Selective delivery of dsRNA to resistance artery endothelial cells

White, K., McGrath, J.C. and Daly, C.J. (2008) Selective delivery of dsRNA to resistance artery endothelial cells. Journal of Vascular Research, 45(Supple), p. 7. (doi: 10.1159/000113929)

Abstract

Introduction: RNA interference (RNAi) has become a powerful tool for silencing gene products in both mammalian and non-mammalian cells. The cardiovascular system is an obvious route for in-vivo delivery and this has now been exploited to selectively target vital organs1. RNAi within the resistance vasculature has not been widely studied due to the problems of transfection, time course and analysis. In this study we have sought to identify the optimal protocol for delivery of dsRNA into the endothelium of isolated perfused segments of mouse mesenteric artery. Methods: 1st branch mesenteric arteries (ext. diameter, ~200 μm) were isolated from mature C57 black mice. Artery segments were mounted between 2 glass micro-pipettes in a pressure myograph (DMT) and equilibrated for 30 minutes at 40 mmHg in MOPS buffer at 37oC prior to experimentation. Liposomes containing Alexa Fluor-555 red fluorescent dsRNA were prepared according to the ‘Block-iTTM kit’ instructions. 2.5 ml of MOPS containing 60 μl of liposomes (0.12 μM dsRNA) was luminally perfused in a closed loop for 3-24 hours. Fluorescence (and thus cellular uptake of fluodsRNA) was examined, using confocal microscopy, at various time points. Results: Vessels were denuded of endothelial cells (ECs) as a consequence of perfusion times of 18-24 hours. However, this EC denudation did not facilitate smooth muscle cell transfection. Following 5 hours of perfusion at 40 mmHg (flow rate of 1.1–1.6 ml/min), fluo-dsRNA was associated with ECs (Fig. 1A). 3D analysis confirmed the presence of dsRNA in the EC nulclei (Fig. 1B). Conclusion: Our preliminary experiments have identified the optimum conditions required to transfect endothelial cells of isolated perfused mouse mesenteric resistance arteries. Pressure and flow are essential components for successful transfection. RNAi of specific endothelial targets (eg. eNOS, ADMA, PDEs etc.) is therefore viable, within a reasonable time frame, using the method described above. 1. Takabatake Y, Isaka Y, Mizui M, Kawachi H, Shimizu F, Ito T, Hori M, Imai E (2005) Gene Therapy 12:965-973

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: McGrath, Professor John and White, Dr Kevin and Daly, Professor Craig
• Authors: White, K., McGrath, J.C., and Daly, C.J.
• College/School: College of Medical Veterinary and Life Sciences > School of Life Sciences
• Journal Name: Journal of Vascular Research
• Journal Abbr.: J. Vasc. Res.
• Publisher: S. Karger AG
• ISSN: 1018-1172
• ISSN (Online): 1423-0135
• Published Online: 26 August 2009