Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.664382
Title: The effect of Epac activation on human coronary artery endothelial cells
Author: Quinn, Rachael
ISNI:       0000 0004 5363 2319
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2014
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Abstract:
The endothelial barrier is essential for vascular function, and its disruption may play a major role in the development of cardiovascular diseases. An increase in cAMP levels tightens the endothelial barrier by enhancing junction formation, and this is, in part, mediated by activation of exchange protein directly activated by cAMP (Epac). Vascular endothelial cells express vascular endothelial cadherin (VE-cadherin), and connexins 37, 40 and 43. Re-distribution of VE-cadherin in human coronary artery endothelial cells (HCAECs), induced by EGTA treatment or blocking VE-cadherin-VE-cadherin interactions through pre-treatment with anti-VE-cadherin primary antibody, triggered a subsequent re-distribution of Cx37 that was reversible. Epac activation with the Epac-selective agonist 8-pCPT induced a re-distribution of VE-cadherin and connexin 37 to sites of cell-cell contact in HCAECs, increasing the co-localisation of these two proteins, as detected by immunocytochemistry. This increased co-localisation was completely blocked by Epac1 siRNA. To test whether the re-distribution of Cx37 induced by Epac activation resulted in the formation of new functional gap junction channels, Lucifer yellow dye transfer was examined in HCAECs under varying conditions. Epac activation in HCAECs enhanced gap junction intercellular communication (GJIC), and this increase was completely blocked by the Epac inhibitor HJC0197. 8-pCPT addition was also shown to induce a transient increase in the intracellular calcium concentration in HCAECs, as detected with the calcium indicator Fluo-4. This calcium transient was independent of protein kinase A (PKA), and occurred in the absence of extracellular calcium, but was inhibited by the presence of the endoplasmic reticulum calcium ATPase inhibitor cyclopiazonic acid (CPA), indicating that Epac primarily mediated the increase, and that intracellular stores were the predominant source of calcium. Furthermore, the calcium transient induced by 8-pCPT was considerably reduced by the Epac inhibitor ESI-09 and completely inhibited by the Epac inhibitor HJC0197. Together, these data suggest that Epac strengthens the endothelial barrier through re-distribution of VE-cadherin and Cx37. In addition, GJIC is enhanced by Epac activation and there is a rise in intracellular calcium, a second messenger that can be transferred to adjacent cells through GJIC. It may be possible, therefore, that these effects of Epac promote endothelial cell- smooth muscle cell communication and therefore Epac could play a role in the regulation of vascular tone.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.664382  DOI: Not available
Keywords: QP Physiology
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