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DNA delivery from an intravascular stent with a denatured collagen-polylactic-polyglycolic acid-controlled release coating: mechanisms of enhanced transfection

Gene Therapy volume 10, pages 1420–1428 (2003)Cite this article

Abstract

We previously demonstrated that DNA–polylactic–polyglycolic acid (PLGA)-coated stents can deliver genes to the arterial wall with reporter expression involving 1% of neointimal cells. The present study investigated a novel formulation utilizing denatured collagen in DNA-stent coatings; denatured collagen was hypothesized to enhance gene transfer due to adhesion molecule interactions and actin-related mechanisms. Arterial smooth muscle cells (SMCs) cultivated on denatured collagen had significantly greater plasmid DNA (β-galactosidase) transfection than SMC grown on native collagen (18.3±1.2 vs 1.0±0.1%, P<0.001). The denatured-collagen effect was completely blocked with anti-αvβ3 integrin antibody. SMCs cultivated on native collagen supplemented with tenascin-C (TN-C), a protein recognized by αvβ3 integrins, showed a 33-fold increase in transfection compared to control (P<0.001); this effect was also blocked with anti-αvβ3 antibody. We observed that cells grown on denatured collagen had marked F-actin-enriched stress fibers and intense perinuclear G actin, compared to those grown on native collagen, which demonstrated F-actin-enriched focal adhesions without perinuclear G-actin localization. Cytochalasin-D, an F actin depolymerizing agent, caused significantly increased SMC transfection in cells cultivated on native collagen compared to control cells (18.0±1.8 vs 3.02±0.9%, P<0.001) further supporting the view that actin-related cytoskeletal changes influence transfection. A denatured-collagen–PLGA composite vascular stent coating similarly resulted in increased plasmid DNA green fluorescent protein (GFP) expression compared to controls (P<0.001) in SMC cultures; the increased transfection was blocked by anti-αvβ3 antibody. Pig coronary studies comparing denatured-collagen–PLGA-coated stents containing plasmid DNA (encoding GFP) to coated stents without DNA demonstrated 10.8% of neointimal cells transfected; this level of expression was almost an order of magnitude greater than previously reported with a DNA delivery stent. It is concluded that denatured collagen incorporated into plasmid DNA-stent coating formulations may increase the level of gene expression in vitro and in vivo because of integrin-related mechanisms and associated changes in the arterial smooth muscle cell actin cytoskeleton.

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Acknowledgements

We thank Ms Jennifer LeBold for her assistance in preparing this manuscript. This research was supported by NIH Grant HL-72108, an American Heart Association Fellowship (IP), and the William J Rashkind Endowment of the Children's Hospital of Philadelphia.

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  1. I Perlstein and J M Connolly: The first two authors contributed equally

Authors and Affiliations

  1. Division of Cardiology, Children's Hospital of Philadelphia, Philadelphia, PA, USA

    I Perlstein, J M Connolly, X Cui, C Song, Q Li, Z Lu, S DeFelice & R J Levy

  2. Division of Cardiology, University of Pennsylvania Health System, Philadelphia, PA

    B Klugherz & R Wilensky

  3. Department of Pediatrics, University of Colorado Health Sciences Center, Section of Critical Care, Denver, CO, USA

    P L Jones

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  1. I Perlstein
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Perlstein, I., Connolly, J., Cui, X. et al. DNA delivery from an intravascular stent with a denatured collagen-polylactic-polyglycolic acid-controlled release coating: mechanisms of enhanced transfection. Gene Ther 10, 1420–1428 (2003). https://doi.org/10.1038/sj.gt.3302043

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