Lactate/Pyruvate Regulation as a Key Determinant for Maintaining Retinal Pigment Epithelium Barrier Function: Insights from Glycolytic Screening

Abstract

Purpose : The retinal pigment epithelium (RPE) is crucial for preserving retinal homeostasis and the integrity of the outer blood-retinal barrier. Failure in maintaining RPE barrier function can lead to severe vision-threatening diseases, including age-related macular degeneration and diabetic macular edema. Despite the recognized importance of glycolysis in glucose homeostasis, its specific role in RPE barrier assembly remains incompletely understood. This study aims to screen the contributions of various glycolytic components in regulating RPE barrier integrity.

Methods : Electric Cell-Substrate Impedance Sensing (ECIS) technology was employed to assess the impact of inhibiting diverse glycolytic enzymes and transporters on RPE barrier functionality. The inhibitors included heptelidic acid for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), NG-52 for phosphoglycerate kinase 1 (PGK1), AP-III for enolase, shikonin for pyruvate kinase M (PKM)-2, galloflavin for lactate dehydrogenase (LDH), AZD3965 for lactate transporter (MCT-1), and MSDC for the mitochondrial pyruvate carrier. Additionally, a cytotoxicity assay was conducted to evaluate RPE cell viability. Statistical analyses involved ANOVA and Tukey post-hoc tests.

Results : The most notable enhancement in electrical resistance of RPE cells was observed with the dose-dependent inhibition of lactate dehydrogenase (LDH) using galloflavin. Consistently, inhibiting lactate efflux with AZD3965 resulted in a dose-dependent reduction in RPE barrier resistance, affirming the critical contribution of lactate in this process. Additionally, inhibiting the mitochondrial pyruvate transporter by MSDC led to a significant decrease in RPE barrier resistance without inducing cytotoxicity. Notably, inhibiting other glycolytic components, including GADPH, PGK1, and PKM2, did not demonstrably alter the barrier functionality of the RPE.

Conclusions : These findings highlight the specific and crucial role of lactate/pyruvate in regulating the integrity of the RPE barrier. Further investigations into the mechanisms underlying lactate's influence on RPE barrier assembly could provide valuable insights for potential therapeutic interventions in vision-threatening diseases associated with RPE dysfunction.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.