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

Lead Research Organisation: Imperial College London
Department Name: Dept of Bioengineering

Abstract

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.

Planned Impact

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.

Publications

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Campbell IC (2018) Quantification of Scleral Biomechanics and Collagen Fiber Alignment. in Methods in molecular biology (Clifton, N.J.)

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Overby DR (2014) Altered mechanobiology of Schlemm's canal endothelial cells in glaucoma. in Proceedings of the National Academy of Sciences of the United States of America

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Reina-Torres E (2017) VEGF as a Paracrine Regulator of Conventional Outflow Facility. in Investigative ophthalmology & visual science

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Sherwood JM (2016) Measurement of Outflow Facility Using iPerfusion. in PloS one

 
Description We have developed technology to accurately measure the hydraulic properties of biological tissues, including not only cultured cell layers as relevant for atherosclerosis (as described in the original proposal), but also eyes as relevant for glaucoma, and lymph nodes and lymphatic vessels as relevant for lymphoedema. These latter applications were unexpected, and the hardware and associated software (which we labelled as 'iPerfusion') has been used to provide preliminary data for two funded proposals from our laboratory as Pi or co-PI (from the Brightfocus Foundation and the US National Institutes of Health), and two additional proposals as co-I (from the Fight for Sight Foundation (UK) and the US National Institutes of Health).
Exploitation Route Our work has already been put to use by other laboratories at Imperial, Ireland, and the US. The iPerfusion system is capable of measuring flows from the nL/min to mL/min range while controlling pressures in the 0.05 mmHg range. We required this precision for the microfluidics system, but as we experienced challenges with other aspects of the project, we realised that the iPerfusion system can be applied in other areas (namely, glaucoma, lymphoedema). We have already reproduced our iPerfusion systems for researchers using it to measure microscale fluid flows at Imperial and UC Dublin. Six other iPerfusion systems have been built for collaborators in the US . The iPerfusion system is ideal for screening drugs and for research into microvascular physiology, where there is a need for precision pressure and flow measurements.
Sectors Education,Pharmaceuticals and Medical Biotechnology

 
Description The iPerfusion system that was developed from the EPSRC funded grant has been reproduced in several leading glaucoma research labs in the US (Duke, GaTech, Jackson Labs), Ireland (Trinity), and UK (Imperial). Several other systems are 'on order'. Each system brings an income of approximately £30k to the Department, recouping costs associated with build time, consumables and continued service.
First Year Of Impact 2013
Sector Education,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description National Glaucoma Research Grant
Amount $100,000 (USD)
Funding ID G2105145 
Organisation BrightFocus Foundation 
Sector Charity/Non Profit
Country United States
Start 07/2015 
End 06/2017
 
Description Project Grant
Amount £168,765 (GBP)
Funding ID 1858 
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2016 
End 07/2019
 
Description R01 Standard Research Grant
Amount $2,100,000 (USD)
Funding ID R01EY022359 
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 09/2016 
End 07/2021
 
Description R21
Amount $275,000 (USD)
Funding ID EY026685 
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 07/2017 
End 07/2019
 
Title iPerfusion 
Description The technology provides a way to precisely control the pressure drop across a biological specimen while measuring the flow rate through the specimen with an accuracy of nL/min 
Type Of Material Improvements to research infrastructure 
Year Produced 2014 
Provided To Others? Yes  
Impact Research developing a molecular therapy to lower intraocular pressure in glaucoma (with collaborators from Trinity College Dublin). 
 
Title iPerfusion code 
Description The model fits pressure-flow perfusion data to various models to extract physiologically relevant parameters. 
Type Of Material Computer model/algorithm 
Year Produced 2014 
Provided To Others? Yes  
Impact Development of a molecular therapy for lowering IOP in glaucoma (with Trinity College Dublin) Development of a system to measure ocular compliance in mice (with Georgia Institute of Technology) 
 
Description Georgia Tech 
Organisation Georgia Institute of Technology
Country United States 
Sector Academic/University 
PI Contribution We have provided 2 versions of the iPerfusion system for use in glaucoma research at Georgia Institute of Technology.
Collaborator Contribution The collaborator provided students who collaborate with us on the technology development.
Impact This is a multi-disciplinary collaboration between bioengineers at Georgia Tech and Imperial College London.
Start Year 2012
 
Description Trinity College Dublin 
Organisation Trinity College Dublin
Department Institute of Neuroscience
Country Ireland 
Sector Hospitals 
PI Contribution We have contributed our iPerfusion system for measurements of ocular resistance in mice and monkeys as part of a collaborative project investigating molecular therapies for the treatment of glaucoma.
Collaborator Contribution The collaborators have contributed £20,000 for the construction of 4 iPerfusion systems. The collaborators have provided students to perfusion experiments using the iPerfusion system in mice. The goal of these studies is to use gene silencing approaches to induce pore formation in Schlemm's canal cells.
Impact This is a multi-diciplinary collaboration, involving genetics and molecular cell biology from Trinity College Dublin with engineering, microfluidics expertise at Imperial College London.
Start Year 2012
 
Title iPerfusion system 
Description licensing of software for measuring microscale pressure/flow relationships in biological systems 
IP Reference  
Protection Copyrighted (e.g. software)
Year Protection Granted 2013
Licensed Yes
Impact identification of physiological and pharmacological factors controlling outflow facility
 
Title iPerfusion code 
Description The software allows extraction of physiologically relevant parameters from pressure-flow perfusion data. 
Type Of Technology Software 
Year Produced 2013 
Impact This software is key to the operation of the iPerfusion system. This system led to several collaborative projects. 
 
Description ICER talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact talk lead to collaborations with others interested in using iPerfusion

After the talk, researchers approached us asking to collaborate.
Year(s) Of Engagement Activity 2013