The lymphatic system has three primary roles: transporting lipids, transporting immune cells, and maintaining fluid balance. Each one of these roles are influenced by the presence of flow. Inflammation increases lymph flow, lipid uptake is enhanced by flow, cancer cell migration increases in the presence of flow, and lymphatic permeability and lymphatic contractility respond to changes in flow. Flow is very important to lymphatic function, and yet, there are no in vitro models that incorporate both luminal (flow along cell lumen) and transmural (flow through cell lumina) flow for lymphatics. To address this need, a microfluidic device has been developed that can incorporate both of these types of flow. This is achieved by driving flow through a channel which creates a pressure gradient that drives fluid through a porous membrane into an adjacent channel. Following several design iterations, the device can be easily fabricated, imaged, and cells can grow and survive in it. Permeability experiments have been performed in static and flow, 0.175 mL/min (0.5 dyne/cm²), cases. The effective permeability of dextran in the static and flow cases was calculated to be 0.0083 μm/s and 2.05 μm/s respectively. While the effective permeability of bodipy in the static and flow cases was calculated to be 0.0053 μm/s and 2.57 μm/s respectively. The static values are similar to values obtained in a transwell study by Dixon et al. As mentioned, lipid uptake is increased in the presence of flow and these numbers suggest the same. In addition to permeability studies, experiments were performed with cancer cells suspended in a collagen gel. Two image processing techniques were used to quantify cancer cell migration. The first technique was used to calculate the number of cells present at the beginning of the experiment and the number of cells that were ever present during the experiment in that particular z slice. The static case yielded a cell flux of 15 additional cells. While the two flow cases, within interstitial flow range, had a flux of 24 and 40 cells. This suggests that flow increases migration in cancer cells and is in agreement with the literature. The second technique was used to show that the cells in the static and flow cases are similarly motile, but the flow case is more directed in the z direction towards the membrane. The future work for this device is quite extensive, but a strong foundation centered around basic capabilities like inducing flow, seeding cells, and imaging has been formed.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 122-135).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 40-47).

The lymphatic system comprises a network of vessels whose primary functions are the maintenance of extracellular fluid balance and the transport of antigen-presenting cells from the periphery to the lymph nodes, thus facilitating immunological surveillance of the tissues and activation of adaptive immunity. In malignant disease, the lymphatics are both a route for dissemination and a reservoir for metastatic cancers such as cutaneous melanoma and breast carcinoma, where lymph node involvement is an early indicator of prognosis. Yet, despite such obvious importance in disease, the fundamental biology of the lymphatics is poorly understood and critical mechanisms such as those underlying trafficking of dendritic and tumour cells have been largely overlooked. The focus of this thesis is lymphatic vessel endothelial hyaluronan receptor LYVE-1, and the regulation of its binding to the extracellular matrix glycosaminoglycan, hyaluronan (HA). Found selectively on the surface of lymphatic vessels, but sharing many of the features of the leukocyte homing receptor CD44, LYVE-1 appears a likely candidate for regulation of lymphatic trafficking during the stage at which cells migrate into the vessel. However, the precise function of LYVE-1 in recent years has remained enigmatic, not least because the native receptor is subject to post-translational modification with sialic acid, with the effect that HA binding is inhibited in lymphatic endothelium. The results of this thesis demonstrate that sialylation of LYVE-1 is not a short term regulatory modification, but rather a longer term mechanism that imposes a requirement for higher order receptor complex formation or HA crosslinking to achieve stable binding. This implies that native LYVE-1 is an active HA binding protein even when sialylated, with implications for our understanding of the role played by the receptor in HA uptake and metabolism, and transmigration of lymphatic endothelium by migratory leukocytes. Like CD44, HA binding to LYVE-1 is dependent on multivalent interactions between receptor and ligand, and may be enhanced by processes that increase the avidity of the interaction. Here for the first time it is shown that the native LYVE-1 molecule on lymphatic endothelium may be activated to bind HA following clustering of the receptor, or presentation of HA in a cross-linked form, such as that resulting from incubation with the HA binding protein TSG-6.

Call number: LD2668 .T4 1962 V47

Le réseau lymphatique permet le drainage des liquides interstitiels, le transport des cellules immunitaires et intervient dans le métabolisme lipidique. La dérégulation de ce système est impliquée dans de nombreuses pathologies comme les lymphœdèmes, le rejet de greffe ou encore l'échappement tumoral. Le cœur est pourvu d'un réseau lymphatique abondant dont l'importance n'est apparue que très récemment dans les pathologies ischémiques. En effet, suite à un infarctus du myocarde, la dysfonction lymphatique observée favorise l'œdème et l'inflammation tissulaire, révélant le réseau lymphatique comme un acteur majeur du développement de cette pathologie. Les processus régulant la formation de vaisseaux lymphatiques chez l'adulte sont largement dépendants de facteurs de croissance aux effets pro- ou anti- lymphangiogéniques. Les cellules immunes et plus particulièrement les macrophages sécrètent un grand nombre de ces facteurs et leur importance dans la réponse lymphangiogénique a été montrée dans différentes conditions physio-pathologiques, telles que la lymphangiogenèse tumorale et inflammatoire. Il a longtemps été admis que les macrophages tissulaires provenaient exclusivement de la différenciation de monocytes sanguins. Cependant, l'existence de macrophages provenant de progéniteurs embryonnaires a été récemment mise en évidence. Dorénavant, les macrophages tissulaires résidents sont perçus comme des populations hétérogènes pouvant prétendre à des différences fonctionnelles. A ce jour, les modifications du réseau lymphatique et les mécanismes permettant sa régulation, au cours de pathologies non ischémiques, n'ont pas été abordées. Ainsi, nous avons entrepris d'identifier et de caractériser les différentes populations de macrophages cardiaques participant à la régulation du réseau lymphatique suite à une surcharge barométrique. Nos résultats ont montré une diminution précoce du réseau lymphatique cardiaque dans le cœur murin hypertrophié au cours d'une surcharge de pression induite par constriction de l'aorte transverse. Cette réduction du réseau est associée à la perte d'une population majoritaire de macrophages cardiaques portant le récepteur 1 à l'acide hyaluronique (Lyve-1). Cette population résidente diminue dans le cœur insuffisant, au profit de macrophages infiltrants dérivés de monocytes sanguins. Par ailleurs, en plus d'être corrélée à la baisse du réseau lymphatique cardiaque, la diminution du nombre de macrophages Lyve-1 est proportionnelle à la détérioration de la fonction cardiaque. La prévention de l'infiltration monocytaire a permis de maintenir le pool de macrophages exprimant Lyve-1, le réseau lymphatique et la fonction cardiaques lors d'une surcharge de pression. La caractérisation par RT-PCR des différentes populations de macrophages récupérés par tri cellulaire nous a permis de montrer que les macrophages Lyve-1 présentent des caractéristiques particulières, avec une forte expression de VEGFR3 et NRP2, molécules de signalisation lymphangiogène. De plus, ces macrophages Lyve-1 à la polarisation mixte et à l'activité phagocytaire importante, expriment de nombreux facteurs pro-lymphangiogéniques (VEGFc, VEGFd, IGF1). Ils ont montré une activité pro-lymphangiogène in vitro sur des explants de canal thoracique et sur des cellules endothéliales lymphatiques et in vivo avec la formation de vaisseaux lymphatiques naissants lors de l'injection de ces macrophages Lyve-1 dans le cœur. Leur localisation à proximité des vaisseaux lymphatiques et leur capacité pro-lymphangiogène leurs confèrent un rôle évident dans le maintien du réseau lymphatique lors d'un remodelage cardiaque de surcharge de pression. Ces travaux ont permis l'identification d'une population originale de macrophages cardiaques pouvant entrer dans la régulation du système lymphatique au cours d'une surcharge de pression. / The lymphatic system has recently emerged as an important regulator of the cardiac interstitial fluid compartment and function. Experimental obstruction of lymphatic vessel leads to cardiac œdema, myocardial stiffness, fibrosis and ventricle dysfunction. Following myocardial infarction, stimulation of lymphangiogenesis was found to reduce fibrosis and inflammation and to improve cardiac function. Macrophages have been largely described as important contributors of lymphangiogenesis in inflammatory situations such as cancer. Recently, genetic fate mapping demonstrated that distinct populations of macrophages coexist in the adult heart. In addition to monocyte derived-macrophages that massively colonize injured heart, a subpopulation of tissue-resident macrophages that originates from embryonic precursors persists into adulthood by means of local self- renewal. To date the distinct involvement of cardiac macrophage subpopulations in cardiac lymphatic remodeling and heart failure progression induced by pressure overload is largely unknown. In our study, we observed that expression of Lyve-1 identifies a resident macrophage subset abundant in mouse heart. This Lyve-1 positive macrophage subset decreased rapidly in cardiac remodeling induced by chronic pressure overload. In addition, the number of cells found in heart was positively correlated with the preservation of cardiac lymphatic network and function after transverse aortic constriction (TAC). Blocking recruitment of monocyte derived macrophages expanded Lyve-1 positive macrophages, attenuated cardiac lymphatic remodeling and contractile dysfunction of pressure overloaded heart. Lyve-1 positive macrophages express pro-lymphangiogenic factors and sustain lymphangiogenesis in vitro and in vivo. In conclusion, resident macrophage subset expressing the Lyve-1 receptor participates to maintain cardiac function after chronic pressure overload by mechanisms that may involve the preservation of cardiac lymphatic system. These results provide insight into the regulation of lymphatic homeostasis by tissue resident macrophage during heart failure induced by pressure overload.

Currently published data on the role of the lymphatic system in amphibians are inadequate and contradictory. Estimates of the rate of formation of lymph and the role of the lymph hearts in returning this fluid to the circulation are not based on actual volume determinations but rather estimates derived from changes in hematocrit using published values of plasma and blood volume. The lymph hearts are known to be vital to the maintenance of normal fluid compartment physiology and to increase their rate of activity during episodes of hypovolemic stress. Yet, significant redistribution of body fluids following hemorrage appears to occur in animals without lymph hearts. In this study, plasma and blood volumes were determined by the dye dilution technique using injected Evan's blue dye to label the plasma. Eight intact and 6 animals with their lymph hearts destroyed were hemorrhaged to 78% and 75% of their initial blood volumes, respectively. Changes in blood volume were measured following the hemorrhage by analysis of Evan's blue washout and hemodilution.

Abstract not available

Collecting microlymphatics play a vital role in promoting lymph flow from the initial lymphatics in the interstitial spaces to the large transport lymph ducts. In most tissues, the primary mechanism for producing this flow is the spontaneous contractions of the lymphatic wall. Individual units, known as lymphangion, are separated by valves that help prevent backflow when the vessel contracts, thus promoting flow through the lymphatic network. Lymphatic contractile activity is inhibited by flow in isolated lymphatics, however there are virtually no in situ measurements of lymph flow in these vessels. Initially, a high speed imaging system was set up to image in situ preparations at 500 fps. These images were then manually processed to extract information regarding lymphocyte velocity (-4 to 10 mm/sec), vessel diameter (25 to 165 um), and particle location. Fluid modeling was performed to obtain reasonable estimates of wall shear stress (-8 to 17 dynes/cm2). One of the difficulties encountered was the time consuming methods of manual particle tracking. Using previously captured images, an image correlation method was developed to automate lymphatic flow measurements and to track wall movements as the vessel contracts. Using this method the standard error of prediction for velocity measurements was 0.4 mm/sec and for diameter measurements it was 7.0 µm. It was found that the actual physical quantity being measured through this approach is somewhere between the spatially averaged velocity and the maximum velocity of a Poiseuille flow model.

Collecting microlymphatics play a vital role in promoting lymph flow from the initial lymphatics in the interstitial spaces to the large transport lymph ducts. In most tissues, the primary mechanism for producing this flow is the spontaneous contractions of the lymphatic wall. Individual units, known as lymphangion, are separated by valves that help prevent backflow when the vessel contracts, thus promoting flow through the lymphatic network. Lymphatic contractile activity is inhibited by flow in isolated lymphatics, however there are virtually no in situ measurements of lymph flow in these vessels. Initially, a high speed imaging system was set up to image in situ preparations at 500 fps. These images were then manually processed to extract information regarding lymphocyte velocity (-4 to 10 mm/sec), vessel diameter (25 to 165 um), and particle location. Fluid modeling was performed to obtain reasonable estimates of wall shear stress (-8 to 17 dynes/cm2). One of the difficulties encountered was the time consuming methods of manual particle tracking. Using previously captured images, an image correlation method was developed to automate lymphatic flow measurements and to track wall movements as the vessel contracts. Using this method the standard error of prediction for velocity measurements was 0.4 mm/sec and for diameter measurements it was 7.0 µm. It was found that the actual physical quantity being measured through this approach is somewhere between the spatially averaged velocity and the maximum velocity of a Poiseuille flow model.