LDN-27219 Modulates High-Glucose-Induced Endothelial Bioenergetic Remodeling and MitoSOX Red Fluorescence

Figures

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  Figure 1. Dose-dependent effects of LDN-27219 on cellular reducing capacity and total protein content under normoglycaemic and high-glucose conditions. EA.hy926 endothelial cells were cultured under normoglycemic or high-glucose conditions and treated for 24 h with increasing concentrations of LDN-27219. (A) Cellular reducing capacity was assessed using the PrestoBlue assay, normalized to total protein content, and expressed relative to the normoglycemic control group, which was set to 100%. (B) Total protein content per well was determined using the Bradford assay and used as a cell-associated biomass-related readout. Data are presented as mean ± SD from three independent biological replicates. Statistical analysis was performed using one-way ANOVA followed by Fisher’s LSD post hoc test. The red dashed line indicates the normoglycemic control reference level. Significant comparisons are indicated in the figure. * p < 0.05 versus the untreated normoglycaemic control.

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  Figure 2. LDN-27219 alters endothelial bioenergetic responses under high-glucose conditions. EA.hy926 endothelial cells were cultured under normoglycemic or high-glucose conditions and treated for 24 h with vehicle or LDN-27219 at 20 µg/mL (48.9 µM). Mitochondrial respiration and glycolytic activity were assessed using the Seahorse XFp Cell Mito Stress Test. (A) Quantification of calculated mitochondrial respiratory parameters after subtraction of non-mitochondrial respiration, including basal respiration, maximal respiration, proton leak, ATP-linked OCR, and spare respiratory capacity. (B,C) Cellular energy phenotype profiles under basal and stressed conditions, showing total OCR, including both mitochondrial and non-mitochondrial OCR, and ECAR relationships across experimental groups. (D) Quantification of metabolic potential expressed as stressed total OCR, including both mitochondrial and non-mitochondrial OCR, and stressed ECAR. Data are presented as mean ± SD from three independent biological replicates. Statistical analysis was performed using one-way ANOVA followed by Fisher’s LSD post hoc test. * p < 0.05 versus normoglycemic control; # p < 0.05 versus high-glucose control.

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  Figure 3. LDN-27219 reduces high-glucose-induced MitoSOX Red fluorescence in endothelial cells. EA.hy926 endothelial cells were cultured under normoglycemic or high-glucose conditions in the absence or presence of LDN-27219 at 48.9 µM, corresponding to 20 µg/mL. Mitochondrial oxidant-associated fluorescence was assessed using MitoSOX Red. (A) Quantification of MitoSOX Red fluorescence intensity normalized to total protein content and expressed relative to the normoglycemic control group, which was set to 100%. Data are presented as mean ± SD from 3 independent biological replicates. Statistical analysis was performed using one-way ANOVA followed by Fisher’s LSD post hoc test. ** p < 0.01. (B) Representative fluorescence images of endothelial cells under the indicated experimental conditions. Scale bar = 20 µm.

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  Figure 4. Selected cytokine and adhesion-marker secretion under low- and high-glucose conditions after LDN-27219 treatment. EA.hy926 endothelial cells were cultured under normoglycemic or high-glucose conditions in the absence or presence of LDN-27219 at 20 µg/mL, corresponding to 48.9 µM. Secretion of MCP-1/CCL2, IL-6, TNFα, and CD62E/E-selectin into the culture supernatant was quantified using a multiplex Luminex assay. Data are presented as mean ± SD from three independent biological replicates. Statistical analysis was performed using one-way ANOVA followed by Fisher’s LSD post hoc test. No statistically significant differences were detected among groups for the analytes shown.