“Nitric oxide synthesis is regulated by endogenously produced inhibitors”

Asymmetric dimethylarginine (ADMA) is an independent risk factor for a range of cardiovascular disease states. In chronic renal failure ADMA is second only to age in predicting mortality. In 1986 ADMA was identified as a competitive inhibitor of nitric oxide (NO) synthesis and subsequently we have elucidated the physiological and pathophysiological effects of ADMA in a number of experimental models of cardiovascular disease.

We identified a family of hydrolase enzymes (dimethylarginine dimethylaminohydrolases, DDAH) that metabolise ADMA and are key regulators of ADMA levels in experimental animals and human. In order to elucidate the mechanisms of action of ADMA we have undertaken both pharmacological and genetic approaches. Using cells isolated from genetically modified experimental animals we have determined the effect of ADMA on cellular motility.

Nitric Oxide Signalling

The Regulation of Nitric Oxide synthesis by Methylarginines. L-arginine is the substrate for nitric oxide synthase (NOS) enzymes. Arginine residues in proteins can be methylated by protein arginine methyl transferases. Following proteolysis of arginine-methylated proteins, methylarginines (ADMA and L-NMMA) accumulate in the cytosol where inhibit NOS activity by competing with arginine at the NOS active site. Inhibitory methylarginines are metabolized by the action of dimethylarginine dimethylaminohydrolase (DDAH).

We have also identified novel function of ADMA in the regulation of cardiomyocyte function by studying cardiomyocytes derived from embryonic stem cells or isolated from genetically modified animals.

We have demonstrated that genetic deletion of DDAH inhibits blood vessel growth in intact animals and impairs vessel function leading to hypertension. In order to explore the therapeutic potential of endogenously produced ADMA we have solved the crystal structure of DDAH and synthesized a novel panel of selective DDAH inhibitors. In vivo these molecules increase survival in experimental models of septic shock.

In order to translate these basic research findings into a deeper understanding of human physiology and pathophysiology we identified functional genetic variants in the human homologues of the genes that we have demonstrated to be responsible for ADMA metabolism in experimental animals. Using these genetic tools we have demonstrated conservation of ADMA metabolizing enzymes in humans and shown that polymorphisms in the human genes increase circulating ADMA levels, elevate blood pressure and effect the rate of renal decline in patients with chronic kidney disease.

Our research programme now aims to build upon our preeminent understanding of the molecular mechanisms that regulate ADMA concentrations, and the established and emerging mechanisms of action of this molecule, to identify novel therapeutic approached for the treatment of a range of cardio-metabolic disorders.

Elevated levels of these inhibitors (ADMA) have been reported in numerous diseases including hypertension, heart failure, renal failure, atherosclerosis, pre-eclampsia and type 2 diabetes. The NO synthase inhibitors are themselves degraded by DDAH (dimethylarginine dimethylaminohydrolase) enzymes. We have cloned these and characterised their distribution and activity in human tissues. From our findings, we propose that regulating the metabolism of endogenous nitric oxide synthase inhibitors significantly regulates NO generation in vivo. We solved the crystal structure of DDAH and then used this information to design novel selective enzyme inhibitors to use as pharmacological tools to probe the physiological role of the enzyme. Recently, we solved the crystal structure of human DDAH bound to such an inhibitor. We have also created a number of mouse lines that conditionally delete or overexpress DDAH genes. Focusing on the cardiovascular system, we have demonstrated that inhibition of DDAH results in impaired vascular NO signalling leading to endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. We plan to further our analysis of DDAH/ADMA in the cardiovascular system, and study its function in additional organ systems. We will also examine non-NO dependent effects of ADMA and non-enzymatic functions of DDAH.

Selected Publications

Ahmetaj-Shala B, Kirkby NS, Knowles R, Al’Yamani M, Mazi S, Wang Z, Tucker AT, Mackenzie L, Armstrong PC, Nüsing RM, Tomlinson JA, Warner TD, Leiper J, Mitchell JA (2015). Evidence that links loss of cyclooxygenase-2 with increased asymmetric dimethylarginine: novel explanation of cardiovascular side effects associated with anti-inflammatory drugs. Circulation, 131(7):633-42. doi: 10.1161/CIRCULATIONAHA.114.011591.

Iannone, L., Zhao, L., Dubois, O., Duluc, L., Rhodes, C. J., Wharton, J., Wilkins, M. R., Leiper, J., & Wojciak-Stothard, B. (2014). MiRNA-21/DDAH1 pathway regulates pulmonary vascular responses to hypoxia. The Biochemical Journal, 462(1), 103-12.

Libri, V., Yandim, C., Athanasopoulos, S., Loyse, N., Natisvili, T., Law, P. P. P., Chan, P. K. K., Mohammad, T., Mauri, M., Tam, K. T. T., Leiper, J., Piper, S., Ramesh, A., Parkinson, M. H., Huson, L., Giunti, P., & Festenstein, R. (2014). Epigenetic and neurological effects and safety of high-dose nicotinamide in patients with friedreich’s ataxia: an exploratory, open-label, dose-escalation study. Lancet, 384(9942), 504-13.

Ghebremariam, Y. T., Lependu, P., Lee, J. C., Erlanson, D. A., Slaviero, A., Shah, N. H., Leiper, J., & Cooke, J. P. (2013). An unexpected effect of proton pump inhibitors: Elevation of the cardiovascular risk factor ADMA. Circulation, 128(8), 845-53.

Caplin, B., Wang, Z., Slaviero, A., Tomlinson, J., Dowsett, L., Delahaye, M., Salama, A., International Consortium for Blood Pressure Genome-Wide Association Studies, Wheeler, D. C., & Leiper, J. (2012). Alanine-glyoxylate aminotransferase-2 metabolizes endogenous methylarginines, regulates NO, and controls blood pressure. Arteriosclerosis, Thrombosis, and Vascular Biology, 32(12), 2892–2900.

Nandi, M., Kelly, P., Torondel, B., Wang, Z., Starr, A., Ma, Y., Cunningham, P., Stidwill, R., & Leiper, J. (2012). Genetic and pharmacological inhibition of dimethylarginine dimethylaminohydrolase 1 is protective in endotoxic shock. Arteriosclerosis, thrombosis, and vascular biology, 32(11), 2589-97.

Pullamsetti, S. S. S., Savai, R., Schaefer, M. B. B., Wilhelm, J., Ghofrani, H. A. A., Weissmann, N., Schudt, C., Fleming, I., Mayer, K., Leiper, J., Seeger, W., Grimminger, F., & Schermuly, R. T. T. (2011). cAMP phosphodiesterase inhibitors increases nitric oxide production by modulating dimethylarginine dimethylaminohydrolases. Circulation, 123(11), 1194-204.

Leiper, J., & Nandi, M. (2011). The therapeutic potential of targeting endogenous inhibitors of nitric oxide synthesis. Nature Reviews Drug Discovery, 10(4), 277–291.

Caplin, B., Nitsch, D., Gill, H., Hoefield, R., Blackwell, S., MacKenzie, D., Cooper, J. A., Middleton, R. J., Talmud, P. J., Veitch, P., Norman, J., Wheeler, D. C., & Leiper, J. M. (2010). Circulating methylarginine levels and the decline in renal function in patients with chronic kidney disease are modulated by DDAH1 polymorphisms. Kidney International, 77(5), 459–467.

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