Evaluation of CaMKII as a therapeutic target for the treatment of diabetic macular oedema

Abstract

Diabetic macular oedema (DMO) is an important cause of vision loss worldwide. It is characterized by hyperpermeability of retinal vessels due to breakdown of the inner blood-retinal barrier. The goal of this thesis was to better understand the mechanisms through which bradykinin (BK) and thrombin (THR) trigger vasopermeability responses in retinal microvascular endothelial cells (RMECs), with particular emphasis on the role of intracellular Ca2+ and the Ca2+/calmodulin-dependent protein kinase, CaMKII. Ca2+ imaging studies revealed that BK and THR elicit biphasic intracellular Ca2+ responses in cultured bovine RMECs. The initial peak phase resulted from endoplasmic reticulum Ca2+ release via the Gαq-PLC-IP3 signalling pathway, while the sustained phase derived from Ca2+ influx via store-operated Ca2+ channels. Receptor pharmacology studies demonstrated that BK acts primarily through the bradykinin B2 receptor, whereas THR signals through protease-activated receptor 1 (PAR1). Experiments to determine CAMKII phosphorylation upon BK and THR exposure were undertaken in bovine and human RMECs. Western blotting studies identified a time-dependent increase in CaMKII activity upon agonist treatment persisting for up to 24 hours. Hyperglycaemic conditions for 21 days did not have a significant effect on basal or agonist induced CaMKII phosphorylation. Increased FITC-dextran leakage was observed in bovine RMEC monolayers challenged with BK and THR and these effects could be largely abolished in the presence of the CaMKII inhibitor, KN-93, but not its inactive analogue, KN-92. To investigate the relevance of these findings to DMO, the effects of systemic administration of KN-93 and KN-92 on retinal vascular leakage in streptozotocin-induced diabetic mice were investigated. These experiments confirmed an important role for CaMKII in mediating retinal vasopermeability responses in the diabetic retina. Taken together, the work in this thesis suggests that CaMKII could represent a novel therapeutic target for DMO treatment, although further studies will be required to confirm this.

Thesis embargoed until 31 July 2025.

Date of Award	Jul 2022
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Fight for Sight (Inc British Eye Research Foundation)
Supervisor	Tim Curtis (Supervisor) & Alan Stitt (Supervisor)