Recruitment of neutrophils into the tissue during inflammation is a crucial component of the immune response. This study aimed to further understand the role of intercellular adhesion molecule-2 (ICAM-2) in this process. Endothelial cell (EC) ICAM-2 has been implicated in neutrophil extravasation however, its precise role in this process is largely unknown. To address this, the current investigation examined the expression and functional role of ICAM-2 in neutrophil-EC interactions in vivo. Analysis of EC ICAM-2 expression was performed in the mouse cremaster muscle using immunofluorescent staining and confocal microscopy. A high EC body expression of ICAM-2 relative to that of EC junctions in post-capillary venules was observed. It was therefore hypothesised that ICAM-2 could potentially be involved in both luminal neutrophil-EC and junctional interactions. This hypothesis was analysed using confocal intravital microscopy (IVM) of cremaster muscles from WT or ICAM-2 KO Lys-eGFP-ki mice (express fluorescent neutrophils) in conjunction with fluorescent labelling of ECs. Neutrophil crawling and transendothelial migration (TEM) dynamics in IL-1β-stimulated post-capillary venules was analysed. A role for ICAM-2 in supporting speed and continuity of crawling and the initiation of TEM was demonstrated. Using functional blocking mAb to MAC-1 in WT and ICAM-2 KOs, the role of ICAM-2 in neutrophil crawling was demonstrated to be governed through a potential interaction with neutrophil MAC-1. It is therefore possible that non-junctional EC ICAM-2 has important roles in regulating neutrophil polarisation during crawling whilst junctional ICAM-2 mediates the opening of EC junctions and/or influencing the site of ‘preferred’ TEM. This study provides the first in vivo evidence for the ability of ICAM-2 to support neutrophil crawling and the initiation of TEM in IL-1β-induced neutrophil extravasation. To extend the above findings in a complex vascular injury model, a cremasteric Shwartzman Reaction, amenable to IVM analysis, was also developed as part of this project.

Sexual dimorphisms exist in the incidence and severity of many diseases, with females demonstrating relative protection from inflammatory conditions. The extent and mechanisms by which excessive leukocyte recruitment underlies these differences are not well established, and better understanding is essential for the development of targeted therapies. Evidence suggests that variances in pathogen-sensing Toll-like receptors (TLRs) underlie sex-differences in leukocyte recruitment. This thesis aimed to investigate sex-differences in trafficking of leukocytes in the zymosan peritonitis murine model of acute inflammation and furthermore evaluate if these differences were accompanied by changes in TLR2 or TLR4 expression. This work shows that female mice recruit fewer classical monocytes and neutrophils during zymosan induced peritonitis. It demonstrates female murine peritoneal macrophages are more numerous, whilst the peritoneal cytokine environments and zymosan-sensing receptors are similar between the sexes. Sex-differences were evident in the circulation as female mice showed reduced neutrophilia and monocytosis versus male counterparts, despite having similar mobilisation from bone marrow (BM) stores. The work further revealed that storage and trafficking of splenic leukocytes during acute inflammation is distinct between the sexes. Male mice have greater splenic stores of neutrophils, classical- and non-classical- monocytes, despite similar spleen sizes, signifying another source of potential pathogenic leukocytes. Furthermore, males but not females mobilise splenic classical monocytes in response to peritonitis. Conversely, neutrophils appear to traffic to the spleen in females, but not males, in this model. Whilst BM neutrophils from males displayed more TLR2 and TLR4 than females, no major differences under basal or inflamed conditions in TLR2 or TLR4 expression were evident on leukocyte subsets. This work demonstrates that males and females have distinct leukocyte trafficking profiles in acute inflammation, and suggests that the spleen, not the BM, plays a role in determining sex-differences in the available pool of immune cells. Such dimorphisms demonstrate the importance of considering gender in assay development, drug design and clinical trials.

Leukocyte recruitment to sites of inflammation is a crucial event in host defense against pathogens and tissue injury. Although there is at present much interest in deciphering the mechanisms of leukocyte transendothelial cell migration, little attention has been paid to the subsequent steps, i.e. leukocyte migration through the pericyte layer and the venular basement membrane. In this context, results from this group previously demonstrated that neutrophils preferentially transmigrate through gaps between adjacent pericytes, regions associated with sites of low matrix protein expression within the vascular basement membrane. The aim of this thesis was to extend these findings by investigating the impact of inflammatory mediators on pericyte morphology and vascular basement membrane deposition using both in vitro and in vivo models. Flow cytometry analysis of pericyte-like C3H/10T1/2 cells and primary lung pericytes revealed the expression of key pro-inflammatory molecules on their surface (including cytokine receptors and adhesion molecules) and the regulation of these molecules upon cytokine stimulation. Using the murine cremaster muscle model it was further demonstrated that key neutrophil chemoattractants (i.e. LTB4, KC, C5a and fMLP) induced neutrophil transmigration that was associated with a change in pericyte morphology (as quantified through enlargement of gaps between adjacent pericytes). These changes in pericyte gap size were neutrophil-dependent and mediated by endogenously generated TNF as demonstrated in neutrophil-depleted mice and TNFR-/- mice, respectively. In addition, TNF appeared to mediate post-inflammatory BM deposition in response to LTB4 and was required for chemoattractant-induced vascular permeability. Hence, the results of the present work have demonstrated the ability of pericytes to respond to both cytokines and chemoattractants, suggesting an active role for pericytes in the regulation of inflammatory responses. In addition, findings provide the first evidence for chemoattractant-induced changes in vascular morphology and barrier functions of venular walls in vivo via the release of endogenous TNF as a secondary mediator, effects that may contribute to the pro-inflammatory properties of these stimuli.

No description available.

No description available.

Diabetic patients have an increased risk of developing both micro- and macrovascular complications. It is thought that the primary lesion in the genesis of these complications is at the level of the vascular endothelium. The effects of diabetes on endothelial function can be monitored in vitro using aortae isolated from streptozotocin-diabetic rats. This project found that two months of untreated diabetes resulted in a profound deficit in maximum endothelium-dependent relaxation to cumulative doses of acetylcholine in phenylepherine precontracted aortae. Treatment of rats with the antioxidants vitamin E and trientine from the induction of diabetes prevented the development of the deficit in endothelium-dependent relaxation, suggesting that increased levels of oxidative stress in diabetes contribute to abnormal endothelial function. Compensation for impaired essential fatty acid metabolism in diabetic rats with evening primrose oil partially prevented the development of defective aortic endothelium-dependent relaxation, implication this hyperglycaemia-induced metabolic disturbance in diabetic endothelial dysfunction. Diabetes is also associated with impaired penis erectile performance and importance in animals and patients. An in vitro preparation was developed to examine the effects of streptozotocin-induced diabetes on chemical stimulation of the endothelium and electrical stimulation of the autonomic nerves of rat corpus cavernosum. Both endothelium and neurally mediated relaxation responses were found to be impaired in two month untreated diabetic rats. Treatment of diabetic rats with the antioxidants α-lipoic acid and trientine prevented the formation of defective endothelium and nerve relaxation responses, thus implicating oxidative stress in the pathogenesis of these impairments. Inhibition of defective polyol pathway activity also prevented the development of reduced endothelium and nitrergic nerve responses, thus suggesting a role for this role for this well studied metabolic abnormality in impaired corpus cavernosum function in experimental diabetes.

Neutrophils are recognised to play a pivotal role at the interface between the innate and adaptive immune responses following their rapid recruitment to inflamed tissues and lymphoid organs. Whilst neutrophil trafficking through blood vessels has been extensively studied, the molecular mechanisms regulating their migration into the lymphatic system are still poorly understood. This thesis therefore aimed to investigate the mechanisms involved in neutrophil migration across the lymphatic endothelium during TNF- or Complete Freund's Adjuvant + antigen (CFA+Ag)-induced inflammation of cremaster muscles in vivo. This work revealed that TNF- or CFA+Ag-stimulation induces a rapid but transient entry of tissue-infiltrated neutrophils into lymphatic vessels, a response associated with the regulation and redistribution of the lymphatic endothelial cell glycocalyx. Interestingly, antigen sensitisation resulted in the production of endogenous TNF within cremaster muscles. Using anti-TNF blocking antibodies and mice deficient in both TNF receptors (p55 and p75), endogenous TNF was demonstrated for the first time to be involved in priming and triggering the migration of neutrophils into tissue-associated lymphatic vessels upon antigen challenge. Additionally, the use of chimeric mice exhibiting neutrophils deficient in both TNFRs demonstrated that TNF directly acts on leukocytes to induce neutrophil migration into lymphatic vessels. Furthermore, the results show that TNF-induced migration of neutrophils into the lymphatic system occurs in a strictly CCR7-dependent manner; blocking CXCR4 or CXCL1 signalling does not affect this response. Finally, both TNF- or CFA+AG-stimulation induced ICAM-1 up-regulation on lymphatic vessels, allowing neutrophils to crawl along the lumen; a response that was demonstrated to be TNF-dependent. These results have provided new insights into the mechanisms that mediate neutrophil migration into lymphatic vessels and their subsequent crawling within these vessels during inflammation. In particular, a new role for TNF as a key regulator of these processes has been demonstrated. Taken together, this work has highlighted potential and effective targets to manipulate the role of neutrophils in adaptive immune responses in vivo.

This thesis presents the development of new optical coherence tomography imaging systems and techniques to improve in vivo 3D microvascular imaging. Specifically these systems and techniques were proposed to address three main problems with 3D Doppler optical coherence tomography imaging: (a) Motion artefacts, (b) angle dependence of the signal, and (c) relatively high minimum detectable velocity of conventional color Doppler algorithms (~500 μm/s). In order to overcome these limitations a multi-pronged strategy was employed: (1) Construction of a retrospectively gated OCT system for the mitigation of periodic motion artefacts. Proof of principle in vivo B-mode imaging of Xenopus Laevis (embryo of African clawed frog) cardiovascular function up to 1000 frames per second (fps) from data acquired at 12 fps. Additionally, 4D imaging of the Xenopus Laevis heart at 45 volumes per second was demonstrated. (2) Construction of a Fourier domain mode locked laser for high speed swept source optical coherence tomography imaging. This laser was capable of reaching sweep rates of 67 kHz and was optimized to function in the SNR limited phase noise regimes upto approximately 55 dB structural SNR. (3) Development of a novel speckle variance image processing algorithm for velocity and angle independent 3D microvascular imaging. The velocity and angle independence of the technique was validated through phantom studies. iii In vivo demonstration of the speckle variance algorithm was performed by imaging the capillary network in the dorsal skin-fold window chamber model, with the results being validated using fluorescence confocal microscopy. In the final part of this thesis, these newly developed technologies were applied to the assessment of anti-vascular and anti-angiogenic therapies in preclinical models, specifically, photodynamic therapy and targeted degradation of HIF-α.

Microfluidic technology is a useful tool to help answer unsolved problems in multidisciplinary fields, including molecular biology, clinical pathology and the pharmaceutical industry.Current microfluidic based devices with diverse structures have been constructed via extensively used soft lithography orphotolithography fabrication methods. A layer-by-layer stacking of 2D planar microchannel arrays can achieve limited degrees of three dimensionality. However, assembly of large-scale multi-tiered structures is tedious, and the inherently planar nature of the individual layers restricts the network’s topological complexity. In order to overcome the limitations of existing microfabrication methodswe demonstrate several novel methods that enable microvasculature networks: electrostatic discharge,global channel deformation and enzymatic sculpting to fabricate complex surface topologies. These methods enable construction of networks of branched microchannels arranged in a tree-like architecture with diameters ranging from approximately 10 μm to 1 mm. Interconnected networks with multiple fluidic access points can be straightforwardly constructed, and quantification of their branching characteristics reveals remarkable similarity to naturally occurring vasculature. In addition, by harnessing enzymatic micromachining we are able to construct nanochannels, microchannels containing embedded features templated by the substrate’s crystalline morphology, and an irregular cross section of microchannel capable of performing isolation and enrichment of cells from whole blood with throughput 1 – 2 orders of magnitude faster than currently possible. These techniques can play a key role in developing an organ-sized engineered tissue scaffolds and high-throughput continuous flow separations.

This thesis presents the development of new optical coherence tomography imaging systems and techniques to improve in vivo 3D microvascular imaging. Specifically these systems and techniques were proposed to address three main problems with 3D Doppler optical coherence tomography imaging: (a) Motion artefacts, (b) angle dependence of the signal, and (c) relatively high minimum detectable velocity of conventional color Doppler algorithms (~500 μm/s). In order to overcome these limitations a multi-pronged strategy was employed: (1) Construction of a retrospectively gated OCT system for the mitigation of periodic motion artefacts. Proof of principle in vivo B-mode imaging of Xenopus Laevis (embryo of African clawed frog) cardiovascular function up to 1000 frames per second (fps) from data acquired at 12 fps. Additionally, 4D imaging of the Xenopus Laevis heart at 45 volumes per second was demonstrated. (2) Construction of a Fourier domain mode locked laser for high speed swept source optical coherence tomography imaging. This laser was capable of reaching sweep rates of 67 kHz and was optimized to function in the SNR limited phase noise regimes upto approximately 55 dB structural SNR. (3) Development of a novel speckle variance image processing algorithm for velocity and angle independent 3D microvascular imaging. The velocity and angle independence of the technique was validated through phantom studies. iii In vivo demonstration of the speckle variance algorithm was performed by imaging the capillary network in the dorsal skin-fold window chamber model, with the results being validated using fluorescence confocal microscopy. In the final part of this thesis, these newly developed technologies were applied to the assessment of anti-vascular and anti-angiogenic therapies in preclinical models, specifically, photodynamic therapy and targeted degradation of HIF-α.