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Vascular Biophysics and Human Function - People

Angela Shore	Peter Winlove	Jacqueline Whatmore	Kenton Arkill	Julian Moger
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Alexandra Kendall	Clare Thorn	Hayley Kyte	Rebecca Sulley	Jessica Mansfield	Sterenn Le Nen Davey	Elly Martin	Rachel Palfrey

Research Interests:
The small blood vessels, the microcirculation, are crucial for tissue health.  Nutrients are supplied to tissues via the microcirculation e.g. oxygen and glucose, and waste products are taken away.  Abnormalities of the microcirculation cause many clinical effects such as blindness in diabetic retinopathy; kidney disease or poor wound healing.  Perhaps somewhat surprisingly, although it plays such a vital role there are many aspects of normal physiology of the microcirculation which are not known.

I have a wide range of research interests which seek to identify normal microvascular function and how these are altered in disease states.  A particular interest of mine is in the regulation of the pressure in the smallest blood vessels, the capillaries.  My group is unique world wide in examining this.  Elevations of capillary pressure are likely to cause activation of endothelial cells, increases in the production of adhesion markers, increased production of basement membrane components an increased movement of fluid across the blood/tissue interface and alter adhesiveness of the blood vessel wall to white cells.  Such changes may contribute to microvascular disease.

Studies in vivo in man directly measure capillary pressure in the capillaries of the skin to establish whether such haemodynamic changes may be involved in the microvascular damage in diabetes or those at risk of diabetes.  The effects of lifestyle or therapeutic interventions on these parameters are also being examined.

In collaboration with Endothelial Cell Biology and Biophysics we are exploring the effects of such perturbations in pressure on endothelial cell metabolism in vitro (see Rebecca Sulley below).

Other interests include exploring ethnicity effects on microvascular function in collaboration with Professor Nish Chaturvedi and Professor Alun Hughes, Imperial College, London.  Risk of cardiovascular disease differs in ethnic groups and our work demonstrates that impairments in the microvasculature also occur and are not explained by traditional risk factors for cardiovascular disease such as obesity and high lipids.
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My research concerns mainly the physical properties of the extracellular matrix and its constituent macromolecules. Concentrating largely on the relationships between tissue structure, mechanical properties and permeability to water and solutes this work aims to clarify the role of physical factors in diseases such as atherosclerosis and arthritis. At the molecular level I am interested in structure-function in elastic proteins. I also work on the physical properties of the plasma membranes of blood-and connective tissue cells. Establishing the molecular basis of their remarkable rheoviscous properties and their contributions both to the mechanical properties of the cell and to processes such as mechanotranduction and signaling is of particular interest. In the course of this research I have been active in the development and application of novel physical methods to physiological problems and have worked extensively on the development of electrochemical and optical sensors for biological investigations. I am currently particularly concerned with the development of techniques of multiphoton microscopy and spectrometry.

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Research Interests:
My research interests focus on understanding the biology of human endothelial cells. These cells form a continuous monolayer lining the inner surface of all blood vessels and lie at the interface of the flowing blood and the underlying vessel wall.  The endothelium is both exquisitely sensitive to blood flow and its associated wall shear stress and is highly metabolically active and thus plays a major role in regulating many essential features of blood vessels including:   

(i)  Control of blood vessel wall permeability by forming a barrier for solutes, macromolecules and blood cells between the vessel lumen and the interstitial space.

(ii)  Contributing to vascular tone by releasing potent vasodilators and vasoconstrictors.

(iii)  Maintaining an anticoagulant and antithrombotic surface in health through the secretion of specific anticoagulant substances.

(iv)  Regulation of inflammation through the controlled spatial-temporal expression of specific adhesion molecules that can interact with circulating white blood cells.

Healthy functioning of the vascular endothelium is essential for maintaining vascular health.  In response to inflammatory stimuli endothelial cells become activated - their barrier functions are disrupted and vasoconstriction, coagulation, proliferation and leukocyte adhesion are enhanced.  Although these responses exist as protective mechanisms, excess stimulation that is observed in many diseases induces these responses to become excessive, resulting in blood damaged vessels and impaired organ function.

Research in my group examines the role of human endothelial cells in the pathogenesis of various diseases, including the role of endothelial cells in:  the development of the vascular complications of diabetes; the accumulation of proinflammatory cells in chronic wounds through altered adhesion molecule expression; metastatic spread of tumour cells and also development of astrocytic scar tissue in multiple sclerosis.
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I am currently the interdisciplinary network postdoctoral fellow assigned to this pioneering project. The concept is to have a fluid process between the pure physical sciences and patient outcome. Besides my personal research interests (below), I have the role of helping combine the sciences. My initial tasks are to aid the new biomedical imaging suite in becoming suitable for handling living tissue. After this I shall be on the front line with the new combined projects between physics and the medical school. I also have the business of finding out what each individual department involved with this project can do, finding context for this in a different department and then the exciting part of doing some pilot experiments.

Microcirculation of Subchondral Bone
Osteoarthritis is a common chronic disease of articular cartilage which itself is an avascular viscoelastic tissue. The chondrocytes within the tissue can be in the order of millimetres from a nutrient source and this is hypothesised to be a factor in disease progression. The dogma is that the only source can be the synovial fluid due to the calcified cartilage between the articular cartilage and the subchondral bone being impermeable. We now believe that this is not the case having carried out extensive quantitative fluorescent microscopy experiments on tracers perfused into intact equine metacarpophalangeal joints. We have also found interesting evidence for fatty acids being transported more rapidly that their external partner albumin. We are currently working on improving imaging techniques for the calcified tissue to improve resolution. It would then be possible to understand the microscopic transport properties of the extracellular matrix around the subchondral bone microcirculation through the calcified layer to the articular cartilage.

Lymphatic Valves
The lymph system is a relatively understudied area. Blood vessels leak fluid into the interstitial tissue and the lymph system is designed to return this fluid back to the blood circulation. Faults with this system result in oedema, for example after breast cancer surgery. We are already completing a collaborative project between the biomedical physics group in physics and the computation fluid flow group in engineering to model the intrinsic pumping mechanism that the collecting lymphatics use to move the fluid. We have found that the valves within the vessel play an important role, but need to be better characterised for some of our computational models to be physiologically valid.

Endothelial cells that line the vessel walls are not always round but have a greater diameter in the direction of fluid flow across them. The first part of my work will be to visualise the shapes of the cells in and around the valves to validate (or not!) our computer flow models.
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My research concerns the development and application of optical techniques to aid the characterisation of biological tissues in both healthy and diseased states. Optical techniques offer many unique advantages over other approaches for studying biological systems, including structural and functional contrast, cellular level spatial resolution, near real-time analysis and non-invasiveness, all at a relatively low-cost.

My work currently focuses on the development of techniques in multiphoton imaging and spectroscopy. I have constructed and am currently managing the Multiphoton Imaging and Spectroscopy Laboratory within the Biophysics Group at the University of Exeter. This is an extremely flexible and expandable system combining several multiphoton mechanisms; Multiphoton Excitation (MPE), sum-frequency generation (SFG), and Coherent Anti-Stokes Raman Scattering (CARS). The simultaneous combination of these modalities offers unrivalled label-free imaging for a wide range of biophysical applications ranging from cell cultures to in-vivo tissue studies. I am currently not aware of a comparable facility in the UK and it will provide a unique tool for research in biophysics, biology and biomedicine.

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Human neutrophil-endothelial interactions in the presence of hyperbaric oxygen

Hyperbaric oxygen therapy (HBO) is the administration of 100% oxygen at a pressure greater than 1 atmosphere absolute. It is commonly used for the treatment of conditions such as decompression sickness and carbon monoxide poisoning, and has also shown great success in the treatment of chronic wounds. It is thought that the benefits of HBO in chronic wound treatment may involve various effects on neutrophils. This is because neutrophils have been strongly implicated in the inflammatory state of chronic wounds due to the accumulation of neutrophils at the wound site and their inadequate oxidative killing of microorganisms. HBO could affect a range of neutrophil functions, including adhesion to endothelial cells of the microvasculature (affecting transmigration of the neutrophils into the wound site), respiratory burst activity and apoptotic clearance, and these will be investigated both in vitro and ex vivo.

For in vitro experiments, human umbilical vein endothelial cell (HUVEC) monolayers, and neutrophils isolated from blood, will be exposed to various oxygen conditions at different pressures, to replicate conditions found in both hypoxic wounds and in typical HBO treatment regimes, as well as various pressure and oxygen controls. For ex vivo experiments, neutrophils will be isolated from volunteers pre- and post- treatment in Plymouth’s hyperbaric medical centre. The pro- and anti-inflammatory effects of HBO on neutrophils and endothelial cells will then be investigated using various techniques, including ELISAs and flow cytometry to quantify adhesion molecule expression, and assays to measure both respiratory burst activity and apoptosis in the neutrophils. The interaction of the two cell types in co-culture will be assessed microscopically, allowing any up- or down-regulation in neutrophil adhesion to endothelial cells to be visualised.
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A study of vasomotion in the microcirculation using Optical Reflectance Spectroscopy (ORS) and Optical Reflectance Spectroscopy (ORS)

The cardiovascular system is designed to deliver oxygen to every cell in the body through the microcirculation. Optical Reflectance Spectroscopy (ORS) is a powerful tool developed in the School of Physics and is used to study oxygen delivery through these vessels less than 50 μm in diameter.  Dysfunction in the microcirculation has been implicated as a cause of poor prognosis in intensive care patients and damage in the eyes, kidneys and nerves of diabetics. Blood flow in the microcirculation may be assisted by the spontaneous rhythmic changes in the diameter of these small vessels known as vasomotion. These oscillations have been linked to both endothelial and sympathetic activity and it has been suggested that vasomotion in muscle may preserves nutritive perfusion not only in the muscle itself but also to neighbouring tissue such as skin. This project aims to make a study of vasomotion in skin using ORS and in underlying muscle using another non-invasive optical technique Near Infrared Spectroscopy (NIRS). Both ORS and NIRS calculate changes in concentration of oxyhaemoglobin and deoxyhaemoglobin in the tissue and from this can be derived changes in blood volume and changes in mean blood oxygenation across the arterial and venous vessels. Frequency analysis of these blood volume oscillations has shown that different origins of vasomotion such as sympathetic and endothelial activity produce different frequencies of oscillation. This project aims to investigate the normal haemdynamics of vasomotion in the microcirculation and it’s response to a period of inadequate perfusion. Changes in vasomotion will be differentiated according to their origin and a combined study of ORS and NIRS will investigate the relationship of vasomotion in the skin and underlying muscle.  Clinical studies intend to study the dysfunctional microcirculation in diabetics and intensive care patients.
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The Actions of Insulin in the Human Microcirculation:  An In Vivo Study

The impaired insulin-induced uptake of glucose to skeletal muscle is a characteristic of insulin resistance.  Insulin resistance is strongly associated with obesity and other metabolic disorders including type two diabetes mellitus, hypertension, dyslipidaemia and an elevated risk of cardiovascular disease.  Insulin is vasoactive; it is able to induce both vasodilatation and vasoconstriction, however, the mechanism by which insulin mediates these effects is poorly understood.  Diabetics suffer from microvascular complications and it is thus plausible that altered vascular function may be associated with insulin resistant states.  Existing research has largely involved the use of animal tissue or large blood vessels, where as only limited research of the microvascular effects of insulin in vivo in humans have been conducted.  This study aims to directly examine the impact of insulin on capillary haemodynamics and to determine if they are altered in insulin resistance for example, in subjects with metabolic syndrome.  This will involve a series of investigations; firstly the effects of locally applied insulin on capillary haemodynamics will be studied by the measurement of capillary pressure and capillary blood cell velocity in both healthy and insulin resistant subjects.  Secondly, the effects of insulin on tissue oxygenation and blood volume in skin and skeletal muscle will be compared in both healthy and insulin resistant subjects.  In addition, the study will investigate the potential underlying mechanisms of insulin by the use of specific inhibitors of for example, nitric oxide, endothelium-derived hyperpolarising factors, prostaglandins and endothelin.
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The Impact of Hydrostatic Pressure on the Metabolic Pathways of Human Microvascular Endothelial Cells

Diabetes affects the body’s ability to uptake glucose into the cells. Previous research has indicated that mechanical stretch leads to increased glucose transport through TGF-β1 dependant up regulation of the glucose transporter GLUT-1 (Gnudi, L. et al, 2003). Diabetic patients commonly experience problems with high blood pressure (hypertension), therefore controlled blood pressure reduces the risks of long term microvascular complications. Raised capillary pressure has been implicated in the formation of diabetic microangiopathy in type-1 diabetes (Fegan, P.G. et al, 2003). In addition capillary compliance is altered in diabetes due to basement membrane thickening.

The aim of the project is to examine whether the introduction of hydrostatic pressures and stretch, similar to those experienced by human microvascular endothelial cells, influences the uptake and metabolism of glucose and fatty acids. These changes could cause oxidative stress that may accelerate diabetic microangiopathy.

A haemodynamic model will be built, that has the ability to independently vary pressure, wall deformation and fluid flow. Human microvascular endothelial cells (HMVECs) will be grown on the inner surface of compliant 4mm diameter silicone tubing before being placed in the haemodynamic model, where the HMVECs will be exposed to pressures, similar to those found in the capillaries, i.e. 15, 25 and 35 mmHg. Stretch of 5% and 10% will also be applied to the tubing, either alone or in combination with pressure changes, to imitate changes in vessel diameter. Glucose and fatty acid uptake as well as oxidation, fatty acid storage and GLUT-1 expression will be examined.
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Non-linear Microscopy

The initial part of this PhD project was to construct a prototype non-linear microscope and use it to image tissues with a high collagen content. This has been completed and the next stage is to transfer imaging to the new multi-photon microscopy suite.

Imaging has been mainly focused on articular cartilage to see if this technique can show early changes related to osteoarthritis. Cartilage has been imaged with both second harmonic generation (SHG) and two photon fluorescence (TPF), with the SHG showing the collagen fibres and the TPF mapping endogenous fluorophores. In these images the cells can clearly be resolved and in the TPF rings of increased intensity can be seen surrounding the cells corresponding to the pericellular matrix. Images at lesion sites show changes in both in the structure of the matrix and the cells. Additional information on the order of the collagen fibres is provided by polarization sensitive SHG microscopy, as the SHG intensity depends on the polarization of the excitation laser beam with respect to the collagen fibres. Therefore polarization sensitive studies of the SHG have been used to see structural changes which occur in cartilage at an osteoarthritic lesion.

Other collagen based tissues such as pericardium, intervertebral disc and arteries are also being investigated. Non-linear microscopy is a very promising technique for these tissues as both the collagen and elastin fibres in these tissues can be clearly seen and distinguished with the collagen fibres producing SHG and the elastin fibres producing TPF at a longer wavelength.
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Biophysical properties of the vascular endothelial cell membrane

Endothelial cells lining the vasculature are exposed to a unique and complex profile of haemodynamic forces imposed by the flowing blood and mechanical strains arising from the deformation of the underlying tissue. The cells respond to these forces both chronically, initiating vasoregulatory responses by signaling to underlying smooth muscle stimulating changes in vessel diameter and local perfusion, as well as through acute angiogenic responses. My project concerns the molecular mechanisms of mechanotransduction through the alteration of biophysical properties of the endothelial cell membrane, focusing in particular on the relationship between structural and biophysical properties and endothelial function. Changes in both the viscosity of the lipid bilayer and the physical properties of the glycocalyx have been hypothesized as methods of mechanotransduction.

Our initial aim is to test these hypotheses in cultured cells exposed to different mechanical forces. Firstly, we will attempt to characterize the spatial and temporal heterogeneity of the endothelial cell membrane by observing the partitioning of fluorescent probes within the membrane and their diffusivity using fluorescence recovery after photobleaching (FRAP). We shall address the fundamental question of reconciling membrane viscosity with the emerging views of a spatially and temporally heterogeneous membrane. We will then attempt to exploit developments in near field and multiphoton microscopy to further probe membrane dynamics. Biophysical properties of both normal cells and cells cultured in a diabetic milieu will be observed with the aim of clarifying some of the abnormal responses associated with vascular disease in diabetic patients.
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The effects of ultrasound on the vascular wall

Ultrasound is used widely for both clinical diagnostic and therapeutic applications.  The need for an understanding of the safety of diagnostic ultrasound and the mechanisms of therapeutic ultrasound has led to experiments which have shown that cells do respond to ultrasound directly.  Many of these experiments have been performed on cells in culture rather than on tissue systems.  Investigations into ultrasound bioeffects have previously mainly concentrated on microbubble formation and cavitation (oscillation and possible collapse of microbubbles) and generation of free radicals.  It is possible that cellular responses to ultrasound are caused by other mechanisms such as direct transduction of physical forces.  Cellular responses to mechanical and electrical forces, and to pressure, have a role in the growth and development of tissue systems.  As a consequence, mechanical forces on the vascular system may have implications in safety of ultrasound and in the context of therapeutic ultrasound.

The work in this study will focus on the response of sections of artery to ultrasound. Muscular artery will be used because of its structure of endothelial cells and smooth muscle cells, both of which are known to be responsive to physical forces.  Preliminary experiments have shown that ultrasound causes a repeatable, reversible contraction of the equine carotid artery wall in vitro.  The effect was still seen when the endothelium was removed, but not when the tissue was dead.  Thermal effects and radiation pressure have been ruled out as possible causes of the effect. 

The study will concentrate on further investigation of this effect and its dependence on ultrasound exposure parameters, the contribution of endothelial cells and smooth muscle cells, and investigation of the mechanisms causing the response.  The results of this study may be of use in obtaining better estimates of the risk associated with diagnostic ultrasound applications.  This work may also contribute to the development of new applications of ultrasound for therapy and research. 
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Nutrition of the intervertebral disc

Back pain is a serial problem within the western world and has been linked to disc degeneration which has in turn been associated with poor nutrition of the disc. It has long been established that the intervertebral disc is the largest avascular structure within the human body. How, and where, the disc exchanges its metabolites has long been a source of interest. It is known that the blood supply to the disc is achieved by vertebral arteries which divide into two vertebral plexuses, but the extent of the capillaries supplying the disc is unknown. By perfusing intact equine tails with a mixture of resin and a radio-opaque iodine compound I hope to discover the morphology of these capillary beds using x-ray. I then hope by using various sized x-ray opaque microspheres placed within the blood supply to not only visualise the microcirculation but to start to understand the flow dynamics around the vertebrae. I will also employ other complimentary techniques. In the first instance I will use fluorescent tracers to investigate and barriers within the microcirculations. These studies will then progress to dynamic MRI whereby contrast agents of manganese and iodine will be used to look at transport processes within the disc. An MRI compatible loading mechanism will also be used to investigate the effects of mechanical loading.

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