viral transduction. In this work we report on therapeutic efficacy of CS from mouse ADSC transduced to express human vascular endothelial growth factor 165 a/a isoform (VEGF165), which showed potency to restore perfusion and protect tissue in a model of limb ischemia.


METHODS
Mouse ADSC (mADSC) isolated from C57 male mice were expanded for CS formation (10(6)cells per CS). Constructs were transduced to express human VEGF165 by baculoviral (BV) system. CS were transplanted subcutaneously to mice with surgically induced limb ischemia and followed by laser Doppler perfusion measurements. At endpoint animals were sacrificed and skeletal muscle was evaluated for necrosis and vessel density; CS with underlying muscle was stained for apoptosis, proliferation, monocytes and blood vessels.


RESULTS
Using BV system and sodium butyrate treatment we expressed human VEGF165 in mADSC (production of VEGF165 reached ≈ 25-27 ng/ml/10(5) cells) and optimized conditions to ensure cells' viability after transduction. Implantation of mock-transduced CS resulted in significant improvement of limb perfusion, increased capillary density and necrosis reduction at 2 weeks post-surgery compared to untreated animals. Additional improvement of blood flow and angiogenesis was observed after transplantation of VEGF165-expressing CS indicating enhanced therapeutic potential of genetically modified constructs. Moreover, we found delivery of mADSC as CS to be superior to equivalent dose of suspended cells in terms of perfusion and angiogenesis. Histology analysis of extracted CS detected limited proliferation and approximately 10 % prevalence of apoptosis in transplanted mADSC. Significant vascularization of CS and infiltration by monocytes were found in both - BV-transduced and control CS indicating graft and host interaction after transplantation.


CONCLUSIONS
Delivery of ADSC by subcutaneous transplantation of CS is effective for stimulation of angiogenesis and tissue protection in limb ischemia with a potential for efficacy improvement by BV transduction to express VEGF165.
Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex, Moscow, 121552, Russia. pmakarevich@mc.msu.ru.|Laboratory of Regenerative Medicine, Medical Research and Educational Centre of Moscow State University, Moscow, 119192, Russia. pmakarevich@mc.msu.ru.
Pavel I
PI
Makarevich
Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex, Moscow, 121552, Russia. mboldyreva@inbox.ru.
Maria A
MA
Boldyreva
Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex, Moscow, 121552, Russia. evgengluhanuk@gmail.com.|Faculty of Medicine, M.V. Lomonosov Moscow State University, Moscow, 119192, Russia. evgengluhanuk@gmail.com.
Evgeny V
EV
Gluhanyuk
Laboratory of Regenerative Medicine, Medical Research and Educational Centre of Moscow State University, Moscow, 119192, Russia. efimenkoan@gmail.com.|Faculty of Medicine, M.V. Lomonosov Moscow State University, Moscow, 119192, Russia. efimenkoan@gmail.com.
Anastasia Yu
AY
Efimenko
Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex, Moscow, 121552, Russia. doctorkote@gmail.com.
Konstantin V
KV
Dergilev
Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex, Moscow, 121552, Russia. dr.eshevchenko@gmail.com.
Evgeny K
EK
Shevchenko
Faculty of Medicine, M.V. Lomonosov Moscow State University, Moscow, 119192, Russia. sharonov@gmail.com.
Georgy V
GV
Sharonov
Faculty of Medicine, M.V. Lomonosov Moscow State University, Moscow, 119192, Russia. julling92@gmail.com.
Julia O
JO
Gallinger
Faculty of Medicine, M.V. Lomonosov Moscow State University, Moscow, 119192, Russia. rodina_polina@list.ru.
Polina A
PA
Rodina
Faculty of Medicine, M.V. Lomonosov Moscow State University, Moscow, 119192, Russia. ss.sarkisyan@gmail.com.
Stepan S
SS
Sarkisyan
Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 300, Taiwan. ychu@mx.nthu.edu.tw.
Yu-Chen
YC
Hu
Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex, Moscow, 121552, Russia. yeparfyon@mail.ru.|Faculty of Medicine, M.V. Lomonosov Moscow State University, Moscow, 119192, Russia. yeparfyon@mail.ru.
Yelena V
YV
Parfyonova
D000818:Animals; D017209:Apoptosis; D016367:Baculoviridae / Q000235:genetics; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D006614:Hindlimb / Q000098:blood supply; D007511:Ischemia / Q000628:therapy*; D008297:Male; D008810:Mice, Inbred C57BL; D055806:Microvessels / Q000502:physiology; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000276:immunology / Q000473:pathology; D009220:Myositis / Q000517:prevention & control; D009336:Necrosis / Q000517:prevention & control; D018919:Neovascularization, Physiologic; D012039:Regional Blood Flow; D033581:Stem Cell Transplantation; D050151:Subcutaneous Fat / Q000473:pathology; D014161:Transduction, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A