The significance of upper glycolytic components in regulating retinal pigment epithelial cellular behavior.

Cell adhesion to the extracellular matrix and its natural outcome of cell spreading, along with the maintenance of barrier activity, are essential behaviors of epithelial cells, including retinal pigment epithelium (RPE). Disruptions in these characteristics can result in severe vision-threatening diseases such as diabetic macular edema and age-related macular degeneration. However, the precise mechanisms underlying how RPE cells regulate their barrier integrity and cell spreading are not fully understood. This study aims to elucidate the relative importance of upper glycolytic components in governing these cellular behaviors of RPE cells. Electric Cell-Substrate Impedance Sensing (ECIS) technology was utilized to assess in real-time the effects of targeting various upper glycolytic enzymes on RPE barrier function and cell spreading by measuring cell resistance and capacitance, respectively. Specific inhibitors used included WZB117 for Glut1 inhibition, Lonidamine for Hexokinase inhibition, PFK158 for PFKFB3/PFK axis inhibition, and TDZD-8 for Aldolase inhibition. Additionally, the viability of RPE cells was evaluated using a lactate dehydrogenase (LDH) cytotoxicity assay. The most significant decrease in electrical resistance and increase in capacitance of RPE cells were observed due to dose-dependent inhibition of Glut1 using WZB117, as well as Aldolase inhibition with TDZD-8. LDH level analysis at 24-72 h post-treatment with WZB117 (1 and 10 µM) or TDZD-8 (1 µM) showed no significant difference compared to the control, indicating that the disruption of RPE functionality was not attributed to cell death. Lastly, inhibition of other upper glycolytic components, including PFKFB3/PFK with PFK158 or Hexokinase with Lonidamine, did not significantly affect RPE cell behavior. This study provides insights into the varied roles of upper glycolytic components in regulating the functionality of RPE cells. Specifically, it highlights the critical roles of Glut1 and Aldolase in preserving barrier integrity and promoting RPE cell adhesion and spreading. Such understanding will guide the development of safe interventions to treat RPE cell dysfunction in various retinal disorders.

Upper glycolytic components contribute differently in controlling retinal vascular endothelial cellular behavior: Implications for endothelial-related retinal diseases.

BACKGROUND: Retinal degenerative diseases such as diabetic retinopathy and diabetic macular edema are characterized by impaired retinal endothelial cells (RECs) functionality. While the role of glycolysis in glucose homeostasis is well-established, its contributions to REC barrier assembly and cell spreading remain poorly understood. This study aimed to investigate the importance of upper glycolytic components in regulating the behavior of human RECs (HRECs). METHODS: Electric cell-substrate impedance sensing (ECIS) technology was employed to analyze the real-time impact of various upper glycolytic components on maintaining barrier functionality and cell spreading of HRECs by measuring cell resistance and capacitance, respectively. Specific inhibitors were used: WZB117 to inhibit Glut1/3, lonidamine to inhibit hexokinases, PFK158 to inhibit the PFKFB3-PFK axis, and TDZD-8 to inhibit aldolases. Additionally, the viability of HRECs was evaluated using the lactate dehydrogenase (LDH) cytotoxicity assay. RESULTS: The most significant reduction in electrical resistance and increase in capacitance of HRECs resulted from the dose-dependent inhibition of PFKFB3/PFK using PFK158, followed by aldolase inhibition using TDZD-8. LDH level analysis at 24- and 48-hours post-treatment with PFK158 (1 µM) or TDZD-8 (1 and 10 µM) showed no significant difference compared to the control, indicating that the disruption of HRECs functionality was not attributed to cell death. Conversely, inhibiting Glut1/3 with WZB117 had minimal impact on HREC behavior, except at higher concentrations (10 µM) and prolonged exposure. Lastly, inhibiting hexokinase with lonidamine did not noticeably alter HREC cell behavior. CONCLUSION: This study illustrates the unique impacts of components within upper glycolysis on HREC functionality, emphasizing the crucial role of the PFKFB3/PFK axis in regulating HREC behavior. Understanding the specific contributions of each glycolytic component in preserving normal REC functionality will facilitate the development of targeted interventions for treating endothelial cell dysfunction in retinal disorders while minimizing effects on healthy cells.

The Warburg effect alters amino acid homeostasis in human retinal endothelial cells: implication for proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) remains a leading cause of blindness despite progress in screening and treatment. Recently, the Warburg effect, a metabolic alteration affecting amino acid (AA) metabolism in proliferating cells, has drawn attention regarding its role in PDR. This study aimed to investigate the impact of the Warburg effect on AA metabolism in human retinal endothelial cells (HRECs) subjected to PDR-associated risk factors and validate the findings in patients with PDR. In vitro experiments exposed HRECs to high glucose (HG) and/or hypoxia (Hyp), known inducers of the Warburg effect. The HG + Hyp group of HRECs exhibited significant differences in non-essential AAs with aliphatic non-polar side chains, mainly driven by elevated glycine concentrations. Pathway enrichment analysis revealed several glycine metabolism-related pathways significantly altered due to the Warburg effect induced by HG + Hyp. Crucially, vitreous humor samples from PDR patients displayed higher glycine levels compared to non-diabetic and diabetic patients without PDR. The odds ratio for PDR patients with glycine levels above the cut-off of 0.0836 µM was 28 (p = 0.03) compared to non-PDR controls. In conclusion, this study provides mechanistic insights into how a specific Warburg effect subtype contributes to glycine accumulation in PDR and supports glycine's potential as a biomarker for PDR pathogenesis.

Relative Importance of Different Elements of Mitochondrial Oxidative Phosphorylation in Maintaining the Barrier Integrity of Retinal Endothelial Cells: Implications for Vascular-Associated Retinal Diseases.

PURPOSE: Mitochondrial dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy and retinopathy of prematurity. Therefore, we aimed to investigate the role of different mitochondrial constituents, specifically those of oxidative phosphorylation (OxPhos), in maintaining the barrier function of RECs. METHODS: Electric cell-substrate impedance sensing (ECIS) technology was used to assess in real time the role of different mitochondrial components in the total impedance (Z) of human RECs (HRECs) and its components: capacitance (C) and the total resistance (R). HRECs were treated with specific mitochondrial inhibitors that target different steps in OxPhos: rotenone for complex I, oligomycin for complex V (ATP synthase), and FCCP for uncoupling OxPhos. Furthermore, data were modeled to investigate the effects of these inhibitors on the three parameters that govern the total resistance of cells: Cell-cell interactions (Rb), cell-matrix interactions (α), and cell membrane permeability (Cm). RESULTS: Rotenone (1 µM) produced the greatest reduction in Z, followed by FCCP (1 µM), whereas no reduction in Z was observed after oligomycin (1 µM) treatment. We then further deconvoluted the effects of these inhibitors on the Rb, α, and Cm parameters. Rotenone (1 µM) completely abolished the resistance contribution of Rb, as the Rb became zero immediately after the treatment. Secondly, FCCP (1 µM) eliminated the resistance contribution of Rb only after 2.5 h and increased Cm without a significant effect on α. Lastly, of all the inhibitors used, oligomycin had the lowest impact on Rb, as evidenced by the fact that this value became similar to that of the control group at the end of the experiment without noticeable effects on Cm or α. CONCLUSION: Our study demonstrates the differential roles of complex I, complex V, and OxPhos coupling in maintaining the barrier functionality of HRECs. We specifically showed that complex I is the most important component in regulating HREC barrier integrity. These observed differences are significant since they could serve as the basis for future pharmacological and gene expression studies aiming to improve the activity of complex I and thereby provide avenues for therapeutic modalities in endothelial-associated retinal diseases.