Dimethylarginine dimethylaminohydrolase I (DDAH I) heterozygous knockout mice display a cardiovascular phenotype consisting of pulmonary and systemic endothelial dysfunction

M Malaki, M Nandi, M Madhani, H Gill, C Smith, J Leiper, P Vallance (P Vallance). Centre for Clinical Pharmacology and Therapeutics, BHF Laboratories, University College London.

Asymmetric dimethylarginine (ADMA) is a naturally occurring endogenous inhibitor of all three isoforms of nitric oxide synthase. Increased levels of ADMA have been associated with conditions such as hypercholesterolaemia, diabetes and hypertension, where there is evidence of endothelial dysfunction. ADMA is metabolised by the enzyme, dimethylarginine dimethylaminohydrolase (DDAH). Two DDAH isoforms are present in mammals (DDAH I and II). Inhibition of DDAH leads to increased ADMA levels and endothelial dysfunction. Reduced expression of DDAH and /or elevated ADMA levels have been reported in human and animal models of pulmonary hypertension ( Arrigoni et al, 2003; Millat et al, 2003; Pullamsetti et al, 2005).

We created a heterozygous knockout DDAH I+/- on a C57Black/SV129 background. Mice of either sex (25g to 35g) were used for the studies. Concentration of ADMA and DDAH expression were determined by high performance liquid chromatography and western blotting, respectively. Vascular function was studied in vitro using standard organ bath pharmacology, and in vivo by measuring systemic and right ventricular pressures. Structural differences in pulmonary vessels were assessed histologically.

The DDAH I+/- had reduced DDAH I protein expression in all tissues examined, and had increased circulating (0.69 ± 0.02uM vs.0.87 ± 0.06uM, p<0.05, n=12 per group) and tissue ADMA levels (2.87 ± 0.24uM vs 3.68 ± 0.31uM, p<0.05, n=12 per group ) compared to their wild type littermates. Vascular studies revealed an impairment of endothelium dependent (ACh) relaxations both in the aorta and pulmonary arteries of DDAH+/- compared to wild types ( aorta,p<0.05, 2- way ANOVA, n=5; pulmonary, p<0.05, 2-way ANOVA, n=7). In vivo there were no significant differences in systemic blood pressures, but the DDAH I+/- mice had higher right ventricular pressures (16.79 ± 0.49 mm Hg vs 14.97 ± 0.62 mm Hg, p<0.001, n=7). Examination of the pulmonary vasculature revealed increased medial smooth muscle area of resistance vessels in DDAH+/- mice consistent with the raised RV pressures.

These studies establish DDAH I as a key determinant of ADMA levels in vivo and demonstrate a casual relationship between ADMA and cardiovascular pathology.

Arrigoni FI et al. (2003 ) Circulation: 107:1195-1201.
Millat LJ et al. (2003 ) Circulation: 108:1493-1498.
Pullamsetti S et al. (2005) FASEB J (Epub).