Investigating the roles of the ADMA-DDAH1 pathway in cerebral artery function and ischaemic stroke pathogenesis

Flynn, Arun (2024) Investigating the roles of the ADMA-DDAH1 pathway in cerebral artery function and ischaemic stroke pathogenesis. PhD thesis, University of Glasgow.

Abstract

Nitric oxide (NO), which is produced by endothelial NO synthase (eNOS) in the cerebral endothelium, is crucial for maintaining cerebrovascular homeostasis in health and disease. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of eNOS and the dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 enzymes regulate ADMA levels. Several studies have shown that ADMA levels are elevated following ischaemic stroke and that loss-of-function DDAH1 polymorphisms are associated with increased stroke risk. However, causal evidence linking elevated ADMA levels and stroke pathogenesis are lacking. Furthermore, in contrast to the systemic circulation, direct evidence showing the functional importance of DDAH1 in the cerebral circulation is also lacking. The overall hypotheses of this thesis are that elevated ADMA plays a causal role in ischaemic stroke pathogenesis by perturbing the eNOS-NO signalling pathway, and that the ADMA-metabolising enzyme DDAH1 plays an important physiological role in regulating endothelial NO signalling in cerebral arteries by controlling ADMA levels.

For the main aims of this thesis, we used a selective DDAH1 inhibitor or endothelial-specific DDAH1 knockout mice to investigate the effect of elevated ADMA levels on stroke outcomes in mice following experimental stroke; and to evaluate the functional importance of DDAH1 for the maintenance of NO-dependent physiological processes (e.g., vasoreactivity and angiogenesis) in mouse cerebral vessels. Firstly, we found that DDAH1 inhibition using the selective DDAH1 inhibitor L-257 disrupts eNOS-derived NO signalling in cerebral arteries in vitro and increases brain ADMA levels in vivo without altering cardiovascular haemodynamics. Importantly, however, the major new finding of this work was that DDAH1 inhibition using L-257 had no effect on ischaemic brain injury or neurological outcomes in mice following focal cerebral ischaemia. These findings indicate that elevated ADMA levels following DDAH1 inhibition does not influence stroke outcomes in this mouse model of stroke. Secondly, using endothelial-specific DDAH1 knockout mice, we found that endothelial DDAH1 is a crucial regulator of cerebral eNOS-NO signalling and resultant endothelial and smooth muscle function, likely through the metabolism of endothelial ADMA, however, we found that it does not appear to play a critical role in ischaemic stroke outcomes. Lastly, using L-257 and endothelial-specific DDAH1 knockout mice, we provide the first evidence that DDAH1 is also critically important for the angiogenic activity of the cerebral endothelium.

In summary, cerebral vascular DDAH1 is a functionally important regulator of cerebral endothelial NO signalling by controlling ADMA levels, however, so far, our work suggests that elevated ADMA levels or DDAH1 dysfunction does not play a causal role in ischaemic stroke pathogenesis. As discussed in this thesis, further studies are required to establish the roles of the ADMA-DDAH1 pathway in ischaemic stroke outcomes.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: This work was also supported by Wellcome [204820/Z/16/Z]. Wellcome ISSF Feasibility Scheme Award.
• Subjects: R Medicine > RC Internal medicine
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Funder's Name: British Heart Foundation (BHF)
• Supervisor's Name: Miller, Dr. Alyson and Leiper, Professor James
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84249
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 23 Apr 2024 12:16
• Last Modified: 23 Apr 2024 12:25
• Thesis DOI: 10.5525/gla.thesis.84249
• URI: https://theses.gla.ac.uk/id/eprint/84249