Vidanapathirana Achini K, Goyne Jarrad M, Williamson Anna E, Pullen Benjamin J, Chhay Pich, Sandeman Lauren, Bensalem Julien, Sargeant Timothy J, Grose Randall, Crabtree Mark J, Zhang Run, Nicholls Stephen J, Psaltis Peter J, Bursill Christina A
Vascular Research Centre, Lifelong Health Theme, South Australian Health and Medical Research Institute, Adelaide, SA 5000, Australia.
Australian Research Council (ARC) Centre of Excellence for Nanoscale BioPhotonics (CNBP), Adelaide, SA 5000, Australia.
Biomedicines. 2022 Jul 27;10(8):1807. doi: 10.3390/biomedicines10081807.
Macrophage-derived nitric oxide (NO) plays a critical role in atherosclerosis and presents as a potential biomarker. We assessed the uptake, distribution, and NO detection capacity of an irreversible, ruthenium-based, fluorescent NO sensor (Ru-NO) in macrophages, plasma, and atherosclerotic plaques. In vitro, incubation of Ru-NO with human THP1 monocytes and THP1-PMA macrophages caused robust uptake, detected by Ru-NO fluorescence using mass-cytometry, confocal microscopy, and flow cytometry. THP1-PMA macrophages had higher Ru-NO uptake (+13%, p < 0.05) than THP1 monocytes with increased Ru-NO fluorescence following lipopolysaccharide stimulation (+14%, p < 0.05). In mice, intraperitoneal infusion of Ru-NO found Ru-NO uptake was greater in peritoneal CD11b+F4/80+ macrophages (+61%, p < 0.01) than CD11b+F4/80− monocytes. Infusion of Ru-NO into Apoe−/− mice fed high-cholesterol diet (HCD) revealed Ru-NO fluorescence co-localised with atherosclerotic plaque macrophages. When Ru-NO was added ex vivo to aortic cell suspensions from Apoe−/− mice, macrophage-specific uptake of Ru-NO was demonstrated. Ru-NO was added ex vivo to tail-vein blood samples collected monthly from Apoe−/− mice on HCD or chow. The plasma Ru-NO fluorescence signal was higher in HCD than chow-fed mice after 12 weeks (37.9%, p < 0.05). Finally, Ru-NO was added to plasma from patients (N = 50) following clinically-indicated angiograms. There was lower Ru-NO fluorescence from plasma from patients with myocardial infarction (−30.7%, p < 0.01) than those with stable coronary atherosclerosis. In conclusion, Ru-NO is internalised by macrophages in vitro, ex vivo, and in vivo, can be detected in atherosclerotic plaques, and generates measurable changes in fluorescence in murine and human plasma. Ru-NO displays promising utility as a sensor of atherosclerosis.
巨噬细胞衍生的一氧化氮（NO）在动脉粥样硬化中起关键作用，并作为一种潜在的生物标志物。我们评估了一种不可逆的、基于钌的荧光NO传感器（Ru-NO）在巨噬细胞、血浆和动脉粥样硬化斑块中的摄取、分布及NO检测能力。在体外，将Ru-NO与人THP1单核细胞和THP1-PMA巨噬细胞孵育会导致大量摄取，通过质谱流式细胞术、共聚焦显微镜和流式细胞术利用Ru-NO荧光进行检测。THP1-PMA巨噬细胞比THP1单核细胞具有更高的Ru-NO摄取量（增加13%，p<0.05），脂多糖刺激后Ru-NO荧光增加（增加14%，p<0.05）。在小鼠中，腹腔注射Ru-NO发现腹膜CD11b+F4/80+巨噬细胞中的Ru-NO摄取量比CD11b+F4/80−单核细胞更大（增加61%，p<0.01）。将Ru-NO注入喂食高胆固醇饮食（HCD）的Apoe−/−小鼠体内，发现Ru-NO荧光与动脉粥样硬化斑块巨噬细胞共定位。当将Ru-NO离体添加到Apoe−/−小鼠的主动脉细胞悬液中时，证实了巨噬细胞对Ru-NO的特异性摄取。将Ru-NO离体添加到每月从喂食HCD或普通饲料的Apoe−/−小鼠采集的尾静脉血样中。12周后，喂食HCD的小鼠血浆Ru-NO荧光信号高于喂食普通饲料的小鼠（37.9%，p<0.05）。最后，在临床指示的血管造影后，将Ru-NO添加到患者（N=50）的血浆中。心肌梗死患者血浆中的Ru-NO荧光低于稳定型冠状动脉粥样硬化患者（-30.7%，p<0.01）。总之，Ru-NO在体外、离体和体内均可被巨噬细胞内化，可在动脉粥样硬化斑块中检测到，并在小鼠和人类血浆中产生可测量的荧光变化。Ru-NO作为一种动脉粥样硬化传感器显示出有前景的应用价值。
