Evidence that links loss of cyclooxygenase-2 with increased asymmetric dimethylarginine: novel explanation of cardiovascular side effects associated with anti-inflammatory drugs

Abstract

Background: Cardiovascular side effects associated with cyclooxygenase-2 inhibitor drugs dominate clinical concern. Cyclooxygenase-2 is expressed in the renal medulla where inhibition causes fluid retention and increased blood pressure. However, the mechanisms linking cyclooxygenase-2 inhibition and cardiovascular events are unknown and no biomarkers have been identified.

Methods and results: Transcriptome analysis of wild-type and cyclooxygenase-2(-/-) mouse tissues revealed 1 gene altered in the heart and aorta, but >1000 genes altered in the renal medulla, including those regulating the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and monomethyl-l-arginine. Cyclo-oxygenase-2(-/-) mice had increased plasma levels of ADMA and monomethyl-l-arginine and reduced endothelial nitric oxide responses. These genes and methylarginines were not similarly altered in mice lacking prostacyclin receptors. Wild-type mice or human volunteers taking cyclooxygenase-2 inhibitors also showed increased plasma ADMA. Endothelial nitric oxide is cardio-protective, reducing thrombosis and atherosclerosis. Consequently, increased ADMA is associated with cardiovascular disease. Thus, our study identifies ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction with nonsteroidal anti-inflammatory drug usage.

Conclusions: We identify the endogenous endothelial nitric oxide synthase inhibitor ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction.

Keywords: endothelium; kidney; nitric oxide; pharmacology; prostaglandins.

Figure 1.

Renal COX-2 expression and effects on renal function. A, Expression of COX-2 in the aorta, renal cortex, and renal medulla of wild-type mice. Changes in levels of serum urea (B) and serum creatinine (C) in wild-type, COX-1^−/−, and COX-2^−/− mice. Data are mean±SEM for n=8 to 14 mice in each group. P values by Kruskal–Wallis with the Dunn post hoc test. COX indicates cyclooxygenase; and SEM, standard error of the mean.

Figure 2.

Transcriptome profiling in cardiovascular tissues from COX-2^−/− mice. A, Number of genes altered in the transcriptome of blood, heart, aorta, and kidney (renal medulla) by >1.2- to >3-fold. B, Focused pathway analysis of genes altered >1.3-fold (q<0.05). qPCR validation of changes in expression of Prmt1 (C), Agxt2 (D), and Ddah1 (E) in the renal medulla of COX-2^−/− mice. Data are from n=7 to 8 mice. Data in C through E are mean±SEM. P values by Mann–Whitney U test. COX indicates cyclooxygenase; qPCR, quantitative polymerase chain reaction; and SEM, standard error of the mean.

Figure 3.

Effect of COX gene deletion ADMA and l-NMMA in plasma. Levels of ADMA (A) and l-NMMA (B) were increased in the plasma of COX-2^−/− mice, but not in COX-1^−/− mice. Data are from n=4 and presented as mean±SEM. P values by Kruskal–Wallis with the Dunn post hoc test. ADMA indicates asymmetrical dimethylarginine; COX, cyclooxygenase; l-NMMA, monomethyl-l-arginine; and SEM, standard error of the mean.

Figure 4.

Effect of parecoxib on blood pressure and methylarginines. Effect of parecoxib on mean arterial blood pressure (A) and plasma ADMA (B) and l-NMMA (C) levels in mice. Data are mean±SEM for n=3 to 7. Data in A were analyzed by Quades 2-way ANOVA. Data in B and C were analyzed by the Mann–Whitney U test. ADMA indicates asymmetrical dimethylarginine; ANOVA, analysis of variance; l-NMMA, monomethyl-l-arginine; and SEM, standard error of the mean.

Figure 5.

Effect of COX-2 gene deletion of eNOS responses in aorta. Acetylcholine (ACh; A) or sodium nitroprusside (SNP; B) induced the relaxation of aorta from wild-type and COX-2^−/− mice. Maximum relaxation responses of the aorta from wild-type and COX-2^−/− mice induced by Ach (C) or SNP (D) in the absence or presence of l-arginine (100 μmol/L). Data are the mean±SEM for n=6 to 10. P values for A and C were by Quades 2-way ANOVA and for B and D were by Mann–Whitney U test. ANOVA indicates analysis of variance; COX, cyclooxygenase; and eNOS, endothelial nitric oxide synthase.