Investigations into eNOS and Phosphomimetic eNOS Gene Delivery to the Vasculature
Sean O Hynes, Sandra Ganly, Faisal Sharif, Leslie Smith, Karl McCullagh, Udo Greiser, Zvonimir Katusic, Timothy O'Brien. University Hospital Galway, Galway, Ireland; National University of Ireland, Galway, Galway, Ireland; Mayo Clinic, Rochester, MN
Background: Endovascular procedures including stenting, denudes endothelium. Removal of this layer, decreases the levels of active endoethelial nitric oxide synthase(eNOS) and bioavailability of NO. Gene delivery of eNOS or an engineered phosphomimetic constitutively active eNOS may prove of benefit to the vasculature.
Design: Two principles are critical to improving outcomes from endovascular procedures. First, to accelerate re-ednothelialisation and second, to prevent neointimal proliferation. We have examined gene therapy approaches to produce beneficial effects in the vasculature. We compared eNOS and phosphomimetic eNOS for their effects on vasomotor activity. Thereafter, we examined the usefulness of eNOS delivery from a stent platform in preventing in-stent restenosis and promoting re-endothelialisation.
Two rabbit models of vascular injury were used. The first was a physiological model where we overexpressed our two candidate transgenes. The second was an injury model where a stent carrying the lead gene was delivered following inflation/deflation injury of the external iliac artery.
Results: Overexpression of phosphomimetic and wild type eNOS both improved vasomotor activity in a rabbit carotid artery model. However, there was no difference between either enzyme. Therefore, wild type eNOS was examined for its beneficial effects following delivery from a stent platform. We then compared viral (adenovirus) versus non-viral (liposome) delivery of wild type eNOS, head-to-head. Both vectors resulted in improved re-endothelialisation, however, only the use of viral vectors decreased neointimal formation.
Conclusions: Our study found that overexpression of an engineered constitutively active form of eNOS is not superior to wild type delivery. This may be due to suboptimal mimicking of pophosphorylation. Interestingly, the vector is critical for delivery of eNOS. When compared head-to-head only the viral vector improved both neointimal and re-endothelialisation results. We speculate that this may be due to the expression of non-viral vectors by macrophages which also express inducible nitric oxide synthase. The direct competition for substrates may be the reason for less effective eNOS function. Whereas, eNOS delivered by virus is expressed in smooth muscle cells and these have no native NOS to compete with the transgene.
Wednesday, March 21, 2012 1:00 PM
Poster Session VI # 45, Wednesday Afternoon