Published Online
on February 11, 2002

A more recent version of this article appeared on March 5, 2002

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Submitted on November 19, 2001
Revised on December 21, 2001
Accepted on December 21, 2001

Vascular Gene Transfer of Phosphomimetic Endothelial Nitric Oxide Synthase (S1177D) Using Ultrasound-Enhanced Destruction of Plasmid-Loaded Microbubbles Improves Vasoreactivity

Claudius Teupe MD, Sergio Richter , Beate Fisslthaler PhD, Voahanginirina Randriamboavonjy PhD, Christian Ihling MD, Ingrid Fleming PhD, Rudi Busse MD, PhD, Andreas M. Zeiher MD, and Stefanie Dimmeler PhD^*

From Molecular Cardiology, Department of Medicine IV, Institut für Kardiovaskuläre Physiologie (B.F., V.R., I.F., R.B., S.D.), University of Frankfurt, and the Department of Pathology (C.I.), University of Freiburg, Germany.

^* To whom correspondence should be addressed. E-mail: dimmeler{at}em.uni-frankfurt.de.

Background—Local gene therapy has enormous potential for the treatment of vascular disease. We determined whether diagnostic ultrasound-mediated destruction of plasmid-loaded albumin microbubbles is a feasible and efficient technique for local vascular gene delivery. For gene transfer, we used a phosphomimetic, active endothelial nitric oxide synthase (eNOS) construct in which Ser1177 was replaced by aspartic acid (S1177D) and exhibits a 2-fold higher basal activity than the wild-type enzyme.

Methods and Results—Gas-filled microbubbles (3.0±1.2 µm) were created by sonication of 5% human albumin in the presence of plasmid DNA encoding for LacZ or eNOS S1177D. Porcine coronary arteries were perfused with DNA-loaded albumin microbubbles in vitro, exposed to diagnostic ultrasound (5 seconds), and incubated for a further 24 hours. Detection of the ß-galactosidase in LacZ-transfected vessels revealed a predominant staining of endothelial cells without any functional impairment of vasoreactivity. Western blotting demonstrated the expression of the eNOS S1177D construct in extracts from the transfected segments. Vascular responsiveness was tested with prostaglandin F₂ and the NOS inhibitor Nnitro-L-arginine. Compared with segments treated with the expression plasmid alone, the contractile response to prostaglandin F₂ was impaired in segments transfected with eNOS S1177D, whereas the contractile response to the administration of Nnitro-L-arginine was markedly enhanced.

Conclusions—Ultrasound-mediated destruction of eNOS S1177D DNA-loaded albumin microbubbles is a feasible and efficient method for vascular gene transfection. Transfection resulted in significant protein expression and enhanced NO-mediated relaxation of bradykinin-stimulated porcine coronary arteries.

Key words: endothelium • echocardiography • gene therapy • nitric oxide synthase

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