目的:在糖尿病性视网膜病变和早产儿视网膜病变等各种致盲性眼病中,线粒体功能障碍是破坏视网膜内皮细胞(REC)屏障完整性的关键。因此,我们旨在研究不同线粒体成分的作用,特别是那些氧化磷酸化(OxPhos),维持REC的屏障功能。
方法:使用电细胞基质阻抗传感(ECIS)技术实时评估不同线粒体成分在人类RECs(HRECs)及其成分的总阻抗(Z)中的作用:电容(C)和总电阻(R)。HRECs用特定的线粒体抑制剂处理,这些抑制剂靶向OxPhos中的不同步骤:鱼藤酮用于复合物I,复杂V(ATP合酶)的寡霉素,和FCCP用于解偶联OxPhos。此外,对数据进行建模以研究这些抑制剂对控制细胞总抗性的三个参数的影响:细胞-细胞相互作用(Rb),细胞-基质相互作用(α),和细胞膜通透性(Cm)。
结果:鱼藤酮(1µM)产生了最大的Z降低,其次是FCCP(1µM),而在寡霉素(1µM)处理后没有观察到Z的减少。然后我们进一步去卷积这些抑制剂对Rb的影响,α,和Cm参数。鱼藤酮(1µM)完全消除了Rb的抗性贡献,治疗后Rb立即变为零。其次,FCCP(1µM)仅在2.5h后消除了Rb的抗性贡献,并增加了Cm,而对α没有显着影响。最后,在所有使用的抑制剂中,寡霉素对Rb的影响最小,实验结束时,该值与对照组相似,对Cm或α没有明显影响。
结论:我们的研究证明了复合物I的不同作用,复数V,和OxPhos偶联以保持HRECs的屏障功能。我们特别表明,复合物I是调节HREC屏障完整性的最重要组成部分。这些观察到的差异是显著的,因为它们可以作为旨在改善复合物I的活性的未来药理学和基因表达研究的基础,从而为内皮相关视网膜疾病的治疗方式提供途径。
Mitochondrial dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy and retinopathy of prematurity. Therefore, we aimed to investigate the role of different mitochondrial constituents, specifically those of oxidative phosphorylation (OxPhos), in maintaining the barrier function of RECs.
Electric cell-substrate impedance sensing (ECIS) technology was used to assess in real time the role of different mitochondrial components in the total impedance (Z) of human RECs (HRECs) and its components: capacitance (C) and the total resistance (R). HRECs were treated with specific mitochondrial inhibitors that target different steps in OxPhos: rotenone for complex I, oligomycin for complex V (ATP synthase), and
FCCP for uncoupling OxPhos. Furthermore, data were modeled to investigate the effects of these inhibitors on the three parameters that govern the total resistance of cells: Cell-cell interactions (Rb), cell-matrix interactions (α), and cell membrane permeability (Cm).
Rotenone (1 µM) produced the greatest reduction in Z, followed by
FCCP (1 µM), whereas no reduction in Z was observed after oligomycin (1 µM) treatment. We then further deconvoluted the effects of these inhibitors on the Rb, α, and Cm parameters. Rotenone (1 µM) completely abolished the resistance contribution of Rb, as the Rb became zero immediately after the treatment. Secondly,
FCCP (1 µM) eliminated the resistance contribution of Rb only after 2.5 h and increased Cm without a significant effect on α. Lastly, of all the inhibitors used, oligomycin had the lowest impact on Rb, as evidenced by the fact that this value became similar to that of the control group at the end of the experiment without noticeable effects on Cm or α.
Our study demonstrates the differential roles of complex I, complex V, and OxPhos coupling in maintaining the barrier functionality of HRECs. We specifically showed that complex I is the most important component in regulating HREC barrier integrity. These observed differences are significant since they could serve as the basis for future pharmacological and gene expression studies aiming to improve the activity of complex I and thereby provide avenues for therapeutic modalities in endothelial-associated retinal diseases.