Effect of increased vascular endothelial growth factor on fluorescence lifetime of human retinal microvascular endothelial cells in vitro

Abstract

Purpose : Vascular endothelial growth factor (VEGF) is known to disrupt tight junction of vascular endothelial cells and induce their proliferation, leading to vascular leakage and neovascularization as seen in diabetic retinopathy. The aim of this study is to investigate the effect of VEGF on cell metabolism and the fluorescence lifetime of retinal microvascular endothelial cells using fluorescence lifetime imaging microscopy (FLIM).

Methods : An immortalized cell line of the human retinal microvascular endothelial cells (HRMEC) were treated either with 10 ng/ml VEGF₁₂₁, only with the vehicle (400 nM hydrogen chloride), or without any addition (control) for 48 hours. Cell metabolic activity and mitochondrial membrane potential were assessed using MTT and JC-10 assays. Mean fluorescence lifetime (t_m) was measured using two-photon microscopy combined with FLIM (excitation at 740 nm, detection with a single channel for 380 nm to 680 nm). t_m of cell nuclei and mitochondria were analyzed separately with SPCImage software.

Results : The MTT assay showed an increase in cellular metabolic activity, and the JC-10 assay detected a significant increase in mitochondrial membrane potential in cells treated with 10 ng/ml VEGF. In FLIM, t_m of the mitochondria was significantly longer than in the nuclei of the same cells under all three conditions. FLIM further revealed that the cells treated with 10 ng/ml VEGF showed longer t_m in mitochondria than control and vehicle-treated cells, where the difference was on the verge of statistical significance (p = 0.08). Comparing the ratios between t_m of mitochondria and cell nuclei, VEGF-treated cells showed a significantly higher ratio (p<0.05).

Conclusions : The results of the MTT and the JC-10 assays suggested that VEGF may increase mitochondrial activity of HRMEC, and FLIM could indicate these changes in a label-free live cell imaging. Elongation of t_m in mitochondria of VEGF-treated cells may suggest the increase of protein-bound nicotinamide adenine dinucleotide (NADH), further indicating the increased function of mitochondrial respiration. VEGF-induced changes in FLIM on retinal cells may provide insight into the clinical application of fluorescence sensing in different chorioretinal pathological conditions, where VEGF plays a significant role.

This is a 2021 ARVO Annual Meeting abstract.