published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

In view of the discrepancies and conflicts produced by previous studies on amphibian lymph hearts, a study was initiated to reinvestigate the pharmacological, temperature, and electrical aspects of lymph heart physiology. Bullfrogs were chosen as the experimental animal, All lymph heart responses to experimentation were physiographically recorded as myograms and electrograms. The results are in agreement with previous studies on some aspects and in conflict on others. From the results obtained, lymph heart muscle appears to possess both skeletal and cardiac muscle properties as evidenced by drug responses and reactions to temperature. The precise components of the electrogram remain unclear. It is suggested that further investigation should be made to better determine the true nature of lymph hearts.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 268-300.

The lymphatic system has fundamental physiological roles in maintaining fluid homeostasis, immune cell trafficking and lipid transport from the small intestine to the venous circulation. Lymphatic vessels are the main functional organ responsible for the diverse transport roles the system plays. Unlike the blood vasculature, the lymphatic system does not have a central pump, such as the heart, and relies on a variety of factors to move lymph through. It was long thought that only external factors, such as skeletal muscle contraction and lymph formation, played a role in the functional transport capacity of these vessels. With the advancement of imaging capabilities (both hardware and software), it has become clear in the past two decades or so that the main factor in driving lymph transport is the ability of these vessels to intrinsically contract whereby each vessel is comprised of a chain of ‘mini pumps’ in series. The functional capacity of these vessels is thus now understood to be primarily determined by this pumping activity that has been shown to be regulated by various mechanical and biochemical cues. Lymphatic vessel dysfunction has been implicated in a variety of diseases including many lipid related pathologies and a neglected tropical disease known as lymphatic filariasis. While it has been possible to study the vessel function in the context of fluid drainage and immune cell trafficking, the capability to understand the role of lymphatic vessels in lipid transport has not been available due to the lack of experimental animal models and acquisition systems. As part of this thesis, we sought to develop an experimental animal model along with hardware and software tools to investigate the interplay between lymphatics and their lipid content. We report the first functional measurements of how vessels respond to elevated lipid loads. We further utilized our engineering expertise to develop an experimental platform allowing us to further understand the parasite known as B. malayi that migrates to and resides in lymphatic vessels.

Ce mémoire de maîtrise porte sur le développement d’un système d’administration de microaiguilles pour livrer des sondes et des capteurs fluorescents dans le contexte du diagnostic et de la surveillance des soins de santé. Bien que parfois négligés en faveur des soins de santé axés sur le traitement, le diagnostic précoce de la maladie et la surveillance préventive des paramètres biologiques peuvent considérablement améliorer les résultats des soins de santé et joueront probablement un rôle plus important dans les années à venir. Cependant, il reste des obstacles importants à cette approche, à savoir le caractère relativement invasif et perturbateur des analyses biologiques. La nécessité de se rendre dans une clinique et de subir un prélèvement de sang (ou de liquide biologique) invasif présente des inconvénients importants par rapport aux traitements classiques, qui consistent souvent de médicaments pouvant être pris à domicile sans douleur. Une solution à ces problèmes réside dans la mise au point de systèmes minimalement invasifs de diagnostic et de suivi médical, idéalement ceux qui peuvent être utilisés à domicile sans nécessiter de personnel qualifié. À cet égard, les microaiguilles sont une technologie au potentiel énorme, car leur petite taille les rend peu invasives et pratiquement indolores, et leur nature simple à usage unique permet potentiellement une administration à domicile par le patient. Particulièrement prometteuses pour les applications de diagnostic et de surveillance sont les microaiguilles en polymère soluble; fabriquées à partir de polymères synthétiques ou biologiques injectables, ces microaiguilles sont solubilisées après la perforation de la peau, libérant ainsi les composés qu’elles contiennent. Bien que prévu initialement pour la livraison d'agents thérapeutiques, en utilisant ces microaiguilles pour livrer des molécules fluorescentes spécifiquement conçues, il est possible de créer un tatouage médical de diagnostic affichant un signal fluorescent précis. En associant cette technologie à un détecteur de fluorescence portable, la surveillance en temps réel d’un large éventail de paramètres biologiques pourrait devenir accessible en dehors du contexte clinique. Afin de fournir un contexte pour le développement de cette technologie, cette mémoire commence par une revue des principes et des avancées majeures récentes dans le domaine des applications diagnostiques des microaiguilles (Chapitre 1). Par la suite, un tatouage par microaiguille est présenté sous la forme d'un capteur de ROS délivré sur la peau, avec des implications diagnostiques pour le vieillissement et la carcinogenèse de la peau liés aux UV, ainsi que pour des affections inflammatoires telles que le psoriasis, comme validation de concept (Chapitre 2). En outre, un autre tatouage par microaiguille est introduit, consistant d’un capteur spécialement adapté ciblant le système lymphatique, permettant la quantification en temps réel du drainage lymphatique, avec des implications pour la détection précoce de plusieurs affections, notamment le lymphœdème (Chapitre 3). / This Master’s thesis concerns the development of a microneedle (MN) delivery system for fluorescent dyes and sensors in the context of diagnostics and healthcare monitoring. While sometimes overlooked in favor of treatment-focused healthcare, early disease diagnosis and preventative monitoring of biological parameters can meaningfully improve healthcare outcomes and will likely play a greater role in coming years. However, significant obstacles to this approach remain, namely the relatively invasive and disruptive nature of biological analyses. The need to travel to a clinic and undergo invasive blood (or biological fluid) sampling presents significant inconveniences relative to common treatments, often consisting of medications that can be taken painlessly at home. A solution to these problems lies in the development of minimally invasive systems for diagnostics and healthcare monitoring, ideally ones which can be used at home without the need for trained personnel. In this regard, MNs are a technology with tremendous potential, as their small size renders them minimally invasive and virtually painless, and their simple, single-use nature potentially allows for at-home administration by the patient. Showing particular promise for diagnostic and monitoring applications are dissolving polymeric MNs; made from injectable synthetic or biological polymers, these MNs are solubilized after breaching the skin, releasing any compound contained within. Though initially envisioned for the delivery of therapeutic agents, by using these MNs to deliver specifically designed fluorescent molecules, it is possible to create a diagnostic medical tattoo displaying a precise fluorescent signal. By pairing this technology with a portable fluorescence detector, real-time monitoring of a wide range of biological parameters could become accessible outside of a clinical setting. To provide context for the development of this technology, this thesis begins with a review of the principles and major recent advances in the field of diagnostic applications of MNs (Chapter 1). Subsequently, a proof-of-concept MN tattoo is introduced in the form of a ROS-sensor delivered to the skin, with diagnostic implications for UV-related skin aging and carcinogenesis, as well as inflammatory conditions such as psoriasis (Chapter 2). Further, another MN tattoo is introduced, consisting of a specifically tailored sensor targeting the lymphatic system, allowing the real-time quantification of lymphatic drainage, with implications in the early detection of several conditions, including lymphedema (Chapter 3).

No description available.

Orientadores: Maria Cristina Cintra Gomes Marcondes, Dora Maria Grassi Kassisse / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T12:12:43Z (GMT). No. of bitstreams: 1 Camargo_EricaAparecidaMariano_M.pdf: 3544834 bytes, checksum: d9515fe1a07bef154d0bed7c21be4a79 (MD5) Previous issue date: 2015 / Resumo: A importância da validação científica de técnicas coadjuvantes a diversos tratamentos de saúde é inquestionável. Assim a influência da drenagem linfática manual na mobilização hidroeletrolítica, na taxa lipolítica, bem como, no sistema nervoso autonômico ainda precisa ser investigada. O objetivo deste trabalho foi avaliar o efeito da drenagem linfática manual na mobilização hidroeletrolítica, na taxa lipolítica e na modulação autonômica da frequência cardíaca em homens e mulheres. Foram estudados 11 homens; 11 mulheres não usuárias de anticoncepcional oral e 12 mulheres usuárias de anticoncepcional oral, com 21,3 ± 2,9 anos de idade, saudáveis, sedentários e eutróficos. As amostras urinárias foram coletadas em um dia, sem intervenção terapêutica, denominado controle e em outro dia com aplicação da drenagem linfática manual. Na urina, foram analisadas osmolaridade, concentração de sódio, fluxo e concentração de glicerol. A variabilidade da frequência cardíaca foi registrada no início, durante e ao final do experimento, nos dias controle e drenagem linfática manual. Os resultados mostram que a drenagem linfática manual promoveu em homens redução dos eletrólitos urinários e aumento do fluxo e em mulheres não usuárias de anticoncepcional oral houve apenas aumento do fluxo. A técnica induziu diluição urinária em homens e mulheres não usuárias de anticoncepcional, sugerindo que os mecanismos são sexo-dependentes. Para usuárias de anticoncepcional oral, a drenagem linfática manual não diferiu do dia controle, onde houve redução dos eletrólitos e aumento do fluxo urinário. A análise da concentração de glicerol urinário mostrou que a drenagem linfática manual não altera a taxa lipolítica nos voluntários dos grupos estudados. Os resultados da modulação autonômica da frequência cardíaca indicaram que a técnica promoveu predomínio simpático no grupo de homens sem alterações nos grupos das mulheres. Conclui-se que a drenagem linfática manual foi eficaz na diluição urinária de homens e mulheres não usuárias de anticoncepcional oral, assim como, foi eficaz em promover predomínio simpático em homens. A técnica não foi eficaz em promover alterações na taxa lipolítica / Abstract: The importance of scientific validation techniques supporting the various health treatments is unquestionable. Thus, the influence of manual lymphatic drainage in the electrolyte mobilization in lipolytic rate as well as in the autonomic nervous system has to be investigated. The objective of this study was to evaluate the effect of manual lymphatic drainage in the electrolyte mobilization in lipolytic rate and autonomic modulation of heart rate in men and women. 11 male patients; 11 women not using oral contraceptives and 12 women used oral contraceptives, with 21.3 ± 2.9 years old, healthy, sedentary and eutrophic. The urine samples were collected in one day, without therapeutic intervention, denominated as the control and on another day with application of manual lymphatic drainage. In the urine, were analyzed osmolality, sodium concentration, flow, and the concentration of glycerol. The heart rate variability was registered at the outset, during and at the end of the experiment, in the days control and manual lymphatic drainage. The results showed that the manual lymph drainage in men promoted reduction of urinary electrolytes and increased flow and in women non-users oral contraceptives there was only increased flow. The technique induced urinary dilution in men and women non-users oral contraceptives, suggesting that the mechanisms are sex-dependent. For oral contraceptive users, manual lymphatic drainage did not differ from control day where there was a reduction of electrolytes and increased urine flow. The analysis of urinary glycerol concentration showed that manual lymphatic drainage does not change the lipolytic rate in voluntary groups studied. The results of the autonomic modulation of heart rate indicated that the technique promoted sympathetic predominance in men group and without changes in women. We conclude that manual lymphatic drainage was effective in urinary dilution of men and women non-users oral contraceptives, as well as, it was effective in promoting sympathetic predominance in men. The technique was not effective in promoting changes in lipolytic rate / Mestrado / Fisiologia / Mestra em Biologia Funcional e Molecular

Le système lymphatique joue un rôle clé dans le transport du cholestérol hors de la paroi artérielle et une dysfonction lymphatique précède l'apparition de l'athérosclérose. Cette dysfonction est associée à une diminution de l’expression du récepteur des lipoprotéines de faible densité (LDLR) et est à priori indépendante des taux de cholestérol circulant. Il a été démontré que les souris dépourvues de la proprotéine subtilisine/kexine de type 9 (PCSK9), une proprotéine qui mène à la dégradation du LDLR, ont une fonction lymphatique améliorée, tandis que les souris dépourvues de LDLR développent une dysfonction lymphatique. Nous visons maintenant à mieux comprendre les mécanismes par lesquels la présence de PCSK9 dans la lymphe ou la modulation du LDLR sur les cellules endothéliales lymphatiques (CEL) affecte la fonction lymphatique. Les CEL sont incubées avec du PCSK9 ou traitées avec un ARN inhibant spécifiquement l’expression du LDLR afin d’évaluer comment la présence de PCSK9 ou la modulation du LDLR affecte l'intégrité des CEL. Nos résultats démontrent que le PCSK9 n’induit pas la sécrétion de cytokines inflammatoires et n'affecte pas l'expression des marqueurs lymphatiques. L’inhibition de l’expression du LDLR entraîne une diminution des marqueurs lymphatiques membranaires endothéliaux. La diminution du LDLR a aussi entrainé une diminution des taux de certains lipides intracellulaires et en particulier des phospholipides, des sphingolipides et des triglycérides. Ces résultats suggèrent qu'une perte du LDLR, mais pas la présence de PCSK9 en circulation, pourrait induire à une altération de l'endothélium lymphatique causée par une diminution de l’expression de protéines membranaires essentielles au bon transport de la lymphe. / The lymphatic system plays a key role in the removal of cholesterol from the artery wall and lymphatic dysfunction is known to occur prior to the onset of atherosclerosis. This dysfunction is associated with reduced expression of the low-density-lipoprotein receptor (LDLR) and is first independent of circulating cholesterol levels. It has been shown that mice lacking proprotein subtilisin/kexin type 9 (PCSK9), a proprotein that degrades LDLR, have improved lymphatic function while mice lacking LDLR had a lymphatic dysfunction. We now aim to better understand the mechanisms by which the presence of PCSK9 in lymph or the modulation of LDLR on lymphatic endothelial cells (LECs) affects lymphatic function. Incubation of LECs with PCSK9 and specific targeting of LDLR expression with silencing RNAs were used to further evaluate how PCSK9 or modulation of LDLR affect the metabolism and integrity of LECs. Our results demonstrate that PCSK9 does not induce the secretion of inflammatory cytokines and does not affect the expression of lymphatic markers. LDLR silencing RNA leads to a decrease of membrane-bound lymphatic endothelial cell markers. A decrease in LDLR expression also led to a decrease in the content of some intracellular lipids and particularly phospholipids, sphingolipids and triglycerides. These results suggest that loss of LDLR expression, but not circulating PCSK9, could lead to alterations in the lymphatic endothelium caused by loss of membrane integrity which could in turn affect lymph transport.

A key step in the development of an adaptive immune response is the activation of naive T cells by dendritic cells (DCs). DCs sample antigens in the periphery and migrate to the lymphoid organs were they provide different signals to T cells: they present antigenic peptides in the context of MHC molecules, express costimulatory or coinhibitory ligands and produce cytokines that influence T cell fate. The integration of these signals will either induce tolerance or lead to the activation and expansion of effector T cells which will mediate the immune response.<p>The costimulatory CD70/CD27 pathway plays important roles in the development of pro-inflammatory Th1 and CTL responses. CD70 expression on DCs has also been described as a molecular switch from tolerance to immunity. Accordingly, its activity is tightly regulated in vivo. The aim of this work was to investigate the mechanisms controlling the expression of CD70 on dendritic cells and CD27 on T lymphocytes.<p>First, we described a cell-extrinsic mechanism of inhibition exerted on DCs by regulatory T cells (Tregs). Indeed, Tregs controlled Th1 priming in vivo and in vitro by downregulating CD70 on DCs. This control involved a transfer of the CD27 receptor to DCs, possibly via the production of CD27-bearing microvesicles by T cells at the immunological synapse. Acquisition of CD27 by DCs induced the internalization of both CD27 and CD70 and probably their lysosomal degradation. As a consequence, DCs were impaired in their ability to efficiently prime Th1 cells. Second, we analyzed CD70 and CD27 expression in the periphery and provided evidence for a cell-intrinsic control of CD27 expression by ectodomain shedding in the gastrointestinal tract.<p>While they efficiently clear infections, inflammatory responses can also be deleterious to the organism. By restraining CD70 expression on DCs, Tregs would promote tolerance and limit inflammation. Interestingly, tolerance is particularly important in the intestines, which are in constant contact with dietary antigens and the commensal microbiota. Accordingly, we propose that a second layer of control of CD27-driven costimulation takes place in the gut :by shedding CD27, T cells would be desensitized for any potential CD70-dependent costimulation.<p>To further investigate the physiological significance of the mechanisms described above, the immune response will be monitored in animals specifically lacking CD27 expression in the Treg population or expressing a nonsheddable CD27 receptor.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

El hígado cirrótico experimenta a lo largo de todo el desarrollo de la enfermedad una serie de cambios estructurales, debidos a la acumulación de matriz extracelular y a la inflamación crónica, que vuelven al tejido hipóxico. La hipoxia tisular provoca la liberación de agentes proangiogénicos, como VEGF-A, que aumentan la irrigación del tejido para compensar la falta de oxígeno. Además, muchas citoquinas sintetizadas por las células inflamatorias o por las células hepáticas estrelladas activadas, también tienen un alto poder angiogénico. Estos nuevos vasos son una puerta de entrada de más infiltrado inflamatorio, estableciéndose así un circuito que se retroalimenta entre angiogénesis e inflamación que lleva a perpetuar la enfermedad. En este contexto, las terapias antiangiogénicas clásicas dirigidas a bloquear VEGF o las vías de los receptores tirosina quinasa, han demostrado ser eficaces en disminuir el grado de angiogénesis, de fibrosis y de inflamación hepática, además de disminuir también la presión portal (Mejias et al., 2009; Tugues et al., 2007). Sin embargo, este tipo de terapias se han visto relacionadas con multitud de efectos adversos, como trombosis o hipertensión, ya que no sólo inhiben la angiogénesis patológica sino que también afectan a la vasculatura sana (Fischer et al., 2007; Wu et al., 2008). De manera que la necesidad de encontrar nuevas dianas terapéuticas más seguras destinadas a modular la angiogénesis en la cirrosis hepática sigue siendo uno de los objetivos de la investigación científica actual. Cuando la cirrosis pasa a ser descompensada, los pacientes cirróticos desarrollan importantes alteraciones en la homeostasis de los líquidos corporales que dan lugar a la acumulación de ascitis. La formación de ascitis es el trastorno más importante que presentan los pacientes cirróticos y constituye un fenómeno extraordinariamente complejo en el que se han descrito la implicación de diversos sistemas neurohormonales y sustancias endógenas capaces de regular el metabolismo renal de sodio y agua o la reactividad vascular (Arroyo and Jiménez, 2000). La probabilidad de fallecimiento a los 2 años en pacientes cirróticos, una vez que desarrollan ascitis, es del 40%. A pesar de la enorme relevancia clínica de este suceso, actualmente aun no se conocen con precisión todos los mecanismos moleculares implicados en la formación y mantenimiento de la ascitis en la cirrosis. El objetivo global de esta tesis doctoral fue el de investigar la implicación de la vasculatura, tanto sanguínea como linfática, en la patogenicidad de la cirrosis hepática. De manera específica, la tesis está dividida en dos estudios dirigidos a: I. Investigar el papel fisiopatológico del factor de crecimiento placentario (PlGF) en la angiogénesis, la inflamación y la fibrosis hepática. II. Caracterizar la funcionalidad y la estructura de la vasculatura linfática en la cirrosis e investigar su contribución a la formación de ascitis. Estudio I: Durante la progresión de gran cantidad de enfermedades hepáticas crónicas tiene lugar un proceso de neoangiogénesis hepática que agrava profundamente dichas patologías. Además, se ha demostrado que diversos factores angiogénicos también son capaces de activar diferentes subpoblaciones de células hepáticas estrelladas, estableciendo así un vínculo entre inflamación, angiogénesis y fibrosis hepáticas (Thabut et al., 2010; Semela et al., 2008). En este contexto, se ha demostrado que la administración de sunitinib y sorafenib a ratas cirróticas, dos inhibidores multidiana de receptores tirosina quinasa que actúan en las vías de señalización de PDGF y VEGF, conseguía disminuir el grado de angiogénesis, de fibrosis y de inflamación hepáticas, además de disminuir también la presión portal (Mejias et al., 2009; Tugues et al., 2007). Sin embargo, este tipo de terapias dirigidas a inhibir VEGF están asociadas a graves efectos adversos, como trombosis, hipertensión o una disminución de la densidad vascular en órganos sanos (Fischer et al., 2007), ya que VEGF también tiene importantes funciones en la angiogénesis fisiológica. Estudios recientes sugieren que las terapias enfocadas a inhibir el PlGF podrían tener un perfil de bioseguridad mucho más óptimo que las terapias antiangiogénicas clásicas (Fischer et al., 2007; Van de Veire et al., 2010). Esto es así porque la acción angiogénica de PlGF se reduce únicamente a situaciones patológicas (Autiero et al., 2003; Van Steenkiste et al., 2009). Resultados I: La cirrosis está asociada a una sobreexpresión de PlGF, tanto en humanos como en modelos experimentales. Cuando se bloquea la actividad de PlGF en cirrosis se consigue disminuir la angiogénesis hepática y en esplácnica, el infiltrado inflamatorio hepático, la fibrosis y la hipertensión portal; todo ello sin efectos adversos. Estudio II: Una de las complicaciones más severas durante la progresión de la cirrosis es la formación de ascitis, que está asociada con un incremento de la morbilidad y la mortalidad (Moore et al., 2003). Algunos de los mecanismos que contribuyen a la aparición y al mantenimiento de la ascitis son: 1) el incremento de la presión hidrostática intravascular, 2) el descenso de la presión oncótica en el plasma, 3) un aumento de la angiogénesis en el territorio esplácnico y 4) la retención renal de sal y agua (Gines et al., 2004; Van Steenkiste et al., 2009; Henriksen et al., 2005). Sin embargo, la función del sistema linfático en la patogénesis de la ascitis no está bien estudiada. En los pacientes cirróticos compensados, el sistema linfático ayuda a prevenir la aparición de ascitis reabsorbiendo el exceso de fluido, tanto en el hígado, como en las demás regiones de la zona esplácnica. Como consecuencia de esto aumenta el flujo linfático (Witte et al., 1969), lo que estimula la aparición de linfangiogénesis hepática (Tugues et al., 2005). A pesar de ello, este mecanismo compensatorio no es capaz de evitar la formación de ascitis en pacientes con cirrosis descompensada y esto hace hipotetizar que existe una disfunción del sistema linfático en los estadíos más avanzados de la enfermedad. Resultados II: En cirrosis, las células endoteliales linfáticas sobreexpresan eNOS y tienen una sobreproducción de NO que lleva a una pérdida de recubrimiento de los vasos linfáticos por células musculares lisas y a una disfunción del sistema linfático. Cuando se bloquea la actividad de eNOS se consigue recuperar el recubrimiento de vasos linfáticos por células musculares lisas, aumenta el drenaje del sistema linfático y disminuye el volumen de ascitis. / Study I: Placental growth factor (PlGF) is associated selectively with pathological angiogenesis, and PlGF blockade does not affect the healthy vasculature. Anti-PlGF is therefore currently being clinically evaluated for the treatment of cancer patients. In cirrhosis, hepatic fibrogenesis is accompanied by extensive angiogenesis. In this study, we evaluated the pathophysiological role of PlGF and the therapeutic potential of anti-PlGF in liver cirrhosis. Results I: PlGF was significantly up-regulated in the CCl4-induced rodent model of liver cirrhosis as well as in cirrhotic patients. Compared with wild-type animals, cirrhotic PlGF -/- mice showed a significant reduction in angiogenesis, arteriogenesis, inflammation, fibrosis, and portal hypertension. Importantly, pharmacological inhibition with anti-PlGF antibodies yielded similar results as genetic loss of PlGF. Notably, PlGF treatment of activated hepatic stellate cells induced sustained extracellular signal-regulated kinase 1/2 phosphorylation, as well as chemotaxis and proliferation, indicating a previously unrecognized profibrogenic role of PlGF. In conclusion, PlGF is a disease-candidate gene in liver cirrhosis, and inhibition of PlGF offers a therapeutic alternative with an attractive safety profile. Study II: The lymphatic network plays a major role in maintaining tissue fluid homoeostasis. Therefore several pathological conditions associated with oedema formation result in deficient lymphatic function. However, the role of the lymphatic system in the pathogenesis of ascites and oedema formation in cirrhosis has not been fully clarified. The aim of this study was to investigate whether the inability of the lymphatic system to drain tissue exudate contributes to the oedema observed in cirrhosis. Results II: The cirrhotic (CH) rats had impaired lymphatic drainage in the splanchnic and peripheral regions compared with the control (CT) rats. LyECs isolated from the CH rats showed a significant increase in eNOS and nitric oxide (NO) production. In addition, the lymphatic vessels of the CH rats showed a significant reduction in smooth muscle cell (SMC) coverage compared with the CT rats. CH rats treated with L-NMMA for 7 days showed a significant improvement in lymphatic drainage and a significant reduction in ascites volume, which were associated with increased plasma volume. This beneficial effect of L-NMMA inhibition was also associated with a significant increase in lymphatic SMC coverage. In conclusion, the upregulation of eNOS in the LyECs of CH rats causes long-term lymphatic remodelling, which is characterised by a loss of SMC lymphatic coverage. The amelioration of this lymphatic abnormality by chronic eNOS inhibition results in improved lymphatic drainage and reduced ascites.