School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457.
Integr Biol (Camb). 2014 May;6(5):511-22. doi: 10.1039/c3ib40265g.
People suffering from Diabetes Mellitus (DM) are prone to an array of vascular complications leading to end organ damage. The hallmark of these vascular complications is endothelium dysfunction, which is caused by endothelial cell (EC) apoptosis. Although the endothelial cell (EC) dysfunction induced by hyperglycaemia and fluid shear stress has been studied, the effects of biological factors in the blood of DM patients on EC integrity have not been reported in the in vitro models that mimic the physiological pulsatile nature of the vascular system. This study reports the development of a hemodynamic lab-on-a-chip system to investigate this issue. The pulsatile flow was applied to a monolayer of endothelial cells expressing a fluorescence resonance energy transfer (FRET)-based biosensor that changes colour from green to blue in response to caspase-3 activation during apoptosis. Plasma samples from healthy volunteers and DM patients were compared to identify biological factors that are critical to endothelial disruption. Three types of microchannels were designed to simulate the blood vessels under healthy and partially blocked pathological conditions. The results showed that EC apoptosis rates increased with increasing glucose concentration and levels of shear stress. The rates of apoptosis further increased by a factor of 1.4-2.3 for hyperglycaemic plasma under all dynamic conditions. Under static conditions, little difference was detected in the rate of EC apoptosis between experiments using plasma from DM patients and glucose medium, suggesting that the effects of hyperglycaemia and biological factors on the induction of EC apoptosis are all shear flow-dependent. A proteomics study was then conducted to identify biological factors, demonstrating that the levels of eight proteins, including haptoglobin and clusterin, were significantly down-regulated, while six proteins, including apolipoprotein C-III, were significantly up-regulated in the plasma of DM patients compared to healthy volunteers. This hemodynamic lab-on-a-chip system can serve as a high throughput platform to assess the risk of vascular complications of DM patients and to determine the effects of therapeutics or other interventions on EC apoptosis.
患有糖尿病（DM）的人容易出现一系列血管并发症，导致终末器官损伤。这些血管并发症的标志是内皮细胞（EC）凋亡引起的内皮功能障碍，尽管已经研究了高血糖和流体切应力引起的内皮细胞（EC）功能障碍，但在体外模拟血管系统生理脉动性质的模型中，尚未报道 DM 患者血液中的生物因素对 EC 完整性的影响。本研究报告了一种血流动力学芯片实验室系统的开发，用于研究这个问题。搏动流应用于单层表达荧光共振能量转移（FRET）基生物传感器的内皮细胞，该传感器在凋亡过程中 caspase-3 激活时颜色从绿色变为蓝色。将来自健康志愿者和 DM 患者的血浆样本进行比较，以确定对内皮破坏至关重要的生物因素。设计了三种微通道来模拟健康和部分阻塞病理条件下的血管。结果表明，随着葡萄糖浓度和切应力水平的增加，EC 凋亡率增加。在所有动态条件下，高血糖血浆使凋亡率进一步增加了 1.4-2.3 倍。在静态条件下，DM 患者血浆和葡萄糖培养基中 EC 凋亡率的实验之间几乎没有差异，这表明高血糖和生物因素对诱导 EC 凋亡的影响均依赖于切应力。然后进行了蛋白质组学研究以鉴定生物因素，结果表明，与健康志愿者相比，在 DM 患者的血浆中，包括触珠蛋白和载脂蛋白 C-III 在内的六种蛋白质的水平显著上调，而包括结合珠蛋白和簇蛋白在内的八种蛋白质的水平显著下调。这种血流动力学芯片实验室系统可以作为一种高通量平台，评估 DM 患者血管并发症的风险，并确定治疗或其他干预措施对 EC 凋亡的影响。
