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。这些细胞创造了一种半透屏障，将血液与体内所有其他组织隔开，任何在血液和组织之间传递的分子都必须穿过这个内皮细胞。因此，内皮细胞处于一个关键的界面上，在那里它扮演着“守门人”的角色，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。内皮屏障的破坏是动脉粥样硬化中动脉壁脂质堆积、组织水肿中的组织肿胀、炎症中的血管渗漏和癌症转移等疾病的发病机制。因此，维持内皮细胞屏障是动态平衡的一个关键方面，但我们对内皮细胞屏障的了解仍然不完全。内皮细胞及其产生的屏障对机械力非常敏感。通常，这些力来自血液流过内皮细胞引起的剪切力或粘性阻力，以及血压施加在壁上的拉伸。结合化学因素，剪切和拉伸调节内皮功能的不同方面(例如，两者都影响对准、收缩和细胞-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，血流紊乱引起的机械力改变被认为是内皮屏障功能障碍的原因，导致动脉壁脂质的渗透和积聚。其他理论描述了脂质渗透如何与内皮细胞所经历的拉伸障碍有关。然而，要研究这些假说，我们需要有一个可靠的工具来测量不同水平的剪切和拉伸时通过内皮的运输速度。在这个项目中，我们开发了一种基于微流体的技术来研究切应力和拉伸如何影响内皮通透性，内皮通透性是控制动脉粥样硬化早期阶段脂质渗入动脉壁的参数。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，同时提供可以实时测量的渗透性的定量读数。微流控平台的设计完全可扩展，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控装置。我们将通过证明该设备能够在没有剪切和拉伸的情况下再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联合剪切和拉伸对内皮通透性的影响。

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.