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PRECLINICAL STUDY

Endothelial Nitric Oxide Synthase Overexpression Provides a Functionally Relevant Angiogenic Switch in Hibernating Pig Myocardium

Christian Kupatt, MD^_*,_*, Rabea Hinkel, DVM^_*, Marie-Luise von Brühl, DVM^_*, Tilmann Pohl, MD^_*, Jan Horstkotte, MD^_*, Philip Raake, MD^_*, Chiraz El Aouni, PhD^_*, Eckehard Thein, DVM, Stefanie Dimmeler, PhD, Olivier Feron, PhD and Peter Boekstegers, MD^_*

^_* Internal Medicine I, Klinikum Grosshadern
Institute of Surgical Research, Ludwig-Maximilians-University of Munich, Munich, Germany
Molecular Cardiology, Department of Medicine IV, University of Frankfurt, Frankfurt, Germany
Unit of Experimental Pharmacology, Catholique University of Leuven, Brussels, Belgium

Manuscript received June 7, 2006; revised manuscript received October 19, 2006, accepted November 27, 2006.

^_* Reprint requests and correspondence: Dr. Christian Kupatt, Internal Medicine I, Klinikum Großhadern, Marchioninistr. 15, 81377 Munich, Germany. (Email: christian.kupatt{at}med.uni-muenchen.de).

Objectives: We investigated whether retroinfusion of liposomal endothelial nitric oxide synthase (eNOS) S1177D complementary deoxyribonucleic acid (cDNA) would affect neovascularization and function of the ischemic myocardium.

Background: Recently, we demonstrated the feasibility of liposomal eNOS cDNA transfection via retroinfusion in a model of acute myocardial ischemia/reperfusion. In the present study, we used this approach to target a phosphomimetic eNOS construct (eNOS S1177D) into chronic ischemic myocardium in a pig model of hibernation.

Methods: Pigs (n = 6/group) were subjected to percutaneous implantation of a reduction stent graft into the left anterior descending artery (LAD), inducing total occlusion within 28 days. At day 28, retroinfusion of saline solution containing liposomal green fluorescent protein or eNOS S1177D cDNA (1.5 mg/animal, 2 x 10 min) was performed. Furthermore, L-nitroarginine-methylester (L-NAME) was applied orally from day 28, where indicated. At day 28 and day 49, fluorescent microspheres were injected into the left atrium for perfusion analysis. Regional functional reserve (at atrial pacing 140/min) was assessed at day 49 by subendocardial segment shortening (SES) (sonomicrometry, percent of ramus circumflexus region).

Results: The eNOS S1177D overexpression increased endothelial cell proliferation as well as capillary and collateral growth at day 49. Concomitantly, eNOS S1177D overexpression enhanced regional myocardial perfusion from 62 ± 4% (control) to 77 ± 3% of circumflex coronary artery–perfused myocardium, unless L-NAME was co-applied (69 ± 5%). Similarly, eNOS S1177D cDNA improved functional reserve of the LAD (33 ± 5% vs. 7 ± 3% of circumflex coronary artery–perfused myocardium), except for L-NAME coapplication (13 ± 6%).

Conclusions: Retroinfusion of eNOS S1177D cDNA induces neovascularization via endothelial cell proliferation and collateral growth. The resulting gain of perfusion enables an improved functional reserve of the hibernating myocardium.

Abbreviations and Acronyms   ANOVA = analysis of variance   cDNA = complementary deoxyribonucleic acid   cGMP = cyclic guanosine monophosphate   eGFP = endothelial green fluorescent protein   eNOS = endothelial nitric oxide synthase   IGF = insulin-like growth factor   LAD = left anterior descending artery   L-NAME = L-nitroarginine-methylester   RCx = ramus circumflexus   SES = subendocardial segment shortening   VEGF = vascular endothelial growth factor

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