Increased sensitivity to eNOS inhibition in vessels from cyclo-oxygeanse-(COX)-2 knock-out mice associates with elevated plasma levels of ADMA and L-NMMA

Blerina Ahmetaj-Shala1, Malak Al-Yamani1,3, Nicholas Kirkby1, James Leiper2, Jane A. Mitchell1. 1Imperial College London, London, UK, 2MRC Clinical Sciences Centre, London, UK, 3King Fahad Academy, Riyadh, Saudi Arabia

Cyclo-oxygenase (COX)-2 selective drugs such as celecoxib are used to treat inflammation and pain and are associated with increased risk of myocardial infarctions and strokes. Since COX-1, but not COX-2, regulates vascular prostacyclin production, the mechanisms associated with COX-2 inhibitors and cardiovascular events remain unknown but may involve the renal medulla where COX-2 is highly expressed1. We have previously shown that the renal medulla of COX-2 knock-out (COX-2-/-) mice has increased mRNA expression of the enzymes protein arginine methyltransferase (PRMT)-1 which synthesises the nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA) and L-NGmono-methylarginine (L-NMMA) and reduced dimethylarginine dimethylaminohydrolase (DDAH) and alanine:glyoxylate aminotransferase 2 (AGXT2) which metabolise the NOS inhibitors. In accordance with this, COX-2-/- mice had elevated levels of circulating ADMA and L-NMMA2, which are associated with renal dysfunction and cardiovascular disease3. Thus, the aim of this study was to investigate whether global loss of COX-2 in COX-2-/- mice is associated with altered eNOS sensitivity of vessels to exogenous ADMA and L-NMMA in vitro.

Using wire myography, we investigated the effects of increasing concentrations of ADMA (1-100µM) and L-NMMA (1-100µM) on the contractile responses of aorta extracted from COX-2-/- mice derived from C57/Bl6 and their wild-type controls. Male and female mice aged 8-12 weeks were used and killed with CO2 followed by cervical dislocation. Data are given as mean±SEM (n animals) and analysis was performed using two-way ANOVA. COX-2-/- aorta had elevated contractile responses to ADMA (Fig. 1a, n=4) and L-NMMA (Fig. 1b, n=4) compared to wild type mice.

In summary, COX-2-/- vessels have increased sensitivity to eNOS inhibition, this together with our earlier findings that ADMA and LNMMA are elevated in COX-2-/-, adds weight to the idea that cardiovascular side-effects associated with COX-2 inhibitors could be mediated by effects on circulating methyl arginine levels and consequent reduced eNOS activity. These novel findings identify ADMA as a potential viable biomarker, and mediate of cardiovascular side effects associated with use of anti-inflammatory drugs.

Fig. 1: Contractile responses of wild-type and COX-2^-/- aorta in the presence of (a)

1. N. S. Kirkby et al. PLoSOne. 8 (7). 2013

2. B Ahmetaj-Shala et al. Circulation. 128 (22). 2013

3. R. H. Boger et al. Cardiovasc Res. 59 (4). 824-33. 2003