Cardioprotection afforded by inducible nitric oxide synthase gene therapy is mediated by cyclooxygenase-2 via a nuclear factor-κB-dependent pathway

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 whereby iNOS gene therapy upregulates COX-2 remains unknown. Accordingly, we used genetically engineered mice to test the hypothesis that the cardioprotection afforded by iNOS gene transfer is mediated by COX-2 upregulation via a nuclear factor (NF)-kappa B-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 in COX-2(-/-) and wild-type groups. WOS gene transfer (confirmed by iNOS immunoblotting and activity assays) markedly reduced infarct size in wild-type mice but failed to do so in COX-2(-/-) mice. In transgenic mice with cardiac-specific expression of a dominant-negative mutant Of I kappa B alpha (I kappa B alpha(S32A,S36A)), the upregulation of phosphorylated I kappa B alpha, activation of NF-kappa B, and cardiac COX-2 protein expression 3 days after WOS gene therapy were abrogated, which was associated with the abolishment of the cardioprotective effects afforded by WOS gene therapy. Conclusions - These data provide strong genetic evidence that COX-2 is an obligatory downstream effector of iNOS-dependent cardioprotection and that NF-kappa B is a critical link between iNOS and COX-2. Thus, iNOS imparts its protective effects, at least in part, by recruiting NF-kappa B, leading to COX-2 upregulation. However, COX-2 does not play an important cardioprotective role under basal conditions (when iNOS is not upregulated).