Store-operated calcium channels regulated by hyperosmolarity as a mechanism underlying endothelial dysfunction in the hypersomolar condition

Hallett, Thomas Gordon

August 2020

Thesis or dissertation

© 2020 Thomas Gordon Hallett. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

Hyperosmolarity is a concomitant factor to the hyperglycaemia observed in diabetes mellitus, a disease known to affect the function of endothelial cells and cause vascular damage. Previous studies have found that hyperglycaemia is capable of reducing the viability of endothelial cells and altering the activity of store-operated Ca2+ entry (SOCE). This study aims to determine the role of hyperosmolarity on the endothelial function and the activity of SOCE and Orai channels.

In this study, hyperosmolarity was simulated (in vitro) via the addition of mannitol at clinically relevant concentrations (19.5, 30 & 60 mM). Physical & biochemical assays were employed to determine the effect this stimulus had on endothelial function. Ca2+ imaging was employed to measure the effect of hyperosmolarity on Orai channel activity and the role of Reactive Oxygen Species was investigated with the use of biochemical dyes (H2DCFDA. Mitosox & Amplex red).

Hyperosmolarity reduced endothelial cell migration (41% fewer migrated cells compared to control P=<0.0001) and increased the number of dead cells per field (160% increase from control P=<0.0001). SOCE was reduced under hyperosmolar conditions in human aortic endothelial cells and endothelial cell line (EA.hy926). SOCE in primary endothelial cells was found to be mediated by Orai channels, which was demonstrated by transfection of Orai siRNAs.
All three types of Orai channels (Orai1-3) were sensitive to hyperosmolarity in the HEK293 cells overexpressing STIM1/Orai with all three subtypes showing a >50% reduction in peak influx compared with untreated cells (P=<0.05 for all groups). Cytosolic reactive oxygen species (ROS) and mitochondrial ROS were increased by hyperosmolarity. Hydrogen peroxide was capable of reducing SOCE in a concentration- dependent manner.

These results suggest that the reduced SOCE by hyperosmolarity may be mediated by an increase in intracellular ROS concentration. The findings in this study suggest that store- operated Orai channels regulated by hyperosmolarity may act as a new underlying mechanism for causing diabetic vascular injury, which allows further therapeutic exploration for diabetic complications.

Hull York Medical School, The University of Hull and the University of York
Xu, Shang-Zhong
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