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

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.

Conventional in vitro biological assays rely largely on static and often non-physiological culture conditions that fail to adequately reproduce the complex microenvironment that strongly influences cellular function in vivo. This shortcoming reduces the predictive potential of in vitro models for in vivo outcomes and often leads to inflated or unnecessary animal experiments in the development of new therapies. Improved in vitro models that incorporate biomechanical and biochemical cues that mimic the in vivo microenvironment would provide better predictive models for human health and disease, improved prioritisation of candidate therapeutics, and a more complete understanding of pharmacological effects, ultimately reducing animal use and accelerating the development of therapeutic ideas from bench to bedside.

In this project, we apply this vision towards developing a more accurate in vitro model for endothelial permeability that controls lipid infiltration into the arterial wall during the early stages of atherosclerosis, the leading cause of cardiovascular disease, heart attack and stroke that affects the lives of tens of millions of people annually. Our in vitro model uses microfluidics to measure the rate of transport of a fluorescent tracer molecule across an endothelial monolayer, thereby providing an estimate of endothelial permeability that can be measured in real-time using a fluorimeter. Our model is scientifically and commercially unique because it overcomes several limitations of previous models (e.g., Huh et al., Science, 2010 v328 p1662) by allowing independent and simultaneous control over two key biomechanical stressors: shear stress and stretch that are known to influence endothelial permeability in vivo. The design of the microfluidics device is scalable to a high-throughput platform to allow parallel experimentation or therapeutic screening, which would find interest in both the academic and industrial communities alike.

Who are the beneficiaries of this research programme and how would they benefit? We have identified nearer-term (2-5 yrs) and longer-term (>5 yrs) beneficiaries.

The nearer-term beneficiaries are:

- academic researchers who are active in the area of cellular mechanobiology and who would use our device or build on our research to further explore the role of mechanical forces in endothelial or epithelial barrier function, as relevant for atherosclerosis, glaucoma, cancer, edema, ventilator induced lung damage, and other diseases;

- commercial partners who would license our intellectual property for commercial exploitation, either directly by providing hardware to reproduce our design in other academic or commercial laboratories or indirectly by exploiting the commercial benefit of therapeutic biomolecules identified using our device;

- animals because building a better in vitro model would reduce and partially replace experimental animal use (the 3 R's) for screening or testing compounds that affect the endothelial permeability barrier.

The longer-term beneficiaries are:

- patients who suffer from diseases related to endothelial dysfunction or altered mechanobiology, including atherosclerosis, glaucoma, cancer, edema, ventilator induced lung damage, and other diseases. Although we do not propose to screen or develop therapeutic biomolecules for this project, this would be a goal for future projects and may eventually lead to new drugs that, for example, may manipulate lipid infiltration and accumulation into the artery wall as a means to intervene and deter lesion development during the early stages of atherosclerosis.