Coronary stents with inducible VEGF/HGF-secreting UCB-MSCs reduced restenosis and increased re-endothelialization in a swine model

Figures

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  Fig. 1. The properties of HGF/U-Ms and VEGF/U-Ms are maintained on stent material. a Schematic illustration of the experiment using the stent with HGF/U-Ms and VEGF/U-Ms to secrete the angiogenic factors in an inducible manner. The stents were coated with polydopamine, fibronectin, and extracellular matrix (ECM), followed by in vitro and in vivo swine experiments. b Human U-Ms secreting HGF and VEGF were seeded onto the pre-coated material sheets to confirm cell viability on the stent material. The cells stained with green fluorescent dye were detected using fluorescence microscopy. U-M human UCB-MSCs, Ctl control. N = 3. c The cells were counted at 7 days post seeding after detachment from the stent material and staining of dying cells using trypan blue (**denotes a p-value < 0.01). d Cell viability was analyzed using a crystal violet assay on the stent 7 days post seeding (** denotes a p-value < 0.01). d, e HGF and VEGF secretion were detected in conditioned media using western blotting. HGF/U-Ms and VEGF/U-Ms were treated with 5 µg of doxycycline for two days in a six-well plate. f MSC markers on the stent material were not altered, even in the HGF- and VEGF-secreting cells. Lane 1:…

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  Fig. 2. The cells remained on the stent after transplantation. a Fluorescent images showing adhesion of the GFP-expressing UCB-MSCs on the stents. b Fluorescent images prior to surgery and stent seeding in the swine model were captured using a microscope, and the SEM images after the end of the experiment were captured using scanning electron microscopy. N = 3

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  Fig. 3. The single conditions of HGF/U-Ms and VEGF/U-Ms alone produced opposite effects on neointima levels 2 weeks after transplantation. a OCT images showing neointimal degrees 2 weeks after stent transplantation. BMS bare material stent, U-M human U-M-coated stent, HGF/U-M stent coated with HGF/U-Ms, VEGF/U-M stent coated with VEGF/U-Ms. b OCT analysis showing neointimal areas in mm2 and in % units. c The microCT results were analyzed at a 50 kV/200 μA spatial resolution with 17-μm units to show the surface smoothness and neointima areas. N = 3

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  Fig. 4. HGF + VEGF/U-Ms enhanced tube formation following HGF + VEGF induction. a Images of tube formation were acquired for the U-Ms (UCB-MSCs), HGF + VEGF/U-Ms (1:1 ratio) without doxycycline (Dox) and with Dox, and HGF + VEGF (5:1 ratio)/U-Ms with Dox. b, c Tube formation was analyzed based on the number of tubes (b) and the number of tube branches (c). N = 3

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  Fig. 5. Stents coated with HGF + VEGF/U-Ms reduced the neointimal area 4 weeks after transplantation. a OCT results showing the neointima degree in the lumens of the coronary arteries 4 weeks after transplantation. b The neointima degree was analyzed in mm2 and the % of the neointima area (*; p-value of 0.019 and 0.040, **; p-value of 0.018 and 0.017). c Representative microCT images of the stents 4 weeks after transplantation. d Image of fibrin staining showing the fibrotic area with a cross-section of the transplanted stents. e The stenosis degrees were assessed as the % of fibrin staining (**p-value of 0.01). f Angiography showing the blood flow in the coronary artery with the transplanted stent. BMS; N = 6, U-M; N = 6, HV/U-M; N = 5

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  Fig. 6. Re-endothelialization was observed in coronary arteries with transplanted stents coated with HGF + VEGF/U-Ms and human MSCs in the swine model. a The coronary arteries transplanted with stem cell-loaded stents were sectioned and stained using anti-vWF (1:200) and anti-Lamin A + C (1:200) antibodies. The samples were harvested 2 weeks after transplantation. b Human-specific Alu was detected using RT-PCR in coronary arteries 2 weeks after transplantation. c Anti-vWF immunostaining showing the re-endothelialized portion of the coronary arteries 4 weeks after transplantation for the stents coated with the HGF + VEGF/U-Ms. d Anti-CD31 (1:50) immunostaining showing the re-endothelialized region of the coronary arteries. N = 3

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  Fig. 7. No tumors were formed in nude mice transplanted with the HGF + VEGF/U-Ms. a Tumor formation was observed 4 weeks after cell transplantation in BALB/c nude mice. MDA-MB-231 cells were used as a positive control. Tumors did not form in the U-M or HGF + VEGF/U-M groups. b The tumor volume was measured weekly. c Mice were sacrificed and dissected 4 weeks after transplantation, and tumor formation was analyzed. N = 4