A isquemia/reperfusão intestinal (I/R-i) se associa ao desenvolvimento de inflamação pulmonar aguda, que pode ser modulada por mediadores inflamatórios presentes na linfa. Avaliamos os efeitos da I/R-i sob a inflamação pulmonar e a participação do sistema linfático. Wistar machos foram submetidos a 45 min de isquemia intestinal e 24, 72 ou 120 h de reperfusão. Outro grupo teve o ducto linfático bloqueado antes da isquemia. Os resultados revelaram maior inflamação pulmonar nos animais reperfundidos por 120 h em relação aos outros períodos de reperfusão estudados. Os animais apresentaram aumento de MPO e permeabilidade. Aumento de VEGF e de IL-1<font face=\"Symbol\">&#946 e diminuição de IL-10 no explante pulmonar. Diminuição de vWf e aumento de integrina <font face=\"Symbol\">&#9461, PECAM-1 e colágeno I e IV no endotélio pulmonar. Os dados indicam que mecanismos temporais modulam a resposta inflamatória decorrente da I/R-i. Mediadores na linfa e na circulação participam do desencadeamento/manutenção da inflamação pulmonar alterando a integridade do endotélio e ativando o pulmão que libera mediadores adicionais. / Intestinal ischemia/reperfusion (i-I/R) is associated with the development of acute lung inflammation, which can be modulated by inflammatory mediators present in the lymph. We evaluated the effects of i-I/R in lung inflammation and the involvement of the lymphatic system. Wistar rats were subjected to 45 min of intestinal ischemia and 24, 72 or 120 h of reperfusion. Another group had the lymphatic duct blocked before ischemia. The results revealed greater lung inflammation in animals reperfused for 120 h in comparison to other periods studied. These animals showed increased MPO and permeability. Increased VEGF and IL-1<font face=\"Symbol\">&#946 and decreased IL-10 in lung explants. Decreased vWf and increased <font face=\"Symbol\">&#9461 integrin, PECAM-1 and collagen I and IV in the pulmonary endothelium. These data indicate that temporal mechanisms modulate the inflammatory response due to i-I/R. Mediators in the lymph and circulation participate in the initiation / maintenance of lung inflammation by altering the integrity of the endothelium and activating the lung which release additional mediators.

A revascularização linfática é um desafio e o estabelecimento de novas estratégias terapêuticas podem melhorar a qualidade de vida de pessoas que sofrem de distúrbios linfáticos. O objetivo deste estudo foi verificar a capacidade de tratamento com VEGFC exógeno na melhoria da vascularização linfática de uma maneira dependente do tempo em membros pélvicos (MP) de camundongos após a remoção do linfonodo inguinal. O linfonodo inguinal esquerdo foi removido cirurgicamente para mimetizar patologias com diminuição da vascularização linfática. Densidade vascular linfática (Vv) e de comprimento (Lv) foram avaliadas por imunohistoquímica, seguidas de estereologia, após a cirurgia com ou sem o tratamento com VEGFC exógeno. O grupo controle não foi manipulado, mas recebeu soro fisiológico em vez de tratamento com VEGFC exógeno. As expressões do VEGFC e FLT4 local foram avaliadas por qPCR. Houve efeito do tempo sobre Vv e Lv no Grupo Cirurgia e diferença significativa entre os grupos Controle e Cirurgia nas três regiões estudadas (região proximal, média e distal) do MP esquerdo (MPE). A Lv mostrou diferença significativa entre os grupos Controle e Cirurgia somente na região média do MPE. A Vv e a Lv para o Grupo Tratamento foram maiores do que os outros grupos em todas as regiões do MPE. A expressão gênica do VEGFC e do FLT4 apresentou efeito do tempo em todas as regiões do MPE para os grupos Cirurgia e Tratamento. Ambas as expressões gênicas do VEGFC e do FLT4 apresentaram diferença significativa entre os grupos Controle e Cirurgia, entre os grupos Cirurgia e Tratamento e entre os grupos Controle e Tratamento. Os resultados mostraram que os camundongos são bons modelos experimentais para o uso de VEGFC exógeno como terapia de revascularização linfática, e o tratamento com VEGFC exógeno aumenta vascularização linfática já após 3 dias de dano linfático. / Lymphatic revascularization is a challenge and the establishment of new therapeutic strategies may improve quality of life from those suffering from lymphatic disorders. The objective of this study was to verify the VEGFC treatment capacity in improving lymphatic vascularization in a time-dependent manner in mouse hind limb (HL) after removal of inguinal lymphnode. The left inguinal lymphnode was surgically removed to mimetize pathologies with decreased lymphatic vascularization. Lymphatic vascular density (Vv) and length (Lv) were evaluated by immunohistochemistry followed by stereology after surgery and/or VEGFC treatment. Control group was not manipulated but received saline instead of VEGFC treatment. VEGFC and FLT4 local expression were assessed by qPCR. There was effect of time over Vv and Lv in the SG and significant difference between CG and SG in the three studied regions (proximal, medium and distal region) of the left HL (LHL). The Lv showed significant difference between CG and SG only in the medium region. The Vv and the Lv for TG were higher than the other groups in all regions of LHL. VEGFC and FLT4 gene expression presented time effect in all regions of the LHL for SG and TG. Both VEGFC and FLT4 gene expression presented significant difference between CG and SG, between SG and TG, and between CG and TG. The results show that mice are good experimental models for VEGFC use as therapy for lymphatic revascularization, and VEGFC treatment increased the lymphatic vasculature already after 3 days of lymphatic damage.

Lymph is primarily composed of fluid and proteins from the blood circulatory system that drain into the space surrounding cells, interstitial space. From the interstitial space, the fluid enters and circulates in the lymphatic system until it is delivered into the venous system. In contrast to the blood circulatory system, the lymphatic system lacks a central pumping organ dictating the predominant driving pressure and velocity of lymph. Transport of lymph via capillaries, pre-collecting and collecting lymphatic vessels relies on the synergy between pressure gradients, local tissue motion, valves and lymphatic vessel contractility. The direction of lymph transport is regulated by bicuspid valves distributed throughout pre-collecting and collecting lymphatic vessels. Effective transport of lymph into the venous system is of prime importance. Disruption of lymph transport, because of impaired lymphatic function, reduced numbers of vessels or valvular insufficiencies can have severe health consequences, including lymphedema for which current clinical therapies are not curative. The lymphatic valves are usually bicuspid, however, congenital malformations in the valve such as single leaflet valve formation and arrested lymphatic valve development are observed and can cause lymphedema. Here we employ 4-week-old mice to study the effects of valves and malformed valves on lymph transport shedding light into some of the potentially underlying consequences of lymphedema. Polyethylene glycol (PEG) coated latex particles were injected into the inguinal lymph node of anesthetized mice. Particle displacement measurements through efferent lymphatic vessels yielded velocity, wall shear stress, vorticity and strain of the efferent lymph flow field carrying lymph from subdermal inguinal lymph nodes. Lymphatic vessel endothelial Prox1 green fluorescent protein (GFP) marker enabled the detection of lymphatic vessel walls and valves. Flow field, flow velocity, flow rate, velocity profiles, wall shear stress, vorticity and strain values were compared in regions downstream of normal and malformed valves in two wild type mice. A Clec2-deficient mouse, which experiences lymphatic development defects and is used as a lymphedema model, was employed to further elucidate the lymphatic valves on transport. The absence of centralized pumping yields highly variable lymphatic flow cycles varying from one to fifteen seconds. The presence of lymphatic valves introduces boundary conditions that yield spatial and temporal flow gradients increasing the degree of complexity of lymph transport. The valves dictate the trajectory of the particles and promote the formation of recirculation zones. Even in the presence of valves, lymph flow commonly reverses. Congenital defects like a single leaflet valve lowers the lymph flow efficiency and promotes higher wall shear stress regions. Furthermore, the absence of functional valves in the Clec2-deficient mouse not displaying lymphedema yielded lymph flow lacking the pulsatility that characterizes normal lymphatic flow.

The leading cause of breast cancer-associated death is metastasis. During metastasis, tumor cells metastasize from primary tumors to distant organs via the circulatory and lymphatic systems. However, in 80% of solid tumors, metastasis via the lymphatic system precedes metastasis via the vascular system. There is a lot of information about metastasis through the circulatory system. However, not much information is available about the tumor cell dissemination through the lymphatic system or the lymphatic microenvironment that aids in this process in breast cancer metastasis. In addition, the molecular properties of tumor cells as they exit the primary tumor into the afferent lymphatics en route to the sentinel lymph nodes (SLNs) are not yet known.<br><div><br></div><div>This project aims to determine why and how tumor cells metastasize to the lymphatic system. The proposal is based on the hypothesis that active migration is needed for tumor cells to spread via the lymphatic vessels. Thus, finding and understanding the molecules that contribute to this can be a breakthrough for breast cancer metastasis therapy.<br></div><div><br></div><div>The goals of this thesis are to 1) Examine the molecular, genetic, and proteomic characteristics of circulatory tumor cells and compare these to the primary tumor and lung metastasis, 2) Examine the role of Toll-like receptors in tumor cell migration to the lymph node, and 3) Identify the difference in protein expression among two different types of breast cancer (Triple-Negative and Luminal A) and understand their aggressive biology.<br></div>

The graduation thesis is focused on Lymphedema patientś problems. It addresses how the patient values his or her own health state. The differences between a patientś health state before therapy and after finishing its reduction phase have been compared. The following aspect have been valued: limited movement, pain and mental stress. All of them were improved after the reduction phase. Thus Hypothesis 1 has been confirmed: "At the end of the Reduction Phase of Lymphedema therapy the overal health state of the patient is improved." Out of above meditioned three aspect, the mental stress improved at a lowest rate. Differences between patients with and without good social background were demonstrated. Patients with bad social background valued mental stress caused by Lymphedema before and after the therapy as significantly higher then patients with good social background. Thus Hypothesis 2 was also confirmed: "Patients with good social background cope with their disease in a better way." In order to test the hypotheses the quantitative research was done. The technique was standardised integration.

Toutes les cellules libèrent plusieurs types de vésicules extracellulaires (VEs) qui transportent protéines, lipides et acides nucléiques. Ces vésicules de petite taille se retrouvent dans tous les fluides biologiques tel que le sang et la lymphe et interagissent avec les cellules environnantes. Le système lymphatique constitue une voie de prédilection pour la mobilisation des accepteurs de cholestérol à partir de la paroi artérielle. Nous avons démontré dans un modèle murin qu’une dysfonction lymphatique précède la formation de la plaque d’athérome et que cette dysfonction touche a priori la capacité de contraction des vaisseaux collecteurs. À la base de toutes ces observations, nous avons émis l’hypothèse que les VEs contribuent à la dysfonction lymphatique liée à l’athérosclérose. Pour répondre à ceci, nous avons mis en place un projet translationnel composé de deux groupes de sujets sains, sans maladie cardiovasculaire, qui se distinguent par la présence d’antécédents familiaux d’accidents cardiovasculaires prématurés chez un parent du premier degré. Nous avons quantifié plusieurs sous-types d’intérêt de VEs en circulation à partir du plasma exempt de plaquettes, par cytométrie en flux ultraspécialisés dans la détection de petites particules (> 100 nm) combiné à d’autres techniques complémentaires standardisées. Ensuite, nous avons évalué la fonction lymphatique grâce à l’imagerie par proche infrarouge après injection du vert indocyanine (ICG). Nos résultats préliminaires sont prometteurs quant aux rôles des VEs et de la dysfonction lymphatique dans le développement de l’athérosclérose et corroborent avec nos observations faites chez la souris. Les sujets avec antécédents familiaux de maladie cardiovasculaire (MCV) présentent des signes de dysfonction lymphatique avant même l’apparition de plaques d’athérome subcliniques. La réponse mécano-sensible de leurs vaisseaux collecteurs paraît défectueuse et est observable de concert avec un profil de VEs qui présument une atteinte lymphatique. Ces résultats restent à être confirmer avec le recrutement de sujets additionnels et l'évaluation de la corrélation avec le score de risque polygénique de développer une MCV, dans l’objectif ultime de faire des VEs et de la fonction lymphatique de nouveaux biomarqueurs dans l’identification précoce des MCV. / All cell types release extracellular vesicles (EVs) that carry different types of cellular cargo, such as proteins, lipids and nucleic acids. These small vesicles are found in all biological fluids including blood and lymph and can interact with neighboring cells. The lymphatic system is a preferred route for the mobilization of cholesterol from the arterial wall. We have demonstrated in a mouse model that lymphatic dysfunction precedes the development of atherosclerosis and that that this dysfunction affects the contraction capacity of the collecting vessels. Based on these observations, we hypothesized that EVs contribute to atherosclerosis-associated lymphatic dysfunction. Therefore, we have initiated a translational study involving two groups of healthy subjects that differ in their risk of cardiovascular disease (CVD). We quantified several subtypes of circulating EVs on platelet-free plasma by ultraspecialized flow cytometry in the detection of small particles (> 100 nm) combined to other state-of-the art complementary techniques. Next, we assessed the lymphatic function using near-infrared imaging and injection of indocyanine green (ICG). Our preliminary results are promising for the role of EVs and lymphatic dysfunction in the development of atherosclerosis and corroborate with our observations made in mice. Individuals at high risk of CVD have signs of lymphatic dysfunction even before the onset of subclinical atherosclerosis. The mechano-sensitive response of their collecting vessels appears to be defective and is observable in concert with a profile of EVs that presume lymphatic damage. These results remain to be confirmed with the recruitment of additional subjects and the assessment of the correlation with the polygenic risk score of developing a CVD, in the hope of making these specific EV subsets and lymphatic function new biomarkers in the early detection of CVD.