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.

Although lymphatic vessel behavior is analogous to hearts (e.g. systole and diastole) and blood vessels (e.g. basal tone), hearts and blood vessels have fundamentally different contractile properties. While summation during contraction is minimized in the heart, summation is necessary for tonic contraction in blood vessels. Because lymphatic vessel behavior mimics cardiac and vascular behavior, we hypothesized that above a critical contraction frequency there is significant summation, evidenced by significantly increased diastolic active tension (i.e. basal tone). We used an isovolumic, controlled-flow preparation to examine the interaction of contraction cycle-time with contraction frequency. Using segments of isolated lymphatic vessels (~1 cm in length and 3-4 mm in diameter) from bovine mesentery, we measured transmural pressure and diameter for end-diastole and end-systole during spontaneous contractions for 10 volume steps. We found time between contractions (beat-to-beat period) decreases with increasing diameter, and total contraction time (vessel twitch length, 11.08 ± 1.54 s) slightly increases with increasing diameter. At the intersection of these relationships, there is a critical period, below which the vessel does not have time to fully relax. Above the diameter at the critical period, diastolic active tension (end-diastolic minus passive vessel tension) significantly increases with increases in diameter (309 to 562% change in slope, p<0.0001), and, below the critical period, diastolic active tension increases with decreases in beat-to-beat period (712 to 2208% change in slope, p<0.0014). Because this transition occurs within a physiological range, it suggests summation may be crucial for lymphatic vessel function as a pump and a conduit.

Proteases play a key role in the cascade of tumor-associated proteolysis leading to extracellular matrix degradation, stromal invasion and blood vessel recruitment and inroad. Protease systems are widely described as implicated in the formation of new blood vessels. Until now, only few datas are available concerning their role in lymphangiogenesis. We successfully transposed the aorta ring assay to a mouse lymphatic thoracic duct assay. By immunochemistry and transmission electron microscopy, we characterized the outgrowing cells as being lymphatic cells that organize into microvessels containing a lumen and that conserved lymphatic endothelial cell features. This quantifiable model responds to several well-known lymphangiogenic factors as the VEGF-C but not to specific angiogenic factors. This model is so suitable to screen growth factors and inhibitors as well as conditioned media. Plasminogen activator inhibitor-1 is a component of the plasminogen cascade and, though it was critical for angiogenesis, it comes out that it is dispensable for lymphatic outgrowth. In sharp contrast, synthetic and physiological inhibitors of matrix metalloproteases inhibit lymphangiogenesis, and thoracic duct rings derived from MMP-2- but not MMP-9-deficient mice showed an impaired lymphatic cell outgrowth. These data identify MMP2 as an important player in lymphangiogenesis and was confirmed by an in vivo experiments. Proteases are thus also implicated in lymphangiogenesis and the lymphatic ring assay seems to be helpful to discover novel genes and mechanisms that underly the lymphangiogenesis process, including by comparing with angiogenesis in a similar system.

Fundamental questions related to the locations of Cerebrospinal Spinal Fluid (CSF) absorption deficit and causes of the pressure gradients that expand the ventricles with hydrocephalus remain largely unanswered. Work in the Johnston lab over a 15 year period has demonstrated that CSF moves through the cribriform plate foramina in association with the olfactory nerves and is absorbed by a network of lymphatic vessels located within the olfactory turbinates. A kaolin-based rat model of communicating hydrocephalus was developed as a collaborative effort with Drs. McAllister, Wagshul and Li. After developing a method to quantify lymphatic CSF uptake in rats, we examined and observed that the movement of a radioactive tracer into the nasal turbinates was significantly reduced in the kaolin-injected animals compared to saline injected controls. However, it was possible that while lymphatic CSF uptake was compromised, other CSF absorption pathways may have compensated. To answer this, we measured the CSF outflow resistance (Rout) and observed it to be significantly greater in the kaolin group compared with animals receiving saline and there was a significant positive correlation between CSF Rout and ventricular volume. Nonetheless, it is not clear how impaired CSF clearance could lead to a dilation of the ventricles since the ventricular and subarachnoid compartments are in communication with one another and pressure would likely increase equally in both. At this point, we came across a theoretical paper that postulated that a drop in periventricular interstitial fluid pressure might provide an intraparenchymal pressure gradient favouring ventricular expansion. In addition, studies in non-CNS tissues indicated that a disruption of beta-1 (β1) integrin-matrix interactions could lower tissue pressure. Based on these suppositions and data, we examined if these concepts had relevance to the brain. For this, we measured pressure in the brain and observed a decline in periventricular pressures to values significantly below those monitored in the ventricular system following the injection of the anti integrin antibodies. Many of the animals developed hydrocephalus over 2 weeks post antibody injection. These data provide a novel mechanism for the generation of intraparenchymal pressure gradients that is likely contributing to ventricular expansion.

The lymphatic system plays an important role in protein and solute transport as well as the immune system. Its functionality is vital to proper homeostasis and fluid balance. Lymphatic fluid (lymph) may be propelled by intrinsic (active) vessel pumping or passively. With regard to the former, nitric oxide (NO) is known to play an important role in lymphatic vessel contraction and vasodilation. Lymphatic endothelial cells (LECs) are sensitive to shear and increases in flow have been shown to cause enhanced production of NO by LECs. Additionally, high concentrations of NO have been experimentally observed in the sinus region of mesenteric lymphatic vessels. The goal of this work was to develop a computational flow and mass transfer model using physiologic geometries obtained from confocal images of a rat mesenteric lymphatic vessel to determine the characteristics of NO transport in the lymphatic flow regime. Both steady and unsteady analyses were performed. Steady models were simulated by prescribing fully developed velocity profiles ranging from 0.5 mm s^-1 to 7 mm s^-1 as the inlet boundary conditions. Unsteady simulations were generated using a velocity profile taken from experimental data from in situ experiments with rats. Production of NO was shear-dependent; basal cases using constant production were also generated. Simulations revealed areas of flow stagnation adjacent to the valve leaflets, suggesting the high concentrations observed here experimentally are due to lack of convection in this region. LEC sensitivity was found to alter the concentration of NO in the vessel, and the convective forces were found to profoundly affect the concentration of NO at a Peclet value greater than or equal to approximately 61. The quasi-steady analysis was able to resolve wall shear stress within 0.15% of the unsteady case. However, the percent error between unsteady and quasi-steady conditions was higher for NO concentration (approximately 6.7%).

Fundamental questions related to the locations of Cerebrospinal Spinal Fluid (CSF) absorption deficit and causes of the pressure gradients that expand the ventricles with hydrocephalus remain largely unanswered. Work in the Johnston lab over a 15 year period has demonstrated that CSF moves through the cribriform plate foramina in association with the olfactory nerves and is absorbed by a network of lymphatic vessels located within the olfactory turbinates. A kaolin-based rat model of communicating hydrocephalus was developed as a collaborative effort with Drs. McAllister, Wagshul and Li. After developing a method to quantify lymphatic CSF uptake in rats, we examined and observed that the movement of a radioactive tracer into the nasal turbinates was significantly reduced in the kaolin-injected animals compared to saline injected controls. However, it was possible that while lymphatic CSF uptake was compromised, other CSF absorption pathways may have compensated. To answer this, we measured the CSF outflow resistance (Rout) and observed it to be significantly greater in the kaolin group compared with animals receiving saline and there was a significant positive correlation between CSF Rout and ventricular volume. Nonetheless, it is not clear how impaired CSF clearance could lead to a dilation of the ventricles since the ventricular and subarachnoid compartments are in communication with one another and pressure would likely increase equally in both. At this point, we came across a theoretical paper that postulated that a drop in periventricular interstitial fluid pressure might provide an intraparenchymal pressure gradient favouring ventricular expansion. In addition, studies in non-CNS tissues indicated that a disruption of beta-1 (β1) integrin-matrix interactions could lower tissue pressure. Based on these suppositions and data, we examined if these concepts had relevance to the brain. For this, we measured pressure in the brain and observed a decline in periventricular pressures to values significantly below those monitored in the ventricular system following the injection of the anti integrin antibodies. Many of the animals developed hydrocephalus over 2 weeks post antibody injection. These data provide a novel mechanism for the generation of intraparenchymal pressure gradients that is likely contributing to ventricular expansion.

Although lymphatic vessel behavior is analogous to hearts (e.g. systole and diastole) and blood vessels (e.g. basal tone), hearts and blood vessels have fundamentally different contractile properties. While summation during contraction is minimized in the heart, summation is necessary for tonic contraction in blood vessels. Because lymphatic vessel behavior mimics cardiac and vascular behavior, we hypothesized that above a critical contraction frequency there is significant summation, evidenced by significantly increased diastolic active tension (i.e. basal tone). We used an isovolumic, controlled-flow preparation to examine the interaction of contraction cycle-time with contraction frequency. Using segments of isolated lymphatic vessels (~1 cm in length and 3-4 mm in diameter) from bovine mesentery, we measured transmural pressure and diameter for end-diastole and end-systole during spontaneous contractions for 10 volume steps. We found time between contractions (beat-to-beat period) decreases with increasing diameter, and total contraction time (vessel twitch length, 11.08 ± 1.54 s) slightly increases with increasing diameter. At the intersection of these relationships, there is a critical period, below which the vessel does not have time to fully relax. Above the diameter at the critical period, diastolic active tension (end-diastolic minus passive vessel tension) significantly increases with increases in diameter (309 to 562% change in slope, p<0.0001), and, below the critical period, diastolic active tension increases with decreases in beat-to-beat period (712 to 2208% change in slope, p<0.0014). Because this transition occurs within a physiological range, it suggests summation may be crucial for lymphatic vessel function as a pump and a conduit.

No description available.

Fluid drainage via the lymphatics prevents swelling due to excess fluid in interstitial space. Since interstitial fluid volume can vary dramatically, the function of lymphangions are rather dynamic so that they can contend with the wide ranges of lymph flow and pressure. Sharing with blood vessels an acute sensitivity to mechanical stresses, lymphangions could be expected to be similarly sensitive to prolonged changes in transmural pressure and flow. The purpose of this research was to quantify the adaptation of lymphangions to prolonged alterations in lymph hydrodynamics and to investigate how adaptation of individual lymphangions acts together within a network to affect the regulation of lymph flow. In project I, bovine postnodal mesenteric lymphatic vessels were partially occluded for three days, which divided the vessel into two segments. Both segments, therefore, were exposed to the same flow but different transmural pressures. In project II, an isometric preparation was employed to study developed wall tension in lymphangions exposed to mesenteric venous hypertension. In project III, an analytical model of a symmetrical lymphatic network was developed to investigate how its pressure-flow relationship emerges from the complex interaction of its pumping lymphangions. Results from this research indicate that lymphangions become stronger pumps when exposed to prolonged increases in transmural pressure, but exposure to prolonged venous hypertension attenuates lymphangion pumping. The main mechanism for lymphangion adaptation is the alteration of preload-dependent maximal tension developed by lymphatic muscle. Lower cytosolic Ca2+ concentration is likely to be the underlying cause for weaker developed tension. The analytical model suggests that the ratio of lymphangion systolic contractility to diastolic stiffness results in a differential response of lymph flow to changes in inlet and outlet pressures. This differential sensitivity to inlet and outlet pressures allows homeostatic responses to both microvascular and venous hypertension, and provides a framework for interpreting the functional implications of chronic lymphangion adaption.

The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Historically, the angiogenesis field has advanced faster and farther than the field of lymphangiogenesis. The discovery of lymphatic markers and the emerging evidence implicating the lymphatic system as a central player in a variety of pathological conditions has attracted research interest and driven the field forward. Research efforts have produced the observation that regulators of the blood endothelium are frequently members of the same protein families of regulators of the lymphatic endothelium. More importantly, these regulators do not act discretely, restricting their regulatory activities to one endothelial cell (EC) type. Two examples of regulators that behave in this manner are the VEGF and the Angiopoietin families of proteins, which have cell-type-dependent effects on EC processes such as migration, proliferation and survival. The study of these regulators therefore requires an in vitro EC system capable of accommodating the simultaneous characterization of the signaling pathways downstream of these shared molecular regulators in venous, arterial and lymphatic endotheliums. To build such an in vitro system, I isolated and validated lymphatic, venous, and arterial ECs derived from vessels of bovine mesentery. The proteomes of the three cell types were comparatively studied using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric identification. The three cell types were used in a subtractive immunization scheme for the production of a monoclonal antibody selectively reactive to a potentially novel surface protein marker of lymphatic ECs. The studies recorded herein all share the common goal of identifying and characterizing unique molecular signatures that distinguish lymphatic ECs from blood ECs, and that may underline the cellular biology of the lymphatic endothelium as distinct from the blood endothelium.