Characterisation of endothelial cell metabolic plasticity and vascular remodelling in the retina

Abstract

Diabetic retinopathy (DR) is the most frequently occurring microvascular complication of diabetes mellitus, where the later stages of the disease are often is chaemia driven which can result in severe vision loss. A key component cell of the retinal neurovascular unit is the vascular endothelium, which is significantly impacted by the diabetic milieu. It has now been established that the endothelium relies on glycolytic metabolism predominantly for ATP generation, however the degree of flux through this pathway in some non-retinal endothelial cell types has been shown to vary in disease conditions. This PhD project has assessed the nature of glycolysis and related plasticity in human retinal microvascular endothelial cells (HRMECs) exposed to in vitro conditions relevant to DR.HRMECs were obtained from human donors and were exposed to either normal glucose conditions of 5 m M D-glucose (NG), 20 m ML-Glucose + 5 m M D-Glucose (HLG), or 25 m M D-Glucose (HDG) for four weeks, along with a hypoxic exposure (1% O2, 5% CO2 )for 24 hours before experiments were performed, mimicking diabetic-like conditions. Assessments of in vitro metabolism were assessed using the Seahorse XF system and qPCR to highlight potential changes in metabolic pathways. Functional assays including 3D-Matrigelexperiments, Clonogenics, proliferation, migration, and barrier integrity were also implemented to ascertain what changes to functionality occur after HDG exposure. Furthermore, the use of pharmacological compounds such as the glycolytic inhibitor 3PO ((2E)-3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one),and Proxison (7-decyl-3-hydroxy-2-(3, 4, 5-trihdroxyphenyl)-4-chromenone) a synthetic flavonoid were also implemented in this study to determine if pharmacological interventions/ modifications to metabolism could be effective in reducing HDG exposure affects. In an attempt to understand angiogenic pathways in more detail, single cell sequencing was also performed on cells recovered from 3D environment. This project together helps to understand the nature of this complex disease and identify potential new targets of therapeutic interventions.

Date of Award	Dec 2021
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	F. Hoffmann-La Roche Ltd & Northern Ireland Department for the Economy
Supervisor	Alan Stitt (Supervisor) & Tim Curtis (Supervisor)