Effect of microRNA-145 to prevent vein graft disease in rabbits by regulation of smooth muscle cell phenotype Motoaki Ohnaka, MD, Akira Marui, MD, PhD,

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Effect of microRNA-145 to prevent vein graft disease in rabbits by regulation of smooth muscle cell phenotype Motoaki Ohnaka, MD, Akira Marui, MD, PhD, Kenichi Yamahara, MD, PhD, Kenji Minakata, MD, PhD, Kazuhiro Yamazaki, MD, PhD, Motoyuki Kumagai, MD, Hidetoshi Masumoto, MD, PhD, Shiro Tanaka, PhD, Tadashi Ikeda, MD, PhD, Ryuzo Sakata, MD, PhD The Journal of Thoracic and Cardiovascular Surgery Volume 148, Issue 2, Pages 676-682.e2 (August 2014) DOI: 10.1016/j.jtcvs.2013.11.054 Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 A, Schematic illustration of the electroporation device and representative photographs of green fluorescent protein immunostaining 3 days after implantation. B, Control and C, microRNA-145 (scale bar, 100 μm). Green fluorescent protein–positive cells were detected in the media of the microRNA-145–transduced grafts. The Journal of Thoracic and Cardiovascular Surgery 2014 148, 676-682.e2DOI: (10.1016/j.jtcvs.2013.11.054) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 A, Representative photographs of vein grafts 4 weeks after implantation. Elastica van Gieson staining. Arrowheads indicate the internal elastic lamina (scale bar, 100 μm). B, Cross-sectional intima/media area ratio 4 weeks after implantation. C, Intimal thickness 2 and 4 weeks after implantation (*P < .05). Neointimal lesion formation was significantly suppressed in the miR-145–transduced group 2 and 4 weeks after implantation. miR-145, microRNA-145. The Journal of Thoracic and Cardiovascular Surgery 2014 148, 676-682.e2DOI: (10.1016/j.jtcvs.2013.11.054) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Smooth muscle (SM)-1 and SM-2 immunostaining of the vein grafts 2 weeks after implantation. Arrowheads indicate internal elastic lamina (scale bar, 100 μm). Staining for SM-2, a mature smooth muscle cell marker, revealed that the SM-2–positive area was barely detectable in the neointima of the control grafts, but SM-2 was positive in the reduced neointimal lesion of the microRNA-145–transduced grafts. The Journal of Thoracic and Cardiovascular Surgery 2014 148, 676-682.e2DOI: (10.1016/j.jtcvs.2013.11.054) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Ki-67 immunostaining of the vein grafts 2 weeks after implantation. A, Control. B, microRNA-145. White arrowheads indicate Ki-67–positive cells; dashed line, internal elastic lamina (scale bar, 50 μm). The frequency of Ki-67–positive cells was much greater in the control grafts, suggesting more proliferative cells were present in the control group. The Journal of Thoracic and Cardiovascular Surgery 2014 148, 676-682.e2DOI: (10.1016/j.jtcvs.2013.11.054) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 A, Serum response factor and B, myocardin mRNA expression in explanted grafts were evaluated by quantitative reverse transcriptase-polymerase chain reaction 3 days postoperatively. The mRNA expression level of myocardin was significantly upregulated in the microRNA-145–transduced grafts (2.03 ± 0.99 vs 0.90 ± 0.46, P < .001, n = 5). The Journal of Thoracic and Cardiovascular Surgery 2014 148, 676-682.e2DOI: (10.1016/j.jtcvs.2013.11.054) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Appendix Figure 1 Schema of the construct of the microRNA-145 (miRNA-145) expression vector, which included microRNA-145 and a green fluorescent protein expression site. The Journal of Thoracic and Cardiovascular Surgery 2014 148, 676-682.e2DOI: (10.1016/j.jtcvs.2013.11.054) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Appendix Figure 2 Left, DsRed fluorescent image and Right, DsRed immunostaining 2 days after electroporation (original magnification ×200). Lu, Lumen side; Ad, adventitia side. Apparent DsRed expression in the lumen side was observed, indicating successful gene transduction after adopting the electric parameters of sample 4 (Appendix Table 1). The Journal of Thoracic and Cardiovascular Surgery 2014 148, 676-682.e2DOI: (10.1016/j.jtcvs.2013.11.054) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Appendix Figure 3 Representative photographs of degenerative vasculature in the preliminary in vivo study using a microRNA-145 expression vector. Electric parameters are described in Appendix Table 2. The vein grafts were explanted 2 weeks after implantation (hematoxylin and eosin staining). Lu, Lumen side; Ad, adventitia side. A, Black arrowheads indicate the vacuolar degeneration in the vasculature (original magnification ×100). B, Between white arrowheads, the disappearance of nuclei was observed in almost all layers of the vasculature (original magnification ×200). The Journal of Thoracic and Cardiovascular Surgery 2014 148, 676-682.e2DOI: (10.1016/j.jtcvs.2013.11.054) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions