naa a22 4500 510776205 CHVBK 20180411083231.0 cr unu---uuuuu 180411e20131001xx s 000 0 eng 10.1007/s12975-013-0261-6 doi (NATIONALLICENCE)springer-10.1007/s12975-013-0261-6 Transgene Delivery to Endothelial Cultures Derived from Porcine Carotid Artery Ex Vivo [Elektronische Daten] [J. Andoh, B. Sawyer, K. Szewczyk, M. Nortley, T. Rossetti, I. Loftus, R. Yáñez-Muñoz, A. Hainsworth] Carotid artery disease is a widespread cause of morbidity and mortality. Porcine models of vascular disease are well established in vivo, but existing endothelial systems in vitro (e.g. human umbilical vein endothelial cells, rat aortic endothelial cultures) poorly reflect carotid endothelium. A reliable in vitro assay would improve design of in vivo experiments and allow reduction and refinement of animal use. This study aimed (1) to develop ex vivo endothelial cultures from porcine carotid and (2) to test whether these were suitable for lentivector-mediated transgene delivery. Surplus carotid arteries were harvested from young adult female Large White pigs within 10min post-mortem. Small sectors of carotid artery wall (approximately 4mm × 4mm squares) were immobilised in a stable gel matrix. Cultures were exposed to HIV-derived lentivector (LV) encoding a reporter transgene or the equivalent integration-deficient vector (IDLV). After 7-14days in vitro, cultures were fixed and labelled histochemically. Thread-like multicellular outgrowths were observed that were positive for endothelial cell markers (CD31, VEGFR2, von Willebrand factor). A minority of cells co-labelled for smooth muscle markers. Sensitivity to cytotoxic agents (paclitaxel, cycloheximide, staurosporine) was comparable to that in cell cultures, indicating that the gel matrix permits diffusive access of small pharmacological molecules. Transgene-expressing cells were more abundant following exposure to LV than IDLV (4.7, 0.1% of cells, respectively). In conclusion, ex vivo adult porcine carotid artery produced endothelial cell outgrowths that were effectively transduced by LV. This system will facilitate translation of novel therapies to clinical trials, with reduction and refinement of in vivo experiments. Springer Science+Business Media New York, 2013 Endothelial cells nationallicence Carotid artery nationallicence Lentiviral vector nationallicence Porcine model nationallicence EGFP : Enhanced green fluorescent protein nationallicence Andoh J. Stroke and Dementia Research Centre, Division of Clinical Sciences, St Georges University of London, Cranmer Terrace, SW17 0RE, London, UK aut Sawyer B. Stroke and Dementia Research Centre, Division of Clinical Sciences, St Georges University of London, Cranmer Terrace, SW17 0RE, London, UK aut Szewczyk K. Microscopy Imaging Unit, Division of Biomedical Sciences, St George's University of London, London, UK aut Nortley M. Renal Transplantation Unit, St George's Healthcare NHS Trust, London, UK aut Rossetti T. School of Biological Sciences, Royal Holloway University of London, Egham, UK aut Loftus I. St George's Vascular Institute, St George's Hospital, Blackshaw Road, London, UK aut Yáñez-Muñoz R. School of Biological Sciences, Royal Holloway University of London, Egham, UK aut Hainsworth A. Stroke and Dementia Research Centre, Division of Clinical Sciences, St Georges University of London, Cranmer Terrace, SW17 0RE, London, UK aut Translational Stroke Research Springer US; http://www.springer-ny.com 4/5(2013-10-01), 507-514 1868-4483 4:5<507 2013 4 12975 https://doi.org/10.1007/s12975-013-0261-6 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s12975-013-0261-6 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Andoh J. Stroke and Dementia Research Centre, Division of Clinical Sciences, St Georges University of London, Cranmer Terrace, SW17 0RE, London, UK aut NATIONALLICENCE 700 1- Sawyer B. Stroke and Dementia Research Centre, Division of Clinical Sciences, St Georges University of London, Cranmer Terrace, SW17 0RE, London, UK aut NATIONALLICENCE 700 1- Szewczyk K. Microscopy Imaging Unit, Division of Biomedical Sciences, St George's University of London, London, UK aut NATIONALLICENCE 700 1- Nortley M. Renal Transplantation Unit, St George's Healthcare NHS Trust, London, UK aut NATIONALLICENCE 700 1- Rossetti T. School of Biological Sciences, Royal Holloway University of London, Egham, UK aut NATIONALLICENCE 700 1- Loftus I. St George's Vascular Institute, St George's Hospital, Blackshaw Road, London, UK aut NATIONALLICENCE 700 1- Yáñez-Muñoz R. School of Biological Sciences, Royal Holloway University of London, Egham, UK aut NATIONALLICENCE 700 1- Hainsworth A. Stroke and Dementia Research Centre, Division of Clinical Sciences, St Georges University of London, Cranmer Terrace, SW17 0RE, London, UK aut NATIONALLICENCE 773 0- Translational Stroke Research Springer US; http://www.springer-ny.com 4/5(2013-10-01), 507-514 1868-4483 4:5<507 2013 4 12975 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer