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

Abstract

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.

Keywords: Age-related macular degeneration (AMD); Barrier integrity; Capacitance; Cell adhesion; Cell spreading; Electric cell-substrate impedance sensing (ECIS); Glycolysis; Resistance.

Figure 1

Overview of glycolytic pathway. TPI triose phosphate isomerase, GA-3-P glyceraldehyde-3-phosphate, DHAP dihydroxyacetone phosphate, 1,3-Bis-PG 1,3-bisphosphoglycerate, 3-PG 3-phosphoglycerate, 2-PG 2-phosphoglycerate, PEP phosphoenolpyruvate, LDH lactate dehydrogenase.

Figure 2

Three-dimensional plots depicting the response of RPE resistance to different glycolytic inhibitors. Panel (A) represents treatment with the vehicle (DMSO), while panels (B,C) show the effects of the Glut1 inhibitor, WZB117, at concentrations of 1 μM and 10 μM, respectively. Panels (D,E) demonstrate the impact of the Hexokinase inhibitor, Lonidamine, at concentrations of 1 μM and 10 μM, respectively. Similarly, panels (F,G) display the effects of the PFKFB3/PFK inhibitor, PFK158, at concentrations of 1 μM and 10 μM, respectively. Lastly, panels (H,I) illustrate the response to the Aldolase inhibitor, TDZD-8, at concentrations of 1 μM and 10 μM, respectively. Treatment initiation occurred after the formation of a confluent monolayer at t = 0 h. Resistance values collected during the experiment were normalized by dividing them by their corresponding resistance values obtained at t = 0 h. Real-time resistance measurements were obtained across various AC frequencies ranging from 250 to 64,000 Hz. Black arrows indicate the endpoint of each inhibitor group. Norm normalized, Freq frequency.

Figure 3

Real-time measurements of total resistance across RPE cells subjected to WZB117 (Glut1 inhibition). (A) The normalized (norm) resistance plot is depicted against time, measured at an AC frequency of 4000 Hz. Vehicle and WZB117 treatments (1 µM and 10 µM) were added to RPE cell groups at t = 0, with resistance measured in real-time post-treatment application. (B) The bar graph represents the normalized resistance acquired at the end of the experiment for each treatment group. (C) The bar graph represents the area under the curve (AUC) of normalized resistance measured from t = 0 until the end of the experiment for each group. Green vertical lines denote distinct time points where LDH levels were collected.

Figure 4

The effects of upper glycolytic inhibitors on RPE cell viability. RPE cell death was assessed by measuring the levels of lactate dehydrogenase (LDH) released in the culture media after treatment with various upper glycolytic inhibitors at (A) 24, (B) 48, and (C) 72 h. Norm normalized, ns no significance.

Figure 5

Real-time measurements of the total capacitance across RPE cells subjected to WZB117 (Glut1 inhibition). (A) The normalized (norm) capacitance plot is depicted against time, measured at an AC frequency of 64,000 Hz. Vehicle and WZB117 treatments (1 µM and 10 µM) were added to RPE cell groups at t = 0, with capacitance measured in real-time post-treatment application. (B) The bar graph represents the normalized capacitance acquired at the end of the experiment for each treatment group. (C) The bar graph represents the area under the curve (AUC) of normalized capacitance measured from t = 0 until the end of the experiment for each group. Green vertical lines denote distinct time points where LDH levels were collected.

Figure 6

Real-time measurements of total resistance across RPE cells subjected to Lonidamine (Hexokinase inhibition). (A) The normalized resistance plot is depicted against time, measured at an AC frequency of 4,000 Hz. Vehicle and Lonidamine treatments (1 µM and 10 µM) were added to RPE cell groups at t = 0, with resistance measured in real-time post-treatment application. (B) The bar graph represents the normalized resistance acquired at the end of the experiment for each treatment group. (C) The bar graph represents the area under the curve (AUC) of normalized resistance measured from t = 0 until the end of the experiment for each group. Green vertical lines denote distinct time points where LDH levels were collected. Norm normalized, ns no significance.

Figure 7

Real-time measurements of the total capacitance across RPE cells subjected to Lonidamine (Hexokinase inhibition). (A) The normalized capacitance plot is depicted against time, measured at an AC frequency of 64,000 Hz. Vehicle and Lonidamine treatments (1 µM and 10 µM) were added to RPE cell groups at t = 0, with capacitance measured in real-time post-treatment application. (B) The bar graph represents the normalized capacitance acquired at the end of the experiment for each treatment group. (C) The bar graph represents the area under the curve (AUC) of normalized capacitance measured from t = 0 until the end of the experiment for each group. Green vertical lines denote distinct time points where LDH levels were collected. Norm normalized, ns no significance.

Figure 8

Real-time measurements of total resistance across RPE cells subjected to PFK158 (PFKFB3/PFK axis inhibition). (A) The normalized resistance plot is depicted against time, measured at an AC frequency of 4000 Hz. Vehicle and PFK158 treatments (1 µM and 10 µM) were added to RPE cell groups at t = 0, with resistance measured in real-time post-treatment application. (B) The bar graph represents the normalized resistance acquired at the end of the experiment for each treatment group. (C) The bar graph represents the area under the curve (AUC) of normalized resistance measured from t = 0 until the end of the experiment for each group. Green vertical lines denote distinct time points where LDH levels were collected. Norm normalized, ns no significance. PFK158 is abbreviated as PFK in (A).

Figure 9

Real-time measurements of the total capacitance across RPE cells subjected to PFK158 (PFKFB3/PFK axis inhibition). (A) The normalized capacitance plot is depicted against time, measured at an AC frequency of 64,000 Hz. Vehicle and PFK158 treatments (1 µM and 10 µM) were added to RPE cell groups at t = 0, with capacitance measured in real-time post-treatment application. (B) The bar graph represents the normalized capacitance acquired at the end of the experiment for each treatment group. (C) The bar graph represents the area under the curve (AUC) of normalized capacitance measured from t = 0 until the end of the experiment for each group. Green vertical lines denote distinct time points where LDH levels were collected. Norm normalized, ns no significance. PFK158 is abbreviated as PFK in (A).

Figure 10

Real-time measurements of total resistance across RPE cells subjected to TDZD-8 (Aldolase inhibition). (A) The normalized (norm) resistance plot is depicted against time, measured at an AC frequency of 4,000 Hz. Vehicle and TDZD-8 treatments (1 µM and 10 µM) were added to RPE cell groups at t = 0, with the resistance measured in the real-time post-treatment application. (B) The bar graph represents the normalized resistance acquired at the end of the experiment for each treatment group. (C) The bar graph represents the area under the curve (AUC) of normalized resistance measured from t = 0 until the end of the experiment for each group. Green vertical lines denote distinct time points where LDH levels were collected.

Figure 11

Real-time measurements of the total capacitance across RPE cells subjected to TDZD-8 (Aldolase inhibition). (A) The normalized (norm) capacitance plot is depicted against time, measured at an AC frequency of 64,000 Hz. Vehicle and TDZD-8 treatments (1 µM and 10 µM) were added to RPE cell groups at t = 0, with capacitance measured in real-time post-treatment application. (B) The bar graph represents the normalized capacitance acquired at the end of the experiment for each treatment group. (C) The bar graph represents the area under the curve (AUC) of normalized capacitance measured from t = 0 until the end of the experiment for each group. Green vertical lines denote distinct time points where LDH levels were collected.