Inhibitory effects of mesenchymal stem cells in intimal hyperplasia after balloon angioplasty Ae-Kyeong Kim, PhD, Min-Hee Kim, Do-Hyung Kim, Ha-Nl Go,

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Inhibitory effects of mesenchymal stem cells in intimal hyperplasia after balloon angioplasty Ae-Kyeong Kim, PhD, Min-Hee Kim, Do-Hyung Kim, Ha-Nl Go, Seung-Woo Cho, PhD, Soong Ho Um, PhD, Dong-Ik Kim, MD, PhD Journal of Vascular Surgery Volume 63, Issue 2, Pages 510-517 (February 2016) DOI: 10.1016/j.jvs.2014.08.058 Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 1 Construction of carotid artery balloon injury model. A, The proximal portion of the carotid artery was punctured with a 16-gauge needle, and then a 2F Fogarty embolectomy catheter was inserted through the puncture site. B, The balloon was inflated for 1 minute at a pressure of 2 atm and then deflated. The inflation and deflation procedure was repeated three times. C, The puncture site was closed with a 7-0 polypropylene suture. D, The balloon-injured carotid artery was coated with a mixture of 7 × 106 human umbilical cord mesenchymal stem cells (HUC-MSCs) and fibrin matrix. Journal of Vascular Surgery 2016 63, 510-517DOI: (10.1016/j.jvs.2014.08.058) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 2 Neointima reduction efficacy of human umbilical cord mesenchymal stem cells (HUC-MSCs). A, Hematoxylin and eosin stain of the harvested carotid artery 2 weeks (four each in treated and nontreated groups), 4 weeks (four each in treated and nontreated groups), and 8 weeks (four each in treated and nontreated groups) after balloon injury (scale bar: 50 μm). B, The intima/media ratio was significantly reduced in the HUC-MSC-treated group relative to the nontreated group (Student t-tests, *P < .05). Journal of Vascular Surgery 2016 63, 510-517DOI: (10.1016/j.jvs.2014.08.058) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 3 Re-endothelialization efficacy of human umbilical cord mesenchymal stem cells (HUC-MSCs). A, Immunohistochemical staining with CD31+ antibody of the native carotid and 2 weeks (four each in treated and nontreated groups), 4 weeks (four each in treated and nontreated groups), and 8 weeks (four each in treated and nontreated groups) after balloon injury (scale bar: 50 μm). B, Percentage of circumferential area of re-endothelialization; 100% represents complete coverage of the lumen by re-endothelialization (Student t-tests, *P < .05). Twenty to 25 sections per harvested balloon-injured carotid artery were evaluated. The image acquisition was performed with ScanScope AT Turbo (Aperio Technologies, Vista, Calif) and ImageScope (version 11.2; Aperio Technologies). The results were analyzed with the Image-Pro Plus (Media Cybernetics, Rockville, Md) program. Journal of Vascular Surgery 2016 63, 510-517DOI: (10.1016/j.jvs.2014.08.058) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 4 Migration of human umbilical cord mesenchymal stem cells (HUC-MSCs). Immunohistochemical staining with monoclonal antihuman nuclear antibody of the harvested carotid artery 3 days, 1 week, 2 weeks, 4 weeks, and 8 weeks after balloon injury in the HUC-MSC-treated group (scale bar: 50 μm). L, Luminal surface of the carotid artery; red, antihuman nuclear antibody; blue (DAPI), nucleus. Journal of Vascular Surgery 2016 63, 510-517DOI: (10.1016/j.jvs.2014.08.058) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 5 Endogenous angiogenic gene expression analysis by quantitative real-time polymerase chain reaction (PCR) at 2 weeks (four each in treated and nontreated groups), 4 weeks (four each in treated and nontreated groups), and 8 weeks (four each in treated and nontreated groups) after balloon injury. There was a 9- to 43-fold increase in the human umbilical cord mesenchymal stem cell (HUC-MSC)-treated group relative to the nontreated group 4 weeks after balloon injury (analysis of variance, *P < .05). AAMP, Angio-associated, migratory cell protein; Ang-1, angiopoietin 1; KDR-1, kinase insert domain receptor 1; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor. Journal of Vascular Surgery 2016 63, 510-517DOI: (10.1016/j.jvs.2014.08.058) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 6 Macrophage distribution. A, Immunohistochemical staining with macrophage at 8 weeks after balloon injury (scale bar: 500 μm). B, The area of macrophages was checked at 2 weeks (four each in treated and nontreated groups), 4 weeks (four each in treated and nontreated groups), and 8 weeks (four each in treated and nontreated groups) after balloon injury. The area of macrophages was increased in the nontreated group relative to the human umbilical cord mesenchymal stem cell (HUC-MSC)-treated group (2 weeks, 31.57% ± 1.15% vs 13.02% ± 1.00%; 4 weeks, 59.11% ± 1.00% vs 11.91% ± 1.53%; 8 weeks, 63.54% ± 5.21% vs 10.99% ± 2.87%) (Student t-tests, *P < .05). Journal of Vascular Surgery 2016 63, 510-517DOI: (10.1016/j.jvs.2014.08.058) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 7 Schematic figure of migration of stem cell from outer surface into the vessel wall and differentiation into vascular smooth muscle cell (VSMC) and endothelial cell (EC). EC-MSC, Endothelial cell derived from mesenchymal stem cell; HUC-MSC, human umbilical cord mesenchymal stem cell. Journal of Vascular Surgery 2016 63, 510-517DOI: (10.1016/j.jvs.2014.08.058) Copyright © 2016 Society for Vascular Surgery Terms and Conditions