Mitochondrial transcription factor A protects human retinal endothelial cell injury induced by hypoxia

Qilian Xie,Xu Cao

doi:10.4314/tjpr.v16i6.8

Abstract

Purpose: To investigate the impact of mitochondrial transcription factor A (TFAM), as a modulator of NF-κB, on proliferation of hypoxia-induced human retinal endothelial cell (HREC), and the probable mechanism.Methods: After exposure to hypoxia (1 % O2) for 5 days, cell proliferation and cell cycle of HREC were measured by MTT (3-（4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay and flow cytometry. Cell signaling and mitochondrial DNA (mtDNA) copies were determined using real-time polymerase chain reaction and Western blot. NF-κB activity was evaluated by luciferase assay.Results: TFAM expression decreased to 40 % in HREC under hypoxic condition (p < 0.05). MTT results revealed that TFAM facilitated HREC proliferation under hypoxia (p < 0.05). Moreover, flow cytometry demonstrated that TFAM promoted HREC proliferation by accelerating cell cycle (p < 0.05). Western blot and luciferase assay exhibited NF-κB activation in HREC after TFAM overexpression (p < 0.05). Finally, real-time PCR results showed that mtDNA and targeted genes of NF-κB were upregulated 3-fold in HREC after TFAM transfection under hypoxia (p < 0.05).Conclusion: These results indicate that NF-κB activated by TFAM protects against hypoxia-induced HREC injury by accelerating cell cycle. The ability of TFAM to enhance NF-κB signaling may be part of the mechanism of hypoxia-induced cell injury. Thus, upregulation of TFAM may help to relieve diabetic retinopathy.Keywords: Mitochondrial transcription factor A, NF-κB, Hypoxia, Human retinal endothelial cell, Diabetic retinopathy

Highlights

Endothelium dysfunction plays an important role in the development of vascular complications in diabetes [1]
Since TFAM was downregulated in human retinal endothelial cell (HREC) under hypoxia environment, we proposed that TFAM was involved in regulating HREC behavior
MTT assay revealed that TFAM upregulation enhanced HREC cell proliferation under hypoxia condition (Figure 2B)

Summary

Introduction

Endothelium dysfunction plays an important role in the development of vascular complications in diabetes [1]. Premature death of the capillary components, including endothelial cell and pericytes, occurs during the vasodegenerative stage of diabetic retinopathy [2]. The injury of retinal microvessels induced by diabetes results in the features of diabetic retinopathy, such as blood-retinal barrier leakage, non-perfusion capillary formation, and neovascularization [3]. Hypoxia is an important factor for the pathogenesis of human retinal endothelial cell (HREC) injury, which may promote intraocular neovascularization via imbalancing angiogenic homeostasis under physiological conditions [4,5]. Retinal mitochondria are compromised in diabetes, leading to mitochondrial DNA (mtDNA) damage [6]. As mtDNA is extremely important in the electron transport chain, it plays a critical role in regulating superoxide and apoptosis [7,8]. Mitochondrial transcription factor A (TFAM), as one of the key factors that binds to the regulatory area of mtDNA, activate the transcription of the target proteins of mtDNA [9]

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