Cardioprotection Afforded by Inducible Nitric Oxide Synthase Gene Therapy Is Mediated by Cyclooxygenase-2 via a Nuclear Factor-κB–Dependent Pathway

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Abstract
Background—Gene therapy with inducible nitric oxide synthase (iNOS) markedly reduces myocardial infarct size; this effect is associated with cyclooxygenase-2 (COX-2) upregulation and is ablated by COX-2 inhibitors. However, pharmacological inhibitors are limited by relative lack of specificity; furthermore, the mechanism wherebyiNOSgene therapy upregulates COX-2 remains unknown. Accordingly, we used genetically engineered mice to test the hypothesis that the cardioprotection afforded byiNOSgene transfer is mediated by COX-2 upregulation via a nuclear factor (NF)-κB–dependent pathway.Methods and Results—Mice received an intramyocardial injection of Av3/LacZ (LacZ group) or Av3/iNOS (iNOS group); 3 days later, myocardial infarction was produced by a 30-minute coronary occlusion followed by 4 hours of reperfusion. Among Av3/LacZ-treated mice, infarct size was similar inCOX-2−/−and wild-type groups.iNOSgene transfer (confirmed by iNOS immunoblotting and activity assays) markedly reduced infarct size in wild-type mice but failed to do so inCOX-2−/−mice. In transgenic mice with cardiac-specific expression of a dominant-negative mutant of IκBα (IκBαS32A,S36A), the upregulation of phosphorylated IκBα, activation of NF-κB, and cardiac COX-2 protein expression 3 days afteriNOSgene therapy were abrogated, which was associated with the abolishment of the cardioprotective effects afforded byiNOSgene therapy.Conclusions—These data provide strong genetic evidence that COX-2 is an obligatory downstream effector of iNOS-dependent cardioprotection and that NF-κB is a critical link between iNOS and COX-2. Thus, iNOS imparts its protective effects, at least in part, by recruiting NF-κB, leading to COX-2 upregulation. However, COX-2 does not play an important cardioprotective role under basal conditions (when iNOS is not upregulated).