Tightening the barrier: developing techniques for obtaining physiological mass transfer properties of endothelium in vitro

Cells in our body require a continuous supply of water, fuel, vitamins, and many other substances, and a continuous removal of waste products. When this supply or removal goes wrong, diseases can result, including the disease that causes heart attacks and strokes. The water and other substances are transported around the body as part of our blood. In order to get to the cells, they therefore need to cross the walls of the blood vessels through which the blood flows. The most difficult step is to get across the sheet of endothelial cells, called the endothelium, that lines the inner surface of all our blood vessels. Although movement across the endothelium is important to us, there are many things about it that we do not understand, so we cannot develop drugs to treat the diseases caused by problems of supply or removal. We don¿t know enough because these processes are hard to investigate. If we try to study them in living animals, then we cannot separate movement across endothelial cells from movement across the layers of our blood vessels that lie immediately under the endothelium. Also, it is impossible to get accurate information about things like blood flow next to the wall or the surface area of the wall. The ideal solution would be to grow endothelial cells out of the body, using methods called cell culture. We could grow the cells until they formed a continuous sheet and then measure how fast water and dissolved substances travel across it. This has been tried many times, but unfortunately the endothelium seems to be up to 1000 times leakier out of the body than in it; we cannot trust the results from such experiments. There is an urgent need to develop better methods. In this proposal, we suggest that the problems occur because the culture conditions are unlike the conditions inside the body. We want to see if making these conditions closer to those in the body will give us an endothelium in culture that behaves properly. We will try: (i) taking care that our cultures are not contaminated by other types of cell that would disrupt the endothelium, (ii) making sure that the substances whose transport we are studying are present at the concentrations at which they occur in the body, in case there movement across the cells gets relatively slower as their concentrations rise (because the pathways get full up), (iii) improving the culture solutions: in particular, culture solutions usually contain a liquid obtained from clotted blood, but clotting may itself release chemicals that increase endothelial leakiness, (iv) exposing the endothelial cells to flow and pressure, just as if they were growing inside our blood vessels, (v) making sure that the cells do not get too much oxygen: cells in culture are normally exposed to air, which contains more oxygen than our blood and may increase leakiness, and (vi) adding the naturally-occurring components of our blood which reduce inflammation (cells in culture can be a little inflamed, and this increases leakiness). If the project were successful, it would reduce the need for animal experiments, and it would give us a cheap, convenient and accurate way of studying how substances cross the endothelium; in the long term, this might allow us to develop new drugs. The project is also interesting because it lies on the boundary between engineering and biology (and would be carried out by engineers and biologists working together); research that crosses boundaries is being encouraged because it often leads to discoveries that could not be made within a single subject.

Exchange of water and solutes between blood and tissue is essential to the growth, maintenance and control of cells. Disorders of such exchange play a key role in many pathological states, particularly atherosclerosis, the disease underlying most heart attacks and strokes. The rate-limiting step in such exchange is transport across the endothelium, a sheet of cells lining the inner surface of blood vessels. Despite its importance, many aspects of this transendothelial mass transfer are poorly understood: even, for example, whether macromolecules travel across endothelial cells or between them. This reflects the technical difficulty to studying such mass transfer. In vivo, tissue components underlying the endothelium always affect transport. It is also difficult to control many variables (eg. Wall shear stress, osmotic gradients, endothelial area), and impossible to modify cell processes with expensive or toxic reagents. Most of these problems remain when using artificially perfused vessels. The ideal solution would be to study mass transfer across monolayers of cultured endothelium. Although this approach has been extensively used, the monolayers are up to 1000x more permeable than in vivo, the transport routes are likely to differ from those in vivo, and culture conditions may modify even the direction of change induced by agonists. There is an urgent need for better models. Our hypothesis is that endothelial monolayers have elevated permeabilities in vitro because they are exposed to nonphysiological conditions and our aim is to obtain realistic permeabilities by modifying these conditions. We propose to test novel methods, and to test novel hypotheses that appear to explain inconsistent results obtained in previous attempts. More specifically, we will investigate effects (i) of growing endothelial monolayers free of contaminating cells, since even small numbers will substantially increase permeability, (ii) of using physiological concentrations of macromolecules whose transport is being investigated, in case saturability of transport pathways gives concentration-dependent permeabilities, (iii) of removing potential permeability-increasing compounds from the serum applied to cells, (v) of applying to the cells chronic, physiologically-realistic mechanical stresses, since their absence in conventional culture may alter phenotype, (v) of culturing cells at physiological Po2 levels, rather than the elevated levels conventionally used, and (vi) of exposing cells to physiological concentrations of endogenous anti-inflammatory compounds, to reduce the activation occurring in culture. The proposal fulfils the EBS Committee remit because it studies mass transfer processes: a classical engineering field: in a biological context (and rests on a collaboration between bioengineers and cell biologists) and because it attempts both to test hypotheses and to improve analytical technologies. It is greatly strengthened by the partnership of the researcher who has obtained the tightest in vitro monolayers to date and who has developed specialised apparatus for assessing mass transfer in vitro; we would be starting from the state of the art. If successful, the project would (i) revolutionise the study of transendothelial mass transfer by developing cheap and convenient methods that also are rigorous and permit interventions not possible in vivo, (ii) reduce the need for animal experiments, and (iii) allow high-throughput development of drugs for controlling transport and hence disease. Subsidiary benefits would be the fostering of collaborations with long-term potential, the introduction of cell techniques and facilities into an engineering department where they would be widely used, and the improvement of culture conditions for studies of endothelial properties other than permeability.