A Biomimetic Microfluidics Platform for High-Throughput Screening of Endothelial Barrier Dysfunction, with Applications to Atherosclerosis

你体内的所有血管都有一层连续的内皮细胞。这些细胞形成了一层半透性屏障，将血液与体内所有其他组织隔开，任何在血液和组织之间流动的分子都必须穿过这层内皮。因此，内皮处于一个关键的界面，它的功能是“看门人”，调节血液和体内所有血管外组织之间的水、细胞和营养物质的运输。内皮屏障的破坏有助于疾病的发病机制，如动脉粥样硬化时动脉壁的脂质积累，水肿时组织肿胀，炎症时血管渗漏，癌症时转移。因此，维持内皮屏障是体内平衡的一个关键方面，但我们对内皮屏障的理解仍然不完整。内皮细胞和它们形成的屏障对机械力非常敏感。通常，这些力是由血流过内皮细胞和血压对血管壁施加的拉伸引起的剪切或粘性阻力引起的。与化学因素结合，剪切和拉伸调节内皮功能的各个方面（例如，两者都影响排列、收缩性和细胞-细胞连接的强度）。内皮细胞的机械敏感性也与动脉粥样硬化有关，动脉粥样硬化是每年影响数百万人的心血管疾病、心脏病发作和中风的主要原因。在动脉粥样硬化中，由于血流紊乱引起的机械力改变被认为有助于内皮屏障功能障碍，导致动脉壁脂质浸润和积聚。其他理论描述了脂质浸润如何与内皮细胞所经历的拉伸干扰有关。然而，研究这些假设需要我们有一个可靠的工具来测量内皮在不同程度的剪切和拉伸下的运输速率。在这个项目中，我们开发了一种基于微流体的技术来研究剪切应力和拉伸如何影响内皮通透性，这是动脉粥样硬化早期控制脂质浸润到动脉壁的参数。我们的设计克服了以前体外模型的几个局限性，允许在生理范围内独立和同时控制剪切和拉伸，同时提供可实时测量的渗透性定量读数。微流体平台的设计是完全可扩展的，以允许高通量筛选或并行实验。在这个项目中，我们将开发和表征微流体装置。我们将通过证明该设备能够在没有剪切和拉伸的情况下重现内皮通透性的标准测量来验证该设备。最后，我们将使用该装置来确定剪切和拉伸联合作用对内皮细胞渗透性的影响。

All blood vessels in your body are lined with a continuous layer of endothelial cells. These cells create a semi-permeable barrier that separates blood from all other tissues in the body, and any molecule passing between blood and tissue must cross this endothelium. The endothelium therefore lies at a critical interface where it functions as "gatekeeper" to regulate the transport of water, cells and nutrients between blood and all extravascular tissues in the body. Disruption of the endothelial barrier contributes to the pathogenesis of disease such as lipid accumulation in the artery wall in atherosclerosis, tissue swelling in oedema, vascular leakage in inflammation, and metastasis in cancer. Thus, maintaining the endothelial barrier is a critical aspect of homeostasis, but our understanding of the endothelial barrier is still incomplete.Endothelial cells and the barrier that they create are exquisitely sensitive to mechanical forces. Typically these forces arise from shear or viscous drag caused by blood flowing over the endothelial cells and stretch imposed on the wall by blood pressure. In combination with chemical factors, shear and stretch regulate diverse aspects of endothelial function (e.g., both affect alignment, contractility, and the strength of cell-cell connections). The mechanical sensitivity of endothelial cells is also involved in atherosclerosis, the leading cause of cardiovascular disease, heart attacks and strokes that affects millions of people annually. In atherosclerosis, altered mechanical forces arising from disturbed blood flow are believed to contribute to dysfunction of endothelial barrier, leading to infiltration and accumulation of lipid in the artery wall. Other theories describe how lipid infiltration may be related to the disturbances in stretch experienced by the endothelium. Investigating these hypotheses, however, requires that we have a reliable tool to measure the rate of transport across the endothelium in response to different levels of shear and stretch. In this project, we develop a micro-fluidics based technology to examine how shear stress and stretch affect endothelial permeability, the parameter controlling lipid infiltration into the artery wall during the early stages of atherosclerosis. Our design overcomes several limitations of previous in vitro models by allowing independent and simultaneous control of both shear and stretch over the physiological range, while providing a quantitative readout of permeability that can be measured in real-time. The design of the microfluidics platform is fully scalable to allow for high-throughput screening or parallel experimentation. In this project, we will develop and characterise the microfluidics device. We will validate the device by demonstrating that it is able to reproduce standard measurements of endothelial permeability in the absence of shear and stretch. Finally, we will use the device to determine the effect of combined shear and stretch on endothelial permeability.