Retinal flavoprotein fluorescence correlates with mitochondrial stress, apoptosis, and chemokine expression

Abstract

Oxidative stress and mitochondrial dysfunction occur before apoptosis in many retinal diseases. Under these conditions, a larger fraction of flavoproteins become oxidized and, when excited by blue-light, emit green flavoprotein fluorescence (FPF). In this study, we evaluated the utility of FPF as an early indicator of mitochondrial stress, pre-apoptotic cellular instability, and apoptosis of human retinal pigment epithelial (HRPE) cells subjected to hydrogen peroxide (H2O2) or monocytes (unstimulated or interferon-γ-stimulated) in vitro and of freshly isolated pieces of human and rat neural retina subjected to H2O2 ex vivo. Increased FPF of HRPE cells exposed to H2O2 correlated with reduced mitochondrial membrane potential (ΔΨm) and increased apoptosis in a time- and dose-dependent manner. HRPE cells co-cultured with monocytes had increased FPF that correlated in a time-dependent manner with reduced ΔΨm, increased apoptosis, and early expression of pro-inflammatory chemokines, interleukin-8 (IL8) and monocyte chemotactic factor-1 (MCP1), which are known to be induced by oxidative stress. Increased FPF, reduced ΔΨm, and upregulation of IL8 and MCP1 occurred as early as 1–2 hours after exposure to stressors, while apoptosis did not occur in HRPE cells until later time points. The antioxidant, N-aceytl-cysteine (NAC), inhibited increased FPF and apoptosis of HRPE cells subjected to H2O2. Increased FPF of human and rat neural retina also correlated with increased apoptosis. This study suggests that FPF is a useful measure of mitochondrial function in retinal cells and tissues and can detect early mitochondrial dysfunction that may precede apoptosis.

In Vitro Evidence for Mycophenolic Acid Dose-Related Cytotoxicity in Human Retinal Cells

“…43 ΔCm have been shown to be an early indicator of instability, even in cells exposed to low levels of oxidants that are insufficient to cause apoptosis. 38,44 In our study, exposure to MPA did not cause significant ΔCm change in either human ARPE-19 or Muller cells after 24 hours. These findings are intriguing as the significant increase in caspase-3/7 activity 24 hours after exposure to 100 mg/mL of MPA suggests increased apoptosis.…”

“…27,29,30,[34][35][36][37][38] Dose-related in vitro toxicity caused by different drugs has been shown using the same methods used in this study. [26][27][28][29] In our experiments, we exposed the human ARPE-19 and Muller cells to MPA for 24 h. Vitreous MPA half-life is not known but plasma half-life is around 17 hours.…”

Fundus autofluorescence lifetimes are increased in non‐proliferative diabetic retinopathy

“…Besides AGEs, increased oxidized flavoprotein levels, secondary to mitochondrial stress, might contribute to an increase of FAF in diabetics, as suggested by Field et al. (, ) as well as to an increase of short‐wavelength FLIO lifetimes.…”

Metabolic Vulnerability in the Neurodegenerative Disease Glaucoma

“…Citrate is a byproduct of the Krebs cycle, so decreased values support the idea that mitochondrial function is impaired in glaucoma patients. Mitochondrial function can be monitored in patient retinas using the green fluorescence emitted by oxidized flavoproteins, a byproduct of oxidative stress (Field et al, 2011). This development may improve diagnosis as well as provide a means to monitor future metabolic therapy.…”