Pharmacological and biomolecular profile of purinergic receptors in vascular endothelium: implications for mechanosensation.

Paz López, Sonia (2023) Pharmacological and biomolecular profile of purinergic receptors in vascular endothelium: implications for mechanosensation. Doctoral thesis, University of East Anglia.

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The endothelium is the monolayer of cells lining the blood vessels in direct contact with blood flow, detecting mechanical, metabolic, and immunological signals in their surroundings through specific receptors. Endothelial cell's function determines vascular tone, blood vessel formation, and immunological response. Calcium signalling, a universal messenger in cellular activities, is one of the main processes governing vascular endothelial function. The purinergic receptors are a fundamental component of the endothelial calcium toolkit, influencing vasodilation, angiogenesis, and inflammatory responses.

P2 receptors respond to extracellular nucleotides, elevating cytosolic calcium concentration through P2X and P2Y receptors. Various P2 receptors have a role in endothelial calcium homeostasis, with P2Y2 and P2X4 being the most studied receptors in this cell type. However, there is also controversy about which is the primary mediator of ATP-evoked responses in endothelial cells. Despite the P2 receptor's crucial roles in a diverse range of endothelial functions, a comprehensive biomolecular and pharmacological characterization of the P2 receptor's contribution to calcium signalling still needs to be performed. This study aims to characterise the transcriptional and functional expression profile of purinergic signalling in endothelial cells, using human microvascular endothelial cells (HMEC-1) as the primary model. Methodologies included transcriptional profiling via RNA sequencing, pharmacological assays using intracellular calcium mobilisation, and protein expression analysis through western blotting. These methods were applied to investigate the roles of P2 receptors in endothelial ATP, VEGF, and Piezo1-evoked calcium responses.

Here we show that the P2Y2 receptors are the primary mediator of the ATP-evoked calcium responses in HMEC-1, and P2Y11 is involved in the VEGF-evoked calcium responses, suggesting a novel mechanism that might comprise adenylyl cyclase activity and Orai1 activation, distinct from the traditional view of P2 receptors' function. In contrast, the role of P2X4 remains ambiguous, with our findings questioning its functional presence in HMEC-1, as the receptor was absent at the protein level. In addition, the second part of this study faced challenges in dynamically characterising Piezo1 responses due to technical limitations.

By elucidating the roles of these receptors, this research contributes to a more comprehensive understanding of endothelial physiology and pathology, providing new routes for therapeutic intervention in cardiovascular diseases. This study improves our understanding of the endothelial calcium toolkit, revealing new facets of purinergic receptors in endothelial cells. However, additional research is needed to understand the key players in the novel proposed mechanisms involving P2Y11's contribution to the VEGF-dependent responses. Moreover, further research should focus on fully comprehending the P2 receptor's specific roles under dynamic conditions and in different endothelial cell types.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Nicola Veasy
Date Deposited: 10 Jul 2024 10:33
Last Modified: 10 Jul 2024 14:00


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