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No description available.

No description available.

The airway epithelium is the first point of contact in the lung for inhaled material, including infectious pathogens and particulate matter, and protects against toxicity from these substances by trapping and clearance via the mucociliary escalator, presence of a protective barrier with tight junctions and initiation of a local inflammatory response. The inflammatory response involves recruitment of phagocytic cells to neutralise and remove and invading materials and is oftern modelled using rodents. However, development of valid in vitro airway epithelial models is of great importance due to the restrictions on animal studies for cosmetic compound testing implicit in the 7th amendment to the European Union Cosmetics Directive. Further, rodent innate immune responses have fundamental differences to human. Pulmonary endothelial cells and leukocytes are also involved in the innate response initiated during pulmonary inflammation. Co-culture models of the airways, in particular where epithelial cells are cultured at air liquid interface with the presence of tight junctions and differentiated mucociliary cells, offer a solution to this problem. Ideally validated models will allow for detection of early biomarkers of response to exposure and investigation into inflammatory response during exposure. This thesis describes the approaches taken towards developing an in vitro epithelial/endothelial cell model of the human airways and identification biomarkers of response to exposure to xenobiotics. The model comprised normal human primary microvascular endothelial cells and the bronchial epithelial cell line BEAS-2B or normal human bronchial epithelial cells. BEAS-2B were chosen as their characterisation at air liquid interface is limited but they are robust in culture, thereby predicted to provide a more reliable test system. Proteomics analysis was undertaken on challenged cells to investigate biomarkers of exposure. BEAS-2B morphology was characterised at air liquid interface compared with normal human bronchial epithelial cells. The results indicate that BEAS-2B cells at an air liquid interface form tight junctions as shown by expression of the tight junction protein zonula occludens-1. To this author’s knowledge this is the first time this result has been reported. The inflammatory response of BEAS-2B (measured as secretion of the inflammatory mediators interleukin-8 and -6) air liquid interface mono-cultures to Escherichia coli lipopolysaccharide or particulate matter (fine and ultrafine titanium dioxide) was comparable to published data for epithelial cells. Cells were also exposed to polymers of “commercial interest” which were in the nanoparticle range (and referred to particles hereafter). BEAS-2B mono-cultures showed an increased secretion of inflammatory mediators after challenge. Inclusion of microvascular endothelial cells resulted in protection against LPS- and particle- induced epithelial toxicity, measured as cell viability and inflammatory response, indicating the importance of co-cultures for investigations into toxicity. Two-dimensional proteomic analysis of lysates from particle-challenged cells failed to identify biomarkers of toxicity due to assay interference and experimental variability. Separately, decreased plasma concentrations of serine protease inhibitors, and the negative acute phase proteins transthyretin, histidine-rich glycoprotein and alpha2-HS glycoprotein were identified as potential biomarkers of methyl methacrylate/ethyl methacrylate/butylacrylate treatment in rats.

Despite their unique structure, function, origin and role in disease, glomerular endothelial cells (GEnC) have been little studied because of difficulty maintaining them in cell culture. This problem has been addressed by the generation of conditionally immortalised GEnC. Endothelial behaviour is potentially dependent on interactions with podocytes, with communication occurring through growth factors including angiopoietins and vascular endothelial growth factor (VEGF). Understanding the effect of these mediators is the first step towards developing a co-culture system simulating the in vivo filtration barrier. In addition the effect of interferon beta (IFNβ) in amelioration of proteinuria in 3 distinct animal models of nephritis led to an investigation into the mechanism of this effect in human podocytes and GEnC in culture Cell culture and co-culture techniques were used to characterise ciGEnC, assess the effects of podocyte-conditioned medium on ciGEnC monolayers and examine the effect of IFNβ oniGEnC and podocyte monolayers.

The vascular system is a complex, highly branched tubular network that is able to supply oxygen and nutrients to all body tissues and remove waste. During growth and tissue repair the vasculature undergoes coordinated expansion through a process known as angiogenesis, which involves the sprouting and branching of new blood vessels from pre-existing vasculature. Vascular endothelial growth factor A (VEGF-A) is the most potent inducer of angiogenesis. VEGF-A regulates blood vessel formation through the activation of three related tyrosine kinase receptors: VEGF receptors (VEGFRs)-l, -2, and -3. The main pro-angiogenic receptor expressed by endothelial cells is VEGFR-2. Upon ligand binding, VEGFR-2 activates multiple signalling pathways, which ultimately leads to the development of new blood vessels. The formation of VEGF-A concentration gradients plays an important role in the regulation of blood vessel growth. Endothelial cells in growing vascular sprouts modify their behaviour in response to their position within a VEGF-A gradient. I show that high doses of VEGF-A (40 ng/ml) remove 80% ofVEGFR-2 from the surface of human umbilical vein endothelial cells (HUVECs) over 2 h, leading to the rapid attenuation of VEGF-A-induced receptor signalling. In contrast, low doses of VEGF-A (2 ng/m!) trigger little receptor internalisation, with 10% of VEGFR-2 being lost over the same period. Although low doses preserve VEGFR-2 surface expression, receptor phosphorylation and p44/42 MAPK activation are down regulated to the same extent as high dose signals. Experiments show that surface-expressed VEGFR-2 remains active after prolonged exposure to 2 ng/ml ofVEGF-A; however, ELISA assays demonstrate that endothelial cells remove/sequester ~ 1 ng/ml VEGF-A from cell media. The termination of VEGF-A-induced signalling is therefore believed to result from either the inactivation or sequestration of extracellular VEGF-A. However, neither sequestration by soluble VEGFR-1 nor proteolysis by plasmin or matrix metalloproteinases can account for the loss of VEGF-A from cell media. Immunofluorescence microscopy shows that significant amounts of extracellular VEGF-A is localised to endothelial cell-cell junctions. However, junctional VEGF-A binding cannot be explained by key VEGF-A receptors or co-receptors, including VEGFR-1, VEGFR-2, neuropilin-1, vascular endothelial cadherin, {3 1 integrin, {3 3 integrin, glypicans-1 -6, and syndecans- 1-4. Proteomic analysis has identified the recently characterised VEGF-AIVEGFRbinding proteins CD146 and multimerin-2 as potential mediators of VEGF-A binding to the surface of endothelial cells. Future work will focus on elucidating their roles as regulators of extracellular VEGF-A distribution and/or concentration.

The endothelium is the primary regulator of microvascular permeability whilst fulfilling other functions by controlling coagulation, inflammation and vascular tone. The glycocalyx covers the luminal side of the endothelia and is an important contributor to the endothelia function. The glomerulus is a highly specialised microcirculation in which the capillary wall, including the endothelium, is uniquely adapted to function as a biological sieve and filters 180 litres of plasma every day. This sieving action of the glomerular capillary is highly efficient and selective: allowing free flow of water and small solutes and highly restrictive to passage of proteins. Alterations in this functions leads to proteinuria and heralds the onset of disease. The glomerular endothelial cells possess transcellular pores or fenestrations to facilitate high hydraulic permeability and like all endothelia, also possess a glycocalyx layer that covers both fenestral, and non-fenestral domains. Unlike the endothelial glycocalyx in systemic vessels, the glomerular endothelial glycocalyx has not been subjected to detailed and direct scientific scrutiny. The aim of this thesis is to examine the role of glomerular endothelial glycocalyx as a barrier to the passage of protein.

Diabetes is commonly associated with chronic hypertension and evidence suggests that blood pressure control, even in the absence of tight glycemic control, can reduce the risk of diabetic complications in type 1 and type 2 diabetes [87, 88]. Hypertension is associated with raised capillary pressure which could provide a link between high blood pressure and the development of diabetic vascular complications in small blood vessels. Long term changes include stiffening of the blood vessels and, as a result, the cells feel less cyclic strain for a given luminal pressure. This leads to the question; is it altered pressure or strain that is important in reducing the risk of diabetic complications? Many existing models for exposing endothelial cells to mechanical forces in an in-vitro environment alter pressure and strain simultaneously, making it impossible to distinguish between the effects of the two potentially independent stimuli. This distinction is particularly relevant when examining the interaction of haemodynamic forces on microvascular endothelial cells, which are exposed to low hydrostatic pressure but significant strains. Part one describes the development of a haernodynarnlc model system that can evaluate independently the impact of pressure (15 to 35mmHg) and strain (0 to 10%), commensurate with the microvasculature on microvascular endothelial cell function. A perfusion model was developed which allows accurate recording of pressures generated inside the system. A method for applying a continuous sinusoidal cyclical strain of 5-10% is discussed and the model is validated under these conditions for 48 hour periods. Part one also examines the growth and attachment of human endothelial cells in compliant tubing and presents a method for obtaining a confluent layer of cells in the tubing. Part two describes preliminary in vitro studies on the effect of hydrostatic pressure on microvascular endothelial cells and other major environmental factors in type 2 diabetes, e.g. hyperinsulinaemia on the metabolic functions of microvascular and vascular endothelial cells, specifically fatty acid uptake. Preliminary studies indicated that chronic physiological insulin exposure inhibited the uptake of free fatty acids in human umbilical endothelial cells (HUVECs). Furthermore chronic exposure to raised levels of insulin reduced free fatty acid uptake further, which is contrary to studies on other cells lines. Extending the study to microvascular cells, it was also shown that acute exposure to pathophysiological insulin levels stimulated free fatty acid uptake in human brain endothelial cell line (hCMECs).

The effects of LC n-3 PUFA, EPA and OHA, on aspects of early stage atherosclerosis including monocyte to endothelial cell adhesion, monocyte to macrophage differentiation, and macrophage cholesterol levels, lipoprotein receptor, and lipid droplet-associated protein expression were investigated. LC n-3 PUFA treatment increased monocyte adhesion to endothelial monolayers and adhesion molecule expression (E-selectin; ICAM-1) in unstimulated HAECs with an opposite effect found in TNF-a stimulated HAECs (ICAM-1; VCAM-l). Adhesion was also decreased upon THP-1 monocyte incubation with LC n-3 PUFA, with no effect on expression of monocyte adhesion molecules. In monocyte to macrophage differentiation immature macro phages were affected at a lower L( n-3 PUFA concentration, and to a greater extent than mature macrophages. Upon L( n-3 PUFA treatment cell-surface expression of the differentiation markers CD11c, CD11b and CD49d on immature and mature macrophages, and (014 mRNA expression on mature macrophages were down- regulated, as was cell-surface expression of the scavenger receptor (036 in immature and mature macrophages, as well as (036 mRNA expression in immature The extent of macrophage differentiation influenced LC n-3 PUFA effects on cholesterol levels and lipoprotein expression. LC n-3 PUFA decreased net cholesterol levels in untreated, immature macrophages, and free cholesterol levels in native LDL treated, immature macro phages; mature macrophages were unaffected. LOX-l and LDLr mRNA expression were also decreased in immature macrophages. DHA increased adipophilin mRNA expression regardless of cell treatment. LC n-3 PUFA treatment during differentiation decreased CD36 cell-surface and mRNA expression in immature macrophages, and CD36 cell-surface and LOX-l mRNA expression in mature macrophages, whereas, post-differentiation treatment reduced LOX-l mRNA expression in immature macrophages with no effect on CD36 expression. LDLr mRNA expression was reduced with treatment during and post- differentiation. Treatment with the LC n-3 PUFA, EPA and DHA, can decrease monocyte adhesion to the endothelium, the expression of monocyte to macrophage differentiation markers, and cholesterol levels within differentiated macrophages.

The vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in angiogenesis and vascular physiology in higher eukaryotes such as vertebrates. Although intracellular signalling pathways linked to VEGFR2 activation have been well characterised, there is a lack of knowledge on membrane and cytosolic effectors that interact with VEGFR2. To address this problem, I have used a novel membrane protein yeast 2-hybrid (Y2H) screen to identify novel factors that interact with membrane VEGFR2. By screening a human primary endothelial cDNA library, six potential VEGFR2-binding proteins were isolated. One candidate was the SlOO calcium-binding protein A6 (SlOOA6). SlOOA6 is an abundant protein present in both the nucleus and cytoplasm of endothelial cells. The interaction between VEGFR2 and SlOOA6 was found to be calcium-dependent. Calcium dependent-translocation of S lOOA6 from the nucleus caused eo-distribution with VEGFR2 in punctate vesicles. Depletion of SI OOA6 protein levels using RNA interference perturbed VEGF-A-stimulated VEGFR2 activation and phosphorylation, cell proliferation, cell migration and angiogenesis. Taken together, the work in this thesis suggests that S lOOA6 is a novel binding partner for VEGFR2 and regulates intracellular signalling and angiogenesis. Thus SlOOA6 could be a new target in the treatment of vascular disease.