Vascular Endothelial Growth Factor Receptor Upregulation in Response to Cell-Based Angiogenic Gene Therapy Terrence M. Yau, MD, MS, Guangming Li, MD,

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Vascular Endothelial Growth Factor Receptor Upregulation in Response to Cell-Based Angiogenic Gene Therapy Terrence M. Yau, MD, MS, Guangming Li, MD, Yaoguang Zhang, MD, Richard D. Weisel, MD, Donald A.G. Mickle, MD, Ren-Ke Li, MD PhD The Annals of Thoracic Surgery Volume 79, Issue 6, Pages 2056-2063 (June 2005) DOI: 10.1016/j.athoracsur.2004.10.066 Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 (A–D) Fetal liver kinase-1 (flk-1) messenger ribonucleic acid (mRNA) expression in rat hearts transplanted with heart cells (HC), vascular endothelial growth factor transfected HC (HC+), skeletal myoblasts (Sk), vascular endothelial growth factor transfected Sk (Sk+) or medium alone controls (Ctrl). Transplantation with HC and Sk induced greater flk-1 expression than Ctrl, and flk-1 mRNA levels in HC+ and Sk+ hearts were markedly elevated over those in HC and Sk. Flk-1 expression peaked at 1 week, earlier than flt-1. Flk-1 levels were greatest in the border zone, intermediate in the scar, and did not differ from control in the normal myocardium. (RNA = ribonucleic acid.) The Annals of Thoracic Surgery 2005 79, 2056-2063DOI: (10.1016/j.athoracsur.2004.10.066) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 (A–D) Fms-like tyrosine kinase-1 (flt-1) messenger ribonucleic acid (mRNA) expression in heart cells (HC), vascular endothelial growth factor transfected HC (HC+), skeletal myoblasts (Sk), vascular endothelial growth factor transfected Sk (Sk+), or control (Ctrl) rats. Transplantation with HC and Sk induced greater flt-1 expression than Ctrl, and flt-1 mRNA levels in HC+ and Sk+ hearts were markedly elevated over those in HC and Sk. Flt-1 expression peaked at 2 weeks, later than flk-1. Flt-1 levels were greatest in the border zone, intermediate in the scar, and did not differ from control in the normal myocardium. (RNA = ribonucleic acid.) The Annals of Thoracic Surgery 2005 79, 2056-2063DOI: (10.1016/j.athoracsur.2004.10.066) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Laser confocal micrographs of serial sections of the border zone of a rat heart transplanted with vascular endothelial growth factor (VEGF) transfected heart cells (×400). The scar is toward the left side of the photomicrograph and the normal myocardium is toward the right. (A) Bromodeoxyuridine (green) staining of transplanted cell nuclei showing transplanted cells predominantly near the scar. (B) Bromodeoxyuridine and 4′,6-Diamidino-2-phenylindole (blue) double staining, showing brighter bromodeoxyuridine-positive 4′,6-Diamidino-2-phenylindole-positive transplanted cells predominantly near the scar and dimmer bromodeoxyuridine-negative 4′,6-Diamidino-2-phenylindole-positive cells (presumably host cells) nearer to the normal myocardium. (C) Bromodeoxyuridine (green, nuclear) staining of transplanted cell nuclei and VEGF (green, cytoplasmic) staining, demonstrating that VEGF expression was predominantly localized to the transplanted cells. (D) 4′,6-Diamidino-2-phenylindole (blue) staining of all cell nuclei, VEGF (green) staining and flk-1 (red) staining, demonstrating that flk-1 expression was predominantly localized to the host cells, in contrast to VEGF expression. The Annals of Thoracic Surgery 2005 79, 2056-2063DOI: (10.1016/j.athoracsur.2004.10.066) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions