需要改进与人类相关的冠状动脉疾病(CAD)临床前模型,以改善转化研究和药物发现。线粒体功能障碍和相关的氧化应激会导致内皮功能障碍,并且是CAD发生和发展的重要因素。内皮集落形成细胞(ECFC)可以来自外周血单核细胞(PBMC),并提供了一种独特的潜在个性化手段,用于研究靶向血管功能重要组成部分的新潜在疗法。我们描述了高通量和共聚焦OperaPhenix®高含量筛选系统在检查线粒体超氧化物(mROS)水平方面的应用,线粒体膜电位,同时建立的细胞系和患者来源的ECFCs中的线粒体面积。与传统的读板器不同,OperaPhenix®是一种集成了自动共聚焦显微镜的成像系统,精确的荧光检测,和多参数算法,在细胞水平上可视化和精确量化目标生物过程。在这项研究中,我们使用mROS产生探针测量人脐静脉内皮细胞(HUVECs)和患者来源的ECFCs中的mROS产生,MitoSOXTM红。暴露于氧化低密度脂蛋白(oxLDL)的HUVEC使mROS水平增加47.7%(p<0.0001)。来自患有CAD的参与者(n=14)的患者来源的ECFCs的汇集组表现出比用oxLDL刺激时没有CAD的患者高30.9%的mROS水平(n=14;p<0.05)。当针对一小组候选化合物进行测试时,该信号被PKT-100衰减(减少36.22%,p=0.03),一种新型P2X7受体拮抗剂。这表明P2X7受体是对抗过量mROS水平的有效靶标。因此,这些发现凸显了MitoSOX-OperaPhenix技术在CAD中用于药物发现工作的潜力。
Improved human-relevant preclinical models of coronary artery disease (CAD) are needed to improve translational research and drug discovery. Mitochondrial dysfunction and associated oxidative stress contribute to endothelial dysfunction and are a significant factor in the development and progression of CAD. Endothelial colony-forming cells (ECFCs) can be derived from peripheral blood mononuclear cells (PBMCs) and offer a unique potentially personalised means for investigating new potential therapies targeting important components of vascular function. We describe the application of the high-throughput and confocal Opera Phenix® High-Content Screening System to examine mitochondrial superoxide (
mROS) levels, mitochondrial membrane potential, and mitochondrial area in both established cell lines and patient-derived ECFCs simultaneously. Unlike traditional plate readers, the Opera Phenix® is an imaging system that integrates automated confocal microscopy, precise fluorescent detection, and multi-parameter algorithms to visualize and precisely quantify targeted biological processes at a cellular level. In this study, we measured mROS production in human umbilical vein endothelial cells (HUVECs) and patient-derived ECFCs using the
mROS production probe, MitoSOXTM Red. HUVECs exposed to oxidized low-density lipoprotein (oxLDL) increased
mROS levels by 47.7% (p < 0.0001). A pooled group of patient-derived ECFCs from participants with CAD (n = 14) exhibited 30.9% higher
mROS levels compared to patients with no CAD when stimulated with oxLDL (n = 14; p < 0.05). When tested against a small group of candidate compounds, this signal was attenuated by PKT-100 (36.22% reduction, p = 0.03), a novel P2X7 receptor antagonist. This suggests the P2X7 receptor as a valid target against excess
mROS levels. As such, these findings highlight the potential of the MitoSOX-Opera Phenix technique to be used for drug discovery efforts in CAD.