The homeobox transcription factor PROX1 is the mammalian ortholog of the Drosophila gene Prospero. Expression of PROX1 in a subset of venous endothelial cells changes their fate to lymphatic endothelial cells (LEC). PROX1 is required for lymphatic development as Prox1 null mice lack all lymphatic vasculature. PROX1 has been shown to have cell-type dependent roles in regulating the cell cycle. We hypothesize that PROX1 functions as a key cell cycle regulator in LECs and promotes their cell cycle progression. In this study, immunocytochemistry, western blotting and luciferase assays were used to characterize PROX1 mediated activation of the mouse Ccne1 promoter. Following deletion of the Prospero 1 domain (PD1∆), the resulting PROX1 protein is localized to both the nucleus and the cytoplasm. We have determined that PROX1 requires both E2F binding sites located in the Ccne1 promoter to activate transcription of the gene. We observed that siRNA knockdown of Prox1 reduced CYCLIN E1 protein levels as well as decreased cellular proliferation in LECs. In contrast, overexpression of a version of PROX1 in which the homeodomain and Prospero domain 2 (HDPD2Δ) were deleted increased CYCLIN E1 protein levels in human umbilical vein endothelial cells (HUVEC), but resulted in the arrest of cells in the G1 phase. We have also established that PROX1 is phosphorylated in primary human LECs. We have shown a role for the PD1 domain in mediating PROX1 subcellular localization and we have observed that the expression of the HDPD2Δ version of PROX1 blocks proliferation in HUVECs. We are the first to demonstrate a role for PROX1 as a transcriptional co-activator and to establish that PROX1 is phosphorylated in LECs.

The homeobox transcription factor PROX1 is the mammalian ortholog of the Drosophila gene Prospero. Expression of PROX1 in a subset of venous endothelial cells changes their fate to lymphatic endothelial cells (LEC). PROX1 is required for lymphatic development as Prox1 null mice lack all lymphatic vasculature. PROX1 has been shown to have cell-type dependent roles in regulating the cell cycle. We hypothesize that PROX1 functions as a key cell cycle regulator in LECs and promotes their cell cycle progression. In this study, immunocytochemistry, western blotting and luciferase assays were used to characterize PROX1 mediated activation of the mouse Ccne1 promoter. Following deletion of the Prospero 1 domain (PD1∆), the resulting PROX1 protein is localized to both the nucleus and the cytoplasm. We have determined that PROX1 requires both E2F binding sites located in the Ccne1 promoter to activate transcription of the gene. We observed that siRNA knockdown of Prox1 reduced CYCLIN E1 protein levels as well as decreased cellular proliferation in LECs. In contrast, overexpression of a version of PROX1 in which the homeodomain and Prospero domain 2 (HDPD2Δ) were deleted increased CYCLIN E1 protein levels in human umbilical vein endothelial cells (HUVEC), but resulted in the arrest of cells in the G1 phase. We have also established that PROX1 is phosphorylated in primary human LECs. We have shown a role for the PD1 domain in mediating PROX1 subcellular localization and we have observed that the expression of the HDPD2Δ version of PROX1 blocks proliferation in HUVECs. We are the first to demonstrate a role for PROX1 as a transcriptional co-activator and to establish that PROX1 is phosphorylated in LECs.

The lymphatic system plays an important role in fluid and protein balance within the interstitial spaces. Its dysfunction could result in a number of debilitating diseases, namely lymphedema. Lymphatic vessels utilize both intrinsic and extrinsic mechanisms to pump lymph. Intrinsic pumping involves the active contraction of vessels, a phenomenon that is regulated in part by nitric oxide (NO) produced by lymphatic endothelial cells (LECs). NO production by arterial endothelial cells has been shown to be sensitive to both shear stress and stretch. Therefore, because of the unique mechanical environment of the LECs, we hypothesize that mechanical forces play an important role in regulation of the lymphatic pumping. Parallel-plate flow chambers and indenter-based cyclic stretch devices were constructed and used to apply mechanical loads to LECs. In addition, high-throughput micro-scale channels were developed and tested for shear experiments to address the need to increase the productivity and high- resolution imaging. Twenty-four hours treatment of LECs with different shear stress conditions showed a shear-dependent elevation in NO production. Moreover, 2.5 folds increase in cumulative NO was observed for stretched cells compared to the unstretched cells over six hours period. In conclusion, the upregulation observed in NO production under mechanical stimuli suggest new regulatory mechanisms that can be pharmaceutically targeted. These results provide an unprecedented insight into lymphatic pumping mechanism.

No description available.

Non-hematopoietic stromal cells (SCs) promote and regulate adaptive immunity through numerous direct and indirect mechanisms. SCs construct and support the secondary lymphoid organs (SLOs) in which lymphocytes crawl on stromal networks and inspect antigen-presenting cells for surface-display of cognate antigens. SCs also secrete survival factors and chemotactic cues that recruit, organize, and facilitate interactions among these leukocytes. They influence antigen access by secreting and ensheathing extracellular matrix-based conduit networks that rapidly convey small, soluble lymph-borne molecules to the SLO core. Furthermore, lymph node stromal cells (LNSCs) directly induce \(CD8^+\) T cell tolerance to peripheral tissue restricted antigens and constrain the proliferation of newly activated T cells in these sites. Thus, stromal-hematopoietic interactions are crucial for the normal functioning of the immune system. LNSCs are extremely rare and difficult to isolate, hampering the thorough study of their biology. In order to better understand these stromal subsets, we sorted fibroblastic reticular cells (FRCs), lymphatic endothelial cells, blood endothelial cells, and podoplanin \(^−CD31^−\) cells (double negative stromal cells; DNCs) to high purity from resting and inflamed murine lymph nodes. We meticulously analyzed the transcriptional profiles of these freshly isolated LNSCs as part of the Immunological Genome Project Consortium. Analysis of the transcriptional profiles of these LNSC subsets indicated that SCs express key immune mediators and growth factors, and provided important insights into the lymph node conduit network, FRC-specialization, and the DNC identity. Examination of hematopoietic and stromal transcription of ligands and cognate receptors suggested complex crosstalk among these populations. Interestingly, FRCs dominated cytokine and chemokine transcription among LNSCs, and were also enriched for higher expression of these genes when compared with skin and thymic fibroblasts, consistent with FRC-specialization. LNSCs that were isolated from inflamed lymph nodes robustly upregulated expression of genes encoding cytokines, chemokines, antigen-processing and presentation machinery, and acute-phase response molecules. Little-explored DNCs showed many transcriptional similarities to FRCs, but importantly did not transcribe interleukin-7. We identified DNCs as consisting largely of myofibroblastic pericytes that express integrin \(\alpha 7\). Together these data comprehensively describe the transcriptional characteristics of four major LNSC subsets isolated from resting and inflamed SLOs, offering many avenues for future study.

No description available.

Le récepteur activateur de NF-κB (RANK), membre de la famille des récepteurs au TNF, est connu pour son rôle dans l’homéostasie de l’os, mais joue aussi un rôle important dans le système immunitaire. J’ai tout d’abord étudié des outils permettant de cibler RANK/RANKL. J’ai caractérisé et comparé l’activité biologique de deux anticorps anti-RANK. J’ai également criblé une librairie de petites molécules pour identifier des inhibiteurs de l’interaction RANK/RANKL. Dans une deuxième partie, je me suis intéressée au rôle du ligand de RANK (RANKL) dans l’homéostasie du ganglion lymphatique. RANKL joue un rôle dans la différenciation des ostéoclastes mais son rôle dans la différenciation d’autres macrophages n’a pas été étudié. Nous avons étudié des souris déficientes pour RANKL dans les cellules marginales réticulaires (MRC) qui expriment RANKL de manière constitutive dans le ganglion adulte. Nous avons observé une diminution de la population de macrophages sous-capsulaires (SSM). Nous avons également montré que les cellules endothéliales lymphatiques (LEC) expriment l’intégrine alpha 2b (ITGA2b) et que cette expression est sensible à la présence de RANKL. / The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation. Firstly I looked for new molecular tools to target RANK/RANKL axis. I characterized and compared the biological activity of two anti-RANK antibodies. Moreover, I screened the Prestwick Chemical Library® of small molecules in order to identify inhibitors of RANK/RANKL interaction. Secondly, I studied the effect of the RANK/RANKL axis in lymph node homeostasis. RANKL is known to promote osteoclast differentiation but whether it also plays a role in the differentiation of other macrophage subsets is not known. We addressed this question by conditionally deleting RANKL from marginal reticular stromal cells (MRCs) that constitutively express RANKL in the lymph node. We observed impaired differentiation of the subcapsular sinus macrophages (SSMs). We also studied lymph node lymphatic endothelial cells (LECs) and showed that integrin alpha 2b (ITGA2b) is expressed by a lymph node subset of LECs and its expression is sensitive to RANKL.

L'athérosclérose est une maladie inflammatoire chronique caractérisée par l'accumulation de cholestérol dans la paroi artérielle et associée à une réponse immunitaire anormale dans laquelle les macrophages jouent un rôle important. Récemment, il a été démontré que les vaisseaux lymphatiques jouent un rôle primordial dans le transport inverse du cholestérol (Martel et al. JCI 2013). L’objectif global de mon stage de maîtrise a été de mieux caractériser la dysfonction lymphatique associée à l’athérosclérose, en étudiant de plus près l’origine physiologique et temporelle de ce mauvais fonctionnement. Notre approche a été d’étudier, depuis l’initiation de l’athérosclérose jusqu’à la progression d’une lésion athérosclérotique tardive, la physiologie des deux constituants principaux qui forment les vaisseaux lymphatiques : les capillaires et collecteurs lymphatiques. En utilisant comme modèle principal des souris Ldlr-/-; hApoB100+/+, nous avons pu démontrer que la dysfonction lymphatique est présente avant même l’apparition de l’athérosclérose, et que cette dysfonction est principalement associée avec un défaut au niveau des vaisseaux collecteurs, limitant ainsi le transport de la lymphe des tissus périphériques vers le sang. De plus, nous avons démontré pour la première fois l’expression du récepteur au LDL par les cellules endothéliales lymphatiques. Nos travaux subséquents démontrent que ce défaut de propulsion de la lymphe pourrait être attribuable à l’absence du récepteur au LDL, et que la dysfonction lymphatique observée précocement dans l’athérosclérose peut être limitée par des injections systémiques de VEGF (vascular endothelial growth factor) –C. Ces résultats suggèrent que la caractérisation fonctionnelle de la capacité de pompage des vaisseaux collecteurs serait une condition préalable à la compréhension de l'interaction entre la fonction du système lymphatique et la progression de l'athérosclérose. Ultimement, nos travaux nous ont amené à considérer de nouvelles cibles thérapeutiques potentielles dans la prévention et le traitement de l’athérosclérose. / Atherosclerosis is driven by the accumulation of cholesterol in the arterial wall, which triggers an inappropriate immune response in which macrophages play an important role. It has now been shown that the lymphatic vessels play an important role in reverse cholesterol transport (Martel et al. JCI 2013). The overall objective of my Master internship was to better characterize lymphatic dysfunction associated with atherosclerosis, studying closely the physiological and temporal origin of this pathological feature. Our approach was to study, from the initiation of atherosclerosis to the progression of the atherosclerotic lesion, the physiology of the two main components that form the lymphatic vessels: the lymphatic capillaries and collectors. Using a mouse model that closely resembles human atherosclerosis (Ldlr-/-; hApoB100+/+) we have demonstrated that lymphatic dysfunction is present before the onset of atherosclerosis, and that this dysfunction is primarily associated with a defect in the collecting vessels, thereby limiting the lymph transport from peripheral tissues to the blood. In addition, we have clearly demonstrated, for the first time to our knowledge, the presence of the LDL receptor on lymphatic endothelial cells. Our subsequent work shows that this reduction in lymph flow could be due to the absence of the LDL receptor, and that lymphatic transport can be restored by systemic injections of VEGF (vascular endothelial growth factor) –C. These results suggest that the functional characterization of the pumping capacity of the collecting vessels would be a prerequisite for the understanding of the interactions between the function of the lymphatic system and the progression of atherosclerosis. Altogether, our work unveils new potential therapeutic targets for the prevention and treatment of atherosclerosis.

No description available.

Les plaquettes sont essentielles au développement lymphatique dès l’embryogenèse et maintiennent la séparation lympho-sanguine au cours de la vie. Elles participent directement à la lymphangiogenèse et à la réparation des vaisseaux en plus d’améliorer l’intégrité lymphatique in vitro. En présence d’apolipoprotéine A-I (apoA-I), les plaquettes consolident les connexions intercellulaires en adhérant à l’endothélium lymphatique. Toutefois, les plaquettes sont absentes de la lymphe, contrairement à leurs vésicules extracellulaires (pEVs) exprimant un protéome similaire. De plus, leur concentration dans la lymphe s’accentue en condition d’inflammation chronique, par exemple lors de l’athérosclérose. En ayant caractérisé les effets de ces vésicules sur l’intégrité lymphatique durant ma maîtrise, nous montrons que les pEVs sont rapidement internalisées par les LECs et aident à préserver l’intégrité lymphatique des effets délétères des constituants de la lymphe tels que les EVs dérivées des globules rouges (rbEVs). In vitro, des concentrations physiologiques de pEVs limitent la production d’espèces réactives d’oxygène (ROS) et diminuent la nécrose des cellules. Comme les pEVs injectées dans le derme de la peau sont prises en charge par les cellules endothéliales des vaisseaux lymphatiques collecteurs et qu’elles voyagent jusqu’aux ganglions afférents, nous croyons qu’une partie des pEVs circulantes dans la lymphe serait cruciale pour le maintien d’une fonction lymphatique adéquate lors de maladies chroniques inflammatoires telles que l’athérosclérose. / Platelets have a protective role in lymphatic function both at the embryogenic stage and throughout life by maintaining blood-lymphatic separation. In addition, platelets enhance the integrity of lymphatic endothelial cells (LECs) in vitro and appear to exert a shielding effect on the lymphatic endothelium by consolidating the connexion between lymphatic endothelial cells when incubated with apolipoprotein A-I (apoA-I). Whereas platelets are absent from lymph, platelet extracellular vesicles (pEVs) are abundantly circulating within lymphatic vessels, and their level increases during chronic inflammation such as atherosclerosis. Having characterized their effect on lymphatic integrity during my Master internship, we show that pEVs are rapidly internalized by LECs, which in turn help preserve the integrity of the lymphatic endothelium when harmful blood constituent such as red blood cell EVs (rbEVs) are infiltrating lymph. In vitro, physiological concentrations of pEVs are limiting the production of reactive oxygen species (ROS) by lymphatic endothelial cells and decreasing their necrosis rate. Furthermore, pEVs injected in the skin interstitium travel through the collecting lymphatics and are rapidly internalized by LECs, which in turn might help preserve the integrity of the lymphatic endothelium. Lymph pEVs might be critical for the maintenance of a proper lymphatic function during chronic inflammatory settings such as atherosclerosis.