Abstract
Purpose :
Damage to the retinal vascular endothelium, also known as the inner blood-retinal barrier (iBRB), is implicated in the progression of retinal degenerative diseases, including diabetic retinopathy (DR) and diabetic macular edema (DME). Moreover, under normal physiological conditions, glycolytic metabolism is critical to the functioning of endothelial cells and iBRB maintenance. Conversely, in hyperglycemic environments, unregulated glycolysis leads to a buildup of glycolytic intermediates, which is thought to contribute to iBRB disruption and retinal cell damage. In this study, we looked to further characterize the roles of upper glycolytic enzymes, those involved in the ATP consumption phase of glycolysis, in the maintenance of the iBRB.
Methods :
Electric cell-substrate impedance sensing (ECIS) technology was used to assess in real-time the role of different glycolytic enzymes in maintaining the barrier functionality of human retinal endothelial cells (HREC). Furthermore, the endothelial cellular viability was assessed through lactate dehydrogenase cytotoxicity assay at 24h, 48h, and 72h time intervals.
Results :
Inhibition with 1 μM and 10 μM heptelidic acid (glyceraldehyde-3-phosphate dehydrogenase (GA3PDH) inhibitor) significantly reduced the resistance (R) and thus the integrity of the HREC barrier (p < .001). 10 μM PFK158 (phosphofructokinase-1 (PFK1) inhibitor) also significantly reduced R (p = .005). Similarly, administration of 10 μM PFK158 reduced HREC viability at the 24h, 48h, and 72h time intervals (p < .001). Administration of 1 μM and 10 μM heptelidic acid reduced HREC viability at the 72h time interval (p < .001). The other inhibitors tested did not significantly reduce HREC barrier resistance or cellular viability.
Conclusions :
Our study specifically shows that the functions of GA3PDH and PFK1 are the most critical upper glycolytic components in regulating HREC barrier integrity. These observed differences could serve as the basis for future pharmacological and gene expression studies aiming to improve the activity of GA3PDH and PFK1, thereby providing avenues for therapeutic modalities in endothelial-associated retinal diseases.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.