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Transgene Delivery to Endothelial Cultures Derived from Porcine Carotid Artery Ex Vivo

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Abstract

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 10 min post-mortem. Small sectors of carotid artery wall (approximately 4 mm × 4 mm 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 7–14 days 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.

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Abbreviations

EGFP:

Enhanced green fluorescent protein

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Acknowledgments

We are grateful to Mr RW Chang FRCS, Mr M Morsy FRCS and Professor G StJ Whitley for valuable advice and access to resources, and to the staff of the Biological Research Facility of St George’s for their expert assistance. We thank Dr AG Clifton FRCRadiol for the helpful comments throughout the project.

Funding

AHH gratefully acknowledges funding from The National Centre for the Replacement, Refinement and Reduction of Animals in Research (www.nc3rs.org.uk), the St George’s Hospital Charity and The Neuroscience Research Foundation. RJYM and AHH acknowledge funding from the South West London Academic Network.

Conflict of Interest

The authors have no conflict of interest.

Author information

Author notes

  1. M. Nortley

    Present address: Oxford Transplant Centre, Churchill Hospital, Old Road, Headington, Oxford, OX3 7LJ, UK

Authors and Affiliations

  1. Stroke and Dementia Research Centre, Division of Clinical Sciences, St Georges University of London, Cranmer Terrace, London, SW17 0RE, UK

    J. Andoh, B. Sawyer & A. H. Hainsworth

  2. Microscopy Imaging Unit, Division of Biomedical Sciences, St George’s University of London, London, UK

    K. Szewczyk

  3. Renal Transplantation Unit, St George’s Healthcare NHS Trust, London, UK

    M. Nortley

  4. School of Biological Sciences, Royal Holloway University of London, Egham, UK

    T. Rossetti & R. J. Yáñez-Muñoz

  5. St George’s Vascular Institute, St George’s Hospital, Blackshaw Road, London, UK

    I. M. Loftus

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  1. J. Andoh
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Correspondence to A. H. Hainsworth.

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Andoh, J., Sawyer, B., Szewczyk, K. et al. Transgene Delivery to Endothelial Cultures Derived from Porcine Carotid Artery Ex Vivo. Transl. Stroke Res. 4, 507–514 (2013). https://doi.org/10.1007/s12975-013-0261-6

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  • DOI: https://doi.org/10.1007/s12975-013-0261-6

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