Halliday, Loryn (2021) The influence of adenosine deaminase 2 on blood outgrowth endothelial cells. (PhD thesis), Kingston University, .
Abstract
Autosomal recessive loss-of-function mutations in Adenosine Deaminase 2 (ADA2) cause deficiency of adenosine deaminase 2 (DADA2), a monogenetic systematic vasculitis, sometimes also associated with immune deficiency or marrow failure. Children with DADA2 exhibit: inflammation, endothelial cell (EC) damage and elevated plasma adenosine levels. ADA2 is typically expressed by leukocytes. Indirect evidence suggests that ADA2 is a growth factor for ECs. It was hypothesised that ADA2 might be expressed by, and functionally important to, EC biology. Recent developments in culture techniques to extract progenitive ECs from whole blood, known as blood outgrowth endothelial cells (BOEC) or endothelial colony-forming cells (ECFC), indicates that ECs can be isolated from a relatively small amount of donor’s blood and represent an individual’s epigenetic makeup. The aim of this thesis was to assess BOECs as an EC model, understand the ADA2 metabolite, adenosine’s signalling mechanism within these cells, and then assess how ADA2 directly impacts EC structure, and function. BOECs were isolated from healthy donors and compared to the endothelial model, HUVECs. Phenotype and functional assays showed that BOECs behaved typically of endothelial cells. Extensive glycocalyx expression and elevated quiescent DDAH2, (a gene involved in the regulation of nitric oxide production), levels in BOEC, suggest that BOECs might be a more relevant EC model, than HUVEC. When investigating the influence of adenosine on BOECs, BOECs expressed more A2B than A2A adenosine receptor (AR) and HUVECs expressed more A2A than A2B AR. Extracellular adenosine inhibited BOEC glycocalyx expression and recovery. At high adenosine levels, adenosine downregulated BOEC DDAH2 expression. Functional assays, proliferation and migration were increased with increasing concentrations of adenosine, and this was mediated by both ARs, but predominantly A2B. Finally, when investigating the ADA2 direct influence on BOECs, we demonstrated that although lower than monocytes, ADA2 mRNA was expressed by BOECs, and ADA2 activity was found in BOEC-derived supernatants. Exogenously added ADA2 inhibited proliferation and migration of BOECs. ADA2 inhibition of BOEC migration was prevented by an A2B AR antagonist and adenylate cyclase inhibitor, suggesting the mechanism is AR pathway-dependent. Exogenous ADA2 also promoted restoration of the glycocalyx and inhibited Tumour necrosis factor alpha (TNF-α) stimulated monocyte adhesion. ADA2 in the presence of 0.5µM adenosine, which is extreme physiological adenosine concentration (as found in mild hypoxia or DADA2 patients) improved HSPG2 expression and glycocalyx restoration; upregulated NOS3 expression; and promoted growth, restored cell migration to control and increased monocyte adhesion. BOECs are ECs thought to repair the vascular endothelium. This work demonstrates an ADA2-dependent mechanism by which BOECs locally influence growth and repair, highlighting another means by which deficiency of ADA2 may lead to vascular dysfunction.
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